萝卜抗真菌蛋白Rs-AFP_2基因PCR定点突变及其在大肠杆菌中的表达  

PCR sited-directed mutagenesis of Raphanus sativus-antifungalprotein_2 gene(Rs-AFP_2) and its expression in E.coli

作　　者：刘柱[1] 胡新文[2] 朱建清[1] 杨志荣[1] 

机构地区：[1]四川大学生命科学学院,四川成都610064 [2]中国热带农科院热带作物生物技术国家重点实验室,海南海口571101

出　　处：《西南农业学报》2002年第3期85-89,共5页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金资助项目 (3 9960 0 65 )

摘　　要：根据已报道的萝卜抗真菌蛋白Rs AFP2 基因序列 ,结合基因及其表达调控中遗传密码的偏爱性 ,人工合成引物 ,利用PCR重叠延伸法 ,使编码序列区的部分核苷酸突变 ,采用嵌套PCR ,迅速克隆到目的基因片段Rs AFPm ,连接到 pGEM Teasy载体上 ,转化大肠杆菌XL1菌株 ,筛选到阳性克隆。序列分析结果表明 ,PCR产物全长 2 40bp ,有一个阅读框 ,编码 2 9个氨基酸的信号肽和 5 1个氨基酸的抗真菌蛋白 ,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Arg相差两个碱基 (CAG→CGA) ,但二者编码产生相同。重新合成引物 ,将切除信号肽的Rs AFP2 基因和Rs AFPm基因分别克隆到pET 2 1b(+ )质粒载体上 ,导入大肠杆菌BL2 1菌株。Tricine SDS PAGE电泳显示工程菌中存在 6KD左右的目的蛋白带 ;软件分析显示 ,突变后pETAFPm的表达产物约为突变前 pETAFPo表达产物的2倍 ;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 ,已有效的提高表达量。Primers were designed according to the reported sequence of Rs AFP 2 gene and the preference of the genetic code in the gene expressions and regulations The splicing by overlap extension was applied to mutate some bases which were in the beginning of the encoding parts The mutated gene was quickly cloned by nested PCR PCR product of Rs AFPm gene was retrived and then inserted into pGEM T easy vector The amplified fragment contained a 240bp open reading frame(ORF),encoding 80 amino acids which include signal peptide of 29 amino acids and mature peptide of 51 amino acids In comparison with the two genes,it was showed that the differences of bases at 9 site(G→A),15 site(G→A),16 site(A→C)and 18 site(G→A)in the encoding parts But both genes have the same sequences of amino acids After removed the signal peptide sequences,the digested products were inserted into pET21b at the sites of Nde I and Xho I The expression vectors pETAFPo and pETAFPm were constructed Each was expressed in E coli BL21 strain,and the protein product was identified by SDS Tecine PAGE The volume of the pETAFPm/BL21 was as two times as that of the pETAFPo/BL21 The inhibition experiments showed that the inhibition acitvity of pETAFPm expression product was much stronger than that of pETAFPo one Thus,the mutated gene had improved the expression validly as a result

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

分 类 号：TQ460.38[化学工程—制药化工] S482.29[农业科学—农药学]