大头金蝇酰基辅酶AΔ9去饱和酶cDNA克隆与原核表达  

cDNA cloning and prokaryotic expression of acyl-CoA delta 9 desaturase from Chrysomya megacephala(Fabricius)

作　　者：张敏[1,2] 张古忍[2] ZHANG Min;ZHANG Gu-Ren(Department of Biochemistry and Molecular Biology,University of South China,Hengyang 421001,Hunan Province,China;Key Laboratory for Biocontrol & Institute of Entomology,Sun Yat-sen University,Guangzhou 510275,China)

机构地区：[1]南华大学生物化学与分子生物学教研室,湖南衡阳421001 [2]中山大学有害生物控制与资源利用国家重点实验室,广州510275

出　　处：《环境昆虫学报》2018年第6期1306-1315,共10页Journal of Environmental Entomology

基　　金：中央高校基本科研业务费专项资金(3165004);南华大学博士启动基金(2014XQD)

摘　　要：为深入研究大头金蝇Chrysomya megacephala (Fabricius)脂肪酸代谢关键功能基因酰基辅酶AΔ9去饱和酶(ACD9des),运用RT-PCR和RACE技术,获得其cDNA全长序列,并对其进行生物信息学分析。大头金蝇ACD9des基因cDNA (GenBank登录号为KF835695)全长1 429 bp,其中开放阅读框(ORF)为1 146 bp,编码381个氨基酸,5'UTR长度为138 bp,3'UTR约为114 bp。ORF编码的蛋白质分子量为43. 47 kD,等电点9. 06,氨基酸序列与其他昆虫酰基辅酶A去饱和酶一致性高达66%-93%,且含有由7个酰基辅酶A去饱和酶蛋白家族特有的保守模式(motif)所构成的指纹(IPR015876)。大头金蝇ACD9des在进化上与葱蝇Delia antiqua最趋于一致。将ACD9des的ORF克隆到原核表达载体p ET-44a(+),并利用Rosetta (DE3)感受态细胞进行ACD9des原核表达。Western Blot分析表明,IPTG诱导表达的特异性蛋白可以与anti-His抗体特异性结合,大小与预期理论值(43. 47 kDa)相符,为ACD9des。该蛋白主要存在于上清溶液中,为可溶性表达。最后利用含250 mM咪唑洗脱液和镍离子亲和层析柱对扩大培养获得的重组蛋白进行了纯化收集。本文的研究结果为大头金蝇功能基因的深入研究提供了坚实的基础。Acyl-CoA delta 9 desaturase (ACD9des) is a key functional gene of fatty acid metabolism. To study the ACD9des from Chrysomya megacephala (Fabricius),the full-length cDNA sequence of ACD9des was cloned by RT-PCR and RACE followed by its bioinformatics analyses. The full-length of ACD9des cDNA (GenBank accession number KF835695) is 1 429 bp,which contains a 1146bp open reading frame (ORF) encoding 381 amino acids. The length of 5'UTR was 138bp and the 3'UTR was about 114 bp. The ORF encodes an ACD9des protein with a molecular weight of 43.47kDa and an isoelectric point of 9. 06,whose amino acid sequence is 66%-93% identical to other ACD9des from insects and contains a conserved fingerprint with 7 unique motifs belonged to acyl-CoA desaturase families. The phylogenetic analysis showed that the ACD9des from C. megacephala is evolutionarily most consistent with Delia antiqua. The ORF of ACD9des was then cloned into a prokaryotic expression vector pET-44a(+) and the expression of ACD9des was performed using Rosetta (DE3) competent cells. Western Blot analysis demonstrated that the specific protein induced by IPTG could specifically bind with anti-His antibody,and its size was consistent with the expected theoretical value (43.47 kDa) of ACD9des. Moreover,the ACD9des protein is mainly detected in the supernatant solution. Purification of the recombinant protein obtained by expansion culture was finally performed by nickel ion affinity chromatography and an equilibration / elution containing 250mM imidazole. Our researches provide a solid foundation for further exploration of fatty acid metabolism in C. megacephala.

关 键 词：大头金蝇 酰基辅酶AΔ9 去饱和酶 ACD9des 基因克隆 原核表达 

分 类 号：Q963[生物学—昆虫学] S433[农业科学—农业昆虫与害虫防治]