毛竹生长过程中纤维素合成酶基因的表达模式和功能分析

发布时间：2018-08-08 12:13

【摘要】：毛竹是中国主要的用材竹种,纤维素的合成是竹材形成的必要条件。纤维素主要由纤维素合成酶(Cellulose synthase,Ces A)合成,并储存在植物的初生壁和次生壁中。因此,研究纤维素合成酶的结构与功能对毛竹生长发育以及纤维素的利用有重要指导意义。本研究以毛竹生长过程中不同时期的5个高度(10cm、30cm、120cm、600cm、1400cm)的毛竹为研究材料,其中10cm和30cm时为毛竹生长初期,120cm为毛竹生长上升期,600cm左右为毛竹生长盛期,1400cm时毛竹开始抽枝展叶,为生长末期,通过生物信息学方法、生物显微镜观察、透射电镜观察、荧光定量PCR、RNA原位杂交、Western Blot、蛋白质的体外表达等方法研究了毛竹纤维素合成酶基因的表达和功能。所得结论如下:(1)毛竹茎秆结构显微观察表明毛竹生长发育分为四个时期,第一个时期,细胞未分化期,没有明显的组织结构,此时的细胞主要以分裂产生更多的细胞为主。第二个时期,原生结构形成期,有典型的维管束结构出现,但密度较大,结构较小,韧皮部细胞分化不明显,纤维细胞和薄壁细胞的界限不明确。第三个时期,维管束结构成熟期,纤维细胞和薄壁细胞的界限明确,有两个典型的后生木质部导管,韧皮部结构明显。第四个时期,纤维细胞木质化时期,可以看到纤维细胞周围一层深色物质。基部的纤维组织最先出现木质化,此时为毛竹生长的上升期,茎秆的高度大约为120cm。(2)毛竹韧皮部细胞超微结构观察发现,毛竹生长发育过程可分为初生壁形成期,次生壁形成期,以及次生壁加厚期。研究表明次生壁随着高度的增加而加厚,上升期(120cm)的毛竹基部可观察到次生壁出现,盛期(600cm)的毛竹中部和基部有次生壁结构,顶部和根部仅有初生壁,末期(14m)毛竹基部次生壁比盛期厚。(3)生物信息学分析表明,毛竹纤维素合成酶家族基因共有16个成员,结构域分析表明,毛竹纤维素合成酶都含有cellulose_synt结构域,N端大多有Zn指结构,毛竹纤维素合成酶超家族成员有48个,分为5个亚家族,分别是CESA、CSLD、CSLE、CSLF、CSLH。系统进化分析表明,Ces A基因极有可能在单、双子叶植物分化前就已经出现分化。祖先状态时,毛竹中的纤维素合成酶基因就比拟南芥和水稻多。物种分化之后,毛竹纤维素合成酶基因也单独发生了多次重复事件,产生了更多的基因拷贝数,这可能和毛竹独特的快速生长特性有关。(4)在前人对毛竹茎秆中纤维素含量的研究基础上,结合显微和超微观察结果以及定量表达结果进行分析发现,在初生壁形成的部位,3个基因(PeCesA6、PeCesA9和PeCesA13)表达最显著。在次生壁形成的部位,毛竹纤维素合成酶基因家族成员的8个基因(PeCesA1、PeCesA2、PeCesA3、PeCesA4、PeCesA5、PeCesA6、PeCesA9、和PeCesA13)表达均非常显著。推测PeCesA6、PeCesA9、PeCesA13对初生壁和次生壁的形成都起到重要作用,而PeCesA1、PeCesA2、PeCesA3、PeCesA4、PeCesA5基因主要参与毛竹次生壁的合成。(5)毛竹纤维素合成酶保守结构域体外表达的蛋白为包涵体,经Western blot检测后确定为该蛋白的表达,表达产物的分子量为33KDa,p I为7.4。
[Abstract]:Phyllostachys pubescens are the main timber species in China. The synthesis of cellulose is a necessary condition for the formation of bamboo. Cellulose is mainly synthesized by Cellulose synthase (Ces A) and stored in the primary wall and secondary wall of the plant. Therefore, the structure and function of cellulose synthase have been studied for the growth and development of Phyllostachys pubescens and the use of cellulose. This study uses 5 height (10cm, 30cm, 120cm, 600cm, 1400cm) of Phyllostachys pubescens in the growth process of Phyllostachys pubescens as the research materials, in which 10cm and 30cm are the initial growth period of Phyllostachys pubescens, 120cm is the growth period of Phyllostachys pubescens, and 600cm is the growth stage of the bamboo, while 1400cm starts to branch out, for the end of growth and through biology. The methods of Informatics, biological microscopy, transmission electron microscopy, fluorescence quantitative PCR, RNA in situ hybridization, Western Blot, protein expression and other methods were used to study the expression and function of the gene of bamboo cellulose synthase. The conclusions are as follows: (1) the growth and development of Phyllostachys pubescens are divided into four periods, the first one is the first one. During the undifferentiated period of the cells, there was no obvious tissue structure. At this time, the cells were mainly divided to produce more cells mainly. Second periods, the primary structure formation period, the typical vascular bundle structure appeared, but the density was large, the structure was small, the phloem cell differentiation was not obvious, and the boundaries of the fibroblast and parenchyma cells were not clear. Third During the period of the mature period of vascular bundle structure, the boundaries of fibroblast and parenchyma cells are clear, there are two typical posterior xylem ducts and the phloem structure is obvious. In the fourth period, the fiber cells lignification period can see the dark substance around the fiber cells. The fibrous tissue of the base is first lignification, at this time it is the growth of Phyllostachys pubescens. The height of the stem is about 120cm. (2) in the phloem ultrastructure of the Phyllostachys pubescens. It is found that the process of growth and development of bamboo can be divided into primary wall formation period, secondary wall formation period, and secondary wall thickening period. The secondary wall thickens with the height increase, and secondary wall can be observed in the base of the up period (120cm). There are secondary wall structures in the middle and base of 600cm, the top and the root are only primary wall, and the secondary wall of the base of bamboo is more thick than that in the end (14m). (3) bioinformatics analysis shows that there are 16 members of the Phyllostachys pubescens cellulose synthase family gene. The analysis of the Phyllostachys pubescens cellulose synthase contains the cellulose_synt domain and the N end. Most of them have Zn finger structure. There are 48 superfamily members of Phyllostachys pubescens cellulose synthase superfamily, which are divided into 5 subfamilies. The phylogenetic analysis of CESA, CSLD, CSLE, CSLF, CSLH. shows that the Ces A gene is very likely to be differentiated before the differentiation of the dicotyledonous plants. The cellulose synthase genes in the forefathers were compared to the southern mustard and water. After species differentiation, the Phyllostachys pubescens cellulose synthetase gene has also been repeated several times, producing more copies of the gene, which may be related to the unique rapid growth characteristics of bamboo. (4) on the basis of previous studies on cellulose content in bamboo stems, combined with microscopic and ultramicro observation results and quantitative expression junctions. The 3 genes (PeCesA6, PeCesA9 and PeCesA13) expressed most significantly at the site of primary wall formation, and 8 genes (PeCesA1, PeCesA2, PeCesA3, PeCesA4, PeCesA5, PeCesA6, PeCesA9, and PeCesA13) were very significant in the parts of the secondary wall formation. PeCesA13 plays an important role in the form of primary wall and secondary wall of Chengdu, while PeCesA1, PeCesA2, PeCesA3, PeCesA4, PeCesA5 genes are mainly involved in the synthesis of the secondary wall of Phyllostachys pubescens. (5) the protein expressed in the conservative domain of bamboo cellulose synthase is inclusion body, which is determined by Western blot as the expression of the protein and the molecule of the expression product. The amount is 33KDa, and P I is 7.4.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S795.7

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