蚬壳花椒种子萌发过程中基因和蛋白质的表达差异研究

Zanthoxylum dissitum Hemsl, belonging to Zanthoxylum genus of Rutaceae family, is a precious and important woody medical plant mainly distributing in southern China. Recently, the resource of Z. dissitum becomes almost endangered because of its obvious low natural seed breeding rate caused by the deep physiological embryo dormancy of its seeds that we have reported before. Although the dormancy and germination mechanism of model-plants' seeds has been well studied in the correlative field of acadmic research, such as a number of related genes and proteins controlling the seed dormancy and germination have been found and reported, the molecular mechanism theroy is still not established very well. Many more highly related genes and proteins need to be found and identified. In addittion, in the seed germination process of the woody plants, there may be some unique internal regulatory pathways and key events due to the obvious difference of dormancy characteristics belonging to the deep physiological embryo dormancy type compared with the model-plants. In order to establish a firm foundation of the theroy about the molecular mechanism of seed germination of Z. dissitum, the project not only intends to get the transcriptome sequence and protein profiling at both spatial and temporal levels to screen out the specific genes and proteins controling the seed germination of Z. dissitum after dormancy broken by using the combination of Solexa expression spectrum analysis technology，nano LC-ESI-MS/MS mass spectrometry analysis and biological imformatics analysis, but also plans to commentate the important regulatory genes and key regulatory pathways in the germinating process of seeds of Z. dissitum according to the changing of the expression spectrum of the genome and other correlative data revealed in the designed experiment. The prospective results of the project would not only be essential in providing important scientific basis for the conservation and utilization of the Z. dissitum, but also be helpful in offering critical theroy and technique reference for the utilization and conservation of other woody medical plants in China.

蚬壳花椒是我国南方重要的珍稀木本药用植物，种子休眠期长，自然萌发率低，资源面临濒危。近年来，学术界对模式植物种子萌发机理研究较多，但分子机理还不是十分清晰，仍需要鉴定更多的与种子萌发相关的基因和蛋白。更重要的是，属于深度休眠型的木本植物与模式植物种子不仅休眠特征明显不同，且萌发过程中更可能存在一些独特的调控途径与关键过程。项目拟结合Solexa表达谱分析技术、nano LC-ESI-MS/MS质谱技术及生物信息学研究方法等，从转录组和蛋白质组两个水平筛选出在蚬壳花椒种子从解休眠到萌发过程中可能存在的特异性基因和蛋白质，并根据表达谱变化解析种子萌发过程中的重要调控基因和主要调控通路，为进一步充分揭示种子萌发的分子机制奠定坚实理论基础。项目成果不仅为蚬壳花椒资源保护性利用提供重要科学依据，而且为其它木本药用植物资源的保护与利用研究提供重要理论和技术参考。

蚬壳花椒是我国南方重要的珍稀木本药用植物，种子休眠期长，自然萌发率低，资源面临濒危。在本研究项目执行期间，形成与蚬壳花椒种子繁育有关的技术专利4项，建设了种质资源收集圃、种子繁育以及林下种植基地共计140亩，并进行了蚬壳花椒林下种植模式的推广种植。与美国肯塔基大学以及斯坦福大学的学术访问交流，有效促进了项目实施方案的形成与研究深入。项目执行期内，课题组一批骨干研究人员学术水平与专业素质得到了明显提升，并培养博士生1名，硕士研究生11人，形成并发表学术论文10篇。通过开展蚬壳花椒萌发种子的转录组、蛋白质组以及相关生理生化研究，在木质藤本植物蚬壳花椒种子萌发的分子机理上取得了重要的理论突破。我们首次系统提出对种子萌发早期具有关键作用的基因以及代谢通路。在萌发早期外源赤霉素GA诱导的可溶性糖抑制SDP1脂肪酶基因转录以降低SDP1脂肪酶活性，同时还抑制油体和过氧化物酶体之间的物理相互作用以减弱过氧化物酶体中的脂肪酸β-氧化，从而显著抑制萌发早期种子存储油脂动员，减轻脂质过氧化伤害。另一方面，GA促进花椒种子成功萌发，主要在于其促进淀粉酶基因表达，将淀粉分解为可溶性单糖或寡糖。可溶性糖在种子萌发早期起关键作用，可溶性糖含量较高不仅有助于抑制种子存储油脂的动员代谢，而且通过磷酸戊糖PPP途径产生更多的还原力NADPH。从而使依赖NADPH的硫氧还系统和谷氧还系统保持良好氧化还原状态，这将有利于促进蛋白质翻译、抗活性氧ROS氧化、抑制蛋白质氧化以及促进二硫键还原以恢复功能蛋白质活性，保证了种子顺利萌发。总之，我们的研究成果揭示了萌发早期种子存储的糖、脂、蛋白质以及激素如何互作来影响早期萌发，并发现了一些在萌发初始阶段决定种子萌发成败的关键基因与通路。研究结果有利于蚬壳花椒植物资源的保护，也为理解并解决其他木质藤本植物种子萌发率低下的问题提供了理论依据和实践参考。