CODEHOP法设计引物克隆色盐杆菌ST307胆碱脱氢酶基因及其序列分析

目的研究色盐杆菌ST307甜菜碱的合成,克隆分析其胆碱脱氢酶betA基因片段。方法采用醇提法提取色盐杆菌ST307中的甜菜碱并检测,用CODEHOP在线程序设计简并引物扩增胆碱脱氢酶betA基因序列,并进行序列分析。结果色盐杆菌ST307细胞中积累甜菜碱,通过PCR获得长度为485 bp的betA基因片段。BLAST序列分析显示该基因序列与多个菌株的betA基因序列具有较高的同源性,最高达83%。核苷酸序列比对及进化树构建结果显示,色盐杆菌ST307胆碱脱氢酶基因序列与Pseudomonas fulva 12-X进化关系最为接近。结论 CODEHOP法设计的简并引物可信性较强,同时betA基因片段的成功克隆将为获得ST307胆碱脱氢酶基因的全序列、研究耐盐机制和遗传改良提供科学依据。

Cloning and Sequence Analysis of Choline Dehydrogenase betA Gene Fragment by CODEHOP from Chromohalobacter sp.ST307

Objective To study the synthesis of betaine from Chromohalobacter sp.ST307 and obtain choline dehydrogenase betA gene fragment for the sequence analysis.Methods Betaine was extracted and detected by alcohol extraction from Chromohalobacter sp.ST307.CODEHOP software was used to online design the degenerate primers to carry out PCR of choline dehydrogenase betA gene obtained and molecular biological softwares were used for the sequence analysis.Results Chromohalobacter sp.ST307 cells accumulated betaine.The betA gene fragment with a length of 485 bp was obtained by PCR.BLAST sequence analysis indicated that the sequence had higher similarity to betA gene of GenBank and the highest similarity reached 83%.Nucleotide phylogenetic analysis showed that betA gene sequence of strain ST307 had a close evolutionary relationship with Pseudomonas fulva 12-X.Conclusion The degenerate primers designed by the CODEHOP software can be used to obtain specific gene fragment with strong credibility.The success of cloning betA gene fragment will provide scientific basis for obtaining all sequences of choline dehydrogenase gene and studying salt-tolerant mechanism and genetic improvement.