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

 从南方红豆杉Taxus wallichiana var.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)。

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

By using PCR and cloning approches，a full-length sequence (1 692 bp) of taxoid 7β hydroxylase gene was obtained from 〖WTBX〗Taxus wallichiana var. mairei genomic 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 var. 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 with ω>1，no positively selected sites are identified; and most of the sites in this gene family are under negative selection with ω<1.