SEFA-PCR法克隆灵芝鲨烯合酶基因启动子及其序列分析

鲨烯合酶是灵芝三萜生物合成的关键酶,灵芝鲨烯合酶基因的表达和活性的高低决定了灵芝中三萜含量的高低。根据已经获得的灵芝鲨烯合酶全长cDNA序列设计一对专一引物,通过PCR扩增得到了灵芝鲨烯合酶基因的基因组全长,序列长1984bp,含有3个内含子。根据其基因组序列设计引物,采用SEFA-PCR的方法,以总DNA为模板,克隆了灵芝鲨烯合酶基因的启动子序列,长1042bp。序列分析发现灵芝鲨烯合酶基因启动子中没有明显的TATA和CAAT框,但是含有CCAAT-bindingfactor、GATA-1、GC-box、TFⅡD等重要的转录因子的结合位点,以及在人和酿酒酵母鲨烯合酶基因启动子中发现的甾醇调节相关的顺式调控元件。

Cloning and sequence analysis of promoter region of squalene synthase gene from Ganoderma lucidum by SEFA-PCR

Squalene synthase is the key enzyme in triterpene biosynthesis of Ganoderma lucidum. There is a positive correlation between the expression level of squalene synthase and the amount of triterpenes produced. The full length of G lucidum squalene synthase gene was cloned by PCR according to the eDNA sequence. The sequence consisted of 1984bp, containing three introns. Using the SEFA-PCR method, the promoter sequence of G lucidum squalene synthase was amplified from G. lucidum genomie DNA. Nueleotide sequence analysis showed that the fragment cloned consisted of 1042bp. The promoter was analyzed using the Softberry software. The results showed that it contained several regulatory elements, such as CCAAT-binding factor, GATA-1, GC-box, TF II D, but the TATA box and the CAAT box could not be found. The promoter contains sterol regulatory elements and is similar to that of human and Saccharomyces cerevisiae.