He mea nui te tipu o te meristem apical (SAM) o te wana mō te hanganga o te kakau. Ngā homoni tipungā giberelinHe mahi nui tā ngā GA (GA) ki te whakarite i te tipu o ngā tipu, engari kāore anō kia tino māramahia tā rātou mahi i roto i te SAM. I konei, i whakawhanakehia e mātou he pūoko koiora ōwehenga o te tohu GA mā te hanga i te pūmua DELLA hei pehi i tana mahi whakahaere nui i roto i te urupare whakawhiti GA me te pupuri i tōna pirau i te wā e mōhiotia ana te GA. E whakaatu ana mātou ka tuhia tika e tēnei pūoko koiora e hangai ana ki te pirau ngā huringa o ngā taumata GA me te rongo pūtau i te wā whanaketanga. I whakamahia e mātou tēnei pūoko koiora hei mahere i te mahi tohu GA i roto i te SAM. E whakaatu ana mātou kei roto i ngā pūtau kei waenganui i ngā primordia okana te nuinga o ngā tohu GA teitei, arā, ko ngā mua o ngā pūtau internode. Mā te whakamahi i ngā huarahi piki me te heke o te mahi, ka whakaatu anō mātou ka whakahaere te GA i te aronga o te papa wehenga pūtau, ka whakatū i te whakaritenga pūtau matua o ngā internode, ā, ka whakatairanga i te whakatakotoranga internode i roto i te SAM.
Kei te tihi o te wana te meristem apical o te wana (SAM), kei roto he āputa o ngā pūtau take e whakaputa ana i ngā okana taha me ngā pona take i roto i te āhua whakarerekē me te auau puta noa i te oranga o te tipu. Kei roto i ia o ēnei waeine tāruarua, i ngā pona tipu rānei, ngā internodes me ngā okana taha i ngā pona, me ngā meristem axillary i ngā axils rau1. Ka rerekē te tipu me te whakaritenga o ngā pona tipu i te wā whanaketanga. I roto i te Arabidopsis, ka pehia te tipu internodal i te wā tipu, ā, ka noho moe ngā meristem axillary i ngā axils o ngā rau rosette. I te wā whakawhiti ki te wā putiputi, ka huri te SAM hei meristem inflorescence, ka whakaputa i ngā internodes roa me ngā puku axillary, ngā manga iti i ngā axils o ngā rau cauline, ā, i muri mai, ngā putiputi kore rau2. Ahakoa kua nui te ahunga whakamua i roto i te mārama ki ngā tikanga e whakahaere ana i te tīmatanga o ngā rau, ngā putiputi, me ngā manga, he iti noa te mōhiotanga mō te putanga mai o ngā internodes.
Mā te mārama ki te tohatoha ā-wāhi o ngā GA ka āwhina i te mārama ake ki ngā mahi a ēnei homoni i roto i ngā kiko rerekē me ngā wāhanga whanaketanga rerekē. Mā te tirohanga ki te whakahekenga o te hanumitanga RGA-GFP i whakaatuhia i raro i te mahi a tōna ake kaiwhakatairanga ka whakaratohia he mōhiohio nui mō te whakahaere i ngā taumata GA katoa i roto i ngā pakiaka15,16. Heoi, he rerekē te whakaaturanga RGA puta noa i ngā kiko17 ā, ka whakahaeretia e GA18. Nō reira, ko te rerekētanga o te whakaaturanga o te kaiwhakatairanga RGA ka hua ake te tauira fluorescence i kitea me te RGA-GFP, nō reira ehara tēnei tikanga i te tauanga. Nō nā tata nei, i whakaatuhia e te tohu fluorescein (Fl) bioactive GA19,20 te kohikohinga o te GA i roto i te endocortex pakiaka me te whakahaere i ōna taumata pūtau e te kawe GA. Nō nā tata nei, i whakaatuhia e te pūoko GA FRET nlsGPS1 he hononga ngā taumata GA ki te roa o te pūtau i roto i ngā pakiaka, ngā miro, me ngā hypocotyls pouri-tipu21. Heoi, e ai ki tā tātou i kite ai, ehara i te mea ko te kukū GA anake te tawhā e whakahaere ana i te mahi tohu GA, nā te mea e whakawhirinaki ana ki ngā tukanga mohiotanga uaua. I konei, i runga i tō mātou māramatanga ki ngā ara tohu DELLA me GA, ka ripoatahia e mātou te whanaketanga me te whakaahuatanga o tētahi pūoko koiora ine-ōrite e hangai ana ki te whakahekenga mō te tohu GA. Hei whakawhanake i tēnei pūoko koiora tauanga, i whakamahia e mātou he RGA tairongo-GA kua whakarerekētia i honoa ki tētahi pūmua fluorescent, ā, kua whakaatuhia whānuitia i roto i ngā kiko, me tētahi pūmua fluorescent kāore e aro ki te GA. Ka whakaatuhia e mātou kāore ngā whakakotahitanga pūmua RGA kua whakarerekētia e whakararuraru i te tohu GA taketake ina whakaatuhia whānuitia, ā, ka taea e tēnei pūoko koiora te ine i te mahi tohu e puta mai ana i te whakaurunga GA me te tukatuka tohu GA e te taputapu ine me te taumira ā-wāhi teitei. I whakamahia e mātou tēnei pūoko koiora hei mahere i te tohatoha ā-wāhi o te mahi tohu GA me te ine i te āhua o te whakahaere a te GA i te whanonga pūtau i roto i te kiriuhi SAM. Ka whakaatuhia e mātou ka whakahaerehia e te GA te whakatakotoranga o te papa wehenga o ngā pūtau SAM kei waenganui i ngā primordia o te okana, ā, nā reira ka tautuhia te whakaritenga pūtau matua o te internode.
Hei whakamutunga, i pātai mātou mēnā ka taea e te qmRGA te ripoata i ngā huringa o ngā taumata GA taketake mā te whakamahi i ngā hypocotyls e tipu ana. I whakaaturia e mātou i mua ko te nitrate e whakaoho ana i te tipu mā te whakanui ake i te hanganga GA, ā, hei utu, ko te whakahekenga o DELLA34. Nō reira, i kitea e mātou ko te roa o te hypocotyl i roto i ngā tipu pUBQ10::qmRGA i whakatipuria i raro i te nui o te nitrate (10 mM NO3−) he roa ake i tērā i roto i ngā tipu i whakatipuria i raro i ngā āhuatanga kore-nitrate (Pikitia Tāpiri 6a). E rite ana ki te urupare tipu, he teitei ake ngā tohu GA i roto i ngā hypocotyls o ngā tipu i whakatipuria i raro i ngā āhuatanga 10 mM NO3− i ngā tipu i whakatipuria i te korenga o te nitrate (Pikitia Tāpiri 6b, c). Nō reira, ka taea hoki e te qmRGA te aroturuki i ngā huringa o te tohu GA i puta mai i ngā huringa taketake i roto i te kukū GA.
Hei mārama mēnā ko te mahi tohu GA i kitea e te qmRGA e whakawhirinaki ana ki te kukū o te GA me te tirohanga GA, e ai ki te whakaaro i runga i te hoahoa o te pūoko, i tātarihia e mātou te whakaaturanga o ngā kaiwhakawhiwhi GID1 e toru i roto i ngā kiko tipu me ngā kiko whakaputa uri. I roto i ngā tipu tipu, i whakaatuhia e te rārangi kairīpoata GID1-GUS he nui te whakaaturanga o te GID1a me te c i roto i ngā cotyledon (Pikitia 3a–c). Hei tāpiri, i whakaatuhia ngā kaiwhakawhiwhi e toru i roto i ngā rau, i te pakiaka taha, i ngā pito pakiaka (haunga te taupoki pakiaka o GID1b), me te pūnaha toto (Pikitia 3a–c). I roto i te SAM o te pua, i kitea e mātou ngā tohu GUS mō GID1b me 1c anake (Pikitia Tāpiri 7a–c). I whakaūhia e te whakarōpūtanga in situ ēnei tauira whakaaturanga, ā, i whakaaturia anō ko te whakaaturanga ōrite o te GID1c i ngā taumata iti i roto i te SAM, engari i whakaatuhia e te GID1b he nui ake te whakaaturanga i te taha o te SAM (Pikitia Tāpiri 7d–l). I whakaatuhia hoki e te whakakotahitanga whakawhiti pGID1b::2xmTQ2-GID1b he whānuitanga whakarōpū o te whakaaturanga GID1b, mai i te iti, te kore rānei o te whakaaturanga i waenganui o te SAM ki te whakaaturanga teitei i ngā rohe o te okana (Pikitia Tāpiri 7m). Nō reira, kāore i te tohatoha ōrite ngā kaiwhakawhiwhi GID1 puta noa i roto i ngā kiko me roto hoki. I ngā whakamātautau i muri mai, i kitea anō e mātou ko te nui rawa o te whakaaturanga o GID1 (pUBQ10::GID1a-mCherry) i whakanui ake i te aro o te qmRGA i roto i ngā hypocotyls ki te tono GA o waho (Pikitia 3d, e). He rerekē, ko te fluorescence i inehia e te qd17mRGA i roto i te hypocotyl kāore i te aro ki te maimoatanga GA3 (Pikitia 3f, g). Mō ngā whakamātautau e rua, i rongoatia ngā tipu ki ngā kukū teitei o te GA (100 μM GA3) hei aromatawai i te whanonga tere o te pūoko, i reira i whakanuia, i ngaro rānei te kaha ki te here ki te kaiwhakawhiwhi GID1. Ngātahi, e whakaū ana ēnei hua he mahi tahi tā te pūoko koiora qmRGA hei pūoko GA me te pūoko GA, ā, e kī ana ka taea e te rerekētanga o te whakaaturanga o te kaiwhakawhiwhi GID1 te whakarerekē nui i te tukunga o te pūoko.
Tae noa ki tēnei wā, kāore anō kia mārama te tohatoha o ngā tohu GA i roto i te SAM. Nō reira, i whakamahia e mātou ngā tipu e whakaatu ana i te qmRGA me te kairīpoata pūtau take pCLV3::mCherry-NLS35 hei tatau i ngā mahere tauanga teitei o te mahi tohu GA, me te arotahi ki te paparanga L1 (epidermis; Pikitia 4a, b, tirohia ngā Tikanga me ngā Tikanga Tāpiri), nā te mea he mahi nui tā L1 ki te whakahaere i te tipu SAM36. I konei, i whakaratohia e te whakaaturanga pCLV3::mCherry-NLS he pūwāhi tohutoro āhuahanga pumau mō te tātari i te tohatoha ā-wāhi o te mahi tohu GA37. Ahakoa e kiia ana he mea nui te GA mō te whanaketanga okana taha4, i kitea e mātou he iti ngā tohu GA i roto i te primordium putiputi (P) mai i te wāhanga P3 (Pikitia 4a, b), engari he rite te mahi a ngā primordium taitamariki P1 me P2 ki tērā i te rohe pokapū (Pikitia 4a, b). I kitea te kaha tohu GA teitei ake i ngā rohe o te okana primordium, mai i P1/P2 (i ngā taha o te rohe) ā, ka eke ki te tihi i P4, tae atu ki ngā pūtau katoa o te rohe taha kei waenganui i te primordia (Pikitia 4a, b me te Pikitia Tāpiri 8a, b). I kitea tēnei kaha tohu GA teitei ake ehara i te mea i roto i te kiriuhi anake engari i roto hoki i ngā paparanga L2 me te L3 o runga (Pikitia Tāpiri 8b). Kāore anō kia rerekē te tauira o ngā tohu GA i kitea i roto i te SAM mā te whakamahi i te qmRGA i roto i te wā (Pikitia Tāpiri 8c–f, k). Ahakoa i whakaitihia te hanganga qd17mRGA i roto i te SAM o ngā tipu T3 mai i ngā rārangi motuhake e rima i tautuhia e mātou i roto i te taipitopito, i taea e mātou te tātari i ngā tauira fluorescence i whiwhihia me te hanganga pRPS5a::VENUS-2A-TagBFP (Pikitia Tāpiri 8g–j, l). I roto i tēnei rārangi whakahaere, he iti noa ngā huringa o te ōwehenga fluorescence i kitea i roto i te SAM, engari i roto i te pokapū SAM i kite mātou i te hekenga mārama me te ohorere o te VENUS e pā ana ki te TagBFP. E whakaū ana tēnei ko te tauira tohu i kitea e te qmRGA e whakaata ana i te whakahekenga o te mRGA-VENUS e whakawhirinaki ana ki te GA, engari e whakaatu ana hoki tera pea ka nui rawa te aro o te qmRGA ki te mahi tohu GA i roto i te pokapū meristem. Hei whakarāpopototanga, e whakaatu ana ā mātou hua i tētahi tauira tohu GA e whakaata ana i te tohatoha o te primordia. Ko tēnei tohatoha o te rohe waenga-primordial (IPR) nā te whakatūnga āta o te mahi tohu GA teitei i waenga i te primordium e whanake ana me te rohe pokapū, i te wā kotahi ka heke te mahi tohu GA i roto i te primordium (Pikitia 4c, d).
Ko te tohatoha o ngā kaiwhakawhiwhi GID1b me GID1c (tirohia i runga ake nei) e kī ana ko te rerekētanga o te whakaaturanga o ngā kaiwhakawhiwhi GA e āwhina ana ki te hanga i te tauira o te mahi tohu GA i roto i te SAM. I whakaaro mātou mēnā kei roto pea te kohikohinga rerekētanga o te GA. Hei tirotiro i tēnei tūponotanga, i whakamahia e mātou te pūoko nlsGPS1 GA FRET21. I kitea te pikinga o te auau whakahohenga i roto i te SAM o te nlsGPS1 i rongoatia ki te 10 μM GA4+7 mō te 100 meneti (Pikitia Tāpiri 9a–e), e tohu ana ka aro te nlsGPS1 ki ngā huringa o te kukū GA i roto i te SAM, pērā i tana mahi i roto i ngā pakiaka21. Ko te tohatoha ā-wāhi o te auau whakahohenga nlsGPS1 i whakaatu i ngā taumata GA iti i roto i ngā paparanga o waho o te SAM, engari i whakaatu i te teitei ake i waenganui me ngā rohe o te SAM (Pikitia 4e me te Pikitia Tāpiri 9a,c). E kī ana tēnei kei te tohatohahia hoki te GA i roto i te SAM me te tauira ā-wāhi e rite ana ki te mea i whakakitea e te qmRGA. Hei huarahi tautoko, i whakamahia hoki e mātou te GA fluorescent (GA3-, GA4-, GA7-Fl) me te Fl anake hei mana whakahaere kino. I tohatohahia te tohu Fl puta noa i te SAM, tae atu ki te rohe pokapū me te primordium, ahakoa he iti ake te kaha (Pikitia 4j me te Pikitia Tāpiri 10d). He rerekē, i kohia katoatia ngā GA-Fl e toru i roto i ngā rohe primordium me ngā nekehanga rerekē i roto i te toenga o te IPR, me te GA7-Fl e kohia ana i roto i te rohe nui rawa atu i roto i te IPR (Pikitia 4k me te Pikitia Tāpiri 10a,b). I whakaatuhia e te ine i te kaha fluorescence he teitei ake te ōwehenga kaha IPR ki te kore-IPR i roto i te SAM i rongoatia e GA-Fl ki te SAM i rongoatia e Fl (Pikitia 4l me te Pikitia Tāpiri 10c). I te taha o ēnei hua, e kī ana kei te kitea te GA i ngā kukū teitei ake i roto i ngā pūtau IPR e tata ana ki te rohe okana. E kī ana tēnei ko te tauira o te mahi tohu SAM GA i puta mai i te whakaaturanga rerekē o ngā kaiwhakawhiwhi GA me te kohikohinga rerekē o te GA i roto i ngā pūtau IPR e tata ana ki ngā rohe okana. Nō reira, i whakaatuhia e tā mātou tātaritanga tētahi tauira ā-wāhi, ā-wā rānei o te tohu GA, me te iti ake o te mahi i waenganui me te primordium o te SAM, me te nui ake o te mahi i roto i te IPR i te rohe taha.
Hei mārama ki te tūranga o te mahi tohu GA rerekē i roto i te SAM, i tātarihia e mātou te hononga i waenga i te mahi tohu GA, te whānui o te pūtau, me te wehenga pūtau mā te whakamahi i te whakaahua wā-tūturu o te SAM qmRGA pCLV3::mCherry-NLS. I runga i te tūranga o te GA i roto i te whakahaere tipu, i tumanakohia he hononga pai ki ngā tawhā whānui o te pūtau. Nō reira, i whakaritea tuatahitia e mātou ngā mahere mahi tohu GA ki ngā mahere o te tere tipu o te mata o te pūtau (hei tohu mō te kaha o te whānui o te pūtau mō tētahi pūtau me ngā pūtau tamāhine i te wehenga) me ngā mahere o te anisotropy tipu, e ine ana i te ahunga o te whānui o te pūtau (e whakamahia ana hoki i konei mō tētahi pūtau me ngā pūtau tamāhine i te wehenga; Pikitia 5a,b, tirohia ngā Tikanga me ngā Tikanga Tāpiri). He rite ā mātou mahere o te tere tipu o te mata o te pūtau SAM ki ngā kitenga o mua38,39, me ngā tere tipu iti i te rohe me ngā tere tipu mōrahi i roto i ngā putiputi e whanake ana (Pikitia 5a). I whakaatuhia e te tātari wāhanga matua (PCA) he hononga kino te mahi tohu GA ki te kaha tipu o te mata o te pūtau (Pikitia 5c). I whakaaturia hoki e mātou ko ngā tuaka matua o te rerekētanga, tae atu ki te whakaurunga tohu GA me te kaha tipu, he orthogonal ki te ahunga i whakatauhia e te whakaaturanga CLV3 teitei, e whakaū ana i te tango i ngā pūtau mai i te pokapū SAM i roto i ngā tātaritanga e toe ana. I whakaū te tātaritanga hononga Spearman i ngā hua PCA (Pikitia 5d), e tohu ana ko ngā tohu GA teitei ake i roto i te IPR kāore i hua ake he whānui ake o te pūtau. Heoi, i whakaatuhia e te tātaritanga hononga he hononga pai iti i waenga i te mahi tohu GA me te anisotropy tipu (Pikitia 5c, d), e kī ana ko te tohu GA teitei ake i roto i te IPR ka awe i te ahunga o te tipu pūtau me te tūranga pea o te papa wehenga pūtau.
a, b Ngā mahere wera o te tipu mata toharite (a) me te anisotropy tipu (b) i roto i te SAM i toharitehia mō ngā tipu motuhake e whitu (i whakamahia hei whakakapi mō te kaha me te ahunga o te whānui o te pūtau). c I whakaurua e te tātari PCA ngā taurangi e whai ake nei: te tohu GA, te kaha o te tipu mata, te anisotropy tipu mata, me te whakaaturanga CLV3. Ko te wāhanga PCA 1 i hono kino ki te kaha o te tipu mata, ā, he hononga pai ki te tohu GA. Ko te wāhanga PCA 2 i hono pai ki te anisotropy tipu mata, ā, he hononga kino ki te whakaaturanga CLV3. Ko ngā ōrau e tohu ana i te rerekētanga i whakamāramahia e ia wāhanga. d Te tātari hononga Spearman i waenga i te tohu GA, te kaha o te tipu mata, me te anisotropy tipu mata i te tauine kiko, haunga te CZ. Ko te tau kei te taha matau ko te uara rho Spearman i waenga i ngā taurangi e rua. Ko ngā whetū e tohu ana i ngā take he tino nui te hononga/kino. e Te whakaaturanga 3D o ngā pūtau Col-0 SAM L1 mā te karuārai confocal. Ko ngā pakitara pūtau hou i hangaia i roto i te SAM (engari ehara i te primordium) i te 10 h ka taehia kia rite ki ō rātou uara koki. Kei te kokonga matau o raro te pae tae. E whakaatu ana te whakaurunga i te ahua 3D e rite ana i te 0 h. I tuaruatia te whakamātautau kia rua ngā wā me ngā hua ōrite. f Ka whakaatuhia e ngā kauwhata pouaka ngā tere wehenga pūtau i roto i te IPR me te kore-IPR Col-0 SAM (n = 10 ngā tipu motuhake). Ka whakaatuhia e te rārangi pokapū te tau waenga, ā, ka tohuhia e ngā rohe pouaka ngā ōrau 25 me te 75. Ka tohuhia e ngā ūkui ngā uara iti rawa me te mōrahi i whakatauhia me te pūmanawa R. I whiwhihia ngā uara P me te whakamātautau-t rua-hiku a Welch. g, h Kauwhata hoahoa e whakaatu ana (g) me pēhea te ine i te koki o te pakitara pūtau hou (magenta) e pā ana ki te ahunga radial mai i te pokapū o te SAM (rārangi iraira mā) (ko ngā uara koki koi anake, arā, 0–90°, ka whakaarohia), me (h) ngā ahunga porowhita/taha me te radial i roto i te meristem. i Ngā histogram auau o te aronga papa wehenga pūtau puta noa i te SAM (kikorangi pouri), IPR (kikorangi waenga), me te kore-IPR (kikorangi mārama). I whiwhihia ngā uara P mā te whakamātautau Kolmogorov-Smirnov e rua ngā hiku. I tuaruatia te whakamātautau me ngā hua ōrite. j Ngā histogram auau o te aronga o te papa wehenga pūtau o te IPR huri noa i te P3 (kākāriki mārama), te P4 (kākāriki waenga), me te P5 (kākāriki pouri). I whiwhihia ngā uara P mā te whakamātautau Kolmogorov-Smirnov e rua ngā hiku. I tuaruatia te whakamātautau me ngā hua ōrite.
Nō reira, i muri mai ka tirotirohia e mātou te hononga i waenga i te tohu GA me te mahi wehenga pūtau mā te tautuhi i ngā pakitara pūtau hou i hangaia i te wā o te whakamātautau (Pikitia 5e). Nā tēnei huarahi i taea ai e mātou te ine i te auau me te ahunga o te wehenga pūtau. He mea whakamiharo, i kitea e mātou he rite tonu te auau o ngā wehenga pūtau i roto i te IPR me te toenga o te SAM (kore-IPR, Pikitia 5f), e tohu ana ko ngā rerekētanga o te tohu GA i waenga i te IPR me ngā pūtau kore-IPR kāore e tino pā ki te wehenga pūtau. Nā tēnei, me te hononga pai i waenga i te tohu GA me te anisotropy tipu, i akiaki i a mātou ki te whakaaro mēnā ka taea e te mahi tohu GA te awe i te aronga o te papa wehenga pūtau. I ine mātou i te aronga o te pakitara pūtau hou hei koki koi e pā ana ki te tuaka radial e hono ana i te pokapū meristem me te pokapū o te pakitara pūtau hou (Pikitia 5e-i) ā, i kitea he ia o te wehenga o ngā pūtau i ngā koki e tata ana ki te 90° e pā ana ki te tuaka radial, me ngā auau teitei rawa i kitea i te 70–80° (23.28%) me te 80–90° (22.62%) (Pikitia 5e,i), e rite ana ki ngā wehenga pūtau i te ahunga porowhita/whakapae (Pikitia 5h). Hei tirotiro i te takoha o te tohu GA ki tēnei whanonga wehenga pūtau, i tātarihia e mātou ngā tawhā wehenga pūtau i roto i te IPR me te kore-IPR motuhake (Pikitia 5i). I kitea e mātou he rerekē te tohatoha o te koki wehenga i roto i ngā pūtau IPR i ērā i roto i ngā pūtau kore-IPR, i roto rānei i ngā pūtau i roto i te SAM katoa, me ngā pūtau IPR e whakaatu ana i te ōwehenga teitei ake o ngā wehenga pūtau taha/porowhita, arā, 70–80° me 80–90° (33.86% me 30.71%, ia, ngā ōwehenga e rite ana) (Pikitia 5i). Nō reira, i whakaatuhia e ā mātou kitenga he hononga i waenga i te tohu GA teitei me te aronga papa wehenga pūtau e tata ana ki te ahunga porowhita, he rite ki te hononga i waenga i te mahi tohu GA me te anisotropy tipu (Pikitia 5c, d). Hei whakaū anō i te tiaki ā-wāhi o tēnei hononga, i inehia e mātou te aronga papa wehenga i roto i ngā pūtau IPR e karapoti ana i te primordium mai i te P3, mai i te mea ko te mahi tohu GA teitei rawa i kitea i tēnei rohe mai i te P4 (Pikitia 4). Kāore i whakaatuhia e ngā koki wehenga o te IPR huri noa i te P3 me te P4 he rerekētanga nui ā-tatauranga, ahakoa i kitea he auau pikinga o ngā wehenga pūtau taha i roto i te IPR huri noa i te P4 (Pikitia 5j). Heoi, i roto i ngā pūtau IPR huri noa i te P5, i tino kitea te rerekētanga o te aronga o te papa wehenga pūtau, me te pikinga tere o te auau o ngā wehenga pūtau whakawhiti (Pikitia 5j). E whakaatu tahi ana ēnei hua ka taea e te tohu GA te whakahaere i te aronga o ngā wehenga pūtau i roto i te SAM, he rite tonu ki ngā pūrongo o mua40,41 ka taea e te tohu GA teitei te whakaoho i te aronga taha o ngā wehenga pūtau i roto i te IPR.
E matapaetia ana kāore ngā pūtau o te IPR e whakaurua ki roto i ngā primordia engari ki roto i ngā internodes2,42,43. Ko te aronga whakawhiti o ngā wehenga pūtau i roto i te IPR ka hua ake pea he whakaritenga noa o ngā rarangi roa whakarara o ngā pūtau epidermal i roto i ngā internodes. Ko ā mātou kitenga i whakaahuatia i runga ake nei e kī ana he wāhi nui pea tā te tohu GA i roto i tēnei tukanga mā te whakahaere i te ahunga o te wehenga pūtau.
Ko te ngaronga o te mahi a ētahi ira DELLA ka hua ake he urupare GA pumau, ā, ka taea te whakamahi i ngā momo ira della hei whakamātautau i tēnei whakapae44. I tātarihia tuatahitia e mātou ngā tauira whakapuakanga o ngā ira DELLA e rima i roto i te SAM. Nā te whakakotahitanga whakawhiti o te rārangi GUS45 i whakaatu ko GAI, RGA, RGL1, me RGL2 (ki te iti rawa) i whakaatuhia i roto i te SAM (Pikitia Tāpiri 11a–d). I whakaatuhia anō e te whakahāngaitanga in situ ka kohia te mRNA GAI i roto i ngā primordia me ngā putiputi e whanake ana (Pikitia Tāpiri 11e). I kitea te mRNA RGL1 me te RGL3 puta noa i te tuanui SAM me ngā putiputi tawhito, engari he nui ake te mRNA RGL2 i te rohe rohe (Pikitia Tāpiri 11f–h). I whakaūhia e te whakaahua confocal o pRGL3::RGL3-GFP SAM te whakapuakanga i kitea e te whakahāngaitanga in situ, ā, i whakaatuhia ko te pūmua RGL3 ka kohia i te wāhanga pokapū o te SAM (Pikitia Tāpiri 11i). Mā te whakamahi i te rārangi pRGA::GFP-RGA, i kitea anō e mātou ka kohia te pūmua RGA i roto i te SAM, engari ka heke tōna nui i te rohe mai i te P4 (Pikitia Tāpiri 11j). He mea nui, ko ngā tauira whakapuakanga o RGL3 me RGA he rite ki te mahi tohu GA teitei ake i roto i te IPR, e kitea ana e te qmRGA (Pikitia 4). Heoi anō, e tohu ana ēnei raraunga kei roto i te SAM ngā DELLA katoa, ā, ko tā rātou whakapuakanga katoa e kapi ana i te SAM katoa.
I muri mai ka tātarihia e mātou ngā tawhā wehenga pūtau i roto i te SAM momo-mohoao (Ler, mana whakahaere) me ngā ira-whakawhiti gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4 della quintuple (ao whānui) (Pikitia 6a, b). He mea whakamīharo, i kitea e mātou he nekehanga nui i roto i te tohatoha o ngā auau koki wehenga pūtau i roto i te SAM ira-whakawhiti della ao whānui ki te momo mohoao (Pikitia 6c). Ko tēnei huringa i roto i te ira-whakawhiti della ao whānui i puta mai i te pikinga o te auau o ngā koki 80–90° (34.71% vs. 24.55%) me, ki te iti iho, 70–80° koki (23.78% vs. 20.18%), arā, e rite ana ki ngā wehenga pūtau whakawhiti (Pikitia 6c). Ko te auau o ngā wehenga kore-whakawhiti (0–60°) i iti ake hoki i roto i te ira-whakawhiti della ao whānui (Pikitia 6c). Ko te auau o ngā wehenga pūtau whakawhiti i tino piki ake i roto i te SAM o te ira-whakawhiti della ao whānui (Pikitia 6b). He nui ake hoki te auau o ngā wehenga pūtau whakawhiti i roto i te IPR i roto i te momo mutant della global ki te momo mohoao (Pikitia 6d). I waho o te rohe IPR, he rite tonu te tohatoha o ngā koki wehenga pūtau o te momo mohoao, engari ko te mutant della global i pai ake ki ngā wehenga tangential pērā i te IPR (Pikitia 6e). I inehia hoki e mātou te whakatakotoranga o ngā wehenga pūtau i roto i te SAM o ngā mutant rima-toru ga2 oxidase (ga2ox) (ga2ox1-1, ga2ox2-1, ga2ox3-1, ga2ox4-1, me te ga2ox6-2), he papamuri mutant GA-kore e kohikohi ai te GA. E ai ki te pikinga o ngā taumata GA, he nui ake te SAM o te pūhui whakaputa uri ga2ox e rima i tērā o Col-0 (Pikitia Tāpiri 12a, b), ā, ki te whakatauritea ki Col-0, i whakaatu te SAM ga2ox e rima i tētahi tohatoha rerekē o ngā koki wehenga pūtau, me te pikinga o te auau koki mai i te 50° ki te 90°, arā, e pai ake ana ki ngā wehenga tānga (Pikitia Tāpiri 12a–c). Nō reira, e whakaatu ana mātou ko te whakahohenga pumau o te tohu GA me te kohikohinga GA ka puta ai ngā wehenga pūtau taha i roto i te IPR me te toenga o te SAM.
a, b Whakaaturanga 3D o te paparanga L1 o te Ler (a) me te della mutant ā-ao (b) SAM kua pania ki te PI mā te whakamahi i te karuārai confocal. Ka whakaaturia, ka taehia hoki ngā pakitara pūtau hou i hangaia i roto i te SAM (engari kāore i te primordium) i roto i te wā 10-hāora kia rite ki ō rātou uara koki. E whakaatu ana te whakaaturanga i te SAM i te 0 hāora. Ka whakaaturia te pae tae i te kokonga matau o raro. E tohu ana te pere i roto i te (b) ki tētahi tauira o ngā kōnae pūtau kua whakarārangihia i roto i te della mutant ā-ao. I tuaruatia te whakamātautau kia rua ngā wā me ngā hua ōrite. Te whakataurite i te tohatoha auau o ngā aronga papa wehenga pūtau i roto i te SAM katoa (d), IPR (e), me te kore-IPR (f) i waenga i te Ler me te della ā-ao. I whiwhihia ngā uara P mā te whakamahi i te whakamātautau Kolmogorov-Smirnov e rua-hiku. f, g Whakaaturanga 3D o ngā whakaahua confocal o te SAM kua pania ki te PI o ngā tipu whakawhiti Col-0 (i) me te pCUC2::gai-1-VENUS (j). E whakaatu ana ngā panera (a, b) i ngā pakitara pūtau hou (engari ehara i te primordia) i hangaia i roto i te SAM i roto i ngā haora 10. I tuaruatia te whakamātautau kia rua ngā wā, ā, he rite tonu ngā hua. h–j Te whakataurite i te tohatoha auau o ngā aronga papa wehenga pūtau kei roto i te SAM katoa (h), IPR (i) me te kore-IPR (j) i waenga i ngā tipu Col-0 me pCUC2::gai-1-VENUS. I whiwhihia ngā uara P mā te whakamahi i te whakamātautau Kolmogorov–Smirnov e rua ngā hiku.
I muri mai ka whakamātauhia e mātou te pānga o te aukati i te tohu GA i roto i te IPR. Hei whakatutuki i tēnei, i whakamahia e mātou te kaiwhakatairanga cotyledon cup 2 (CUC2) hei akiaki i te whakaaturanga o te pūmua gai-1 kino matua kua honoa ki a VENUS (i roto i te rārangi pCUC2::gai-1-VENUS). I roto i te SAM momo-mohoao, ka akiakihia e te kaiwhakatairanga CUC2 te whakaaturanga o te nuinga o ngā IPR i roto i te SAM, tae atu ki ngā pūtau rohe, mai i te P4 ki mua, ā, i kitea he whakaaturanga motuhake rite i roto i ngā tipu pCUC2::gai-1-VENUS (tirohia i raro). Kāore i tino rerekē te tohatoha o ngā koki wehenga pūtau puta noa i te SAM, i te IPR rānei o ngā tipu pCUC2::gai-1-VENUS i tērā o te momo mohoao, ahakoa i kitea ohoreretia e mātou ko ngā pūtau kāore he IPR i roto i ēnei tipu ka wehea i te auau teitei ake o te 80–90° (Pikitia 6f–j).
Kua whakaarohia ko te ahunga o te wehenga pūtau e whakawhirinaki ana ki te āhua o te SAM, inā koa te ahotea kume i puta mai i te piko o te kiko46. Nō reira i pātai mātou mēnā i whakarerekētia te āhua o te SAM i roto i te tipu della global mutant me te pCUC2::gai-1-VENUS. E ai ki te pūrongo o mua12, he nui ake te rahi o te della global mutant SAM i tērā o te momo mohoao (Pikitia Tāpiri 13a, b, d). Ko te whakaranu in situ o te CLV3 me te STM RNA i whakaū i te whānui o te meristem i roto i ngā della mutants, ā, i whakaatu anō i te whānui taha o te niche pūtau take (Pikitia Tāpiri 13e, f, h, i). Heoi, he rite te piko o te SAM i roto i ngā iraira e rua (Pikitia Tāpiri 13k, m, n, p). I kite mātou i te pikinga rite o te rahi i roto i te gai-t6 rga-t2 rgl1-1 rgl2-1 della quadruple mutant me te kore he huringa o te piko ki te momo mohoao (Pikitia Tāpiri 13c, d, g, j, l, o, p). I pāngia anō hoki te auau o te aronga wehenga pūtau i roto i te mutant della quadruple, engari he iti iho i tērā i roto i te mutant della monolithic (Pikitia Tāpiri 12d–f). Ko tēnei pānga horopeta, me te kore pānga ki te piko, e kī ana ko te mahi RGL3 toenga i roto i te mutant Della quadruple ka whakawhāiti i ngā huringa o te aronga wehenga pūtau i puta mai i te ngaronga o te mahi DELLA, ā, ka puta ngā huringa o ngā wehenga pūtau taha hei whakautu ki ngā huringa o te mahi tohu GA kaua ki ngā huringa o te āhua o SAM. E ai ki te whakaahuatanga i runga ake nei, ko te kaiwhakatairanga CUC2 te mea e akiaki ana i te whakaaturanga IPR i roto i te SAM e tīmata ana i te P4 (Pikitia Tāpiri 14a, b), ā, i te rerekētanga, he iti ake te rahi o te pCUC2::gai-1-VENUS SAM engari he nui ake te piko (Pikitia Tāpiri 14c–h). Ko tēnei huringa o te āhua o te pCUC2::gai-1-VENUS SAM ka hua ake he tohatoha rerekē o ngā ahotea miihini ki te momo mohoao, e tīmata ana ngā ahotea porowhita teitei i te tawhiti poto ake mai i te pokapū SAM47. Heoi anō, ko ngā panonitanga o te āhua o te pCUC2::gai-1-VENUS SAM ka puta pea i ngā panonitanga o ngā āhuatanga miihini ā-rohe i puta mai i te whakaaturanga ira whakawhiti48. I ngā take e rua, ka taea e tēnei te whakakore i ngā pānga o ngā panonitanga o te tohu GA mā te whakanui ake i te tūponotanga ka wehea ngā pūtau i te aronga porowhita/whakapae, e whakamārama ana i ā mātou kitenga.
Ina huihuia, e whakaū ana ā mātou raraunga he mahi nui tā te tohu GA teitei ake i roto i te aronga taha o te papa wehenga pūtau i roto i te IPR. E whakaatu ana hoki rātou he pānga anō hoki te piko o te meristem ki te aronga o te papa wehenga pūtau i roto i te IPR.
Ko te aronga whakawhiti o te papa wehenga i roto i te IPR, nā te nui o te mahi tohu GA, e kī ana ka whakarite mua a GA i tētahi kōnae pūtau radial i roto i te kiriuhi i roto i te SAM hei tautuhi i te whakaritenga pūtau ka kitea i muri mai i roto i te internode epidermal. Ina hoki, he maha ngā wā i kitea ai aua kōnae pūtau i roto i ngā whakaahua SAM o ngā mutants della global (Pikitia 6b). Nō reira, hei tūhura atu i te mahi whanaketanga o te tauira ā-wāhi o te tohu GA i roto i te SAM, i whakamahia e mātou te whakaahua wā-wā hei tātari i te whakaritenga ā-wāhi o ngā pūtau i roto i te IPR i roto i ngā tipu momo-mohoao (Ler me Col-0), ngā mutants della global, me ngā tipu whakawhiti pCUC2::gai-1-VENUS.
I kitea e mātou i whakaatu a qmRGA i piki ake te mahi tohu GA i roto i te IPR mai i te P1/P2, ā, i eke ki te taumata teitei rawa atu i te P4, ā, i noho tonu tēnei tauira i roto i te wā (Pikitia 4a–f me te Pikitia Tāpiri 8c–f, k). Hei tātari i te whakaritenga ā-wāhi o ngā pūtau i roto i te IPR me te tohu GA e piki haere ana, i tapaina e mātou ngā pūtau Ler IPR i runga ake nei, i ngā taha hoki o te P4, e ai ki tā rātou whanaketanga i tātarihia i muri i te 34 hāora i muri i te kitenga tuatahi, arā, neke atu i te rua ngā wā plastid, e āhei ai mātou ki te whai i ngā pūtau IPR i te whanaketanga primordium mai i te P1/P2 ki te P4. E toru ngā tae rerekē i whakamahia e mātou: kowhai mō ngā pūtau i whakaurua ki roto i te primordium tata ki te P4, kakariki mō te hunga i roto i te IPR, me te papura mō te hunga i whai wāhi ki ngā tukanga e rua (Pikitia 7a–c). I te t0 (0 hāora), 1–2 ngā paparanga o ngā pūtau IPR i kitea i mua i te P4 (Pikitia 7a). E ai ki te whakaaro, i te wā i wehea ai ēnei pūtau, i mahia e rātou mā te papa wehenga whakawhiti (Pikitia 7a–c). I whiwhihia ngā hua ōrite mā te whakamahi i te Col-0 SAM (me te arotahi ki te P3, ko tōna rohe e takai ana he rite ki te P4 i roto i te Ler), ahakoa i roto i tēnei momoira i huna wawetia e te takai i hangaia i te rohe putiputi ngā pūtau IPR (Pikitia 7g–i). Nō reira, ko te tauira wehenga o ngā pūtau IPR ka whakarite i mua i ngā pūtau ki ngā rarangi radial, pērā i ngā internodes. Ko te whakaritenga o ngā rarangi radial me te taunga o ngā pūtau IPR i waenga i ngā okana e whai ake nei e kī ana ko ēnei pūtau he tūpuna internodal.
I konei, i whakawhanakehia e mātou he pūoko koiora tohu GA ōwehenga, qmRGA, e āhei ai te mahere tauanga o te mahi tohu GA i puta mai i ngā kukū o te GA me te kaiwhakawhiwhi GA i te wā e whakaitihia ana te pokanoa ki ngā ara tohu taketake, ā, mā reira ka whakaratohia he mōhiohio mō te mahi GA i te taumata pūtau. Hei whakatutuki i tēnei, i hangaia e mātou he pūmua DELLA kua whakarerekētia, mRGA, kua ngaro te kaha ki te here i ngā hoa taunekeneke DELLA engari kei te aro tonu ki te proteolysis i puta mai i te GA. Ka aro atu te qmRGA ki ngā huringa o waho me o roto i ngā taumata GA, ā, ko ōna āhuatanga pūoko hihiri ka taea te aromatawai i ngā huringa ā-wāhi me te ā-wā i roto i te mahi tohu GA i te wā whanaketanga. He taputapu tino ngāwari hoki te qmRGA nā te mea ka taea te urutau ki ngā kiko rerekē mā te whakarerekē i te kaiwhakatairanga i whakamahia mō tōna whakaaturanga (mēnā e tika ana), ā, i te mea he āhua tiakina o te ara tohu GA me te kaupapa PFYRE puta noa i ngā angiosperms, tera pea ka taea te whakawhiti ki ētahi atu momo22. E rite ana ki tēnei, i whakaaturia hoki he whakarerekētanga ōrite i roto i te pūmua raihi SLR1 DELLA (HYY497AAA) hei pehi i te mahi aukati tipu o SLR1, me te whakaiti noa i tōna pirau e whakahaerehia ana e GA, he rite ki te mRGA23. He mea nui, i whakaatuhia e ngā rangahau tata nei i roto i te Arabidopsis he whakarerekētanga kotahi o te waikawa amino i roto i te rohe PFYRE (S474L) i whakarerekē i te mahi whakawhiti o te RGA me te kore e pā ki tōna kaha ki te taunekeneke me ngā hoa taupū whakawhiti50. Ahakoa he tino tata tēnei whakarerekētanga ki ngā whakakapinga waikawa amino e 3 kei roto i te mRGA, e whakaatu ana ā mātou rangahau he whakarerekētanga ēnei whakarerekētanga e rua i ngā āhuatanga motuhake o DELLA. Ahakoa he maha ngā hoa taupū whakawhiti e herea ana ki ngā rohe LHR1 me SAW o DELLA26,51, ko ētahi waikawa amino kua tiakina i roto i te rohe PFYRE ka āwhina pea ki te whakau i ēnei taunekeneke.
Ko te whanaketanga o ngā pito o te pūtau he āhuatanga matua i roto i te hoahoa tipu me te whakapai ake i te hua. I whakaatuhia e te qmRGA te kaha tohu GA teitei ake i roto i ngā pūtau tupuna pito o te pūtau IPR. Mā te whakakotahi i te whakaahua tauanga me te ira, i whakaaturia e mātou ko ngā tauira tohu GA e whakatakoto ana i ngā papa wehenga pūtau porowhita/whakapae i roto i te kiriuhi SAM, e hanga ana i te whakaritenga wehenga pūtau e hiahiatia ana mō te whanaketanga pito o te pūtau. He maha ngā kaiwhakahaere o te aronga o te papa wehenga pūtau kua tautuhia i te wā whanaketanga52,53. He tauira mārama tā mātou mahi mō te whakahaere a te mahi tohu GA i tēnei tawhā pūtau. Ka taea e DELLA te taunekeneke me ngā matatini pūmua kua takai mua41, nō reira ka taea e te tohu GA te whakahaere i te aronga papa wehenga pūtau mā te awe tika i te aronga microtubule cortical40,41,54,55. I whakaaturia ohoreretia e mātou i roto i te SAM, ko te hononga o te kaha tohu GA teitei ake ehara i te roa o te pūtau, i te wehenga rānei, engari ko te anisotropy tipu anake, e rite ana ki te pānga tika o te GA ki te ahunga o te wehenga pūtau i roto i te IPR. Heoi, kāore e taea e mātou te whakakore ko tēnei pānga he pānga ā-kore, hei tauira, nā te whakangawari o te pakitara pūtau i puta mai i te GA56. Ka puta te ahotea ā-pūnaha57,58 i ngā panonitanga o te pakitara pūtau, ka taea hoki e tēnei te awe i te aronga o te papa wehenga pūtau mā te awe i te aronga o ngā microtubules cortical39,46,59. Ko ngā pānga whakakotahi o te ahotea ā-pūnaha i puta mai i te GA me te whakahaere tika i te aronga o ngā microtubule e te GA, tera pea ka uru ki te whakaputa i tētahi tauira motuhake o te aronga wehenga pūtau i roto i te IPR hei tautuhi i ngā internodes, ā, me whai atu rangahau hei whakamātautau i tēnei whakaaro. Waihoki, kua whakamāramahia e ngā rangahau o mua te hiranga o ngā pūmua e taunekeneke ana a DELLA, arā, ko TCP14 me te 15, ki te whakahaere i te hanganga internode60,61, ā, ka taea e ēnei āhuatanga te takawaenga i te mahi a te GA me BREVIPEDICELLUS (BP) me PENNYWISE (PNY), e whakahaere ana i te whanaketanga internode, ā, kua whakaaturia he awe ki te tohu GA2,62. I te mea ka taunekeneke ngā DELLA me ngā ara tohu brassinosteroid, ethylene, jasmonic acid, me te abscisic acid (ABA)63,64, ā, ka taea e ēnei homoni te awe i te aronga o te microtubule65, ko ngā pānga o te GA ki te aronga o te wehenga pūtau ka taea hoki te whakahaere e ētahi atu homoni.
I whakaatuhia e ngā rangahau cytological tōmua e hiahiatia ana ngā rohe o roto me o waho o te Arabidopsis SAM mō te whanaketanga o te internode2,42. Ko te meka e whakahaere kaha ana te GA i te wehenga pūtau i roto i ngā kiko o roto12 e tautoko ana i te mahi takirua a te GA ki te whakahaere i te rahi o te meristem me te internode i roto i te SAM. Ko te tauira o te wehenga pūtau aronga e whakahaeretia ana hoki i roto i te kiko o roto o te SAM, ā, he mea nui tēnei whakahaere mō te tipu o te kakau52. He mea whakamere ki te tirotiro mēnā he wāhi anō tā te GA ki te whakarite i te papa wehenga pūtau i roto i te whakaritenga o roto o te SAM, kia rite ai te whakatakotoranga me te whanaketanga o ngā internode i roto i te SAM.
I whakatipuria ngā tipu i roto i te vitro i roto i te oneone, i te 1x rānei o te reo Murashige-Skoog (MS) (Duchefa) i tāpirihia ki te 1% sucrose me te 1% agar (Sigma) i raro i ngā āhuatanga paerewa (16 hāora te mārama, 22 °C), engari mō ngā whakamātautau hypocotyl me te tipu pakiaka i whakatipuria ai ngā tipu ki runga i ngā pereti poutū i raro i te mārama tonu me te 22 °C. Mō ngā whakamātautau nitrate, i whakatipuria ngā tipu ki runga i te reo MS kua whakarerekētia (reo tipu bioWORLD) i tāpirihia ki te nitrate e tika ana (0, 10 mM KNO3 rānei), 0.5 mM NH4-succinate, 1% sucrose me te 1% A-agar (Sigma) i raro i ngā āhuatanga rā roa.
Ko te GID1a cDNA i whakaurua ki roto i te pDONR221 i honoa anō ki te pDONR P4-P1R-pUBQ10 me te pDONR P2R-P3-mCherry ki roto i te pB7m34GW hei whakaputa i te pUBQ10::GID1a-mCherry. Ko te IDD2 DNA i whakaurua ki roto i te pDONR221 i honoa anō ki roto i te pB7RWG266 hei whakaputa i te p35S:IDD2-RFP. Hei whakaputa i te pGID1b::2xmTQ2-GID1b, i whakakahangia tuatahitia tētahi maramara 3.9 kb i mua o te rohe waehere GID1b me tētahi maramara 4.7 kb kei roto ko te cDNA GID1b (1.3 kb) me te terminator (3.4 kb) mā te whakamahi i ngā primers i te Ripanga Tāpiri 3, kātahi ka whakaurua ki roto i te pDONR P4-P1R (Thermo Fisher Scientific) me te pDONR P2R-P3 (Thermo Fisher Scientific), ā, i te mutunga ka honoa anō ki te pDONR221 2xmTQ268 ki roto i te pūwhakapūtanga pGreen 012567 mā te whakamahi i te Gateway cloning. Hei whakaputa i te pCUC2::LSSmOrange, ko te raupapa whakatairanga CUC2 (3229 bp i mua atu i te ATG) ā, i whai ake ko te raupapa waehere o te mOrange nui kua nekehia e Stokes (LSSmOrange)69 me te tohu whakarōpū karihi N7 me te whakamutunga whakawhiti NOS i huihuia ki roto i te ira arotahi kanamycin pGreen mā te whakamahi i te pūnaha whakakotahitanga kongakonga Gateway 3 (Invitrogen). I whakaurua te ira takirua tipu ki roto i te momo Agrobacterium tumefaciens GV3101, ā, i whakaurua ki roto i ngā rau Nicotiana benthamiana mā te tikanga whakaurunga Agrobacterium me te tikanga tuku putiputi ki roto i te Arabidopsis thaliana Col-0. I wehea te pUBQ10::qmRGA pUBQ10::GID1a-mCherry me te pCLV3::mCherry-NLS qmRGA mai i ngā uri F3 me F1 o ngā whakawhiti.
I mahia te whakawhānau RNA in situ ki runga i ngā pito o te wana e 1 cm te roa72, i kohia, ā, i whakapiri tonutia ki roto i te otinga FAA (3.7% formaldehyde, 5% waikawa acetic, 50% ethanol) i whakamataohia ki te 4 °C. I muri i ngā maimoatanga korehau e 2 × 15 meneti, i hurihia te mea whakapiri, ā, i whakatōngia ngā tauira i te pō. Ko ngā cDNA GID1a, GID1b, GID1c, GAI, RGL1, RGL2, me RGL3 me ngā probe antisense ki ō rātou 3′-UTR i hangaia mā te whakamahi i ngā primers e whakaaturia ana i te Ripanga Tāpiri 3 e ai ki te whakaahuatanga a Rosier et al.73. I kitea ngā whakamātautau i tapaina ki te digoxigenin mā te whakamahi i ngā paturopi digoxigenin (whakarewatanga 3000-fold; Roche, nama rarangi: 11 093 274 910), ā, i pania ngā wāhanga ki te otinga 5-bromo-4-chloro-3-indolyl phosphate (BCIP, whakarewatanga 250-fold)/nitroblue tetrazolium (NBT, whakarewatanga 200-fold).
Te wā tuku: 10-Pepuere-2025