摘要
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.
出处
《北京林业大学学报》
CAS
CSCD
北大核心
2004年第4期25-29,共5页
Journal of Beijing Forestry University
基金
国家转基因植物与产业化专项基金 (J0 0 A 0 0 8 0 9)
"863"国家项目 ( 2 0 0 1AA2 12 161)资助
关键词
盐藻
烯醇酶
基因克隆
简并PCR
RACE
D. salina, enolase, gene cloning, modified degenerate PCR technology, RACE
