SlSAUR58/59调控番茄坐果以及果实早期发育的分子机理研究

Tomato, a fleshy fruit, is widely grown in the world, and accounts for a large portion of agricultural production and consumption. However, the abortion or abnormal growth of the ovaries often occurs due to the unsuccessful pollination/fertilization under protected culture. Therefore, it is important to study the molecular mechanisms regulating fruit set and early fruit development by auxin in tomato. Based on the isolation of novel members of small auxin-up RNAs in Lycopersicon esculentum, the expression profiles of SlSAUR58, SlSAUR59 and some other related SlSAURs during fruit set and development are characterized using qRT-PCR and in situ hybridization. The relationship between the transcription levels of SlSAUR genes with the fruit development is analyzed based on the results of hormone treatments and temperature stresses. The functions of SlSAUR58 and SlSAUR59 are identified by overexpression and RNAi technique during fruit set and early fruit development. Meanwhile, their target genes are screened using RNA-Seq and qRT-PCR analysis, and the differential expression characters of downstream genes are also analyzed. The interaction between SlSAUR58/59 and other auxin response genes or transcription factors is proved using ChIP assay and DNA-binding analysis. The different or redundant role of SlSAUR58 and SlSAUR59 in auxin signal transduction is also studied. Finally, the molecular model of their regulation on fruit development will be established. The results will provide the basis for exploring the molecular mechanisms of auxin action on fruit development. The project also can provide the knowledge to develop the novel pathenocarpic germplasm by genetic engineering in tomato.

针对设施番茄生产中易发生的授粉受精不良，坐果率下降等问题，开展番茄果实发育的调控机理研究。在业已分离番茄SlSAUR家族新基因基础上，利用细胞学技术以及qRT-PCR方法研究SlSAUR58与SlSAUR59在番茄果实发育过程的表达模式；通过激素与逆温处理试验研究SlSAUR基因的表达水平与番茄果实发育的关系；利用SlSAUR58/59超量表达以及RNAi技术研究SlSAUR58/59在番茄坐果与早期果实发育过程的生物学功能；利用转录组测序RNA-Seq结合qRT-PCR方法鉴定SlSAUR58/59作用的目标基因；并利用ChIP分析验证互作关系；旨在阐明SlSAUR58/59在生长素诱导的果实发育信号途径的作用，建立其调控番茄果实发育的分子模式，为深入解析生长素调控果实发育的分子机理提供研究基础，同时为利用遗传工程技术创建单性结实番茄新种质提供理论依据。

生长素是最早被鉴定出来的一类植物激素，其信号传导机制一直是植物生物学研究的热点。SAURs是能被生长素在很短的时间内诱导表达的、数量最多的一类生长素原初反应基因。番茄是一种世界上广泛种植的重要蔬菜作物以及生殖发育模式植物。番茄雄蕊发育、果实坐果与发育有赖于植物多种内源激素的协同作用。本项目在分离番茄生长素早期响应基因SlSAUR基因家族99个成员基础上，对其基因结构、进化与表达特征进行了分析，获得一批表达特异的SlSAURs基因；探索建立了番茄高效的CISPR/Cas9基因编辑技术；利用qRT-PCR、原位杂交以及启动子活性分析等方法研究了SAUR58/59以及相近SAUR家族基因成员在番茄生殖发育过程的表达模式以及组织表达特性；利用基因过表达以及抑制或敲除技术研究了SlSAUR58/59以及在雄蕊与雌蕊中特异表达的SlSAUR54等基因在番茄生长以及生殖发育过程的生物学功能；SlSAUR58可以调控番茄的营养生长以及果实的膨大过程，而SlSAUR59与SlSAUR54下调均影响花粉发育从而降低座果率，同时SlSAUR54可诱导兼性单性结实；说明不同SlSAUR基因即使序列相似，但功能上也存在分化。通过对SlSAUR58同源基因SAUR32的功能分析证实不同物种间SAUR基因功能较保守，利用RNA-seq及qRT-PCR明确了SlSAUR58参与调控的下游激素信号传导基因；通过蛋白质杂交以及激素处理实验证实了SlSAUR58可以同细胞分裂素信号传导基因ARR6与ARR15互作，说明其通过参与细胞分裂素信号途径参与调控番茄发育过程；最终建立了SlSAUR58通过参与生长素与细胞分裂素等激素信号互作从而调控番茄发育过程的分子模型。为深入阐明生长素调控植物果实发育过程分子机理提供研究基础，也为番茄坐果及果实性状的遗传改良提供新途径。