刺梨果刺形成的细胞起源及分子机理研究

Rosa roxburghii Tratt is one of the important fruits in Guizhou province. However there are lots of spines in the fruit surface, this makes inconvenient for cultivation, management, harvesting, transportation, processing and so on in planting. The variety of Guinong 5 was used as an experiment material and eight different periods were selected, SAM and individually 5d, 10d, 20d, 30d and 45d after pollination. With the methods of the lens and scanning electron microscope, the morphology changes of fruit spine were observed. Further we could obtain the fruit spine development and origin of cells by microscopic observation of paraffin section in tissues and cells; Meanwhile using the key genes of fruit spine GL and Tu in cucumber (Cucumis sativus Linn), and the main regulatory genes of trichome GL1, TTG1 and GL3 in Arabidopsis thaliana, we can obtain the homologous genes RrGL, RrTu, RrGL1, RrTTG1 and RrGL3 in Rosa roxburghii Tratt. The gene functions can also be verified by transformation with arabidopsis thaliana and cucumber. The results of this project mainly clarify the cytological process of fruit spine development and regulation mechanism, and will provide theoretical base for the improvement of Rosa roxburghii Tratt fruit quality .

刺梨是贵州省农村经济发展的重要支柱产业，但大多数栽培刺梨果实表面被有果刺，给栽培管理、鲜果采收、运输、加工等都带来不方便。本项目以刺梨贵农5号花分化期的茎尖组织、花原基和果实形成期授粉后5d、10d、20d、30d、45d的刺梨果实为材料，通过体视镜和扫描电镜观察果刺形态变化，进一步通过石蜡切片进行组织细胞的显微观察，研究刺梨果刺的发育形成过程，探明果刺的细胞来源；同时，利用黄瓜果刺形成的关键基因GL和Tu以及拟南芥表皮毛主要调控基因GL1、TTG1和GL3，克隆刺梨中的同源基因RrGL、RrTu、RrGL1、RrTTG1和RrGL3，并转化拟南芥和黄瓜进行功能验证。本研究对阐明刺梨果刺发育的细胞学过程和调控机理具有重要意义，为刺梨的果实品质改良提供遗传基础。

刺梨的果刺给栽培管理和果实采收带来不方便。本项目将研究重点集中于刺梨果刺的起源和果刺调控的分子机制。首先通过对花和果实发育过程中果刺的形态和细胞学结构的观察，探明果刺形成的起源。结果表明，在花芽形成初期，雄蕊原基下基部内的细胞慢慢形成粗而短的近似椭圆形的结构，迅速分裂变大。椭圆形的细胞开始变细、变长，尖端呈“针”状型结构，初步形成刺结构，后果刺的生长随着果实的发育逐渐变大变硬。进一步以拟南芥表皮毛关键基因GL3、EGL3、TTG1和GL1为切入点，通过同源克隆，得到刺梨中同源基因RrGL3、RrEGL3、RrTTG1和RrGL1的cDNA全长，通过生信分析、时空表达、亚细胞定位、蛋白互作以及拟南芥的遗传转化等实验，系统地对RrGL3、RrEGL3、RrTTG1和RrGL1进行功能研究和分析，结果表明这些基因均定位在细胞核内，其中RrEGL3、RrTTG1和RrGL1在刺梨果刺中高表达。通过拟南芥转化实验表明RrGL1、RrEGL3和RrTTG1的功能相对保守，调控拟南芥的表皮毛发育并形成RrGL1- RrEGL3-RrTTG1的BMW类复合物；最后对刺梨茎、叶、花、幼果和成熟果等5个组织分别进行转录组分析，得到与MYB相关的基因并挖掘EST-SSR的相关信息，为调控刺梨果刺发育和分子辅助育种提供了理论基础。综合本项目的结果，本研究对阐明刺梨果刺发育的细胞学过程和分子调控机理具有重要意义，为培育无果刺的刺梨品种提供遗传基础。