苏云金芽胞杆菌cry1Ia32基因克隆和原核表达的研究  

Cloning and expression of cry1Ia32 gene from Bacillus thuringiensis isolate

作　　者：宋萍[1] 南宫自艳[1] 张杰[1] 王立宇[1] 王勤英[1] 

机构地区：[1]河北农业大学植物保护学院,河北保定071000

出　　处：《河北农业大学学报》2015年第5期48-52,共5页Journal of Hebei Agricultural University

基　　金：国家自然科学基金青年基金(31301718);河北省自然科学基金(C2014204031)

摘　　要：根据GenBank中cry1I类基因序列设计特异性引物,以Bt SC-13菌总DNA为模板PCR扩增得到2 151bp的cry1Ia全长基因。该基因编码蛋白由717个氨基酸组成,相对分子量为80.9kDa,等电点为6.57,编码蛋白与Cry1Ia1(CAA44633)亲缘关系最高达99.03%的序列同源性,该蛋白被Btδ-内毒素国际命名委员会正式命名为Cry1Ia32(登录号为JX944039)。cry1Ia32基因通过表达载体pET-21b在大肠杆菌中高效表达分子量为81kDa的蛋白,诱导表达的Cry1Ia32蛋白对棉铃虫和甜菜夜蛾的2龄幼虫具有较高杀虫活性,LC50值分别为0.025mg/mL和0.011mg/mL,为抗虫转基因植物研究提供了新的基因。A full‐length cry1Iagene fragment was obtained by PCR amplification with a pair of primers designed according to cry1I‐type gene sequences and total DNA from Bacillus thuringiensis SC‐13 isolate .The encoded protein was composed of 717 amino acid residues with a calculated molecular weight of 80 .9 kDa ,the iso‐electric point of the protein was 6 .57 . This gene was designated as Cry1 Ia32 with accession number JX944039 by International No‐menclature Committee of Bt .The amino acid sequence of Cry1Ia32 was with the highest iden‐tity of 99 .03% to Cry1Ia1（CAA44633） ,the difference was seven amino acids .cry1Ia32 gene was introduced into expression vector pET‐21b and transformed into Escherichia coli BL21 （DE3） .81 kDa Cry1Ia32 protein was successfully expressed .And expressed‐Cry1Ia32 protein showed high toxicity to second instar larvae of Helicoverpa armigera and Spodoptera ex‐igua .LC50 values were 0 .025mg/mL and 0 .011mg/mL respectively .

关 键 词：苏云金芽胞杆菌 CRY1 Ia32基因 基因表达 杀虫活性 

分 类 号：S435.121.4[农业科学—农业昆虫与害虫防治]