牛樟芝倍半萜合酶基因克隆及在不同培养基中的表达

牛樟芝(Antrodia camphorata)是一种珍稀食药用菌,能产生具有抗癌活性的倍半萜类化合物。对牛樟芝基因组进行分析,获得倍半萜合酶基因序列并设计特异引物,提取在Glu培养基(麦芽浸粉6 g/L,酵母提取物3 g/L,葡萄糖40 g/L)上生长的牛樟芝菌丝体的RNA,利用RT-PCR技术克隆得到倍半萜合酶基因AcTPS1。AcTPS1基因c DNA全长为969 bp,编码323个氨基酸,根据系统进化树可知AcTPS1氨基酸序列与其他9种真菌倍半萜合酶聚为一类。AcTPS1拥有典型倍半萜合酶的结构域(RRSRSATAEAYACFIW),之后检测AcTPS1在不同培养基上的菌丝中的表达结果显示不同碳源中,只有葡萄糖作为碳源时该基因表达,不同氮源中,以番茄浸粉和酪蛋白胨为氮源时该基因表达。说明AcTPS1是一类诱导型表达的基因。为利用发酵培养以及异源表达手段获得牛樟芝活性化合物提供参考。

Clone and Expression of Sesquiterpene Synthase Gene of Antrodia camphorata in Different Media

Antrodia camphorata was rare edible and medicinal fungi and that could produce sesquiterpene compounds with anti cancer activity. In this study, A. camphorata of genome was analyzed, then sesquiterpene synthase gene sequence was obtained and specific primers were designed according to this sequence. The RNA of A. camphorata mycelium grew on the Glu medium (malt powder 6 g/L, yeast extract 3 g/L, glucose 40 g/L) was extracted. The sesquiterpene synthase gene of AcTPS1 was cloned by RT-PCR which full length of cDNA was 969 bp, this sequence could encode 323 amino acids. According to phylogenetic tree, the amino acid sequence of AcTPS1 was clustered with other 9 sesquiterpene synthases which had a typical sesquiterpene synthase domain (RRSRSATAEAYACFIW). The expression level of AcTPS1 in mycelia on different media was shown, gene was expressed only when glucose was used as the carbon source among the different carbon sources, the gene also expressed when tomato powder and casein peptone as the nitrogen source in different nitrogen sources. Indicated that AcTPS1 is a number of inducible type of expression genes. This research make an important theoretical foundation to obtain active compounds from A. camphora using fermentation cultivation method and heterologous expression measurement.