Cloning and Prokaryotic Expression of Sweet Sorghum Succinic Semialdehyde Dehydrogenase SbSSADH

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

Abstract

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

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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