陆地棉耐盐相关基因GhSAMS的克隆及表达

doi:10.3724/SP.J.1006.2011.01012

摘要/Abstract

摘要： 为了挖掘棉花耐盐相关基因，我们根据陆地棉耐盐性抑制消减文库中的一个S-腺苷甲硫氨酸合成酶基因的同源EST设计引物，利用RACE结合RT-PCR技术克隆陆地棉S-腺苷甲硫氨酸合成酶基因的cDNA全长，命名为GhSAMS。该cDNA全长1 576 bp，ORF为1 182 bp，编码393个氨基酸的多肽。生物信息学分析表明GhSAMS蛋白与拟南芥、盐地碱蓬、水稻中该蛋白的相似性分别为91%、93%和93%。系统发育树结果显示GhSAMS与盐地碱蓬中该蛋白的亲缘关系最近。Real-time PCR分析结果表明，GhSAMS的表达受盐胁迫诱导，在盐敏感材料中诱导被推迟，而且，该基因表达水平在耐盐材料中9835中明显高于在盐敏感材料中S9612中。我们构建了原核表达载体pET28-GhSAMS，经IPTG诱导，实现了GhSAMS在大肠杆菌中的表达，为进一步开展GhSAMS的遗传转化工作奠定了有益基础。

关键词: 陆地棉, 盐胁迫, S-腺苷甲硫氨酸合成酶基因, 原核表达

Abstract: As one of main abiotic stresses in nature, salt stress does great harm to plants, and seriously affect plant growth and development. Simultaneously, the crops cultivated in the saline land undergo a wide range of yield decline. To excavate salt-tolerance gene, we cloned the cDNA of S-adenosyl-L-methionine synthetase gene from Gossypium hirsutum by RACE and RT-PCR, which was named GhSAMS, with the cDNA full length of 1 576 bp, ORF of 1 182 bp, and coding 393 amino acid residues. Bioinformatics analysis showed GhSAMS has the similarity of 91%, 93%, and 93% with Arabidopsis thaliana, Suaeda salsa, and Oryza sativa, respectively. Phylogenetic analysis showed GhSAMS was the closest to Suaeda salsa,and Real-time PCR suggested that GhSAMS was induced by salt stress, while the induction was postponed in salt sensitivity material. It showed lower gene expression level on salt sensitive material Zhong S9612 relative to salt resistance material Zhong 9835. At the same time, we established protokaryotic expression vector pET28-GhSAMS and transformed GhSAMS into E. coli after IPTG induction, showing a successful gene expression.

Key words: Gossypium hirsutum, Salt stress, S-adenosyl-L-methionine synthetase, Prokaryotic expression

周凯, 宋丽艳, 叶武威*, 王俊娟, 王德龙, 樊保香. 陆地棉耐盐相关基因GhSAMS的克隆及表达[J]. 作物学报, 2011, 37(06): 1012-1019.

ZHOU Kai, SONG Li-Yan, YE Wu-Wei-*, WANG Jun-Juan, WANG De-Long, FAN Bao-Xiang. Cloning and Expression of GhSAMS Gene Related to Salt-tolerance in Gossypium hirsutum L.[J]. Acta Agron Sin, 2011, 37(06): 1012-1019.

参考文献

[1]Guo Y H, Yu Y P, Wang D, Wu C A, Yang D G, Huang J G, Zheng C C. GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5. New Phytologist, 2009, 183: 62–75
[2]Huang B, Jin L, Liu J Y. Identification and characterization of the novel gene GhDBP2 encoding a DRE-binding protein from cotton (Gossypium hirsutum). J Plant Physiol, 2008, 165: 214–223
[3]Yang Y-W(杨郁文), Ni W-C(倪万潮), Zhang B-L(张保龙), Shen X-L(沈新莲), Zhang X-G(张香桂), Xu Y-J(徐英俊), Yao S(姚姝). Molecular cloning and expression analysis of a serine/threonine protein kinase gene in upland cotton. Cotton Sci (棉花学报), 2006, 18(3): 140–144 (in Chinese with English abstract)
[4]Wu C A, Yang G D, Meng Q W, Zheng C C. The cotton GhNHX1 gene encoding a novel putative tonoplast Na+/H+ antiporter plays an important role in salt stress. Plant Cell Physiol, 2004, 45: 600–607
[5]Chen Y-J(陈亚娟). Isolation and Characterization of GaP5CS and GaTPS in Gossypium arboretum L. MS Dissertation of Chinese Academy of Agricultural Sciences, 2009 (in Chinese with English abstract)
[6]Baker J, Steele C, Dure L III. Sequence and characterization of 6 lea proteins and their genes from cotton. Plant Mol Biol, 1988, 11: 277–291
[7]Yang S F, Hoffman N E. Ethylene biosynthesis and its regulation in higher plants. Ann Rev Plant Physiol, 1984, 35: 155–189
[8]Tabor C W, Tabor H. Methionine adenosyltransferase (S-adenosylmethionine synthetase) and S-adenosylmethionine decarboxylase. Adv Enzymol Related Areas Mol Biol, 1984, 56: 251–282
[9]Sánchez-Aguayo I, Rodriguez-Galan J M, García R, Torreblanca J, Pardo J M. Salt stress enhances xylem development and expression of S-adenosyl-L-methionine synthase in lignifying tissues of tomato plants. Planta, 2004, 220: 278–285
[10]Markham G D, Hafner E W, Tabor C W, Tabor H. S-adenosylmethionine synthetase from Escherichia coli. J Biol Chem, 1980, 255: 9082
[11]Peleman J, Boerjan W, Engler G, Seurinck J, Botterman J, Alliotte T, Montagu M V, Inzé D. Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetasee. Plant Cell, 1989, 1: 81–93
[12]Thomas D, Surdin-Kerjan Y. SAM1, the structural gene for one of the S-adenosylmethionine synthetases in Saccharomyces cerevisiae. J Biol Chem, 1987, 362: 16704–16709
[13]Schröder G, Eichel J, Breinig S, Schröder J. Three defferentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization. Plant Mol Biol, 1997, 33: 211–222
[14]Breusegem F V, Dekeyser R, Gielen J, Montagu M V, Caplan A. Characterization of a S-adenosylmethionine synthetase gene in rice. Plant Physiol, 1994, 105: 1463–1464
[15]Zhaki A, shoseyov O, Weiss D. A petunia cDNA encoding S-adenosylmethionine synthetase. Plant Physiol, 1995, 108: 841–842
[16]Doorsselaere J V, Gielen J, Montagu M V, Inzé D. A cDNA encoding S-adenosyl-L-methionine synthetase from poplar. Plant Physiol, 1993, 102: 1365–1366
[17]Larsen P B, Woodson W R. Cloning and nucleotide sequence of an S-adenosylmethionine synthetase cDNA from Carnation. Plant Physiol, 1991, 96: 997–999
[18]Wen C M, Wu M, Goh C J, Pua E C. Cloning and nucleotide sequence of a cDNA encoding S-adenosyl-L-methionine synthetase from mustard (Brassica juncea). Plant Physiol, 1995, 107: 1021–1022
[19]Espartero J, Pintor-Toro J A, Pardo J M. Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress. Plant Mol Biol, 1994, 25: 217–227
[20]Ma X L, Wang Z L, Qi Y C, Zhao Y X, Zhang H. Isolation of S-adenosylmethionine synthetase gene from Suaeda salsa and its differential expression under NaCl stress. J Integ Plant Biol, 2003, 45: 1359–1365
[21]Qi Y C, Wang F F, Zhang H, Liu W Q. Overexpression of suadea salsa S-adenosylmethionine synthetase gene promotes salt tolerance in transgenic tobacco. Acta Physiol Plant, 2009, 32: 263–269
[22]Ye W-W(叶武威), Liu J-D(刘金定). Technique and application on salt-tolerance appraisal of cotton germplasm resources. China Cotton (中国棉花), 1998, 25(9): 34–38 (in Chinese)
[23]Salzman R A, Fujita T, Salzman K Z, Hasegawa P M, Bressan R A. An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Mol Biol Rep, 1999, 17: 11–17
[24]Jiang J-X(蒋建雄), Zhang T-Z(张天真). Extraction of total RNA in cotton tissues with CTAB-acidic phenolic method. Cotton Sci (棉花学报), 2003, 15(3): 166–167 (in Chinese with English abstract)
[25]Ye W-W(叶武威), Zhao Y-L(赵云雷), Wang J-J(王俊娟), Fan B-X(樊保相). Construction of SSH library on root system of salinity-tolerance variety (G. hirsutum L.) under the stress of salinity. Cotton Sci (棉花学报), 2009, 21(5): 339–345 (in Chinese with English abstract)
[26]Levitt J. Responses of Plants to Environmental Stresses: Chilling, Freezing, and High Temperature Stresses. New York: Academic Press, 1980
[27]Shen F-F(沈法富), Yu Y-J(于元杰), Bi J-J(毕建杰), Liu F-Z(刘凤珍), Yin C-Y(尹承佾). A diallel analysis of salt tolerance in upland cotton. Acta Agron Sin (作物学报), 2001, 27(1): 50–54 (in Chinese with English abstract)
[28]Hua Y(化烨). GsSAMS Gene Transformation into Alfalfa and Cultivation of Transformation New Lines. MS Dissertation of Northeast Agricultural University, 2009 (in Chinese with English abstract)
[29]Yang J-L(杨金丽), Zhao X-M(赵小明), Yin H(尹恒), Zhang H-Y(张洪艳), Du Y-G(杜昱光). Analysis of proteins interacted with OIPK by yeast two-hybrid method. Chin J Appl Environ Biol (应用与环境生物学报), 2010, 16(4): 474–477 (in Chinese with English abstract)
[30]Qi Y-C(戚元成), Ma L(马雷), Wang F-F(王菲菲), Liu W-Q(刘卫群). Overexpression of S-adenosylmethionine synthetase promote polyamine biosynthesis in transgenic tobacco. Plant Physiol Commun (植物生理学通讯), 2009, 45(8): 791–793 (in Chinese with English abstract)
[31]Fan J-P(樊金萍), Bai Xi(柏锡), Li Yong(李勇), Ji W(纪巍), Wang X(王希), Cai H(才华), Zhu Y-M(朱延明). Cloning and function analysis of gene SAMS from Glycine soja. Acta Agron Sin (作物学报), 2008, 34(9):1581–1587 (in Chinese with English abstract)

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