对甜菜夜蛾具有杀虫活性的Bt菌株cry1Ca基因研究

根据已报道的14个cry1Ca基因,设计能够扩增cry1Ca全长基因的简并引物。利用该引物对本实验室分离的472株Bt菌株进行筛选。PCR扩增发现22株菌含有cry1Ca基因。对22株菌株进行cry基因多样性分析,结果获得5种类型酶切图谱,表明这些菌株分为5种类型。SDS-PAGE结果显示22株菌均表达约130ku的蛋白。Western Blotting结果证实这些株菌中cry1Ca基因均正常表达。提取菌株的晶体蛋白,经胰蛋白酶活化,对甜菜夜蛾Spodoptera exigua(Hübner)]进行初步生测,结果表明22株菌中活化的Cry蛋白对甜菜夜蛾均表现出很强的毒杀作用。进一步从5个类型的菌中各挑选一株毒力较高的菌株进行LC_(50)的测定,结果证实它们均具有很高毒力,其中,T1-E12(0.087μg/g)、B16-C8(0.103μg/g)、T1-B8(0.202μg/g)的活性与阳性对照菌株G03(0.090μg/g)相当,具有生产应用潜力。本研究为新型高效Bt菌株的挖掘奠定了基础。

Analysis of cry1Ca gene from Bacillus thuringiensis strains with high insecticidal activity against Spodoptera exigua

One pair of degenerate primers,designed based upon the 14 known sequences of cry 1Ca genes,was used to identify cry 1Ca genes from 472 wild isolates of Bacillus thuringiensis screened in our laboratory,and there were 22 isolates containing cry 1Ca genes.Five patterns of restriction fragment were obtained by the analysis of cry gene diversity depended on PCR-RFLP,indicating that 22 B .thuringiensis isolates could be classified as five types.SDS-PAGE analysis results showed that a main protein band with approximate 130 ku molecular mass was detected in each strain.Western Blotting analysis results also demonstrated that Cry1Ca proteins were expressed normally in all of the 22 strains.The preliminary bioassay was conducted with trypsin-activated Cry proteins ex-tracted from 22 Bt isolates against the neonate larvae of Spodoptera exigua (Hübner).The results showed that all of these strains hade high insecticidal activity against target larvae.The LC50 values of the five selected Bt isolates representing five types against S .exigua were measured,and the results indicated that the toxicity of the five strains was very high;the insecticidal activity of the strains T1-E12 (0.087 μg/g),B16-C8 (0.103 μg/g)and T1-B8 (0.202 μg/g)was similar to that of the positive strain G03 (0.090 μg/g),and these strains have great potential for commercialization.Our findings will lay the foundation for discovery of novel B .thuringiensis isolates.