应用RNA沉默技术获得抗PRSV和PLDMV病毒的转基因番木瓜两性株的研究

Papaya production is seriously limited by Papaya ringspot virus (PRSV) worldwide and Papaya leaf-distortion mosaic virus (PLDMV) in Eastern Asia. An efficient transformation method for developing papaya lines with transgenic resistance to these viruses and commercially desirable traits, such as hermaphroditism, is crucial to shorten the breeding program for this fruit crop. Transgenic plants expressing double-stranded RNA(dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this proposal, a chimeric construct with two short inverted repeat (IR) under control of the 35S promoter and terminator of Cauliflower mosaic vius will be generated. Each IR contains highly conserved sequence of PRSV and PLDMV replicase. The two short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgenic assembly process. The somatic embryos derived from adventitious roots of in vitro multiple shoots of hermaphroditic plants of papaya cultivars "shuluo No.1" will be transformed by Agrobacterium carrying the two short IRs with highly conserved sequence of PRSV and PLDMV replicase. .The proposed research will be innovative because it represents the first comprehensive effort to generation of hermaphrodite transgenic papaya lines with robust RNAi-based resistance to mixed infection of two viruses in papaya plants expressing separate short hairpins from single transgene. The transgene design in the proposal has at least three advantages. First, instead of using viral CP coding sequence to construct the trangene, we choose to use highly conserved regions of replicase genes. Viral CPs are considered to be more variable than viral replicase. As a result, RNAi triggered by highly conserved replicase sequences should be effective against more strains of a virus. Another advantage of our transgene design is that it confers resistance to two different viruses with one single transgene. The third advantage is that the transgenic papaya will be achieved from the proposal will be hermaphrodite papaya lines which have commercially desirable characters. And the transformation method proposed here can significantly shorten the timeconsuming breeding programs of the generation of elite cultivars of papaya hybrids..Our proposed studies are significant because, upon their completion, we will achieve the hermaphrodite transgenic papaya with robust resistance to PRSV and PLDMV. It will not only solve the problem of papaya production, but also contribute to deepen our understanding of papaya breeding through RNA silencing technology.

番木瓜（Carica papaya L.）是人们喜爱的果蔬兼药用的热带水果。番木瓜环斑病毒（PRSV）正引起毁灭性严重病害，番木瓜叶扭曲花叶病毒（PLDMV）也在威胁着目前转基因番木瓜的推广和应用。这两种病毒病害严重威胁着番木瓜种植业和加工产业的发展。因此本项目拟以对病毒的复制起关键作用并在各病毒株系中比较保守的病毒复制酶基因为靶标基因，根据RNA沉默的原理构建同时包含PRSV和PLDMV复制酶基因高度保守片段的反向重复结构的植物干扰表达载体，以番木瓜两性株的组培苗根尖诱导的体胚为转化材料，通过农杆菌介导法快速获得稳定、高抗、谱抗、安全性高并且具有果实种子少、果肉厚等商业化所需特性的抗病毒转基因番木瓜两性株新种质。研究结果将解决当前番木瓜生产和发展的实际问题，并为利用RNA沉默技术在番木瓜育种中的运用提供理论依据。

番木瓜是人们喜爱的果蔬兼药用的热带水果。番木瓜环斑病毒正引起毁灭性严重病害，番木瓜叶扭曲花叶病毒也在威胁着目前转基因番木瓜的推广和应用，这两种病毒病害严重威胁着番木瓜种植业和加工产业的发展。本项目通过人工合成目的片段的方法，构建包含这两种病毒的病毒复制酶基因及Hc-Pro蛋白基因的高度保守序列的反向重复序列的植物干扰表达载体pPTN-LS；运用本生烟瞬时表达系统验证所构建的RNAi载体pPTN-LS能有效同时沉默两种病毒Nib和Hc-Pro基因的表达。以两性株侧芽为外植体，建立栽培种番木瓜台农2号两性株再生体系；以组培苗的不定根为外植体，建立该品种体胚发生的转化体系；通过农杆菌介导法将构建的RNAi载体pPTN-LS导入番木瓜台农2号，经PCR及Southern杂交证明已获得10株转基因植株，正在扩繁转基因植株准备做大田实验；通过运用实质性等同原则，对插入三个短的反向重复结构的抗病毒转基因大豆种子与对应的非转基因大豆种子进行比较，结果显示，这些抗病毒转基因大豆的营养成分与相对应的非转基因实质性等同，并且大部分的营养成分和抗营养成分的测定值都在已报道的其他商业大豆范围内。该结果暗示，反向重复序列的插入不会导致抗病毒转基因大豆发生具有重要生物学意义的改变，为利用RNA沉默技术在番木瓜育种中的运用提供理论依据。