罗汉果性别分化分子遗传机制研究

It seriously blocked breed improvement that seedling sex of siraitia grosvenorri could not be determinated. Understanding mechanism of sex differentiation play a role to sex determination of siraitia grosvenorri. It remain unknown how sex differentiation of siraitia grosvenorri was determinated. We found that flower development processes of siraitia grosvenorri were in accord with Cucumis sativus. Cucumis sativus were beyond the same family with siraitia grosvenorri, and its sex differentiation of were regulated by hormonal. Additionally, gibberellins content of bisexual and male flower stems were more than of female flower stems. Therefore，we supposed that sex differentiation of siraitia grosvenorri were relative with hormonal also, and their genetic regulating hypothesis is proposed. Gibberellins induced expression of 1-Aminocyclopropane-1-carboxylate synthase genes to regulate ethylene biosynthesis, and ethylene induced sex differentiation of siraitia grosvenorri. Using female, male and hermaphrodite near-isogenic line deriving from five backcross generation of the mutant hermaphrodite clone, transcription，cloning, spatial and temporal expressing characteristic, and functional identification of sex genes were carried out by methods of Solexa high-throughput RNA sequencing，rapid-amplification of cDNA ends, real-time fluorescence quantitative PCR and Agrobacterium-mediated genetic transformation, in order to explore interaction relations of hormonal，small RNA and 1-Aminocyclopropane-1-carboxylate synthase genes, and reveal genetic regulating molecular mechanism of sex differentation in siraitia grosvenorri. That will lay foundations for seedling sex determination of siraitia grosvenorri and revealing mysteries of sexual differentation in gonochoristic medicinal plants of Cucurbitaceae such as Trichosanthes kirilowi.

苗期性别无法鉴定严重阻碍罗汉果品种遗传改良。明确性别分化机制在罗汉果苗期性别鉴定过程中起关键作用，但罗汉果性别如何分化问题不清，我们前期研究发现罗汉果花发育与性别受赤霉素和乙烯调控的同科植物黄瓜一致，且其两性花、单雄花分生茎赤霉素含量高于单雌花分生茎的，故认为其性别分化也与激素相关，据此提出赤霉素诱导1-氨基环丙烷-1-羧酸合酶基因（ACS）表达调控乙烯生成决定罗汉果性别的遗传机制假设。我们拟以芽变无性系回交五代构建的雌性、雄性和两性近似等位基因系为材料，采用Solexa高通量测序、cDNA末端快速扩增、实时荧光定量PCR和农杆菌遗传转化技术手段，进行罗汉果性别分化相关基因转录组与小RNA测序筛选、cDNA全长克隆、时空表达特性分析和功能遗传转化鉴定研究，探索激素、小RNA和ACS互作问题，以揭示罗汉果性别分化分子遗传机制，为其苗期性别鉴定和栝楼等雌雄异株药用植物性别分化奥秘揭示奠定基础。

罗汉果雌雄异株，一方面有性繁殖后代雄株远多于雌株，育种群体种植占用土地、资金多且管理难度大，另一方面因性别调控机制认识缺乏，经多方面研究仍未能建立有效的性别早期鉴定方法剔除雄株，使得育种工作举步维艰。因此，本项目以两性、雌性、雄性株系及其有性繁殖后代群体为材料，性别遗传分离分析发现，“两性株系×雄株系”后代雄株∶雌株∶两性株为6∶3∶1，“雌株系×雄株系”后代雄株∶雌株∶两性株为7∶3∶0；外源激素诱导性别转变发现，性别分化受乙烯和细胞分裂素拮抗调控，雌、雄株乙烯反应敏感性差异明显，建立起诱导单性花成两性花方法；雌、雄株茎尖转录组、表达谱和microRNA高通量测序分析，获得一批性激素乙烯和细胞分裂素合成及信号转导途径的候选性别基因，并RACE和PCR克隆了与同科植物黄瓜、甜瓜性别基因同源的SgACS1G、SgACS2、SgACS7B基因cDNA全长，SgACS1G、SgACS2、SgEIL1、SgERF110、SgGyno-hAT基因DNA全长，以及SgEIL1基因启动子区，发现该启动子区存在性别连锁SNP突变；BSA结合QTL-seq性别定位分析，在Chr3拟染色体29～30 cM区域检测到一个候选性别位点，发现性激素合成及信号转导途径基因仅SgEIL1位于该区域；雌、雄株营养生长、花性分化期和激素诱导二级蔓茎尖、茎段、叶片、花蕾表达谱测序分析基因时空表达规律发现，SgEIL1偏雌株表达，在花蕾尤其花蕊表达量最高，显示其参与花蕊发育；基因遗传转化拟南芥鉴定功能表明，SgACS7B参与乙烯合成使植株变矮小，SgACS7A和SgEIL1则未引起植株表型变异；利用对乙烯反应敏感性差异，建立起茎端和叶片早期鉴定性别的方法。这些结果初步阐释了罗汉果性别生理、分子调控机制，为基因编辑调控性别、性别分子标记开发提供了重要基因信息，也为育种中自交系培育和子代性别鉴定提供了方法。