植物mRNAs在寄生体系中的长距离运输及功能研究

Besides functioning in the local cells, mRNAs (messenger RNAs) can be translocated to distant organs as signaling agents to mediate plant development. Due to the difficulties in distinguishing the long-distance transported mRNAs from those synthesized locally, only few mRNAs have been characterized by grafting. Previous studies and our preliminary work revealed that parasitic plants can import mRNAs from their hosts, and the sequence divergence makes it easy to distinguish the host mRNAs from the parasite mRNAs, and this provides us an ideal system for studying RNA trafficking. Previous methods used to detect the transported mRNAs in large scale also suffered from low sensitivity and specificity, and limited further investigation in their functions. In this project, we firstly identify the profiles of the transported mRNAs in three parasitic systems, Cuscuta australis grown on soybean, C. pentagona grown on tomato, and C. chinensis grown on oilseed rape, under different developmental stages and stress conditions with the next generation sequencing technology. By comparing the transported mRNAs from the three parasitic systems, the mRNAs with possible important functions can be identified from the common ones. The transport distance, integrity, and stability will be analyzed along the growing dodder stems using RT-PCR, 5'-RACE and qPCR, respectively. The possible roles as long distance signaling agents allowing developmental coordination of parasitic plants with their hosts will be lastly investigated by detecting their tissue-specific distribution using in situ RT-PCR and immunohistochemistry. This project utilizes the parasitic system to investigate mRNAs trafficking and the results will not only present an important foundation for further functional study of transported mRNAs but also provides new insights into the non-cell-autonomous signaling macromolecules.

近年报道mRNAs可作为长距离运输的信号大分子，因其难与本地转录的mRNAs相区分，多用嫁接法进行研究。我们的前期工作表明寄生植物也能转运寄主的mRNAs，且其序列由于物种的差异可以较为容易地被鉴定出来，因此寄生体系为研究mRNAs的长距离运输提供了理想的材料。本研究以寄生在不同发育阶段的大豆和拟南芥上的菟丝子为研究对象，利用二代深度测序技术、qPCR、5'-RACE等手段，结合生物信息学和分子进化分析，全面鉴定菟丝子不同组织中转运寄主mRNAs的种类和差异；选取功能重要的分子，检测其在菟丝子内的运输距离、完整性和稳定性。本项目以寄生体系来研究长距离运输的mRNAs，可为其功能研究提供新契机，并为非细胞自身合成的生物大分子的信号传递提供新思路。

近年报道mRNAs可作为长距离运输的信号大分子,因其难与本地转录的mRNAs相区分,多用嫁接法进行研究。我们的前期工作表明寄生植物也能转运寄主的mRNAs,且其序列由于物种的差异可以较为容易地被鉴定出来,因此寄生体系为研究mRNAs的长距离运输提供了理想的材料。本研究首先对生长在大豆上的南方菟丝子种子、幼苗、预吸器、距离吸器近端和远端的茎段、花、花蕾和果实进行了转录组分析，发现长距离转运的mRNA种类丰富,有5967种。但丰度普遍很低,平均每个mRNA仅有3.9条reads被检出。在组织分布上，近吸器茎组织中检出大豆mRNA最多。其次，从寄生在不同发育阶段和胁迫条件下(促使表达谱更为丰富)的大豆、番茄和拟南芥上的菟丝子茎转录组中,全面鉴定菟丝子转运不同寄主mRNAs的种类和差异;并通过与寄主体内的丰度进行比较，分析了影响转运的因素，发现mRNA的运输具有一定的选择性，而非随机运输。选取功能重要的20个mRNA分子,使用RT-PCR检测其在菟丝子内的运输距离、完整性和稳定性，但得不到目的产物，因此推测转运后mRNA在完整性和稳定性方面都比较差。将不同寄主中转运mRNA的种类进行比较，发现共同转运的mRNA较少，由此认为mRNA的运输对寄生植物的影响较小。本项目的开展为同一植物体内mRNA的长距离运输的功能提供了重要参考。