巴西橡胶树单染色体微克隆文库构建的研究

Rubber is Chinese important strategic material. Hevea brasiliensis is one of the most important sources of natural rubber, is an important economic crop for tropical and subtropical areas in China too. It was difficulty to construct some linkage groups or genetic maps of rubber tree with the molecular mark method, and the reported linkage genetic maps of rubber tree were limited which were obtained from its total DNA. These linkage groups or linkage markers of rubber tree were instability, few and unevenly distributed, the chromosomal positions and their distribution characteristics of many important genes from rubber tree were not be determined and so on. These problems could be resolved effectively if the single chromosomal DNA library of rubber tree was constructed. This project mainly about the single chromosome microdissection for the 36 chromosomes of the rubber tree (Hevea brasiliensis,2n=36), these DNA libraries of the full set single-chromosomes would be constructed,the Functional Analysis would be done for some cloning sequences from these constructed chromosomal libraries, the location on chromosome for some important genes which had been cloned should be definited, and the existing linkage groups of rubber tree would be classified. This project would provide an effective tool for screening specific segments of chromosomes, specific probes or specific markers for the single-chromosome or its segment from Hevea brasiliensis, constructing high-density molecular linkage maps for single-chromosome, isolating and cloning of functional genes，positioning and the constructing of Physical Map. It would be applied effectively to molecular assisted breeding for the rubber tree. Therefore, this project has a certain theoretical significance and practical application prospects.

橡胶是我国重要的战略物质。巴西橡胶树是一种重要的产胶植物，也是我国热带、亚热带地区重要的经济作物。用分子标记方法构建橡胶树的连锁群或遗传图谱难度大，现有的橡胶树连锁遗传图少而且是从总基因组中获得，并有连锁群不稳定，连锁标记少而分布不均匀，无法确定许多重要基因在染色体上的位置和分布特点。构建橡胶树单染色体文库，可以有效解决上述问题。本项目对巴西橡胶树2n=36条染色体进行单染色体微分离，构建橡胶树的全套单染色体DNA文库，并对其部分克隆子序列进行功能分析，明确橡胶树已克隆的重要基因在染色体上的位置，进而对现有的橡胶树连锁群进行归类。本项目可为筛选橡胶树染色体特异片段、单染色体或其区段的特异探针或特异标记，构建单条染色体高密度分子标记连锁图谱、功能基因的分离及基因组物理图谱构建等提供有效工具，有效地应用于橡胶树分子辅助育种。因此，本项目研究具有一定的理论创新意义和良好的实际应用前景。

本研究针对用分子标记方法构建橡胶树的连锁群或遗传图谱难度大，现有的橡胶树连锁遗传图少而且不稳定，连锁标记少而分布不均匀，无法确定许多重要基因在染色体上的位置和分布特点提出问题，提出从单条染色体入手，以构建橡胶树单染色体文库来有效解决上述问题的新思路。主要研究成果如下：以巴西橡胶树热研7-33-97为材料，建立并优化了巴西橡胶树单染色体显微分离的技术体系。通过借助显微操作仪的玻璃针分离法，对热研7-33-97全套单条染色体进行了分离，分离出巴西橡胶树热研7-33-97的全套染色体中18条，并成功构建出巴西橡胶树单染色体微克隆文库的实验体系，已对分离出的单染色体进行了克隆、转化，建立巴西橡胶树热研7-33-97较高质量的全套单染色体15条的DNA文库，文库克隆子数目在4.9×104~4.97×105个范围，插入片段的平均长度在282bp~650bp范围，文库覆盖率为1.0倍~7倍；并随机对15个染色体文库的部分阳性克隆子进行了测序，同时对三条单染色体尝试进行了高通量测序（其中5号染色体是用Ion torrent技术，4号及8号染色体是用Illumina平台技术对其完成了高通量测序）；通过BLAST在线序列分析及BLAST 2 go软件对部分与EST库有同源性的序列进行了基因注释分析，得到了部分GO术语注释结果，其注释的功能多为与橡胶树耐割性、抗冻性、死皮病、耐盐、抗旱等相关，少数序列与参与半胱氨酸、脯氨酸前体合成等。这些研究成果可为巴西橡胶树热研7-33-97全基因组测序组装提供参考依据，为筛选出巴西橡胶树染色体特异探针或特异标记、部分重要的功能基因在染色体组上的定位、增加遗传图谱和物理图谱的饱和度等方面提供基础性资料，进而可有效的应用于分子辅助橡胶树育种中。