棉花肉桂醇脱氢酶基因GhCAD6的克隆及正义、反义与RNAi干扰载体的构建

肉桂醇脱氢酶(CAD)催化木质素单体合成的最后一步反应,将肉桂醛还原生成相应的肉桂醇.采用PCR方法从棉花中克隆了GhCAD6基因1 569 bp的基因组序列,序列分析表明GhCAD6基因由5个外显子和4个内含子组成.为了研究GhCAD6基因的功能,构建由棉纤维组织特异性启动子E6驱动的GhCAD6基因正义、反义表达载体;同时克隆了GhCAD6基因的一段417 bp高度保守的NBD区序列,构建了GhCAD6基因的RNAi干扰载体,并将上述载体转入农杆菌菌株LBA4404中.为通过转基因技术深入研究GhCAD6基因在棉纤维发育和木质素代谢途径中的作用创造条件.

Molecular Cloning of GhCAD6 Gene and Construction of Its Sense,Anti-sense and RNAi Expression Vectors

Cinnamyl alcohol dehydrogenase (CAD) (EC 1.1.1.195) catalyses the final step in lignin precursor synthesis reducing the cinnamyl aldehydes to the corresponding alcohols in the presence of NADPH.In this paper,we report the molecular cloning a length of 1 569 bp sequence from genomic DNA of GhCAD6 by PCR.The genomic DNA of GhCAD6 contains five exons and four introns.To investigate the function of GhCAD6 gene,the sense and anti-sense expression vectors of GhCAD6 were constructed,in which the coding region of the gene was placed under the E6 promoter in either sense or anti-sense orientation.In addition,especial 417 bp fragments of GhCAD6 NBD were also cloned,and this fragment was inserted into plant vector to construct RNAi expression vector of GhCAD6.These constructed vectors were then transformed into Agrobacterium LBA4404.These constructed vectors provided an effective tool for the further study of GhCAD6 gene function.