桑寄生顽拗性种子保存及脱水敏感性死亡机理研究

Taxillus chinensis (DC.）Danser is a well known traditional chinese medicine but whose breeding is very difficult. The seeds of Taxillus chinensis (DC.）Danser are typical recalcitrant which are fast inactivation and very sensitive to desiccation, what's more, the seeds are the only reproductive materials. Recently, there are many studies focus on the preservation of recalcitrant seeds, however, successful cases is little, the main reason is there is little known about the mechanism of the death of desiccation sensitivity. The key scientific problem of the death of recalcitrant seeds is desiccation sensitivity. The aim of this research project is to realize the short and long term aim of preservation of Taxillus chinensis (DC.）Danser seeds by studying the effects of dehydration, low temperature and ultra-low temperature on seeds preservation. Through the comprehensive study of the forms of death during seed dehydration process, morphological structure and antioxidant system to screen the critical period of desiccation sensitivity. By high-throughput Solexa sequencing, the key genes associated with desiccation sensitivity will be mined and full-length of these genes will be cloned by RACE. Besides, RT-PCR will be used to verify expression of spatiotemporal correlation process of the seeds dehydration during the death process and reveal the mechanism of seed death during dehydration process. The study not only provides theory basis for the key technology in the preservation of Taxillus chinensis (DC.）Danser seeds, but also new knowledge and technology for other recalcitrant seed's preservation.

桑寄生是地位重要而又繁育困难的特殊药材，其种子为典型的顽拗性种子，失活快，脱水敏感性强。而种子是唯一的繁殖材料，严重制约其繁育。目前顽拗性种子保存研究多，但成功实例较少，其根本原因是对脱水敏感性引起的死亡机理知之甚少。种子脱水敏感性是引起顽拗性种子死亡的关键科学问题。本研究拟通过对桑寄生种子进行脱水、低温与超低温保存的系统研究，实现短期和长期保存种质资源的目标；围绕桑寄生种子脱水过程中细胞的死亡形式、形态结构及抗氧化系统的综合研究，筛选种子脱水敏感性关键时期；通过Solexa高通量测序，挖掘与脱水敏感性相关的关键基因并进行RACE全长克隆，采用RT-PCR的方法验证种子脱水死亡过程中关联基因的时空表达规律，揭示脱水过程中种子死亡的机理。本研究成果不仅为研制桑寄生种子保存关键技术提供理论依据，还为其它顽拗性种子的保存提供新的知识与技术。

桑寄生种子是典型的顽拗性种子，对脱水和低温极其敏感，长期保存困难，自然状态下保存7天后就失去了活力。本项目对桑寄生种子的生物学特性、脱水与低温保存进行了系统的研究，使桑寄生种子寿命可延长至120天左右；围绕桑寄生种子脱水过程中细胞的形态结构及抗氧化系统的综合研究，筛选了种子脱水敏感性关键时期；利用高通量测序技术，对未脱水的桑寄生种子（CK），脱水敏感时期16小时（Tac-16）和36小时（Tac-36）进行了转录组分析，一共得到386,542,846条高质量序列。利用Trinity软件，组装得到164,546转录本（对应114,971基因）。通过与NR，UniProt，GO，KEGG pathway和COG数据库进行比对，并对组装得到的转录本进行了详细的注释。在CK，Tac16和Tac-36样本中，分别检测到60,695, 56,027和66,389 转录本 (>1 FPKM) 。通过与CK进行比较，在Tac-16中发现了2,102 个上调 和 1,344 个下调的转录本，而在Tac-36中发现了1,649 个上调和 2,135个下调的转录本。这些差异表达的转录本包含一些与已知的脱水过程相关的基因，比如RD22，热休克蛋白（HSP），转录因子（MYB，WRKY和乙烯反应性转录因子）。研究结果显示HSP和核糖体蛋白（ribosomal proteins）属于桑寄生顽拗性种子早期响应脱水过程的重要基因。原始数据已提交到NCBI SRA数据库（SRA309567）。本研究系统地对桑寄生种子的生物学特性进行研究，也是第一次利用转录组技术研究桑寄生顽拗性种子脱水敏感性的机理，所得结果对于桑寄生种子的繁育、保存及脱水过程中的基因调控具有重要的指导意义，并拓宽了我们对于顽拗性种子脱水及萌发过程中基因变化的认识。