梨果实萼片宿存与脱落关键基因鉴定及其功能分析研究

Fruit quality is becoming more and more attractive, and it is always the key factor of fruit value. However, the typic Chinese pear 'Kuerlexiangli' always produces a mass of fruit with calyx,which shows worse quality, compared with the fruit without calyx. Obviously, this characteristic has brought negative effect on economic benefit of pear production, and inhibit the sustainable development of pear industry. Basing on our previous morphological, physiological and RNA-Seq research on this phenomenon, in this study, 'Kuerlexiangli', the variety possess high rate of calyx survival should be treated with our own technique to produce fruit with ans without calyx,and be as research meterial. Data from RNA-Seq will be further analyze,and iTRAQ coupled with LC?MS/MS technique will also enployed to screen protein profiles. Besides, analysis on differential expression of transcriptome , proteome, as well as gene annotation, expression pattern, related metabolism pathway will be further performed to confirm differentially expressed genes and their involvement in metabolism pathway, to screen critical genes that control calyx development in pear fruit. Meanwhile, transgenic plants for gene function validation, responding mode of spatio-temporal, protein-protein interactions, subcellular location of candidate genes will be also employed to investigate the molecular mechanism of pear fruit calyx development. Results from this study will provide a important scientific basis for regulating fruit quality as well as provide gene source for realizing directional improvement fructification characters of pear.

由于品种特性原因，我国的传统特色梨品种'库尔勒香梨'，常易形成大量萼片宿存的"宿萼果"，其外观及内在品质严重下降，远不及"脱萼果"。在对水果品质要求日益提高的市场环境下，这种特性已然对梨生产的经济效益造成了不良影响，成为梨产业可持续发展的瓶颈之一。在已开展形态解剖、生理机制、基因表达谱研究的基础上，本研究以宿萼严重的'库尔勒香梨'为材料，采用脱萼专利技术进行宿/脱萼处理，进一步对前期基因表达谱数据进行分析，筛选宿\脱萼果转录水平表达差异基因；同时应用 iTRAQ 及LC?MS/MS 分析技术比较宿\脱萼果蛋白差异表达谱；通过基因及蛋白差异表达谱的关联分析、基因注释、表达模式和相关代谢通路分析，鉴定控制萼片发育的关键基因，并通过转基因功能验证、时空响应模式、蛋白互作及亚细胞定位研究，阐明相关基因参与萼片宿存与脱落的代谢途径及其功能，为实现梨果实性状的定向遗传改良提供基因资源。

针对当前梨生产中普遍存在宿萼果（俗称“公梨”）比脱萼果（“母梨”） 石细胞多、外观及风味品质差、且严重影响市场竞争力和经济效益这一突出产业问题，本项目以宿萼现象表现严重的‘库尔勒香梨’等主栽品种为研究试材，在已开展的形态解剖及生理机制研究基础上，通过专利技术获得宿存/脱落果实，并应用 iTRAQ 及LC−MS/MS 分析技术进行蛋白差异表达研究，筛选出60个与‘库尔勒香梨’脱萼相关的差异蛋白，发现与光合作用有关的蛋白质可能在花萼脱落过程中起重要作用，光合功能降低的样品更容易脱落；进一步结合转录组表达谱的关联分析、基因注释、表达模式和相关代谢通路分析，鉴定参与果实萼片宿存与脱落的关键基因及其时空表达特性，开展了功能验证研究，明确差异表达基因及其参与调控的代谢途径。筛选出并克隆得到控制果实萼片宿存与脱落的关键基因，PsIDA和JOINTLESS的全长，并将其构建到GFP融合表达载体pCAMBIA1302，通过基因枪轰击转化BY-2(N. tabacum cv. Bright Yellow 2)，用激光共聚焦扫描显微镜观察发现PsIDA-GFP融和蛋白主要在细胞核和细胞膜上有很强的表达，而JOINTLESS蛋白定位于细胞核内。上述研究结果及关键基因的克隆，为梨果实品质的调控提供了重要的科学依据，也为实现梨果实性状的定向遗传改良提供基因资源。