南方红豆杉紫杉烷7β-羟基化酶基因全长序列的克隆和进化分析

Molecular Cloning and Evolutionary Analysis of the Full-length Sequence of the Taxus wallichiana var. mairei Taxoid 7-beta-hydroxylase Gene

从南方红豆杉Taxus wallichianavar.mairei的新鲜嫩叶中提取基因组DNA作为模板,利用三组特异引物进行PCR扩增,然后克隆测序得到紫杉烷7β-羟基化酶的基因全长。该基因编码区起始密码子为ATG,终止密码子为TGA,全长1 692 bp;碱基组成为490 A(29.0%),351 C(20.7%),362 G(21.4%)和489 T(28.9%)。将紫杉烷7β-羟基化酶基因全长序列与细胞色素P450基因家族的其它三个成员进行比对,发现它与紫杉烷2α-羟基化酶基因、紫杉烷10β-羟基化酶基因及紫杉烷13α-羟基化酶基因的一致性分别为74%、68%及76%。它们的外显子和内含子的连接区均具保守的GT-AG结构,内含子区的变异性明显高于外显子区。进一步以红豆杉属的13个紫杉烷羟基化酶基因家族成员为对象,利用位点间可变ω(非同义替换率dN和同义替换率dS的比值)模型对该基因家族的适应性进化进行分析。分支模型、位点模型以及分支-位点模型的分析表明:紫杉烷羟基化酶基因家族的少数分支处于正选择压力下(ω>1),但未检测到正选择位点;而绝大部分位点受强烈的负选择作用(ω<1)。

By using PCR and cloning approches, a full-length sequence （ 1 692 bp） of taxoid 7β- hydroxylase gene was obtained from Taxus wallichiarta var. mairei genomie DNA. The gene sequence contains 490 adenine （29.0%） , 351 cytosine （20. 7% ） , 362 guanine （21.4%） and 489 thymine （28.9%） , using ATG and TGA as start and stop codons. Senquence comparisons reveal that the T. wallichiana vat. mairei taxoid 7β-hydroxylase gene is highly similar to other P450 gene family members. It shows a identity of 74% with taxoid 2α-hydroxylase, 68% with taxoid 10β-hydroxylase, and 76% with taxoid 13α-hydroxylase. The joint region between introns and extons possess the conserved GT-AG, and introns exhibit higher variability than extrons. An evolutionary analysis was further performed on the P450 gene family members under models allowing ω （nonsynonymous/synonymsous rate ratio） to vary among sites. Under the branch model, the site model and the branch-site model developed by Yang et al, the results show that although several lineages are under positive selection withω 〉 1, no positively se-lected sites are identified; and most of the sites in this gene family are under negative selection with ω〈 1.