毛竹SAUR、DELLA基因的鉴定、克隆及功能分析

发布时间：2018-05-29 17:20

本文选题：毛竹 + SAUR　；参考：《中国林业科学研究院》2017年博士论文

【摘要】：毛竹(Phyllostachys edulis)是中国最重要的经济竹种之一,适宜的条件下,竹笋能够在短时间内从0 m长到20 m,经组织解剖发现,这一过程包括细胞分裂和细胞伸长。对模式植物拟南芥(Arabidopsis thaliana)等的研究发现:赤霉素(gibberellic acid,GA)和生长素(indole-3-acetic acid,IAA)在促进植物细胞分裂和伸长方面具有重要功能。IAA早期响应基因SAUR在促进细胞伸长方面发挥了重要作用;GA途径中的转录因子DELLA蛋白通过参与调控下游相关基因调节植物生长。本研究中:通过透射电镜(transmission electron microscope,TEM)对毛竹竹笋显微结构进行分析;基于毛竹基因组数据库进行SAUR和DELLA基因鉴定和生物信息分析;利用荧光实时定量(fluorescence quantification real-time PCR,qRT-PCR)、基因克隆、遗传转化、亚细胞定位、原核表达等分子生物学方法对SAUR和DELLA基因功能进行初步研究。主要研究结果如下:1.利用TEM对毛竹不同高度竹笋顶部细胞发育状况进行观察,在前期竹笋细胞较小,以细胞分裂为主;在发育中后期以细胞伸长为主,并伴随着细胞的逐渐发育成熟。基于毛竹基因组数据库对毛竹SAUR基因进行鉴定,得到38个毛竹SAUR(PheSAUR)基因,并对38个PheSAUR基因的编码区长度、编码蛋白氨基酸残基数、启动子元件分析及等电点等进行生物信息分析;多数PheSAUR基因没有内含子;对38个PheSAUR蛋白系统发育分析发现有多个姐妹对形成;对毛竹、水稻(Oryza sativa)及拟南芥的SAUR蛋白系统发育分析发现多数PheSAUR蛋白与水稻OsSAUR蛋白聚到一起,亲缘关系更近;在PheSAUR、AtSAUR及Os SAUR蛋白中发现了3个保守性很高的基序。在毛竹基因组数据库中鉴定得到1个毛竹DELLA基因,该基因没有内含子,并且丢失了部分保守结构域。2.用qRT-PCR对不同PheSAUR基因的表达量进行分析,PheSAUR2、PheSAUR6、PheSAUR18、PheSAUR20、PheSAUR26、PheSAUR35等基因在实生苗中表现出组织特异性表达现象;多数PheSAUR基因在毛竹实生苗外施IAA后表达量上调。克隆得到6个毛竹PheSAUR基因,亚细胞定位研究发现,PheSAUR4定位到细胞膜上,PheSAUR28定位到细胞质中,PheSAUR29和PheSAUR34同时定位到细胞膜和细胞核中,毛竹PheSAUR蛋白可能参与到IAA信号接收和传递过程中;通过转化拟南芥,35S:PheSAUR29-GFP在含有GA3的培养基上生长更快,PheSAUR29基因可能参与到GA途径中;接着对毛竹实生苗进行外施GA3和烯效唑(S3307)处理并分析PheSAUR4和PheSAUR29基因的表达量,两个基因均在外施GA3后上调表达,在外施S3307后下调表达,表明这两个基因均可能参与到GA途径中;同时发现了许多响应GA3的基因(PheSAUR7、PheSAUR13、PheSAUR16、PheSAUR17、PheSAUR18等)。此外,构建了PheSAUR4和PheSAUR29原核表达载体并探索得到两个蛋白的原核表达条件。3.克隆得到毛竹DELLA基因(1 857 bp),没有内含子,GC含量为69.04%,编码618个氨基酸,其蛋白含有DELLA蛋白全部典型的保守结构域;与不同物种的DELLA蛋白进化分析发现该蛋白与水稻SLR1蛋白同源性最高,因此将克隆得到的该基因命名为PheSLR1;同时克隆得到PheSLR1基因上游1 933 bp的启动子序列,并鉴定出GA和光响应元件;经qRT-PCR分析,PheSLR1在竹笋生长过程中表达量上升,在实生苗外施GA3后上调表达,外施S3307后下调表达;通过S3307处理的毛竹种子表现出发育缓慢的现象,PheSLR1在S3307处理的芽中的表达量较GA3和DMSO(对照)更低;亚细胞定位分析发现PheSLR1蛋白定位到细胞核中;用qRT-PCR方法对PheSLR1蛋白潜在下游基因(PheGA20ox、PheCesA1、PheCesA2、PheGASA6等)的表达量进行分析,这些基因多在竹笋发育过程中上调表达;此外,通过原核表达及纯化得到PheSLR1蛋白。本研究基于毛竹基因组数据库进行SAUR和DELLA基因的鉴定工作,并初步探索了PheSAUR29和PheSLR1等基因的功能,为这些基因参与毛竹竹笋发育机理研究提供了依据。
[Abstract]:Phyllostachys edulis is one of the most important economic bamboo species in China. Under suitable conditions, bamboo shoots can grow from 0 m to 20 m in a short time. By histological anatomy, this process includes cell division and cell elongation. Studies on the model plant Arabidopsis (Arabidopsis thaliana) have been found: gibberellin (gibberellic acid, GA) and so on. Indole-3-acetic acid (IAA) plays an important role in promoting the division and elongation of plant cells..IAA early response gene SAUR plays an important role in promoting cell elongation; the transcription factor DELLA protein in the GA pathway regulates the growth of the plant by regulating the downstream related genes. In this study, transmission electron microscopy (trans) (trans) Mission electron microscope, TEM) analyzed the microstructure of bamboo shoot of bamboo; based on the bamboo genome database for SAUR and DELLA gene identification and biological information analysis, using real-time fluorescence quantitative (fluorescence quantification real-time PCR, qRT-PCR), gene clout, genetic transformation, subcellular localization, prokaryotic expression and other molecular organisms The primary study on the function of SAUR and DELLA gene was studied. The main results were as follows: 1. the cell development of bamboo shoots at different height of bamboo was observed by TEM. In the early stage, the cell division was smaller and the cell division was dominant in the early stage of development, and the growth of the cells was gradually developed. 38 bamboo SAUR (PheSAUR) genes were identified by the genome database. The length of the coding region, the amino acid residue of the encoded protein, the analysis of the promoter element and the isoelectric point of the 38 PheSAUR genes were analyzed. Most of the PheSAUR genes had no introns, and the development of 38 PheSAUR proteins was analyzed. Many of the existing sisters were formed; the phylogenetic analysis of the SAUR protein of bamboo, rice (Oryza sativa) and Arabidopsis found that most of the PheSAUR protein was gathered together with the rice OsSAUR protein, and the relationship was closer; 3 very high conserved sequences were found in PheSAUR, AtSAUR and Os SAUR protein. In the bamboo genome database, 1 were identified. The gene of Phyllostachys pubescens DELLA, the gene has no introns, and a partial conserved domain.2. has been lost to analyze the expression of different PheSAUR genes with qRT-PCR. PheSAUR2, PheSAUR6, PheSAUR18, PheSAUR20, PheSAUR26, PheSAUR35, and other genes show tissue specific expression in the seedlings; most PheSAUR genes are outside the seedlings of bamboo. 6 Phyllostachys pubescens PheSAUR gene was cloned and 6 Phyllostachys pubescens were cloned. The subcellular localization study found that PheSAUR4 was located on the cell membrane, PheSAUR28 was located in the cytoplasm, PheSAUR29 and PheSAUR34 were located at the cell membrane and nucleus. The bamboo PheSAUR protein may be involved in the reception and transmission of IAA signal; by transforming the pseudo south. Mustard, 35S:PheSAUR29-GFP grows faster on the medium containing GA3, and PheSAUR29 gene may be involved in the GA pathway; then GA3 and entrazoles (S3307) are applied to the seedlings of Phyllostachys pubescens and the expression of PheSAUR4 and PheSAUR29 genes are analyzed. The two genes are up to be expressed after external GA3, and the expression is downregulated after external application of S3307. Two genes may be involved in the GA pathway, and many genes that respond to GA3 (PheSAUR7, PheSAUR13, PheSAUR16, PheSAUR17, PheSAUR18, etc.) are also found. In addition, PheSAUR4 and PheSAUR29 prokaryotic expression vectors are constructed and the prokaryotic expression condition of two proteins is explored to obtain the DELLA gene of bamboo (1857 BP), without introns. The content of 69.04%, encoding 618 amino acids, its protein contains all the typical conservative domain of DELLA protein, and the evolution analysis of DELLA protein of different species found that the protein has the highest homology with the rice SLR1 protein, so the cloned gene is named PheSLR1; and clon obtains the promoter sequence of the 1933 BP upstream of the PheSLR1 gene. GA and light response elements were identified. After qRT-PCR analysis, the expression of PheSLR1 increased during the growth of bamboo shoots, up expression after GA3 was applied to the seedlings, and after S3307 was down regulated. The growth of bamboo seeds treated by S3307 was slow, and the expression of PheSLR1 in S3307 treated buds was lower than that of DMSO (control). The cell location analysis found that PheSLR1 protein was located in the nucleus; the expression of the potential downstream genes of PheSLR1 protein (PheGA20ox, PheCesA1, PheCesA2, PheGASA6, etc.) was analyzed by qRT-PCR method. These genes were up to be expressed in the process of bamboo shoot development; in addition, PheSLR1 protein was obtained through the expression and purification of the prokaryotic cells. This study was based on the hair. The bamboo genome database was used for the identification of SAUR and DELLA genes, and the functions of PheSAUR29 and PheSLR1 were preliminarily explored, which provided a basis for these genes to participate in the study of bamboo shoot development mechanism.
【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S795.7;Q943.2

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