苏云金芽胞杆菌等离子复合体诱变提高抗鳞翅目害虫的杀虫毒力

Improvement of the virulence of Bacillus thuringiensis to lepidopteran pests by ARTP and NTG mutagenesis

摘要:

【目的】苏云金芽胞杆菌(Bacillus thuringiensis,Bt) D18对鳞翅目、鞘翅目等农业害虫具有杀虫毒力,本研究拟通过复合诱变育种,筛选出杀虫毒力更高的突变菌。【方法】经四轮室温常压等离子体(ARTP)诱变和一轮ARTP-NTG复合诱变后,镜检形态观察与生物毒力测定筛选高毒力菌株,SDS-PAGE检测Cry蛋白,qRT-PCR定量cry基因的表达,揭示突变菌株毒力提高的原因。【结果】复合诱变筛选到2株高毒力突变菌株An-L5-1和An-L5-7,与出发菌株Bt D18相比,An-L5-1和An-L5-7的生长衰亡期略微提前;芽胞形成能力增强;Cry蛋白表达量分别提高了80.47%和88.31%;对小菜蛾和黏虫的杀虫毒力显著增强。【结论】突变菌株An-L5-1和An-L5-7杀虫活性提高主要是由于毒力基因cry1Aa、cry2Aa和调控基因sigK的表达显著上调。

关键词:苏云金芽胞杆菌;杀虫晶体蛋白;室温常压等离子体;复合诱变

Abstract:

[Objective] Bacillus thuringiensis(Bt) D18 is virulent to agricultural pests of Lepidoptera and Coleoptera.This study aims to improve the virulence of D18 by compound mutagenesis.[Methods] After four rounds of atmospheric and room temperature plasma (ARTP) mutagenesis and one round of ARTP-N-methyl-N'-nitro-N-nitrosoguanidine (ARTP-NTG) mutagenesis,highly virulent mutants were screened out by microscopy and virulence bioassay.To reveal the reason for the increase of virulence,we detected the Cry proteins by SDS-PAGE and key cry genes by qRT-PCR.[Results] Two highly virulent mutants,An-L5-1 and An-L5-7 were screened out,compared with the wild strain D18,An-L5-1 and An-L5-7 demonstrated slightly early decline,improved ability of sporulation,80.47% and 88.31% rise of Cry protein expression,and significantly stronger virulence to Plutella xylostella and Mythimna seperata.[Conclusion] The increase in the virulence of An-L5-1 and An-L5-7 was mainly attributed to the significantly up-regulated expression of cry1Aa,cry2Aa,and sigK.

Key words:Bacillus thuringiensis;insecitcidal crystal protein;ARTP;compound mutagenisis