Bt工程菌HD8Ea2的构建及Cry8Ea2蛋白对华北大黑鳃金龟中肠微生物的影响
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摘要
华北大黑鳃金龟(Holotrichia oblita)属于鞘翅目金龟甲总科(Scarabaeoidae),其幼虫对农、林、牧草等都有为害,在我分布广泛,种群数量大,危害严重。
苏云金杆菌(Bacillus thuringiensis,简称Bt)是革兰氏阳性细菌(G~+),在其生活史中能产生多种杀虫晶体蛋白,是目前应用最为广泛而有效的一种生物杀虫剂。本研究从全国各地的土样中筛选得到含cry8类基因的野生型菌株GW11,该菌株对华北大黑鳃金龟具有杀虫活性,LC_(50)为6.43×10~6cell/mL。Cry8Ea2蛋白是一种新型苏云金杆菌杀虫晶体蛋白。本研究通过穿梭载体pSXY422b将cry8Ea2导入Bt无晶体突变菌株HD73~-(cry~-)中,获得工程菌HD8Ea2。Cry8Ea2蛋白在工程菌中获得表达。通过分子筛层析,成功获得纯化的130kDa活性毒蛋白。用改进的生物活性测定方法进行Cry8Ea2蛋白对华北大黑鳃金龟一龄幼虫的杀虫活性测定,LC_(50)为0.2679μg/mL。
本研究应用16S rDNA PCR-RFLP方法,对正常生长的华北大黑鳃金龟二龄幼虫和饲喂Cry8Ea2蛋白的二龄幼虫中肠微生物进行了研究。从正常生长和饲喂Cry8Ea2蛋白的二龄幼虫中分别得到300个含有16S rDNA的克隆,分别随机挑选24个克隆进行PCR-RFLP的鉴定,结果发现:源自正常二龄幼虫的的24个克隆呈现17种不同带型;源自饲喂Cry8Ea2蛋白的二龄幼虫的24个阳性克隆呈现5种不同带型。
Having damage to agriculture, forestry, pastured and other at the stage of larvae, Holotrichia oblita belongs to coleopteran of Scarabaeoidae. Holotrichia oblita is of widely distribution, large population and serious harm, and is a important plant pest in China.
As a Gram-positive bacterium Bacillus thuringiensis can produce a variety of insecticidal crystal proteins in its life cycle, and is one of the most effective and widely used microbial insecticides at present. In this study, a wild-type strain named GW11 haboring cry8 gene was screened from soil samples collected from diffent areas of China. Insecticidal activity of GW11 to H. oblita was tested, and the LC_(50) is 6.43×10~6 cell/mL. Bt Cry8Ea2 protein is a new type of insecticidal crystal protein. cry8Ea2 was cloned into E. coli-Bt shuttle vector pSXY422b, resulting recombinant pScry8Ea2, and pScry8Ea2 was transformed into the Bt acrystalliferous mutant, generating engineered Bt strain HD8Ea2. Cry8Ea2 protein was successfully expressed in HD8Ea2 strain. By molecular sieve chromatography, 130 kDa of toxin protein Cry8Ea2 was purified. Using improved bioassay method, activity of the Cry8Ea2 toxin was evaluated, and the LC_(50) value to first instars larvae of H. oblita was 0.2679μg/mL.
H. oblita was normally feed in control group, and was feeded Cry8Ea2 protein in treatment group. Midgut microorganisms was analyzed by 16S rDNA gene amplification and PCR-RFLP analysis, 300 clones harboring 16S rDNA of bacteria from each group larvae were got, and 24 clones were randomly selected to perform PCR-RFLP analysis. 17 PCR-RFLP polymorphic bands appeared in control group, and 5 PCR-RFLP polymorphic bands in treatment group.
苏云金杆菌(Bacillus thuringiensis,简称Bt)是革兰氏阳性细菌(G~+),在其生活史中能产生多种杀虫晶体蛋白,是目前应用最为广泛而有效的一种生物杀虫剂。本研究从全国各地的土样中筛选得到含cry8类基因的野生型菌株GW11,该菌株对华北大黑鳃金龟具有杀虫活性,LC_(50)为6.43×10~6cell/mL。Cry8Ea2蛋白是一种新型苏云金杆菌杀虫晶体蛋白。本研究通过穿梭载体pSXY422b将cry8Ea2导入Bt无晶体突变菌株HD73~-(cry~-)中,获得工程菌HD8Ea2。Cry8Ea2蛋白在工程菌中获得表达。通过分子筛层析,成功获得纯化的130kDa活性毒蛋白。用改进的生物活性测定方法进行Cry8Ea2蛋白对华北大黑鳃金龟一龄幼虫的杀虫活性测定,LC_(50)为0.2679μg/mL。
本研究应用16S rDNA PCR-RFLP方法,对正常生长的华北大黑鳃金龟二龄幼虫和饲喂Cry8Ea2蛋白的二龄幼虫中肠微生物进行了研究。从正常生长和饲喂Cry8Ea2蛋白的二龄幼虫中分别得到300个含有16S rDNA的克隆,分别随机挑选24个克隆进行PCR-RFLP的鉴定,结果发现:源自正常二龄幼虫的的24个克隆呈现17种不同带型;源自饲喂Cry8Ea2蛋白的二龄幼虫的24个阳性克隆呈现5种不同带型。
Having damage to agriculture, forestry, pastured and other at the stage of larvae, Holotrichia oblita belongs to coleopteran of Scarabaeoidae. Holotrichia oblita is of widely distribution, large population and serious harm, and is a important plant pest in China.
As a Gram-positive bacterium Bacillus thuringiensis can produce a variety of insecticidal crystal proteins in its life cycle, and is one of the most effective and widely used microbial insecticides at present. In this study, a wild-type strain named GW11 haboring cry8 gene was screened from soil samples collected from diffent areas of China. Insecticidal activity of GW11 to H. oblita was tested, and the LC_(50) is 6.43×10~6 cell/mL. Bt Cry8Ea2 protein is a new type of insecticidal crystal protein. cry8Ea2 was cloned into E. coli-Bt shuttle vector pSXY422b, resulting recombinant pScry8Ea2, and pScry8Ea2 was transformed into the Bt acrystalliferous mutant, generating engineered Bt strain HD8Ea2. Cry8Ea2 protein was successfully expressed in HD8Ea2 strain. By molecular sieve chromatography, 130 kDa of toxin protein Cry8Ea2 was purified. Using improved bioassay method, activity of the Cry8Ea2 toxin was evaluated, and the LC_(50) value to first instars larvae of H. oblita was 0.2679μg/mL.
H. oblita was normally feed in control group, and was feeded Cry8Ea2 protein in treatment group. Midgut microorganisms was analyzed by 16S rDNA gene amplification and PCR-RFLP analysis, 300 clones harboring 16S rDNA of bacteria from each group larvae were got, and 24 clones were randomly selected to perform PCR-RFLP analysis. 17 PCR-RFLP polymorphic bands appeared in control group, and 5 PCR-RFLP polymorphic bands in treatment group.
引文
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