松褐天牛泛素基因的克隆及表达  

Cloning and Expression of the Ubiquitin Gene of Monochamus alternatus

作　　者：林同[1,2] 张宇宏[1] 常润磊[1] 张琪[1] 温秀军[1] 

机构地区：[1]华南农业大学林学院,广州510642 [2]华南农业大学经济林研究中心,广州510642

出　　处：《林业科学》2009年第9期101-105,共5页Scientia Silvae Sinicae

基　　金：广东省自然科学基金项目(8151064201000068);华南农业大学经济林研究中心资助项目(4400-32990190);华南农业大学校长基金项目(4400-208012)

摘　　要：设计一对简并引物,应用RT-PCR技术,从松褐天牛细胞中克隆泛素基因的编码区,GenBank登录号EU433567。序列分析表明:该编码区的长度为228bp,编码76个氨基酸,编码蛋白的相对分子质量为8.49ku,理论等电点为5.83。同源性比较发现,松褐天牛泛素基因与其他10种昆虫泛素基因在氨基酸水平上具有94%～98%的相似性。基于核苷酸水平上的系统发育树显示松褐天牛与斜纹夜蛾、草地贪夜蛾遗传距离较近;通过同源建模获得了松褐天牛泛素基因编码蛋白的理论三维结构。将泛素基因与pET-32a(+)连接,构建原核表达载体pET-32a-ub,经IPTG诱导,松褐天牛泛素基因在大肠杆菌BL21(DE3)中高效表达,并经Westernblotting分析验证该表达,为研究泛素在松褐天牛体内的功能奠定基础。The coding sequence of the ubiquitin gene from Monochamus alternatus was cloned with a pair of degenerated primers by using RT-PCR（GenBank Accession No. EU433567） . The length of this opening reading-frame（ORF） was 228 bp,encoding a protein of 76 amino acids with molecular mass of 8.49 ku and theoretical isoelectric point of 5.83. Multiple sequence alignment indicated that M. alternatus ubiquitin was very similar to those of the homologous proteins of other eukaryotic species and shared 94 % - 98 % identity with other eukaryotic ubiquitins at amino acid level. Phylogenetic tree based on the nucleotide sequence indicated that M. alternatus had close relationship with Spodoptera litura. The theoretical three-dimensional structure of the ubiquitin gene was generated by homology modeling. Using pET-32a （ ＋ ） as a fused expressive vector, a recombinant plasmid which contained ubiquitin gene was constructed. Western blotting indicated that the M. alternatus ubiquitin gene was expressed successfully in the BL21 （DE3） strain of E. coli induced by IPTG. This work is a basis for futher studying the function of ubiquitin in M. alternatus.

关 键 词：松褐天牛 泛素 基因克隆 序列分析 原核表达 

分 类 号：S718.7[农业科学—林学]