双价杀虫蛋白基因在荧光假单胞菌中的表达及增效  

EXPRESSION AND SYNERGISM OF TWO CRY INSECTICIDAL PROTEIN GENES IN PSEUDOMONAS FLUORESCENS'

作　　者：丁之铨[1] 张杰[1] 宋福平[1] 黄大昉[2] 李季伦[3] 

机构地区：[1]中国农业科学院植物保护研究所,北京100004 [2]中国农业科学院生物技术研究中心,北京100081 [3]中国农业大学生物学院,北京100094

出　　处：《微生物学报》2000年第6期573-578,共6页Acta Microbiologica Sinica

基　　金：国家"8 63"项目资助!( 1 0 1 -0 3 -0 1 -0 1 )&&

摘　　要：利用广宿主质粒载体 pJMS6α lac将苏云金芽胞杆菌 (Bacillusthuringiensis)杀虫晶体蛋白基因cry1Ac和cry2Aa基因分别及一起进行克隆 ,将重组质粒导入能在多种作物上定殖、对植物病菌有良好抑菌和防治作用的荧光假单胞菌 (Pseudomonasfluorescens)P30 3菌株 ,分别得到工程菌株IPP1 0 1、IPP2 0 1和IPP2 0 2。PCR RFLP和Southernblot检测均证明目的基因已经导入了工程菌。SDS PAGE电泳显示工程菌中存在明显的Cry1Ac蛋白带 ;透射电镜观察发现含cry1Ac基因的两个菌株IPP1 0 1和IPP2 0 2中杀虫蛋白形成了典型的菱形晶体和蛋白包含体 ,而在野生P30 3菌株中均无这些结构。这些结果说明 ,工程菌中cry1Ac基因得到了很好表达。室内杀虫试验表明 :工程菌对棉铃虫初孵幼虫的致死中浓度 (LC50 ) ,只含cry1Ac的IPP1 0 1为 0 0 0 81 2mL/g饲料 ,只含cry2Aa的IPP2 0 1为 0 0 2 60 4mL/g饲料 ,含双基因的IPP2 0 2为 0 0 0 1 86mL/g饲料 ;HD73为 0 0 0 1 70mL/g饲料。cry1Ac和cry2Aa双基因表达产物具有显著增效作用 ,共毒系数达 332 8。Several engineered \%Pseudomonas fluorescens\%(Pf) strains were constructed mainly based on a \%Pseudomonas\% plasmid pJMS6α lac and two insecticidal crystal protein genes of \%Bacillus thuringiensis, cry1Ac and cry2Aa , and the host Pf strain, P303, which was with highly antifungal activity to some plant disease fungi and colonizing ability on a wide range of plants. The DNA introduction was confirmed by PCR\|RFLP and Southern blot. The 132kD insecticidal protein was detected in IPP101 and IPP202 by SDS\|PAGE and rhombic insecticidal protein crystals of them were observed through electromicroscope, also indicating that cry1Ac gene was highly expressed. The results of insecticidal bioassay indicated that IPP101 was more toxic than IPP201, and IPP202 was the most toxic among the 3 strains. LC\-\{50\} to the neonates of cotton boll worm(\%Helicoverpa armigera\%) were 0.02604,0.00812 and 0.00186mL/g feed, consecutively. In IPP202, two gene products showed significant synergism, with the co\|toxicity coefficient of 332.8.

关 键 词：荧光假单胞菌 杀虫晶体蛋白基因 表达 

分 类 号：Q786[生物学—分子生物学]