陆地棉GhMYB9基因克隆及结合特性分析
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摘要
MYB转录因子蛋白普遍存在于植物中,广泛参与植物的生长发育和代谢调控。为研究GhMYB9基因对棉纤维的调控机制,使用PCR方法,从棉花中克隆1个R2R3-MYB基因GhMYB9(Gen Bank登录号:AF336286.1);利用生物信息学方法分析其氨基酸序列;构建酵母表达载体。结果表明,该基因c DNA全长1 145 bp,开放阅读框长795 bp,编码264个氨基酸,预测其蛋白分子量约为29.624 k D,等电点为9.13。推测的氨基酸序列中含有2个高度保守的SANT结构域。GhMYB9基因组包含2个外显子和1个内含子。进化树分析表明,GhMYB9和Gh MYB聚在同一分支(HQ234875.1)。酵母单杂交试验结果表明,GhMYB9转录因子能够特异结合AC顺式作用元件。本研究结果为进一步阐明GhMYB9的生物学功能奠定了基础。
The MYB transcription factor protein is generally existed in plants and involved in growth and metabolism regulation. In order to analyze the regulatory mechanism of GhMYB9 on cotton fiber,a gene coding for R2R3- MYB,designated as GhMYB9( Gen Bank No. AF336286. 1) was isolated from cotton by PCR,the amino acid sequence was analyzed by bioinformatics methods and the yeast expression vector was constructed. The results showed that the full length of GhMYB9 c DNA is 1 145 bp,including an ORF of 795 bp. This c DNA sequence encoded a polypepide of 264 amino acid residues with a predicted molecular mass of 29. 624 k D and a basic isoelectric point of 9. 13,and contained two highly conserved SANT domains. The genomic DNA of GhMYB9 contains three exons and one intron. Phylogenetic analysis indicated that GhMYB9 clustered with Gh MYB( HQ234875. 1). The yeast one hybrid experiment results showed that GhMYB9 transcription factor can specifically bind AC elements. Thse results provide a strong foundation for further study on function of GhMYB9 gene in cotton.
The MYB transcription factor protein is generally existed in plants and involved in growth and metabolism regulation. In order to analyze the regulatory mechanism of GhMYB9 on cotton fiber,a gene coding for R2R3- MYB,designated as GhMYB9( Gen Bank No. AF336286. 1) was isolated from cotton by PCR,the amino acid sequence was analyzed by bioinformatics methods and the yeast expression vector was constructed. The results showed that the full length of GhMYB9 c DNA is 1 145 bp,including an ORF of 795 bp. This c DNA sequence encoded a polypepide of 264 amino acid residues with a predicted molecular mass of 29. 624 k D and a basic isoelectric point of 9. 13,and contained two highly conserved SANT domains. The genomic DNA of GhMYB9 contains three exons and one intron. Phylogenetic analysis indicated that GhMYB9 clustered with Gh MYB( HQ234875. 1). The yeast one hybrid experiment results showed that GhMYB9 transcription factor can specifically bind AC elements. Thse results provide a strong foundation for further study on function of GhMYB9 gene in cotton.
引文
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[2]王娜,张振葆,黄凤珠,李洪有,张素芝.WRKY转录因子参与植物非生物胁迫应答的研究进展[J].核农学报,2014,28(10):1819-1827
[3]郭丽雪,王永强,孟宪鹏,赵俊丽,郭娴,张寒霜.棉花纤维发育相关转录因子的研究进展[J].河北农业科学,2011,15(6):50-52,60
[4]刘春华,高剑瑞,王鹏,夏超,董晓秋,刘兵,赵丹,韦如俊,兰海.玉米转录因子基因Zm PTF1在转基因烟草中的表达[J].核农学报,2015,29(4):692-698
[5]Ernst H A,Leggio L L,Olsen A N,Skriver K.NAC transcription factors:structurally distinct,functionally diverse[J].Trends in Plant Science,2005,10(2):79-87
[6]Jakoby M,Weisshaar B,W Droge-Laser W,Vicente-Carbajosa J,Tiedemann J,Kroj T,Parcy F.b ZIP transcription factors in Arabidopsis[J].Trends in Plant Science,2002,7(3):106-111
[7]王磊,高晓清,朱苓华,周永力,黎志康.植物WRKY转录因子家族基因抗病相关功能的研究进展[J].植物遗传资源学报,2011,12(1):80-85
[8]Rushton P J,Somssich I E,Ringler P,Shen Q X J.WRKY transcription factors[J].Trends in Plant Science,2010,15(5):247-258
[9]Song C P,Agarwal M,Ohta M,Guo Y,Halfter U,Wang P C,Zhu J K.Role of an Arabidopsis AP2/EREBP-Type transcriptional repressor in abscisic acid and drought stress responses[J].Plant Cell,2005,17(8):2384-2396
[10]王传增,余贤美,董庆龙,张安宁,刘伟,董飞,王淑贞,王长君.桃已知MADS-box转录因子的生物信息学及花发育表达分析[J].核农学报,2015,29(5):849-858
[11]Ambawat S,Sharma P,Yadav N R,Yadav R C.MYB transcription factor genes as regulators for plant responses:an overview[J].Physiology and Molecular Biology of Plants,2014,19(3):307-321
[12]陈俊,王宗阳.植物MYB类转录因子研究进展[J].植物生理与分子生物学学报,2002,28(2):81-88
[13]房栋.棉纤维初始发育相关基因及转录因子的克隆与鉴定[D].南京:南京农业大学,2006
[14]刘翔,左开井,张飞,许洁婷,黎颖,赵凌侠,唐克轩.MYB类转录因子在植物腺毛发育中的作用研究进展[J].上海交通大学学报(农业科学版),2010,28(2):188-194
[15]Larkin J C,Oppenheimer D G,Pollock S,Marks M D.Arabidopsis GLABROUS1 gene requires downstream sequences for function[J].The Plant cell,1993,5(12):1739-1784
[16]Serna L,Martin C.Trichomers:different regulatory networks lead to convergent structures[J].Trends Plant Science,2006,11(6):274-280
[17]Loguercio L L,Scott H C,Trolinder N L,Wilkins T A.Hmg-co A reductase gene family in cotton(Gossypium hirsutum L.):unique structural features and differential expression of hmg2 potentially associated with synthesis of specific isoprenoids in developing embryos[J].Plant and Cell Physiology,1999,40(7):750-761
[18]陈全家,倪志勇,刘静静,冯文林,刘超,陈章良,康定明,曲延英.海岛棉Gb MYB25基因的克隆及表达分析[J].中国农业大学学报,2015,20(1):1-7
[19]Suo J F,Liang X E,Pu L,Zhang Y,Xue Y.Identification of Gh MYB109 encoding a R2R3 MYB transcription factor that expressed specifically in fiber initials and elongating fibers of cotton(Gossypium hirsutum L.)[J].Biochimica et Biophysica Acta,2003,1630(1):25-34
[20]Wu Y R,Machado A C,White R G,Llewellyn D J,Dennis E S.Expression profiling identifies genes expressed early during lint fiber initiation in cotton[J].Plant and Cell Physiology,2006,47(1):107-127
[21]Zhang T,Hu Y,Jiang W,Fang L,Guan X,Chen J,Zhang J,Saski C A,Scheffler B E,Stelly D M,Hulse-Kemp A M,Wan Q,Liu B,Liu C,Wang S,Pan M,Wang Y,Wang D,Ye W,Chang L,Zhang W,Song Q,Kirkbride R C,Chen X,Dennis E,Llewellyn D J,Peterson D G,Thaxton P,Jones D C,Wang Q,Xu X,Zhang H,Wu H,Zhou L,Mei G,Chen S,Tian Y,Xiang D,Li X,Ding J,Zuo Q,Tao L,Liu Y,Li J,Lin Y,Hui Y,Cao Z,Cai C,Zhu X,Jiang Z,Zhou B,Guo W,Li R,Chen Z J.Sequencing of allotetraploid cotton(Gossypium hirsutum L.acc.TM-1)provides a resource for fiber improvement[J].Nature Biotechnology,2015,33(5):531-537
[22]陈全家.棉纤维发育相关基因转录组学、表达谱分析研究[D].北京:中国农业大学,2014
[23]Machado A,Wu Y R,Yang Y M,Llewellyn D J,Dennis E S.The MYB transcription factor Gh MYB25 regulates early fiber and trichome development[J].The Plant Journal,2009,59(1):52-62
[24]Sumita O,Santosh K,Khan B M.Over-expression of a subgroup 4R2R3 type MYB transcription factor gene from Leucaena leucocephala reduces lignin content in transgenic tobacco[J].Plant Cell Reports,2013,32(1):161-171
[25]王诺菡,于霁雯,吴嫚,马启峰,李兴丽,裴文锋,李海晶,黄双领,张金发,喻树迅.棉花Gh MYB0基因的克隆、表达分析及功能鉴定[J].作物学报,2014,40(9):1540-1548
[26]曹丹琴,杨健,关晓弯,张永娟,龚凌燕,张水明.石榴种皮木质素合成相关转录因子基因Pg MYB的克隆与表达[J].西北植物学报,2015,35(1):23-29
[27]唐芳,王敏杰,杨海峰,赵树堂,卢孟柱.拟南芥MYB基因对次生维管系统发育的影响[J].林业科学研究,2010,23(2):170-176
[28]倪志勇,吕萌,范玲.棉花咖啡酰辅酶A-O-甲基转移酶基因的克隆及特征分析[J].西北植物学报,2010,30(6):1083-1091
[29]于月华,倪志勇,梁小莉,刘真芳,陈全家,高文伟.棉花转录因子基因Gh MYB的克隆及特征分析[J].棉花学报,2015,27(1):31-38
[30]秦秋琳.银杏中与木质素合成和苯丙氨酸代谢相关的转录调控因子的克隆与研究[D].上海:复旦大学,2007
[31]Zhu L,Shan H,Chen S M,Jiang J F,Gu C S,Zhou G Q,Chen Y,Song A P,Chen F D.The heterologous expression of the chrysanthemum R2R3-MYB transcription factor Cm MYB1 alters lignin composition and represses flavonoid synthesis in Arabidopsis thaliana[J].PLo S One,2013,8(6):e65680
[2]王娜,张振葆,黄凤珠,李洪有,张素芝.WRKY转录因子参与植物非生物胁迫应答的研究进展[J].核农学报,2014,28(10):1819-1827
[3]郭丽雪,王永强,孟宪鹏,赵俊丽,郭娴,张寒霜.棉花纤维发育相关转录因子的研究进展[J].河北农业科学,2011,15(6):50-52,60
[4]刘春华,高剑瑞,王鹏,夏超,董晓秋,刘兵,赵丹,韦如俊,兰海.玉米转录因子基因Zm PTF1在转基因烟草中的表达[J].核农学报,2015,29(4):692-698
[5]Ernst H A,Leggio L L,Olsen A N,Skriver K.NAC transcription factors:structurally distinct,functionally diverse[J].Trends in Plant Science,2005,10(2):79-87
[6]Jakoby M,Weisshaar B,W Droge-Laser W,Vicente-Carbajosa J,Tiedemann J,Kroj T,Parcy F.b ZIP transcription factors in Arabidopsis[J].Trends in Plant Science,2002,7(3):106-111
[7]王磊,高晓清,朱苓华,周永力,黎志康.植物WRKY转录因子家族基因抗病相关功能的研究进展[J].植物遗传资源学报,2011,12(1):80-85
[8]Rushton P J,Somssich I E,Ringler P,Shen Q X J.WRKY transcription factors[J].Trends in Plant Science,2010,15(5):247-258
[9]Song C P,Agarwal M,Ohta M,Guo Y,Halfter U,Wang P C,Zhu J K.Role of an Arabidopsis AP2/EREBP-Type transcriptional repressor in abscisic acid and drought stress responses[J].Plant Cell,2005,17(8):2384-2396
[10]王传增,余贤美,董庆龙,张安宁,刘伟,董飞,王淑贞,王长君.桃已知MADS-box转录因子的生物信息学及花发育表达分析[J].核农学报,2015,29(5):849-858
[11]Ambawat S,Sharma P,Yadav N R,Yadav R C.MYB transcription factor genes as regulators for plant responses:an overview[J].Physiology and Molecular Biology of Plants,2014,19(3):307-321
[12]陈俊,王宗阳.植物MYB类转录因子研究进展[J].植物生理与分子生物学学报,2002,28(2):81-88
[13]房栋.棉纤维初始发育相关基因及转录因子的克隆与鉴定[D].南京:南京农业大学,2006
[14]刘翔,左开井,张飞,许洁婷,黎颖,赵凌侠,唐克轩.MYB类转录因子在植物腺毛发育中的作用研究进展[J].上海交通大学学报(农业科学版),2010,28(2):188-194
[15]Larkin J C,Oppenheimer D G,Pollock S,Marks M D.Arabidopsis GLABROUS1 gene requires downstream sequences for function[J].The Plant cell,1993,5(12):1739-1784
[16]Serna L,Martin C.Trichomers:different regulatory networks lead to convergent structures[J].Trends Plant Science,2006,11(6):274-280
[17]Loguercio L L,Scott H C,Trolinder N L,Wilkins T A.Hmg-co A reductase gene family in cotton(Gossypium hirsutum L.):unique structural features and differential expression of hmg2 potentially associated with synthesis of specific isoprenoids in developing embryos[J].Plant and Cell Physiology,1999,40(7):750-761
[18]陈全家,倪志勇,刘静静,冯文林,刘超,陈章良,康定明,曲延英.海岛棉Gb MYB25基因的克隆及表达分析[J].中国农业大学学报,2015,20(1):1-7
[19]Suo J F,Liang X E,Pu L,Zhang Y,Xue Y.Identification of Gh MYB109 encoding a R2R3 MYB transcription factor that expressed specifically in fiber initials and elongating fibers of cotton(Gossypium hirsutum L.)[J].Biochimica et Biophysica Acta,2003,1630(1):25-34
[20]Wu Y R,Machado A C,White R G,Llewellyn D J,Dennis E S.Expression profiling identifies genes expressed early during lint fiber initiation in cotton[J].Plant and Cell Physiology,2006,47(1):107-127
[21]Zhang T,Hu Y,Jiang W,Fang L,Guan X,Chen J,Zhang J,Saski C A,Scheffler B E,Stelly D M,Hulse-Kemp A M,Wan Q,Liu B,Liu C,Wang S,Pan M,Wang Y,Wang D,Ye W,Chang L,Zhang W,Song Q,Kirkbride R C,Chen X,Dennis E,Llewellyn D J,Peterson D G,Thaxton P,Jones D C,Wang Q,Xu X,Zhang H,Wu H,Zhou L,Mei G,Chen S,Tian Y,Xiang D,Li X,Ding J,Zuo Q,Tao L,Liu Y,Li J,Lin Y,Hui Y,Cao Z,Cai C,Zhu X,Jiang Z,Zhou B,Guo W,Li R,Chen Z J.Sequencing of allotetraploid cotton(Gossypium hirsutum L.acc.TM-1)provides a resource for fiber improvement[J].Nature Biotechnology,2015,33(5):531-537
[22]陈全家.棉纤维发育相关基因转录组学、表达谱分析研究[D].北京:中国农业大学,2014
[23]Machado A,Wu Y R,Yang Y M,Llewellyn D J,Dennis E S.The MYB transcription factor Gh MYB25 regulates early fiber and trichome development[J].The Plant Journal,2009,59(1):52-62
[24]Sumita O,Santosh K,Khan B M.Over-expression of a subgroup 4R2R3 type MYB transcription factor gene from Leucaena leucocephala reduces lignin content in transgenic tobacco[J].Plant Cell Reports,2013,32(1):161-171
[25]王诺菡,于霁雯,吴嫚,马启峰,李兴丽,裴文锋,李海晶,黄双领,张金发,喻树迅.棉花Gh MYB0基因的克隆、表达分析及功能鉴定[J].作物学报,2014,40(9):1540-1548
[26]曹丹琴,杨健,关晓弯,张永娟,龚凌燕,张水明.石榴种皮木质素合成相关转录因子基因Pg MYB的克隆与表达[J].西北植物学报,2015,35(1):23-29
[27]唐芳,王敏杰,杨海峰,赵树堂,卢孟柱.拟南芥MYB基因对次生维管系统发育的影响[J].林业科学研究,2010,23(2):170-176
[28]倪志勇,吕萌,范玲.棉花咖啡酰辅酶A-O-甲基转移酶基因的克隆及特征分析[J].西北植物学报,2010,30(6):1083-1091
[29]于月华,倪志勇,梁小莉,刘真芳,陈全家,高文伟.棉花转录因子基因Gh MYB的克隆及特征分析[J].棉花学报,2015,27(1):31-38
[30]秦秋琳.银杏中与木质素合成和苯丙氨酸代谢相关的转录调控因子的克隆与研究[D].上海:复旦大学,2007
[31]Zhu L,Shan H,Chen S M,Jiang J F,Gu C S,Zhou G Q,Chen Y,Song A P,Chen F D.The heterologous expression of the chrysanthemum R2R3-MYB transcription factor Cm MYB1 alters lignin composition and represses flavonoid synthesis in Arabidopsis thaliana[J].PLo S One,2013,8(6):e65680