绿僵菌Argonaute基因片段原核表达载体的构建及其表达  

Construction and Expression of the Prokaryotic Expression Vector of Argonaute Gene Related Segment from Metarhizium Anisopliae

作　　者：汪章勋[1,2] 孟慧敏[2] 周权[2] 

机构地区：[1]安徽农业大学植物保护学院,安徽合肥230036 [2]安徽农业大学微生物防治省重点实验室,安徽合肥230036

出　　处：《安徽科技学院学报》2013年第6期39-43,共5页Journal of Anhui Science and Technology University

基　　金：国家自然科学基金(31272096);安徽省自然科学基金(1308085QC61)

摘　　要：绿僵菌是一类广泛应用于生物防治的昆虫病原真菌。研究表明小RNA能够调控基因的表达,其中Argonaute基因在整个小RNA通路中发挥重要的作用。本研究通过RT-PCR的方法从绿僵菌中获得了Argonaute基因的部分功能片段,构建了其重组原核表达载体,将重组载体转化至大肠杆菌进行诱导表达;采用镍柱亲和纯化重组的目的蛋白并通过Western blot技术鉴定。结果发现:通过RT-PCR的方法获得长度约为950bp的基因片段;重组原核表达载体经诱导表达后,SDS-PAGE检测发现分子量约为34kDa的目的蛋白条带;诱导5h后蛋白的表达量最高,采用镍柱亲和层析纯化重组蛋白,经Western blot技术检测,重组蛋白可与His-tag抗体发生特异性反应。该纯化重组蛋白的获得为将来绿僵菌Argonaute蛋白抗体的制备,并进一步通过该抗体获得绿僵菌体内的小RNA及其靶基因提供了基础。As a class of entomopathogenic fungi, M. anisopliae is widely used in biological control, studies have found that small RNA can regulate gene expression, in which Argonaute gene plays an important role in the small RNA pathway. In the present study, the partial fragment of Argonaute gene was obtained by RT -PCR method from M. anisopliae. Its prokaryotic expression vector was constructed, and then was transformed into E. coli for induced expression. The recombinant protein was purified in Ni -Agarose column and identified in Western blot analysis. The results show that the gene fragments with a length of about 950bp were obtained by RT - PCR meth- od. After induced expression, a protein band with molecular mass about 34 kDa was detected by SDS - PAGE a- nalysis. The highest expression level of induced protein was found after 5h induction. The recombinant protein was successfully purified by Ni - Agarose column and identified by Western blot analysis with His - tag antibody. The obtainment of purified recombinant protein will provide a foundation for the preparation Argonaute protein antibodies, and further acquiring small RNA and its target genes through the antibody in vivo.

关 键 词：绿僵菌 Argonaute基因 原核表达 

分 类 号：S476.12[农业科学—农业昆虫与害虫防治]