论文部分内容阅读
本研究以木薯为材料, 通过RT-PCR克隆出了MeCREB基因, 核酸序列分析表明, 所克隆的基因与phytozome数据库收录的MeCREB基因 (Manes.04G058300.1) 序列基本一致.MeCREB基因全长968 bp, 其中开放性阅读框 (ORF) 为747 bp, 编码248个氨基酸, 预测得出MeCREB蛋白是一个不稳定的亲水性蛋白, 编码蛋白质的分子量为26.26 kD, 理论等电点为5.99, GRAVY为-0.667.构建MeCREB-pGEX-6p-1表达载体, 在大肠杆菌中进行诱导表达, 并对影响重组蛋白表达的4个主要因素 (诱导温度, 诱导起始菌量, IPTG浓度及诱导时间) 进行优化, 确定GST-MeCREB融合蛋白的最适表达条件.结果表明:重组工程菌生长至OD600=0.8时加入终浓度为0.1 mmol/L的IPTG, 在28℃下诱导6 h, 最适合GST-MeCREB融合蛋白的表达.本研究为高效诱导获得GST-MeCREB融合蛋白用于下游实验提供了最优方案, 并为进一步研究MeCREB基因的生物学功能奠定了基础.“,”MeCREB gene was cloned by RT-PCR from cassava in this study. The analysis of nucleic acid sequence indicated that the cloned gene was consistent with the sequence of MeCREB gene (Manes. 04 g058300. 1) recorded in the phytozome database. The MeCREB gene was 968 bp with an open reading frame (ORF) of 747 bp which encoded 248 amino acids. MeCREB protein was predicted to be an unstable hydrophilic protein, encoding a protein of 26. 26 kD. The theoretical isoelectric point of MeCREB was 5. 99, and the GRAVY was-0. 667. The MeCREB-pGEX-6 p-1 expression vector was constructed and induced to express in E. coli. Also, the expression conditions such as temperature, bacterium concentration, IPTG concentration and induction time were optimized by Me C REB-p GEX-6 p-1 to confirm the optimal conditions for the expression of GST-MeCREB fusion protein. The results showed that the conditions of 28℃, OD600=0. 8, 0. 1 mmol/L concentration of IPTG and 6 hours' culture were the most suitable for the expression of GST-MeCREB fusion protein. This study provided an optimal scheme for efficiently getting the GST-MeCREB fusion protein using for the downstream experiments, and laid the foundation for the further study on the biological functions of MeCREB genes.