改进简并PCR法克隆盐生杜氏藻烯醇酶基因

Cloning enolase gene of Dunaliella salina with modified degenerate PCR technology.

该文针对基因同源性克隆中存在的问题 ,采用改进简并PCR法对盐生杜氏藻具抗逆功能的烯醇酶基因 .改进后的同源引物简并度从 10 0 0倍以上降低至 10 0倍以下 ,利用RT PCR成功地扩增出一条 6 30bp的盐藻cDNA片段 ,该片段与其他物种的烯醇酶基因具有很高的相似性 .在此基础上进行 5’和 3’RACE获得了盐藻烯醇酶基因的全长cDNA ,其长度为 1734bp .序列分析表明 ,该基因与莱茵衣藻烯醇酶基因的相同性达 83% ,相似性达 89% ,且在进化上与莱茵衣藻的亲缘关系最近 .Southern杂交结果显示 。

An anti-adversity gene (enolase gene) of D. salina is cloned by modified degenerate PCR technology. Traditional homogene cloning method is modified in two aspects: one is designing homo-primers at the sites with minimum degeneracy instead of maximum homology, the other is replacing some N bases with inosine in degenerate codes which reduce the degeneracy from above 1000 times to below 100 times. A pair of degenerate primer is designed as 5’--GCI HTN CAR GAR TTY ATG AT--3’/5’--CAT IAC NCC CCA NCC--3’,thus a cDNA fragment of D. salina with a length of 630 bp is obtained, which has highly similarity with enolases from other species. A 1 734 bp full-length enolase cDNA of D. salina (GeneBank accession No. 245549) is acquired by 5’RACE and 3’RACE technology, which has 83% identity and 89% similarity with that of Chlamydomonas. Analysis of southern blotting suggests that the enolase cDNA is from D.salina and that it is possible to have single copy.