苏云金芽孢杆菌伴孢晶体20kb DNA中cry1Ac基因的克隆、高效表达和生物活性研究  

Cloning and Superexpression of Cry1Ac Gene from 20kb DNA Associated with Bacillus thuringiensis Cry1A Crystal Protein

作　　者：胡宏源[1] 夏立秋[1] 史红娟[1] 孙运军[1] 高必达[2] 丁学知[1] 

机构地区：[1]湖南师范大学生命科学学院微生物学系,长沙410081 [2]湖南农业大学植物保护学院,长沙410128

出　　处：《生物工程学报》2004年第5期656-661,共6页Chinese Journal of Biotechnology

基　　金：国家"8 63计划"(No.2 0 0 2AA2 45 0 2 1);国家自然科学基金 (No .3 0 2 70 0 3 7);湖南省自然科学基金 (No.0 3JJY3 0 2 5 )~~

摘　　要：已经证实苏云金芽孢杆菌 (Bacillusthuringiensis,Bt)伴孢晶体结合 2 0kbDNA ,但其序列特异性及作用有待进一步研究阐明。研究了选择性溶解Bt 4 0 718菌株Cry1类原毒素所形成的菱形伴孢晶体 ,从中抽提出与其结合的 2 0kbDNA。经NdeⅠ酶切消化后亚克隆构建文库 ,通过PCR RFLP及测序筛选出含cry1Ac基因的转化子。然后设计引物PCR扩增出cry1Ac基因的ORF并与pET30a连接 ,转化E .coliBL2 1(DE3) ,高效表达了 14 1kD蛋白。表达蛋白占总蛋白量的 5 0 %以上 ,且 90 %以上以包涵体形式存在。利用穿梭载体pHT30 4构建表达质粒pHTX4 2 ,电转化Bt无晶体突变株XBU0 0 1,获得重组菌株HTX4 2 ,经SDS PAGE分析 ,cry1Ac基因得到强表达 ,蛋白质定量分析显示目的蛋白量占总蛋白量的 79 2 8% ,且其在细胞中累积达细胞干重的 6 4 13% ,比文献报道的 2 5 %左右高了 1倍以上。原子力显微镜 (AtomicForceMicroscopy ,AFM)检测显示 ,目的基因在大肠杆菌 (E .coli)中表达的包涵体呈不规则形状且较小 ,而在无晶体突变株中表达的晶体呈典型菱形晶体 ,大小约为 1 2 μm× 2 0 μm。生测结果显示 ,包涵体与晶体对小菜蛾 (Plutellaxylostella)幼虫均有高效杀虫活性。本研究为构建高效杀虫工程菌及进一步阐明Bt伴孢晶体中 2 0kbDNA分?The Cry1A Crystal Protein from Bacillus thuringiensis is associated with DNA, but the role and sequences of these DNA molecules are unknown. Cry1A bipyramidal crystals from B. thuringiensis strain 4 0718 was selectively dissolved and associated DNA was extracted from protoxin. The DNA was digested with Nde Ⅰ to obtain 3 to 5 kb fragments and then the fragments were subcloned into pMD18 T vector, screening of recombinants were done by PCR RFLP and sequencing. The ORF of cry 1Ac gene was amplified by primers designed and then subcloned. The 3 5 kb Bam HⅠ and Sal Ⅰ fragments of pMDX35 was inserted into the pET30a vector, giving 8 9 kb recombinant plasmid, pETX35 ETX35 strain were obtained by transformed pETX35 into Bl21 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies. Quantitative protein analysis indicated that the amount of 141 kD protein was above the level of 51 36% of total cellular protein. Plasmid pHTX42 constructed from shuttle vector pHT304 was transformed B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant HTX42 The recombinant protein was found with a molecular mass of 130 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Scanning analysis indicated that the expressed protein accounted up to 79 28% of total cellular proteins and accumulated in the cells mounted up to 64 13% of cellular dry weight. Under Atomic Force Microscopy (AFM), typical bipyramidal crystals from HTX42 strain were found with a size of 1 2 μm×2 0 μm. Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against the larvae of Plutella xylostella . On such a base, constructing insecticidal recombinant and analyzing the source, structure, and function of the 20 kb DNA can be further achieved.

关 键 词：苏云金芽孢杆菌 伴孢晶体 20kbDNA CRY1AC基因 表达 小菜蛾 克隆 

分 类 号：Q786[生物学—分子生物学]