水稻細(xì)胞壁降解過程中的多糖變化及OsCslF6基因的遺傳轉(zhuǎn)化研究
發(fā)布時間：2018-09-03 10:32
【摘要】：降低木質(zhì)纖維素轉(zhuǎn)化可再生的燃料成本的首要障礙在于植物的抗降解能力,即細(xì)胞壁受到化學(xué)處理和酶解時所產(chǎn)生的抵抗力。當(dāng)前,細(xì)胞壁抗體工具庫是足夠大而廣泛的,通過抗體對抗原的特異性結(jié)合,能夠檢測植物細(xì)胞壁多糖的主要種類的表位。本研究通過讓細(xì)胞壁多糖導(dǎo)向的單克隆抗體作為探針來具體分析細(xì)胞壁經(jīng)過預(yù)處理及酶解過程中細(xì)胞壁纖維素、半纖維素、果膠多糖的結(jié)構(gòu)變化,結(jié)合吸光度法己糖、戊糖、糖醛酸以及氣相質(zhì)譜測定半纖維素單糖含量的變化,揭示細(xì)胞壁各組分之間的聯(lián)系。主要結(jié)果如下:1.堿預(yù)處理對纖維素的作用不明顯,而酸預(yù)處理卻能去除部分纖維素,與直接酶解的相比,預(yù)處理后酶解對纖維素的作用更加顯著。酸堿預(yù)處理酶解都能夠完全去除半纖維素和果膠的表位,說明酸堿預(yù)處理酶解對初生壁降解作用明顯。2.直接酶解只能夠去除木葡聚糖的表位,同時也能夠揭示在薄壁組織中掩蓋的木聚糖,而對果膠的表位并沒有明顯作用。3.在細(xì)胞壁的降解過程中,薄壁組織最先被降解,韌皮部隨后被降解,除韌皮部外的維管束是最后被降解的組織。4.在細(xì)胞壁的降解過程中,果膠是首先被去除的細(xì)胞壁成分,其次是木葡聚糖,最后是木聚糖,說明木聚糖的抗降解能力強(qiáng)于果膠以及木葡聚糖。5.結(jié)合單糖釋放量以及己糖、戊糖的釋放量,相比較于野生型日本晴材料(NPB),Osfc16材料在酸、堿預(yù)處理下都有更高的酶解效率,兩個材料的戊糖、己糖及糖醛酸釋放量趨勢基本一致�；旌咸擒真I葡聚糖(MLG)是禾本科細(xì)胞壁半纖維素的重要成分。由于MLG含量的高低對人類膳食纖維的營養(yǎng)健康、生產(chǎn)生物乙醇可發(fā)酵的多糖、在啤酒釀造過程密切相關(guān),MLG的合成機(jī)制受到大量的關(guān)注。MLG合成基因大多屬于在糖基轉(zhuǎn)移酶(glycotransferease,GT)家族中的纖維素合酶(CesA like,CSL)超家族中,其中CslF家族和CslH家族是兩個禾本科特定的亞家族,有研究表明它們參與MLG的生物合成。在水稻中,CslF和CslH家族分別含有8個成員和3個成員,已有研究表明,CslF6基因是MLG合成中最重要的一個基因。本文就水稻CslF6基因功能進(jìn)行了初步研究,主要結(jié)果如下:1.構(gòu)建了不同熒光蛋白標(biāo)簽(GFP和mCherry)OsCslF6基因超表達(dá)載體和互補(bǔ)載體,通過農(nóng)桿菌介導(dǎo)轉(zhuǎn)入水稻和擬南芥,篩選陽性轉(zhuǎn)基因材料。這些材料將在后續(xù)研究中進(jìn)行水稻細(xì)胞壁半纖維素MLG的含量、結(jié)構(gòu)的分析,為進(jìn)一步了解OsCslF6在水稻MLG合成中的功能提供了材料基礎(chǔ)。2.提取T0代OsCslF6超表達(dá)轉(zhuǎn)基因水稻葉片的膜蛋白,通過GFP抗體,檢測到的較弱的具有OsCslF6蛋白的條帶,支持OsCslF6基因在質(zhì)膜上調(diào)控MLG合酶的合成。3通過把35S啟動子以及自身啟動子的OsCslF6基因載體農(nóng)桿菌介導(dǎo)的轉(zhuǎn)基因方法轉(zhuǎn)入擬南芥,通過qRT-PCR分析擬南芥轉(zhuǎn)基因植株的基因表達(dá)。與野生型相比,OsCslF6超表達(dá)轉(zhuǎn)基因植株株高偏矮小,生育期延長。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S511
本文編號：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S511
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 王艷婷;徐正丹;彭良才;;植物細(xì)胞壁溝槽結(jié)構(gòu)與生物質(zhì)利用研究展望[J];中國科學(xué):生命科學(xué);2014年08期
，本文編號：2219723
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