油菜人工小染色体的构建

Plant artificial minichromosome could be used as an independent platform for foreign gene expression, and good materials for studying structure and function of chromosomes. Plant artificial minichromosome has been successfully obtained in maize, Arabidopsis, barley and rice. But so far, the stable transgenic lines with a pair of minichromosomes and site specific insertion of foreign genes into the minichromosome have not been achieved. Brassica napus is one of the most important oil crops in the world. The studies on its artificial minichromosome have not been reported. In this study, the construct with Arabidopsis telomeric sequence will be used to transform Brassica napus, and different artificial minichromosomes will be produced by telomere-mediated chromosomal truncation. The truncations will be identified and characterized by multicolor FISH, Southern hybridization and primer extension telomere rapid amplification. Transgenic lines with a pair of artificial minichromosomes will be obtained by crossing and selfing, which will be new germplasms of Brassica napus. We will also prove that the new foreign genes can be inserted into the artificial minichromosome by transferring a new site-specific recombination vector. This study will provide a new approach and materials for molecular breeding and studying structure and function of chromosome in Brassica napus. Therefore this research is of important in breeding theory and application of rapeseed.

植物人工小染色体有望成为外源基因表达的独立平台，并为研究染色体结构和功能提供优良遗传材料。植物人工小染色体的创制已在玉米、拟南芥、大麦和水稻中获得成功，但至今尚未获得稳定的具有单对小染色体的株系，也未实现将外源基因稳定整合在小染色体上。油菜人工小染色体的构建还未见报道。本研究拟利用含有拟南芥端粒序列重复的载体转化油菜，获得端粒介导的多个不同的人工小染色体，并通过多色FISH、Southern杂交及引物延伸端粒快速扩增方法提供油菜染色体截短和端粒序列重新合成的直接证据。通过进一步的选育（自交，杂交）获得具有单对人工小染色体的油菜新种质；同时利用新构建的定点重组载体，证明新的外源基因能够插入人工小染色体上，为油菜进一步分子育种及染色体结构与功能的研究提供新的技术途径和新材料。本研究具有重要的应用前景和理论意义。

利用农杆菌介导法，将带有拟南芥端粒重复序列的植物载体pWY86-5转化到甘蓝型油菜。荧光原位杂交分析结果表明在一个细胞中同时存在多个荧光信号，部分位于染色体末端，部分位于染色体内部。Southern杂交及PETRA分析表明，在转基因油菜中发生了端粒介导的染色体切割。通过自交或杂交能够获得带有小染色体的后代，小染色体减数分裂行为观察表明，转基因植株后代中的小染色体能够稳定遗传。通过特异性位点特异重组，我们验证了外源基因能够定点整合到油菜小染色体上。综上，本研究成功地在油菜中构建了人工小染色体，为进一步进行油菜分子育种及染色体结构与功能的研究提供了新的技术途径和新材料。