利用愈伤组织转绿技术与TALEN技术建立水稻高效叶绿体转化系统的研究

叶绿体转化技术在农业育种或医药领域具有独特的优越性和广阔的应用前景。烟草等双子叶植物叶绿体转化系统之所以成熟的一个重要原因是这些植物可利用绿色叶片或绿色愈伤组织作为受体，外源基因导入成熟叶绿体比较容易，而且在筛选过程中可以依赖于绿色组织反复再生获得同质化植株。然而水稻愈伤组织中的前质体还未发育成叶绿体，且水稻叶片再生较难，这些限制是建立稳定的水稻叶绿体转化系统的主要障碍。本研究目的是对水稻愈伤组织绿化组培技术进行研究，克服水稻愈伤组织不含成熟叶绿体带来的障碍，以及利用TALEN定点敲除系统对叶绿体基因组定点断开，再用同源重组方法将外源基因定点整合至叶绿体基因组断点处，从而完善提高同源重组效率的技术手段。拟通过基因枪转化，以绿色愈伤组织为靶目标，利用现代分子遗传学手段分析转化植株，建立新型的水稻叶绿体转化系统，为创新水稻育种材料和提高水稻光合效率途径提供理论依据。

Chloroplast transformation technology has become a valuable tool for the creation and cultivation of plants, especially for dicotyledons, and chloroplasts offer a viable alternative to conventional bioreactor systems. Tobacco has been the most extensively used plant for chloroplast transformation, mostly because leaf blades were served as target tissues which contained a large number of mature chloroplasts, and the ability to obtain homoplastomic plants via subsequent rounds of regeneration using leaves as explants. The major obstacle to the extension of chloroplast transformation technology to monocotyledonous cereal crop such as rice is that the proplastids rather than chloroplasts existed in the rice callus, a nearly exclusively explant for the rice regeneration in tissue culture. In order to overcome the barriers of rice chloroplast transformation existed in the tissue culture, in this project, we will study the tissue culture technologies related to callus greening of rice, and use the method of TALEN system combined with homologous recombination to improve integration efficiency of foreign gene to chloroplast genome. The establishment of effective chloroplast transformation in rice provides theoretical basis for hybrid rice breeding and improvement of photosynthetic efficiency of rice.