【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。更多还原

【Abstract】 Acyl-CoA delta 9 desaturase( ACD9 des) is a key functional gene of fatty acid metabolism.To study the ACD9 des from Chrysomya megacephala( Fabricius),the full-length c DNA sequence of ACD9 des was cloned by RT-PCR and RACE followed by its bioinformatics analyses. The full-length of ACD9 des cDNA( GenBank accession number KF835695) is 1 429 bp,which contains a 1 146 bp open reading frame( ORF) encoding 381 amino acids. The length of 5’UTR was 138 bp and the 3’UTR was about 114 bp. The ORF encodes an ACD9 des protein with a molecular weight of 43. 47 kDa and an isoelectric point of 9. 06,whose amino acid sequence is 66%-93% identical to other ACD9 des from insects and contains a conserved fingerprint with 7 unique motifs belonged to acyl-CoA desaturase families. The phylogenetic analysis showed that the ACD9 des from C. megacephala is evolutionarily mostconsistent with Delia antiqua. The ORF of ACD9 des was then cloned into a prokaryotic expression vector pET-44 a( +) and the expression of ACD9 des 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 ACD9 des. Moreover,the ACD9 des 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 250 m M imidazole. Our researches provide a solid foundation for further exploration of fatty acid metabolism in C. megacephala.更多还原