摘要
利用RT-PCR和RACE技术,以小菜蛾(Plutella xylostella)3龄幼虫的cDNA为模板,克隆获得泛素延伸蛋白基因Px-ubi(GenBank No.FJ527489),核苷酸序列全长537 bp,其中开放阅读框全长387 bp,编码129个氨基酸残基,预测分子质量为16.7 ku,等电点为9.1,与已知真核生物泛素延伸蛋白的同源性为92%～98%。RT-PCR结果显示Px-ubi在小菜蛾不同生长发育时期均有表达,其中卵期、3龄期和预蛹期含量较高,而成虫期含量较低。同源建模显示,Px-ubi的3D结构主要由1个α螺旋、4个β折叠以及β转角和不规则卷曲组成,4个β折叠两两反向平行排列在α螺旋的一侧。将该基因重组到表达载体pET-32a(+)中,获得重组载体pET32a-Px-ubi,并转入原核细胞中表达。SDS-PAGE和Western blot分析表明,经IPTG诱导后,Px-ubi能在大肠杆菌BL21中高效表达,电泳检测到1条约16.7 ku的外源蛋白,与预测的蛋白分子质量吻合。
Ubiquitin is a small protein that widely exists in all eukaryote whose basic function is through the ubiquitin-proteasome pathway （UPP）to let the targeting protein degradation effective and highly selectivity. A cDNA fragment encoding ubiquitin-52aa extension protein from the third instar larvae of Plutella xylostella was amplified through RT-PCR and RACE method. The ubiquitin-52aa extension gene in P.xylostella, named Px-ubi, was 537 bp in total length and 387 bp in ORF. It encoded a peptide of 129 amino acid residues （GeneBank No.FJ527489）, in which the relative quality of molecules is 16.7 ku and the isoelectric point is 9.1.Multiple sequence alignment indicated that Px-ubi was similar to the homologous proteins of other eukaryotic species and it shared 92% to 98% amino acid sequence identity with other species. The theoretical three dimensional structure of Px-ubi was displayed by homology modeling. Through the RT-PCR, it was found that Px-ubi was expressed at different times in the P.xylostella, and its content was higher in the egg stage, third instar larvae and pre-pupal stage than in the adult stage. The Px-ubi was then inserted into expression pET-32a （＋） and transformed into E.coli DE3. Western blotting indicated that the Px-ubi was expressed successfully in the BL21 strain of Escherichia coli induced with IPTG. The results provide some helpful information for the further study of the function of ubiquitin gene in insect.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2011年第1期54-60,共7页
Journal of Huazhong Agricultural University
基金
教育部霍英东教育基金会高等院校青年教师基金项目(111027)
关键词
小菜蛾
泛素
基因
克隆
原核表达
Plutella zylostella
ubiquitim gene
cloning
expression profile
