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为了明确斜纹夜蛾Spodoptera litura对溴氰菊酯产生抗性的分子机理,本研究利用RT-PCR技术和RACE方法获得了1个斜纹夜蛾羧酸酯酶基因的全长cDNA序列,命名为Slest2。序列分析表明,该cDNA全长1796bp(GenBank登录号:DQ445461),5′和3′UTR区分别长63和119bp,开放阅读框编码一个由537个氨基酸残基组成的羧酸酯酶蛋白。通过对氨基酸同源性分析表明,该羧酸酯酶与其他物种的酯酶均具有很高的氨基酸相似性,并具有多个在不同酯酶蛋白家族中均保守的区域。采用实时定量PCR技术比较了Slest2在斜纹夜蛾抗、感品系中的表达水平。当以cDNA为模板检测mRNA转录水平时发现,Slest2在抗性品系中的转录水平是敏感品系的46.85倍;以基因组DNA为模板检测Slest2基因的拷贝数时发现,Slest2在抗、感性品系中的拷贝数无显著差异(前者为后者的1.16倍)。这些结果表明,抗性与敏感品系具有相似的Slest2基因拷贝数,但它们在抗性品系中的转录水平显著升高。由此推测Slest2基因的转录水平升高与斜纹夜蛾对溴氰菊酯的抗药性密切相关。
In order to clarify the molecular mechanism of the resistance of Spodoptera litura to deltamethrin, we obtained a full-length cDNA sequence of the carboxylesterase gene of Spodoptera litura by RT-PCR and RACE, and named it Slest2 . Sequence analysis showed that the full length cDNA of 1796bp (GenBank accession number: DQ445461) was 63 and 119 bp in 5 ’and 3’ UTR regions, respectively. The open reading frame encoded a carboxylesterase protein consisting of 537 amino acid residues. The amino acid homology analysis showed that the carboxylesterase had high amino acid similarity with esterases of other species and had multiple conserved regions in different esterase protein families. The expression of Slest2 in the resistant and susceptible strains of Spodoptera litura was compared by real-time quantitative PCR. When using cDNA as a template to detect mRNA transcription level, we found that Slest2 transcription level in the resistant strain was 46.85 times that of the susceptible strain. Using genomic DNA as a template to detect the copy number of Slest2 gene, we found that Slest2 transcription level in the resistant and susceptible strains No significant difference in copy number (the former is 1.16 times the latter). These results indicate that the resistant and susceptible lines have similar Slest2 gene copy numbers, but their transcription levels in the resistant lines are significantly elevated. It is speculated that the Slest2 gene transcription level and Spodoptera litura resistance to deltamethrin are closely related.