苏云金芽胞杆菌cry1Ac与烟草几丁质酶tchiB双价基因克隆表达及其杀虫增效作用研究

将苏云金芽胞杆菌(Bacillus thuringiensis，Bt)4.0718菌株质粒上的cry1Ac基因和烟草几丁质酶tchiB基因 (去掉信号肽或去信号肽再加肠激酶位点)构建了重组基因。经过双酶切和亚克隆，将带有cry1Ac基因上游启动序列和下游终止序列的重组基因片段克隆至穿梭载体pHT315，分别构建重组质粒pHUAccB6、pHUAccB7，在大肠杆菌中扩增后，将两个重组质粒分别电转化苏云金芽胞杆菌无晶体突变株XBU001中，获得重组菌株HAccB6和HAccB7。经液体双相胞晶分离提取离心后，将晶体和上清液分别进行SDS-PAGE分析，双价基因重组与cry1Ac基因在无晶体突变株中表达量相比较，几丁质酶活性提高5.2倍，双价重组蛋白表达量显著提高，主要产生130kDa蛋白条带。经定量分析：双价重组目的晶体蛋白占总蛋白量的61.38%；Cry1Ac蛋白占总蛋白量的42%。发酵上清液经60％硫酸铵沉淀，显示出一条分子量为18kDa新蛋白条带。经原子力显微镜和电子显微镜观察，表达后的重组蛋白呈菱形或椭原形晶体，其规格约为1.5×3.0μm；经生测分析，重组菌株HAccB6和 HAccB7毒力相近，与HAc菌株比较毒力提高4.5倍，对棉铃虫（Helicourpa armigora）具有高效杀虫活性，其3d LC₅₀值分别为9.1μg/mL和11.34μg/mL。研究结果表明，烟草几丁质酶与cry1Ac双价基因重组表达产物具有杀虫增效作用。

Cloning and expression of the cry1Ac-tchiB fusion gene from Bacillus Thuringinesis and Tobacco and its insecticidal synergistic effect

A new fusion gene cry1Ac-tchiB was constructed to enhance the toxicity of crystal proteins, the cry1Ac gene of Bacillus thuringiensis strain 4.0718 was combined with a tchiB (deleted signal peptide and Enterokinase site sequence). In this process, the Enterokinase site sequence was inserted into the midst of these two genes. Then the combined fragment carrying the upstream promoter region and the downstream terminator region of cry1Ac gene were cloned into the shuttle vector pHT315. And after a series of enzyme digestions and subclonings two new expression vector pHUAccB6 and pHUAccB7 were obtained. The two vectors were transformed into B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant strain HAccB6 and HAccB7. The fusion gene was expressed and the expression product was detected by SDS-PAGE. The result indicated that the recombinant Cry1Ac-tchiB protein accumulated to the level of 61.38% of total bacterial proteins. Cry1Ac protein accumulated to the level of 42% of total bacterial proteins. Chitinase activities is 5.2 time more than that of the control strain. Under Atomic Force Microscopy and SEM of crystals protein, there were some bipy ramidal crystals with a size of 1.5 x 3.0 microm. Bioassay showed that the fusion crystals from recombinant strain HAccB6 and HAccB7 were high toxic against third-instar larvae of Helicourpa armigora with the LC50 (after 72h) value of 9.10 micro/mL and 11.34 micro/mL.The constructed fusion proteins had more toxicity than Cry1Ac crystal proteins. The study will enhances the toxicity of B. thuringiensis Cry toxins protein and makes a ground for constructing the fusion genes of B. thuringiensis cry gene and other foreign toxin genes and the recombinant strain with high toxicity.