柑橘衰退病毒基因RNAi载体的构建

发布时间：2018-10-11 18:39

【摘要】：基于转化病毒基因介导抗性,通过在植物表达载体p CAMBIA 2301上插入启动子内含子终止子的方式,构建一种含有发夹结构的、通用型强的RNAi载体骨架结构;以柑橘衰退病毒(citrus tristeza virus,CTV)的p25、p20和p23基因保守序列为模板设计正向片段和反向片段,先后与骨架载体连接,成功构建了3个RNAi载体(分别命名为ds2301 p25、ds2301 p20和ds2301 p23),并将载体ds2301 p23注射入墨西哥莱蒙叶片。制作p23基因的地高辛标记探针,用Northern杂交检测是否有si RNA产生。结果表明:用Northern杂交可以检测到p23特异的si RNA,在墨西哥莱蒙叶片中瞬时表达的农杆菌ds2301 p23可以发生RNAi,表达有效的si RNA。本研究中构建的骨架载体可以广泛用于RNAi载体的构建,含有柑橘衰退病毒基因片段的3个载体可以用于基因功能分析及具有CTV抗性的柑橘种质资源获得。
[Abstract]:Based on the transformation virus gene mediated resistance, a universal RNAi vector skeleton structure with hairpin structure was constructed by inserting the promoter intron terminator into the plant expression vector p CAMBIA 2301. Using the conserved sequence of p25p20 and p23 genes of Citrus recession virus (citrus tristeza virus,CTV) as template, forward and reverse fragments were designed and connected with skeleton vector successively. Three RNAi vectors (ds2301 p25ds2301 p20 and ds2301 p23) were successfully constructed, and the vector ds2301 p23 was injected into the leaves of Lemont, Mexico. A digoxigenin labeled probe of p23 gene was made and si RNA production was detected by Northern hybridization. The results showed that the transient expression of p23 specific si RNA, in the leaf of Agrobacterium tumefaciens ds2301 p23 in Lemmon, Mexico, could be detected by Northern hybridization. The RNAi, expression of p23 could be found in the leaves of Agrobacterium tumefaciens. The framework vector constructed in this study can be widely used in the construction of RNAi vector, and three vectors containing the gene fragment of Citrus recession virus can be used to analyze the gene function and obtain citrus germplasm resources with CTV resistance.
【作者单位】：湖南农业大学园艺园林学院;国家柑橘改良中心长沙分中心;
【基金】：国家自然科学基因项目(300900972,3157211) 公益性行业(农业)科研专项(201203076 06) 湖南省研究生科研创新项目(CX2013B290)
【分类号】：Q943.2;S436.66

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