Aux/IAA家族基因GhAux1沉默促进棉花幼苗的生长
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摘要
为明确Aux/IAA家族基因GhAux1在棉花幼苗生长发育中的功能,利用病毒诱导的基因沉默(VIGS)技术构建了重组表达载体pTRV-GhAux1,转化农杆菌并注射到棉花幼苗子叶,采用qRT-PCR检测GhAux1沉默效率,并对GhAux1沉默植株的表型、内源生长素(IAA)含量和赤霉素(GA_4)含量以及GA合成关键酶基因的表达进行了分析。结果表明,成功建立了VIGS体系,注射含pTRV-GhAux1的菌液后15 d,GhAux1在沉默植株中的表达量仅为空载体对照的26%;注射含pTRV-GhAux1的菌液后24、27、30 d,GhAux1沉默植株的株高较空载体对照分别显著增加了18%、27%、20%,其中注射后27 d达到极显著水平;注射含pTRV-GhAux1的菌液后21、24、27 d,GhAux1沉默植株的节间内源GA_4含量比空载体对照分别增加了33%、20%、33%,且GA合成关键酶基因GhGA20ox1、GhGA20ox2和GhGA3ox1的表达量均显著或极显著增加,而IAA含量仅在注射后27 d显著增加。表明GhAux1参与IAA与GA的互作,可调控GA合成途径下游基因的表达,抑制棉花幼苗的生长。
To explore the function of Aux/IAA family gene GhAux1 in the growth and development of cotton seedlings,we constructed the recombinant expression vector pTRV-GhAux1,transformed it into Agrobacterium and further inoculated it into the cotyledons of cotton seedlings by virus-induced gene silencing(VIGS) method.The silencing efficiency of GhAux1 in silenced cotton seedlings was detected by qRT-PCR.Plant phenotypes,the content of endogenous auxin(IAA) and gibberellin(GA_4) and the expression level of key genes for GA synthesis were investigated.The result showed that,the expression level of GhAux1 in GhAux1-silenced plants was only 26% of that in pTRV control plants at 15 days after inoculation.The GhAux1-silenced plants were successfully obtained.The height of GhAux1-silenced plants was increased significantly by 18%,27% and 20% at 24,27 and 30 days after inoculation respectively compared to pTRV control plants,and the difference was extremely significant at 27 d.Silencing of GhAux1 increased the level of endogenous GA_4 by 33%,20% and 33% at 21,24 and 27 days after inoculation respectively compared to pTRV control plants.Correspondingly,the expression level of GhGA20 ox1,GhGA20 ox2 and GhGA3 ox1 which encoded GA biosynthesis key enzymes showed significant or extremely significant up-regulation in GhAux1-silenced plants.The endogenous IAA content was increased significantly only at 27 days after inoculation.These results suggest that GhAux1 may play a negative role during the growth and development of cotton seedlings by regulating the expression of genes in GA biosynthesis pathway.
To explore the function of Aux/IAA family gene GhAux1 in the growth and development of cotton seedlings,we constructed the recombinant expression vector pTRV-GhAux1,transformed it into Agrobacterium and further inoculated it into the cotyledons of cotton seedlings by virus-induced gene silencing(VIGS) method.The silencing efficiency of GhAux1 in silenced cotton seedlings was detected by qRT-PCR.Plant phenotypes,the content of endogenous auxin(IAA) and gibberellin(GA_4) and the expression level of key genes for GA synthesis were investigated.The result showed that,the expression level of GhAux1 in GhAux1-silenced plants was only 26% of that in pTRV control plants at 15 days after inoculation.The GhAux1-silenced plants were successfully obtained.The height of GhAux1-silenced plants was increased significantly by 18%,27% and 20% at 24,27 and 30 days after inoculation respectively compared to pTRV control plants,and the difference was extremely significant at 27 d.Silencing of GhAux1 increased the level of endogenous GA_4 by 33%,20% and 33% at 21,24 and 27 days after inoculation respectively compared to pTRV control plants.Correspondingly,the expression level of GhGA20 ox1,GhGA20 ox2 and GhGA3 ox1 which encoded GA biosynthesis key enzymes showed significant or extremely significant up-regulation in GhAux1-silenced plants.The endogenous IAA content was increased significantly only at 27 days after inoculation.These results suggest that GhAux1 may play a negative role during the growth and development of cotton seedlings by regulating the expression of genes in GA biosynthesis pathway.
引文
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[4] 刘晓柱,李银凤,赵燕,等.生长素对荠菜心皮发育的影响[J].河南农业科学,2018,47(8):88-94.
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[10] DENG W,YAN F,LIU M,et al.Down-regulation of SlIAA15 in tomato altered stem xylem development and production of volatile compounds in leaf exudates[J].Plant Signaling & Behavior,2012,7(8):911-913.
[11] KLOOSTERMAN B,VISSER R,BACHEM C.Isolation and characterization of a novel potato Auxin/Indole-3-Acetic Acid family member (StIAA2) that is involved in petiole hyponasty and shoot morphogenesis[J].Plant Physiology & Biochemistry,2006,44(11/12):766-775.
[12] HAN X,XU X,FANG D D,et al.Cloning and expression analysis of novel Aux/IAA family genes in Gossypium hirsutum[J].Gene,2012,503(1):83-91.
[13] 何钟佩.农作物化学控制实验指导[M].北京:北京农业大学出版社,1993.
[14] 曲玲,李彦龙,安巍,等.病毒诱导的基因沉默在茄科植物基因功能研究中的应用进展[J].河南农业科学,2018,47(7):8-19.
[15] 王新博,任永哲,岳慧芳,等.基于VIGS技术的小麦干旱响应基因TaEF-1α的功能研究[J].华北农学报,2018,33(2):72-78.
[16] 李瑞雪,王钰婷,胡飞,等.桑树PDS基因VIGS转化体系的构建与鉴定[J].南方农业学报,2018,49(7):1432-1438.
[17] 王丽,穆春,张明才,等.GhCPS基因沉默对棉花幼苗生长和内源激素含量的影响[J].棉花学报,2014,26(3):189-196.
[18] ULLAH A,SUN H,HAKIM,et al.A novel cotton WRKY-gene,GhWRKY6-like,improves salt tolerance by activating the ABA signaling pathway and scavenging of reactive oxygen species[J].Physiologia Plantarum,2018,162(4):439-454.
[19] SHABAN M,AHMED M M,SUN H,et al.Genome-wide identification of lipoxygenase gene family in cotton and functional characterization in response to abiotic stresses[J].BMC Genomics,2018,19(1):599.
[20] LIU S D,HU Q N,LUO S,et al.Expression of wild-type PtrIAA14.1,a poplar Aux/IAA gene causes morphological changes in Arabidopsis[J].Frontiers in Plant Science,2015,6:388.
[21] WANG H,JONES B,LI Z G,et al.The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis[J].Plant Cell,2005,17(10):2676-2692.
[22] SU L Y,BASSA C,AUDRAN C,et al.The auxin Sl-IAA17 transcriptional repressor controls fruit size via the regulation of endoreduplication-related cell expansion[J].Plant and Cell Physiology,2014,55(11):1969-1976.
[23] NIGAM D,SAWANT S V.Identification and analyses of AUX-IAA target genes controlling multiple pathways in developing fiber cells of Gossypium hirsutum L[J].Bioinformation,2013,9(20):996-1002.
[24] LIU M,CHEN Y,CHEN Y,et al.The tomato ethylene response factor Sl-ERF.B3 integrates ethylene and auxin signaling via direct regulation of Sl-Aux/IAA27[J].New Phytologist,2018,219(2):631-640.
[25] SONG Y,YOU J,XIONG L.Characterization of OsIAA1gene,a member of rice Aux/IAA family involved in auxin and brassinosteroid hormone responses and plant morphogenesis[J].Plant Molecular Biology,2009,70(3):297-309.
[26] REED J W,WU M F,REEVES P H,et al.Three auxin response factors promote hypocotyl elongation[J].Plant Physiology,2018,178(2):864-875.
[27] WOLBANG C M,CHANDLER P M,SMITH J J,et al.Auxin from the developing inflorescence is required for the biosynthesis of active gibberellins in barley stems[J].Plant Physiology,2004,134(2):769-776.
[28] FRIGERIO M,ALABADí D,PéREZ-GóMEZ J,et al.Transcriptional regulation of gibberellin metabolism genes by auxin signaling in Arabidopsis[J].Plant Physiology,2006,142(2):553-563.
[29] OZGA J A,YU J,REINECKE D M.Pollination-,development-,and auxin-specific regulation of gibberellin 3beta-hydroxylase gene expression in pea fruit and seeds[J].Plant Physiology,2003,131(3):1137-1146.
[30] 石建斌,王宁,周红,等.陆地棉中赤霉素合成途径关键酶基因的时空表达变化[J].华北农学报,2018,33(4):9-16.
[2] GALLAVOTTI A.The role of auxin in shaping shoot architecture[J].Journal of Experimental Botany,2013,64(9):2593-2608.
[3] FENDRYCH M,LEUNG J,FRIML J.TIR1/AFB-Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls[J].Elife,2016,5:e19048.
[4] 刘晓柱,李银凤,赵燕,等.生长素对荠菜心皮发育的影响[J].河南农业科学,2018,47(8):88-94.
[5] LEYSER O.Auxin signaling[J].Plant Physiology,2018,176(1):465-479.
[6] LUO J,ZHOU J J,ZHANG J Z.Aux/IAA gene family in plants:Molecular structure,regulation,and function[J].International Journal of Molecular Sciences,2018,19(1):E259.
[7] YANG X,LEE S,SO J H,et al.IAA1 protein is encoded by AXR5 and is a substrate of SCFTIR1[J].Plant Journal,2004,40(5):772-782.
[8] KIM B C,SOH M C,KANG B J,et al.Two dominant photomorphogenic mutations of Arabidopsis thaliana identified as suppressor mutations of hy2[J].Plant Journal,1996,9(4):441-456.
[9] ROGG L E,LASSWELL J,BARTEL B.A gain-of-function mutation in IAA28 suppresses lateral root development[J].The Plant Cell,2001,13(3):465-480.
[10] DENG W,YAN F,LIU M,et al.Down-regulation of SlIAA15 in tomato altered stem xylem development and production of volatile compounds in leaf exudates[J].Plant Signaling & Behavior,2012,7(8):911-913.
[11] KLOOSTERMAN B,VISSER R,BACHEM C.Isolation and characterization of a novel potato Auxin/Indole-3-Acetic Acid family member (StIAA2) that is involved in petiole hyponasty and shoot morphogenesis[J].Plant Physiology & Biochemistry,2006,44(11/12):766-775.
[12] HAN X,XU X,FANG D D,et al.Cloning and expression analysis of novel Aux/IAA family genes in Gossypium hirsutum[J].Gene,2012,503(1):83-91.
[13] 何钟佩.农作物化学控制实验指导[M].北京:北京农业大学出版社,1993.
[14] 曲玲,李彦龙,安巍,等.病毒诱导的基因沉默在茄科植物基因功能研究中的应用进展[J].河南农业科学,2018,47(7):8-19.
[15] 王新博,任永哲,岳慧芳,等.基于VIGS技术的小麦干旱响应基因TaEF-1α的功能研究[J].华北农学报,2018,33(2):72-78.
[16] 李瑞雪,王钰婷,胡飞,等.桑树PDS基因VIGS转化体系的构建与鉴定[J].南方农业学报,2018,49(7):1432-1438.
[17] 王丽,穆春,张明才,等.GhCPS基因沉默对棉花幼苗生长和内源激素含量的影响[J].棉花学报,2014,26(3):189-196.
[18] ULLAH A,SUN H,HAKIM,et al.A novel cotton WRKY-gene,GhWRKY6-like,improves salt tolerance by activating the ABA signaling pathway and scavenging of reactive oxygen species[J].Physiologia Plantarum,2018,162(4):439-454.
[19] SHABAN M,AHMED M M,SUN H,et al.Genome-wide identification of lipoxygenase gene family in cotton and functional characterization in response to abiotic stresses[J].BMC Genomics,2018,19(1):599.
[20] LIU S D,HU Q N,LUO S,et al.Expression of wild-type PtrIAA14.1,a poplar Aux/IAA gene causes morphological changes in Arabidopsis[J].Frontiers in Plant Science,2015,6:388.
[21] WANG H,JONES B,LI Z G,et al.The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis[J].Plant Cell,2005,17(10):2676-2692.
[22] SU L Y,BASSA C,AUDRAN C,et al.The auxin Sl-IAA17 transcriptional repressor controls fruit size via the regulation of endoreduplication-related cell expansion[J].Plant and Cell Physiology,2014,55(11):1969-1976.
[23] NIGAM D,SAWANT S V.Identification and analyses of AUX-IAA target genes controlling multiple pathways in developing fiber cells of Gossypium hirsutum L[J].Bioinformation,2013,9(20):996-1002.
[24] LIU M,CHEN Y,CHEN Y,et al.The tomato ethylene response factor Sl-ERF.B3 integrates ethylene and auxin signaling via direct regulation of Sl-Aux/IAA27[J].New Phytologist,2018,219(2):631-640.
[25] SONG Y,YOU J,XIONG L.Characterization of OsIAA1gene,a member of rice Aux/IAA family involved in auxin and brassinosteroid hormone responses and plant morphogenesis[J].Plant Molecular Biology,2009,70(3):297-309.
[26] REED J W,WU M F,REEVES P H,et al.Three auxin response factors promote hypocotyl elongation[J].Plant Physiology,2018,178(2):864-875.
[27] WOLBANG C M,CHANDLER P M,SMITH J J,et al.Auxin from the developing inflorescence is required for the biosynthesis of active gibberellins in barley stems[J].Plant Physiology,2004,134(2):769-776.
[28] FRIGERIO M,ALABADí D,PéREZ-GóMEZ J,et al.Transcriptional regulation of gibberellin metabolism genes by auxin signaling in Arabidopsis[J].Plant Physiology,2006,142(2):553-563.
[29] OZGA J A,YU J,REINECKE D M.Pollination-,development-,and auxin-specific regulation of gibberellin 3beta-hydroxylase gene expression in pea fruit and seeds[J].Plant Physiology,2003,131(3):1137-1146.
[30] 石建斌,王宁,周红,等.陆地棉中赤霉素合成途径关键酶基因的时空表达变化[J].华北农学报,2018,33(4):9-16.
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