陆地棉NAC转录因子基因GhSNAC1的克隆及其抗旱耐盐分析
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摘要
NAC (NAM/ATAF/CUC)家族是植物中特有的一类转录因子,其功能涉及植物生长发育调控、激素信号转导、逆境胁迫响应等重要生理过程。本研究利用反转录PCR (reverse transcription PCR, RT-PCR)技术从陆地棉(Gossypium hirsutum)中克隆了1个逆境胁迫响应(stress-responsive) NAC类转录因子基因,命名为GhSNAC1 (GenBank No. KU759894)。该基因长度为1 104 bp,预测编码蛋白含有299个氨基酸,分子量为33.9 kD,等电点为6.18。蛋白序列分析表明,GhSNAC1具有典型的NAC转录因子特征,N端含有保守的NAC结构域。进化分析表明,GhSNAC1与拟南芥(Arabidopsis thaliana) ATAF1 (Arabidopsis transcription activation factor 1)高度相似。qRT-PCR结果显示,GhSNAC1受到干旱、高盐、低温和黄萎病菌(Verticillium dahliae)胁迫诱导,提示该基因可能参与生物和非生物胁迫响应。在烟草(Nicotiana tabacum)中过表达GhSNAC1,分析干旱或盐胁迫条件下转基因和野生型烟草株系种子的萌发率和幼苗生长差异,实验结果显示,在100 mmol/L NaCl或200 mmol/L甘露醇胁迫下,野生型烟草的种子萌发受到明显抑制,萌发率低于50%,而转基因株系显著优于野生型,其种子萌发率均在80%以上;野生型烟草幼苗弱小,而转基因株系长势强,其单株鲜重和根长分别是野生型的2倍和1.5倍以上。上述结果表明,过表达GhSNAC1可显著提高转基因烟草的抗旱耐盐能力。本研究为深入探究GhSNAC1抗旱耐盐的分子机制提供了基础资料。
NAC(NAM/ATAF/CUC) family is a class of transcription factors unique to plants, and plays crucial roles on developmental regulation, hormone signal transduction, biotic and abiotic stress responses. In the present study, a stress-responsive NAC gene, named as GhSNAC1(GenBank No. KU759894), was isolated from upland cotton(Gossypium hirsutum) via reverse transcription PCR(RT-PCR). The sequence analysis indicated that GhSNAC1 was 1 104 bp in length and encoded a protein of 299 amino acids with a relative molecular mass of 33.9 kD and an isoelectric point of 6.18. GhSNAC1 contained a conserved NAC domain in the N-terminus, indicating that it was a typical NAC transcription factor. The phylogenetic analysis indicated that GhSNAC1 had extremely high similarity with ATAF1(Arabidopsis transcription activation factor 1) from Arabidopsis thaliana. qRT-PCR anaylsis showed that GhSNAC1 was induced by treatments of drought, salt,cold and Verticillium dahliae which suggested that GhSNAC1 might be involved in responses to biotic and abiotic stresses. GhSNAC1 was then overexpressed in transgenic tobacco(Nicotiana tabacum) plants.Transgenic lines and wild type(WT) were used to evaluate the germination and seedling growth under either salt(100 mmol/L NaCl) or drought(200 mmol/L mannitol) stress. The results indicated that drought and salt stresses significantly inhibited the germination of WT seeds, and the germination rate of WT was below 50%,while the germination of transgenic lines was above 80%. The WT seedlings were small and weak. However,the transgenic lines were vigorous. The fresh weight per plant and root length of transgenic lines were more than twice and 1.5 times of those of wild type, respectively. Collectively, the above results suggested that overexpression of GhSNAC1 in tobacco could improve tolerance to drought and salt stresses. The present study laid a foundation for further exploring the molecular mechanism of drought and salt tolerance of GhSNAC1.
NAC(NAM/ATAF/CUC) family is a class of transcription factors unique to plants, and plays crucial roles on developmental regulation, hormone signal transduction, biotic and abiotic stress responses. In the present study, a stress-responsive NAC gene, named as GhSNAC1(GenBank No. KU759894), was isolated from upland cotton(Gossypium hirsutum) via reverse transcription PCR(RT-PCR). The sequence analysis indicated that GhSNAC1 was 1 104 bp in length and encoded a protein of 299 amino acids with a relative molecular mass of 33.9 kD and an isoelectric point of 6.18. GhSNAC1 contained a conserved NAC domain in the N-terminus, indicating that it was a typical NAC transcription factor. The phylogenetic analysis indicated that GhSNAC1 had extremely high similarity with ATAF1(Arabidopsis transcription activation factor 1) from Arabidopsis thaliana. qRT-PCR anaylsis showed that GhSNAC1 was induced by treatments of drought, salt,cold and Verticillium dahliae which suggested that GhSNAC1 might be involved in responses to biotic and abiotic stresses. GhSNAC1 was then overexpressed in transgenic tobacco(Nicotiana tabacum) plants.Transgenic lines and wild type(WT) were used to evaluate the germination and seedling growth under either salt(100 mmol/L NaCl) or drought(200 mmol/L mannitol) stress. The results indicated that drought and salt stresses significantly inhibited the germination of WT seeds, and the germination rate of WT was below 50%,while the germination of transgenic lines was above 80%. The WT seedlings were small and weak. However,the transgenic lines were vigorous. The fresh weight per plant and root length of transgenic lines were more than twice and 1.5 times of those of wild type, respectively. Collectively, the above results suggested that overexpression of GhSNAC1 in tobacco could improve tolerance to drought and salt stresses. The present study laid a foundation for further exploring the molecular mechanism of drought and salt tolerance of GhSNAC1.
引文
郭亚宁,窦玲玲,马启峰,等.2017.陆地棉GhNAC63基因的克隆及功能分析[J].农业生物技术学报,25(2):173-185.(Guo Y N,Dou L L,Ma Q F,et al.2017.Cloning and functional analysis of GhNAC63 gene in upland cotton(Gossypium hirsutum)[J].Journal of Agricultural Biotechnology,25(2):173-185.)
杨勇,田新权,刘会利,等.2017.陆地棉NAC转录因子基因GhNAC6的克隆、表达和耐盐性分析[J].棉花学报,29(2):138-146.(Guo Y,Tian X Q,Liu H L,et al.2017.Cloning,expression and salt-tolerance analysis of the NAC transcription factor gene GhNAC6 in upland cotton[J].Cotton Science,29(2):138-146.)
赵灿,刘任重,柳展基,等.2015.亚洲棉全基因组中NAC类转录因子基因的鉴定与分析[J].石河子大学学报(自然科学版),33(4):397-404.(Zhao C,Liu R Z,Liu Z J,et al.Genome-wide identification and analysis of NACtranscription factor gene family in Gossypium arboretum L[J].Journal of Shihezi University(Natural Science),2015,33(4):397-404.)
Aida M,Ishida T,Fukaki H,et al.1997.Genes involved in organ separation in Arabidopsis:An analysis of the cupshaped cotyledon mutant[J].Plant Cell,9(6):841-857.
Delessert C,Kazan K,Wilson I W,et al.2005.The transcription factor ATAF2 represses the expression of pathogenesis-related genes in Arabidopsis[J].Plant Journal,43(5):745-757.
Fan K,Wang M,Miao Y,et al.2014.Molecular evolution and expansion analysis of the NAC transcription factor in Zea mays[J].PLoS One,9(11):e111837.
He X,Zhu L,Xu L,et al.2016.GhATAF1,a NAC transcription factor,confers abiotic and biotic stress responses by regulating phytohormonal signaling networks[J].Plant Cell Reports,35(10):2167-2179.
Huang G Q,Li W,Zhou W,et al.2013.Seven cotton genes encoding putative NAC domain proteins are preferentially expressed in roots and in responses to abiotic stress during root development[J].Plant Growth Regulation,71(2):101-112.
Kong X,Luo Z,Dong H,et al.2013.Gene expression profiles deciphering leaf senescence variation between earlyand late-senescence cotton lines[J].PLoS One,8(7):e69847.
Kumar S,Stecher G,Tamura K.2016.MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology and Evolution,33(7):1870-1874.
Larkin M A,Blackshields G,Brown N P,et al.2007.Clustal W and Clustal X version 2.0[J].Bioinformatics,23(21):2947-2948.
Le D T,Nisjiyama R,Watanabe Y,et al.2011.Genome-wide survey and expression analysis of the plant-specific NAC transcription factor family in soybean during development and dehydration stress[J].DNA Research,18(4):263-276.
Li F,Fan G,Lu C,et al.2015.Genome sequence of cultivated upland cotton(Gossypium hirsutum TM-1)provides insights into genome evolution[J].Nature Biotechnology,33(5):524-530.
Li F,Fan G,Wang K,et al.2014.Genome sequence of the cultivated cotton Gossypium arboretum[J].Nature Genetics,46(6):567-572.
Li W,Zhou Z,Meng Y,et al.2009.Modeling boll maturation period,seed growth,protein,and oil content of cotton(Gossypium hirsutum L.)in China[J].Field Crops Research,112(2-3):131-140.
Liu Z J,Li F,Wang L G,et al.2018.Molecular characterization of a stress-induced NAC gene,GhSNAC3,from Gos?sypium hirsutum[J].Journal of Genetics,97(2):539-548.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using Real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,25(4):402-408.
Meng C,Cai C,Zhang T,et al.2009.Characterization of six novel NAC genes and their responses to abiotic stresses in Gossypium hirsutum L[J].Plant Science,176(3):352-359.
Nuruzzaman M,Manimekalai R,Sharoni A M,et al.2010.Genome-wide analysis of NAC transcription factor family in rice[J].Gene,465(1-2):30-44.
Nuruzzaman M,Sharoni A M,Kikuchi S.2013.Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants[J].Frontiers in Microbiology,4:248.
Olsen A N,Ernst H A,Leggio L L,et al.2005.NAC transcription factors:Structurally distinct,functionally diverse[J].Trends in Plant Science,10(2):79-87.
Paterson A H,Wendel J F,Gundlach H,et al.2012.Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibers[J].Nature,492(7429):423-427.
Shah S T,Pang C,Fan S,et al.2013.Isolation and expression profiling of GhNAC transcription factor genes in cotton(Gossypiumhirsutum L.)during leaf senescence and in response to stresses[J].Gene,531(2):220-234.
Shah S T,Pang C,Hussain A,et al.2014.Molecular cloning and functional analysis of NAC family genes associated with leaf senescence and stresses in Gossypium hirsutum L[J].Plant Cell,Tissue and Organ Culture(PCTOC),117(2):167-186.
Shang H,Li W,Zou C,et al.Analyses of the NAC transcription factor gene family in Gossypium raimondii Ulbr.:Chromosomal location,structure,phylogeny,and expression patterns[J].Journal of Integrative Plant Biology,2013,55(7):663-676.
Shang H,Wang Z,Zou C,et al.2016.Comprehensive analysis of NAC transcription factors in diploid Gossypium:Sequence conservation and expression analysis uncover their roles during fiber development[J].Science China Life Sciences,59(2):142-153.
Sun H,Hu M,Li J,et al.2018.Comprehensive analysis of NAC transcription factors uncovers their roles during fiber development and stress response in cotton[J].BMCPlant Biology,18(1):150.
Souer E,van Houwelingen A,Kloos D,et al.1996.The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries[J].Cell,85(2):159-170.
Takasaki H,Maruyama K,Takahashi F,et al.2015.SNAC-As,stress-responsive NAC transcription factors,mediate ABA-inducible leaf senescence[J].Plant Journal,84(6):1114-1123.
Tian X,Ruan J X,Huang J Q,et al.Characterization of gossypol biosynthetic pathway[J].Proceedings of the National Academy of Sciences of the USA,115(23):E5410-E5418.
Tran L S,Nakashima K,Sakuma Y,et al.2004.Isolation and functional analysis of Arabidopsis stress-inducible NACtranscription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress1 promoter[J].Plant Cell,16(9):2481-2498.
Wang K,Wang Z,Li F,et al.2012.The draft genome of a diploid cotton Gossypium raimondii[J].Nature Genetics,44(10):1098-1103.
Wang W,Yuan Y,Yang C,et al.2016.Characterization,expression,and functional analysis of a novel NAC gene associated with resistance to verticillium wilt and abiotic stress in cotton[J].G3:Genes,Genomes,Genetics,6(12):3951-3961.
Wang X,Basnayake B M,Zhang H,et al.2009.The Arabidop?sis ATAF1,a NAC transcription factor,is a negative regulator of defense responses against necrotrophic fungal and bacterial pathogens[J].Molecular Plant-Microbe Interactions,22(10):1227-1238.
Wu Y,Deng Z,Lai J,et al.2009.Dual function of Arabidopsis ATAF1 in abiotic and biotic stress responses[J].Cell Research,19(11):1279-1290.
Zhang J,Huang G Q,Zou D,et al.2018.The cotton(Gossypi?umhirsutum)NAC transcription factor(FSN1)as a positive regulator participates in controlling secondary cell wall biosynthesis and modification of fibers[J].New Phytologist,217(2):625-640.
Zhang T,Hu Y,Jiang W,et al.2015.Sequencing of allotetraploid cotton(Gossypium hirsutum L.acc.TM-1)provides a resource for fiber improvement[J].Nature Biotechnology,33(5):531-537.
Zhao F,Ma J,Li L,et al.2016.GhNAC12,a neutral candidate gene,leads to early aging in cotton(Gossypium hir?sutum L)[J].Gene,576(1 Pt 2):268-274.
杨勇,田新权,刘会利,等.2017.陆地棉NAC转录因子基因GhNAC6的克隆、表达和耐盐性分析[J].棉花学报,29(2):138-146.(Guo Y,Tian X Q,Liu H L,et al.2017.Cloning,expression and salt-tolerance analysis of the NAC transcription factor gene GhNAC6 in upland cotton[J].Cotton Science,29(2):138-146.)
赵灿,刘任重,柳展基,等.2015.亚洲棉全基因组中NAC类转录因子基因的鉴定与分析[J].石河子大学学报(自然科学版),33(4):397-404.(Zhao C,Liu R Z,Liu Z J,et al.Genome-wide identification and analysis of NACtranscription factor gene family in Gossypium arboretum L[J].Journal of Shihezi University(Natural Science),2015,33(4):397-404.)
Aida M,Ishida T,Fukaki H,et al.1997.Genes involved in organ separation in Arabidopsis:An analysis of the cupshaped cotyledon mutant[J].Plant Cell,9(6):841-857.
Delessert C,Kazan K,Wilson I W,et al.2005.The transcription factor ATAF2 represses the expression of pathogenesis-related genes in Arabidopsis[J].Plant Journal,43(5):745-757.
Fan K,Wang M,Miao Y,et al.2014.Molecular evolution and expansion analysis of the NAC transcription factor in Zea mays[J].PLoS One,9(11):e111837.
He X,Zhu L,Xu L,et al.2016.GhATAF1,a NAC transcription factor,confers abiotic and biotic stress responses by regulating phytohormonal signaling networks[J].Plant Cell Reports,35(10):2167-2179.
Huang G Q,Li W,Zhou W,et al.2013.Seven cotton genes encoding putative NAC domain proteins are preferentially expressed in roots and in responses to abiotic stress during root development[J].Plant Growth Regulation,71(2):101-112.
Kong X,Luo Z,Dong H,et al.2013.Gene expression profiles deciphering leaf senescence variation between earlyand late-senescence cotton lines[J].PLoS One,8(7):e69847.
Kumar S,Stecher G,Tamura K.2016.MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology and Evolution,33(7):1870-1874.
Larkin M A,Blackshields G,Brown N P,et al.2007.Clustal W and Clustal X version 2.0[J].Bioinformatics,23(21):2947-2948.
Le D T,Nisjiyama R,Watanabe Y,et al.2011.Genome-wide survey and expression analysis of the plant-specific NAC transcription factor family in soybean during development and dehydration stress[J].DNA Research,18(4):263-276.
Li F,Fan G,Lu C,et al.2015.Genome sequence of cultivated upland cotton(Gossypium hirsutum TM-1)provides insights into genome evolution[J].Nature Biotechnology,33(5):524-530.
Li F,Fan G,Wang K,et al.2014.Genome sequence of the cultivated cotton Gossypium arboretum[J].Nature Genetics,46(6):567-572.
Li W,Zhou Z,Meng Y,et al.2009.Modeling boll maturation period,seed growth,protein,and oil content of cotton(Gossypium hirsutum L.)in China[J].Field Crops Research,112(2-3):131-140.
Liu Z J,Li F,Wang L G,et al.2018.Molecular characterization of a stress-induced NAC gene,GhSNAC3,from Gos?sypium hirsutum[J].Journal of Genetics,97(2):539-548.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using Real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,25(4):402-408.
Meng C,Cai C,Zhang T,et al.2009.Characterization of six novel NAC genes and their responses to abiotic stresses in Gossypium hirsutum L[J].Plant Science,176(3):352-359.
Nuruzzaman M,Manimekalai R,Sharoni A M,et al.2010.Genome-wide analysis of NAC transcription factor family in rice[J].Gene,465(1-2):30-44.
Nuruzzaman M,Sharoni A M,Kikuchi S.2013.Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants[J].Frontiers in Microbiology,4:248.
Olsen A N,Ernst H A,Leggio L L,et al.2005.NAC transcription factors:Structurally distinct,functionally diverse[J].Trends in Plant Science,10(2):79-87.
Paterson A H,Wendel J F,Gundlach H,et al.2012.Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibers[J].Nature,492(7429):423-427.
Shah S T,Pang C,Fan S,et al.2013.Isolation and expression profiling of GhNAC transcription factor genes in cotton(Gossypiumhirsutum L.)during leaf senescence and in response to stresses[J].Gene,531(2):220-234.
Shah S T,Pang C,Hussain A,et al.2014.Molecular cloning and functional analysis of NAC family genes associated with leaf senescence and stresses in Gossypium hirsutum L[J].Plant Cell,Tissue and Organ Culture(PCTOC),117(2):167-186.
Shang H,Li W,Zou C,et al.Analyses of the NAC transcription factor gene family in Gossypium raimondii Ulbr.:Chromosomal location,structure,phylogeny,and expression patterns[J].Journal of Integrative Plant Biology,2013,55(7):663-676.
Shang H,Wang Z,Zou C,et al.2016.Comprehensive analysis of NAC transcription factors in diploid Gossypium:Sequence conservation and expression analysis uncover their roles during fiber development[J].Science China Life Sciences,59(2):142-153.
Sun H,Hu M,Li J,et al.2018.Comprehensive analysis of NAC transcription factors uncovers their roles during fiber development and stress response in cotton[J].BMCPlant Biology,18(1):150.
Souer E,van Houwelingen A,Kloos D,et al.1996.The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries[J].Cell,85(2):159-170.
Takasaki H,Maruyama K,Takahashi F,et al.2015.SNAC-As,stress-responsive NAC transcription factors,mediate ABA-inducible leaf senescence[J].Plant Journal,84(6):1114-1123.
Tian X,Ruan J X,Huang J Q,et al.Characterization of gossypol biosynthetic pathway[J].Proceedings of the National Academy of Sciences of the USA,115(23):E5410-E5418.
Tran L S,Nakashima K,Sakuma Y,et al.2004.Isolation and functional analysis of Arabidopsis stress-inducible NACtranscription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress1 promoter[J].Plant Cell,16(9):2481-2498.
Wang K,Wang Z,Li F,et al.2012.The draft genome of a diploid cotton Gossypium raimondii[J].Nature Genetics,44(10):1098-1103.
Wang W,Yuan Y,Yang C,et al.2016.Characterization,expression,and functional analysis of a novel NAC gene associated with resistance to verticillium wilt and abiotic stress in cotton[J].G3:Genes,Genomes,Genetics,6(12):3951-3961.
Wang X,Basnayake B M,Zhang H,et al.2009.The Arabidop?sis ATAF1,a NAC transcription factor,is a negative regulator of defense responses against necrotrophic fungal and bacterial pathogens[J].Molecular Plant-Microbe Interactions,22(10):1227-1238.
Wu Y,Deng Z,Lai J,et al.2009.Dual function of Arabidopsis ATAF1 in abiotic and biotic stress responses[J].Cell Research,19(11):1279-1290.
Zhang J,Huang G Q,Zou D,et al.2018.The cotton(Gossypi?umhirsutum)NAC transcription factor(FSN1)as a positive regulator participates in controlling secondary cell wall biosynthesis and modification of fibers[J].New Phytologist,217(2):625-640.
Zhang T,Hu Y,Jiang W,et al.2015.Sequencing of allotetraploid cotton(Gossypium hirsutum L.acc.TM-1)provides a resource for fiber improvement[J].Nature Biotechnology,33(5):531-537.
Zhao F,Ma J,Li L,et al.2016.GhNAC12,a neutral candidate gene,leads to early aging in cotton(Gossypium hir?sutum L)[J].Gene,576(1 Pt 2):268-274.