控制番茄长节间主效QTL的克隆与机理解析

The internode is the critical factor to affect the plant architecture. An idea plant architecture provides not only a base for the yield production but also the saving of time, labor and cost. Dwarf mutants as called ‘Green Revolution Gene’ have been widely used for the genetic improvement in crops including rice, wheat, sorghum, maize etc. Presently many researches have been conducted to explore these dwarf genes and to understand their regulatory mechanism. Whereas, less information have been reported in the model plant tomato. In our group, one accession named as P502 (Solanum lycopersicum) possessing long internode was found from amount of germplasms during a long term of breeding program. Genetic analysis and mapping proved that this trait is controlled by one major QTL and further mapped to an interval around 214.14kb in tomato genome. In this project, candidates involved in this region will be further analyzed by combing the informatics based on a deep re-sequencing（50X）of accession P502 with long internode. The function of these confirmed candidates will be verified further by CRISPR-Cas9 and complementary experiments. The first gene conferring long internode will be cloned in tomato to understand its structural features and possible genetic evolution, and to illuminate the regulatory mechanism under this locus. The obtained results will provide not only a theoretical base for deciphering the regulatory network of the internode length but also a technical support for genetic improvement of tomato plant internode and its control in the practice.

植株的节间长度是株型构成的关键因子，理想株型不仅是作物高产的基础，而且还可省时、省工、省力，节约成本。被誉为“绿色革命基因”的矮化突变基因已广泛应用于水稻、小麦、高粱、玉米等大田作物。目前，众多研究集中在矮化基因的挖掘及其调控机制，而对番茄这一重要园艺作物节间的研究还鲜有报道。项目组在长期从事育种的过程中，从大量资源中发现了一份长节间特异资源P502，通过前期遗传与作图分析，确定其受主效QTL控制并将其定位在番茄基因组214.14kb的区间内。本项目拟在对长节间材料P502深度（50X）重测序的基础上，结合生物信息学获得该位点的候选基因，利用基因编辑技术、互补实验验证其功能，将首次克隆番茄控制节间长度的基因，明确其结构特征和可能的遗传演化，解析其在调控节间长度中的作用机制。研究结果不仅能够为完善植物节间长度的调控网络提供理论基础，也将为番茄株型遗传改良及生产调控提供一定的技术支撑。

通过表型分析，发现番茄长节间突变体P502与野生型05T606相比，表现整个苗期节间均伸长的特性，且每一节间都显著增长；结合内源GA测定和外源GA喷施实验，突变体P502能响应外源GA3和PAC，且生物活性的GAs含量增加，为GA敏感型突变体。细胞学观察表明，P502节间细胞较野生型05T606显著伸长。利用普通节间自交系Heinz 1706和紧凑型自交系P1609分别与突变体P502杂交，构建了两个组合的六世代遗传群体（P1、P2、F1、B1、B2、F2），通过对节间伸长性状进行主基因+多基因的数量性状遗传模型分析，结果表明长节间受主基因调控。利用Heinz 1706与P502构建的F2群体，采用BSA混池分析法，将目标位点定位在2号染色体上，进一步的QTL初定位分析检测到一个主效位点，可解释表型变异率的73.6%；进一步扩大F2群体并加密分子标记，最终将目标位点锁定在2号染色体上的标记CAPS17和InDel6之间75.8-kb范围内；分析候选区间内10个基因的序列差异，发现只有基因solyc02g080120.1编码区序列存在一个碱基差异（G-T），可能为目标基因的候选基因。solyc02g080120.1（EI）基因编码的SlGA2ox7蛋白与拟南芥AtGA2ox7和AtGA2ox8同源，且碱基突变（G-T）引起了保守结构域PcbC家族中氨基酸（G-V）的变异。通过过表达转基因互补验证，将野生型cDNA全长导入长节间突变体P502后，转基因植株表现矮小，节间变短，且能响应外源GA3，说明过表达EI基因能抑制节间伸长，使植株矮化。亚细胞定位结果表明，SlGA2ox7蛋白位于细胞核、细胞膜和内质网中。qRT-PCR结果表明，EI基因的表达具有组织特异性，在器官叶中的表达量较高，茎和根中的表达量较低。EI基因的突变未引起自身表达量变化，但引起了GA代谢通路中其他基因的表达量发生了不同程度的变化。