苏云金芽孢杆菌Cry1Aa和Cry1Ca结构域交换对晶体形态和杀虫活性影响

通过体外重组的方法,实现了苏云金芽孢杆菌杀虫晶体蛋白Cry1Aa和Cry1Ca的功能性结构域Ⅰ、Ⅱ和Ⅲ的互换,得到了6株苏云金杆菌重组菌株BT-ACC,BT-AAC,BT-ACA,BT-CAA,BT-CCA和BT-CAC。SDS-PAGE和Westernblot分析表明,重组菌株BT-CAA和BT-CCA能表达产生135kDa左右的杂交晶体蛋白Cry1CAA和Cry1CCA,但其蛋白表达量较野生型Cry1Aa和Cry1Ca低。用牛胰蛋白酶对杂交晶体蛋白Cry1CAA、Cry1CCA及野生型Cry1Aa和Cry1Ca进行消化,证明所有晶体蛋白都能产生65kDa的活性毒素。电镜观察发现,野生菌株BT-Cry1Aa和BT-Cry1Ca形成典型的菱形晶体,而重组菌株BT-CCA和BT-CAA则形成球形或颗粒状杂交晶体。纯化晶体的生物测定显示,杂交晶体蛋白Cry1CAA和Cry1CCA对甜菜夜蛾的毒力比野生型晶体蛋白降低3~5倍,对棉铃虫的毒力比野生型晶体蛋白降低了190~260倍。研究结果表明,苏云金杆菌晶体蛋白不同结构域的相互作用会影响杂交晶体蛋白的表达、晶体形态和杀虫活性。

Domain swapping of Cry1Aa and Cry1Ca from Bacillus thuringiensis influence crystal formation and toxicity

Bacillus thuringiensis has been successfully used for agricultural pest and medical insect control with its significant benefits based on environmental and safety considerations. However, the deficiency of this pesticide, such as limited spectrum of insecticidal activity, low toxicity to the targets and the inducement of insect resistance, results in the urgent need to exploit new resources of B. thuringiensis or to modify known strains by genetic engineering. In this study, six recombinant Bacillus thuringiensis BT-ACC, BT-AAC, BT-ACA, BT-CAA, BT-CCA and BT-CAC were constructed through the domain swapping between crystal protein Cry1Aa and Cry1Ca. SDS-PAGE and Western blot revealed that only the recombinant BT-CAA and BT-CCA produced a 135kDa chimeric protein Cry1CAA and Cry1CCA respectively, but the production level was lower than the native protein CrylAa and CrylCa. These chimeric crystal proteins could be activated by trypsin, giving a 65kDa protease-resistant core toxin as the native crystal proteins Cry1Aa and Cry1Ca. Electron microscopy study indicated that the two chimeric proteins could be produced and accumulated as spherical or granules crystals during sporulation of BT-CAA and BT-CCA, whereas native Cry1Aa and Cry1Ca were accumulated as bipyramidal crystals. Unexpectedly, the toxicity of purified chimeric crystal proteins Cry1CAA and Cry1 CCA was 3 - 5 times lower to Spodoptera exigua, and 190 - 260 times lower to Helicoverpa armigera than that of native Cry1Aa and Cry1Ca. These data suggested that the domain swapping of different crystal proteins might influence the formation and toxicity to targets of chemeric crystal proteins.