昆虫学报 ›› 2016, Vol. 59 ›› Issue (12): 1332-1339.doi: 10.16380/j.kcxb.2016.12.006

• 研究论文 • 上一篇    下一篇

中华蜜蜂SRP9基因的克隆、序列分析及原核表达

吴鹏杰1, 金红岩2, 徐进1, 武江利1, 李雨时1, 郭岳琴1, 姚军1, 徐书法1,*, 吴杰1,*   

  1. (1. 中国农业科学院蜜蜂研究所, 农业部授粉昆虫生物学重点实验室, 北京 100093; 2. 中国农业科学院北京畜牧兽医研究所, 北京 100193)
  • 出版日期:2016-12-20 发布日期:2016-12-20

Molecular cloning, sequence analysis and prokaryotic expression of SRP9 gene in the Chinese honeybee, Apis cerana cerana (Hymenoptera: Apidae)

WU Peng-Jie1, JIN Hong-Yan2, XU Jin1, WU Jiang-Li1, LI Yu-Shi1, GUO Yue-Qin1, YAO Jun1, XU Shu-Fa1,*, WU Jie1,*   

  1. (1. Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture, Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, China; 2. Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing 100193, China)
  • Online:2016-12-20 Published:2016-12-20

摘要: 【目的】探究中华蜜蜂Apis cerana cerana信号识别颗粒9(signal recognition particle 9, SRP9)基因的序列, 获得纯化的重组蛋白,为探究其蛋白功能奠定基础。【方法】利用RT-PCR方法,以中华蜜蜂的cDNA为模板扩增中华蜜蜂SRP9基因编码区,并通过软件MEGA5.1构建系统发育树,运用PrediceProtein和SWISS-MODEL等软件预测分析蛋白结构;用大肠杆菌Escherichia coli表达系统进行原核表达;采用尿素洗脱法进行重组蛋白纯化。【结果】扩增得到的中华蜜蜂SRP9基因编码区长为237 bp,命名为AccSRP9。该基因编码78个氨基酸,编码蛋白的相对分子量为9.36 kD,等电点为9.17。系统进化树分析表明,AccSRP9与西方蜜蜂Apis mellifera的SRP9和小蜜蜂Apis florea的SRP9聚成一支。蛋白质二级结构预测发现其含有2个α-螺旋区和3个β-折叠区。利用同源建模获得蛋白质的三维结构。将AccSRP9连入表达载体pGEX-6P-1并在BL21(DE3)中进行原核表达,发现该重组蛋白表达在包涵体中,经蛋白纯化,最终获得了N端带GST标签的重组蛋白pGEX-6P-1-SRP9。【结论】本研究成功克隆了中华蜜蜂SRP9基因AccSRP9,对其序列进行了分析,并获得了带有标签的重组蛋白,为进一步研究该基因的功能提供参考和材料。

关键词: 中华蜜蜂, SRP9, 基因克隆, 序列分析, 结构预测, 原核表达

Abstract: 【Aim】 The study aims to clone the SRP9 gene from the Chinese honeybee, Apis cerana cerana, to analyze its sequence, and to gain the purified recombinant protein, so as to lay the foundation for the study of its protein function. 【Methods】 The cDNA sequence of the SRP9 gene was cloned from A. c. cerana by RT-PCR. Then the phylogenetic tree was constructed by MEGA5.1, and the protein structure was predicted and analyzed by software PrediceProtein and SWISS-MODEL. The SRP9 gene of A. c. cerana was expressed in Escherichia coli. The recombinant protein was purified by urea elution. 【Results】 Sequence analysis revealed an open reading frame (ORF) of 237 bp, which was named AccSRP9 and encodes 78 amino acids. The deduced relative molecular weight of the encoded protein is 9.36 kD and the isoelectric point is 9.17. Phylogenetic analysis showed that the SRP9 proteins of A. c. cerana, A. mellifera and A. florea cluster together. Protein secondary structure prediction showed that AccSRP9 contains two α-helix and three β-fold regions. By using homology modeling, the predicted three-dimensional structure of AccSRP9 was obtained. The AccSRP9 ORF was ligated into the expression vector pGEX-6P-1 and then transformed to E. coli BL21(DE3) for prokaryotic expression and the recombinant protein was purified. The results showed that the protein was expressed in inclusion bodies. Using the method of washing inclusion bodies, the N-terminal GST tagged recombinant protein pGEX-6P-1-SRP9 was obtained. 【Conclusion】 This study successfully cloned the SRP9 gene AccSRP9 from A. c. cerana, analyzed its sequences and obtained the purified recombinant protein, providing referencs and materials for further study of the functions of the gene.

Key words:  Apis cerana cerana; SRP9,  gene cloning, sequence analysis;  structure prediction, prokaryotic expression