番茄RNA解旋酶基因的抗病功能及机理研究

RNA helicases are involved in every step of RNA metabolism, including transcription, pre-mRNA splicing, translation, ribosome biogenesis and assembly, nucleocytoplasmic transport and RNA decay. Because of their multiple functions, RNA helicases have been the research hotspot in life sciences. RNA helicase is one of the largest families, with abundant members and various functions in Arabidopsis thaliana, Oryza sativa, etc. Previous studies have shown that the RNA helicases are mainly involved in plant growth and development, diverse physiological processes and responses to stresses. Nevertheless, the role of RNA helicases in plant disease resistance is still largely unknown, especially in vegetable plants. Tomato virus diseases are widely distributed during tomato growth, and significantly reduce the quality and yield of tomato. So far, the function of tomato RNA helicases was poorly known, especially its role in responses to tomato virus diseases. In this project, we aim to systemically study the function of two RNA helicase genes SlRH-a and SlRH-b screened by Agilent microarray analysis in tomato. Initially, we will examine the expression profiles of SlRH-a and SlRH-b in different tissues. Secondly, the responses of SlRH-a and SlRH-b will be studied in tomato treated with defense-related molecules such as SA, MeJA, and ETH. Thirdly, the transgenic lines will generated, with over-expression or RNA interference of genes to study the function of SlRH-a and SlRH-b in controlling plant defense to deceases caused by tomato virus. Together, this study will provide valuable information in understanding the mechanisms of RNA helicases in tomato defense responses, and provide theoretical basis on breeding the resistant tomato plants.

RNA解旋酶是一类参与RNA结构调控并影响RNA合成、翻译启始、核糖体组装、核mRNA输出及mRNA降解的蛋白质新家族。有关植物RNA解旋酶的研究近年来成为生命科学领域的研究热点。研究表明，RNA解旋酶家族在拟南芥、水稻等模式植物中成员繁多，参与调控生长发育、生理生化以及胁迫响应过程。但是，RNA解旋酶在植物抗病中的功能及作用机理研究较少。番茄病毒病是番茄生产中的重要病害之一，分布十分广泛，严重影响了番茄的品质和产量。目前，对于番茄RNA解旋酶基因的研究仍处于初始阶段，它们与番茄病毒病之间的关系报道甚少。本课题拟研究通过Agilent芯片筛选出的番茄抗病信号相关RNA解旋酶基因SlRH-a和SlRH-b，分析并验证它们在抗病信号下的表达调控特点；分析超表达和RNAi株系在抗病中的表型差异，确定抗病功能，并对二者可能的抗病作用机制进行探索，以期为番茄抗病新品种的选育提供新途径。

本课题对通过Agilent芯片筛选出的抗病信号相关的番茄RNA解旋酶基因SlRH-a和SlRH-b进行了功能验证，并初步探讨其在防御激素条件下的作用机理。SlRH-a和SlRH-b均具有两个Domain组成的核心区域，保守基序II分别是DEIH和DEID，属于DExD/H-box型RNA解旋酶；SlRH-a基因在成熟萼片和果实中表达量较高，而SlRH-b基因在成熟叶中表达量最高，在主茎、成熟萼片、侧茎和果实中表达量次之，二者呈现不同的组织表达模式；SlRH-a和SlRH-b基因受MeJA的诱导非常明显，且SA和ETH处理后均能使两基因的表达量升高，40℃高温处理后两基因的表达量呈下调趋势，说明两基因表达受激素和高温信号的调节，并且呈现一定的时空规律；在两基因启动子区域中包含了一些重要的响应激素、胁迫和光信号等顺式作用元件；通过遗传转化获得了转番茄RNA解旋酶SlRH-a和SlRH-b的转基因烟草株系，分别进行了防卫响应激素和高温处理的表型鉴定，随后对转基因株系和野生型进行了芯片分析，结果显示抗病信号相关激素处理后表达下调的差异基因多于上调的，经MeJA处理后筛选的DEGs明显少于经SA或ETH处理后筛选的DEGs，暗示经过SA和ETH两种激素处理后可以诱导更广泛的基因变化，抗病信号激素处理后抑制了更多基因的表达；GO分析发现差异表达基因参与多种过程，其中参与防御响应过程基因数量较多，比如PR1、PTO、PTI5、RFA1和RPA1等，后期将着重研究RNA解旋酶SlRH-a和SlRH-b与上述防御响应关键基因的互作关系。本研究将有助于从RNA解旋酶角度及防御信号激素调控角度揭示SlRH-a和SlRH-b在抗病领域及相关胁迫过程中的表达机制，对新基因资源的发掘及抗逆分子育种等领域的研究都具有重要的科学意义和应用价值。