甜高粱琥珀酸半醛脱氢酶SbSSADH基因克隆及原核表达

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.013

摘要/Abstract

摘要：

γ-氨基丁酸(γ-aminobutyric acid，GABA)代谢旁路是 TCA循环的一个代谢支路，广泛存在于动植物和微生物中，植物GABA代谢旁路与植物生物和非生物胁迫响应相关。琥珀酸半醛脱氢酶(SSADH)是GABA旁路的一个关键酶，对GABA途径在生物体内发挥功能起着至关重要的作用。通过检索MaizeGDB和Gramene数据库获得甜高粱SbSSADH基因信息，利用同源克隆的方法获得了甜高粱SbSSADH基因。序列分析结果表明，SbSSADH基因与玉米、甘蔗在核酸序列的相似度为94.77%，但前端约200 bp的信号肽部分差异较大。通过以pET-28a为表达载体构建SbSSADH原核表达系统，Rosetta菌株为表达菌，表达出约50 kD的SSADH蛋白，将纯化的蛋白进行酶活分析，纯化蛋白具有琥珀酸半醛脱氢酶活性，并且受酶抑制剂ATP和AMP的抑制。

关键词: 甜高粱, SSADH, 蛋白表达, 酶活测定

Abstract:

Gamma-aminobutyric acid(GABA)shunt is a metabolism bypass of tricarboxylic acid cycle(TCA)and widely existed in animals, plants and micro-organism. The activity of the GABA shunt is drastically enhanced in response to biotic and abiotic stress. Succinic semialdehyde dehydrogenase(SSADH), which can oxidize succinic semialdhyde into succinate, is one kind of key enzymes in the metabolic pathway of the γ-aminobutyric acid(GABA)shunt, and plays an important role in biological functioned of GABA shunt in organisms. Objective：This study aimed to construct the prokaryotic expression vector of Sweet Sorghum SSADH and express the soluble protein in E.coli. With the primers designed according to the homologous genes sequence provided by MaizeGDB and Gramene databases and mRNA from sweet sorghum as a template, SSADH gene cDNA was cloned with RT-PCR. Sequence analysis showed that SbSSADH gene have 94.77% similarity with Corn and Sugarcane SSADH gene, but the forward 200 bp signal peptide sequence of SSADH gene are different. The construct harboring the truncated SSADH gene fragment without the signal peptide sequence of SbSSADH were transformed into E. coli Rosetta cell, respectively. The expression and purification of proteins were detected with SDS-PAGE, and the enzyme activity of soluble protein SSADH was analyzed with enzymatic dynamics analysis. Results showed that we cloned the cDNA of SSADH from sweet sorghum, constructed the pET-28a-SSADH prokaryotic expression vector, and obtained the soluble protein：the result of enzymatic kinetics analysis indicated that the expressed protein has activity of Succinic semialdehyde dehydrogenase and activity have been inhibited by AMP and ATP. In this study the SSADH prokaryotic expression vector was constructed successfully and recombinant expression soluble protein of SSADH has Succinic semialdehyde dehydrogenase activity.

Key words: Sweet Sorghum, SSADH, protein expression, enzyme assay

王龙海, 杨泽伟, 朱莉, 黄大昉, 郎志宏. 甜高粱琥珀酸半醛脱氢酶SbSSADH基因克隆及原核表达[J]. 生物技术通报, 2015, 31(7): 83-90.

Wang Longhai, Yang Zewei, Zhu Li, Huang Dafang, Lang Zhihong. Cloning and Prokaryotic Expression of Sweet Sorghum Succinic Semialdehyde Dehydrogenase SbSSADH[J]. Biotechnology Bulletin, 2015, 31(7): 83-90.

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