昆虫学报2012，Vol.55Issue(8)：885-894,10.
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
摘要
Abstract
[ Aim ] The main aim of this research was to clone and align the full-length cDNA and promoter sequences of desatA 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 desatA 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 desatA 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 desalA 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.关键词
家蚕/野桑蚕/脂肪酸脱氢酶/全长cDNA/启动子/原核表达Key words
Bombyx mori/ Bombyx mandarina/ fatty acid desaturase/ full-length cDNA/ promoter/ prokaryotic expression分类
生物科学引用本文复制引用
陈全梅,程道军,马振刚,胡晓明,查幸福,赵萍..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[J].昆虫学报,2012,55(8):885-894,10.基金项目
国家重大基础研究规划(“973”计划)项目(2012CB114600) （“973”计划）
国家高技术研究发展计划(“863”计划)项目(2011AA00306) （“863”计划）
国家自然科学基金重点项目(30972147) （30972147）
