DNA甲基转移酶RNAi干涉载体的构建及其鉴定

Construction and Evaluation of DNAMethyltransferase Vector for RNA Interference

DNA甲基化(DNA Methylation)是真核生物基因组最常见的DNA共价修饰形式,影响蛋白质-DNA的相互作用,在基因表达的调控上起着重要作用。RNAi(RNA interference)干涉是关闭特定基因功能的新技术,在植物功能基因组、植物发育及生理代谢途径调控等方面有着广泛应用。本文根据植物DNA甲基转移酶(DNMTs)的保守序列设计引物,首先从拟南芥总基因组DNA中克隆出DNA甲基转移酶基因保守片段,然后以此保守片段为模板扩增长度约570bp的靶序列用于构建RNAi载体。根据RNAi作用机制,将570bp靶序列正向、反向连接到pHANNIBAL载体上,然后将带有此反向重复结构的完整ORF框连接到植物表达载体pGreeII上,经过酶切鉴定和测序分析证实DNA甲基转移酶RNAi重组表达载体构建成功。转化红豆杉细胞表明该干涉载体具有生物学功能,为研究受DNA甲基化调控的性状改良打下基础。

DNA Methylation,the most common form of covalent modification in eukaryotic cell,influencing the interaction between proteins and DNA,plays an important role in the regulation of gene expression.As a new technology for knocking out specific genes,RNAi （RNA interference）is applied extensively to plant functional genomics,development,the regulation of physiological metabolic pathway and so on.in this paper,RNAi expression vectorof DNA methyltransferases （DNMTs） designed based on the conservative sequence DNMTs were constructed,which lays a foundation forthe DNA Methylation regulation of characterimprovement.firstly,the conservative fragment of DNMTs was cloned from the genomic DNA of Arabidopsis.And then a target sequence,about 570bp,was amplified from the received conservative sequence and then the target sequence was inserted into the pHANNIBAL vector.Later the complete ORF contained the inverted repeat was linked to the plant expression vectorPgreenII.By the analysis of both restriction enzyme digestion and sequencing,the accomplishment of the DNMTs-RNAi recombinant expression vectorwas confirmed.The results of further transformation of Taxus cell showed that the RNAi recombinant expression vector had biology function laying a foundation for genetic modification of DNA methylation controlled character.