柑橘CsPRP4基因RNAi载体的构建

富含脯氨酸蛋白4(PRP4,proline-rich protein 4)是一类响应胁迫并在细胞发育过程中起作用一种细胞壁蛋白。用RT-PCR克隆柑橘CsPRP4基因的正、反义cDNA序列,分别连接到T载体pMD–19T上;经限制性核酸内切酶2次双酶切,将CsPRP4基因的正、反义片段定向连接至双元质粒pFGC5941查耳酮合成酶A(CHSA)内含子的两端,构成反向重复序列;经菌液PCR和测序验证,成功构建了CsPRP4基因的RNA干涉载体;经农杆菌介导法转化柑橘,转基因植株具有明显不同于对照的表现型,研究为进一步阐明CsPRP4的功能奠定了基础。

Construction the RNAi vector of citrus PRP4 gene

PRP4(proline-rich protein 4)were kinds of proteins that accumulate in the cell wall,response to environmental stress,and have subsequently been shown to be temporally regulated during plant development.The sense and anti-sense fragments of cDNA of CsPRP4 were amplified from citrus by RT-PCR,and cloned into pMD-19T respectively.Plasmids containing sense/anti-sense CsPRP4,and binary vector pFGC5941 were twice double digested by endonuclease,followed by ligation of T4 ligase.Resulting in RNAi vector of CsPRP4 with sense and antisense fragment flanking the intron of chalcone synthase A gene.Verified by PCR testing and sequencing,the RNAi vector of CsPRP4 was successfully constructed.Antibiotic resistant buds were regenerated via agrobacterium-mediated transformation of citrus explants,the phenotype of the transgenic plants showed significant different against control.Our work provides a way towards the elucidation of the function of CsPRP4.