棉卷叶野螟泛素基因的克隆、序列分析及原核表达

本研究用RT-PCR方法，克隆了棉卷叶野螟Haritalodes derogata (Fabricius)泛素基因编码区，GenBank登录号为EU580145。序列分析表明，该编码区长228 bp，编码76个氨基酸，推测的编码蛋白的相对分子质量和等电点分别为8.53 kD和5.83。同源性比较发现，棉卷叶野螟泛素基因与其他10种昆虫泛素基因在氨基酸水平上具有93%以上的相似性。系统发育树显示棉卷叶野螟与斜纹夜蛾Spodoptera litura (Fabricius)遗传距离较近，通过同源建模获得了该棉卷叶野螟基因编码蛋白的理论三维结构。将棉卷叶野螟泛素基因与pET-32a(+)连接，构建原核表达载体pET-32a-ub，经IPTG诱导，棉卷叶野螟泛素基因在大肠杆菌BL21(DE3) 中高效表达。本研究成功克隆了棉卷叶野螟泛素基因的编码区，并经Western blotting分析证明实现了该基因的原核表达，为进一步研究其在该昆虫体内的作用机理奠定了基础。

Cloning, sequence analysis and prokaryotic expression of the ubiquitin gene of Haritalodes derogata (Fabricius) (Lepidoptera: Pyralidae)

Ubiquitin plays a very important role in regulating non-lysosomal ATP-dependent protein degradation.The coding sequence of Haritalodes derogata(Fabricius)ubiquitin gene was cloned and determined(GenBank accession no.EU580145).The opening reading frame(ORF)is 228 bp in length,encoding 76 amino acids with the molecular weight of 8.53 kD and theoretical isoelectric point of 5.83.Multiple sequence alignment indicated that H.derogata ubiquitin shared more than 93% identity with those of other ten insect species at the amino acid level,indicating high homology among them.Phylogenetic tree indicated that H.derogate has close relationship with Spodoptera litura(Fabricius).The theoretical three-dimensional structure of this gene was displayed by homology modeling.Using pET-32a( )as a fused expression vector,a recombinant plasmid pET-32a-ub containing the coding sequence of H.derogata ubiquitin gene was constructed.Western blotting indicated that the H.derogata ubiquitin gene was expressed successfully in the BL21(DE3)strain of Escherichia coli induced with IPTG.The results may serve as basis for further studying the function of ubiquitin gene in this insect.