【作者】 陶雷；

【导师】 王秋玉；

【作者基本信息】 东北林业大学 ， 遗传学， 2013， 博士

【摘要】 植物体细胞胚胎发生是指植物体细胞在特定条件下未经性细胞融合而发育成胚状体的形态发生过程。它在快速繁殖、人工种子、种质保存、基因工程等领域具有广阔的应用前景和巨大的经济价值。体细胞胚发生是细胞全能性的具体体现,重演了合子胚形态发生的过程,为此深入地研究体细胞胚发生与发育的机制对于揭示细胞分化、发育、形态发生、分子生物学等机制有重要意义。本论文以刺五加体细胞胚发生为研究内容,对刺五加体细胞胚发生形态观察、生理生化进行了研究；利用质壁分离处理刺五加合子胚诱导体细胞胚发生,对胼胝质的动态变化与体细胞胚发生的关系进行研究;同时,以刺五加体细胞胚早期发育三个时期为试材,采用Solexa高通量测序技术对刺五加体细胞胚的转录组进行了分析,在此基础上,对三个时期进行了数字基因表达谱分析,对差异表达基因进行功能分析及分类,并筛选与刺五加早期体细胞胚发生相关基因,探讨刺五加的体细胞胚早期发生机制,为进一步研究刺五加体细胞胚发生提供分子资源。主要结果如下：1.刺五加体细胞胚发生经历球形胚、心形胚、鱼雷胚、子叶胚,最后发育成体胚苗,测定了刺五加胚性愈伤组织、球形胚、鱼雷胚、子叶胚、体胚苗中的可溶性糖、淀粉、可溶性蛋白含量,同时,对超氧化物歧化酶、过氧化物酶活性进行测定。刺五加胚性愈伤组织中可溶性糖含量最高,随着体细胞胚的发育,含量逐渐降低；淀粉在整个发育时期有2个高峰,球形胚和子叶胚时期；随着体细胞胚的发育,可溶性蛋白含量呈上升趋势,由胚性愈伤组织到球形胚时期上升速度较快；从胚性愈伤组织到成熟体胚苗发育过程中,SOD活性逐渐升高；胚性愈伤组织中POD活性最高,其活性表现出先下降后上升的趋势。这些物质的变化可能与体细胞胚的分化、发育相关。2.质壁分离处理产生的胼胝质与体细胞胚发生呈正相关。①质壁分离处理刺五加合子胚诱导体细胞胚的最佳试剂是1.0M甘露醇,其最佳处理时间是12小时。②质壁分离处理过程中和体细胞胚发生过程中,胼胝质含量都有很大的变化。质壁分离处理12小时的外植体材料中,胼胝质含量较高,体细胞胚的发生数目最多。处理外植体材料中的胼胝质在体细胞胚发生过程中先下降,然后随着体细胞胚发生而升高。③利用激光共聚焦显微镜观察胼胝质沉积情况,质壁分离处理刺五加合子胚时,胼胝质主要沉积在细胞膜周围。质壁分离处理诱导体细胞胚发生过程中,胼胝质在不同组织沉积有较大的差别,在表皮和维管组织中有大量的沉积,而在其它组织中沉积很少。④胚性愈伤组织中可观察到大量胼胝质的沉积,而非胚性愈伤组织中观察不到胼胝质的沉积。⑤胼胝质抑制剂(2-DDG)的加入,降低了体细胞胚的诱导率和每个外植体产生的体细胞胚数,体细胞胚发生率仅为11.5%。3.刺五加早期体细胞胚转录组测序总共进行58,327,688次读数,最后共获得75,803个Unigene。这些Unigene被COC、GO、KEGG进行注释。数字基因表达谱表明刺五加不同发育阶段(EC, YEC, GE)基因表达差异情况。每个样本获得5.6M以上的tags,获得了不同发育阶段差异表达的基因,对差异表达基因进行GO和Pathway分析,筛选到一些与刺五加早期体细胞胚发生相关基因。更多还原

【Abstract】 Plant somatic embryogenesis means the process of morphogenesis of somatic cell developing into embryoid without sexual cell fusion under given conditions. It has broad application prospect and huge economic values in rapid propagation, artificial seeds, germplasm conservation, plant genetic engineering and other fields. Somatic embryogenesis is not only a concrete manifestation of totipotence of cells, but also a reappearance process of the zygotic embryo morphogenesis, so the study on somatic embryogenesis and developmental mechanisms has a very important significance for revealing the molecular biology mechanisms of cell differentiation, cell development and morphogenesis.In this study, the somatic embryogenesis of Eleutherococcus senticosus was studied on the basis of the morphological observation, physiological and biochemical characteristics test. The relationship between dynamic changes of callose and somatic embryogenesis was analyzed through plasmolyzing the zygotic embryos of Eleutherococcus senticosus to induce somatic embryogenesis; meanwhile, through Solexa high-throughput sequencing technology, transcriptome of somatic embryo, which are in three periods of early development of Eleutherococcus senticosus, were analyzed. Further more, the digital gene expression profiles of those materials were analyzed, including function analysis and classification of differentially expressed genes, as well as screening genes related to early somatic embryogenesis of Eleutherococcus senticosus. The purpose is to explore mechanism of early somatic embryogenesis, which will provide molecular resources for further study on somatic embryogenesis of Eleutherococcus senticosus. The main results as follows:1. The somatic embryogenesis of Eleutherococcus senticosus includes five phases: globular, heart-shaped, torpedo, cotyledon embryo and finally somatic embryo seedling. Soluble sugars, starch, soluble protein content, superoxide dismutase, peroxidase activity in embryonic calli, globular, torpedo, cotyledon embryos, and seedlings were measured. The content of soluble sugar was the highest in embryonic calli and gradually reduced with developing of somatic embryos; there are two peaks of starch content during the whole development:in globular and cotyledon embryo period. Soluble protein content increased along the development of somatic embryos, with higher speed when somatic embryos developing from embryonic calli to globular embryos; SOD activity increased gradually from embryonic calli to somatic embryos seedlings; The POD activity was the highest in embryonic calli, which showed tend of down first and up later. The changes of these substances might be related with the differentiation and development of somatic embryos.2. There was positive correlation between somatic embryogenesis and callose produced after plasmolysis treatment.①The best reagent is1.0M mannitol and the optimum treatment time was12hours for zygotic embryos of Eleutherococcus senticosus treated by plasmolysis.②The callose content changed a lot during plasmolysis treatment and somatic embryogenesis, with the trends of decrease first and then increase with somatic embryogenesis. Callose content was higher and the number of somatic embryos was the biggest after12hours treatment of plasmolysis.③Confocal laser scanning microscope was used to observe callose deposition, which showed great difference in different tissues during somatic embryogenesis after being treated by plasmolysis. There were a large number of callose deposition in the epidermis and vascular tissue mainly around the cytomembrane, while very little in other tissues.④A number of callose deposition can be observed in embryonic calli, but not in non-embryonic calli.⑤Adding of2-DDG decreased the induction of somatic embryos and the numbers of somatic embryos produced by each explants, while incidence rate of somatic embryo was only11.5%.3.58,327,688sequencing reads were obtained by transcriptome sequencing of Eleutherococcus senticosus, which were eventually assembled into75,803unigenes. In order to understand these gene functions, they were annotated with Clusters of Orthologous Groups (COG), gene orthology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Digital gene expression (DGE) profile showed differences of gene expression at different development stages (EC, YEC, and GE). The differentially expressed genes were analyzed by GO and Pathway. More than5.6million tags per sample were obtained and a large number of differentially expressed genes at different stages of somatic embryogenesis were identified, which included some genes related with early somatic embryogenesis.更多还原