Serratia sp．NDW3 GADH 小亚基基因ga2 dh 的克隆及表达分析

摘要

[Objective] To clone and identify GADH small subunit gene ga2dh.[Method] Changes in soluble phosphorus content , GADH activity and ga2dh gene expression level during the phosphate solubi-lizing process of Serratia sp.NDW3 from rice rhizosphere were studied .The ga2dh gene was cloned and expressed in Escherichia coli BL21, and bioinformatic analysis was performed .[Result] The relative expression of ga2dh gene in the process of phosphate solubilizing by Serratia sp.NDW3 reached maxi-mum at 12 h, GADH activity reached maximum at 24 h, and the soluble phosphorus content stabilized after 36 h.The ga2dh gene sequence of 781 bp was obtained from Serratia sp.NDW3 by cloning.The similarity between ga2dh gene and Serratia sp.SCBI sequences was 99.62% based on bioinformatic analysis.The protein encoded by ga2dh belonged to the superfamily of gluconate dehydrogenase subunit 3 and was composed of three α-helices.The amino acid sequence was consisted of intracellular , extracellu-lar and transmembrane regions.The expression of ga2dh gene in E.coli BL21 significantly increased GADH activity.[Conclusion] The main mechanism of phosphate solubilizing by Serratia sp.NDW3 is through the direct oxidation pathway .The small subunit encoded by ga2dh gene not only plays an impor-tant role in GADH activation , but also is part of the transmembrane structure of GADH .%【目的】克隆葡萄糖酸脱氢酶（ GADH）小亚基基因ga2dh并进行鉴定。【方法】研究水稻根际细菌Serratia sp．NDW3溶磷过程中菌株溶磷量、GADH活性与ga2dh基因表达量的变化，对ga2dh基因进行克隆和生物信息学分析，并检测ga2dh基因在大肠埃希菌BL21中的表达。【结果】 Serratia sp．NDW3溶磷过程中ga2dh基因的相对表达量在12 h最大，GADH活性在24 h达到最大值，NDW3溶磷量在36 h后趋于稳定。从Serratia sp．NDW3菌株中克隆获得了781 bp的ga2dh基因序列，生物信息学分析发现该序列与Serratia sp．SCBI菌株的基因相似性为99.62％，编码的蛋白属于葡萄糖酸脱氢酶亚基3超家族，主要由3个α－螺旋构成，且氨基酸序列中包含有位于胞内、胞外和跨膜的区域。 ga2dh基因在大肠埃希菌BL21体内表达，能够使得菌体GADH的活性显著增加。【结论】Serratia sp．NDW3菌株溶磷的主要机制依赖于直接氧化途径，ga2dh基因编码的小亚基不仅对GADH活性起重要作用，也是介导GADH跨膜结构的重要组成部分。