家蚕和野桑蚕脂肪酸脱氢酶desat4全长cDNA和启动子的克隆及其原核表达

Cloning of full-length cDNA and promoter sequences of fatty acid desaturase gene desat4 from silkworms,Bombyx mori and B. mandarina,and its prokaryotic expression

【目的】获得家蚕Bombyx mori和野桑蚕Bombyx mandarina脂肪酸脱氢酶基因desat4的cDNA及其启动子序列,并应用原核表达系统获得目的蛋白质,为进一步研究该基因的功能提供基础。【方法】利用RACE技术克隆家蚕和野桑蚕desat4基因全长cDNA序列,基于家蚕全基因组序列设计引物克隆它们的启动子,并采用原核表达技术对该基因进行异源表达。【结果】克隆得到家蚕与野桑蚕desat4基因全长cDNA,长度分别为1717 bp和1718 bp,ORF长度为1059 bp,编码352个氨基酸残基,结构上有3个组氨酸保守区和4次跨膜结构域,说明该蛋白质是膜蛋白。同源性分析发现Desat4氨基酸序列与烟草天蛾Manduca sexta MsexKPSE脱氢酶拥有88.9%相似性。克隆获得的家蚕和野桑蚕desat4基因启动子序列长度分别为1808 bp和870 bp,该区域包含有热休克因子HSF(heat shock factor)结合位点、NIT2结合位点和CdxA(caudal type homeobox transcription factor A)结合位点等等,并未发现有典型的TATA-box,但在靠近5'-UTR区域发现了转录起始因子特征序列。时期表达谱分析显示desat4基因从刚产下卵到成虫(蛾)的36个不同发育时间点都有持续稳定的表达。应用原核表达系统成功地表达了Desat4蛋白质,并用温和变性剂十二烷基-β-D-麦芽吡喃糖苷(dodecy l-β-D-maltoside,DDM)就能很好地促进该蛋白质的溶解。【结论】本研究成功地克隆了家蚕和野桑蚕desat4基因全长cDNA及其启动子序列并进行了序列比对分析,构建了desat4基因的原核表达载体并成功表达,为进一步研究该基因的功能提供参考。

[ Aim ] The main aim of this research was to clone and align the full-length cDNA and promoter sequences of desat4 gene from silkworms, Bombyx mori and B. mandarina, and construct prokaryotic expression vector to obtain a membrane protein Desat4 for further functional study. [ Methods] Full-length cDNA of the desat4 gene from silkworm was obtained through RACE technique, the encoded protein was expressed in Escherichia coli expression system, and its promoter sequences were cloned based on genome sequences of silkworm. [ Results ] The full-length cDNA of the desat4 gene from B. mori and B. mandarina was 1 717 bp and 1 718 bp, respectively. Their open reading frame （ORF） is 1 059 bp in length and encodes 352 amino acid residues with four transmembrane helixes and three conserved histidine clusters, which are essential for desaturase catalytic activity. The deduced amino acid sequence shares 88. 9% similarity to that of the fatty acid desaturase MsexKPSE （GenBank no. CAJ27975） of Manduca sexta. There is no typical TATA-box in promoter sequences, but there is a transcriptional initiator as well as other transcription factor binding sites, including HSF, NIT2, CdxA and so on. The membrane protein Desat4 was expressed in E. coli expression system and solubilized with mild detergent DDM. [ Conclusion ] The full-length cDNA and promoter sequences of desat4 gene from B. mori and B. mandarina were successfully cloned and comparatively analyzed. The membrane protein was expressed in vitro by pET system, thus providing a foundation for further functional study.