紫杉烷14β-羟基化酶基因片段的克隆及其植物表达载体的构建

Cloning a DNA Fragment of Taxane 14β-hydroxylase Gene and Constructi on of its Plant Expresstion Vectors for Plant System

采用CTAB法提取中国红豆杉基因组DNA,利用PCR技术克隆得到紫杉烷14β-羟基化酶(简称14OH)基因前半段,测序结果表明DNA片段序列为915bp,与报道的14OH基因同源性为98.3%。然后将获得的DNA片段(915bp)正反向插入到二元载体pZh01的GUS两端,构建成14OHRNAi植物表达载体pZ14a,又选其中与羟基化酶家族其他成员同源性低的部分(390bp),正反向插入到二元载体pZh01的GUS两端,构建成14OHRNAi植物表达载体pZ14b。同时将克隆得到的DNA片段(915bp)反向插入到二元载体pZh01中,构建成14OH反义RNA植物表达载体pZ14c。经PCR和酶切图谱分析鉴定,确认载体构建成功。

A DNA fragment with the length of 915bp of 14β-hydroxylase （14OH） gene was cloned from the genome DNA that was extracted from young leaves and cells of Taxus chinensis, The DNA fragment was verified by sequencing after it was inserted into pCF-T easy vector. Compared with the nucleotide sequence in the Genebank, the cloned DNA fragment showed 98.3% identity with the reported taxane 14β-hydroxylase. To constructe the 14β-hydroxylase RNAi expression vector, pZ14a, two copies of the 915bp fragment were further inserted into a binary vector, pZh01, in an inverted-repeat orientation at both ends of GUS. And two copies of a 390bp fragment, whose sequence having low identity with other members in the family of taxane hydroxylase, were inserted into pZh01 in an inverted-repeat orientation at both ends of GUS to construct the second 14β-hydroxylase RNAi vector, pZ14b. And the 915bp DNA fragment was inserted into pZh01 in an inverte orientation to construct the 14β-hydroxylase antisense RNA vector, pZ14c. PCR and restriction enzyme analyses confirmed correctness of the vectors.