黄瓜单性结实主效基因的克隆与功能研究

Parthenocarpy is kind of fruit developmental mode that production of fruit without pollination or fertilization of ovules. It is an excellent agronomic trait that can ensures yield stability of horticultural crops in unfavorable pollination conditions. In-depth study of parthenocarpy could improve the understanding of regulation mechanism of fruit development and help to enhance the fruit setting rate of horticultural crops. The rich parthenocarpic germplasm of cucumber offer an opportunity to investigate molecular mechanism of parthenocarpy. In previous study, we have have firstly finished the physical mapping of the major parthenocarpic QTL in cucumber. In this study, we intend to clone the target gene of the QTL which we named PARTH2, and identified the molecular function of the gene. First, fine mapping of PARTH2 will be conducted by whole genome resequencing of NIL populations. Thus the candidate gene of PARTH2 could be predicted and cloned. Transgenic technology will be used to identify the functions of the candidate genes to confirm the target gene. The subcellular location and mRNA/protein expression profiles of PARTH2 will be studied by single cell system, qRT-PCR and western blotting. The interaction protein of PARTH2 will be screened by the Y2H library. The protein regulatory network of PARTH2 and its interaction proteins will be studied by combined analysis with the results of iTRAQ study of parthenocarpy. This study will lay an important foundation for revealing the molecular regulation mechanism of parthenocarpy, and is able to provide assistance for improving the parthenocarpic characteristics of cucumber and other horticultural crops.

单性结实指子房不依赖于授粉/受精而坐果的果实发育方式，在不利于授粉的栽培条件下能够保证作物产量，是一种重要的农艺性状。深入研究单性结实分子机制对于完善果实发育的调控模型以及作物单性结实性的改良具有重要意义。黄瓜具有丰富的单性结实表型，是研究单性结实的重要作物。前期研究中，我们首次完成了黄瓜单性结实主效QTL的物理定位，本项目拟在此基础上开展其主控基因PARTH2的克隆和功能研究：构建NIL群体，采用群体重测序方法实现主效QTL精细定位，进而完成定位区段内的PARTH2候选基因的预测；对PARTH2候选基因进行克隆和转基因功能分析，确认目标基因并研究其亚细胞定位及表达特征；利用酵母双杂交文库技术筛选鉴定PARTH2的互作蛋白，结合已完成的iTRAQ蛋白质组学研究结果分析其蛋白调控网络。本研究将为揭示单性结实分子调控机理奠定了重要基础，对指导黄瓜和其它园艺作物单性结实性的育种改良有重要意义。

单性结实对于果实产量和品质都用重要的影响，发掘单性结实基因改良黄瓜单性结实性具有非常重要的意义。前期研究中，本团队已完成了对该主效位点Parth2.1的初步定位。本项目研究在此基础上，完成了Parth2.1的精细定位（<37kb，仅1个候选基因），并首次开发了黄瓜强单性结实性状的育种标记，同时成功克隆了该位点的目标基因CsPAT1，该基因编码Ca2+ATPase钙泵蛋白；由于第6个内含子有26bp的碱基缺失，第7个内含子有4bp的碱基插入， CsPAT1基因mRNA发生可变剪切，形成了转录本PAT-EC1-4和PAT-EC1-11其中PAT-EC1-4编码蛋白的Ca2+ATPase功能域部分丢失，而PAT-EC1-11编码蛋白的Ca2+ATPase功能域完全丢失；亚细胞定位结果显示PAT-EC1-4与未突变的等位基因编码的蛋白均定位于烟草叶片原生质体的叶绿体中，而PAT-EC1-11蛋白不但在叶绿体中有大量表达，在围绕细胞核的内质网中也有表达。在拟南芥中分别异位表达PAT-EC1-4和PAT-EC1-11后，均可造成未授粉果荚的生长，证明了CsPAT1参与了单性结实调控。我们发现在单性结实黄瓜果实中钙调素类似蛋白基因CsaCML25大量表达,而在非单性结实黄瓜中过量表达CsCML25可以诱导强单性结实，因此推测由于CsPAT1基因的Ca2+ATPase功能缺失造成了胞质游离钙浓度增高，从而激活了下游钙信号应答因子CsCML25的大量表达，最终诱导形成单性结实。.项目对黄瓜单性结实的重要位点Parth1.1、Parth3.1、Parth7.1进行了遗传定位研究，为强单性结实的多位点调控机制研究奠定了基础。同时开展了低温诱导单性结实种植资源的鉴定和转录调控机制研究，为环境调控单性结实的分子机理研究奠定了基础。.