杨树木质素合成酶hct基因的克隆及核苷酸序列分析

Cloning and sequence analysis of lignin biosynthetic enzyme hct gene in Poplar

根据毛果杨全序列(AC185363.2)中唯一具有转移酶功能的保守区设计引物,以正在分化的2年生欧美杨107次生木质部总RNA为模板经RT-PCR扩增出hct基因片段,与pMD20-T载体连接,重组质粒经特异引物扩增、限制性内切酶酶切和测序鉴定。结果表明,扩增片段长度为709bp,包含一个708bp的开放阅读框,与毛果杨全序列及HCT2(EU603314)的同源性均为98%,编码的氨基酸序列与毛果杨HCT2氨基酸(ACC63883.1)的同源性为98%,断定我们克隆的cDNA为hct,属于转移酶超家族,GenBank登录号为FM202091,该重组质粒命名为pMD20-T-hct。

Based on the full sequence conserved domain（transferase）of Populus trichocarpa（accession number：AC185363.2）,a pair of primers was designed and used to amplify a fragment of hct（shikimate/quinate hydroxycinnamoyltransferase）gene by RT-PCR from poplar（Populus×euramericana cv.‘74/76 ）developing second xylem RNA,then cloned into pMD20-T vector,identified by PCR amplification,restriction enzyme cutting, sequencing and named pMD20-T-hct.The sequence of the amplified DNA fragment was 709bp, containing a 708bp ORF （open reading frame）. The identity of the fragment with Populus trichocarpa complete sequence and HCT2 both are 98%. Alignment with amino acid ofPopulus triehocarpa HCT2 showed that their homology was 98%. Therefore, we confirmed that the cloned DNA fragment was hct, belonged to transferase superfamily. It was submitted to GenBank and the accession number was FM202091 and named PMD20-T-hct.