小麦籽粒胚乳基因sbe2b cDNA的克隆与原核表达条件的优化

Cloning and Optimizing Expression of sbe2b cDNA from Wheat Endosperm in E.coil

本研究采用RT-PCR技术克隆了小麦胚乳基因sbe2b全长cDNA序列。序列分析表明,该序列与已报道的sbe2bcDNA序列(登录号:AY740401)同源性为98%。同时构建了原核生物表达载体pET28(a+)-sbe2b,对不同的IPTG诱导浓度和诱导时间等条件的优化结果表明,37℃8h能诱导sbe2b融合蛋白的最大量表达,在0.5mmol/LIPTG浓度下可以成功表达出sbe2b蛋白。为进一步体外研究sbe2b的物理化学性质及催化机理奠定基础。

In this paper,we cloned the whole cDNA sequence of sbe2b from wheat endosperm by RT-PCR sequence analysis showed that it had 98%homology with reported sequence of sbe2b cDNA in GenBank（accession No.：AY740401）.The sbe2b cDNA were inserted pET28a（＋） vector by restriction endonuclease digestion,electrophoresis,gel reclamation DNA ligation and so on molecular biological technologies,and then transformed it to E.coil BL21（DE3）.The sbe2b fusion protein was expressed and the expression conditions were optimized.The results showed that the expression level of sbe2b fusion protein reached the highest when cultured for 8 hours at 37℃,and 0.5 mmol/L IPTG can induced the expression of sbe2b in prokaryotic expression system effectively.The experimental results provide insights into study on the physical and chemical properties of sbe2b in vitro.