【作者】 赵辉；

【导师】 彭明； 崔百明；

【作者基本信息】 华南热带农业大学 ， 遗传育种， 2007， 硕士

【摘要】 巴西橡胶(Hevea brasiliensis)是重要的热带经济作物,是具有不可替代性的重要工业原料和军事物资,但我国橡胶自给严重不足,低温寒害是限制我国橡胶发展的主要原因。常规育种只能部分解决抗寒问题,不能从根本上解决橡胶寒害的瓶颈。基因工程育种成为橡胶树生物技术研究、品种改良的核心内容。巴西橡胶树体外再生技术在20世纪70年代已经成功,但目前其组织培养技术仍然比较落后,至今只有少数无性系能够实现体外形态发生,且大多数品系体外植株再生的效率非常低下,这已经成为建立高效遗传转化体系,从分子水平进行橡胶树遗传改造的重要障碍。相比于模式植物和粮食等经济作物来说,橡胶分子生物学研究严重滞后。项目以海南目前大规模推广种植的橡胶树新品系热研7-33-97为植物材料,通过农杆菌(Agribacterium tumefaciens)介导法,分别选择花药和内珠被脱分化愈伤,以及长期继代增殖的胚性愈伤为转化受体进行转化,采取导入抗寒调控基因和关键性抗寒基因相结合的策略,将新疆小拟南芥抗冻调节基因——CBF基因及其下游功能基因——cor15a转入橡胶中,以期完善组织培养技术,获得抗寒转基因植株,探索提高橡胶抗寒性的基因工程策略,建立稳定、高效的遗传转化体系。研究主要解决了以下问题:1、优化了橡胶热研7-33-97品种花药组织培养配方。2、成功建立了橡胶胚性愈伤长期继代保存和增殖技术(已申请专利,申请号200710084622.7,申请日2007年2月12日),国内外还未见相关报道。为提高橡胶树组培效率,建立新的高效的转化技术路线,以及为橡胶花药悬浮培养和橡胶原生质体培养长期提供大量的好材料提供了技术基础。3、在橡胶花药组培和胚性愈伤长期继代增殖技术的基础上,利用花药脱分化愈伤和经长期继代增殖的胚性愈伤拟行农杆菌转化,实验表明胚性愈伤是更适宜转化的受体,在转化的侵染、共培养、抑菌、筛选、成胚等各环节均表现出显著的优越性。胚性愈伤经农杆菌转化后形成的抗性胚,经PCR检测已有部分转基因体胚存在。开拓了橡胶转化技术新的技术路线。4、探索和优化了橡胶农杆菌转化技术各环节的技术指标。5、为研究和开发利用通过向橡胶中导入抗寒基因来提高橡胶的抗寒性提供了一条新的途径和方法,具有一定的理论和应用前景。更多还原

【Abstract】 Rubber tree (Hevea brasiliensis) is an important economic crop in tropics. It has very important value especially in the fields of industry and military. At present, the consumption of natural rubber in China increases annually, and the shortage increases annually. Cold injury is the key factor that restricts the growth and distribution of rubber. Routine breeding of rubber tree can only partially, but not radically settle the problem. Gene engineering breeding becomes the core content of rubber tree biotechnological research and variety improvement.The technique of somatic embryogenesis in Hevea brasiliensis was succeeded in 1970s. But now the technique is dropped behind because that so far, only a few the clones can produce somatic embryogenesis through this technique, and the rate of the in vitro plant regeneration in most of the clones is very low. This becomes the main obstacle in the way to found the high genetic transformation system for reconstructing the inheritance of Hevea in molecule level. Compare of studies on model plants and economic crops, there are lags in advances in molecular biology of rubber tree.In this research item,we chosed the rubber variety of RY 7-33-97 widely generalized in Hainan, China. CBF gene, its downstream COR15a gene and regulating element CRT/DRE in promoter of COR15a gene, were transfered to firsthand calli from anther or inner integument culture and the long-term proliferated embryogenic calli via somatic embryogenesis by Agrobacterium tumefaciens to develop cold endurance rubber trees and to establish of genetic transformation system in Hevea including efficient culture via somatic embryogenesis. The results were shown as follow.1. The anther culture media of RY 7-33-97 was optimized.2. The long-term proliferation technique of Hevea embryonic calli was successfully established. The technique could increase the efficient of Hevea tissue culture, carve out a new way to found the high Hevea genetic transformation system and chronically afford plentiful good material for Hevea embryogenic cell suspension culture and protoplast culture.3. On the basis of Hevea anther culture and long-term proliferation technique of Hevea embryonic calli, we used the firsthand calli of Hevea anther and long-term proliferated embryonic calli to transfer by Agrobacterium tumefaciens. The result showed that embryonic calli were consumedly superior to the firsthand calli in transformation. And we have gained transformed embryoids via long-term proliferated embryonic calli. The study caved out a new way to found the high Hevea genetic transformation system.4. The study explored or optimized the index of every step of transformation mediated by Agrobacterium tumefaciens.5.This research laid a foundation for researching on fouction of CBF1、CBF3、ApCOR15a and provided a new method for cold endurance, presenting theorical significance and applicative prospect.更多还原