抗真菌蛋白Rs-AFP_2基因定点突变及其对大肠杆菌中表达的影响  

Sited-directed Mutagenesis of Raphanus Sativus-antifungal Protein_2 (RS-AFP_2) Gene and Its Effect on Expression in the E.Coli

作　　者：朱建清[1] 刘柱[1] 胡新文[2] 杨志荣[1] 

机构地区：[1]四川大学生命科学学院 [2]中国热带农业科学院热带作物生物技术国家重点实验室,海口571101

出　　处：《高技术通讯》2003年第4期18-23,共6页Chinese High Technology Letters

基　　金：国家自然科学基金(39960065)资助项目。

摘　　要：为了研究遗传密码子对表达调控的影响,利用PCR重叠延伸法,对萝卜抗真菌蛋白Rs-AFP2基因编码序列区的部分核苷酸进行沉默突变,构建突变体Rs-AFPm。序列分析表明,PCR产物全长240bp,有一个阅读框,编码的蛋白由29个氨基酸的信号肽和51个氨基酸的抗真菌蛋白组成。突变体与突变前的Rs-AFP2基因相比,在编码区第3号氨基酸Lys相差一个碱基(TTG→TTA),第5号氨基酸Gln相差一个碱基(CAG→CAA),第6号稀有密码子Arg相差两个碱基(CAG→CGA)。重新合成引物,将切除信号肽的Rs-AFP2基因和Rs-AFPm基因与原核表达载体pET-21b(+)分别重组到大肠杆菌BL21菌株。IPTG诱导后,二者均得到了表达。软件分析显示,突变前pETAFPo表达产物占全菌蛋白的3％,突变后pETAFPm的表达产物占全菌蛋白含量的8％;表达蛋白主要以包涵体的形式存在,包涵体经超声波破碎后,蛋白质复性,抑菌结果表明,pETAFPm表达产物的抑菌半径大于pETAFP2表达产物的抑菌半径。这些都说明改造后的Rs-AFPm基因与Rs-AFP2基因相比,已有效地提高表达量。In order to research on effect of genetic codon in the gene expression and regulation, the splicing by overlap extension was used to the silence mutation in the partial nucleotides of Rs-AFP2 (Raphanus sativus-antifungal Protein2) gene . The mutant, which included Rs-AFPm gene, was constructed. Sequence analysis showed that the PCR product was 240bp. It had an open reading frame, encoding signal peptide of 29 arnino acids and mature peptide of 51 amino acids. By comparison between the Rs-AFP2 one and the mutated Rs-AFPm gene , there were five bases differences in the Lys3 amino acid (TTG→TTA), the Gln5 (CAG→CAA), and the rear codon Arg6 (CAG→CGA). After removing the signal peptide, the digested products were inserted into pET-21b ( + ). The expression vectors were expressed in B121 strain after induction with IPTG. Software analysis showed that the target protein of pETAFPo occupied 3 % and that of mutated pETAFPm occupied 8 % in the whole bacterial protein. The target protein was mainly existed in inclusion bodies. The inclusion bodies were broke and then re-folded. The inhibition experiments showed that antifungal radius of pETAFPm was bigger than that of pETAF-Po. All these showed Rs-AFPm gene had validly increased the expression volume in contrast with Rs-AFP2 gene.

关 键 词：萝卜抗真菌蛋白2 Rs-AFP2 定点突变 密码偏爱性 基因表达 大肠杆菌 遗传密码子 基因突变 

分 类 号：Q75[生物学—分子生物学]