赤霉素对麻疯树雌雄花分化和发育调控的机制研究

Jatropha curcas L is thought as a promised idea biofuel material. It is a sexual dimorphism monoecism plant. It is reported that the small ratio of female to male flowers (1/10—1/30) was one of the critical factors to response for the low seed yield of J. curcas. Previous studies suggested that the application of exogenous gibberellins acid (GA) could increased both the number of female flower the ratio of female flower to male flower. In the previous studies, the gene expression profile analysis showed that GA would promoted the carpels differentiation in female flowers and/or the pollen maturance in male flowers. In this study, the inflorescence buds before the initiation of sex-differentiation and that after the finish of sex-differentiation will be treated with exogenous GA and GA-biosynthesis inhibitor at different concentrations, respectively. The gene expression profiles will be constructed from inflorescence buds of different treatment, and GA-regulated genes will be selected based on the analysis of differently expressed genes. The GA-regulated genes related to sex development will be preliminarily screened out from GA-regulated genes based on the sex related genes selected from gene expression profiles constructed from the flower buds at different development stages of sex differentiation and the flower buds at different development stages of micro- and macro-spore genesis and male and female gametophytes development. The GA-regulated genes related to sex development will further be identified, based on the concentration of endogenous GA in flower buds at different development stages and the expression of preliminarily selected GA-regulated genes related to sex development in male, female and/or bisexual flower that developed from the inflorescence buds treated with GA and GA-biosynthesis inhibitor, and in the flower buds at different development stages of flower. The functions of these GA-regulated genes related to sex development will be intensive studied, and threw light on the mechanism that GA apply to regulate the sex development of flower in J. curcas.

麻疯树是一种很有开发前途的优良生物能源树种,雌雄异花同株。在自然情况下，麻疯树的雌、雄花比例很低（1/10—1/30），这也是麻疯树种子产量低下的一个主要原因。外施适量浓度的赤霉素可增加雌花的数量和雌、雄花比例。分析麻疯树花性别发育不同时期的基因表达谱发现赤霉素可能在雌花的心皮分化和雄花花粉粒成熟期起重要作用。本研究将用不同浓度的外源赤霉素及其生物合成抑制剂分别处理性别分化前的和完成性别分化的花序芽，建立不同处理花序芽的基因表达谱，分析差异表达基因，找出赤霉素调控基因,结合前期工作筛选出的性别发育相关基因,初步确定受赤霉素调控的性别发育相关基因。分析这些基因在由处理花序芽发育而来的雄花,雌花（和）或两性花中的表达情况,结合花发育不同时期这些基因表达情况及內源赤霉素含量,确定受赤霉素调控的性别发育相关基因,深入研究这些基因的功能,从而深入而全面揭示赤霉素影响麻疯树花性别发育机制。

有研究表明外施赤霉素可以增加麻疯树雌花数目和雌雄花比例，而其中机制还不清楚。本项目分析了麻疯树花发育不同时期的花蕾中内源激素含量变化,结合花发育不同时期转录组数据分析来研究赤霉素对麻疯树花发育影响的机制。分析了麻疯树雄花小孢子发生和雄配子体发育过程主要阶段的花芽的蛋白质组，对蛋白质组和转录组进行了联合分析。克隆了与麻疯JcGASA1和JcCAL-like1这两个基因，并转化烟草验证其在花发育中的作用；对这两个基因进行了启动子元件分析，通过酵母单杂筛选其激素响应元件互作的转录因子。通过酵母单杂和酵母双杂来筛选与JcGASA6互作的上下游基因。结果如下：.1）生长素有助于雄花花粉粒成熟期的发育。GA4和GA7更有利于雌花的分化和发育；GA1有利于雄花小孢子母细胞减数分裂和游离小孢子发育，以及雌花胚乳成熟期的发育；GA3参与了雄花和雌花的大小孢子母细胞减数分裂。茉莉酸参加了雄花花粉粒成熟期和雌花胚囊成熟期发育。同时还发现了一批可能参与花发育的激素合成相关基因和激素信号途径基因。.2）赤霉素可能通过调空JcCAL A-Like，JcGASA6和 JcGAST1基因的表达来影响花的发育。JcCAL A-Like和 JcGAST1这两个基因的转录受赤霉素、脱落酸、茉莉酸甲酯和水杨酸这四种植物激素的调控。赤霉素负调JcCAL A-Like的转录，而茉莉酸正调其转录，而JcGAST1刚好相反。JcGASA6和JcGAST1除了是赤霉素信号途径下游组分，同时也是多种激素（尤其是茉莉酸）信号途径的cross-talking 的节点。.3）开花相关基因JcFLX、脱落酸受体JcPYL9 和JcAP2能从转录水平正调JcGASA6基因的表达。JcCAL A-Like和JcGAST1的表达受AT-hook motif nuclear-localized protein 9、bZIP transcription factor 11和transcription factor RF2a、AT-hook motif nuclear-localized protein 19和zinc finger A20 and AN1 domain-containing stress-associated protein等转录因子的调控。.本研究为进一步全面和更深入研究赤霉素调控花发育的机制奠定了基础。