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Author: 裴胜强
Degree Level: 硕士
Issued Date: 2017-05
Degree Grantor: 中国科学院大学
Place of Degree Grantor: 北京
Supervisor: 拟南芥,HD-ZIP转录因子,表皮细胞,粘液质,结晶化纤维素
Keyword: 工学
Subject: A Study on the Mechanisms of HDG2 transcription factor in Regulating Arabidopsis Seed Coat Mucilage Structure
Alternative Title: 生物工程
Major: 纤维生物质是地球上储量最丰富的可再生资源,是人类生产和生活中能源、纤维、建筑材料和造纸原料等的主要来源。在植物所积累的太阳能中,90%以上是以纤维生物质形式存在于细胞壁。深入解析植物细胞壁的组成结构及其生物合成机制,并通过基因工程手段改变细胞壁的组分、结构及不同组分间的连接方式,进而创制可高效转化的纤维生物质原料,是当今植物细胞壁领域的研究热点。 拟南芥种子表皮细胞在分化过程中的特定时期能够合成和分泌大量的粘液质多糖(主要为鼠李半乳糖醛酸聚糖I)至胞外,与少量的纤维素和半纤维素成分(木聚糖和葡甘露聚糖等)一起构成一类特化的次生细胞壁。拟南芥种皮粘液质对于种子的萌发或生长发育并非必需,并且具有表型容易观察,分离提取方便等优点,因此拟南芥种皮粘液可作为研究细胞壁多糖生物合成、功能与调控的理想模式体系。 通过前期实验鉴定出一个在拟南芥种皮发育中特异表达的转录因子基因HOMODOMAIN GLABROUS 2(HDG2),属同源异型盒蛋白HD-Zip IV亚家族成员。本论文中针对HDG2参与拟南芥种皮粘液质结构调控的功能展开深入研究。取得主要结果如下:1)通过实时荧光定量PCR(qRT-PCR)、原位杂交和启动子GUS活性分析表明HDG2 在正在发育的种皮中特异表达,其表达峰值出现在授粉后10天(10 DPA);2)通过酵母系统分析了HDG2的转录激活活性及其转录激活域的位置,根据HDG2 蛋白的保守结构域(HD、LZ、START 和 SAD)设置了截短试验,证明了HDG2具有转录激活活性,且其转录激活活性位于亮氨酸拉链(LZ)结构域;3)对T-DNA 插入突变体hdg2-2和hdg2-3的粘液质层进行钌红染色发现,其粘液质释放正常,总量也无明显变化,但与野生型(WT)相比,hdg2突变体的粘液质层明显变得容易脱落,在轻微振荡下出现显著变薄的缺陷表型;4)取授粉后4天至13天(4 DPA-13 DPA)的hdg2-3突变体种子,进行树脂切片分析,发现hdg2-3突变体与WT各发育时期的表皮细胞发育正常,粘液质合成、分泌以及总量均与与WT无异;5)分析hdg2-3种皮粘液质的含量、组成和结构,发现 hdg2-3 种皮粘液质总量与WT无显著性差异;单糖组成成分表明hdg2-3内外层粘液质中影响粘液质结构的各单糖成分(如木糖、甘露糖等)无显著性变化;6)种子原位免疫组化分析结果表明,hdg2-3突变体种皮粘液质中结晶化纤维素含量较WT显著降低,结晶化纤维素含量测定进一步证实了免疫组化分析结果,据此推断导致hdg2-3突变体粘液质缺陷表型是由结晶化纤维素含量减少所致;7)进一步分析HDG2影响结晶化纤维素的分子机制,对不同时期hdg2-3突变体及WT果荚进行qRT-PCR分析,发现影响纤维素合成的关键基因CESA5的表达量在hdg2-3突变体中显著降低,进一步通过凝胶迁移率阻滞(EMSA)和酵母单杂交实验证实了HDG2蛋白可以结合CESA5启动子的L1-box结构域;此外,利用原生质体转录激活分析显示HDG2能够激活CESA5基因的表达。 综上,本论文鉴定了一个调控种皮粘液质结构维持的HDG2转录因子,通过一系列的生理和生化证据表明HDG2通过直接激活CESA5的表达,进而调控种皮粘液质纤维素的合成或组装,维持种皮粘液质正常结构。反之,HDG2突变抑制CESA5的表达,种皮粘液质纤维素的合成或组装受阻,导致种皮粘液质层极易脱落,最终呈现出种皮粘液质层显著变薄的缺陷表型。这将有助于加深对种皮粘液质中纤维素合成与组装的分子机制和调控网络的认识,并为将来通过人工分子设计可高效转化的细胞壁奠定理论基础。
Abstract: As the most abundant renewable resource on the earth, plant cell wall has numerous applications in various aspects of human living and industry servering as the main source of energy, fiber, building materials and papermaking materials. Thus, in-depth analysis of the composition, structure and biosynthesis mechanisms of plant cell wall, and optimization of the composition, structure and connections between different components by genetic engineering to improve the saccharification efficiency , is hot topics in the research of plant cell wall. Arabidopsis thaliana seed coat cells synthesize and secrete large amounts of mucilage ploysaccarides (mainly composed of rhamnogalactoside I, RG I) to the extracellular at specific satges during the seed coat differentiation.Besides pectic RG I, mucilage also consists a small amount of cellulose and hemicellulose (e.g. heteroxylansand glactoglucomannan) components, thus mucilage represnet a specialized cell wall. Seed coat mucilage is not necessary for the germination or growth of plants, in addition, seed coat mucilage can be easily extracted and the defect phenotype can be readily identified, so it can be utilized as an ideal model system for the study of biosynthesis, modification and regulation of cell wall polysaccharides. Previous study identified a transcription factor gene, HOMODOMAIN GLABROUS 2 (HDG2) . HDG2 belongs to homeobox subfamily HD-Zip IV. This study mainly focused on the mechanism of HDG2 in the regulation of seed coat mucilage structure.The main results are as follows: 1) Real-time quantitative PCR (qRT-PCR), in situ hybridization and promoter GUS activity analysis showed that HDG2 was specifically expressed in the seed coat, and its peak was observed at 10 days after pollination (10 DPA). 2) The transcriptional activation activity of HDG2 and its transcriptional activation domain was analyzed in yeast cell. The truncation test was performed according to the conserved domain of HDG2 protein (HD, LZ, START and SAD). The results indicated that HDG2 had transcriptional activation activity, and its transcriptional activation is located in the leucine zipper (LZ) domain. 3) Ruthenium red staining was carried out for T-DNA insertion mutants of HDG2(hdg2-2 and hdg2-3). The results showed that seed coat mucilage of hdg2 can be normally released, and no significant changes in total amount was observed. However, the mucilage layers of hdg2 was easily shaked off, leaving thinner adherent inner mucilage than that of the wild type (WT). 4) The hdg2 and WT seeds were dissected from 4 DPA-13 DPA and observed by resin embedding and slicing, the results showed that epidermal cells of hdg2-3 seed were normally developed and the secretion and the amount of mucilage displayed no significant differences compared to the WT. 5) The content, composition and structure of hdg2-3 mucilage was analyzed, and it was found that the total amount of hdg2-3 mucilage had no significant differences with that of the WT. The composition of monosaccharide showed that the contents of monosaccharides,which affect the mucilage structure (e.g. xylose, mannose) had no significant differences with the WT. 6) In situ immunohistochemical analysis showed that the crystalline cellulose content was significantly reduced in hdg2-3 compare with the WT, which was confirmed by the quantification of crystalline cellulose contents. Thus it can be conclueded that the decrease of crystalline cellulose led to the mucilage defect in hdg2-3. 7) To further confirm if HDG2 is involved in the modulation of crystalline cellulose, qRT-PCR analysis of hdg2 and WT seeds at different stages was performed. The results showed that CESA5, a key gene affecting cellulose synthesis in seed coat mucilage, was significantly decreased in hdg2-3. EMSA and yeast one hybridization analysis confirmed that HDG2 protein could bind to the L1-box motif located in CESA5 promoter. In addition, protoplast transcriptional activation assay showed that HDG2 could activate the expression of CESA5. In summary, the molecular mechanisms of HDG2 transcription factor in the regulation of mucilage structure were characterzide in this study. A series of physiological and biochemical evidence suggested that HDG2 regulates the synthesis or assembly of cellulose by direct activation of CESA5 to maintain the normal structure of the seed coat mucilage. In contrast, HDG2 mutations inhibit the expression of CESA5, the synthesis or assembly of crystalline cellulose is blocked and finally showing a defective phenotype of the seed coat mucilage. The results obtained will help to deepen our understanding of the molecular mechanisms and regulation networks underlying cellulose synthesis and assembly, which lay a theoretical foundation for the future custom-design of the cell wall by genetic engineering means.
English Abstract: 中文
Language: 中文
Department: 植物代谢工程团队
Available Date: 2019-07-01
DOC Type: 学位论文
Content Type: 学位论文
Appears in Collections:植物代谢工程团队_学位论文

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description.institution: 中国科学院大学

Recommended Citation:
裴胜强. HDG2转录因子调控拟南芥种皮粘液质结构的机制研究[D]. 北京. 中国科学院大学. 2017.
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