编码L-苏氨酸-O-3-磷酸盐脱羧酶基因的克隆和解析

目的]为探索维生素B12生物合成的限速步骤奠定基础。方法]提取费氏丙酸杆菌染色体DNA,利用Cassette和Cassette Primer的LA PCR技术克隆cobD的上游基因。将扩增的目的基因片段克隆至pGEM-T Easy Vector上,经PstⅠ酶切鉴定后对阳性克隆质粒进行测序,并应用Blast对所得碱基序列进行解析。结果]经LA PCR扩增获得全长为1 085 bp的目的基因。经PstⅠ酶切鉴定,cobD基因成功克隆到pGEM-T Easy Vector上。测序结果表明,所得的基因为编码L-苏氨酸-O-3-磷酸盐脱羧酶的基因,与GenBank中cobD基因编码的氨基酸序列同源性为100%。结论]在厌氧条件下,费氏丙酸杆菌具有与鼠伤寒沙门氏菌相似的合成途径。

Cloning and Analysis of the Gene for Encoding L-Threonine-O-3-Phosphate Decarboxylase

Objective] The research aimed to lay the foundation for discussing the speed-limiting process for the biosynthesis of vitamine B12.Method] Chromosome DNA was extracted from Propionibacterium freudenreichii and the upstream gene of cobD was cloned by using LA PCR technology of Cassette and Cassette Primer.The amplified target gene fragment was cloned to pGEM-T Easy Vector.The positive cloned plasmid was sequenced after the identification of PstⅠ enzyme digestion and the obtained base sequence was analyzed by using Blast software.Result] The target gene with the whole length of 1 085 bp was obtained through LA PCR amplification.The identification results of Pst Ⅰ enzyme digestion showed that cobD gene was successfully cloned to pGEM-T Easy Vector.The sequencing results showed that the obtained gene was the gene that encoded L-threonine-O-3-phosphate decarboxylase and its homology with that of amino acid sequence encoded by cobD gene on GenBank website was 100%.Conclusion] Under anaerobic conditions,P.freudenreichii had the similar synthesis pathway with Salmonella typhimurium.