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Title:
南荻GT43基因家族参与木聚糖合成的功能研究
Author: 王小玉
Degree Level: 博士
Issued Date: 2016
Degree Grantor: 中国科学院大学
Place of Degree Grantor: 北京
Supervisor: 周功克 ; 胡瑞波
Keyword: 南荻 ; 木聚糖 ; 糖基转移酶43家族 ; 次生细胞壁 ; 种皮粘液质
Subject: 理学
Alternative Title: Functional Characterization of Miscanthus lutarioriparius GT43 Gene Family in Xylan Biosynthesis
Major: 生物化学与分子生物学
Abstract: 木聚糖(xylan)是自然界中第二丰富的多糖,是双子叶植物次生细胞壁和草本植物初生及次生细胞壁中半纤维素的主要成分。木聚糖主要由β-(1-4)连接的木糖残基组成主链骨架,以及被乙酰基、葡萄糖醛酸、甲基化的葡萄糖醛酸和阿拉伯糖等残基取代的侧链组成。双子叶植物的木聚糖在其还原性末端还含有4-β-D-Xyl-(1–4)-β-D-Xyl-(1–3)-α-L-Rha-(1–2)-α-D-GalA-(1–4)-D-Xyl四糖序列。 在模式植物拟南芥中,利用不规则木质部(irregular xylem, irx)突变体,对木聚糖生物合成方面的研究已经取得了较大进展。来自糖基转移酶43家族的IRX9/IRX9L和IRX14/IRX14L,形成功能非冗余的两组基因对,已被证明参与木聚糖主链的延伸过程。然而,在草本植物特别是芒属植物中木聚糖的生物合成过程却了解甚少。芒草是一种多年生的纤维类草本能源植物,具有光合效率高、水肥需求低、适应性广和生物质产量高等优点,可以为新一代生物燃料乙醇生产提供优质的木质纤维素原料,在世界范围内引起了广泛关注。木聚糖是纤维类能源植物生产生物燃料乙醇的主要原材料之一, 因此阐明其生物合成和修饰的分子调控机制,对通过基因工程手段改良纤维类能源植物的细胞壁组成和结构具有重要的理论价值和应用价值。 本研究从芒属能源植物南荻中克隆到7个GT43家族基因,并对其功能进行了深入的研究,结果表明这7个基因分别是拟南芥IRX9或IRX14的同源基因。首先对9种具有代表性的植物和南荻中的GT43蛋白进行系统进化分析,发现这些GT43蛋白被分为3个亚家族,分别是IRX9、IRX9L和IRX14/IRX14L。实时荧光定量PCR检测发现7个基因在所有检测组织中均有表达,但mRNA原位杂交实验证明MlGT43A-B和MlGT43F-G主要是在次生壁增厚的细胞中表达,而MlGT43C-E则在薄壁组织和厚壁组织细胞中均有表达。亚细胞定位分析发现7个MlGT43蛋白全定位于高尔基体。在拟南芥irx9突变体中过表达MlGT43A-E而非MlGT43F和MlGT43G基因,可以完全或部分恢复irx9突变体表型,包括植株高度、茎中不规则木质部细胞以及木糖含量;然而过表达MlGT43F和MlGT43G而非MlGT43A-E基因,可以恢复拟南芥irx14突变体表型,这些结果表明GT43基因在南荻中进化出功能不同的两组,其中MlGT43A-E行使IRX9同源基因功能,而MlGT43F和MlGT43G具有IRX14同源基因功能。然而,南荻中IRX14同源基因发生了功能分化,因为MlGT43基因均不能恢复irx14种子粘液质缺失表型,说明MlGT43并不参与维持种皮细胞粘液质多糖中木聚糖的合成。此外,瞬时转录激活实验分析发现MlGT43A和MlGT43B可以不同程度地被三个调控次生细胞壁合成的主要转录开关MlSND1、MlMYB46 或MlVND7激活,而IRX9L同源基因(MlGT3C-E)则不能被这些转录因子有效激活。 本研究结果首次用遗传证据证明南荻GT43基因在参与木聚糖合成过程中形成功能非冗余的两类,这一点是进化保守的;但在种皮粘液质合成方面却出现了功能分化。本研究进一步加深了对植物木聚糖生物合成的认识,为将来遗传改良草本植物木聚糖的组成和结构奠定了基础。
English Abstract: Xylans are the second most abundant polysaccharides in nature, and represent a major hemicellulosic component in secondary cell walls of dicots and all walls of grasses. Xylans consist of a linear polymer of β-(1-4)-linked xylose backbone and substituted with acetyl, glucuronic acid (GlcA), 4-O-methylglucuronic acid (Me-GlcA), and arabinose residues. Dicot xylans contain the tetrasaccharide 4-β-D-Xyl-(1–4)-β-D-Xyl-(1–3)-α-L-Rha-(1–2)-α-D-GalA-(1–4)-D-Xyl at their reducing ends. In the model plant Arabidopsis, much progress has been gained in xylan biosynthesis especially in the studies utilizing the irregular xylem (irx) mutants. Irregular Xylem 9 (IRX9)/9L and IRX14/14L from glycosyltransferase (GT) family 43, which form two functionally nonredundant groups, have been proved to play crucial roles in xylan backbone elongation. However, xylan biosynthesis in grasses especially in Miscanthus is poorly understood. Miscanthus is a perennial lignocellulosic herbaceous energy plant with superior characteristics such as high photosynthetic efficiency, low fertilizer and water demand, wide adaptability and high biomass yield. It has attracted much concern worldwide as a superior lignocellulosic feedstock for next-generation bioethanol production. Xylans are the main source of feedstock for next-gerneration biofuel production, thus it is of important theoretical value to gain a complete understanding of the biochemical mechanism underlying xylan biosynthesis and modification, which may lay the foundation for future genetic modification of lignocellulosic biomass to be better tailored for various economically important applications, including the more efficient utilization of xylan for biofuel production. In this study, we identified seven putative GT43 genes from M. lutarioriparius, a promising bioenergy crop, in-depth studies indicated that they are functional orthologues of Arabidopsis IRX9 or IRX14. Firstly, Phylogenetic analysis of GT43 proteins from nine representative plant species and Miscanthus revealed that these proteins were classified into three major clades, namely IRX9, IRX9L and IRX14/IRX14L. Quantitative real-time RT-PCR analysis revealed that all MlGT43 genes exhibited broad expression patterns across the tissues examined. mRNA in situ hybridization experiment unambiguously demonstrated that MlGT43A-B and MlGT43F-G were preferentially expressed in cells undergoing secondary wall thickening, while MlGT43C-E were expressed in both sclerenchyma and parenchyma cells. Subcellular localization analysis indicated all seven MlGT43 proteins were localized to Golgi apparatus. Overexpression of MlGT43A-E genes but not MlGT43F and MlGT43G in Arabidopsis irx9 fully or partially rescued the mutant defects, including plant height, irregular xylem cells in stem cross sections and xylose contents, whereas overexpression of MlGT43F and MlGT43G but not MlGT43A-E complemented the defects of irx14. These results suggest that MlGT43 genes have evolved into two distinct functional groups, with MlGT43A-E are orthologous to IRX9, while MlGT43F and MlGT43G are orthologous to IRX14. Nevertheless, the functional divergence of IRX14 orthologues in M. lutarioriparius has occurred as none of MlGT43 genes could rescue the mucilage defects of irx14 seeds, suggesting that MlGT43 could not synthesize the xylan which is involved in maintaining the structure of seed coat mucilage. Furthermore, transient transactivation analyses of MlGT43A-E reporters demonstrated that MlGT43A and MlGT43B were differentially activated by MlSND1, MlMYB46 or MlVND7 orthologues, three key transcriptional switches governing secondary cell wall biosynthesis. By contrast, the Miscanthus IRX9L orthologues (MlGT43C-E) were not significantly transactivated by these transcription factors. Our results provide the first line of genetic evidence demonstrating that Miscanthus has evolved to retain two functionally non-redundant groups of MlGT43 genes involved in xylan biosynthesis but diverged in seed coat mucilage biosynthesis. This study further deepen our understanding of xylan biosynthesis, and lay a foundation for future genetic modification the composition and structure of grass xylan.
Language: 中文
Department: 植物代谢工程团队
Available Date: 2016
DOC Type: 学位论文
Content Type: 学位论文
URI: http://ir.qibebt.ac.cn/handle/337004/9763
Appears in Collections:植物代谢工程团队_学位论文

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Recommended Citation:
王小玉. 南荻GT43基因家族参与木聚糖合成的功能研究[D]. 北京. 中国科学院大学. 2016.
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