胼胝质酶基因表达调控与枸杞育性关系的研究

Male sterility and heterosis play an important role in increasing food production and quality improvement process, The first male sterile wolfberry 'Ningqi 5'，the hybrid has many excellent features such as large fruit, good quality, early mature, and fresh eating. It has important practical significance to study the sterile mechanism for solving wolfberry production problem of single varieties,slow update and low yield. In this study, the male sterile varieties'Ningqi 5' and a normal fertile varieties 'Ningqi 1' are used materials. Based on the event that an anther callase gene (LG1) was silenced in male sterile varieties at transcriptional level, through separating LG1 gene promoter and constructing bud cDNA library, yeast one-hybrid system and transcriptome sequencing (RNA-Seq) technology are used to screen LG1 upstream regulatory transcription factors , all these factors are positively tested with real-time quantitative RT-PCR, then the full-length gene are cloned by the RACE method. Finally, using yeast one-/two- hybrid experiments, the factors activity on LG1 gene transcriptional starting is analyzed as well as the relationships among them. Thereby, it is expected to reveal the molecular mechanism of male sterile regulation，and provide theoretical reference for wolfberry breeding and germplasm innovative using.

雄性不育和杂种优势利用在提高粮食产量和品质改良过程中发挥着重要作用，首个枸杞雄不育品种'宁杞5号'具有杂种果粒大、品质佳、成熟早以及适合鲜食等优良特性。研究枸杞雄性不育机理对于解决目前枸杞生产中存在的品种更新慢、产量低、品质单一的问题具有重要的现实意义。本研究基于雄性不育枸杞花药胼胝质酶基因（LG1）在转录水平上的表达沉默事件，以雄性不育枸杞'宁杞5号'和正常可育枸杞品种'宁杞1号'为材料，通过分离LG1基因启动子，构建花蕾cDNA文库，运用酵母单杂交系统和转录组测序（RNA-Seq）技术对LG1上游的转录调控因子进行差异性筛查，实时定量RT-PCR分析验证，RACE法克隆基因全长，最后利用酵母单/双杂交试验研究分析转录因子对LG1基因启动表达的控制作用以及它们之间的相互关系，以此揭示雄性不育枸杞育性调控的分子机理，为枸杞育种和种质创新利用提供理论参考。

首个枸杞雄不育品种‘宁杞5号’（N5）具有杂种果粒大、品质佳、成熟早以及适合鲜食等优良特性，本研究基于雄性不育枸杞花药胼胝质酶基因（LG1）在转录水平上的表达沉默事件，以雄性不育枸杞N5和正常可育枸杞品种N1为材料，通过分离LG1基因启动子，构建花蕾cDNA文库，运用酵母单杂交系统和转录组测序（RNA-Seq）技术对LG1上游的转录调控因子进行差异性筛查，并实时定量RT-PCR分析验证，以此揭示雄性不育枸杞育性调控的分子机理。通过研究，克隆枸杞花药特异表达的胼胝质酶基因启动子2163bp，建立了枸杞酵母单杂交技术体系一套，筛选到与启动子相互作用的调控蛋白27个，获得枸杞花药发育关键时期的转录因子4个及其全长序列。该研究成果对于植物花器官发育研究及植物育性的基因调控关系网络建立具有重要的科学意义。