嵌合基因、保守片段序列特征对RNA介导多病毒抗性的影响及其机理研究

Viruses mixed infection often cause crops significant yield loss and quality decline. Based on RNA silencing, the multi-virus resistant transgenic plants can be obtained by transformation with the chimeric gene or the conservative sequence of virus genes. The transgene sequence characteristics have important influence on the silencing efficiency . In this study, the coat protein (CP) genes and the genes which have the function of silencing suppressor of Potato virus Y (PVY), Cucumber mosaic virus （CMV）and Tobacco mosaic virus (TMV) are to be cloned by Reverse Transcription-PCR（RT-PCR). The hairpin RNA (hpRNA) expressing vectors are to be constructed，which contains the 3' end or middle fragments of the cloned genes with the length of 300bp and the high ΔGloc or the low H-B index. Based on silencing the Green Fluorescent Protein (GFP) fusion protein expression by Agrobacterium tumfaciens-mediated transient expression technology, the cDNA sequences presenting potential high silencing efficiency are to be screened. Then, the RNA interference (RNAi) vectors of chimeric gene presenting different sequence characteristics are to be constructed by the use of the screened cDNA sequences. Additionally, the short hpRNA (shRNA) or the artificial miRNA (amiRNA) plant expression vectors are to be constructed, from 21bp high-sequence-similarity cDNA sequences, which correspond with siRNA or miRNA characteristics derived from genome sequence of PVY and Tobacco etch virus (TEV). These constructed vectors are to be introduced into Agrobacterium tumefaciens and transformed into tobaccos. Through the virus resistance assay, molecular biology analysis, and bioinformatics analysis of obtained transgenic plants, the effect of the sequence characteristics of chimeric gene and conservative fragment on RNA-mediated multi-virus resistance is to be investigated. After that, the effect mechanism is to be excavated in terms of the targeted sites, the formation and accumulation of siRNA, the amplification of silencing signal, and the function of silencing suppressor, etc. Through these research, the efficient genetic engineering strategies of multiple anti-virus will be explored.

病毒混合侵染常造成作物的重大产量损失和品质下降。基于RNA沉默原理，转化多病毒的嵌合基因或保守片段，可获得多抗病毒转基因作物。转基因的序列特征对沉默效率有着重要影响。本研究，拟以马铃薯Y病毒、黄瓜花叶病毒、烟草花叶病毒的外壳蛋白基因和具有沉默抑制子功能的基因为靶序列，在筛选获得高沉默效率cDNA序列的基础上，构建具有不同序列特征的嵌合基因的RNAi载体；以马铃薯Y病毒和烟草蚀纹病毒的全基因组序列为基础，寻找基因组中符合siRNA或miRNA特征、相似性较高的21bp的cDNA序列，构建小发夹RNA和人工miRNA结构的表达载体；将构建的载体转化烟草，对获得的转基因植株进行抗性鉴定和分子生物学、生物信息学分析，研究嵌合基因和保守片段的序列特征对RNA介导多病毒抗性的影响，从靶向位点、siRNA形成和积累、沉默信号的放大、沉默抑制子的作用等方面探讨影响的机理,探寻高效的多抗病毒基因工程策略。

病毒混合侵染常造成作物的重大产量损失和品质下降。基于RNA沉默原理，转化多病毒的嵌合基因或保守片段，可获得多抗病毒转基因作物。转基因的序列特征可能对沉默效率有着重要影响。本研究，以马铃薯Y病毒（PVY）、黄瓜花叶病毒（CMV）、烟草花叶病毒（TMV）的外壳蛋白基因和具有沉默抑制子功能的基因为靶序列，在筛选获得高沉默效率cDNA序列的基础上，构建了嵌合基因单发夹和双发夹、不同病毒基因排列顺序、类“SHUTR”结构等具有不同序列特征的RNAi表达载体，通过瞬时侵染或转基因稳定表达，研究嵌合基因的序列特征对RNA介导的多病毒抗性的影响。结果表明：嵌合基因双发卡较单发夹可介导更好抗病毒效果；不同病毒序列连接方式对RNA介导的多病毒抗性有一定的影响，由病毒沉默抑制组成的序列介导的抗病效率高于由病毒CP基因组成的序列，且以TMV-P126在5’端、CMV-2b在中间、PVY-HC-Pro在3’端的序列抗病效率为最高；类“SHUTR”嵌合基因序列能介导对病毒的抗性，不同结构间存在差异，“双链”区域位于中间的效率最高。不同序列特征的嵌合基因介导的抗病性差异，可能与靶向基因的功能、靶序列的二级结构、小分子RNA（small RNA）的剪切效率等有关。以马铃薯Y病毒（PVY）和烟草蚀纹病毒（TEV）的全基因组序列为基础，寻找基因组中符合siRNA或miRNA特征、相似性较高的21bp的cDNA序列，构建小发夹RNA和人工miRNA结构的表达载体；通过瞬时侵染或转基因稳定表达，研究了保守片段的序列特征对RNA介导多病毒抗性的影响。结果表明：序列相似性在85.71%以上单shRNA或miRNA均可介导对两种病毒的抗性，但是抗性与序列相似性并不呈完全正相关；shRNA有义链5' 端碱基性质、碱基错配的位置等对抗性有着明显的影响；多sRNA的组合可以显著提高抗性水平，尤以siRNA和miRNA组合为最佳。此外，还对温度、沉默抑制子等其他影响RNA沉默效率的因素进行了研究，解析其作用机理。上述研究结果，为我们深入了解RNA沉默的机理，更加高效地利用RNA沉默策略，培育高抗或多抗病毒转基因植物提供了依据。