基于miRNA和蛋白质组的桑寄生顽拗性种子脱水敏感性分子机制研究

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 and restricting its industrialization. The key scientific problem of the death of recalcitrant seeds is desiccation sensitivity. Fortunately, We've got some transcriptional factors, Dehydration-responsive proteinRD22, Heat shock protein and other important genes which are closely related to the desiccation response on the prophase research. But , how the genes at the post-transcriptional level? If they can be regulated ? how to regulate? all is worth us to explore. Therefore, on the basis of the previous research, this research intends to study on the recalcitrant seeds of Taxillus chinensis (DC.）Danser: ①The determination of indexes on physiological and biochemical level to find out the result of the change of physiology and biochemistry; ②extracting the total RNA, use the new generation of Solexa high-throughput sequencing method and then to analyze the differential expression of microRNAS; ③extracting the total protein and then to make sure the differentially expressed proteins by iTRAQ; ④through the omics integration method, to compare the differential expression of genes which involved in transcription and translation level and the relevant miRNA regulation; ⑤using the quantitative PCR method to further verify the expression of related genes; and then to further reveal the molecular mechanism of the desiccation sensitivity and interaction from the system level. The study not only provides theory basis for the key technology in the preservation and breeding of Taxillus chinensis (DC.）Danser seeds, but also new knowledge and technology for other recalcitrant seed's preservation and breeding.

桑寄生是地位重要但繁育困难的特殊中药材，其种子为典型的顽拗性种子，失活快，脱水敏感性强；而种子是唯一繁殖材料，严重制约其产业化，脱水敏感性是引起顽拗性种子死亡的关键科学问题。课题组前期研究挖掘到了与桑寄生种子脱水响应密切相关的转录因子、干旱响应蛋白、热休克蛋白等重要基因，但这些基因转录后翻译水平如何？是否可以调控？值得我们去探索。因此本研究在原有基础上拟进行①生理生化指标测定，深入分析桑寄生种子脱水敏感的生理生化机制；②通过高通量测序分析种子microRNA的差异表达；③通过蛋白质组iTRAQ技术确定种子差异表达蛋白；④利用组学整合手段比较相关基因在转录、翻译层次的表达差异及相关miRNA调控；⑤运用定量PCR法进一步验证相关基因的表达。通过系统研究揭示桑寄生种子脱水敏感性的分子机制及调控规律，不仅为桑寄生种子的保存与繁育提供理论依据，还为其它科属顽拗性种子的保存与繁育提供新的知识与技术。

桑寄生 Taxillus chinensis (DC.）Danser，植物学名“广寄生” ，是传统的常用大宗中药材， 具有补肝肾、 祛风湿、 强筋骨、 安胎等功效，为历版《中国药典》收载品种。主产于广西、广东，其中广西是国内开发桑寄生保健品最早的省区。随着需求量的增加及生境不断遭受到破坏，资源储量大幅减少。其种子为典型的顽拗性种子，失活快，脱水敏感性及低温敏感性强。而种子是唯一的繁殖材料，严重制约其繁育，是我国地位重要而又繁育困难的特殊药材。目前顽拗性种子保存研究多，但成功实例较少，其根本原因是对低温敏感性引起的死亡机理知之甚少。种子低温敏感性和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，种子由原来的 7 天可以保存到 120 天，极大地延长了种子的寿命，实现了桑寄生种子短期保存种质资源的目标，为生产繁殖赢得了时间；桑寄生种子对脱水非常敏感，其活力和发芽率与含水量显著相关。新鲜种子的发芽率为 86%（活力为 99%），脱水 16h 后发芽率迅速下降到 39%（活力为 66%，含水量为 35.17%），36h 后含水量会下降至 24.93%，种子活力变为 15%，而发芽率仅为 6%，40h 种子含水量为23.47%时，种子基本丧失了活力，只有 9%，而发芽率为 0。本次脱水处理40 h后，桑寄生种子各生理指标也变化明显，随着脱水时间的延长，含水量逐渐降低，可溶性糖含量、淀粉含量、可溶性蛋白含量、细胞色素氧化酶含量以及种子浸出液相对电导率都是先升高再降低的趋势，说明脱水后期种子都受到了不同程度的影响。miRNA -seq和蛋白质组分析表明，脱水显著影响了相关蛋白及靶基因的表达以及调控，从而影响了种子萌发及长期保存。本研究主要对桑寄生种子的脱水敏感过程进行相关研究，同时借助组学技术研究桑寄生顽拗性脱水敏感性的机理，所得结果对于桑寄生种子的繁育、保存及低温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。