【作者】 曲春浦；

【导师】 刘桂丰； 刘关君；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2013， 博士

【摘要】 小黑杨(Populus simonii×Populus nigra)是杨柳科杨属乔木,是荒山荒地造林绿化的先锋树种,又是著名的抗旱、耐寒、耐瘠薄树种,同时也是北方主推的造林树种。目前,对小黑杨的研究多集中于生态学、造林学和育种学等方面,在小黑杨种子萌发机理方面的研究尚未见报道。大多数开花植物通过有性生殖产生的种子来繁衍后代,种子的萌发是从休眠胚时期到产生一个能够进行光合自养的个体植株的一系列转变过程,从浸种到成苗涉及诸多迅速而复杂的生理生化过程。因为在此阶段最易受到各种伤害,萌发被认为是植物一生中最重要的时期。因此,种子萌发等过程对林木的育苗生产具有重要意义。采用分子生物学手段了解和揭示林木种子萌发过程中基因表达的变化规律及生理学变化特征可以为促进种子萌发、提高种子抗性等研究提供理论依据。对种子萌发过程的分子机理研究一直是种子生物学研究的重要方向之一,而近年来基因组学的出现为解决这些问题提供了一种更方便有效的手段。但是当前种子萌发的基因组学研究主要集中在拟南芥及农作物等少数测序的模式植物和经济作物上,作为重要的木木模式植物的杨树在此方面的研究还缺少相关报道。开展小黑杨种子萌发的数字基因表达谱分析工作,不仅对探讨小黑杨萌发的机理、完善种子生物学研究、解决林木种子萌发难题具有非常重要的意义,而且对于深入理解复杂基因组植物的分子遗传学特征及指导其育种研究也具有重要价值。本研究采用高通量的数字基因表达谱(DGE)技术,测定了干种子期、快速吸水期、缓速吸水期、胚轴伸长期、子叶伸展期、幼苗期等六个时期的表达谱,分别获得了在小黑杨成熟种子中特异/优势表达的基因和小黑杨种了萌发过程不同时期表达变化差异显著的基因,并对这两部分基因进行了分析,结果如下：(1)通过对成熟种子与幼苗表达谱的比较,获得了1789个在小黑杨成熟种子中特异/优势表达的基因。对这1789个基因在丰度、家族数量以及GO富集方面的分析发现这些基因主要参与了脂代谢、转运、氧化还原、ATP合成、己糖合成、高尔基体转运以及转录调控等相关过程。(2)通过对成熟种子、快速吸水期种子、缓速吸水期种-子、胚轴伸长期种子以及子叶伸展期幼苗的表达谱的比较分析,获得了6496个差异表达基因。这些基因按其表达模式可分为12类,主要参与了细胞骨架重建、细胞多糖类代谢、转运、蛋白质降解、氨基酸代谢以及氧化磷酸化等生物学过程。(3)翻译以及氧化磷酸化途径等生物学过程所富集的基因在小黑杨种子萌发的大部分过程中其普遍以较高水平转录。说明这类基因参与了对小黑杨种子萌发过程中大部分阶段的调控作用。(4)细胞多糖代谢相关基因、脂代谢相关基因、光合作用相关基因以及单羧酸代谢类相关基因的mRNA仅在1个或2个时期表达量显著提高,说明这类基因参与了对小黑杨种子萌发过程中个别时期的调控作用。(5)对种子萌发全过程差异基因进行分析,共发现64个显著性富集的Pathway途径,分属于共21种不同的代谢途径,分别富集于不同时期；对相邻时期差异基因富集情况进一步分析表明,这些显著富集的途径与呼吸作用、贮藏物质分解及细胞原料类物质合成以及核酸代谢有关。(6)对种子萌发过程中呼吸强度、蛋白质含量、DNA含量、氨基酸含量以及可溶性多糖含量等的生理指标检测结果分析表明,种子萌发过程中存在着两个种子贮藏物质急速变化期——快速吸水期和胚轴伸长期,同时在这两个时期呼吸速率也显著发生改变。(7)对可能影响呼吸强度、蛋白质含量、DNA含量、氨基酸含量以及可溶性多糖含量等的生理指标的限速酶家族进行分析,结果表明,21个基因贮藏物质分解类限速酶在转录水平上与生理指标变化表现为一定的联系。本研究通过6个时期的DGE分析,结合呼吸速率、可溶性糖及可溶性蛋白等生理指标的变化分析,从转录组学角度系统地揭示了小黑杨种子从萌发到幼苗阶段6个时期各类基因的表达模式,从而为进一步分析各基因家族在种子萌发过程中的作用提供了宝贵的实验依据。本研究中得到了大量参与小黑杨种子萌发过程的基因,这些基因中不仅包含大量已知功能的基因,更重要的是还包括许多未知功能的新基因。这些基因的发现为进一步从遗传学和分子生物学角度揭示小黑杨种子萌发过程的分子机理提供了可靠依据。更多还原

【Abstract】 Populus simonii×Populus nigra is a lofty arbor, which belongs to Salicaceae and Populus, and it is a vanward species of afforestation in mountain waste and wasteland and a famous industrial timber species. The study of Populus simonii×Populus nigra mainly focuses on ecology, silviculture and breeding. But little is known about the seed germination of Populus simonii×Populus nigra in molecular mechanism.Most flowering plants reproduce by seed for multiplication with sexual reproduction. Seed germination is the transition of the quiescent embryo into a new photosynthetically active plant. Germination is a complex physiological and biochemical process during imbibed mature seed to seedlings in a rapid conversion and prepare for seedling growth. Because of its susceptibility to injury, disease and so on, germination is considered to be the most critical phase in the plant life cycle. It is one important issues of plant cell biology to study the molecular mechanisms of seed germination. The emergences of genomics provide a good means for studying the mechanisms of seed germination in recent years. At present, except on model plant Arabidopsis and other minority of model plants and crops, few works have been performed on Populus which is as an important mode of woody plant. The genomics research on Populus simonii×Populus nigra seed germination has very important significant not only for exploring the mechanism of germination and improving seed biology research but also for deeply understanding of molecular genetics of complex genomic plants. It also has great value for guiding breeding work.In this study, we identified Populus simonii×Populus nigra seed-specific/preferential genes and differentially expressed genes in Populus simonii×Populus nigra seed during germination via DGE. The main results are listed as follows:(1) By comparing the DGE of Populus simonii×Populus nigr mature seed with ten-day-old seedling, we identified1789genes specifically or preferentially expressed in Populus simonii×Populus nigr mature seed. Through our analysis of these1789genes in abundance, family number, and GO enrichment, we found that most of them were involved in lipid metabolic process, transport, redox, ATP synthesis, hexose metabolic process, Golgi vesicle-mediated transport and transcription factors. We speculate that the genes specificially expressed in seed of Populus simonii×Populus nigr might be involved in the regulation of seed germination in early phase.(2) Through comparison mature seed, rapid water-uptake phase, slow water-uptake phase, radical elongation phase and postgermination phase expressed genes, we obtained6496differentially expressed genes. These genes were divided into12clusters according to their expression patterns, and most of them were involved in cell skeleton organization, cellular polysaccharide metabolic process, transport and protein degradation, amino acid activation and oxidative phosphorylation. These genes may play important roles during seed germination in Populus simonii×Populus nigr.(3) Genes enriched in translation and oxidative phosphorylation pathways and other biological processes in the Populus seed germination is express in high level in most of the germination process, and these genes may play a key role in Populus simonii×Populus nigr seed germination.(4) Genes expression related polysaccharide metabolism, lipid metabolism-related genes, photosynthesis, as well as single-carboxylic acid metabolism was significantly increased only in one or two periods, these results show that these genes play a regulation role in thePopulus seed germination in a specific period in the Populus seed germination.(5) Analysis of the differentially genes expressed in the whole germination,64pathway were enriched significant and belongs to21different metabolic pathways and respectively occurred in different periods; Futher analysis the enriched differential gene in adjacent period show that these gene involved pathway related respiration, storage the substance decomposes synthesis and cell raw material as well as nucleic acid metabolism.(6) To test the respiration rate, protein content, DNA content, amino acid content and soluble polysaccharide content, the results shows that there are two period, rapid water-uptake phase and radical elongation, that seed storage substances changes rapidly during seed germination, while respiration rate in both periods also significantly changed.(7) Analysis of the rate-limiting enzyme coded by the gene family that effect respiratory strength, protein content, DNA content, amino acid content and soluble polysaccharide content, the results showed that there are some relevant between21genes belong to storage material decomposition rate-limiting enzyme in transcriptional level and physiological changes.In conclusion, a large number of genes involved in seed germination were identified through DGE analysis of six different experimental samples in Populus simonii×Populus nigr. Some of them have been found to function in reproduction. We also found a number of novel candidate genes that may play roles in the process. Our results provide a basis for further analysis of seed germination in Populus simonii xPopulus nigr.In the study we got a large numbers gene invoved in the process of seed germination of Populus simonii×Populus nigr, these genes not only contains a large number of known genes, more important is also include many novel genes. These genes will largely contribute to study of the molecular mechanism of seed germination process on genetics and molecular biology.更多还原