反向PCR克隆黑根霉R306Δ6-脂肪酸脱饱和酶基因

Cloning of Δ6-Fatty Acid Desaturase Gene from Rhizopus nigricans R306 with Reverse PCR

利用简并引物扩增出Δ6-脂肪酸脱饱和酶基因的核心序列,设计与核心序列互补的反向引物,以5′端带磷酸基团的引物进行反转录cDNA,然后在12℃下环化cDNA,并以此作为模板进行反向PCR,引物的延伸方向自核心区向环化分子的未知序列进行,扩增出核心区的上下游序列。应用该方法扩增并测定了黑根霉R306的Δ6-脂肪酸脱饱和酶基因全序列。并对酶的低温适应机制在氨基酸序列上进行了分析。

Core sequence of △6-fatty acid desaturase gene was amplified using degenerate primer, and a reverse primer complementary to the core sequence was designed, reverse transcription of cDNA was carried out using primer with 5 end phosphate radical then cyclized the cDNA under 12 ℃ , the cyclized cDNA was taken as template to carry out the reverse PCR along the extensive direction of the primer from the core region to the unknown sequence of the cyclized molecule, and amplified the upstream and downstream sequence of the core region. Applying these methods the full sequence of △6-fatty acid desaturase gene from Rhizopus nigricans was measured. Finally the mechanism of low temperature adaptability of the enzyme was analyzed based on amino acid sequence.