北京植物园苏云金芽胞杆菌菌株的分离鉴定

Isolation of Bacillus thuringiensis strains and identification of cry genes from the samples collected in Beijing Botanical Garden

[目的]从北京植物园采集的土壤样品中分离高毒力的苏云金芽胞杆菌。[方法]采用温度法筛选Bt菌株,比对大质粒DNA图谱,区分不同类型的Bt菌株,PCR-RFLP方法对cry1～cry4 0类基因型进行鉴定,SDS-PAGE分析杀虫晶体蛋白,测定Bt分离株对大猿叶甲、小菜蛾幼虫的杀虫活性,筛选出高毒力的菌株。[结果]从149份土壤样品中分离出147株Bt,分为12种类型,含有菱形、方形、球形和不规则等晶体类型;6株菌中分别含有cry1Aa、cry1Ab、cry1Ac、cry1Ah、cry1Ba、cry1Be、cry1Ia、cry1La、cry2Ab和cry7Aa等基因类型,其余6株中不含有已知基因。有9株表达约130ku蛋白,1株表达约70ku蛋白,2株表达约150ku蛋白,有3株既表达130ku蛋白又表达60ku蛋白;发现一株对大猿叶甲具有较高毒力的菌株ZWY-7,1株对小菜蛾有高毒力的菌株ZWY-9,2株菌都没有检测到已知基因型。[结论]北京植物园中Bt分布广泛,类型多样,其中发现两株高毒力的菌株,有望获得新的杀虫基因。

[Objective]To isolate highly insecticidal Bacillus thuringiensis strains from soil samples collected in Beijing Botanical Garden.[Methods]B.thuringiensis strains were isolated from soil samples using temperature method.Comparison of plasmid DNA pattern by agarose gel electrophoresis was conducted to discriminate the types of the isolates.cry1-cry40 type genes were identified via PCR-RFLP method.SDS-PAGE was used to analyze insecticidal crystal proteins.Insecticidal activity of these isolates against Colaphellus bowringi larvae and Plutella xylostella larvae was assayed.[Results]One hundred and forty-nine soil samples were collected and 147 B.thuringiensis isolates were obtained.The types of the isolates were divided into 12 types,and these isolates contained bipyramidal,square,spherical and irregular crystal,respectively.cry1Aa,cry1Ab,cry1Ac,cry1Ah,cry1Ba,cry1Be,cry1Ia,cry1La,cry2Ab and cry7Aa type genes were found in 6 isolates,but the other cry gene-types were unknown.SDS-PAGE results showed that 9 isolates could express Cry proteins of 130 ku,1 of 70 ku,2 of 150 ku,and 3 of both 130 ku and 60 ku.Bioassay results of 12 isolates indicated that the isolate ZWY-7 was toxic to C.bowringi larvae,and the isolate ZWY-9 had highly insecticidal activity to P.xylostella larvae,but their cry gene-types were unknown.[Conclusion]The B.thuringiensis strains spread widely in Beijing Botanical Garden,belonging to different types.We found 2 Bt strains which were highly insecticidal,and it is possible to find more novel genes.