Murakoze gusura Nature.com. Verisiyo ya mushakisha ukoresha ifite ubufasha buke kuri CSS. Kugira ngo ubone uburambe bwiza, turakugira inama yo gukoresha mushakisha ivuguruye (cyangwa uzimye uburyo bwo guhuza na Internet Explorer). Hagati aho, kugira ngo dukomeze gushyigikira, tuzerekana urubuga rudafite imiterere na JavaScript.
Bitandukanye n'inyamaswa zo mu bwoko bwa vertebrates, udukoko twinshi dukekwaho kutagira imisemburo ya steroide y’igitsina gabo. Muri Anopheles gambiae, ecdysone steroid 20-hydroxyecdysone (20E) isa nkaho yahindutse kugira ngo igenzure iterambere ry'igi iyo ikozwe n'abagore2 no gutera igihe cyo guhuza iyo ikorewe mu mibonano mpuzabitsina n'abagabo3. Kubera ko iterambere ry'igi no guhuza ari ingenzi mu myororokere, gusobanukirwa uburyo imibu y'abagore ya Anopheles ihuza ibi bimenyetso by'imisemburo bishobora koroshya itegurwa rya gahunda nshya zo kurwanya malariya. Aha, turahishura ko iyi mikorere yo kubyara igenzurwa na steroide zitandukanye z'igitsina binyuze mu rusobe rw'imisemburo ikora/ikora ya ecdysteroid. Twabonye ecdysone y'abagabo ifite oxidized, 3-dehydro-20E (3D20E), irinda ababyeyi binyuze mu guhagarika kwakira imibonano mpuzabitsina y'abagore nyuma yo kwimura no gukora imibonano mpuzabitsina binyuze mu gukuraho phosphorylation. By'umwihariko, kwimura 3D20E byanatumye habaho igaragara ry'uturemangingo tw'imyororokere dukomeza gukura kw'igi mu gihe cya Plasmodium yandura, bigatuma ubuzima bw'abagore banduye burushaho kuba bwiza. 20E ikomoka ku bagore ntabwo itera uburyo bwo gukora imibonano mpuzabitsina, ariko bituma abantu bahura n’abahuje igitsina batera amagi nyuma y’uko 20E-inhibiting kinases ihagaritswe. Kumenya iyi hormone ya steroidi y’udukoko tw’igitsina gabo n’uruhare rwayo mu kugenzura uburyo abagore bakira, kubyara no gukorana na Plasmodium bigaragaza ko bishoboka kugabanya uburyo imibu itera malariya mu kubyara.
Abandura malariya n'impfu byongeye kwiyongera4 bitewe n'ubudahangarwa bw'imiti yica udukoko mu mibu ya Anopheles, ari na yo yonyine itera indwara ya malariya mu bantu. Imiterere y'iyi mibu ni intego ikomeye cyane yo kurwanya malariya kuko abagore bahura rimwe gusa5; gutuma iyi ndwara yo guhuza imwe itagira ubwandu byaba bifite ubushobozi bukomeye bwo kugabanya umubare w'imibu mu murima.
Abagore bacika intege mu mibonano mpuzabitsina nyuma yo guhabwa imisemburo ya steroid ifite tite nyinshi iturutse ku bagabo. Ubushakashatsi bwagaragaje ko impamvu ituma bigorana mu kongera kwegerana ari 20-hydroxyecdysone (20E), imisemburo ya steroid izwi cyane nk'igenzura ry'ingendo zo gushonga mu cyiciro cy'ibibuno. Ubushobozi bw'abagabo bwo gukora no kohereza 20E bwahindutse cyane cyane mu bwoko bwa Anopheles bugize Cellia7, ikwirakwira muri Afurika kandi ikaba irimo ibintu biteje akaga cyane bya malariya, harimo na Anopheles gambiae. Ibi ni ingenzi cyane kuko muri ubu bwoko abagore nabo bakora 20E nyuma ya buri funguro ry'amaraso, kandi 20E ituma habaho uruziga rw'amaraso (reba reba 8). Ariko, nta byinshi bizwi ku buryo abagore bahuza ibimenyetso bivuye mu masoko abiri atandukanye ya ecdysone (gutanga amaraso ku bagabo no kuyatanga) badashyize mu kaga ubushobozi bwabo bwo kwegerana. Mu by'ukuri, niba 20E ikorwa n'abagore itera kutihanganira ibitsina, ibi bizatuma abantu bonsa bakiri isugi barwara, imyitwarire ikunze kugaragara muri iyi mibu5.
Igisobanuro gishoboka ni uko abagabo ba A. gambiae bohereza ecdysone yahinduwe y’abagabo, igatuma habaho uruhererekane rw’ibimenyetso mu buryo bw’imyororokere y’abagore, bigatuma igitsina cyabo kidakora neza. Nyamara, nubwo inyamaswa zo mu rutirigongo zigira imisemburo myinshi ya steroid, nka estrogen na androgen (yasuzumwe mu gitabo cya 9), nk'uko tubizi, steroidi zishingiye kuri androgenic ntizagaragaye mu dukoko.
Twatangiye kumenya imiterere y’imisemburo ya steroid mu gahu k’abagabo gafasha mu gukora imibonano mpuzabitsina (MAG) ya A. gambiae ikuze mu gushaka steroide zishobora guhinduka. Dukoresheje chromatography y’amazi ifite imikorere myiza ihujwe na tandem mass spectrometry (HPLC-MS/MS) aho gukoresha uburyo butari bwihariye bwakoreshejwe mbere, twabonye ecdysone (E) na 20E muri uru rugingo, twemeza ibyavuye mu bushakashatsi bwabanje. Ariko, icyitegererezo cyari cyiganjemo steroide za phosphorylated oxidized, zihuye na formula ya 3-dehydro-20E-22-phosphate (3D20E22P)12 (Ishusho ya 1). Izindi ngero zirimo 3-dehydro-20E (3D20E) na 20E-22-phosphate (20E22P). Ingufu z’ikimenyetso cya HPLC-MS/MS cya 3D20E22P zari ebyiri z’ingero ziruta iy’ingero ya dephosphorylated, 3D20E, n’izindi eshatu z’ingero ziruta iy’ingero ya E na 20E (Ishusho ya 1). Nubwo mu bindi bice by’umubiri n'inzira yo hasi y'imyororokere (LRT; Extended Data Fig. 1a). Twanasesenguye ecdysteroids mu bagabo n'abagore baherutse gufungwa (munsi y'umunsi 1) maze tubona 3D20E na 3D20E22P gusa muri MAG; E, 20E na 20E22P byari biri mu bagabo n'abagore bombi (Extended Data Fig. 1b). Aya makuru agaragaza ko abagabo bakuru ba A. gambiae bakora imisemburo myinshi ihindura imisemburo muri MAG zabo idakorwa n'abagore.
MAG na LRT y'ingore (harimo atria, seminal vesicles, na parovarium) byakuwe mu bagabo b'inkumi bafite iminsi 4 (iminsi 4) n'abagore b'inkumi n'abakundanye (0.5, 3, na 12 hpm). Ecdysone muri izi ngingo yasesenguwe na HPLC-MS/MS (ikigereranyo ± sem; t-test idahuye, two-sided, false discovery rate (FDR) yakosowe; NS, ntabwo ari ingenzi; *P < 0.05, **P < 0.01 . 3D20E: amasaha 3 ugereranije n'amasaha 0.5, P = 0.035; amasaha 12 ugereranije n'amasaha 3, P = 0.0015; amasaha 12 ugereranije n'amasaha 0.5, P = 0.030. 3D20E22P: amasaha 3 ugereranije n'amasaha 0.5, P = 0.25; amasaha 12 ugereranije n'amasaha 3, P = 0.0032; Amasaha 12 ugereranije n'amasaha 0.5, P = 0.015). Amakuru aturuka ku bintu bitatu by’ibinyabuzima. Agace k’agasongero ka buri ecdysone y’ingenzi kabazwe kandi gashyirwa ku rugero rusanzwe hakurikijwe umubare w’imibu. Ecdysone igaragazwa n’ibara rikurikira: E, icyatsi kibisi; 20E, umuhondo; 20E22P, umuhengeri; 3D20E, ubururu; 3D20E22P, umutuku. Ishusho y’imbere yongera igipimo ku murongo wa y kugira ngo yerekane urugero rwo hasi rwa ecdysone.
Kugira ngo dukore ubushakashatsi niba 3D20E22P na 3D20E byimurirwa mu gihe cyo guhuza igitsina, twasesenguye LRT z'ingore mu bihe bitandukanye nyuma yo guhuza igitsina. Nubwo ecdysone itabonetse mu nkumi, twabonye ingano nini ya 3D20E22P muri LRT nyuma yo guhuza igitsina (amasaha 0.5 nyuma yo guhuza igitsina, hpm), igabanuka uko igihe kigenda gihita, mu gihe urugero rwa 3D20E rwiyongereye cyane (Ishusho ya 1). Dukoresheje 3D20E yakozwe mu buryo bwa shimi nk'ibipimo ngenderwaho, twasanze urugero rw'iyi hormone ya steroid mu guhuza igitsina cya LRTs rwari ruri hejuru byibuze inshuro 100 kurusha 20E (Imbonerahamwe y'amakuru yagutse ya 1). Bityo, 3D20E22P ni ecdysone ikomeye y'abagabo yimurirwa muri LRT y'ingore mu gihe cyo guhuza igitsina, kandi imiterere yayo ya dephosphorylated, 3D20E, iba nyinshi cyane nyuma gato yo guhuza igitsina. Ibi bigaragaza uruhare runini rwa ecdysone ya nyuma mu buzima bw'igitsina gore nyuma yo guhuza igitsina.
Nyuma yo gukora amakuru mashya ya RNA sequencing (RNA-seq) (Ishusho ya 2a), dukoresheje umuyoboro wa bioinformatics wubatswe ku giti cyacu, twashatse ecdysone kinase (EcK), ecdysone oxidase (EO), na ecdysone encoding 20E-modified phosphatase gene.EPP) igaragara mu ngingo z'imyororokere. Twabonye gene imwe ya EPP hamwe na gene ebyiri zishobora kuba za EcK (EcK1 na EcK2), ariko ntitwashoboye kubona gene nziza ya EO. By’umwihariko, gene za EPP zagaragaye ku rugero rwo hejuru (98.9th percentile) muri Gambia MAG ariko ntabwo ari muri LRTs z’abagore (Ishusho ya 2b), bitandukanye n’ibyo twari twiteze kuva dephosphorylation ya 3D20E22P yabayeho muri iyi ngingo y’abagore. Kubwibyo, twizera ko EPP y’abagabo ishobora kwimurirwa mu gihe cyo gushyingiranwa. Mu by’ukuri, twakoresheje label ya isotope ihamye mu mubiri kugira ngo dupfuke poroteyine y’abagore nyuma yo gushyingiranwa, enzyme yagaragajwe na MS muri atrium y’abagore (Ishusho ya 2c na Imbonerahamwe y'inyongera ya 1). Kuba hariho EPP muri MAG na LRT y'ingore yahuje (ariko atari isugi) byemejwe hakoreshejwe uturemangingo tw'umubiri twihariye (Ishusho ya 2d).
a, Umuyoboro wa bioinformatics wubatswe ku giti cye wo gushakisha mu ngingo z'imyororokere za buri gitsina kugira ngo urebe imiterere ya EcKs, EOs, na EPPs. Imibare iri iruhande rw'utwambi igaragaza umubare w'abagabo n'abagore kuri buri ntambwe. Iri sesengura ryagaragaje gene imwe ya EPP (EPP) na gene imwe ya EcK (EcK1) igaragara mu bagabo, na gene imwe ya EcK (EcK2) igaragara mu bagabo bombi ariko ntitange gene ya EO.b, Ikarita igereranya imiterere ya gene ya candidate mu bugimbi (V) no mu mating (M) Anopheles gambiae na Anopheles albicans.Spca, fertilization; MAGs, accessory glands mu bagabo; Ibindi bice by'umubiri, harimo amabere, amababa, amaguru, ibinure, n'ingingo z'imbere mu gitsina byombi, ndetse n'intanga ngabo mu bagore. EcK2 igaragara cyane muri MAG na atria yo muri Gambia, mu gihe EPP iboneka gusa muri MAG.c, isesengura rya Proteomic ry'itsinda ry'abagabo basohora intanga mu kigore kuri 3, 12 na 24 hpm, bigaragaza poroteyine 67 nyinshi cyane. Abagore barerewe ku ndyo irimo 15N kugira ngo bashyireho (no gupfuka) poroteyine zose. Abagabo badafite tag bashyizwe hamwe n'abagore bafite tag, naho abagore ba LRT bagabanyijwe kuri 3, 12 na 24 hpm kugira ngo bakore isesengura rya proteomic (reba Imbonerahamwe y'inyongera ya 1 kugira ngo ubone urutonde rwuzuye rwa poroteyine zisohora intanga). Insight, EPP, Eck1 na EcK2 byabonetse muri MAG y'abagabo badafite igitsina binyuze mu isesengura rya proteomic ry'izi ngingo.d, EPP yabonetse na western blot muri MAG na LRT y'abagore bahuje igitsina, ariko ntabwo ari mu bagore badafite igitsina cyangwa abagabo cyangwa abandi bagore. umubiri. Uturemangingo twasuzumwe icyarimwe hakoreshejwe anti-actin (igenzura ry’umubyigano) na anti-EPP. Abagabo bose ni isugi. Reba Ishusho y'inyongera ya 1 kugira ngo umenye amakuru aturuka kuri gel. Western blots yakozwe kabiri, haboneka ibisubizo bisa.
Imikorere ya ecdysteroid phosphophosphatase ya EPP yemejwe nyuma yo gucukurwa na HPLC-MS/MS hamwe na 3D20E22P yakuwe muri MAG (Extended Data Fig. 2a). Byongeye kandi, ubwo twacecekeshaga EPP dukoresheje RNA-mediated interference (RNAi), twabonye igabanuka rikomeye ry'imikorere ya phosphatase mu ngingo z'imyororokere z'izi mboro (Ishusho ya 3a), kandi igitsina gore cyahuje n'igitsina gabo cyacecetsewe na EPP cyagaragaje ko hari igice gito cya dephosphorylated 3D20E (Ishusho ya 3b) nubwo igice cya gene cyacecetsewe (Extended Data Fig. 2b,c). Mu buryo bunyuranye, ntabwo twabonye impinduka zikomeye ku gipimo cya 20E22P/20E mu mibu imwe, bishobora kwerekana ko enzyme yihariye kuri 3D20E22P (Ishusho ya 3b).
a, Kugabanuka k'imikorere ya phosphatase muri MAG guterwa no guceceka kwa EPP hakoreshejwe EPP RNA (dsEPP) ifite imigozi ibiri cyangwa GFP RNA (dsGFP) ifite imigozi ibiri. Muri buri tsinda hakoreshejwe ibipimo makumyabiri bya MAG (P = 0.0046, ikizamini cya t gikozwe ku ruhande rumwe, impande ebyiri), bihagarariwe n'utudomo dutandukanye.b, Ingore zahujwe n'ingabo za EPP zifite igipimo gito cyane cya 3D20E ya dephosphorylated kuri 3 hpm (P = 0.0043, ikizamini cya t kidafite imigozi ibiri, impande ebyiri), mu gihe urugero rwa 20E nta ngaruka rwagize (P = 0.063, nta migozi ibiri). ikizamini cya t, gifite impande ebyiri). Amakuru agaragazwa nk'ikigereranyo cya ± sem giturutse ku biyaga bitatu by'abagore 13, 16 na 19 buri kimwe. c, Abagore bahuje igitsina n'abagabo bacecetse EPP bari bafite igipimo cyo kongera guhuza igitsina (P = 0.0002, ikizamini cya Fisher, gifite impande ebyiri). Abagore babanje guhatirwa guhuza igitsina kugira ngo barebe ko bahuje igitsina; Nyuma y'iminsi 2, bahuye n'izindi ntanga ngabo zifite intanga zahinduwe kugira ngo bapime igipimo cyo kongera gukururana hakoreshejwe uburyo bwa PCR bwo gusuzuma transgene.d, igitsina gore cyagaburiwe amaraso cyahuje n'igitsina gabo cyacecetse EPP cyagabanutse cyane mu burumbuke (P < 0.0001; ikizamini cya Mann-Whitney, impande ebyiri) n'umubare w'igi wagabanutseho gato (P = 0.088, ikizamini cya Mann-Whitney, impande ebyiri), mu gihe igipimo cyo kubyara kitagize icyo kivuze (P = 0.94, ikizamini cya Fisher's nyacyo, impande ebyiri). Mu bice byose, n ihagarariye umubare w'imibu yigenga mu buryo bw'umubiri. NS, ntabwo ari ingenzi.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Hanyuma, twasuzumye niba ecdysone dephosphorylation ari ingenzi mu gutera imbaraga zo guhuza igitsina cy’ingore. Ikigaragara ni uko abagore bahuje igitsina n’abagabo badafite EPP bongeye guhuza igitsina ku nshuro nyinshi cyane (44.9%) kurusha abagore bagenzurwa (10.4%) iyo bahuye n’izindi igitsina cy’ingore (cyahinduwe mu jisho) (Ishusho ya 3c). Twabonye kandi igabanuka rikomeye ry’uburumbuke (Ishusho ya 3d, ibumoso) n’igabanuka rito ry’umubare w’intanga ngabo ziterwa n’izi igitsina (Ishusho ya 3d, hagati), mu gihe igipimo cy’intanga ngabo ziterwa (ikindi gisubizo cyatanzwe n’abagore bitewe no guhuza igitsina )) kitagizweho ingaruka (Ishusho ya 3d, iburyo). Bitewe n’uburyo EPP yagaragaye ku buryo bwihariye bwa 3D20E22P, ibi bisubizo bigaragaza ko gukora kwa 3D20E na EPP mu gihe cyo guhuza igitsina bishobora kugira uruhare runini mu kubuza abagore kwakira igitsina gabo, imyitwarire yari isanzwe iterwa no guhuza igitsina kwa 20E. Kubwibyo, iyi hormone yihariye y’abagabo nayo igira ingaruka zikomeye ku burumbuke bw’igitsina cy’ingore.
Hanyuma, twagereranije ibikorwa bya 20E na 3D20E mu igerageza ry’inshinge ku nkumi zikuze mu mibonano mpuzabitsina hakoreshejwe 3D20E yakozwe mu buryo bwa chimique (Ishusho ya 4a–c) na 20E igurishwa. Twabonye ko 3D20E yari ifite akamaro kanini kurusha 20E mu guhagarika ubushobozi bw’abagore bwo guhuza ibitsina ku bipimo byombi (Ishusho ya 4d). Ikigaragara ni uko kimwe cya kabiri cy’urwego rw’umubiri rwa 3D20E muri LRT (1,066 pg nyuma yo gutera urukingo ugereranyije na 2,022 pg nyuma yo gutera urukingo) byateye igice cy’abagore badashobora kwihanganira imiterere y’umubiri cyari hejuru inshuro 20 ugereranyije n’urwego rw’umubiri rwa 20E (361 pg nyuma yo gutera urukingo) amasaha 24 nyuma yo gutera urukingo ku rugero rwo hejuru rw’ibipimo 18 pg nyuma yo gutera urukingo; Imbonerahamwe y'amakuru yagutse ya 1). Ibi bihuye n'igitekerezo cy'uko kwimura 20E mu mibonano mpuzabitsina bidatera ibihe byo gutandukana, kandi byongera kugaragaza 3D20E nk'ikintu gikomeye mu gutuma umubano w'ababyeyi n'abana urushaho kuba mwiza. 3D20E nayo yari ifite imbaraga zirenze 20E mu isuzuma ry'amagi ku bagore b'inkumi (Ishusho ya 4e), bigaragaza ko igipimo gisanzwe cyo gutera amagi twabonye nyuma yo guceceka igice cya EPP cyatewe n'uko hari ibikorwa bya 3D20E bisigaye bikivuka bitewe n'ibintu by'abagore bikomoka ku gutera amagi.
(a,b) 3D20E yakozwe mu buryo bwa shimi iva kuri 20E (a) ifite impinduka/ubushobozi bwinshi cyane (amakuru agaragazwa nk'ikigereranyo cya ± sem giturutse ku bikorwa bitatu byigenga bya synthesis) (b).c, Mass spectrum (igice cyo hasi) ihuye neza na ecdysone iboneka muri LRT y'ingore yahuje (igice cyo hejuru).d, Ugereranije na 20E (0.63 µg, P = 0.02; 0.21 µg, P < 0.0001; Fisher's exact test, two-side) na 10% ethanol (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Fisher's exact test, two-side), mu gihe 20E yari hejuru cyane kuruta control gusa ku rugero ruri hejuru (0.63 µg, P = 0.0002; 0.21 µg, P = 0.54; Fisher's exact test, 2-side).e, inshinge za 3D20E zateje igipimo cyo gutera inda mu nkumi zikiri isugi kirenze 10% by’igenzura rya ethanol (0.21 µg, P < 0.0001; 0.13 µg, P = 0.0003; Isuzuma ry’ukuri rya Fisher, impande ebyiri), mu gihe 20E ugereranije n’isuzuma gusa ku gipimo cyo hejuru (0.21 µg, P = 0.022; 0.13 µg, P = 0.0823; Isuzuma ry’ukuri rya Fisher, impande ebyiri).3D20E yateje igipimo cyo gutera inda kirenze 20E ku gipimo cyo hejuru (0.21 µg, P = 0.0019; 0.13 µg, P = 0.075; Isuzuma ry’ukuri rya Fisher, impande ebyiri). Mu duce twose, n ihagarariye umubare w’ingero z’imibu idakoresha ibinyabuzima. NS, ntabwo ari ingenzi.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. Amakuru aturuka ku makopi atatu.
Mu bushakashatsi bwabanje, twasanze ko imisemburo ya steroide mu mibonano mpuzabitsina ituma MISO (Mating-Induced Stimulator of Oogenesis 11) ikora, ari yo gene y’abagore irinda abagore ba A. gambiae kwandura P. falciparum. Ikiguzi cy’ubuzima giterwa na 13, parasite yica cyane ya malariya ku bantu. Dukurikije akamaro ka MISO ku buzima bw’imyororokere bwa Anopheles mu bice bikunze kugaragaramo malariya, twahisemo kumenya imisemburo ya 3D20E cyangwa 20E itera iyi gene gukora. Twasanze nubwo inshinge za 20E zitera cyane cyangwa cyane zimwe mu misemburo ya nuclear hormone (HR), nka HR3 na HR4, hamwe n’ibipimo bisanzwe bya steroide, nka yolkogenic genes Vg14, 15, 16, MISO yatewe cyane na 3D20E (Ishusho ya 3). Bityo, ihererekanya ry’iyi hormone ya steroide ya androgenic rigaragara nk’aho ritera uburyo bwo kurinda abagore ingaruka ziterwa n’ubwandu bw’udukoko. Byongeye kandi, 3D20E igira ingaruka zitandukanye kuri isoforms zombi za E receptor EcR, itera EcR-A kandi igahagarika EcR-B, kandi irushaho gutera izindi gene zitera kwegerana, harimo na HPX15, zigira ingaruka ku burumbuke bw'abagore. Ibi bishobora gusobanura ubugumba bugaragara ku bagore bahura n'abagabo bacecekeshejwe na EPP (Ishusho ya 3 y'amakuru). Aya makuru agaragaza ko hariho inzira zo mu nzira zikoreshwa cyane n'imisemburo ibiri ya ecdysone ishobora kuba intandaro y'imikorere yihariye y'igitsina.
Hanyuma, twagerageje imikorere ya za gene ebyiri za EcK zagaragaye mu muyoboro wacu wa bioinformatics. Gucecekesha EcK1 cyangwa EcK2 byateje impfu nyinshi mu bagabo (Extended Data Fig. 4a), bigaragaza ko ecdysone phosphorylation, bityo ikaba idakora, ari ingenzi kugira ngo umuntu akomeze kubaho. Kubera ko EcK2 yagaragaye ku rugero rwo hejuru kurusha EcK1 kandi yabonetse muri MAG na proteomics (Ishusho ya 2b, c n'Imbonerahamwe y'Inyongera ya 2), twemeje imikorere yayo ya ecdysteroid kinase tuyishyiramo 20E, byatumye phosphorylation 20E22P (Ishusho ya Extended Data Fig. 2). 4b). Mu gihe twakoreshaga 3D20E nk'ishingiro, ntitwashoboye kubona 3D20E22P (Extended Data Fig. 4c), bigaragaza ko 20E aho kuba 3D20E ishobora kuba ari yo ntego ya EcK2.
Dukurikije isesengura ryacu rya RNA-seq, EcK2 nayo yagaragaye cyane muri LRT y'ingore zikiri isugi, aho yazimijwe nyuma yo gushyingiranwa (Ishusho ya 2b). Twemeje aya makuru maze tumenya ko EcK2 itagize ingaruka ku kunyobwa amaraso (Ishusho ya Extended Data Fig. 5a). Twongereye igerageza ryacu rya mbere rya MS, twasanze impera ya 20E22P yari ifitanye isano rya hafi n'impera ya 20E (amasaha 22-26 nyuma yo kurya amaraso; Ishusho ya Extended Data Fig. 5b). Guceceka kwa EcK2 mu nkumi zikiri isugi byatumye habaho ukwiyongera inshuro 3 k'ikigereranyo cya 20E na 20E22P mu masaha 26 nyuma yo kurya amaraso (Ishusho ya Extended Data Figures 2c na 5c), byemeza ko EcK2 inatera fosifori 20E mu nkumi. Ikigaragara ni uko inkumi zikiri isugi za EcK2 zakomeje kwakira imibonano mpuzabitsina yose (Ishusho ya Extended Data Fig. 5d,e), bikomeza bigaragaza ko umusaruro w'inkumi wa 20E udatuma gushyingiranwa gucika intege. Ibihe by'inda. Ariko, izi nkumi zari zifite igipimo cyo gutera amagi cyiyongereye cyane ugereranije n'ibindi bipimo, aho abarenga 30% by'inkumi zitera amagi (Extended Data Fig. 5f). Niba inshinge ebyiri za Eck2 RNA (dsEcK2) zatewe nyuma yo konka amaraso, ntabwo konka kwabayeho, icyo gihe uburebure bwa 20E bitewe no kunywa amaraso bwari bwaragabanutse. Muri rusange, ibi bisubizo bishyigikira icyitegererezo 20E yakoze nyuma yo konka amaraso gishobora gutera konka, ariko gusa iyo urwego rwo gutera konka (EcK2 ndetse n'ibindi bintu) rwazimijwe no gutera konka. Nta n'imwe muri 20E cyangwa 3D20E yabujije EcK2 ku nkumi zikiri nto (Extended Data Fig. 5g), bigaragaza ko hari izindi mpamvu zituma iyi kinase ihagarara. Ariko, igipimo cya 20E nyuma yo konka amaraso nticyari gihagije kugira ngo gitere kubabara mu gutera konka, ariko cyatewe neza n'urugero ruri hejuru rwa 3D20E ihererekanywa mu mibonano mpuzabitsina.
Ibisubizo byacu bitanga ibisobanuro by'ingenzi ku buryo bugenga iterambere ry'imyororokere rya A. gambiae. Hagaragaye icyitegererezo aho abagabo bahindutse kugira ngo bakore 3D20E, ecdysone yahinduwe yihariye y'abagabo yemeza ko abagore babyara binyuze mu gutuma abagore badakomeza gukururana. Muri icyo gihe, izi vectors za malariya nazo zashyizeho uburyo bwiza bwo gukora 3D20E mu bagore mu rwego rwo gusubiza ihererekanya ry'imibonano mpuzabitsina rya EPP yihariye y'abagabo. Uko tubizi, uru ni rwo rugero rwa mbere rw'imisemburo ya steroid yiganjemo abagabo n'abagore ikora umurimo udasanzwe kandi w'ingenzi mu dukoko. Imikorere ya ecdysone yihariye y'abagabo yavuzwe ariko ntiyerekanwe neza. Urugero, igitekerezo cya 18 kidashidikanywaho cyane ni uko iyi mirimo ishobora gukorwa n'intangiriro ya 20E E1. Birazwi neza ko muri Drosophila, monandry iterwa no ihererekanya ry'imibonano mpuzabitsina rya peptide nto z'igitsina19,20 zikorana n'imitsi iyobora inzira y'imyororokere y'abagore binyuze muri receptors za peptide z'igitsina21,22. Hakenewe andi masomo kugira ngo hamenyekane aho ihererekanya riherereye. imiyoboro y'ibimenyetso igenzurwa na 3D20E mu nkokokazi za A. gambiae no kumenya niba iyi miyoboro ishobora kubikwa hagati y'imibu na Drosophila.
Bitewe n'uruhare runini rwa 3D20E ku burumbuke bw'abagore n'imyitwarire yabo byagaragajwe mu bushakashatsi bwacu, inzira zigana ku gukora no gukora 3D20E zitanga amahirwe mashya yo mu ngamba zo kurwanya imibu mu gihe kizaza, nko kubyara igitsina gabo gihanganye mu ngamba z'ikoranabuhanga ry'udukoko duto. Koresha mu kurekura udukoko cyangwa kwigana 3D20E mu buryo bw'isugi. Imikorere yihariye ya 3D20E ku bagabo ishobora kuba yarahindutse ubwo A. gambiae n'izindi nkomoko za Cellia zagiraga ubushobozi bwo gufunga amasohoro yazo mu dukingirizo tw'inyongera, kuko ibi bituma habaho kohereza neza imisemburo myinshi na enzymes zikora imisemburo. Ku rundi ruhande, gushyira mu bikorwa monandry ya 3D20E bitanga uburyo ku bagore (binyuze mu kugaragaza cyane MISO) kugira ngo bakomeze ubuzima bwabo bwo kubyara mu bice bifite ubwandu bwinshi bwa malariya, ibyo bikaba bigira uruhare mu buryo butaziguye mu kwandura Plasmodium. Bitewe n'uko 20E y'abagore byagaragaye ko igira ingaruka zikomeye ku kubaho no gukura kwa P. falciparum mu mibu y'abagore ya Anopheles,24 inzira z'imisemburo ya steroid y'abagabo n'abagore ubu ni ingenzi mu mibanire y'imibu n'udukoko.
Ubwoko bwa A. gambiae G3 bwororerwaga mu buryo busanzwe bw’udukoko (26-28 °C, ubushyuhe buri hagati ya 65-80%, igihe cy’izuba ryinshi mu masaha 12:12). Imbuto zagaburirwaga ibiryo by’amafi by’ifu (TetraMin Tropical Flakes, Koi Pellets na Tetra Pond Sticks ku gipimo cya 7:7:2). Imibu ikuze yagaburirwaga umuti wa dextrose 10% n’amaraso y’umuntu buri cyumweru (ibice by’amaraso byigwa). Imibu y’isugi yabonetse mu gutandukanya igitsina mu cyiciro cy’imbwa nyuma yo gusuzuma impera hakoreshejwe mikorosikopi. Imboro zitwaye transgene ya DsRed zavuzwe mbere.
Igerageza ryo guhatira guhuza intanga ryakozwe hakurikijwe amabwiriza yavuzwe mbere. Ku bijyanye no guhuza intanga karemano, intanga z’ingore z’iminsi 4 zashyizwe mu gipimo cya 1:3 hamwe n’intanga z’abahungu z’inkumi zikuze mu mibonano mpuzabitsina mu gihe cy’amajoro abiri. Ku bijyanye n’igerageza aho intanga z’ingabo zatewe inshinge za dsEPP, guhuza intanga byahuriranye n’iminsi 3-4 nyuma yo gutera inshinge, ubwo ibikorwa bya phosphatase byahagararaga cyane (Ishusho ya 2b).
Uduce tw’imibu, ibisigaye by’umubiri (igice cy’umubiri), cyangwa umubiri wose byaciwemo 100% bya methanol hanyuma bihuzwa n’icyuma gifata (amasaro y’ikirahure ya mm 2, 2.400 rpm, amasegonda 90). Ingano y’uturemangingo n’ingano ya methanol byari ibi bikurikira: igice cy’umubiri, 50 muri 1.000 µl; MAG, 50–100 80 µl; LRT y’ingore, 25–50 80 µl. Umuvuduko washyizwemo kabiri wa methanol hamwe n’ingano imwe ya methanol. Imyanda y’uturemangingo yakuweho hakoreshejwe centrifugation. Methanol yo mu bicuruzwa byombi yahujwe yumishwa munsi y’amazi atemba azote, hanyuma yongera gushyirwamo ingano ikurikira ya 80% ya methanol mu mazi: igice cy’umubiri, 50 µl; MAG na LRT y’ingore, 30 µl.
Ingero zasesenguwe kuri spectrometero y'uburemere (ID-X, Thermo Fisher) ihujwe n'igikoresho cya LC (Vanquish, Thermo Fisher). 5 µl y'icyitegererezo yatewe ku nkingi ya 3 µm, 100 × 4.6 mm (Inspire C8, Dikma) iguma kuri 25 °C. Ibyiciro byo kugenda bya LC byari A (amazi, 0.1% aside formic) na B (acetonitrile, 0.1% aside formic). Uburemere bwa LC bwari ubu bukurikira: 5% B mu gihe cy'umunota 1, hanyuma byongera kugera kuri 100% B mu minota 11. Nyuma y'iminota 8 kuri 100%, ongera uhuze inkingi kuri 5% B mu minota 4. Igipimo cy'amazi cyari 0.3 ml min-1. Ionization mu isoko ya MS ikorwa hakoreshejwe electrospray ionization ishyushye mu buryo bwiza na bubi.
Spektrometero y'uburemere ipima amakuru mu ntera ya m/z kuva kuri 350 kugeza kuri 680 kuri resolution ya 60.000 mu buryo bwuzuye bwa MS. Amakuru ya MS/MS yabonetse kuri [M + H]+ (intego zose), [M - H2O + H]+ (intego zose), na [M - H]- (intego za phosphorylated). Amakuru ya MS/MS yakoreshejwe kugira ngo yemeze imiterere ya ecdysone y'intego zitari zisanzweho. Kugira ngo hamenyekane ecdysteroids zitari zigamije, amakuru ya MS/MS ku ntambwe zose za HPLC zifite ubwinshi bwa >15% yarasesenguwe. Isuzuma hakoreshejwe imirongo isanzwe yakozwe uhereye ku bipimo byuzuye (20E, 3D20E) kugira ngo hamenyekane ingano cyangwa igabanuka ry'icyitegererezo kimwe cyihariye (izindi ntego zose) kugira ngo hamenyekane ingano zihwanye n'ingano zabonetse mu mugabo umwe. Kuri 3D20E, isuzuma ryakozwe hakoreshejwe igiteranyo cy'inyongera zikurikira: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-. Amakuru yakuwemo kandi apimwa hakoreshejwe Tracefinder (version 4.1). Amakuru ya MS/MS yasesenguwe hakoreshejwe Xcalibur (version 4.4). MS spectra ya E, 20E na 3D20E yagereranyijwe n'ibipimo ngenderwaho. 3D20E22P yasesenguwe hakoreshejwe reagent ya Girard. 20E22P yasesenguwe hakoreshejwe ikigereranyo cya m/z.
3D20E22P yasukuwe na MAG. Gusukura byakozwe ku gipimo cyo gusesengura hakoreshejwe chromatograph y'amazi ikora neza cyane (Acquity, Waters) hamwe n'icyuma gipima uburemere bwa quadrupole (QDa, Acquity, Waters) mu buryo bumwe bwa LC nk'isesengura rya HPLC-MS/MS. Gukusanya ibice byatangiye igihe m/z ijyanye na 3D20E22P yabonekaga mu gihe kimwe cyo kubika nk'uko byagenwe mbere. Ubuziranenge bw'ibinyabutabire byakuwemo bwagenzuwe na HPLC-MS/MS nk'uko byasobanuwe haruguru.
RNA yose yakuwe mu ngingo 10-12 z'imyororokere cyangwa izindi ngingo z'umubiri (zidafite umutwe) hakoreshejwe reagent ya TRI (Thermo Fisher) hakurikijwe amabwiriza y'uwakoze. RNA yavuwe na TURBO DNase (Thermo Fisher). cDNA yakozwe hakoreshejwe Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) hakurikijwe amabwiriza y'uwakoze. Primers za reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) zasohotse mbere24 cyangwa zakozwe hakoreshejwe Primer-BLAST26, hibandwa ku bicuruzwa bifite ingano ya 70-150 bp kandi bivangira exon-exon junctions cyangwa Primer pair primers zitandukanye exons. cDNA ingero kuva kuri eshatu kugeza kuri enye za biological replicates zavanzwe inshuro enye mu mazi kuri RT-qPCR. Isuzuma ryakozwe muri 15 µl replica reactions zirimo 1 × PowerUp SYBR Green Master Mix (Thermo Fisher), primers, na 5 µl ya diluted cDNA. Reactions zakozwe kuri Sisitemu ya QuantStudio 6 Pro ya PCR mu gihe nyacyo (Thermo Fisher) n'amakuru byakusanyijwe kandi binasesengurwa hakoreshejwe Igishushanyo n'Isesengura (version 2.4.3). Nkuko byagaragajwe muri ubu bushakashatsi, ingano ijyanye n'ingirabuzimafatizo ya ribosomal RpL19 (AGAP004422), aho isura yayo itahindutse cyane mu gihe cyo kurya amaraso 27 cyangwa guhuza 3.
Ubuziranenge bwa RNA bwagenzuwe hakoreshejwe Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). Amasomero ya Illumina paired-end yarateguwe kandi akorerwa muri Broad Institute of MIT na Harvard. Urutonde rw'ibipimo byahujwe na genome ya A. gambiae (ubwoko bwa PEST, verisiyo ya 4.12) hakoreshejwe HISAT2 (verisiyo ya 2.0.5) hamwe n'ibipimo bisanzwe. Ibipimo bifite ubuziranenge bwa mapping (MAPQ) amanota <30 byakuweho hakoreshejwe Samtools (verisiyo ya 1.3.1). Umubare w'ibipimo byashyizwe kuri genes wabazwe hakoreshejwe htseq-count (verisiyo ya 0.9.1) hamwe n'ibipimo bisanzwe. Umubare w'ibipimo bisanzwe wabazwe kandi hasesengurwa itandukaniro rya genes hakoreshejwe paki ya DESeq2 (verisiyo ya 1.28.1) muri R (verisiyo ya 4.0.3).
Ibimenyetso bya Ecdysone-modifiing gene byamenyekanye binyuze mu gushakisha ku nyuguti za A. gambiae hakoreshejwe algorithm ya PSI-BLAST (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), hakoreshejwe agaciro gasanzwe Ibipimo bifite urukurikirane rwa poroteyine y'ibibazo: kuva kuri Bombyx mori (Accession No. NP_001038956.1), Musca domestica (Accession No. XP_005182020.1, XP_005175332.1 na XP_011294434.1) na Microplitis demolitor (Accession No. XP_008552646.1 na XP_008552645.1) EcK kuva kuri B. mori (Accession No. NP_001036900), Drosophila melanogaster (Accession No. NP_651202), Apis mellifera (Inomero yo kwinjira XP_394838) na Acyrthosiphon pisum (Inomero yo kwinjira XP_001947166); na EPP kuva kuri B. mori (Inomero yo kwinjira XP_001947166) NP_001177919.1 na NP_001243996.1) na EO ya D. melanogaster (Inomero yo kwinjira NP_572986.1) (intambwe ya 1). Hanyuma, filter irakora hashingiwe ku mikorere ya mRNA nyinshi (ibice >100 / kilobase exons kuri miliyoni ya mapping reads (FPKM) cyangwa >85%) mu ngingo z'imyororokere (LRT y'abagore cyangwa MAG) muri Gambia (intambwe ya 2). Kugira ngo tunoze imiterere yihariye, twahisemo enzymes zisanzwe zigaragara no mu ngingo z’imyororokere za A. albimanus, ubwoko bwa anopheles budakora cyangwa ngo bwohereze ecdysone mu gihe cyo guhuza. Genes zakozwe mu buryo bwa “candidate” zahinduwe hashingiwe ku miterere mike (<100 FPKM cyangwa <85th percentile) mu ngingo z’imyororokere za A. albimanus (intambwe ya 3). Nk’urufunguzo rwa nyuma (intambwe ya 4), genes zisanzwe zigomba kuzuza nibura kimwe muri ibi bikurikira: (1) kwiyongera cyane nyuma yo guhuza (P < 0.05) hakurikijwe isesengura rya genes zitandukanye na (2) mu ngingo zitabyara (< 85 % cyangwa <100 FPKM).
Twahinduye uburyo bwavuzwe mbere bwa 28,29,30 kugira ngo tugere ku bipimo bya isotopi by’ibinyabuzima byose. Muri make, Saccharomyces cerevisiae yo mu bwoko bwa II (YSC2, Sigma) yageragejwe mu ishingiro rya azote y’umusemburo (BD Difco, DF0335) irimo (wt/vol) 2% ya glucose (G7528, Sigma), 1.7% idafite aside amine na ammonium sulfate. culture medium) na 5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) nk'isoko yonyine ya azote. Umusemburo wabonetse hakoreshejwe centrifugation maze imbu z’imibu zigaburirwa ifu kugeza igihe zivutse. Inyongera ku ifu y’amafi (0.5 mg kuri imbu 300) kugira ngo hirindwe urupfu rwa kane rw’intambwe. Ingore ni zo zonyine zakoreshejwe mu igerageza ryo guhuza imbu n’ingabo z’imigabo zitarashyirwa mu byiciro kugira ngo zisesengure proteome y’ingabo yakuwe mu gihe cyo guhuza imbu.
Inka z'inkumi zifite iminsi 4-6 zifite tagi ya N 15 zategetswe kuryamana n'ingabo z'inkumi zitari zifite tagi zijyanye n'imyaka yazo. Kuryamana neza byemejwe no kubona uduce tw'inkuru dukoreshwa muri microscopy ya epifluorescence. Kuri 3, 12, na 24 hpm, atria y'ingore 45-55 zavutse zaciwemo 50 µl ya ammonium bicarbonate buffer (pH 7.8) hanyuma zihuzwa n'agace gato. Homogenate yashyizwemo centrifuge maze supernatant ivangwa na 50 µl ya 0.1% RapiGest (186001860, Amazi) muri 50 mM ammonium bicarbonate. Supernatant na pellet bya buri gipimo byafatiwe mu rubura rwumye maze byoherezwa nijoro muri laboratwari ya MacCoss muri Kaminuza ya Washington, aho icyitegererezo cyo gutegura LC-MS/MS cyarangiye. Ongera ushyire pellet muri 50 µl ya 0.1% RapiGest muri 50 mM. Ammonium bicarbonate na sonicate mu bwogero bw'amazi. Igipimo cya poroteyine cya pellet na supernatant cyapimwe hakoreshejwe BCA assay, ingero zagabanijwe hakoreshejwe 5 mM dithiothreitol (DTT; Sigma), zivanze na 15 mM iodoacetamide (Sigma) hanyuma zishyirwa kuri 37 °C (1:0 50) mu gihe cy'isaha 1 hamwe na trypsinization: trypsin: substrate ratio). RapiGest yakuwemo 200 mM HCl, hakurikiraho incubation kuri 37 °C mu minota 45 na centrifugation kuri 14.000 rpm mu minota 10 kuri 4 °C kugira ngo haveho imyanda. Ingero zogejwe hakoreshejwe dual-mode solid-phase extraction (Oasis MCX cartridges, Waters) hanyuma zongerwamo 0.1% formic acid kugira ngo haboneke 0.33 µg µl-1. Proteome zitanditseho MAG nazo zasesenguwe muri virgin. igitsina gabo. Hasesenguwe kopi ebyiri z’isesengura kuri buri cyitegererezo. Hanyuma, 1 µg ya buri kimwe yasesenguwe hakoreshejwe inkingi ya silica ya cm 25 ya fused silica 75-μm ifite umutego wa silica Kasil1 (PQ) wa cm 4 urimo Jupiter C12 resin reversed-phase (Phenomenex) na chromatography y’amazi y’iminota 180. Ingero z'ibipimo - MS/MS yakoreshejwe kuri spectrometero ya Q-Exactive HF mass (Thermo Fisher) ikoresheje nanoACQUITY UPLC System (Waters). Amakuru ajyanye no kubona amakuru yakozwe kuri buri gikorwa yahinduwe muri format ya mzML hakoreshejwe Proteowizard (version 3.0.20287) no gukoresha Comet31 (version 3.2) ukurikije database ya FASTA irimo poroteyine kuva kuri Anopheles gambiae (version 54 ya VectorBase), Anopheles coluzzi Hakozwe ubushakashatsi kuri Mali-NIH (version 54 ya VectorBase), Saccharomyces cerevisiae (Uniprot, Werurwe 2021), A. gambiae RNA-seq, na translations eshatu z'ibintu bizwi byanduza abantu. FDRs zahujwe na peptide map zagenwe hakoreshejwe Percolator32 (version 3.05) zifite ikigereranyo cya 0.01, maze peptides zivangwa mu buryo bwo kumenya poroteyine hakoreshejwe poroteyine parsimony muri Limelight33 (verisiyo ya 2.2.0). Ubucucike bwa poroteyine bungana bwapimwe hakoreshejwe uburyo bwa normalized spectral abundance factor (NSAF) bwabazwe kuri buri poroteyine muri buri cyiciro nkuko byasobanuwe mbere. NSAF ugereranije na buri poroteyine yapimwe mu bipimo byavuye mu bipimo bibiri bitandukanye by’ibinyabuzima. 15N label yapfutse proteome y’ingore neza, nubwo ingano nto ya poroteyine idafite label yashoboraga kugaragara mu bipimo bya virgins byanditseho. Twanditse uburyo bwo kugabanya poroteyine y’abagabo (spectra 1-5) mu bipimo bya virgins gusa mu bipimo bya tekiniki, aho ingero za virgins zakozwe nyuma y’ibipimo bya virgins byashyizwe mu byiciro, bitewe na HPLC "gutwara". Rimwe na rimwe poroteyine zabonetse nk' 'umwanda' uturutse mu bigero bya virgins byanditseho urutonde mu mbonerahamwe y'inyongera ya 1.
Peptide ebyiri za antigenic, QTTDRVAPAPDQQQ (muri isotype PA) na MESGTTTPSGDSEQ (muri isotype PA na PB) muri Genscript. Peptide ebyiri zahujwe, hanyuma zihuzwa na poroteyine KLH itwara hanyuma ziterwa mu nkwavu zo muri Nouvelle-Zélande. Inkwavu zaratanzwe nyuma yo guterwa ku nshuro ya kane, maze IgG yose ikurwamo hakoreshejwe uburyo bwo gusukura affinity. IgG ikomoka ku nkwavu yihariye ya EPP yakoreshejwe mu gukwirakwiza umwijima mu burengerazuba.
Ku birebana n’udukoko two mu burengerazuba, MAG (n = 10, aho n ihagarariye umubare w’imibu yigenga mu buryo bw’ibinyabuzima) na LRT y’ingore (n = 30) ikomoka ku bagabo b’inkumi bafite iminsi 4 n’inkumi z’inkumi cyangwa iz’inkumi (<10 nyuma yo gushyingiranwa), poroteyine igabanya ubukana (50 mM Tris, pH 8.0; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 × protease inhibitor cocktail (Roche)) yongewemo ukwayo. Ingero zahinduwe homogenized ako kanya nyuma yo gucibwa hakoreshejwe beader (2 mm ikirahure cy’amasaro, 2,400 rpm, amasegonda 90). Imyanda idashonga yakuweho hakoreshejwe centrifugation kuri 20.000 g kuri 4 °C. Poroteyine zapimwe hakoreshejwe Bradford assay (Bio-Rad). Hanyuma, 20 µg ya poroteyine ya MAG, 40 µg ya poroteyine ya LRT, na 20 µg ya poroteyine nini isigaye. Byahinduwe kandi bitandukanyijwe na 10% Bis-Tris NuPAGE hakoreshejwe MOPS buffer. Poroteyine zoherejwe mu membrane ya polyvinylidene fluoride hakoreshejwe sisitemu yo kohereza iBlot2 (Thermo Fisher). Impande zogejwe kabiri muri 1 × PBS-T (0.1% Tween-20 muri PBS) hanyuma zigafungwa muri Odyssey blocking buffer (Li-Cor) mu gihe cy'isaha 1 kuri 22°C. Impande zogejwe ijoro ryose kuri 4°C hakoreshejwe antibody yihariye ya polyclonal anti-EPP polyclonal primary antibody (1:700 muri blocking buffer) na antibody ya rat anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000). Impande zogejwe na PBS-T hanyuma zishyirwamo antibody za kabiri (donkey anti-rabbit 800CW na goat anti-rat 680LT (Li-Cor), zombi 1:20,000) muri blocking buffer irimo 0.01% SDS na 0.2% Tween -20 mu gihe cy'isaha 1 kuri 22 °C. Uturemangingo twogejwe na PBS-T hanyuma dufatwa amashusho hifashishijwe Odyssey CLx scanner. Amashusho yakusanyijwe kandi atunganywa muri Image Studio (version 5.2). Umugozi wihariye ujyanye na isoform ya EPP-RA (82 kDa) ntiwabonetse.
Uduce twa EPP (nk'uko isoform AGAP002463-RB irimo histidine phosphatase domain, NCBI conserved domain search 34) na EcK2 (AGAP002181) twashyizwe muri pET-21a(+) plasmid (Novagen Millipore Sigma); primers ziri ku rutonde rwa Extended Data Table 2. Ibikoresho umunani bya GS4 (biri ku murongo umwe) byashyizwemo mbere y’uko C-terminal 6xHis tag ya pET-21a(+)-EcK2 ikorwa. Poroteyine zihuza zakozwe hakoreshejwe uburyo bwa NEBEExpress E. coli protein synthesis reaction (New England BioLabs). Poroteyine zihuza zatunganyijwe hakoreshejwe NEBEExpress Ni spin columns (New England BioLabs). Poroteyine igenzura Dihydrofolate reductase (DHFR) yakozwe hakoreshejwe ADN template yavuye muri NEBEExpress Cell-Free E. coli Protein Synthesis Kit. Poroteyine zabitswe muri 50% glycerol muri PBS kuri -20 °C mu gihe cy’amezi 3.
Umusaruro wa fosfatase wa EPP n'ibikomoka ku ngirabuzimafatizo byapimwe hakoreshejwe 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). Ingufu yo gukora imyitozo yari irimo 25 mM Tris, aside asetiki ya 50 mM, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, na 1 mM DTT. Ingirabuzimafatizo zahujwe mu buryo bumwe mu buryo buhuye kandi ibisigazwa by'uturemangingo byakuweho hakoreshejwe centrifugation. Tangira imyitozo wongeramo enzyme cyangwa tissue extract kuri 2.5 mg ml-1 pNPP. Uruvange rw'imyitozo rwashyizwe mu bushyuhe bw'icyumba mu mwijima, kandi ingano ya pNP yahinduwe iva muri pNPP yapimwe hakoreshejwe gupima ubushobozi bwo gufata umwuka kuri 405 nm mu bihe bitandukanye.
Ku bijyanye n'imikorere ya EcK muri vitro, poroteyine yashyizwemo 0.2 mg 20E cyangwa 3D20E muri 200 µl buffer (pH 7.5) irimo 10 mM HEPES–NaOH, 0.1% BSA, 2 mM ATP na 10 mM MgCl2 mu gihe cy'amasaha 2 kuri 27 °C. Uburyo bwo gukora bwahagaze hongerwamo 800 µl methanol, hanyuma ikonjeshwa kuri -20 °C mu gihe cy'isaha 1, hanyuma ishyirwa kuri centrifuge kuri 20.000 g mu gihe cy'iminota 10 kuri 4 °C. Hanyuma supernatant yasesenguwe na HPLC-MS/MS. Kugira ngo poroteyine zakoreshejwe mu itsinda rigenzura zishyuhe, poroteyine zashyizwemo 50% bya glycerol muri PBS mu gihe cy'iminota 20 kuri 95 °C.
Ku bijyanye n'imikorere ya EPP muri vitro, poroteyine yashyizwemo 3D20E22P (ingana n'ingano yabonetse muri pairs 18 za MAG, yasukuwe na HPLC-MS/MS) muri 100 µl buffer (pH 7.5) irimo 25 mM Tris, aside asetiki 50 mM, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, na 1 mM DTT mu gihe cy'amasaha 3 kuri 27 °C. Igisubizo cyahagaze hongerwamo 400 µl methanol hanyuma ikonjeshwa kuri -20 °C mu gihe cy'isaha 1, hanyuma ishyirwa kuri centrifuge kuri 20.000 g mu gihe cy'iminota 10 kuri 4 °C. Supernatant yasesenguwe na HPLC-MS/MS.
Uduce twa PCR twa EPP (362 bp), EcK1 (AGAP004574, 365 bp) na EcK2 (556 bp) twongerewe muri cDNA yakozwe mu mikungugu idafite imitwe y’abagabo n’abagore. Agace ka PCR k’igenzura rya eGFP (495 bp) kongerewe muri pCR2.1-eGFP yavuzwe mbere; PCR primers ziri mu mbonerahamwe ya 2 y’amakuru yagutse. Igice cya PCR cyashyizwe hagati ya promoters za T7 zahinduwe kuri plasmid ya pL4440. Plasmid constructs zakuwe muri NEB 5-α competent E. coli (New England Biolabs) kandi zemezwa hakoreshejwe ADN sequencing mbere yo gukoreshwa (reba amakuru y’inyongera 1 kugira ngo ubone sequence y’ishyirwa). Primers zahujwe na promoters ya T7 (Extended Data Table 2) zakoreshejwe kugira ngo hongerwe ishyirwa muri plasmid ishingiye kuri pL4440. Ingano y’ibicuruzwa bya PCR yemejwe na agarose gel electrophoresis.dsRNA yakuwe muri templates za PCR hakoreshejwe Megascript T7 Transcription Kit (Thermo Fisher) kandi itunganywa hakurikijwe amabwiriza y’uwakoze hamwe n’impinduka zavuzwe mbere.
Ku gutera dsRNA, 1.380 ng bya dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) byatewe ku gipimo cya 10 ng nl-1 mu gituza cy'abagabo cyangwa abagore bakuru (Nanoject III, Drummond) mu munsi 1 nyuma yo gucika. Igipimo cyo gucika kw'uturemangingo twa Gene cyagenwe nibura mu buryo butatu bw'ibinyabuzima hakoreshejwe gukuramo RNA, gukora cDNA, na RT-qPCR. Ku gutera ecdysone, abagore b'abakobwa b'iminsi 4 cyangwa ab'iminsi 6 batewe amaraso na 0.13, 0.21, cyangwa 0.63 µg ya 20E cyangwa 3D20E (Nanoject III, Drummond) ku gipimo cya 1.3, 2.1, hakurikijwe imiterere y'igerageza cyangwa 6.3 ng nl-1. Shyira 100 nl ya 10% (vol/vol) ethanol mu mazi; 100 nl ya 3D20E22P muri 10% ya etanoli (ingana na 75% by'ingano yabonetse muri MAG ebyiri). Imibu yashyizwe mu itsinda ry'abatewe inshinge ku buryo butunguranye.
Mu gupima uko amagi y’ingore afite iminsi 3 yahabwaga amaraso y’umuntu adahari. Kuramo imibu yagabuwe igice cyangwa itaragaburirwa. Bitewe n’uburyo yavuwe, ingore zashyirwaga mu bikombe bitandukanye byo kubyara mu gihe cy’iminsi ine nibura amasaha 48 nyuma yo kurya amaraso. Amagi yabazwe hifashishijwe icyuma gipima amagi (Stemi 508, Zeiss); ku ngore zarazwe, amagi yavutsemo ibirahure yafatwaga nk’afite uburumbuke.
Ku igeragezwa ryo guhuza, abagore bemerewe nibura iminsi 2 bitewe n’uburyo bavurwa kugira ngo bagire ubudahangarwa ku guhuza, kandi ingabo z’ingore zihuye n’imyaka yo mu gasozi zashyizwe mu kato kamwe. Nyuma y’amajoro abiri, uduce tw’ingore twatewe intanga twaciwe maze ADN y’uturemangingo irekurwa binyuze mu gukonjesha no gukaraba mu gikoresho kirimo 10 mM Tris-HCl, 1 mM EDTA, na 25 mM NaCl (pH 8.2). Ingero zashyizwemo Proteinase K (0.86 µg µl-1) mu gihe cy’iminota 15 kuri 55 °C, hagakurikiraho iminota 10 kuri 95 °C. Amategura ya ADN y’uturemangingo yagabanijwe inshuro 10 hanyuma agenzurwa na qPCR mu buryo bwa chromosome Y; primers ziri mu mbonerahamwe ya Extended Data Table 2. Kuba nta buryo bwa Y bugaragaza ko nta buryo bwa Y buhuza.
Ku isuzuma ryo kongera guhuza, abagore bashyizwe mu byiciro byasuzumwe niba hari udukingirizo two guhuza kugira ngo bemeze ko bahuje igitsina kandi bamara iminsi 2 badashobora guhuza igitsina mu gihe abagabo batari bahari, nk'uko byasobanuwe mbere 36. Imboro z'abagabo zirimo intanga za DsRed zashyizwe mu tuzu tw'abagore. Nyuma y'amajoro abiri, utunyangingo tw'imisemburo twakuwe mu ngore, maze ADN y'uturemangingo irategurwa nk'uko byasobanuwe haruguru maze hagenzurwa qPCR transgene ya DsRed; primers ziri mu mbonerahamwe ya 2. Kuba nta transgene ya DsRed byagaragaje ko nta kongera guhuza byabayeho.
3D20E yakozwe nk'uko byasobanuwe mbere 37. Muri make, 10 mg ya 20E (Sigma-Aldrich) yashongeshejwe muri 10 ml y'amazi, hakurikiraho kongeramo 30 mg ya platine y'umukara (mu buryo bw'ifu, Sigma-Aldrich). Umugezi woroshye wa O2 wakomeje gushyirwa mu mvange y'imvange, ivangwa ku bushyuhe bw'icyumba. Nyuma y'amasaha 6, hongewemo 30 mL ya methanol kugira ngo ihagarike imvange. Imvange yashyizwemo centrifuge kugira ngo ikureho uduce twa catalyst. Supernatant yakuwe mu mwuka ikamera mu cyuma mu bushyuhe bw'icyumba. Imvange yumye yashongeshejwe muri 10% ya ethanol na methanol kugira ngo ikoreshwe mu isesengura rya HPLC-MS/MS. Igipimo cyo guhindura (kuva kuri 20E kugera kuri 3D20E) cyari hafi 97% (Ishusho ya 4b), kandi imiterere ya MS ya 3D20E yakozwe yahuye n'iyabonetse mu nkumi zashakanye (Ishusho ya 4c).
Iyi nkuru irimo ibisobanuro birambuye ku bizamini by’ibarurishamibare byakozwe. GraphPad (verisiyo ya 9.0) yakoreshejwe mu gukora ikizamini nyacyo cya Fisher, ikizamini cya Mantel-Cox, n’ikizamini cya t cy’umunyeshuri. Ibizamini bya Cochran-Mantel-Haenszel byakozwe hakoreshejwe inyandiko ya R yihariye (iboneka kuri https://github.com/duopeng/mantelhaen.test). Ikwirakwizwa ry’amakuru ryageragejwe ku buryo busanzwe hakoreshejwe ikizamini cya Shapiro-Wilk gifite igipimo cy’agaciro cya 0.05. Iyo amakuru yananiranaga ikizamini cy’uko ibintu bihagaze, ikizamini cya Mann-Whitney cyakozwe. Amakuru y’abarokotse yasesenguwe hakoreshejwe ikizamini cya Mantel-Cox. Pake ya DESeq2 (verisiyo ya 1.28.1) yakoreshejwe mu gukora isesengura ry’itandukaniro ry’imvugo ya RNA-seq. Akabari katambitse ku gishushanyo kagaragaza igipimo cy’ikigereranyo. Agaciro k’agaciro ka P = 0.05 kakoreshejwe nk’igipimo cy’ibizamini byose.
Kugira ngo ubone amakuru arambuye ku gishushanyo mbonera cy'ubushakashatsi, reba incamake ya Raporo y'Ubushakashatsi bw'Inyamaswa ijyanye n'iyi nkuru.
Amakuru ya MS proteomic yashyizwe muri ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) binyuze muri PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) hamwe n'ikimenyetso cy'amakuru PXD032157.
Amakuru ya RNA-seq abikwa muri Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) hakurikijwe inyandiko y’uruhererekane GSE198665.
Andi makuru yakozwe cyangwa yasesenguwe mu gihe cy'ubushakashatsi bukorwa ubu ashobora kuboneka ku banditsi babifite mu gihe cy'ubusabe. Iyi nkuru itanga amakuru y'inkomoko.
De Loof, A. Ecdysteroids: Steroids zo mu gitsina zititaweho? Igitsinagabo: Agasanduku k'umukara. Ubumenyi bw'Indwara.13, 325–338 (2006).
Redfern, CPF 20-hydroxyecdysone n'iterambere ry'intanga ngore muri Anopheles stephens.J. Indwara Physiology.28, 97–109 (1982).