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为验证电子克隆技术获取茶树功能候选基因的可行性,本研究以可可的咖啡碱合成酶基因BCS1为探针,对构建的茶树EST数据库进行本地BLAST检索,获得与BCS1具有高度同源性的茶树EST序列26条。将26条茶树EST序列经程序CAP(contig assembly program)拼接,得到2条含有独立ORF的EST簇(Contig),分别命名为基因TCSnew1和TCSnew2。将这2个基因与GenBank中由实验方法克隆得到的茶树咖啡机合成酶基因TCS的cDNA进行核酸序列、推导氨基酸序列比对,及构建系统发育树进行3个基因推导氨基酸序列间的同源性分析。结果发现,茶树远缘物种的兴趣序列作为探针用于电子克隆获取茶树功能候选基因与实验克隆技术相同,是一条可行的技术途径。
In order to verify the feasibility of electronic cloning technology to obtain candidate genes of tea tree function, we constructed a EST database of tea EST using BCS1 as probe, and obtained BLAST EST with high homology with BCS1 Sequence 26. Twenty-six ESTs were cloned by contig assembly program (CAP). Two ESTs (Contig) containing independent ORFs were obtained and named as TCSnew1 and TCSnew2 respectively. The two genes were cloned from GenBank cloned cDNA of TCS from the tea plant coffee machine and the deduced amino acid sequence alignment and phylogenetic tree were used to deduce the deduced amino acid sequence homology analysis. The results showed that the sequence of interest of tea distant species as a probe for electronic cloning access to tea tree functional candidate genes and experimental cloning technology is the same, is a viable technical approach.