菌B49乙醛乙醇脱氢酶基因克隆中的引物设计  

Primers' design of cloning aldehyde-alcohol dehydrogenase fragment for bacteria B49

作　　者：林海龙[1] 任南琪[1] 郑国香[1] 张坤[1] 段志洁[1] 

机构地区：[1]哈尔滨工业大学市政环境工程学院,哈尔滨150090

出　　处：《哈尔滨工业大学学报》2008年第6期865-869,共5页Journal of Harbin Institute of Technology

基　　金：国家自然科学基金资助项目(30470054)

摘　　要：为研究乙醛乙醇脱氢酶基因及其在乙醇发酵生物制氢中的重要作用,采用clustalw对12个菌种的乙醛乙醇脱氢酶蛋白质序列进行多重比对分析,共得到6个保守区,以这6个保守区设计引物,共选用9对简并引物,命名为ADHJ1、ADHJ2、ADHJ3、ADHJ4、ADHJ5、ADHJ6、ADHJ7、ADHJ8、ADHJ9.用这9对引物对B49总DNA进行简并PCR,共有两对引物的扩增得到预期的结果.其PCR产物经pMD18-T克隆转化至大肠杆菌DH5α中,经筛选后测序.结果表明,这两段DNA推导的氨基酸序列与Bacillus cereus的乙醛乙醇脱氢酶基因的相似性最高为69%,证实所克隆的序列为乙醛乙醇脱氢酶基因DNA片段.尽管采用clustalw软件设计ADHJ5简并引物的简并度分别达1048576倍和98304倍,引物长度分别为30 bp和26 bp,但扩增结果特异性强.说明采用clustalw软件设计的简并引物可信度高,同时,也说明简并度不是合理评判简并引物质量的标准.In order to clone the aldehyde-alcohol dehydrogenase gene and study its important role in the ethanol fermentation bio-hydrogen, multiple alignment analyses were conducted on twelve aldehyde-alcohol dehydrogenase protein sequences of different bacteria using clustalw software, and six blocks were obtained. Then degenerate primers were designed according the six blocks, and nine degenerate primers of the aldehyde-alcohol dehydrogenase gene were acquired, named ADHJ1, ADHJ2, ADHJ3, ADHJ4, ADHJ5, ADHJ6, ADHJ7, ADHJ8 and ADHJ9. The high-biohydrogen bacteria B49 genome DNA as the template and nine degenerate primers as primers were used to degenerate PCR, two of them achieved anticipative results. Their PCR products were transformed into E. coli DH5α through being linked with pMD18 - T vector and sequenced after filtration. Similarity alignment showed that the products were the most similar to the aldehyde-alcohol dehydrogenase gene of Bacillus cereus , and the identities was 69%. The results indicate that these degenerate primers designed by the clustalw software can be used to obtain the specific gene fragment. Although the degeneracy of ADHJ5 primers are 1048576 and 98304 respectively, the length of ADHJ5 primers are 30 bp and 26 bp, the PCR result is very good. The results show that these degenerate primers designed by this software can be used to obtain specific PCR products.

关 键 词：乙醛乙醇脱氢酶基因 clustalw 生物制氢细菌 简并引物 

分 类 号：Q93[生物学—微生物学]