橡胶树胚状体花青素合成调控因子的发掘及转基因可视化筛选系统的构建

The long breeding period (around 30 years) is the main restricting factors of rubber tree varieties update in rubber tree (Hevea brasiliensis). Breeding new rubber tree varieties by genetic engineering is an important way to accelerate the update of natural rubber varieties..NptII, gus/gfp were used as reporter genes in the selection system of genetic transformation system of Hevea brasiliensis, their selection efficiency was low. Anthocyanin as a reporter gene can be seen by the naked eye without substrate and special equipment. The published regulators controlled anthocyanin synthesis are from root, leaf, flower and fruit except somatic embryo. Base on that there will be complete gene regulation pathway of anthocyanin synthesis in Hevea embryoids, this project will clone the anthocyanin accumulation key regulatory factors R2R3-MYB and bHLH, exploring the high expression embryo specific promoter, under the control of embryo high expression promoter, R2R3-MYB and bHLH express specifically in embryo. Construct the efficient screening system by taking anthocyanin as selectable marker, screening red transformed embryos, eliminating white non transformed embryos in transient expression and resistant embryo differentiation these two stages, providing technical support for the rubber tree genetic modification and varieties update.

巴西橡胶树（Hevea brasiliensis）育种周期长达30年，是橡胶树品种更新的主要制约因素，通过基因工程手段培育橡胶树新品种，是加快天然橡胶品种更新的重要途径。.橡胶树遗传转化筛选体系以NptII为筛选标记，gus/gfp为报告基因，筛选效率低。花青素作为报告基因，具有无需底物及特殊设备、肉眼可视的特点。已报道的花青素合成调控因子来自除体胚外的根、叶、花、果等组织。本项目将在橡胶树胚状体存在花青素合成完整基因调控路径的理论基础上，首次从胚状体克隆花青素累积R2R3-MYB和bHLH调控因子，并发掘橡胶树胚状体特异高表达启动子，构建由胚状体高表达启动子驱动R2R3-MYB和bHLH表达载体，在瞬时表达和抗性胚分化、阳性胚增殖三个阶段，直接筛选红色转化胚状体，剔除白色非转化胚状体，构建胚状体高累积花青素的可视化转基因高效筛选系统，为橡胶树转基因改良、品种更新提供技术支撑。

巴西橡胶树（Hevea brasiliensis）高度杂合，育种周期长达30年，品种更新换代缓慢。通过基因工程手段培育橡胶树新品种，是加快天然橡胶品种更新的重要途径。.橡胶树遗传转化筛选体系以NptII为筛选标记，gus/gfp为报告基因，筛选效率低。花青素作为报告基因，具有无需底物及特殊设备、肉眼可视的特点。本项目克隆了橡胶树花青素累积R2R3-MYB和bHLH调控因子，并发掘橡胶树胚状体特异高表达启动子（糖基磷脂酰肌醇启动子PGAPr），构建由PGAPr驱动的R2R3-MYB表达载体，转化愈伤组织和胚状体，结果表明，对于愈伤转化体系，可在共培养后的愈伤筛选阶段通过肉眼直接筛选出红色纯合、嵌合转化愈伤，对于胚状体转化体系，可在共培养后抗性胚诱导阶段直接筛选出红色纯合、嵌合转化胚状体，剔除黄色非转化愈伤/白色非转化胚状体，实现早期精准筛选，大大提高筛选效率，且对愈伤增殖无不良影响，PGAPr驱动的R2R3-MYB表达载体部分嵌合和纯合抗性胚状体发育至子叶型，初步证明花青素可用于橡胶树愈伤/胚状体高累积花青素的可视化转基因高效筛选，为橡胶树转基因改良、品种更新提供技术支撑。.项目发现花青素对胚状体发育有毒害作用，胚状体特异启动子与35S组成型启动子相比，显著降低毒害作用，35S阳性转化子在球形胚阶段发育一段时间后就停止发育，未能发育至成熟子叶型胚状体。而PGAPr驱动的R2R3-MYB表达载体部分嵌合和纯合抗性胚状体发育至子叶型，因此，使用愈伤特异启动子或PGAPr有望解决这一应用限制。花青素累积需要光照和时间，时间需要15天以上，而共培养仅3-7天，因此，共培养阶段无法通过花青素筛选出转化子。