Imibono ehlukene yokuvikela izitshalo inikeza isiqondiso esibalulekile semfundiso yokuchaza amaphethini e-metabolism ekhethekile yezitshalo, kodwa izibikezelo zazo ezibalulekile zisazovivinywa. Lapha, sisebenzise ukuhlaziywa kwe-tandem mass spectrometry (MS/MS) okungachemile ukuze sihlole ngokuhlelekile i-metabolome yezinhlobo ezincishisiwe zikagwayi ezivela ezitshalweni ngazinye kuya kubantu kanye nezinhlobo ezihlobene eduze, futhi sacubungula inani elikhulu lemibono ye-mass spectrometric esekelwe kuma-compound spectra kulwazi. Uhlaka lokuhlola izibikezelo eziyinhloko zokuzivikela okuhle kakhulu (OD) kanye nemibono ehambayo (MT). Ingxenye yolwazi ye-metabolomics yezitshalo iyahambisana nemfundiso yoku-OD, kodwa iphikisana nokubikezela okuyinhloko kwemfundiso yoku-MT mayelana ne-metabolomics dynamics ebangelwa yizilwane ezidla uhlaza. Kusukela ku-micro kuya esikalini sokuziphendukela kwemvelo esikhulu, isignali ye-jasmonate yabonakala njengesici esiyinhloko se-OD, kuyilapho isignali ye-ethylene ihlinzeka ngokulungiswa kahle kwempendulo ethile ye-herbivore echazwe yinethiwekhi yamangqamuzana ye-MS/MS.
Ama-metabolite akhethekile anezakhiwo ezahlukahlukene angabahlanganyeli abaphambili ekuzivumelaniseni nezitshalo nemvelo, ikakhulukazi ekuvikeleni izitha (1). Ukuhlukahluka okumangalisayo kwe-metabolism ekhethekile etholakala ezitshalweni kuye kwavuselela amashumi eminyaka ocwaningo olujulile ngezici zayo eziningi zemisebenzi yemvelo, futhi kwenze uhlu olude lwemibono yokuvikela izitshalo, okuyintuthuko yokuziphendukela kwemvelo kanye nemvelo yokuxhumana kwezitshalo nezinambuzane. Ucwaningo oluqanjiwe lunikeza isiqondiso esibalulekile (2). Kodwa-ke, le mibono yokuvikela izitshalo ayizange ilandele indlela ejwayelekile yokucabanga okucatshangelwayo, lapho izibikezelo ezibalulekile zazisezingeni elifanayo lokuhlaziya (3) futhi zahlolwa ngokuhlola ukuze kuthuthukiswe umjikelezo olandelayo wentuthuko yemfundiso (4). Imikhawulo yobuchwepheshe ikhawulela ukuqoqwa kwedatha ezigabeni ezithile ze-metabolic futhi ikhipha ukuhlaziywa okuphelele kwama-metabolite akhethekile, ngaleyo ndlela kuvimbele ukuqhathaniswa kwezigaba ezihlukene okubalulekile ekuthuthukisweni kwemfundiso (5). Ukuntuleka kwedatha ephelele ye-metabolomics kanye nemali efanayo yokuqhathanisa ukuhamba komsebenzi kokucubungula isikhala se-metabolic phakathi kwamaqembu ezitshalo ahlukene kuvimbela ukuvuthwa kwesayensi kwensimu.
Intuthuko yakamuva emkhakheni we-tandem mass spectrometry (MS/MS) metabolomics ingachaza ngokuphelele izinguquko ze-metabolic ngaphakathi naphakathi kwezinhlobo ze-system clade ethile, futhi ingahlanganiswa nezindlela zokubala ukuze kubalwe ukufana kwesakhiwo phakathi kwalezi zingxube eziyinkimbinkimbi. Ulwazi lwangaphambilini lwe-chemistry (5). Ukuhlanganiswa kobuchwepheshe obuthuthukisiwe ekuhlaziyeni nasekubhaleni kunikeza uhlaka oludingekayo lokuhlolwa kwesikhathi eside kwezibikezelo eziningi ezenziwe yimibono yemvelo kanye nokuziphendukela kwemvelo yokuhlukahluka kwe-metabolic. UShannon (6) wethula i-information theory okokuqala esihlokweni sakhe esiyinhloko ngo-1948, ebeka isisekelo sokuhlaziywa kwezibalo kolwazi, okusetshenziswe emikhakheni eminingi ngaphandle kokusetshenziswa kwayo kwasekuqaleni. Ku-genomics, i-information theory isetshenziswe ngempumelelo ukulinganisa ulwazi olugcinwa ngokulandelana (7). Ocwaningweni lwe-transcriptomics, i-information theory ihlaziya izinguquko eziphelele ku-transcriptome (8). Ocwaningweni lwangaphambilini, sisebenzise uhlaka lwezibalo lwe-information theory ku-metabolomics ukuchaza ubuchwepheshe be-metabolic bezinga lezicubu ezitshalweni (9). Lapha, sihlanganisa ukuhamba komsebenzi okusekelwe ku-MS/MS nohlaka lwezibalo lwethiyori yolwazi, olubonakala ngokuhlukahluka kwe-metabolic ngohlobo lwemali oluvamile, ukuze siqhathanise izibikezelo ezibalulekile zethiyori yokuvikela izitshalo ye-metabolome ebangelwa yizilwane ezidla uhlaza.
Izinhlaka zemfundiso yokuzivikela kwezitshalo zivame ukuhlanganisa futhi zingahlukaniswa ngezigaba ezimbili: lezo ezizama ukuchaza ukusatshalaliswa kwama-metabolite athile ezitshalo ngokusekelwe emisebenzini yokuzivikela, njengokuzivikela okuhle kakhulu (OD) (10), ukuqondiswa okuhambayo (MT) (11)) kanye nemfundiso yokubukeka (12), kanti ezinye zifuna izincazelo zemishini zokuthi izinguquko ekutholakaleni kwezinsiza zithinta kanjani ukukhula kwezitshalo kanye nokuqongelela kwama-metabolite akhethekile, njenge-carbon: i-nutrient balance hypothesis (13), i-growth rate hypothesis (14), kanye ne-growth and differentiation equilibrium hypothesis (15). Amaqoqo amabili emibono asemazingeni ahlukene okuhlaziya (4). Kodwa-ke, imibono emibili ehilela imisebenzi yokuzivikela ezingeni lokusebenza ibusa ingxoxo mayelana nokuzivikela okwakha izitshalo nokungaguquki: i-OD theory, ecabanga ukuthi izitshalo zitshala imali ekuvikeleni kwazo kwamakhemikhali okubizayo kuphela lapho kudingeka, isibonelo, lapho zigwinywa Lapho isilwane sotshani sihlasela, ngakho-ke, ngokusho kwamathuba okuhlasela esikhathini esizayo, inhlanganisela enomsebenzi wokuzivikela inikezwa (10); I-MT hypothesis iphakamisa ukuthi akukho axis yokushintsha kwe-metabolite eqondisiwe, kodwa i-metabolite iyashintsha ngokungahleliwe, ngaleyo ndlela idale ithuba lokuvimbela "inhloso yokunyakaza" kwe-metabolic yokuhlasela i-herbivores. Ngamanye amazwi, le mibono emibili yenza izibikezelo eziphikisanayo mayelana nokuguqulwa kwe-metabolic okwenzeka ngemva kokuhlaselwa kwe-herbivores: ubudlelwano phakathi kokuqongelela kwe-metabolites okuqondisiwe okukodwa nomsebenzi wokuzivikela (OD) kanye nezinguquko ze-metabolic ezingaqondisiwe (MT) (11).
Izimvo ze-OD kanye ne-MT azibandakanyi nje kuphela izinguquko ezibangelwa yi-metabolome, kodwa futhi nemiphumela yemvelo kanye nokuziphendukela kwemvelo yokuqongelela kwalezi metabolites, njengezindleko eziguquguqukayo kanye nezinzuzo zalezi zinguquko ze-metabolic endaweni ethile yemvelo (16). Nakuba zombili izimvo ziqaphela umsebenzi wokuzivikela wama-metabolites akhethekile, okungenzeka ukuthi ubize noma ungabi mkhulu, isibikezelo esiyinhloko esihlukanisa izimvo ze-OD kanye ne-MT sisekuqondeni kwezinguquko ze-metabolic ezibangelwa yizo. Ukubikezela kwethiyori ye-OD kuthole ukunakwa okukhulu kokuhlola kuze kube manje. Lezi zivivinyo zifaka phakathi ucwaningo lwemisebenzi yokuzivikela eqondile noma engaqondile yezicubu ezahlukene zamakhemikhali athile ezindaweni zokugcina izitshalo kanye nezimo zemvelo, kanye nezinguquko esigabeni sokuthuthuka kwezitshalo (17-19). Kodwa-ke, kuze kube manje, ngenxa yokuntuleka komsebenzi kanye nohlaka lwezibalo lokuhlaziywa okuphelele komhlaba wonke kokuhlukahluka kwe-metabolic kwanoma yisiphi isidalwa, umehluko omkhulu wokubikezela phakathi kwalezi zinkolelo-mbono ezimbili (okungukuthi, isiqondiso sezinguquko ze-metabolic) kusazohlolwa. Lapha, sinikeza ukuhlaziywa okunjalo.
Esinye sezici ezibaluleke kakhulu zama-metabolite athile ezitshalweni ukuhlukahluka kwawo okwedlulele kwesakhiwo kuwo wonke amazinga kusukela ezitshalweni ezilodwa, inani labantu kuya ezinhlotsheni ezifanayo (20). Izinguquko eziningi zobuningi kuma-metabolite akhethekile zingabonakala esikalini sabantu, kuyilapho umehluko omkhulu wekhwalithi uvame ukugcinwa ezingeni lezinhlobo (20). Ngakho-ke, ukuhlukahluka kwe-metabolic yezitshalo kuyisici esiyinhloko sokuhlukahluka kokusebenza, okubonisa ukuzivumelanisa nezimo ezindaweni ezahlukene, ikakhulukazi lezo zindawo ezinezindlela ezahlukene zokuhlasela yizinambuzane ezikhethekile kanye nezilwane ezidla uhlaza ezivamile (21). Kusukela esihlokweni sikaFraenkel (22) esiqavile ngezizathu zokuba khona kwama-metabolite athile ezitshalweni, ukusebenzisana nezinambuzane ezahlukahlukene kuye kwabhekwa njengezingcindezi ezibalulekile zokukhetha, futhi lokhu kusebenzisana kukholakala ukuthi kuye kwabumba izitshalo ngesikhathi sokuziphendukela kwemvelo. Indlela ye-metabolic (23). Umehluko phakathi kwezinhlobo ezahlukene ekuhlukeni kwama-metabolite akhethekile ungase futhi ubonise ibhalansi yomzimba ehlotshaniswa nokuzivikela kwezitshalo okwakhayo nokungabangelwa yizindlela zokulwa nokhula, njengoba lezi zinhlobo ezimbili zivame ukuhlobana kabi (24). Nakuba kungaba wusizo ukugcina ukuzivikela okuhle ngaso sonke isikhathi, izinguquko ze-metabolic ezihambisana nokuzivikela zinikeza izinzuzo ezicacile ekuvumeleni izitshalo ukuthi zabelane ngezinsiza ezibalulekile kwezinye izimali ezisetshenziswayo (19, 24), futhi zigweme isidingo sokusebenzisana. Umonakalo we-collective (25). Ngaphezu kwalokho, lokhu kuhlelwa kabusha kwama-metabolite akhethekile abangelwa yizinambuzane ezidla uhlaza kungaholela ekusabalaleni okubhubhisayo kubantu (26), futhi kungabonisa ukufundwa okuqondile kwezinguquko zemvelo ezinkulu esignalini ye-jasmonic acid (JA), engagcinwa kubantu. Izimpawu ze-JA eziphakeme neziphansi ziyizimpikiswano phakathi kokuzivikela ezidla uhlaza kanye nokuncintisana nezinhlobo ezithile (27). Ngaphezu kwalokho, izindlela ze-biosynthetic ze-metabolite ezikhethekile zizolahlekelwa ngokushesha futhi ziguqulwe ngesikhathi sokuziphendukela kwemvelo, okuholela ekusabalaleni kwe-metabolic okuxakile phakathi kwezinhlobo ezihlobene eduze (28). Lezi zinhlobo ze-polymorphisms zingasungulwa ngokushesha ekuphenduleni amaphethini ashintshayo e-herbivore (29), okusho ukuthi ukuguquguquka kwemiphakathi ye-herbivore kuyisici esiyinhloko esiqhuba ukungafani kwe-metabolic.
Lapha, sixazulule ngqo izinkinga ezilandelayo. (I) Izinambuzane ezidla uhlaza ziyilungisa kanjani kabusha i-metabolome yesitshalo? (Ii) Yiziphi izingxenye zolwazi eziyinhloko ze-metabolic plasticity ezingalinganiswa ukuze kuhlolwe izibikezelo zethiyori yokuzivikela yesikhathi eside? (Iii) Ukuthi kufanele kuhlelwe kabusha i-metabolome yesitshalo ngendlela ehlukile kumhlaseli, uma kunjalo, iyiphi indima edlalwa yi-hormone yesitshalo ekuhleleni impendulo ethile ye-metabolic, futhi yiziphi i-metabolites ezifaka isandla ekuqondeni kohlobo lokuzivikela? (Iv) Njengoba izibikezelo ezenziwe yizinkolelo-mbono eziningi zokuzivikela zinganwetshwa kuwo wonke amazinga ezicubu zezinto eziphilayo, sibuze ukuthi impendulo ye-metabolic ebangelwayo ivumelana kangakanani kusukela ekuqhathanisweni kwangaphakathi kuya ekuqhathanisweni kwezinhlobo ezahlukene? Ngenxa yalokhu, sifunde ngokuhlelekile i-metabolome yamaqabunga e-nicotine kagwayi, okuyisitshalo esiyimodeli yezemvelo esinomuthi ocebile we-metabolism, futhi sisebenza kahle ngokumelene nezibungu zezilwane ezimbili ezidla uhlaza, i-Lepidoptera Datura (Nks) (Inolaka kakhulu, idliwa kakhulu) Ku-Solanaceae kanye ne-Spodoptera littoralis (Sl), izimpethu zamaqabunga ekotini ziwuhlobo "lohlobo", kanye nezitshalo eziphethe i-Solanaceae kanye nezinye izizukulwane zezinye izizukulwane kanye nemindeni Ukudla kwezitshalo. Sihlaziye i-MS/MS metabolomics spectrum futhi sakhipha izincazelo zezibalo zethiyori yolwazi ukuze siqhathanise imibono ye-OD kanye ne-MT. Dala amamephu okucacisa ukuze kwembulwe ukuthi ungubani we-metabolites ebalulekile. Ukuhlaziywa kwandiswe kubantu bomdabu be-N. nasi kanye nezinhlobo zikagwayi ezihlobene eduze ukuze kuhlaziywe kabanzi i-covariance phakathi kokubonisana kwama-hormone ezitshalo kanye nokungeniswa kwe-OD.
Ukuze sibambe imephu iyonke mayelana nokwakheka kwe-plasticity kanye nesakhiwo se-metabolome yamaqabunga kagwayi odla uhlaza, sisebenzise ukuhlaziywa kanye nomsebenzi wokubala othuthukisiwe ngaphambilini ukuze siqoqe ngokuphelele futhi sisuse idatha ye-MS/MS spectra ezimele enesinqumo esiphezulu kusuka ezitshalweni zezitshalo (9). Le ndlela engahlukanisiwe (ebizwa ngokuthi i-MS/MS) ingakha i-compound spectra engadingeki, engasetshenziswa kuzo zonke izihlaziyi zezinga le-compound ezichazwe lapha. Lawa ma-metabolite ezitshalo angahlanganisiwe anezinhlobo ezahlukene, aqukethe amakhulu kuya ezinkulungwaneni zama-metabolite (cishe ama-500-1000-s/MS/MS lapha). Lapha, sibheka ukwakheka kwe-metabolic ohlakeni lwethiyori yolwazi, futhi silinganise ukuhlukahluka kanye nobungcweti be-metabolome ngokusekelwe ku-Shannon entropy yokusatshalaliswa kwemvamisa ye-metabolic. Sisebenzisa ifomula esetshenziswe ngaphambilini (8), sibale isethi yezinkomba ezingasetshenziswa ukulinganisa ukuhlukahluka kwe-metabolome (inkomba ye-Hj), ukukhethekile kwephrofayili ye-metabolic (inkomba ye-δj) kanye nokucaciswa kwe-metabolic kwe-metabolite eyodwa (inkomba ye-Si). Ngaphezu kwalokho, sisebenzise i-Relative Distance Plasticity Index (RDPI) ukuze silinganisele ukukhiqizwa kwe-metabolome kwezilwane ezidla uhlaza (Isithombe 1A) (30). Ngaphakathi kwalolu hlaka lwezibalo, siphatha i-MS/MS spectrum njengeyunithi yolwazi oluyisisekelo, futhi sicubungula ubuningi be-MS/MS kumephu yokusatshalaliswa kwemvamisa, bese sisebenzisa i-Shannon entropy ukulinganisa ukuhlukahluka kwe-metabolome kuyo. Ukuqokwa kwe-metabolome kulinganiswa ngokucaciswa okumaphakathi kwe-spectrum eyodwa ye-MS/MS. Ngakho-ke, ukwanda kobuningi bezinye izigaba ze-MS/MS ngemuva kokungeniswa kwe-herbivore kuguqulwa kube ukuqokwa kwe-spectral, i-RDPI kanye nokuqokwa, okungukuthi, ukwanda kwe-δj index, ngoba kukhiqizwa ama-metabolite akhethekile kakhulu futhi kukhiqizwa i-Si index ephezulu. Ukwehla kwe-Hj diversity index kubonisa ukuthi inani le-MS/MS ekhiqizwe lincishisiwe, noma ukusatshalaliswa kwemvamisa yephrofayili kuyashintsha ngendlela engalingani, ngenkathi kunciphisa ukungaqiniseki kwayo konke. Ngokusebenzisa i-Si index calculation, kungenzeka ukugqamisa ukuthi yiziphi i-MS/MS ezibangelwa yizilwane ezithile ezidla uhlaza, ngokuphambene nalokho, yiziphi i-MS/MS ezingaphenduli ekuqalisweni, okuyisibonakaliso esibalulekile sokuhlukanisa ukubikezela kwe-MT ne-OD.
(A) Izincazelo zezibalo ezisetshenziselwa i-herbivorous (H1 kuya ku-Hx) i-MS/MS-inducibility (RDPI), ukuhlukahluka (inkomba ye-Hj), ukukhethekile (inkomba ye-δj) kanye nokucaciswa kwe-metabolite (inkomba ye-Si). Ukwanda kwezinga lokukhethekile (δj) kubonisa ukuthi, ngokwesilinganiso, kuzokhiqizwa ama-metabolite amaningi athile adla uhlaza, kuyilapho ukwehla kokuhlukahluka (Hj) kubonisa ukwehla kokukhiqizwa kwama-metabolite noma ukusatshalaliswa okungalingani kwama-metabolite kumephu yokusatshalaliswa. Inani le-Si lihlola ukuthi i-metabolite iqondene nesimo esithile (lapha, idla uhlaza) noma igcinwe ngokuphambeneyo ezingeni elifanayo. (B) Umdwebo womqondo wokubikezela kwethiyori yokuzivikela usebenzisa i-axis yethiyori yolwazi. Ithiyori ye-OD ibikezela ukuthi ukuhlaselwa kwe-herbivore kuzokwandisa ama-metabolite okuzivikela, ngaleyo ndlela kwandise i-δj. Ngesikhathi esifanayo, i-Hj iyancipha ngoba iphrofayili ihlelwa kabusha maqondana nokungaqiniseki okuncishisiwe kolwazi lwe-metabolic. Ithiyori ye-MT ibikezela ukuthi ukuhlaselwa kwe-herbivore kuzobangela izinguquko ezingezona eziqondisweni ku-metabolome, ngaleyo ndlela kwandise i-Hj njengesibonakaliso sokwanda kokungaqiniseki kolwazi lwe-metabolic futhi kubangele ukusatshalaliswa okungahleliwe kwe-Si. Siphinde saphakamisa imodeli exubile, i-MT engcono kakhulu, lapho amanye ama-metabolites anenani eliphezulu lokuzivikela azokhushulwa khona ikakhulukazi (inani eliphezulu le-Si), kanti amanye abonisa izimpendulo ezingahleliwe (inani eliphansi le-Si).
Sisebenzisa izincazelo zethiyori yolwazi, sichaza ithiyori ye-OD ukuze sibikezele ukuthi izinguquko ze-metabolite ekhethekile ezibangelwa uhlaza esimweni esingabangelwa zizoholela ku-(i) ukwanda kokucaciswa kwe-metabolic (inkomba ye-Si) ukushayela ukucaciswa kwe-metabonomic (inkomba ye-δj) Ukwanda) kwamaqembu athile e-metabolite akhethekile anenani eliphezulu lokuzivikela, kanye (ii) ukwehla kokuhlukahluka kwe-metabolome (inkomba ye-Hj) ngenxa yokushintsha kokusatshalaliswa kwemvamisa ye-metabolic kuya ekusakazweni komzimba kwe-leptin eningi. Ezingeni le-metabolite eyodwa, kulindeleke ukusatshalaliswa kwe-Si okuhleliwe, lapho i-metabolite izokwandisa inani le-Si ngokuya ngenani layo lokuzivikela (Isithombe 1B). Ngale ndlela, sichaza ithiyori ye-MT ukuze sibikezele ukuthi ukuvusa kuzoholela ku-(i) izinguquko ezingaqondile kuma-metabolite okuholela ekwehleni kwenkomba ye-δj, kanye (ii) ukwanda kwenkomba ye-Hj ngenxa yokwanda kokungaqiniseki kwe-metabolic. Noma ukungahleliwe, okungalinganiswa yi-Shannon entropy ngesimo sokuhlukahluka okujwayelekile. Ngokuphathelene nokwakheka kwe-metabolic, ithiyori ye-MT izobikezela ukusatshalaliswa okungahleliwe kwe-Si. Uma sibheka ukuthi ama-metabolite athile angaphansi kwezimo ezithile ngaphansi kwezimo ezithile, kanti ezinye izimo azingaphansi kwezimo ezithile, futhi inani lawo lokuzivikela lincike endaweni ezungezile, siphinde saphakamisa imodeli yokuzivikela exubile, lapho i-δj ne-Hj zisatshalaliswa khona kabili eceleni kwe-Si. Ukwanda kuzo zonke izinkomba, amaqembu athile kuphela e-metabolite, anamanani aphezulu okuzivikela, azokwandisa i-Si ikakhulukazi, kanti amanye azoba nokusatshalaliswa okungahleliwe (Isithombe 1B).
Ukuze sihlole ukubikezela kwethiyori yokuzivikela echazwe kabusha ku-axis yencazelo yethiyori yolwazi, siphakamise izibungu ze-herbivore ezingochwepheshe (Nksz) noma ezivamile (Sl) emaqabungeni e-Nepenthes pallens (Isithombe 2A). Sisebenzisa ukuhlaziywa kwe-MS/MS, sithole ama-spectra angu-599 angewona awe-redundant MS/MS (ifayela ledatha S1) kusuka ku-methanol extracts yezicubu zamaqabunga eziqoqwe ngemva kokudla kwezibungu. Sisebenzisa izinkomba ze-RDPI, Hj, kanye ne-δj ukuze sibone ukulungiselelwa kabusha kokuqukethwe kolwazi kumafayela okucushwa kwe-MS/MS kwembula amaphethini athakazelisayo (Isithombe 2B). Umkhuba jikelele ukuthi, njengoba kuchazwe yincazelo yolwazi, njengoba izibungu ziqhubeka nokudla amaqabunga, izinga lakho konke ukuhlelwa kabusha kwe-metabolic liyanda ngokuhamba kwesikhathi: emahoreni angu-72 ngemva kokudla kwezibungu, i-RDPI iyanda kakhulu. Uma kuqhathaniswa nokulawula okungonakalanga, i-Hj yancishiswa kakhulu, okwakubangelwa izinga elikhuphukile lobuchwepheshe bephrofayili ye-metabolic, eyalinganiswa yi-δj index. Lo mkhuba obonakalayo uhambisana nezibikezelo zethiyori ye-OD, kodwa awuhambisani nezibikezelo eziyinhloko zethiyori ye-MT, ekholelwa ukuthi izinguquko ezingahleliwe (ezingezona ezendlela) emazingeni e-metabolite zisetshenziswa njenge-defensive camouflage (Isithombe 1B). Nakuba okuqukethwe kwe-excitor yomlomo (OS) kanye nokuziphatha kokudla kwalezi zilwane ezimbili ezidla uhlaza kuhlukile, ukudla kwazo okuqondile kuholele ezinguqukweni ezifanayo eziqondisweni ze-Hj kanye ne-δj ngesikhathi sokuvuna samahora angu-24 kanye namahora angu-72. Umehluko kuphela owenzekile emahoreni angu-72 e-RDPI. Uma kuqhathaniswa nalokho okubangelwa ukudla kwe-Ms, i-metabolism iyonke ebangelwa ukudla kwe-Sl yayiphezulu.
(A) Umklamo wokuhlola: ingulube evamile (S1) noma izinyoni ezidla uhlaza (Nksz) zondliwa ngamaqabunga akhishwe usawoti ezitshalweni ze-pitcher, kuyilapho kwezitshalo ezidla uhlaza ezilingisiwe, i-OS ye-Ms (W + OSM) isetshenziselwa ukuphatha ukubhobozwa kwezikhala zamaqabunga ezijwayelekile. Izibungu ze-S1 (W + OSSl) noma amanzi (W + W). Ukulawula (C) kuyiqabunga elingonakali. (B) Ukungakwazi ukubekezelela (RDPI uma kuqhathaniswa neshadi lokulawula), ukuhlukahluka (inkomba ye-Hj) kanye nenkomba yokukhethekile (inkomba ye-δj) ebalwe kumephu ekhethekile ye-metabolite (599 MS/MS; ifayela ledatha S1). Ama-Asterisk abonisa umehluko omkhulu phakathi kokudla okuqondile kwezilwane ezidla uhlaza kanye neqembu lokulawula (ukuhlolwa kwe-t komfundi okunokuhlolwa kwe-t okubhangqiwe, *P<0.05 kanye ***P<0.001). ns, akubalulekile. (C) Inkomba yesikhathi sokulungiswa kwe-main (ibhokisi eliluhlaza okwesibhakabhaka, i-amino acid, i-organic acid kanye noshukela; ifayela ledatha S2) kanye ne-spectrum ekhethekile ye-metabolite (ibhokisi elibomvu 443 MS/MS; ifayela ledatha S1) ngemuva kokwelashwa okulingisiwe kwe-herbivory. Ibhendi yombala ibhekisela esikhawulweni sokuzethemba esingu-95%. I-asterisk ikhombisa umehluko obalulekile phakathi kokwelashwa kanye nokulawula [ukuhlaziywa kwe-quadratic of variance (ANOVA), kulandelwe umehluko obalulekile kaTukey (HSD) wokuqhathanisa okuningi kwe-post hoc, *P<0.05, **P<0.01 kanye no-*** P <0.001]. (D) Ukukhethekile kwezindawo zokuhlakaza kanye namaphrofayili e-metabolite akhethekile (amasampula aphindaphindwayo anezindlela ezahlukene zokwelapha).
Ukuze sihlole ukuthi ukuvuselelwa okubangelwa ukhula lwezitshalo ezingeni le-metabolome kubonakala yini ezinguqukweni ezingeni lama-metabolite ngamanye, siqale sagxila kuma-metabolite afundwe ngaphambilini emaqabungeni e-Nepenthes pallens anokumelana nokhula lwezitshalo okuqinisekisiwe. Ama-phenolic amide angama-hydroxycinnamamide-polyamine conjugates aqoqana ngesikhathi senqubo yokhula lwezinambuzane futhi aziwa ngokunciphisa ukusebenza kwezinambuzane (32). Siseshe izandulela ze-MS/MS ehambisanayo futhi sadweba ama-curve awo e-cumulative kinetic (Isithombe S1). Akumangazi ukuthi ama-phenol derivatives angahilelekile ngqo ekuvikeleni ama-herbivores, njenge-chlorogenic acid (CGA) kanye ne-rutin, ancishiswa ngemuva kwe-herbivory. Ngokuphambene nalokho, ama-herbivores angenza ama-phenol amide abe namandla kakhulu. Ukondla okuqhubekayo kwama-herbivores amabili kuholele cishe ku-excitation spectrum efanayo yama-phenolamide, futhi leli phethini lalisobala kakhulu ekwakhiweni kwe-de novo kwama-phenolamide. Isenzakalo esifanayo sizobonakala lapho kuhlolwa indlela ye-17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), ekhiqiza inani elikhulu lama-diterpenes acyclic anemisebenzi yokulwa nokudla uhlaza (33), lapho uNks Feeding with Sl ebangele khona iphrofayili efanayo yokubonakaliswa (Isithombe S1)).
Ububi obungaba khona bokuhlolwa kokudla okuqondile kwezilwane ezidla uhlaza umehluko wesilinganiso sokusetshenziswa kwamaqabunga kanye nesikhathi sokudla kwezilwane ezidla uhlaza, okwenza kube nzima ukuqeda imiphumela ethile yezilwane ezidla uhlaza ebangelwa amanxeba kanye nezilwane ezidla uhlaza. Ukuze sixazulule kangcono ukucaciswa kwezinhlobo zezilwane ezidla uhlaza kwempendulo ye-metabolic yamaqabunga ebangelwa, silingise ukondla izibungu ze-Ms kanye ne-Sl ngokusebenzisa ngokushesha i-OS eqoqwe kabusha (OSM kanye ne-OSS1) ku-W ejwayelekile yokubhoboza yezindawo zamaqabunga ezihambisanayo. Le nqubo ibizwa ngokuthi ukwelashwa kwe-W + OS, futhi ilinganisa ukungeniswa ngokubeka isikhathi esifanele sokuqala kwempendulo ebangelwe yizilwane ezidla uhlaza ngaphandle kokubangela imiphumela edidayo yomehluko ngesilinganiso noma ubuningi bokulahlekelwa kwezicubu (Isithombe 2A) (34). Sisebenzisa ipayipi lokuhlaziya kanye nokubala le-MS/MS, sithole ama-spectra angu-443 e-MS/MS (ifayela ledatha i-S1), elalihambisana nama-spectra aqoqwe ngaphambilini kusuka ekuhlolweni kokudla okuqondile. Ukuhlaziywa kwethiyori yolwazi yale sethi yedatha ye-MS/MS kubonise ukuthi ukuhlelwa kabusha kwama-metabolome akhethekile amaqabunga ngokulingisa ama-herbivore kubonise izimbangela ezithile ze-OS (Isithombe 2C). Ikakhulukazi, uma kuqhathaniswa nokwelashwa kwe-OSS1, i-OSM ibangele ukuthuthukiswa kokukhethekile kwe-metabolome emahoreni ama-4. Kubalulekile ukuqaphela ukuthi uma kuqhathaniswa nesethi yedatha yokuhlola yokudla okuqondile kwe-herbivore, i-metabolism kinetics ebonwe esikhaleni esinezinhlangothi ezimbili kusetshenziswa i-Hj kanye ne-δj njengezixhumanisi kanye nokuqondiswa kokukhethekile kwe-metabolome ekuphenduleni ukwelashwa kwe-herbivore okulingisiwe ngokuhamba kwesikhathi. Ukwanda okuqhubekayo (Isithombe 2D). Ngesikhathi esifanayo, silinganise okuqukethwe kwama-amino acid, ama-organic acid kanye noshukela (ifayela ledatha S2) ukuze sihlole ukuthi lokhu kwanda okuqondisiwe kobuchwepheshe be-metabolome kungenxa yokulungiswa kabusha kwe-metabolism yekhabhoni ephakathi ekuphenduleni ama-herbivore alingisiwe (Isithombe S2). Ukuze sichaze kangcono leli phethini, siqhubeke nokuqapha i-metabolism accumulation kinetics yezindlela ze-phenolamide kanye ne-17-HGL-DTG ezixoxwe ngazo ngaphambilini. Ukungeniswa kwe-OS kwezilwane ezidla uhlaza kuguqulwa kube iphethini yokuhlelwa kabusha okuhlukile ngaphakathi kwe-metabolism ye-phenolamide (Isithombe S3). Ama-phenolic amide aqukethe i-coumarin kanye ne-caffeoyl moieties abangelwa ngokukhethekile yi-OSS1, kuyilapho ama-OSM eqala ukungeniswa okuthile kwe-ferulyl conjugates. Endleleni ye-17-HGL-DTG, ukungeniswa kwe-differential OS ngemikhiqizo ye-malonylation kanye ne-dimalonylation engezansi kutholakale (Isithombe S3).
Okulandelayo, sifunde i-transcriptome plasticity ebangelwa yi-OS sisebenzisa isethi yedatha ye-microarray yesikhathi, elingisa ukusetshenziswa kwama-OSM ukwelapha amaqabunga amaqabunga esitshalo se-rosette ezitshalweni ezidla uhlaza. I-kinetics yesampula ihlangana ngokuyisisekelo ne-kinetics esetshenziswe kulolu cwaningo lwe-metabolomics (35). Uma kuqhathaniswa nokulungiswa kabusha kwe-metabolome lapho i-metabolic plasticity ikhula khona ngokuhamba kwesikhathi, sibona ukuqhuma kwe-transcription yesikhashana emaqabunga okubangelwa yi-Ms, lapho i-transcriptome inducibility (RDPI) kanye ne-specialization (δj) ku-1 Kwakukhona ukwanda okukhulu kwamahora, kanye nokuhlukahluka (Hj) kulesi sikhathi, ukubonakaliswa kwe-BMP1 kwehliswe kakhulu, kwalandelwa ukukhululeka kwe-transcriptome specialization (Isithombe S4). Imindeni yezakhi zofuzo ze-metabolic (njenge-P450, i-glycosyltransferase, kanye ne-BAHD acyltransferase) ihlanganyela enkambisweni yokuhlanganisa ama-metabolite akhethekile avela kumayunithi esakhiwo atholakala ku-metabolism eyinhloko, kulandela imodeli yokuqala ye-high-specialization. Njengesifundo secala, indlela ye-phenylalanine yahlaziywa. Ukuhlaziywa kuqinisekisile ukuthi izakhi zofuzo eziyinhloko ekusetshenzisweni kwe-phenolamide zibangelwa kakhulu yi-OS ezitshalweni ezidla uhlaza uma kuqhathaniswa nezitshalo ezingakhangwa, futhi zihambisana kakhulu namaphethini azo okuveza. I-transcription factor MYB8 kanye nezakhi zofuzo zesakhiwo i-PAL1, i-PAL2, i-C4H kanye ne-4CL ezingenhla kwale ndlela zibonise ukuqaliswa kwangaphambi kwesikhathi kokubhalwa kwe-transcription. Ama-acyltransferases adlala indima ekuhlanganisweni kokugcina kwe-phenolamide, njenge-AT1, i-DH29, kanye ne-CV86, abonisa iphethini yokwenyuka kwesikhathi eside (Isithombe S4). Okubonwe ngenhla kubonisa ukuthi ukuqaliswa kwangaphambi kwesikhathi kochwepheshe be-transcriptome kanye nokuthuthukiswa kwakamuva kochwepheshe be-metabolomics kuyimodi ehlanganisiwe, okungenzeka ukuthi kungenxa yesistimu yokulawula ehambisanayo eqala impendulo enamandla yokuzivikela.
Ukulungiswa kabusha kokubonakaliswa kwama-hormone ezitshalo kusebenza njengesendlalelo sokulawula esihlanganisa ulwazi lwe-herbivorous ukuze kuhlelwe kabusha i-physiology yezitshalo. Ngemva kokulingisa i-herbivore, silinganise ukuguquguquka okuqongelelekayo kwezigaba ezibalulekile zama-hormone ezitshalo futhi sabona ukuhlangana kwesikhashana phakathi kwazo [i-Pearson correlation coefficient (PCC)> 0.4] (Isithombe 3A). Njengoba bekulindelekile, ama-hormone ezitshalo ahlobene ne-biosynthesis axhumene ngaphakathi kwenethiwekhi ye-herbivore co-expression. Ngaphezu kwalokho, ukucaciswa kwe-metabolic (inkomba ye-Si) kufakwe kule nethiwekhi ukuze kuqokonyiswe ama-hormone ezitshalo abangelwa ukwelashwa okuhlukene. Izindawo ezimbili eziyinhloko zokuphendula okuqondile kwe-herbivorous zidwetshwe: eyodwa iseqenjini le-JA, lapho i-JA (ifomu layo elisebenzayo ngokwebhayoloji i-JA-Ile) kanye nezinye izinto ezisuselwa ku-JA zibonisa amaphuzu aphezulu kakhulu e-Si; enye i-ethylene (ET). I-Gibberellin ibonise ukwanda okulinganiselwe kokucaciswa kwe-herbivore, kuyilapho amanye ama-hormone ezitshalo, njenge-cytokinin, i-auxin, ne-abscisic acid, ayenokucaciswa okuphansi kwe-induction kwe-herbivores. Uma kuqhathaniswa nokusebenzisa i-W + W kuphela, ukukhuliswa kwenani eliphakeme lama-derivatives e-JA ngohlelo lokusebenza lwe-OS (W + OS) kungaguqulwa kube yinkomba eqinile yama-JA. Ngokungalindelekile, i-OSM ne-OSS1 enokuqukethwe okuhlukile kwe-encitor kwaziwa ukuthi kubangela ukuqongelela okufanayo kwe-JA ne-JA-Ile. Ngokungafani ne-OSS1, i-OSM ibangelwa ngqo futhi ngamandla ama-OSM, kuyilapho i-OSM1 ingakhulisi impendulo yamanxeba ayisisekelo (Isithombe 3B).
(A) Ukuhlaziywa kwenethiwekhi yokubonisana okusekelwe ekubalweni kwe-PCC kokulingiswa kwe-kinetics ye-hormone yesitshalo ebangelwa ukhula. I-node imelela i-hormone yesitshalo esisodwa, kanti ubukhulu be-node bumelela inkomba ye-Si ethize ku-hormone yesitshalo phakathi kokwelashwa. (B) Ukuqongelela kwe-JA, i-JA-Ile kanye ne-ET emaqabunga okubangelwa ukwelashwa okuhlukene okuboniswe ngemibala ehlukene: i-apricot, i-W + i-OSM; eluhlaza okwesibhakabhaka, i-W + i-OSl; emnyama, i-W + i-W; grey, i-C (ukulawula). Ama-asterisk abonisa umehluko omkhulu phakathi kokwelashwa nokulawula (i-ANOVA enezindlela ezimbili elandelwa ukuqhathaniswa okuningi kwe-Tukey HSD ngemuva kwe-hoc, *** P <0.001). Ukuhlaziywa kwethiyori yolwazi lwe-(C)697 MS/MS (ifayela ledatha S1) ku-JA biosynthesis kanye ne-spectrum yokuqonda okuphazamisekile (irAOC kanye ne-irCOI1) kanye (D)585 MS/MS (ifayela ledatha S1) ku-ETR1 enesignali ye-ET ephazamisekile Ukwelashwa okubili kwe-herbivore okulingisiwe kwabangela imigqa yezitshalo kanye nezitshalo zokulawula ezingenalutho (EV). Ama-asterisk abonisa umehluko omkhulu phakathi kokwelashwa kwe-W+OS kanye nokulawulwa okungonakalanga (i-ANOVA enezindlela ezimbili elandelwa ukuqhathaniswa okuningi kwe-Tukey HSD ngemuva kwe-hoc, *P<0.05, **P<0.01 kanye ***P<0.001). (E) Amagrafu ahlakazekile okuphikisana okuhlakazekile nokukhethekile. Imibala imelela izinhlobo ezahlukene eziguqulwe izakhi zofuzo; izimpawu zimelela izindlela ezahlukene zokwelapha: unxantathu, u-W + OSS1; unxande, u-W + OSM; indilinga u-C
Okulandelayo, sisebenzisa uhlobo oluguqulwe ngokwezakhi zofuzo lwe-Nepenthes encishisiwe (irCOI1 kanye ne-sETR1) ezinyathelweni ezibalulekile ze-JA kanye ne-ET biosynthesis (irAOC kanye ne-irACO) kanye nokubona (irCOI1 kanye ne-sETR1) ukuhlaziya imetabolism yalawa ma-hormone amabili ezitshalo kuma-herbivores Umnikelo ohlobene wokuhlela kabusha. Ngokuhambisana nokuhlolwa kwangaphambilini, siqinisekisile ukungeniswa kwe-herbivore-OS ezitshalweni ezingenalutho (EV) (Isithombe 3, C kuya ku-D) kanye nokwehla okuphelele kwenkomba ye-Hj okubangelwa yi-OSM, kuyilapho inkomba ye-δj ikhuphuka. Impendulo ivelele kakhulu kunempendulo ebangelwa yi-OSM1. Igrafu yemigqa emibili esebenzisa i-Hj kanye ne-δj njengezixhumanisi ikhombisa ukuqedwa kwemithetho ethile (Isithombe 3E). Umkhuba osobala kakhulu ukuthi ezinhlotsheni ezingenayo isignali ye-JA, ukuhlukahluka kwe-metabolome kanye nezinguquko ezikhethekile ezibangelwa ama-herbivores cishe kuqedwe ngokuphelele (Isithombe 3C). Ngokuphambene nalokho, umbono we-ET othule ezitshalweni ze-sETR1, yize umphumela ojwayelekile ezinguqukweni ze-metabolism yezitshalo ezidla uhlaza uphansi kakhulu kunowokusayina kwe-JA, unciphisa umehluko kuma-indices e-Hj kanye ne-δj phakathi kokuvusa kwe-OSM kanye ne-OSS1 (Isithombe 3D kanye nesithombe S5). . Lokhu kubonisa ukuthi ngaphezu komsebenzi oyinhloko wokudluliselwa kwesignali ye-JA, ukudluliselwa kwesignali ye-ET kusebenza futhi njengokulungiswa kahle kwempendulo ye-metabolic yezinhlobo ezithile zezilwane ezidla uhlaza. Ngokuhambisana nalo msebenzi wokulungiswa kahle, bekungekho ushintsho ekungaguquki kwe-metabolome okuphelele ezitshalweni ze-sETR1. Ngakolunye uhlangothi, uma kuqhathaniswa nezitshalo ze-sETR1, izitshalo ze-irACO zabangela ukuguquguquka okufanayo kwezinguquko ze-metabolic ezibangelwa yizilwane ezidla uhlaza, kodwa zabonisa amaphuzu ahlukene kakhulu e-Hj kanye ne-δj phakathi kwezinselele ze-OSM kanye ne-OSS1 (Isithombe S5).
Ukuze sithole ama-metabolite akhethekile aneqhaza elibalulekile ekuphenduleni kwezinhlobo ezithile zezilwane ezidla uhlaza futhi silungise ukukhiqizwa kwazo ngezibonakaliso ze-ET, sisebenzise indlela ye-MS/MS eyakhiwe ngaphambilini. Le ndlela incike endleleni ye-bi-clustering ukuze ithole kabusha umndeni we-metabolic kusuka ku-MS/MS fragments [umkhiqizo we-dot ojwayelekile (i-NDP)] kanye ne-similarity score ngokusekelwe ekulahlekelweni okungathathi hlangothi (i-NL). Isethi yedatha ye-MS/MS eyakhiwe ngokuhlaziywa kwemigqa ye-ET transgenic yakhiqiza ama-MS/MS angu-585 (ifayela ledatha i-S1), okwaxazululwa ngokuwahlanganisa abe amamojula ayisikhombisa amakhulu e-MS/MS (i-M) (Isithombe 4A). Amanye ala mamojula agcwele ama-metabolite akhethekile achazwe ngaphambilini: isibonelo, i-M1, i-M2, i-M3, i-M4 kanye ne-M7 acebile kuma-phenol derivatives ahlukahlukene (i-M1), ama-flavonoid glycosides (i-M2), ushukela we-acyl (i-M3 kanye ne-M4), kanye ne-17-HGL-DTG (i-M7). Ngaphezu kwalokho, ulwazi oluthile lwe-metabolic (inkomba ye-Si) ye-metabolite eyodwa kumojuli ngayinye luyabalwa, futhi ukusatshalaliswa kwayo kwe-Si kungabonakala ngokwemvelo. Ngamafuphi, ama-spectra e-MS/MS abonisa ukucaciswa okuphezulu kwe-herbivory kanye ne-genotype abonakala ngamanani aphezulu e-Si, kanti izibalo ze-kurtosis zibonisa ukusatshalaliswa koboya ekhoneni lomsila kwesokudla. Ukusatshalaliswa okukodwa okunjalo kwe-colloid okunciphile kwatholakala ku-M1, lapho i-phenol amide ibonise ingxenye ephezulu kakhulu ye-Si (Isithombe 4B). I-herbivorous inducible 17-HGL-DTG okukhulunywe ngayo ngaphambilini ku-M7 ibonise amaphuzu aphakathi kwe-Si, okubonisa izinga eliphakathi lokulawulwa komehluko phakathi kwezinhlobo ezimbili ze-OS. Ngokuphambene nalokho, ama-metabolite amaningi akhethekile akhiqizwa ngokuqongelela, njenge-rutin, i-CGA, kanye noshukela we-acyl, aphakathi kwamaphuzu aphansi kakhulu e-Si. Ukuze kuhlolwe kangcono ubunzima besakhiwo kanye nokusatshalaliswa kwe-Si phakathi kwama-metabolite akhethekile, kwakhiwa inethiwekhi yama-molecule yemojuli ngayinye (Isithombe 4B). Isibikezelo esibalulekile sethiyori ye-OD (efingqiwe ku-Figure 1B) ukuthi ukuhlelwa kabusha kwama-metabolite akhethekile ngemva kokuhluma kufanele kuholele ekushintsheni kwendlela eyodwa kuma-metabolite anenani eliphezulu lokuzivikela, ikakhulukazi ngokwandisa ukucaciswa kwawo (ngokungafani nokusatshalaliswa okungahleliwe) Imodi) I-metabolite yokuzivikela ebikezelwe yithiyori ye-MT. Iningi lama-phenol derivatives aqoqwe ku-M1 ahlobene nokusebenza nokwehla kokusebenza kwezinambuzane (32). Lapho siqhathanisa amanani e-Si ​​kuma-metabolite e-M1 phakathi kwamaqabunga abangelwayo namaqabunga ayingxenye yezitshalo zokulawula ze-EV emahoreni angama-24, sibonile ukuthi ukucaciswa kwe-metabolic kwama-metabolite amaningi ngemva kwezinambuzane ezihlumayo kunesimo esikhulayo esiphawulekayo (Figure 4C). Ukwanda okuqondile kwenani le-Si kutholakale kuphela kuma-phenolamides okuzivikela, kodwa akukho ukwanda kwenani le-Si okutholakale kwamanye ama-phenol kanye nama-metabolite angaziwa akhona kule module. Lena imodeli ekhethekile, ehlobene nethiyori ye-OD. Izibikezelo eziyinhloko zezinguquko ze-metabolic ezibangelwa ama-herbivore ziyavumelana. Ukuze sihlole ukuthi ngabe lokhu kuhlukahluka kwe-phenolamide spectrum kubangelwa yi-ET ethile ye-OS, sidwebe inkomba ye-metabolite Si futhi sabangela inani lokubonisa umehluko phakathi kwe-OSM ne-OSS1 ku-genotypes ye-EV ne-SETR1 (Isithombe 4D). Ku-sETR1, umehluko obangelwa yi-phenamide phakathi kwe-OSM ne-OSS1 wehliswe kakhulu. Indlela ye-bi-clustering nayo yasetshenziswa kudatha ye-MS/MS eqoqwe ezinhlotsheni ezingenayo i-JA eyanele ukuphetha amamojula amakhulu e-MS/MS ahlobene nokukhethekile kwe-metabolic okulawulwa yi-JA (Isithombe S6).
(A) Imiphumela yokuqoqana kwe-585 MS/MS ngokusekelwe ku-shared fragment (NDP similarity) kanye nokulahlekelwa okungathathi hlangothi okwabiwe (NL similarity) kuholela ekutheni i-module (M) ihambisane nomndeni we-compound owaziwayo, noma ngokwakheka kwe-Metabolite okungaziwa noma okungagaywanga kahle. Eceleni kwe-module ngayinye, kuboniswa ukusatshalaliswa kwe-metabolite (MS/MS) specific (Si). (B) Inethiwekhi yama-molecule e-Modular: Ama-Node amelela i-MS/MS kanye nemiphetho, amaphuzu e-NDP (obomvu) kanye ne-NL (oluhlaza okwesibhakabhaka) e-MS/MS (okunqunyiwe,> 0.6). Inkomba ye-metabolite specificity index (Si) efakwe umbala ngokusekelwe ku-module (kwesobunxele) futhi ibekwe ku-molecular network (kwesokudla). (C) I-Module M1 yesitshalo se-EV esimweni esihlanganisiwe (sokulawula) kanye nesimo esibangelwe (i-herbivore elingisiwe) emahoreni angama-24: umdwebo wenethiwekhi yama-molecule (Inani le-Si usayizi we-node, i-phenolamide yokuzivikela igqanyiswe ngombala oluhlaza okwesibhakabhaka). (D) Umdwebo wenethiwekhi yama-molecule e-M1 womugqa we-spectrum i-SETR1 one-EV kanye ne-ET ephazamisekile: i-phenolic compound emelelwe yi-node yesiyingi esiluhlaza, kanye nomehluko obalulekile (inani le-P) phakathi kokwelashwa kwe-W + OSM kanye ne-W + OSS1 njengosayizi we-Node. I-CP, i-N-caffeoyl-tyrosine; I-CS, i-N-caffeoyl-spermidine; I-FP, i-N-ferulic acid ester-uric acid; I-FS, i-N-ferulyl-spermidine; I-CoP, N', N “-Coumarolyl-tyrosine; I-DCS, N', N”-dicaffeoyl-spermidine; I-CFS, N', N”-caffeoyl, i-feruloyl-spermidine; I-Lycium barbarum ku-wolfberry Son; I-Nick. O-AS, ushukela we-O-acyl.
Siphinde sandisa ukuhlaziywa kusukela kuhlobo olulodwa lwe-genotype ye-Nepenthes oluncishisiwe kuya kubantu bemvelo, lapho izinguquko ezinamandla zangaphakathi ezithile emazingeni e-JA adla uhlaza kanye namazinga athile e-metabolite ziye zachazwa ngaphambilini kubantu bemvelo (26). Sebenzisa le sethi yedatha ukumboza ama-germplasm angu-43. Lawa ma-germplasm aqukethe izinhlobo ezingu-123 zezitshalo ezivela kuma-N. pallens. Lezi zitshalo zithathwe embewini eqoqwe ezindaweni ezahlukene zokuhlala e-Utah, eNevada, e-Arizona, naseCalifornia (Isithombe S7), sibale ukuhlukahluka kwe-metabolome (lapha okubizwa ngokuthi izinga labantu) ukuhlukahluka kwe-β) kanye nokukhethekile okubangelwa yi-OSM. Ngokuhambisana nezifundo zangaphambilini, sibone ushintsho olubanzi lwe-metabolic eceleni kwe-Hj kanye ne-δj axes, okubonisa ukuthi ama-germplasm anomehluko omkhulu ekuqineni kwempendulo yawo ye-metabolic kuma-herbivores (Isithombe S7). Le nhlangano ikhumbuza okubonwe ngaphambilini mayelana nobubanzi obuguquguqukayo bezinguquko ze-JA ezibangelwa ama-herbivores, futhi igcine inani eliphakeme kakhulu kubantu bemvelo (26, 36). Ngokusebenzisa i-JA ne-JA-Ile ukuhlola ukuhlangana kwezinga eliphelele phakathi kwe-Hj ne-δj, sithole ukuthi kukhona ukuhlangana okuhle okuphawulekayo phakathi kwe-JA kanye ne-metabolome β diversity kanye ne-specialization index (Isithombe S7). Lokhu kusikisela ukuthi ukungafani okubangelwa ukhula lwezitshalo ekuqalisweni kwe-JA okutholakale ezingeni labantu kungase kube ngenxa ye-polymorphisms ebalulekile ye-metabolic ebangelwa ukukhethwa kwezinambuzane ezidla uhlaza.
Izifundo zangaphambilini zikhombisile ukuthi izinhlobo zikagwayi ziyahlukahluka kakhulu ngohlobo kanye nokuncika okuhlobene ekuzivikeleni okubangelwayo nokwakhiweni kwe-metabolic. Kukholakala ukuthi lezi zinguquko ekudlulisweni kwesignali yokulwa nokudla utshani kanye namakhono okuzivikela kulawulwa yingcindezi yenani lezinambuzane, umjikelezo wokuphila kwezitshalo, kanye nezindleko zokukhiqiza ukuzivikela endaweni lapho uhlobo oluthile lukhula khona. Sifunde ukuhambisana kokuguqulwa kabusha kwe-metabolome yamaqabunga okubangelwa yizilwane ezidla uhlaza zezinhlobo eziyisithupha zeNicotiana ezidabuka eNyakatho Melika naseNingizimu Melika. Lezi zinhlobo zihlobene kakhulu neNepenthes eNyakatho Melika, okungukuthi uNicolas Bociflo. La, N. nicotinis, Nicotiana n. utshani obuncishisiwe, Nicotiana tabacum, linear tobacco, tobacco (Nicotiana spegazzinii) kanye ne-tobacco leaf tobacco (Nicotiana obtusifolia) (Isithombe 5A) (37). Eziyisithupha zalezi zinhlobo, okuhlanganisa nezinhlobo eziphawulwe kahle i-N. sicela, ziyizitshalo zonyaka ze-petunia clade, kanti i-obtusifolia N. ziyizitshalo ezihlala isikhathi eside ze-sister clade Trigonophyllae (38). Ngemva kwalokho, ukuqaliswa kwe-W + W, W + OSM kanye ne-W + OSS1 kwenziwa kulezi zinhlobo eziyisikhombisa ukuze kufundwe ukuhlelwa kabusha kwe-metabolic ezingeni lezinhlobo zokudla kwezinambuzane.
(A) Umuthi we-phylogenetic we-bootstrap osekelwe ku-maximum likelihood [yokwenziwa kwe-nuclear glutamine (38)] kanye nokusatshalaliswa kwendawo kwezinhlobo eziyisikhombisa ezihlobene eduze ze-Nicotiana (imibala ehlukene) (37). (B) Isakhiwo sokusabalala kokwehlukahluka okukhethekile kwamaphrofayili e-metabolic ezinhlobo eziyisikhombisa ze-Nicotiana (939 MS/MS; ifayela ledatha S1). Ezingeni lezinhlobo, ukwehlukahluka kwe-metabolome kuhlobene kabi nezinga lokukhetha. Ukuhlaziywa kokuxhumana kwezinhlobo phakathi kokuhlukahluka kwe-metabolic kanye nokukhethekile kanye nokuqoqwa kwe-JA kuboniswe kuMfanekiso 2. S9. Umbala, izinhlobo ezahlukene; unxantathu, i-W + OSS1; unxande, i-W + OSM; (C) I-Nicotiana JA kanye ne-JA-Ile dynamics zihlelwe ngokwe-OS excitation amplitude (i-ANOVA enezindlela ezimbili kanye ne-Tukey HSD post-multiple comparison, * P <0.05, ** P <0.01 kanye * ** Ukuze kuqhathaniswe i-W + OS kanye ne-W + W, P <0.001). Ibhokisi lesakhiwo (D) sokuhlukahluka kanye (E) ukukhethekile kohlobo ngalunye ngemva kokulingisa i-herbivorous kanye ne-methyl JA (MeJA). Inkanyezi ikhombisa umehluko obalulekile phakathi kwe-W + OS kanye ne-W + W noma i-lanolin kanye ne-W (Lan + W) noma i-Lan kanye ne-MeJA (Lan + MeJa) kanye nokulawula i-Lan (ukuhlaziywa kwezindlela ezimbili kokuhlukahluka, kulandelwe ukuqhathaniswa okuningi kwe-HSD post hoc kukaTukey, *P<0.05, **P<0.01 kanye ***P<0.001).
Sisebenzisa indlela ye-dual cluster, sithole amamojula angu-9 e-939 MS/MS (ifayela ledatha S1). Ukwakheka kwe-MS/MS okuphinde kwahlelwa kabusha ngezindlela ezahlukene kushintshashintsha kakhulu phakathi kwamamojula ahlukene phakathi kwezinhlobo (Isithombe S8). Ukubuka ngeso lengqondo i-Hj (okubizwa lapha ngokuthi i-γ-diversity yezinga lezinhlobo) kanye ne-δj kwembula ukuthi izinhlobo ezahlukene zihlangana zibe amaqembu ahlukene kakhulu esikhaleni se-metabolic, lapho ukuhlukaniswa kwezinga lezinhlobo kuvame ukugqama kakhulu kunokuvusa inkanuko. Ngaphandle kwe-N. linear kanye ne-N. obliquus, zibonisa ububanzi obukhulu bemiphumela yokungenisa (Isithombe 5B). Ngokuphambene nalokho, izinhlobo ezifana ne-N. purpurea kanye ne-N. obtusifolia zinempendulo ye-metabolic engabonakali kangako ekwelashweni, kodwa i-metabolome ihlukahlukene kakhulu. Ukusatshalaliswa kwezinhlobo ezithile kwempendulo ye-metabolic ebangelwayo kuholele ekuxhumaneni okungalungile okukhulu phakathi kokukhethekile kanye nokuhlukahluka kwe-gamma (PCC = -0.46, P = 4.9×10-8). Izinguquko ezibangelwa yi-OS emazingeni e-JA zihlobene kahle nokukhethekile kwe-metabolome, futhi zihlobene kabi nokuhlukahluka kwe-gamma ye-metabolic okuboniswa uhlobo ngalunye (Isithombe 5B kanye nesithombe S9). Kubalulekile ukuqaphela ukuthi izinhlobo ezibizwa ngokuthi “izinhlobo zempendulo yesignali” ku-Isithombe 5C, njenge-Nepenthes nematodes, i-Nepenthes nepenthes, i-Nepenthes acute, kanye ne-Nepenthes attenuated, zibangele izimpawu ezibalulekile ngemizuzu engama-30. Ukuqubuka kwamuva kwe-OS-specific JA kanye ne-JA-Ile, kuyilapho amanye amabhaktheriya abizwa ngokuthi “ayiphenduli isignali”, njenge-Nepenthes mills, i-Nepenthes powdery kanye ne-N. obtusifolia abonisa kuphela ukungeniswa kwe-JA-Ile Edge ngaphandle kwanoma yikuphi ukucaciswa kwe-OS (Isithombe 5C). Ezingeni le-metabolic, njengoba kushiwo ngenhla, kuma-Nepenthes attenuated, izinto eziphendula isignali zibonise ukucaciswa kwe-OS futhi zandisa kakhulu i-δj, ngenkathi zinciphisa i-Hj. Lo mphumela we-OS-specific priming awutholakalanga ezinhlotsheni ezihlukaniswe njengezinhlobo zesignali ezingasebenzi (Isithombe 5, D kanye no-E). Ama-metabolite athile e-OS abiwa kaningi phakathi kwezinhlobo eziphendula isignali, futhi la maqoqo esignali ahlangana nezinhlobo ezinezimpendulo ezibuthakathaka zesignali, kuyilapho izinhlobo ezinezimpendulo ezibuthakathaka zesignali zibonisa ukuncika okuncane (Isithombe S8). Lo mphumela ubonisa ukuthi ukungeniswa kwe-JA okuqondene ne-OS kanye nokulungiswa kabusha kwe-OS okuqondene ne-metabolome engezansi kuhlanganiswe ezingeni lezinhlobo.
Okulandelayo, sisebenzise i-lanolin paste equkethe i-methyl JA (MeJA) ukwelapha izitshalo ukuze sihlole ukuthi lezi zindlela zokuhlanganisa zivinjelwe yini ukutholakala kwe-JA esetshenziswa yi-JA yangaphandle, ezoba ku-cytoplasm yezitshalo. Ukuqothulwa okusheshayo yi-JA. Sithole umkhuba ofanayo wokushintsha kancane kancane kusuka ezinhlotsheni eziphendula isignali kuya ezinhlotsheni ezingaphenduli isignali okubangelwa ukuhlinzekwa okuqhubekayo kwe-JA (Isithombe 5, D no-E). Ngamafuphi, ukwelashwa kwe-MeJA kwahlela kabusha kakhulu ama-metabolome ama-nematode aqondile, i-N. obliquus, i-N. aquaticus, i-N. pallens, kanye ne-N. mikimotoi, okwaholela ekwandeni okukhulu kwe-δj kanye nokwehla kwe-Hj. N. purpurea kubonise ukwanda kwe-δj kuphela, kodwa hhayi i-Hj. N. obtusifolia, eye yabonakala ngaphambilini ukuthi iqoqa amazinga aphansi kakhulu e-JA, nayo ayiphenduli kahle ekwelashweni kwe-MeJA maqondana nokulungiswa kabusha kwe-metabolome. Le miphumela ikhombisa ukuthi ukukhiqizwa kwe-JA noma ukudluliselwa kwesignali kuvinjelwe ngokomzimba ezinhlotsheni ezingaphenduli isignali. Ukuze sihlole lo mbono, sifunde izinhlobo ezine (ama-N. pallens, ama-N. mills, ama-N. pink kanye nama-N. microphylla) abangelwa yi-W + W, W + OSM kanye ne-W + OSS1 Transcriptome (39). Ngokuhambisana nephethini yokuguqulwa kwe-metabolome, izinhlobo zihlukaniswe kahle esikhaleni se-transcriptome, phakathi kwazo i-N. attenuated ibonise i-RDPI ephezulu kakhulu ebangelwa yi-OS, kuyilapho i-N. gracilis yayinephansi kakhulu (Isithombe 6A). Kodwa-ke, kutholakale ukuthi ukuhlukahluka kwe-transcriptome okubangelwa yi-N. oblonga kwakuphansi kakhulu phakathi kwezinhlobo ezine, ngokuphambene nokuhlukahluka okuphezulu kwe-metabonomic kwe-N. oblonga okuboniswe ngaphambilini ezinhlotsheni eziyisikhombisa. Izifundo zangaphambilini zibonise ukuthi isethi yezakhi zofuzo ezihlobene nezimpawu zokuzivikela zakuqala, kufaka phakathi izimpawu ze-JA, kuchaza ukucaciswa kwezimpendulo zokuzivikela zakuqala ezibangelwa yizitho ezihlobene nohlaza ezinhlotsheni ze-Nicotiana (39). Ukuqhathanisa izindlela zokubonisa i-JA phakathi kwalezi zinhlobo ezine kwembule iphethini ethakazelisayo (Isithombe 6B). Iningi lezakhi zofuzo kule ndlela, njenge-AOC, i-OPR3, i-ACX kanye ne-COI1, libonise amazinga aphezulu okungeniswa kulezi zinhlobo ezine. Kodwa-ke, i-gene ebalulekile, i-JAR4, iguqula i-JA ibe uhlobo lwayo olusebenzayo ngokwebhayoloji lwe-JA-Ile transcripts eziqoqiwe, futhi izinga layo lokubhalwa liphansi kakhulu, ikakhulukazi ku-N. mills, i-Nepenthes pieris kanye ne-N. microphylla. Ngaphezu kwalokho, umbhalo wesinye i-gene AOS kuphela ongatholakalanga ku-N. bifidum. Lezi zinguquko ekubonakalisweni kwezakhi zofuzo zingaba yimbangela yezinhlobo ezingavamile ezibangelwa ukukhiqizwa kwe-JA okuphansi ezinhlotsheni ze-signal anergic kanye nokungeniswa kwe-N. gracilis.
(A) Ukuhlaziywa kwethiyori yolwazi ngokuhlelwa kabusha kwezimpendulo zokuqala zokubhalwa kwezinhlobo ezine zikagwayi ezihlobene eduze ezithathwe imizuzu engama-30 ngemuva kokungeniswa kwezitshalo ezidla utshani. I-RDPI ibalwa ngokuqhathanisa amaqabunga abangelwa yi-OS yezitshalo ezidla utshani nokulawulwa kwezilonda. Imibala ikhombisa izinhlobo ezahlukene, kanti izimpawu zikhombisa izindlela zokwelapha ezahlukene. (B) Ukuhlaziywa kokubonakaliswa kwezakhi zofuzo ezindleleni zokubonisa i-JA phakathi kwezinhlobo ezine. Indlela ye-JA elula iboniswa eduze kwesakhiwo sebhokisi. Imibala ehlukene ikhombisa izindlela ezahlukene zokucubungula. Inkanyezi ikhombisa ukuthi kukhona umehluko omkhulu phakathi kokwelashwa kwe-W + OS kanye nokulawulwa kwe-W + W (kokuhlolwa kwe-t koMfundi kokwehluka okubili, *P<0.05, **P<0.01 kanye ***P<0.001). I-OPDA, i-12-oxophytodienoic acid; I-OPC-8: 0,3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid.
Engxenyeni yokugcina, sifunde ukuthi ukuguqulwa kwezinhlobo ezithile zezinambuzane ze-metabolome yezinhlobo ezahlukene zezitshalo kungaba kanjani ukumelana nezilwane ezidla uhlaza. Ucwaningo lwangaphambilini lugcizelele uhlobo lweNicotiana. Ukumelana kwazo ne-Ms kanye nezibungu kuhluke kakhulu (40). Lapha, sifunde ukuxhumana phakathi kwale modeli kanye nokuqina kwazo kwe-metabolic. Sisebenzisa izinhlobo ezine zikagwayi ezingenhla, futhi sihlola ukuxhumana phakathi kokwehlukahluka kanye nokukhethekile kwe-metabolome okubangelwa yizilwane ezidla uhlaza kanye nokumelana kwezitshalo ne-Ms kanye ne-Sl, sithole ukuthi ukumelana, ukwehlukahluka kanye nokukhethekile ku-generalist Sl All kuhlobene kahle, kuyilapho ukuhlobana phakathi kokumelana nabesifazane abangochwepheshe kanye nokukhethekile kubuthakathaka, futhi ukuhlobana nokuhlukahluka akubalulekile (Isithombe S10). Ngokuphathelene nokumelana ne-S1, zombili i-N. chinensis kanye ne-N. gracilis ezincishisiwe, ezaziboniswe ngaphambilini ukuthi zibonisa amazinga okudluliselwa kwesignali ye-JA kanye nokuqina kwe-metabolome, zazinezimpendulo ezihluke kakhulu ekufakweni kwe-herbivore, futhi zibonise ukumelana okuphezulu okufanayo. Ubulili.
Eminyakeni engamashumi ayisithupha edlule, inkolelo-mbono yokuvikela izitshalo inikeze uhlaka lwenkolelo-mbono, olusekelwe kulokho abacwaningi ababikezele khona inani elikhulu lokuvela kanye nemisebenzi ye-metabolites ekhethekile yezitshalo. Iningi lalezi zinkolelo-mbono alilandeli inqubo evamile yeziphetho ezinamandla (41). Ziphakamisa izibikezelo ezibalulekile (3) ezingeni elifanayo lokuhlaziya. Lapho ukuhlolwa kwezibikezelo ezibalulekile kuvumela ukuthi kuhlaziywe izinkolelo-mbono ezithile, lokhu kuzokwenza ukuthi insimu iqhubekele phambili. Sekela, kodwa wenqabe ezinye (42). Esikhundleni salokho, inkolelo-mbono entsha yenza izibikezelo emazingeni ahlukene okuhlaziya futhi yengeza ungqimba olusha lokucatshangelwa okuchazayo (42). Kodwa-ke, izinkolelo-mbono ezimbili eziphakanyisiwe ezingeni lokusebenza, i-MT kanye ne-OD, zingachazwa kalula njengezibikezelo ezibalulekile zezinguquko ezikhethekile ze-metabolic ezibangelwa yizilwane ezidla uhlaza: inkolelo-mbono ye-OD ikholelwa ukuthi izinguquko "esikhala" se-metabolic ezikhethekile ziqondisa kakhulu. Inkolelo-mbono ye-MT ikholelwa ukuthi lezi zinguquko ngeke zibe zendlela futhi zitholakale ngokungahleliwe esikhaleni se-metabolic, futhi zivame ukuba nama-metabolites anenani eliphezulu lokuzivikela. Ukuhlolwa kwangaphambilini kwezibikezelo ze-OD kanye ne-MT kuhlolwe kusetshenziswa isethi encane yamakhemikhali "okuzivikela" angaphambili. Lezi zivivinyo ezigxile ku-metabolite zivimbela ikhono lokuhlaziya ubukhulu kanye nomzila wokulungiswa kabusha kwe-metabolome ngesikhathi sokukhula kwezitshalo, futhi azivumeli ukuhlolwa ngaphakathi kohlaka oluhambisanayo lwezibalo ukuthi kudinge izibikezelo ezibalulekile ezingacatshangelwa ngokuphelele. Linganisa izinguquko ku-metabolome yezitshalo. Lapha, sisebenzise ubuchwepheshe obusha ku-metabolomics ngokusekelwe ku-MS yokubala futhi senza ukuhlaziywa kwe-deconvolution MS ngohlobo olujwayelekile lwezincazelo zethiyori yolwazi ukuze sihlole umehluko phakathi kwalokhu okubili okuphakanyisiwe ezingeni le-metabolomics yomhlaba wonke. Isibikezelo esiyinhloko sale thiyori. Ithiyori yolwazi isetshenziswe emikhakheni eminingi, ikakhulukazi kumongo wocwaningo lwe-biodiversity kanye nokugeleza kwezakhamzimba (43). Kodwa-ke, ngokwazi kwethu, lesi yisicelo sokuqala esisetshenziselwa ukuchaza isikhala solwazi lwe-metabolic yezitshalo nokuxazulula izinkinga zemvelo ezihlobene nezinguquko zesikhashana ze-metabolic ekuphenduleni izinkomba zemvelo. Ikakhulukazi, ikhono lale ndlela lisekhonweni layo lokuqhathanisa amaphethini ngaphakathi naphakathi kwezinhlobo zezitshalo ukuze kuhlolwe ukuthi izilwane ezidla uhlaza ziguquke kanjani kusuka ezinhlotsheni ezahlukene kuya kumaphethini e-macroevolutionary aphakathi kwezinhlobo emazingeni ahlukene okuziphendukela kwemvelo. I-Metabolism.
Ukuhlaziywa kwezingxenye eziyinhloko (i-PCA) kuguqula isethi yedatha ye-multivariate ibe yindawo yokunciphisa ubukhulu ukuze umkhuba oyinhloko wedatha uchazwe, ngakho-ke uvame ukusetshenziswa njengendlela yokuhlola ukuhlaziya isethi yedatha, njenge-deconvolution metabolome. Kodwa-ke, ukunciphisa ubukhulu kuzolahlekelwa ingxenye yokuqukethwe kolwazi kusethi yedatha, futhi i-PCA ayikwazi ukunikeza ulwazi oluningi mayelana nezici ezihambisana kakhulu nemfundiso yemvelo, njengokuthi: ama-herbivore ahlela kanjani ukuhlukahluka emikhakheni ekhethekile (isibonelo, ukuceba, ukusatshalaliswa) Nobuningi) ama-metabolite? Yimaphi ama-metabolite ayizibikezelo zesimo esibangelwa yi-herbivore ethile? Ngokombono wokucacisa, ukuhlukahluka kanye nokungaguquki, okuqukethwe kolwazi lwephrofayili ye-metabolite ethile yeqabunga kuyabola, futhi kutholakala ukuthi ukudla ama-herbivore kungavuselela i-metabolism ethile. Ngokungalindelekile, sabona ukuthi, njengoba kuchaziwe ezinkombeni zethiyori yolwazi ezisetshenzisiwe, isimo se-metabolic esiphumelayo sinokuhlangana okukhulu ngemva kokuhlaselwa kwezilwane ezimbili ezidla uhlaza (i-generalist edla ebusuku i-Sl) kanye nochwepheshe we-Solanaceae uNks. Nakuba ukuziphatha kwazo kokudla kanye nokuhlushwa kuhluke kakhulu. I-Fatty acid-amino acid conjugate (FAC) initiator ku-OS (31). Ngokusebenzisa i-herbivore OS ukwelapha amanxeba okubhoboza ajwayelekile, ukwelashwa kwe-herbivore okulingisiwe nakho kubonise ukuthambekela okufanayo. Le nqubo ejwayelekile yokulingisa impendulo yezitshalo ekuhlaselweni kwe-herbivore isusa izici ezididayo ezibangelwa izinguquko ekuziphatheni kokudla kwezilwane ezidla uhlaza, okuholela emazingeni ahlukahlukene omonakalo ngezikhathi ezahlukene (34). I-FAC, eyaziwa njengembangela eyinhloko ye-OSM, inciphisa i-JAS kanye nezinye izimpendulo ze-hormone yezitshalo ku-OSS1, kuyilapho i-OSS1 inciphisa izikhathi ezingamakhulu (31). Kodwa-ke, i-OSS1 ibangele amazinga afanayo okuqongelela kwe-JA uma kuqhathaniswa ne-OSM. Kuye kwaboniswa ngaphambilini ukuthi impendulo ye-JA kuma-Nepenthes anciphile ibucayi kakhulu kwi-OSM, lapho i-FAC ingagcina khona umsebenzi wayo noma ngabe ixutshwe ngamanzi angu-1:1000 (44). Ngakho-ke, uma kuqhathaniswa ne-OSM, yize i-FAC ku-OSS1 iphansi kakhulu, yanele ukudala ukuqubuka kwe-JA okwanele. Izifundo zangaphambilini zibonise ukuthi amaprotheni afana ne-porin (45) nama-oligosaccharides (46) angasetshenziswa njengezinkomba zama-molecule ukuqala izimpendulo zokuzivikela kwezitshalo ku-OSS1. Kodwa-ke, akukacaci ukuthi lezi zinto ezibangela i-OSS1 zinesibopho sokuqongelela kwe-JA okubonwe ocwaningweni lwamanje.
Nakuba kunezifundo ezimbalwa ezichaza iminwe yeminwe ehlukile ye-metabolic ebangelwa ukusetshenziswa kwezinhlobo ezahlukene ze-herbivores noma i-JA noma i-SA yangaphandle (i-salicylic acid) (47), akekho oye waphazamisa ukuphazamiseka kwezinhlobo ze-herbivore kunethiwekhi yotshani bezitshalo kanye nemiphumela yako kulwazi lomuntu siqu. Lokhu kuhlaziywa kuqinisekisile ukuthi ukuxhumana kwenethiwekhi ye-hormone yangaphakathi namanye ama-hormone ezitshalo ngaphandle kwama-JA kwakheka ukucaciswa kokuhlelwa kabusha kwe-metabolic okubangelwa yi-herbivores. Ikakhulukazi, sithole ukuthi i-ET ebangelwa yi-OSM yayinkulu kakhulu kunalokho okubangelwa yi-OSS1. Le ndlela ihambisana nokuqukethwe kwe-FAC okuningi ku-OSM, okuyisimo esidingekayo nesanele sokubangela ukuqhuma kwe-ET (48). Esimweni sokusebenzisana phakathi kwezitshalo nezilwane ezidla uhlaza, umsebenzi wokubonisa we-ET ku-metabolite dynamics yezitshalo ezithile usaqhubeka ngokungahleliwe futhi uhlose iqembu elilodwa kuphela elihlanganisiwe. Ngaphezu kwalokho, izifundo eziningi zisebenzise ukusetshenziswa kwe-ET noma izandulela zayo noma izithibi ezahlukene ukuze kufundwe ukulawulwa kwe-ET, phakathi kwazo lezi zicelo zamakhemikhali zangaphandle zizokhiqiza imiphumela emibi eminingi engaqondile. Ngokwazi kwethu, lolu cwaningo lumele ukuhlolwa kokuqala okukhulu okuhlelekile kwendima ye-ET ekusetshenzisweni kwe-ET ukukhiqiza nokubona izitshalo ezingasebenzi kahle ze-transgenic ukuze kuhlanganiswe amandla e-metabolome yezitshalo. Ukungeniswa kwe-ET okuqondene ne-Herbivore kungagcina kuguqule impendulo ye-metabolome. Okubaluleke kakhulu ukuphathwa kwe-transgenic kwezakhi zofuzo ze-ET biosynthesis (ACO) kanye ne-perception (ETR1) okwembule ukuqongelela kwe-phenolamides okuqondene ne-herbivore. Kuye kwaboniswa ngaphambilini ukuthi i-ET ingalungisa ukuqongelela kwe-nicotine okubangelwa yi-JA ngokulawula i-putrescine N-methyltransferase (49). Kodwa-ke, ngokombono womshini, akucaci ukuthi i-ET ilungisa kanjani ukufakwa kwe-phenamide. Ngaphezu komsebenzi wokudluliselwa kwesignali we-ET, i-metabolic flux ingaphinde ihanjiswe ku-S-adenosyl-1-methionine ukulawula ukutshalwa kwezimali kuma-polyaminophenol amides. I-S-adenosyl-1-methionine iyi-ET futhi iyindlela ejwayelekile yendlela ye-polyamine biosynthetic. Indlela i-ET signal elawula ngayo izinga le-phenolamide idinga ukufundwa okwengeziwe.
Isikhathi eside, ngenxa yenani elikhulu lama-metabolite akhethekile anesakhiwo esingaziwa, ukunaka okukhulu ezigabeni ezithile ze-metabolic bekungakwazi ukuhlola ngokuqinile izinguquko zesikhathi zokuhlukahluka kwe-metabolic ngemva kokuxhumana kwezinto eziphilayo. Njengamanje, ngokusekelwe ekuhlaziyweni kwethiyori yolwazi, umphumela oyinhloko wokutholwa kwe-MS/MS spectrum ngokusekelwe kuma-metabolite angachemile ukuthi ama-herbivore adla noma alingisa ama-herbivore ayaqhubeka nokunciphisa ukuhlukahluka kwe-metabolic jikelele kwe-metabolome yamaqabunga ngenkathi ekhulisa izinga lawo lobuchwepheshe. Lokhu kwanda kwesikhashana kokucaciswa kwe-metabolome okubangelwa ama-herbivore kuhlotshaniswa nokwanda okuhambisanayo kokucaciswa kwe-transcriptome. Isici esinikela kakhulu kulokhu okukhethekile kwe-metabolome okukhulu (okunenani eliphakeme le-Si) yi-metabolite ekhethekile enomsebenzi we-herbivorous ochazwe ngaphambilini. Le modeli iyahambisana nokubikezela kwethiyori ye-OD, kodwa ukubikezela kwe-MT okuhlobene nokungahleliwe kokuhlelwa kabusha kwe-metabolome akuhambisani. Kodwa-ke, le datha ihambisana nokubikezela kwemodeli exubile (i-MT engcono kakhulu; Umfanekiso 1B), ngoba amanye ama-metabolite angacaciswanga anemisebenzi yokuzivikela engaziwa angase alandele ukusatshalaliswa kwe-Si okungahleliwe.
Iphethini ephawulekayo eqoqwe kabanzi yilolu cwaningo ukuthi kusukela ezingeni lokuziphendukela kwemvelo okuncane (isitshalo esisodwa kanye nenani likagwayi) kuya esikalini esikhulu sokuziphendukela kwemvelo (izinhlobo zikagwayi ezihlobene eduze), amazinga ahlukene okuhleleka kokuziphendukela kwemvelo "asesivikeloni esingcono kakhulu". Kunomehluko omkhulu emakhonweni ezilwane ezidla uhlaza. UMoore et al. (20) kanye noKessler noKalske (1) baphakamise ngokuzimela ukuguqula amazinga amathathu okusebenza kwezinto eziphilayo ahlukaniswe nguWhittaker (50) ekuqaleni abe yizinguquko zesikhathi ezihlanganisiwe nezibangelwa ukuhlukahluka kwamakhemikhali; laba babhali abazange bafingqe. Izinqubo zokuqoqwa kwedatha ye-metabolome enkulu azichazi ukuthi kungabalwa kanjani ukuhlukahluka kwe-metabolic kusuka kule datha. Kulesi sifundo, ukulungiswa okuncane ekuhlukanisweni kokusebenza kukaWhittaker kuzocabangela ukuhlukahluka kwe-α-metabolic njengokwehlukahluka kwe-MS/MS spectra esitshalweni esithile, kanye nokuhlukahluka kwe-β-metabolic njengokwejwayelekile kwe-intraspecific metabolism yeqembu labantu Isikhala, kanye nokuhlukahluka kwe-γ-metabolic kuzoba ukwandiswa kokuhlaziywa kwezinhlobo ezifanayo.
Isignali ye-JA ibalulekile ezinhlobonhlobo zezimpendulo ze-metabolism ye-herbivore. Kodwa-ke, kukhona ukuntuleka kokuhlolwa okuqinile kwenani lomnikelo wokulawulwa kwe-JA biosynthesis ekuhlukeni kwe-metabolome, nokuthi ngabe isignali ye-JA iyindawo evamile yokuhlukahluka kwe-metabolic okubangelwa ukucindezeleka esikalini esiphezulu sokuguquka kwe-macroevolution akukacaci. Sibonile ukuthi isimo se-herbivorous se-Nepenthes herbivorous esibangela ukukhethekile kwe-metabolome futhi ukuhlukahluka kokukhethekile kwe-metabolome ngaphakathi kwenani lezinhlobo ze-Nicotiana kanye naphakathi kwezinhlobo ze-Nicotiana ezihlobene eduze kuhlobene kahle ngokuhlelekile nokusayina kwe-JA. Ngaphezu kwalokho, lapho isignali ye-JA iphazamiseka, ukucaciswa kwe-metabolic okubangelwa yi-genotype eyodwa ye-herbivore kuzokhanselwa (Isithombe 3, C no-E). Njengoba izinguquko ze-metabolic spectrum zezinhlobo ze-Nepenthes ezincishisiwe ngokwemvelo ziningi kakhulu, izinguquko ekuhlukeni kwe-metabolic β kanye nokucaciswa kulokhu kuhlaziywa zingabangelwa kakhulu ukukhungatheka okukhulu kwezigaba ze-compound ezicebile nge-metabolite. Lezi zigaba ze-compound zibusa ingxenye yephrofayili ye-metabolome futhi ziholela ekuxhumaneni okuhle nezimpawu ze-JA.
Ngenxa yokuthi izindlela ze-biochemical zezinhlobo zikagwayi ezihlobene kakhulu nazo zihlukile kakhulu, ama-metabolites akhonjwa ngqo ngesici sekhwalithi, ngakho-ke kuyahlaziya kakhulu. Ukucubungula kwethiyori yolwazi lwephrofayili ye-metabolic ebanjiwe kwembula ukuthi ukungeniswa kwe-herbivorous kwandisa ukuhweba phakathi kokwehlukahlukana kwe-gamma ye-metabolic kanye nokukhethekile. Isignali ye-JA idlala indima ebalulekile kulokhu kuhwebelana. Ukwanda kokukhethekile kwe-metabolome kuhambisana nokubikezela okuyinhloko kwe-OD futhi kuhlobene kahle nesignali ye-JA, kuyilapho isignali ye-JA ihlobene kabi nokuhlukahlukana kwe-gamma ye-metabolic. Lawa mamodeli akhombisa ukuthi amandla e-OD ezitshalo anqunywa kakhulu yi-plasticity ye-JA, kungakhathaliseki ukuthi kusezingeni lokuguquguquka okuncane noma kusezingeni elikhulu lokuziphendukela kwemvelo. Ukuhlolwa kokusetshenziswa kwe-JA yangaphandle okuvimbela amaphutha e-JA biosynthesis kwembula ukuthi izinhlobo zikagwayi ezihlobene eduze zingahlukaniswa zibe izinhlobo eziphendula isignali kanye nezingaphenduli isignali, njengendlela yazo ye-JA kanye ne-metabolome plasticity ebangelwa yizimila. Izinhlobo ezingaphenduli isignali azikwazi ukuphendula ngenxa yokungakwazi kwazo ukukhiqiza i-JA engokwemvelo futhi ngenxa yalokho zingaphansi kwemikhawulo ye-physiological. Lokhu kungase kubangelwe izinguquko kwezinye izakhi zofuzo ezibalulekile endleleni yokubonisana ye-JA (i-AOS ne-JAR4 ku-N. crescens). Lo mphumela uqokomisa ukuthi la maphethini okuguquka kwezinhlobo ezahlukene angaqhutshwa kakhulu yizinguquko ekuqondeni kwangaphakathi kwama-hormone kanye nokusabela.
Ngaphezu kokuxhumana phakathi kwezitshalo nezilwane ezidla uhlaza, ukuhlolwa kokwehlukahlukana kwe-metabolic kuhlobene nazo zonke intuthuko ebalulekile yethiyori ocwaningweni lokuzivumelanisa nezinto eziphilayo nendawo kanye nokuvela kwezimpawu eziyinkimbinkimbi ze-phenotypic. Njengoba inani ledatha elitholwa yizinsimbi ze-MS zanamuhla likhula, ukuhlolwa kwe-hypothesis ngokuhlukahlukana kwe-metabolic manje kungadlula umehluko we-metabolite yomuntu ngamunye/isigaba futhi kwenze ukuhlaziywa komhlaba wonke ukuze kwembulwe amaphethini angalindelekile. Enkambisweni yokuhlaziywa okukhulu, isingathekiso esibalulekile umqondo wokuqonda amamephu anencazelo angasetshenziswa ukuhlola idatha. Ngakho-ke, umphumela obalulekile wokuhlanganiswa kwamanje kwe-metabolomics ye-MS/MS engachemile kanye nethiyori yolwazi ukuthi inikeza i-metric elula engasetshenziswa ukwakha amamephu ukuze uphequlule ukwehlukahlukana kwe-metabolic ezikalini ezahlukene ze-taxonomic. Kuyisidingo esiyisisekelo sale ndlela. Ucwaningo lokuziphendukela kwemvelo okuncane/okukhulu kanye ne-ecology yomphakathi.
Ezingeni lokuziphendukela kwemvelo okukhulu, umongo wenkolelo-mbono yokuziphendukela kwemvelo kwezitshalo nezinambuzane ka-Ehrlich noRaven (51) ukubikezela ukuthi ukuhlukahluka kokwehlukahlukana kwe-metabolic phakathi kwezinhlobo zezitshalo kuyimbangela yokuhlukahlukana kwezinhlobo zezitshalo. Kodwa-ke, eminyakeni engamashumi amahlanu kusukela kwashicilelwa lo msebenzi obalulekile, lo mbono awukaze uhlolwe (52). Lokhu kungenxa kakhulu yezici ze-phylogenetic zezici ze-metabolic ezifanayo kuzo zonke izinhlobo zezitshalo ezikude. Ukungatholakali kungasetshenziswa ukuqinisa izindlela zokuhlaziya okuqondiwe. Ukuhamba komsebenzi kwamanje kwe-MS/MS okucutshungulwa yi-information theory kulinganisa ukufana kwesakhiwo se-MS/MS kwama-metabolite angaziwa (ngaphandle kokukhethwa kwe-metabolite kwangaphambilini) futhi kuguqula la ma-MS/MS abe yisethi ye-MS/MS, ngakho-ke ku-metabolism yobungcweti Lawa mamodeli e-macro-evolutionary aqhathaniswa ngesilinganiso sokuhlukaniswa. Izinkomba zezibalo ezilula. Inqubo ifana nokuhlaziywa kwe-phylogenetic, okungasebenzisa ukulungiswa kokulandelana ukuze kulinganiswe izinga lokuhlukahlukana noma ukuguquka kwezinhlamvu ngaphandle kokubikezela kwangaphambilini.
Ezingeni le-biochemical, umbono wokuhlola kaFirn noJones (53) ukhombisa ukuthi ukwehlukahlukana kwe-metabolic kugcinwa emazingeni ahlukene ukuze kuhlinzekwe ngezinto zokusetshenziswa ukuze kusebenze imisebenzi ye-biologies yama-metabolites angahlobene ngaphambili noma afakwe esikhundleni sawo. Izindlela zethiyori yolwazi zinikeza uhlaka lapho lezi zinguquko zokuziphendukela kwemvelo ezithile ze-metabolite ezenzeka ngesikhathi sokuqeqeshwa kwe-metabolite zingalinganiswa njengengxenye yenqubo yokuhlola yokuziphendukela kwemvelo ehlongozwayo: ukuzivumelanisa nokusebenza kwe-biologistic kusuka ekucacisweni okuphansi kuya ekucacisweni okuphezulu Ama-metabolites avinjelwe endawo ethile.
Sekukonke, ezinsukwini zokuqala zebhayoloji yama-molecular, kwasungulwa imibono ebalulekile yokuvikela izitshalo, futhi izindlela ezisuselwa ku-deductive hypothesis zibhekwa kabanzi njengezindlela zodwa zentuthuko yesayensi. Lokhu kungenxa kakhulu yemikhawulo yobuchwepheshe yokulinganisa yonke i-metabolome. Nakuba izindlela ezisuselwa ku-hypothesis ziwusizo kakhulu ekukhetheni ezinye izindlela ezibangela, ikhono lazo lokuthuthukisa ukuqonda kwethu amanethiwekhi e-biochemical lilinganiselwe kakhulu kunezindlela zokubala ezitholakalayo njengamanje kwisayensi yesimanje esebenzisa idatha eningi. Ngakho-ke, imibono engenakubikezelwa ingaphezu kakhulu kwedatha etholakalayo, ngakho-ke umjikelezo wefomula/wokuhlola ocatshangelwayo wentuthuko ensimini yocwaningo awunakuqedwa (4). Sibona kusengaphambili ukuthi ukuhamba komsebenzi wokubala kwe-metabolomics okulethwe lapha kungavuselela isithakazelo ezindabeni zakamuva (kanjani) nezokugcina (kungani) zokuhlukahluka kwe-metabolic, futhi kufake isandla enkathini entsha yesayensi yedatha eqondiswa ngokwethiyori. Le nkathi yahlola kabusha imibono ebalulekile eyaphefumulela izizukulwane zangaphambilini.
Ukondla izilwane ezidla uhlaza ngqo kwenziwa ngokukhuphula i-larva yesibili noma i-Sl larva eqabungeni elilodwa lesitshalo esinombala okhanyayo lesitshalo esisodwa esiqhakaza i-rose, kanye nezitshalo eziyi-10 eziphindaphindayo ngesitshalo ngasinye. Izibungu zezinambuzane zaboshwa ngama-clamp, kwathi izicubu zeqabunga ezisele zaqoqwa emahoreni angama-24 nama-72 ngemva kokutheleleka futhi zaqandiswa ngokushesha, kwathi ama-metabolites akhishwa.
Lingisa ukwelashwa okunokhula ngendlela ehambisana kahle kakhulu. Indlela iwukusebenzisa amasondo ephethini yendwangu ukubhoboza imigqa emithathu yameva ohlangothini ngalunye lwe-midrib yamaqabunga amathathu anwetshwe ngokugcwele esitshalo ngesikhathi sokukhula kwe-garland yendwangu, bese ufaka ngokushesha u-1:5 Diluted Ms. Noma sebenzisa iminwe egilavu ​​​​ukufaka i-S1 OS enxebeni lokubhoboza. Vuna futhi ucubungule iqabunga njengoba kuchaziwe ngenhla. Sebenzisa indlela echazwe ngaphambilini ukukhipha ama-metabolites ayinhloko nama-hormone ezitshalo (54).
Uma kusetshenziswa i-JA yangaphandle, amaqabunga amathathu e-petiole esitshalo ngasinye kweziyisithupha eziqhakaza ama-rose ohlobo ngalunye aphathwa nge-20μl ye-lanolin paste equkethe i-150μg MeJA (Lan + MeJA), kanye ne-20μl ye-lanolin kanye nokwelashwa kwamanxeba (Lan + W), noma sebenzisa i-lanolin emsulwa engu-20μl njengokulawula. Amaqabunga avunwa emahoreni angu-72 ngemva kokwelashwa, aqandiswe ku-nitrogen ewuketshezi, futhi agcinwa ku--80°C kuze kube yilapho esetshenziswa.
Imigqa emine ye-JA kanye ne-ET transgenic, okuyi-irAOC (36), i-irCOI1 (55), i-irACO kanye ne-sETR1 (48), itholakale eqenjini lethu locwaningo. I-irAOC ibonise kakhulu ukwehla kwamazinga e-JA kanye ne-JA-Ile, kuyilapho i-irCOI1 yayingazweli kuma-JA. Uma kuqhathaniswa ne-EV, ukuqongelela kwe-JA-Ile kwanda. Ngokufanayo, i-irACO izonciphisa ukukhiqizwa kwe-ET, futhi uma kuqhathaniswa ne-EV, i-sETR1, engazweli ku-ET, izokwandisa ukukhiqizwa kwe-ET.
I-photoacoustic laser spectrometer (i-Sensor Sense ETD-300 real-time ET sensor) isetshenziselwa ukwenza ukulinganisa kwe-ET ngaphandle kokuphazamisa. Ngokushesha ngemva kokwelashwa, ingxenye yamaqabunga yanqunywa yadluliselwa ebhodleleni lengilazi elivaliwe elingu-4-ml, futhi isikhala sekhanda savunyelwa ukuba siqongeleleke zingakapheli amahora ama-5. Ngesikhathi sokulinganisa, ibhodlela ngalinye lahlanzwa ngomfudlana wamalitha ama-2/ihora womoya ohlanzekile imizuzu engu-8, owawudlule ngaphambilini nge-catalyst enikezwe yi-Sensor Sense ukususa i-CO2 namanzi.
Idatha ye-microarray yanyatheliswa ekuqaleni ku-(35) futhi yagcinwa ku-National Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (inombolo yokufinyelela i-GSE30287). Idatha ehambisana namaqabunga abangelwa ukwelashwa kwe-W + OSM kanye nokulawula okungonakalanga yakhishwa kulolu cwaningo. Ukuqina okuluhlaza kuyi-log2. Ngaphambi kokuhlaziywa kwezibalo, isisekelo saguqulwa futhi salungiswa saba yi-percentile yaso engama-75 kusetshenziswa iphakheji yesofthiwe ye-R.
Idatha yokuqala yokulandelana kwe-RNA (i-RNA-seq) yezinhlobo ze-Nicotiana ithathwe ku-NCBI Short Reading Archives (SRA), inombolo yephrojekthi yi-PRJNA301787, eyabikwa nguZhou et al. (39) futhi iyaqhubeka njengoba kuchaziwe ku-(56). Idatha eluhlaza ecutshungulwa yi-W + W, W + OSM kanye ne-W + OSS1 ehambisana nezinhlobo ze-Nicotiana ikhethiwe ukuze ihlaziywe kulolu cwaningo, futhi icutshungulwa ngale ndlela elandelayo: Okokuqala, ukufundwa kwe-RNA-seq eluhlaza kwaguqulwa kwaba yifomethi ye-FASTQ. I-HISAT2 iguqula i-FASTQ ibe yi-SAM, kanti i-SAMtools iguqula amafayela e-SAM abe amafayela e-BAM ahleliwe. I-StringTie isetshenziselwa ukubala ukubonakaliswa kwezakhi zofuzo, futhi indlela yayo yokubonakaliswa ukuthi kunezingcezu ngezingcezu eziyinkulungwane ngezingcezu eziyisigidi zokubhala ezilandelanayo.
Ikholomu ye-Acclaim chromatographic (150 mm x 2.1 mm; usayizi wezinhlayiya 2.2μm) esetshenziswe ekuhlaziyeni kanye nekholomu yokulinda engu-4 mm x 4 mm iqukethe izinto ezifanayo. I-gradient elandelayo ye-binary isetshenziswa ohlelweni lwe-Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC): imizuzu engu-0 kuya ku-0.5, i-isocratic 90% A [amanzi ahlanzekile, i-0.1% (v/v) acetonitrile kanye ne-0.05% formic acid], i-10% B (i-Acetonitrile kanye ne-0.05% formic acid); imizuzu engu-0.5 kuya ku-23.5, isigaba se-gradient singu-10% A kanye no-90% B, ngokulandelana; imizuzu engu-23.5 kuya ku-25, i-isocratic 10% A kanye no-90% B. Izinga lokugeleza lingu-400μl/min. Kuzo zonke izihlaziyi ze-MS, faka ikholomu eluent ku-quadrupole kanye ne-time-of-flight analyzer (qTOF) analyzer efakwe umthombo we-electrospray osebenza kwimodi ye-ionization enhle (i-capillary voltage, 4500 V; i-capillary outlet 130 V; Izinga lokushisa lokomisa 200°C; ukomisa umoya 10 amalitha/min).
Yenza ukuhlaziywa kwezingcezu ze-MS / MS (okubizwa ngokuthi i-MS / MS) okungabalulekile noma okungahlukaniseki kudatha ukuze uthole ulwazi lwesakhiwo mayelana nephrofayili ye-metabolic etholakalayo iyonke. Umqondo wendlela ye-MS/MS engakhethi uncike eqinisweni lokuthi i-quadrupole inefasitela elikhulu kakhulu lokuhlukaniswa kwesisindo [ngakho-ke, cabanga ngazo zonke izimpawu ze-mass-to-charge ratio (m/z) njengezingcezu]. Ngenxa yalesi sizathu, ngenxa yokuthi ithuluzi le-Impact II alikwazanga ukudala ukuthambekela kwe-CE, kwenziwa ukuhlaziywa okuningana okuzimele kusetshenziswa amanani akhuphukile e-collision-induced dissociation collision energy (CE) ionization. Ngamafuphi, qala uhlaziye isampula nge-UHPLC-electrospray ionization/qTOF-MS usebenzisa imodi ye-single mass spectrometry (izimo zokuqhekeka eziphansi ezikhiqizwa ukuqhekeka komthombo), ukuskena kusuka ku-m/z 50 kuya ku-1500 ngemvamisa yokuphindaphinda engu-5 Hz. Sebenzisa i-nitrogen njengegesi yokushayisana ekuhlaziyweni kwe-MS/MS, bese wenza izilinganiso ezizimele ku-voltage ezine ezahlukene zokuhlukana okubangelwa ukushayisana: 20, 30, 40, kanye no-50 eV. Kuyo yonke inqubo yokulinganisa, i-quadrupole inefasitela elikhulu kakhulu lokuhlukaniswa kwesisindo, kusukela ku-m/z 50 kuya ku-1500. Lapho ukuhlolwa kobubanzi be-m/z kanye nomzimba wangaphambili kusethwe ku-200, ububanzi besisindo buvuselelwa ngokuzenzakalelayo yisofthiwe yokusebenza yethuluzi kanye ne-0 Da. Skena izingcezu zesisindo njengakwimodi yesisindo esisodwa. Sebenzisa i-sodium formate (i-isopropanol engu-50 ml, i-200 μl formic acid kanye nesisombululo samanzi esingu-1 ml se-1M NaOH) ukuze kulinganiswe isisindo. Usebenzisa i-algorithm yokulinganisa enembile kakhulu kaBruker, ifayela ledatha liyalinganiswa ngemuva kokusebenzisa i-spectrum ephakathi esikhathini esinikeziwe. Sebenzisa umsebenzi wokuthumela wesofthiwe ye-Data Analysis v4.0 (Brook Dalton, Bremen, Germany) ukuguqula amafayela edatha eluhlaza abe yifomethi ye-NetCDF. Isethi yedatha ye-MS/MS igcinwe kusizindalwazi esivulekile se-metabolomics i-MetaboLights (www.ebi.ac.uk) ngenombolo yokungena. MTBLS1471.
Ukuhlanganiswa kwe-MS/MS kungabonakala ngokuhlaziywa kokuxhumana phakathi kwezimpawu zekhwalithi ye-MS1 ​​ne-MS/MS zamandla okushayisana aphansi naphezulu kanye nemithetho esanda kusetshenziswa. Iskripthi se-R sisetshenziselwa ukuqaphela ukuhlaziywa kokuxhumana kokusatshalaliswa kwesandulela kumkhiqizo, kanti iskripthi se-C# (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) sisetshenziselwa ukusebenzisa imithetho.
Ukuze kuncishiswe amaphutha amahle abangelwa umsindo wangemuva kanye nokuhlangana okungamanga okubangelwa ukuthola izici ezithile ze-m/z kumasampula ambalwa kuphela, sisebenzisa umsebenzi "we-filled peak" wephakheji ye-R i-XCMS (yokulungisa umsindo wangemuva). Kufanele isetshenziswe ukufaka esikhundleni sokuqina "kwe-NA" (i-peak engatholakali). Uma kusetshenziswa umsebenzi we-fill peak, kusenamanani amaningi "okuqina kwe-0" kusethi yedatha azothinta ukubalwa kokuhlangana. Bese, siqhathanisa imiphumela yokucubungula idatha etholwe lapho kusetshenziswa umsebenzi we-filled peak nalapho umsebenzi we-filled peak ungasetshenziswa, bese sibala inani lomsindo wangemuva ngokusekelwe enanini elilinganisiwe elilungisiwe, bese sishintsha lawa manani okuqina kwe-0 ngenani langemuva elibaliwe. Siphinde sacabangela izici kuphela amandla azo adlula kathathu inani langemuva futhi sazibheka "njengeziqongo zangempela." Ekubalweni kwe-PCC, kucatshangelwa kuphela izimpawu ze-m/z zesampula precursor (MS1) kanye namasethi edatha ezingxenyeni ezineziqongo zangempela okungenani eziyisishiyagalombili.
Uma ukuqina kwesici sekhwalithi yokwandulela kulo lonke isampula kuhlobene kakhulu nokuqina okuncishisiwe kwesici sekhwalithi efanayo esibhekene namandla aphansi noma aphezulu okushayisana, futhi lesi sici singabhalwanga njenge-isotope peak yi-CAMERA, singachazwa kabanzi. Ngemuva kwalokho, ngokubala wonke amabhangqa angaba khona omkhiqizo wokwandulela ngaphakathi kwemizuzwana emi-3 (iwindi lesikhathi sokugcinwa esilinganiselwe sokugcina okuphezulu), ukuhlaziywa kokuhambisana kwenziwa. Kuphela uma inani le-m/z liphansi kunenani lokwandulela kanye nokuqhekeka kwe-MS/MS kwenzeka endaweni efanayo yesampula kusethi yedatha njenge-precursor lapho ithathwe khona, lapho ibhekwa khona njenge-fragment.
Ngokusekelwe kule mithetho emibili elula, sikhipha izingcezu ezicacisiwe ezinamanani e-m/z amakhulu kune-m/z yesandulela esikhonjiwe, futhi ngokusekelwe endaweni yesampula lapho isandulela sivela khona kanye nesaqhekeza esicacisiwe. Kungenzeka futhi ukukhetha izici zekhwalithi ezikhiqizwe yizingcezu eziningi ezitholakala emthonjeni ezikhiqizwe kwimodi ye-MS1 ​​njengezandulela ezikhethiwe, ngaleyo ndlela zikhiqize ama-compound e-MS/MS angafuneki. Ukuze kuncishiswe lokhu kuphindaphindeka kwedatha, uma ukufana kwe-NDP kwe-spectra kudlula u-0.6, futhi kungengxenye ye-chromatogram "pcgroup" echazwe yi-CAMERA, sizozihlanganisa. Okokugcina, sihlanganisa yonke imiphumela emine ye-CE ehlotshaniswa nesandulela kanye nezingcezu ku-spectrum yokugcina ehlanganisiwe ehlukanisiwe ngokukhetha i-peak enamandla aphezulu phakathi kwazo zonke iziqongo ezikhethiwe ezinenani elifanayo le-m/z emandleni ahlukene okushayisana. Izinyathelo zokucubungula ezilandelayo zisekelwe emqondweni we-spectrum ehlanganisiwe futhi zicabangela izimo ezahlukene ze-CE ezidingekayo ukuze kwandiswe amathuba okuqhekeka, ngoba ezinye izingcezu zingatholakala kuphela ngaphansi kwamandla athile okushayisana.
I-RDPI (30) yasetshenziswa ukubala ukungakwazi ukuguquguquka kwephrofayili ye-metabolic. Ukuhlukahluka kwe-metabolic spectrum (inkomba ye-Hj) kutholakala ngobuningi bezinto ezingaphambi kwe-MS/MS ezisebenzisa i-Shannon entropy yokusatshalaliswa kwemvamisa ye-MS/MS kusetshenziswa i-equation elandelayo echazwe nguMartínez et al. (8). I-Hj = −∑i = 1mPijlog2(Pij) lapho i-Pij ihambelana nemvamisa ehlobene ye-i-th MS/MS kusampula ye-j-th (j = 1, 2,…, m) (i = 1, 2, …, m) t).
Ukucaciswa kwe-Metabolic (inkomba ye-Si) kuchazwa njengokubonakaliswa kobunikazi be-MS/MS enikeziwe maqondana nemvamisa phakathi kwamasampula acatshangwayo. Ukucaciswa kwe-MS/MS kubalwa njengo-Si = 1t (∑j = 1tPijPilog2PijPi)
Sebenzisa ifomula elandelayo ukukala inkomba ye-δj ethize ye-metabolome yesampula ngayinye ye-j, kanye nesilinganiso sokucaciswa kwe-MS/MS δj = ∑i = 1mPijSi
Ama-spectra e-MS/MS aqondaniswe ngamabili, futhi ukufana kubalwa ngokusekelwe kumaphuzu amabili. Okokuqala, usebenzisa i-NDP ejwayelekile (eyaziwa nangokuthi indlela yokuxhumana kwe-cosine), sebenzisa i-equation elandelayo ukuthola ukufana kwesigaba phakathi kwe-spectra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 lapho i-S1 ne-S2 Ngokufanayo, ku-spectrum 1 kanye ne-spectrum 2, kanye ne-WS1, i kanye ne-WS2, i imele isisindo ngokusekelwe ekuqineni kwe-peak ukuthi umehluko we-i-th common peak phakathi kwama-spectra amabili ungaphansi kuka-0.01 Da. Isisindo sibalwa kanje: W = [peak intensity] m [quality] n, m = 0.5, n = 2, njengoba kuphakanyiswe yi-MassBank.
Kwasetshenziswa indlela yesibili yokufaka amaphuzu, eyayihilela ukuhlaziya i-NL eyabiwe phakathi kwe-MS/MS. Kulokhu, sisebenzise uhlu lwe-NL olungama-52 oluvame ukuhlangana ngesikhathi senqubo yokuqhekeka kwe-MS ngokulandelana, kanye ne-NL ecacile kakhulu (ifayela ledatha i-S1) elake lachazwa ngaphambilini nge-spectrum ye-MS/MS yama-metabolites esibili ezinhlobo ze-Nepenthes ezibuthakathaka (9, 26). Dala i-vector binary engu-1 no-0 ye-MS/MS ngayinye, ehambisana nesikhathi samanje kanye nokungabikho kwe-NL ethile ngokulandelana. Ngokusekelwe ekufaneni kwebanga le-Euclidean, i-NL similarity score ibalwa kumbhangqwana ngamunye wama-vector binary NL.
Ukuze senze i-dual clustering, sisebenzise iphakheji ye-R i-DiffCoEx, esekelwe ekwandisweni kwe-Weighted Gene Co-expression Analysis (WGCNA). Sisebenzisa ama-matrices e-NDP kanye ne-NL scoring e-MS/MS spectra, sisebenzise i-DiffCoEx ukubala i-comparative correlation matrix. I-binary clustering yenziwa ngokusetha ipharamitha ethi “cutreeDynamic” ibe yindlela = “hybrid”, cutHeight = 0.9999, deepSplit = T, kanye ne-minClusterSize = 10. Ikhodi yomthombo we-R ye-DiffCoEx ilandwe kusuka kufayela elingeziwe 1 nguTesson et al. (57); Iphakheji yesofthiwe ye-R WGCNA edingekayo ingatholakala ku-https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Ukuze senze ukuhlaziywa kwenethiwekhi yama-molecule ye-MS/MS, sibale ukuxhumeka kwe-spectral okubhangqiwe ngokusekelwe ezinhlotsheni zokufana kwe-NDP ne-NL, futhi sasebenzisa isofthiwe ye-Cytoscape ukubona ngeso lengqondo i-topology yenethiwekhi sisebenzisa ukwakheka kwezinto eziphilayo kuhlelo lokusebenza lwesandiso se-algorithm yokwakheka kwe-CyFilescape yFiles.
Sebenzisa i-R version 3.0.1 ukwenza ukuhlaziywa kwezibalo kudatha. Ukubaluleka kwezibalo kuhlolwe kusetshenziswa ukuhlaziywa kwezindlela ezimbili kokuhlukahluka (i-ANOVA), kulandelwe ukuhlolwa kwe-post-hoc kukaTukey okuneqiniso okuphawulekayo (i-HSD). Ukuze kuhlaziywe umehluko phakathi kokwelashwa okunokhula kanye nokulawula, ukusatshalaliswa kwemisila emibili kwamaqembu amabili amasampula anokuhlukahluka okufanayo kuhlaziywe kusetshenziswa ukuhlolwa kwe-t komfundi.
Ukuze uthole izinto ezengeziwe zalesi sihloko, sicela ubheke ku-http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
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Qaphela: Sicela kuphela ukuthi unikeze ikheli lakho le-imeyili ukuze umuntu omncomayo ekhasini azi ukuthi ufuna abone i-imeyili nokuthi akuyona ugaxekile. Ngeke sithathe noma yimaphi amakheli e-imeyili.
Lo mbuzo usetshenziselwa ukuhlola ukuthi uyisivakashi yini futhi uvimbele ukuthunyelwa kogaxekile okuzenzakalelayo.
Ithiyori yolwazi inikeza uhlobo lwemali olujwayelekile lokuqhathanisa ama-metabolome akhethekile kanye nokubikezela imibono yokuzivikela ekuhlolweni.
Ithiyori yolwazi inikeza uhlobo lwemali olujwayelekile lokuqhathanisa ama-metabolome akhethekile kanye nokubikezela imibono yokuzivikela ekuhlolweni.
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