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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 (DNA Methylation) is the most common form of DNA covalent modification of eukaryotic genome, affecting the protein-DNA interaction, plays an important role in the regulation of gene expression. RNAi (RNAi) interference is a new technology to turn off the function of a specific gene and has been widely used in plant functional genomics, plant development and regulation of physiological metabolic pathways. Based on the conserved sequences of plant DNA methyltransferases (DNMTs), primers were designed. First, a DNA methyltransferase gene fragment was cloned from Arabidopsis thaliana genome DNA, and then the conserved fragment was used as a template to amplify a fragment of about 570 bp The target sequence is used to construct the RNAi vector. According to the mechanism of RNAi, the target sequence of 570bp was ligated to the pHANNIBAL vector in the forward and reverse direction, and then the complete ORF box with this inverted repeat structure was ligated to the plant expression vector pGreeII. DNA digestion and sequencing confirmed the DNA Methyltransferase RNAi recombinant expression vector was constructed successfully. The transformation of Taxus chinensis cells indicates that the interference vector has the biological function and lays the foundation for the study of the improvement of the traits regulated by DNA methylation.