甘薯近缘野生种Ipomoea triloba L.干旱响应基因ItHAP2的克隆及表达分析
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摘要
避旱性是植物重要的干旱响应机制,其重要表现就是早花。甘薯近缘野生种I.triloba(三裂叶薯)中包含大量的优良抗旱基因,本研究室前期对甘薯栽培种高系14号、I.triloba以及二者的体细胞杂种KT1三者进行了PEG干旱胁迫转录组测序,并通过Weighted Gene Co-expression Network Analysis(WGCNA)方法从测序数据中分析得到了两个和抗旱性关联度较高的干旱响应基因模块。本研究利用实时荧光定量PCR的方法对模块中的基因进行了干旱胁迫表达验证,筛选并克隆到一个与雌雄配子体融合相关的基因ItHAP2,该基因表达受到PEG胁迫的强烈诱导且在I.triloba的花中具有较高表达量。ItHAP2基因的开放阅读框为2 058 bp,基因组序列为5 688 bp,具有17个外显子和16个内含子。ItHAP2蛋白具有HAP2-GCS1特征结构域以及一个跨膜结构域,亚细胞定位于细胞膜,综合分析表明ItHAP2基因同I.triloba的避旱性早花有关。本研究进一步阐明了I.triloba具有良好抗旱性的分子基础以及I.triloba相较于甘薯栽培种在自然条件下更易开花结实的特性。
Drought avoidance is an important drought-response mechanism of plants and a key behavior is early flowering. Sweetpotato wild relative I. triloba contains large amount of drought-resistance genes. Previously, we carried out the RNA sequencing of PEG-drought-stressed sweetpotato cultivar Kokei No.14, I. triloba and their somatic hybrid KT1 and two gene modules that were highly correlated with drought resistance were obtained from the sequencing data by Weighted Gene Co-expression Network Analysis(WGCNA). In this study, quantitative real-time PCR was used to verify the drought response of the genes in these modules and a gene associated with the fusion of male and female gametophyte ItHAP2 was selected and cloned. The expression of ItHAP2 was strongly induced by PEG stress and abundantly detected in the flower of I. triloba. ItHAP2 had an open reading frame of 2 058 bp and genomic sequence of 5 688 bp which contained 17 exons and 16 introns. ItHAP2 protein had a characteristic domain of HAP2-GCS1 and a transmembrane domain and its subcellular localization was on the plasma membrane. Comprehensive analysis revealed that ItHAP2 was related with the early flowering of I.triloba during drought escape. This study further illustrated the molecular foundation behind the superior drought escape of I. triloba and why the flowering and fructification properties of I. triloba were better than those of the regular sweetpotato cultivars in natural environment.
Drought avoidance is an important drought-response mechanism of plants and a key behavior is early flowering. Sweetpotato wild relative I. triloba contains large amount of drought-resistance genes. Previously, we carried out the RNA sequencing of PEG-drought-stressed sweetpotato cultivar Kokei No.14, I. triloba and their somatic hybrid KT1 and two gene modules that were highly correlated with drought resistance were obtained from the sequencing data by Weighted Gene Co-expression Network Analysis(WGCNA). In this study, quantitative real-time PCR was used to verify the drought response of the genes in these modules and a gene associated with the fusion of male and female gametophyte ItHAP2 was selected and cloned. The expression of ItHAP2 was strongly induced by PEG stress and abundantly detected in the flower of I. triloba. ItHAP2 had an open reading frame of 2 058 bp and genomic sequence of 5 688 bp which contained 17 exons and 16 introns. ItHAP2 protein had a characteristic domain of HAP2-GCS1 and a transmembrane domain and its subcellular localization was on the plasma membrane. Comprehensive analysis revealed that ItHAP2 was related with the early flowering of I.triloba during drought escape. This study further illustrated the molecular foundation behind the superior drought escape of I. triloba and why the flowering and fructification properties of I. triloba were better than those of the regular sweetpotato cultivars in natural environment.
引文
Cao Q.H.,Zhang A.,Ma D.F.,Li H.M.,Li Q.,and Li P.,2009Novel interspecific hybridization between sweetpotato(Ipomoea batatas(L.)Lam.)and its two diploid wild relatives,Euphytica,169(3):345-352
Daryanto S.,Wang L.X.,and Jacinthe P.A.,2015,Global synthesis of drought effects on food legume production,PLo S One10(6):e0127401
Daryanto S.,Wang L.X.,and Jacinthe P.A.,2016,Global synthesis of drought effects on maize and wheat production,PLo SOne,11(5):e0156362
Franke D.M.,Ellis A.G.,Dharjwa M.,Freshwater M.,Fujikawa M.,Padron A.,and Weis A.E.,2006,A steep cline in flowering time for Brassica rapa in southern California:population-level variation in the field and the greenhouse,Int.JPlant Sci.,167(1):83-92
Franks S.J.,2011,Plasticity and evolution in drought avoidance and escape in the annual plant Brassica rapa,New Phytol.190(1):249-257
Franks S.J.,Sim S.,and Weis A.E.,2007,Rapid evolution of flowering time by an annual plant in response to a climate fluctuation,Proc.Natl.Acad.Sci.USA,104(4):1278-1282
Hirai M.,Arai M.,Mori T.,Miyagishima S.Y.,Kawai S.,Kita K.Kuroiwa T.,Terenius O.,and Matsuoka H.,2008,Male fertility of malaria parasites is determined by GCS1,a planttype reproduction factor,Curr.Biol.,18(8):607-613
Huang J.C.,and Sun M.,2000,Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas(Convolvulaceae)as revealed by inter-simple sequence repeat(ISSR)and restriction analysis of chloroplas DNA,Theor.Appl.Genet.,100(7):1050-1060
Jia L.C.,Zhai H.,He S.Z.,Yang Y.F.,and Liu Q.C.,2017,Analysis of drought tolerance and genetic and epigenetic variations in a somatic hybrid between Ipomoea batatas(L.)Lam and I.triloba L.,J.Integr.Agr.,16(1):36-46
Johnson M.A.,Besser V.K.,Zhou Q.,Smith E.,Aux G.,Patton D.,Levin J.Z.,and Preuss D.,2004,Arabidopsis hapless mutations define essential gametophytic functions,Genetics168(2):971-982
Kazan K.,and Lyons R.,2016,The link between flowering time and stress tolerance,J.Exp.Bot.,67(1):47-60
Kooyers N.J.,2015,The evolution of drought escape and avoidance in natural herbaceous populations,Plant Sci.,234:155-162
Krannich C.T.,Maletzki L.,Kurowsky C.,and Horn R.,2015,Network candidate genes in breeding for drought tolerant crops,Int.J.Mol.Sci.,16(7):16378-16400
Liu L.F.,Ma Z.M.,Yao H.L.,and Zhang S.S.,2011,The genetic analysis on special materials of natural flowering and self fruitfulness in sweet potato,Huabei Nongxuebao(Acta A-griculturae Boreali-Sinica),26(S):24-27(刘兰服,马志民,姚海兰,张松树,2011,甘薯自然开花自交结实特异材料的遗传分析,华北农学报,26(S):24-27)
Liu Y.J.,Tewari R.,Ning J.,Blagborough A.M.,Garbom S.,Pei J.M.,Grishin N.V.,Steele R.E.,Sinden R.E.,Snell W.J.,and Billker O.,2008,The conserved plant sterility gene HAP2functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes,Genes Dev.,22(8):1051-1068
Mckay J.K.,Richards J.H.,and Mitchell-Olds T.,2003,Genetics of drought adaptation in Arabidopsis thaliana:I.Pleiotropy contributes to genetic correlations among ecological traits,Mol.Ecol.,12(5):1137-1151
Mori T.,Kuroiwa H.,Higashiyama T.,and Kuroiwa T.,2006,GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization,Nat.Cell Biol.,8(1):64
Olesen J.E.,Trnka M.,Kersebaum K.C.,Skjelv覽g A.O.,Seguin B.,Peltonen-Sainio P.,Rossi F.,Kozyra J.,and Micale F.,2011,Impacts and adaptation of European crop production systems to climate change,Eur.J.Agron.,34(2):96-112
Paccard A.,Fruleux A.,and Willi Y.,2014,Latitudinal trait variation and responses to drought in Arabidopsis lyrata,Oecologia,175(2):577-587
Sherrard M.E.,and Maherali H.,2006,The adaptive significance of drought escape in Avena barbata,an annual grass,Evolution,60(12):2478-2489
Song K.,Kim H.C.,Shin S.,Kim K.H.,Moon J.C.,Kim J.Y.,and Lee B.M.,2017,Transcriptome analysis of flowering time genes under drought stress in maize leaves,Front.Plant Sci.,8:267
Strasser R.,Bondili J.S.,Schoberer J.,Svoboda B.,Liebminger E.,Glossl J.,Altmann F.,Steinkellner H.,and Mach L.,2007,Enzymatic properties and subcellular localization of Arabidopsisβ-N-acetylhexosaminidases,Plant Physiol.,145(1):5-16
Su Z.,Ma X.,Guo H.H.,Sukiran N.L.,Guo B.,Assmann S.M.,and Ma H.,2013,Flower development under drought stress:morphological and transcriptomic analyses reveal acute responses and long-term acclimation in Arabidopsis,Plant Cell,25(10):3785-3807
Besser V.K.,Frank A.C.,Johnson M.A.,and Preuss D.,2006,Arabidopsis HAP2(GCS1)is a sperm-specific gene required for pollen tube guidance and fertilization,Development,133(23):4761-4769
Wang F.Q.,Hou F.Y.,Qin Z.,Li A.X.,Dong S.X.,Wang Q.M.and Zhang L.M.,2015,Progress on the molecular biology o the abiotic stress tolerance in sweet potato,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology)34(1):221-226(王福琴,侯夫云,秦桢,李爱贤,董顺旭,王庆美,张立明,2015,甘薯耐非生物胁迫的分子生物学研究进展,基因组学与应用生物学,34(1):221-226)
Zhang J.Y.,Cruz de Carvalho M.H.,Torres-Jerez I.,Kang Y.Allen S.N.,Huhman D.V.,Tang Y.H.,Murray J.,Sumner LW.,and Udvardi M.K.,2014,Global reprogramming of tran scription and metabolism in Medicago truncatula during pro gressive drought and after rewatering,Plant Cell Environ.,37(11):2553-2576
Zhao X.,Zhang Q.T.,Fu Y.F.,Yang C.X.,Zeng L.J.,Zhu P.,Xu an S.Q.,and Li S.Y.,2006,Studies on flower characteristi in close plant of sweet potato,Xi'nan Shifan Daxue Xuebao(Journal of Southwest China Normal University(Natura Science)),31(3):143-147(赵昕,张启堂,付玉凡,杨春贤曾令江,朱鹏,宣善勤,李斯勇,2006,甘薯近缘野生植物的开花特性研究,西南师范大学学报(自然科学版),31(3)143-147)
Zhuang Y.L.,Ren G.J.,Yue G.D.,Li Z.X.,Qu X.,Hou G.H.,Zhu Y.,and Zhang J.R.,2007,Effects of water-deficit stress on the transcriptomes of developing immature ear and tassel in maize,Plant Cell Rep.,26(12):2137-2147
Daryanto S.,Wang L.X.,and Jacinthe P.A.,2015,Global synthesis of drought effects on food legume production,PLo S One10(6):e0127401
Daryanto S.,Wang L.X.,and Jacinthe P.A.,2016,Global synthesis of drought effects on maize and wheat production,PLo SOne,11(5):e0156362
Franke D.M.,Ellis A.G.,Dharjwa M.,Freshwater M.,Fujikawa M.,Padron A.,and Weis A.E.,2006,A steep cline in flowering time for Brassica rapa in southern California:population-level variation in the field and the greenhouse,Int.JPlant Sci.,167(1):83-92
Franks S.J.,2011,Plasticity and evolution in drought avoidance and escape in the annual plant Brassica rapa,New Phytol.190(1):249-257
Franks S.J.,Sim S.,and Weis A.E.,2007,Rapid evolution of flowering time by an annual plant in response to a climate fluctuation,Proc.Natl.Acad.Sci.USA,104(4):1278-1282
Hirai M.,Arai M.,Mori T.,Miyagishima S.Y.,Kawai S.,Kita K.Kuroiwa T.,Terenius O.,and Matsuoka H.,2008,Male fertility of malaria parasites is determined by GCS1,a planttype reproduction factor,Curr.Biol.,18(8):607-613
Huang J.C.,and Sun M.,2000,Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas(Convolvulaceae)as revealed by inter-simple sequence repeat(ISSR)and restriction analysis of chloroplas DNA,Theor.Appl.Genet.,100(7):1050-1060
Jia L.C.,Zhai H.,He S.Z.,Yang Y.F.,and Liu Q.C.,2017,Analysis of drought tolerance and genetic and epigenetic variations in a somatic hybrid between Ipomoea batatas(L.)Lam and I.triloba L.,J.Integr.Agr.,16(1):36-46
Johnson M.A.,Besser V.K.,Zhou Q.,Smith E.,Aux G.,Patton D.,Levin J.Z.,and Preuss D.,2004,Arabidopsis hapless mutations define essential gametophytic functions,Genetics168(2):971-982
Kazan K.,and Lyons R.,2016,The link between flowering time and stress tolerance,J.Exp.Bot.,67(1):47-60
Kooyers N.J.,2015,The evolution of drought escape and avoidance in natural herbaceous populations,Plant Sci.,234:155-162
Krannich C.T.,Maletzki L.,Kurowsky C.,and Horn R.,2015,Network candidate genes in breeding for drought tolerant crops,Int.J.Mol.Sci.,16(7):16378-16400
Liu L.F.,Ma Z.M.,Yao H.L.,and Zhang S.S.,2011,The genetic analysis on special materials of natural flowering and self fruitfulness in sweet potato,Huabei Nongxuebao(Acta A-griculturae Boreali-Sinica),26(S):24-27(刘兰服,马志民,姚海兰,张松树,2011,甘薯自然开花自交结实特异材料的遗传分析,华北农学报,26(S):24-27)
Liu Y.J.,Tewari R.,Ning J.,Blagborough A.M.,Garbom S.,Pei J.M.,Grishin N.V.,Steele R.E.,Sinden R.E.,Snell W.J.,and Billker O.,2008,The conserved plant sterility gene HAP2functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes,Genes Dev.,22(8):1051-1068
Mckay J.K.,Richards J.H.,and Mitchell-Olds T.,2003,Genetics of drought adaptation in Arabidopsis thaliana:I.Pleiotropy contributes to genetic correlations among ecological traits,Mol.Ecol.,12(5):1137-1151
Mori T.,Kuroiwa H.,Higashiyama T.,and Kuroiwa T.,2006,GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization,Nat.Cell Biol.,8(1):64
Olesen J.E.,Trnka M.,Kersebaum K.C.,Skjelv覽g A.O.,Seguin B.,Peltonen-Sainio P.,Rossi F.,Kozyra J.,and Micale F.,2011,Impacts and adaptation of European crop production systems to climate change,Eur.J.Agron.,34(2):96-112
Paccard A.,Fruleux A.,and Willi Y.,2014,Latitudinal trait variation and responses to drought in Arabidopsis lyrata,Oecologia,175(2):577-587
Sherrard M.E.,and Maherali H.,2006,The adaptive significance of drought escape in Avena barbata,an annual grass,Evolution,60(12):2478-2489
Song K.,Kim H.C.,Shin S.,Kim K.H.,Moon J.C.,Kim J.Y.,and Lee B.M.,2017,Transcriptome analysis of flowering time genes under drought stress in maize leaves,Front.Plant Sci.,8:267
Strasser R.,Bondili J.S.,Schoberer J.,Svoboda B.,Liebminger E.,Glossl J.,Altmann F.,Steinkellner H.,and Mach L.,2007,Enzymatic properties and subcellular localization of Arabidopsisβ-N-acetylhexosaminidases,Plant Physiol.,145(1):5-16
Su Z.,Ma X.,Guo H.H.,Sukiran N.L.,Guo B.,Assmann S.M.,and Ma H.,2013,Flower development under drought stress:morphological and transcriptomic analyses reveal acute responses and long-term acclimation in Arabidopsis,Plant Cell,25(10):3785-3807
Besser V.K.,Frank A.C.,Johnson M.A.,and Preuss D.,2006,Arabidopsis HAP2(GCS1)is a sperm-specific gene required for pollen tube guidance and fertilization,Development,133(23):4761-4769
Wang F.Q.,Hou F.Y.,Qin Z.,Li A.X.,Dong S.X.,Wang Q.M.and Zhang L.M.,2015,Progress on the molecular biology o the abiotic stress tolerance in sweet potato,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology)34(1):221-226(王福琴,侯夫云,秦桢,李爱贤,董顺旭,王庆美,张立明,2015,甘薯耐非生物胁迫的分子生物学研究进展,基因组学与应用生物学,34(1):221-226)
Zhang J.Y.,Cruz de Carvalho M.H.,Torres-Jerez I.,Kang Y.Allen S.N.,Huhman D.V.,Tang Y.H.,Murray J.,Sumner LW.,and Udvardi M.K.,2014,Global reprogramming of tran scription and metabolism in Medicago truncatula during pro gressive drought and after rewatering,Plant Cell Environ.,37(11):2553-2576
Zhao X.,Zhang Q.T.,Fu Y.F.,Yang C.X.,Zeng L.J.,Zhu P.,Xu an S.Q.,and Li S.Y.,2006,Studies on flower characteristi in close plant of sweet potato,Xi'nan Shifan Daxue Xuebao(Journal of Southwest China Normal University(Natura Science)),31(3):143-147(赵昕,张启堂,付玉凡,杨春贤曾令江,朱鹏,宣善勤,李斯勇,2006,甘薯近缘野生植物的开花特性研究,西南师范大学学报(自然科学版),31(3)143-147)
Zhuang Y.L.,Ren G.J.,Yue G.D.,Li Z.X.,Qu X.,Hou G.H.,Zhu Y.,and Zhang J.R.,2007,Effects of water-deficit stress on the transcriptomes of developing immature ear and tassel in maize,Plant Cell Rep.,26(12):2137-2147