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E75是昆虫蜕皮级联反应中的早期转录因子之一。本研究运用RT-PCR和RACE技术,首次获得了斜纹夜蛾Spodoptera lituraE75D基因,命名为Sli-E75D(GenBank登录号:JQ266225),其开放阅读框全长1836bp,编码611个氨基酸残基,3’非翻译区全长为358bp,同时获得其5’非翻译区共341bp。经核苷酸序列比对分析,E75在鳞翅目昆虫间保守性较高,尤其3’非翻译区具有高保守性,但由于启动子不同,E75异构体mRNA5’端序列存在差异;经氨基酸序列比对,Sli-E75D与棉贪夜蛾Spodoptera littoralis、烟草天蛾Manduca sexta、家蚕Bombyx mori E75D的一致性分别为98.4%,79.3%和76.5%。基于E753’非翻译区的高保守性,利用PITA和RNAhybird程序预测了最有可能调控鳞翅目昆虫E75基因的3种miRNAs:miR-14,miR-33和miR-87。构建pET28a-Sli-E75D表达载体,分别转化BL21(DE3)和Transetta(DE3)菌株,检测大肠杆菌Escherichia coli稀有密码子对E75D原核表达的影响,SDS-PAGE结果显示,转化Transetta(DE3)菌株的pET28a-Sli-E75D可高效表达大小约74.79kD(含预测的67.19kDSli-E75D,7.6kDT7·Tag和His·Tag)的重组蛋白,与其理论分子质量基本吻合,而转化BL21(DE3)菌株的pET28a-Sli-E75D质粒只见微量重组蛋白表达。由于Transetta(DE3)菌株可补充大肠杆菌6种稀有密码子的tRNA,较BL21(DE3)更适合于E75D的外源表达。qPCR检测了斜纹夜蛾从末龄幼虫到成虫发育过程中各时间点Sli-E75的相对表达水平:Sli-E75在6龄幼虫期的表达量较低,从预蛹开始,表达量急剧升高,并在蛹中期达到最高峰,之后迅速下降,但成虫期表达水平又出现回升。这些结果有助于深入研究E75在昆虫蜕皮级联反应中的作用。
E75 is one of the early transcription factors in insect molting cascade. In this study, Spodoptera lituraE75D gene, named as Sli-E75D (GenBank accession number: JQ266225), was obtained for the first time by RT-PCR and RACE. The full length open reading frame of the Spodoptera litura E75D gene is 1836bp, encoding 611 amino acid residues, The total length of the untranslated region was 358 bp, and 341 bp was obtained from its 5 ’untranslated region. Nucleotide sequence alignment showed that E75 was highly conserved among Lepidoptera insects, especially in the 3’-untranslated region. However, due to different promoters, there were differences in the 5 ’end sequences of E75 isoforms; Amino acid sequence alignment showed that the identities of Sli-E75D and Spodoptera littoralis, Manduca sexta and Bombyx mori E75D were 98.4%, 79.3% and 76.5%, respectively. Based on the high conservation of the E753 ’untranslated region, three miRNAs most likely to regulate lepidopteran E75 gene were predicted using the PITA and RNAhybird programs: miR-14, miR-33 and miR-87. The pET28a-Sli-E75D expression vector was constructed and transformed into BL21 (DE3) and Transetta (DE3) strains. The Escherichia coli E75D prokaryotic expression vector was detected by SDS-PAGE. The transformants of Transetta pET28a-Sli-E75D could efficiently express recombinant protein of about 74.79kD (including the predicted 67.19kDSli-E75D, 7.6kDT7 Tag and His-Tag), which was consistent with the theoretical molecular mass of pET28a-Sli-E75D. The Sli-E75D plasmid only showed trace recombinant protein expression. Transetta (DE3) strain is more suitable for exogenous expression of E75D than BL21 (DE3) because it complements the six rare codons of E. coli. The relative expression level of Sli-E75 at different time points from the late-stage larvae to the adult was detected by qPCR. The expression level of Sli-E75 in the 6th instar larvae was low, , And peaked in mid-pupa, then dropped rapidly, but the expression level in adult stage rose again. These results help to further investigate the role of E75 in insect molting cascade.