Bt Cry1Ac抗性粉纹夜蛾离体细胞与敏感细胞mRNA的差异显示

Differential Display of mRNA between the Bt Cry1Ac Resistant and Susceptible Cells of Trichoplusia ni

为了研究昆虫对Bt的抗性机制 ,以苏云金芽孢杆菌Cry1Ac活性毒素对粉纹夜蛾离体细胞连续筛选 80代 ,获得了高抗性细胞。采用DDRT PCR技术比较了该抗性细胞与非选择敏感细胞mRNA的差异 ,经反向RNA斑点印迹杂交确证了 5个片段为两种细胞的显著差异表达序列标签(ESTs)。序列分析结果表明 ,敏感细胞特有的三种ESTs都位于cDNA的非编码区 ,两种ESTs(GenBank注册号 :S1 ,CF32 2 4 1 5 ;S3,CF32 2 4 1 7)与数据库中EST没有显著同源性 ,另一种S2(GenBank注册号 :CF32 2 4 1 6)与已登录的某些昆虫的ESTs具有一定的同源性。从抗性细胞特有的R1 (GenBank注册号 :CF32 2 4 1 3)推导出的氨基酸序列与磷酸烯醇式丙酮酸羧激酶有 60 %～63%的同源性 ,从抗性细胞特有的R2 (GenBank注册号 :CF32 2 4 1 4)推导出的氨基酸序列与黏蛋白类有约 30 %的同源性。这些差异表达基因可能与抗性的形成相关。

In order to get more information about the mechanism of insect resistance to Bacillus thuringiensis (Bt) toxin, the high level of resistant Trichoplusia ni cell line BTI-TN-5B1-4 against Bt toxin Cry1Ac had been achieved by continuous selection for 80 generations. Differential display of mRNA between the resistant cells selected by Cry1Ac and the non-selected susceptible cells was analyzed with DDRT-PCR technique,and 5 bands were proved to be special expressed sequence-tags (EST) by RNA rev-Northern hybridization using DIG labeled second strand cDNA probes.Sequence analysis of ESTs was performed.two (GenBank accession:CF 322415,CF322417) of three special bands from the susceptible cells had no homology with the known ESTs,but the other (GenBank accession:CF322416) was similar to the ESTs of some insects.The deduced amino sequence from a special EST (GenBank accession:CF322413) for the resistant cells shared 60%～63% homology with phosphoenolpyruvate carboxykinase,and that from the other EST (GenBank accession:CF322414) shared 30% homology with mucin.The differently expressed genes might be associated with the selected-resistance of Trichoplusia ni cell line against Bt Cry1Ac.;