CgSVP基因对低温解除春兰花芽休眠的调控作用机理

Cymbidium goeringii, which is also named spring orchid, is a very famous traditional orchid plant in China, with a high ornamental value. They need 5-10 ℃ cold condition to release flower bud dormancy and promote flowering. However, the mechanism underlying its cold-dependent regulation of flowering is unclear. Our previous study on flowering physiology indicated that the flower bud will enter the growth retardation period in two months after floral initiation, and at least 40 days at low temperature can relieve this growth inhibition. There is a significant spatial and temporal difference between this process and the vernalization pathway, which probably resulted from a specific regulatory mechanism. Though comparative transcriptome analysis, we found that the expression of a new SVP gene, CgSVP, is strongly suppressed and overexpression of CgSVP leads to a significant delay in flower development and opening, which indicated that CgSVP is involved in cold-induced release of flower bud dormancy of C. goeringii. On this basis, this project will deeply analyze the functional mechanism of CgSVP using the technical methods of developmental biology and molecular biology, to reveal the biological function of CgSVP and determine protein-protein interaction of CgSVP. Combining with the response to upstream low-temperature signals and the regulation of downstream flowering genes, we will finally clarify the regulatory mechanism of CgSVP during the process of low-temperature induced flowering, which would provide valuable data and practical guidance for flowering regulation and molecular breeding of C.goeringii and other orchid plants as well.

春兰是我国特色传统兰花的典型代表，具有极高的观赏价值。春兰需要5-10℃低温环境解除花芽休眠促进开花，但其分子机制尚不清楚。申请人前期工作发现，春兰在成花起始两个月后将进入生长停滞期，至少40天低温处理才能解除花芽生长抑制。此过程与春化途径有显著时空差异，可能存在物种特异的调控机制。通过转录组比较分析，新发现一个CgSVP基因在低温处理春兰休眠花芽后表达受到强烈抑制，而该基因的过表达则导致开花明显延迟。这表明CgSVP参与低温解除花芽休眠后促进开花过程。本项目将结合发育生物学和分子生物学手段，通过分析CgSVP基因功能、鉴定CgSVP上游调控因子以及相互作用蛋白，研究CgSVP对春兰独特的低温依赖开花过程的调控作用及其分子机理，为今后利用基因工程技术调控春兰以及其它兰科植物开花提供理论基础。

SVP基因可感受外界环境温度，在植物成花转变、花芽分化和花器官发育等阶段发挥重要作用，且不同SVP基因可能通过结合不同蛋白从而实现基因功能的分化。春兰花器官分化存在特异的开花调控机制，需要经历冬季5-10℃低温环境打破 “花芽休眠”状态，促进花芽继续发育，但其分子调控机制尚不清楚。项目结合发育生物学和分子生物学手段，开展以下四个方面的研究工作：（1）通过兰花体内瞬时高表达技术，结合拟南芥表型互补辅助验证，分析评估转基因植物的表型，揭示CgSVP在低温解除花芽休眠过程中的负调控作用；（2）通过酵母筛库获得SVP互作蛋白，通过候选基因的酵母双杂交验证、体外pull-down和体内双分子荧光互补验证获得其互作蛋白，AP1和SOC1；（3）开发了一套国兰原生质体高效转化体系，并通过原生质体内高表达CgSVP2和CgSVP3，揭示他们对下游开花效应基因的调控作用，发现SVP基因之间存在反馈调控，且 CgSVP可以负调控FT、SOC1和AP1基因的表达。在低温条件下，低温抑制了SVP的表达，解除了其对开花的抑制作用，从而促进春兰花发育。通过以上研究，建立一个从CgSVP对上游低温信号的响应到对下游花开基因调控的分子模型。通过项目实施，发表SCI研究论文1篇（JCR 2区）；建立墨兰原生质体高效转化体系一套，申请国家发明专利1项；培养研究生1名，入站博士后1名；两名技术人员分别晋升中级和正高级职称。