反向PCR方法克隆腾冲嗜酸两面菌的分子伴侣基因  

Cloning of Chaperonin Gene from Acidianus tengchongrensis Using Inverse PCR Method

作　　者：马晴[1] 张渝英[2] 

机构地区：[1]北京师范大学生命科学学院,北京100875 [2]中国科学院微生物研究所,北京100080

出　　处：《微生物学通报》2005年第4期112-115,共4页Microbiology China

基　　金：国家自然科学基金资助项目(No.3997003)

摘　　要：根据已知的Sulfolobus属的分子伴侣基因,设计简并引物,用PCR的方法从腾冲嗜酸两面菌(Acidianus tengchongensis)基因组DNA中分别克隆到了分子伴侣α亚基和β亚基的约500bp的基因片段。以它们为探针进行Southern杂交,确定了合适的限制性内切酶。以确定的限制性内切酶消化的基因组DNA环化物为模板,进行反向PCR反应,引物的延伸方向由已知序列出发沿环化分子向未知区域进行,扩增产物经测序表明为α亚基和β亚基基因。根据所得序列分别设计两对引物进行PCR,测序结果表明得到了α亚基和β亚基的完整基因。DNA fragments of α subunit and β subunit were amplified by PCR from the genome of Acidianus tengchongensia with the degenerated primers designed from the consensus amino acid sequence of Sulfolobus chaperonins. The two DNA fragments about 500bp were used as probe for southern hybridization to determine the suitable restriction endonuclease. Restriction endonuclease digested genomic DNA was circularized by self-ligation, and complete sequences of α subunit and β subunit were obtained by inverse PCR. The primers oriented in the reversed direction of the usual orientation to amplify the DNA sequence that flank the known region. The complete genes of α subunit and β subunit were PCR amplified from genomic DNA using two pairs primers.

关 键 词：腾冲嗜酸两面菌 反向PCR 分子伴侣 

分 类 号：Q933[生物学—微生物学]