水稻细胞壁降解过程中的多糖变化及OsCslF6基因的遗传转化研究
发布时间：2018-09-03 10:32
【摘要】：降低木质纤维素转化可再生的燃料成本的首要障碍在于植物的抗降解能力,即细胞壁受到化学处理和酶解时所产生的抵抗力。当前,细胞壁抗体工具库是足够大而广泛的,通过抗体对抗原的特异性结合,能够检测植物细胞壁多糖的主要种类的表位。本研究通过让细胞壁多糖导向的单克隆抗体作为探针来具体分析细胞壁经过预处理及酶解过程中细胞壁纤维素、半纤维素、果胶多糖的结构变化,结合吸光度法己糖、戊糖、糖醛酸以及气相质谱测定半纤维素单糖含量的变化,揭示细胞壁各组分之间的联系。主要结果如下:1.碱预处理对纤维素的作用不明显,而酸预处理却能去除部分纤维素,与直接酶解的相比,预处理后酶解对纤维素的作用更加显著。酸碱预处理酶解都能够完全去除半纤维素和果胶的表位,说明酸碱预处理酶解对初生壁降解作用明显。2.直接酶解只能够去除木葡聚糖的表位,同时也能够揭示在薄壁组织中掩盖的木聚糖,而对果胶的表位并没有明显作用。3.在细胞壁的降解过程中,薄壁组织最先被降解,韧皮部随后被降解,除韧皮部外的维管束是最后被降解的组织。4.在细胞壁的降解过程中,果胶是首先被去除的细胞壁成分,其次是木葡聚糖,最后是木聚糖,说明木聚糖的抗降解能力强于果胶以及木葡聚糖。5.结合单糖释放量以及己糖、戊糖的释放量,相比较于野生型日本晴材料(NPB),Osfc16材料在酸、碱预处理下都有更高的酶解效率,两个材料的戊糖、己糖及糖醛酸释放量趋势基本一致。混合糖苷键葡聚糖(MLG)是禾本科细胞壁半纤维素的重要成分。由于MLG含量的高低对人类膳食纤维的营养健康、生产生物乙醇可发酵的多糖、在啤酒酿造过程密切相关,MLG的合成机制受到大量的关注。MLG合成基因大多属于在糖基转移酶(glycotransferease,GT)家族中的纤维素合酶(CesA like,CSL)超家族中,其中CslF家族和CslH家族是两个禾本科特定的亚家族,有研究表明它们参与MLG的生物合成。在水稻中,CslF和CslH家族分别含有8个成员和3个成员,已有研究表明,CslF6基因是MLG合成中最重要的一个基因。本文就水稻CslF6基因功能进行了初步研究,主要结果如下:1.构建了不同荧光蛋白标签(GFP和mCherry)OsCslF6基因超表达载体和互补载体,通过农杆菌介导转入水稻和拟南芥,筛选阳性转基因材料。这些材料将在后续研究中进行水稻细胞壁半纤维素MLG的含量、结构的分析,为进一步了解OsCslF6在水稻MLG合成中的功能提供了材料基础。2.提取T0代OsCslF6超表达转基因水稻叶片的膜蛋白,通过GFP抗体,检测到的较弱的具有OsCslF6蛋白的条带,支持OsCslF6基因在质膜上调控MLG合酶的合成。3通过把35S启动子以及自身启动子的OsCslF6基因载体农杆菌介导的转基因方法转入拟南芥,通过qRT-PCR分析拟南芥转基因植株的基因表达。与野生型相比,OsCslF6超表达转基因植株株高偏矮小,生育期延长。
[Abstract]:The main obstacle to reducing the cost of converting lignocellulose into renewable fuel is the plant's ability to resist degradation, that is, the resistance of the cell wall to chemical treatment and enzymatic hydrolysis. At present, the cell wall antibody tool library is large enough and wide enough to detect the major epitopes of plant cell wall polysaccharides by the specific binding of antibodies to antigens. In this study, the structure changes of cell-wall cellulose, hemicellulose and pectin polysaccharides were analyzed by using monoclonal antibody directed by cell wall polysaccharides as a probe. The content of hemicellulose monosaccharide was determined by uronic acid and gas-phase mass spectrometry to reveal the relationship between the components of cell wall. The main results are as follows: 1. The effect of alkali pretreatment on cellulose is not obvious, but acid pretreatment can remove part of cellulose. Compared with direct enzymatic hydrolysis, the effect of enzymatic hydrolysis on cellulose is more obvious. The enzymatic hydrolysis of acid-base pretreatment could completely remove the epitopes of hemicellulose and pectin, which indicated that the enzymatic hydrolysis of acid-base pretreatment had obvious effect on the degradation of primary wall. Direct enzymatic hydrolysis could only remove the epitopes of xylodextran and reveal the covering up of xylan in parenchyma, but had no obvious effect on the epitope of pectin. In the process of cell wall degradation, parenchyma was first degraded, phloem was then degraded, vascular bundle except phloem was the last degraded tissue. In the process of cell wall degradation, pectin is the first cell wall component to be removed, followed by xylodextran, and finally xylan, which indicates that xylan has stronger anti-degradation ability than pectin and xylodextran .5. Combined with the amount of monosaccharide, hexose and pentose, compared with the wild Japanese fine material (NPB), Osfc16 had higher enzymatic hydrolysis efficiency under the pretreatment of acid and alkali. The release trend of pentose, hexose and uronic acid of the two materials was basically the same. Mixed glucoside dextran (MLG) is an important component of hemicellulose in gramineous cell wall. Due to the effect of MLG content on the nutrition and health of human dietary fiber, the polysaccharides that can be fermented by the production of biological ethanol can be produced. The biosynthesis mechanism of MLG is closely related to the brewing process. MLG synthesis genes mostly belong to the superfamily of cellulose synthase (CesA like,CSL) in the family of glycosyltransferases (glycotransferease,GT), and most of them belong to the superfamily of cellulosic synthase (CesA like,CSL) in the glycosyltransferase (glycotransferease,GT) family. The CslF family and the CslH family are two specific subfamilies of Gramineae, which have been shown to be involved in the biosynthesis of MLG. In rice, CSLF and CslH family have 8 members and 3 members, respectively. It has been shown that CslF6 gene is the most important gene in MLG synthesis. The function of rice CslF6 gene was studied in this paper. The main results are as follows: 1. The superexpression vectors and complementary vectors of OsCslF6 gene with different fluorescent protein tags (GFP and mCherry) were constructed and transformed into rice and Arabidopsis thaliana mediated by Agrobacterium tumefaciens to screen positive transgenic materials. These materials will be used to analyze the content and structure of hemicellulose MLG in rice cell wall in the follow-up study, which provides a material basis for further understanding the function of OsCslF6 in rice MLG synthesis. The membrane proteins of transgenic rice leaves were extracted from T0 generation OsCslF6 and the weak bands with OsCslF6 protein were detected by GFP antibody. To support the regulation of the synthesis of MLG synthase by OsCslF6 gene on the plasma membrane. 3. The expression of MLG synthase in Arabidopsis thaliana was analyzed by qRT-PCR by means of Agrobacterium tumefaciens-mediated transfer of 35s promoter and OsCslF6 gene vector of its own promoter into Arabidopsis thaliana (Arabidopsis thaliana). Compared with wild type, OsCslF6 transgenic plants were shorter in height and longer in growth period.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511
本文编号：2219723
[Abstract]:The main obstacle to reducing the cost of converting lignocellulose into renewable fuel is the plant's ability to resist degradation, that is, the resistance of the cell wall to chemical treatment and enzymatic hydrolysis. At present, the cell wall antibody tool library is large enough and wide enough to detect the major epitopes of plant cell wall polysaccharides by the specific binding of antibodies to antigens. In this study, the structure changes of cell-wall cellulose, hemicellulose and pectin polysaccharides were analyzed by using monoclonal antibody directed by cell wall polysaccharides as a probe. The content of hemicellulose monosaccharide was determined by uronic acid and gas-phase mass spectrometry to reveal the relationship between the components of cell wall. The main results are as follows: 1. The effect of alkali pretreatment on cellulose is not obvious, but acid pretreatment can remove part of cellulose. Compared with direct enzymatic hydrolysis, the effect of enzymatic hydrolysis on cellulose is more obvious. The enzymatic hydrolysis of acid-base pretreatment could completely remove the epitopes of hemicellulose and pectin, which indicated that the enzymatic hydrolysis of acid-base pretreatment had obvious effect on the degradation of primary wall. Direct enzymatic hydrolysis could only remove the epitopes of xylodextran and reveal the covering up of xylan in parenchyma, but had no obvious effect on the epitope of pectin. In the process of cell wall degradation, parenchyma was first degraded, phloem was then degraded, vascular bundle except phloem was the last degraded tissue. In the process of cell wall degradation, pectin is the first cell wall component to be removed, followed by xylodextran, and finally xylan, which indicates that xylan has stronger anti-degradation ability than pectin and xylodextran .5. Combined with the amount of monosaccharide, hexose and pentose, compared with the wild Japanese fine material (NPB), Osfc16 had higher enzymatic hydrolysis efficiency under the pretreatment of acid and alkali. The release trend of pentose, hexose and uronic acid of the two materials was basically the same. Mixed glucoside dextran (MLG) is an important component of hemicellulose in gramineous cell wall. Due to the effect of MLG content on the nutrition and health of human dietary fiber, the polysaccharides that can be fermented by the production of biological ethanol can be produced. The biosynthesis mechanism of MLG is closely related to the brewing process. MLG synthesis genes mostly belong to the superfamily of cellulose synthase (CesA like,CSL) in the family of glycosyltransferases (glycotransferease,GT), and most of them belong to the superfamily of cellulosic synthase (CesA like,CSL) in the glycosyltransferase (glycotransferease,GT) family. The CslF family and the CslH family are two specific subfamilies of Gramineae, which have been shown to be involved in the biosynthesis of MLG. In rice, CSLF and CslH family have 8 members and 3 members, respectively. It has been shown that CslF6 gene is the most important gene in MLG synthesis. The function of rice CslF6 gene was studied in this paper. The main results are as follows: 1. The superexpression vectors and complementary vectors of OsCslF6 gene with different fluorescent protein tags (GFP and mCherry) were constructed and transformed into rice and Arabidopsis thaliana mediated by Agrobacterium tumefaciens to screen positive transgenic materials. These materials will be used to analyze the content and structure of hemicellulose MLG in rice cell wall in the follow-up study, which provides a material basis for further understanding the function of OsCslF6 in rice MLG synthesis. The membrane proteins of transgenic rice leaves were extracted from T0 generation OsCslF6 and the weak bands with OsCslF6 protein were detected by GFP antibody. To support the regulation of the synthesis of MLG synthase by OsCslF6 gene on the plasma membrane. 3. The expression of MLG synthase in Arabidopsis thaliana was analyzed by qRT-PCR by means of Agrobacterium tumefaciens-mediated transfer of 35s promoter and OsCslF6 gene vector of its own promoter into Arabidopsis thaliana (Arabidopsis thaliana). Compared with wild type, OsCslF6 transgenic plants were shorter in height and longer in growth period.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511
【参考文献】
相关期刊论文 前1条
1 王艳婷;徐正丹;彭良才;;植物细胞壁沟槽结构与生物质利用研究展望[J];中国科学:生命科学;2014年08期
，本文编号：2219723
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