红花栝楼鲨烯合酶基因的克隆及其序列分析

Cloning and sequence analysis of squalene synthase gene from Trichosanthes rubriflos

目的克隆红花栝楼鲨烯合酶（squalene synthase,SS）的基因并进行序列分析,为进一步研究葫芦科植物三萜合成通路关键酶SS的正选择位点与功能的关联性分析奠定基础。方法根据绞股蓝与罗汉果SS基因c DNA比对分析的结果,设计SS的5’端简并引物,采用3’RACE扩增红花栝楼SS全长c DNA基因。结果获得红花栝楼SS c DNA全长共1 466个核苷酸,其中包括一个含有1 254个核苷酸的开放读码框,编码417个氨基酸残基。通过NCBI的Blast比对,发现红花栝楼SS基因编码的氨基酸序列与已知植物SS基因编码的氨基酸序列同源性为78%~94%,核苷酸的同源性为74%~93%。结论成功克隆了红花栝楼SS的全长c DNA,为进一步研究红花栝楼SS基因结构、基因表达、基因突变提供了基础,并为葫芦科植物三萜合成通路关键酶SS的正选择位点与功能的关联性分析提供了数据支持。

Objective To clone the full length c DNA encoding squalene synthase（SS） from Trichosanthes rubriflos and to carry on its sequence analysis, so as to lay the foundation for the further study on the positively selected sites and function correlation analysis of SS which is the key enzyme for triterpene synthesis pathway. Methods According to the c DNA comparison on SS gene from Gynostemma pentaphyllum and Siraitia grosvenorii, 5＇-upstream degenerate primers of the c DNA of SS gene from T. rubriflos were designed and the full length c DNA of SS gene from T. rubriflos was amplified by 3＇RACE kit. Results The full length c DNA of SS gene from T. rubriflos composed of 1 466 nucleotides was obtained. The open reading frame（ORF） of SS gene from T. rubriflos was 1 254 bp in length, corresponding to a predicted polypeptide of 417 amino acid residues. The results of homologous alignment analysis in Gen Bank demonstrated that the c DNA sequence of SS gene from T. rubriflos had 78%—94% similarity on the nucleotide sequence compared with SS from known plant and 74%—93% similarity on the deduced amino acid sequence compared with SS gene from other plants. Conclusion The full length c DNA of SS gene from T. rubriflos has been cloned, which not only lays a foundation for the further study on the gene expression, gene structure, and gene mutation, but also provides the important data base for the association study between the positively selected sites and function correlation analysis of which is the key enzyme for triterpene synthesis pathway.