转基因罗汉果单性结实雌性系的创制、表现及主要性状的遗传研究

Siraitia grosvenorii is a kind of precious medical and edible economic crop in China, characterized by male and female reproductive organs occurring on different individuals. Therefore, artificial pollination should be conducted to get enough fruits, which wasted plenty of time, labor and cost,and caused products supply falling short of market demand. On the other hand, people's harvest aims have been focused on fruits, where seeds contain none mogrosides yet influence processing and flavor. Parthenocarpy may occurr with only female plant and have none seeds for being uncoupled from fruits development.So creation of parthenocarpic female lines of siraitia grosvenorii should be the key pick to overcome these problems. Several key research aspects would be carried out as follows: Parthenocarpic transgenic Luohanguo female plants would be obtained by the regulation of genes for auxin synthesis (iaaM) or responsiveness (rolB) driven by ovule-specific promoter DefH9 or the INNER NO OUTER (INO).Main characters such as quality, yield, the adaptability to temperature and day length etc. be investigated for parthenocarpic transgenic female line, also the microstructure and inner hormon of fruits from their intiation to ripening would be studied by comparsion.Based on the study of the accumulaiton law for morgrosides in developing fruits,combining iTRAQ (isobaric tag for relative and absolute quantitation) labelling and 2D LC - MS/MS technology, quantitative proteomic differences of fruits at the critical stages would be analyzed to reveal the changes of pathway related to secondary metabolism, then temporal and spatial expression of some genes corresponding to key enzyme be studied by using real-time fluorescent quantitative PCR and in situ hybridization and immuno-labelling electron microscopy. In order to find out whether transgenic parthenocarpy generation could be applied in cultivation, artificial pollination seeds would be harvested from parthenocarpic trangenic female line,and genetic studies on the production of fruit without fertilization and other main characters would be made.As a result,parhthenocarpic female lines of siraitia grosvenorii with comprehensive good agronomic characters would be selected out via comparison of different female lines and adaptability experiment to different ecological conditions such as day lenth and temperature etc.

罗汉果是我国重要特色药食兼用经济作物，因雌雄异株需要大量人工辅助授粉，生产成本高，产品供不应求，是其产业化发展中存在的主要问题；又因以果实为收获目标，种子不含甜苷，但影响加工和风味。单性结实只需雌株且果实无籽，故单性结实罗汉果的创制是解决问题的关键。拟克隆子房特异启动子DefH9、INO，生长素合成或响应基因iaaM、rolB，构建嵌合基因载体，转化罗汉果雌株，获得单性结实种质；调查转基因罗汉果单性结实雌性系的品质、产量、温光适应性等生物学性状，比较其果实发育的显微结构特征与内源激素的时序变化；研究单性结实罗汉果果苷的积累规律，以及关键时期iTRAQ标记的蛋白质组定量差异，并采用实时荧光定量PCR和超微免疫定位技术，进行关键酶基因的时空表达研究与功能分析；考察人工授粉杂交后代雌株的单性结实性及其它重要性状的遗传规律，经株系比较和温光适应性试验，评选综合性状优良的罗汉果单性结实雌性系。

罗汉果是是重要药食兼用经济植物，因雌雄株栽培中需要大量人工授粉，劳动成本高；因以罗汉果甜苷为最终收获目标，其种子占果实极大比重、富含油脂，却几乎不含甜苷，影响加工、风味和贮运。选育无需人工授粉且无籽的新品种是罗汉果种植和加工中亟需解决的科技难题。本项目将子房特异启动子和生长素信号途径关键基因与pBI121-Gus连接，构建过量表达载体，转化罗汉果雌株，获得单性结实种质，研究重要性状的遗传表现，结果如下：.1、克隆子房特异启动子（DefH9和2A11）和生长素信号途径关键酶基因（iaaM和rolB）；将二者的嵌合基因连入pBI121-Gus构建过量表达载体；根癌农杆菌介导转化罗汉果雌株，鉴定获得转基因罗汉果单性结实雌株系：AI系、AR系、DI系和DR系。.2、相比于普通罗汉果（CK1），转基因罗汉果单性结实雌性系营养生长正常，植株花芽、花、幼果和果实发育中总IAA的含量提高，或对IAA的敏感性提高，花器发育正常，花量增多，兼性单性结实，果实无籽或少籽，果皮厚，小于或接近有籽果实大小，果形变异较大，果肉细胞数量增加，总苷及罗汉果苷V含量提高，总产更高，适应性更强。.3、与普通有籽罗汉果相比，转基因罗汉果果实发育及甜苷组成含量表现相似变化规律， 10天、30天、50天和70天是罗汉果苷主要成分转化的关键时期，以T0代DI 系4个时期的果实为材料，进行蛋白质组差异分析，共鉴定差异表达蛋白192个，涉及132个代谢通路，以参与细胞和代谢过程、应激反应、发育过程、生物学调节为主，为揭示转基因罗汉果单性结实果实发育和品质形成机理提供了基础。.4、对转基因罗汉果雌性系授以不同遗传背景的花粉，获得系列杂交种子，为其深入研究和遗传改良提供了基础。.5、单性结实罗汉果花器发育正常，只开雌花，不存在昆虫传粉引起种间杂交和基因漂移，果实无籽或少籽，减少了种子所致遗传漂移，表现良好的生态安全性；除没有种子油脂味以外，转基因罗汉果重要营养成分没有显著变化，表现良好的食品药品安全性。