發(fā)布時間：2018-05-29 17:20

  本文選題：毛竹 + SAUR�。� 參考：《中國林業(yè)科學(xué)研究院》2017年博士論文

【摘要】：毛竹(Phyllostachys edulis)是中國最重要的經(jīng)濟竹種之一,適宜的條件下,竹筍能夠在短時間內(nèi)從0 m長到20 m,經(jīng)組織解剖發(fā)現(xiàn),這一過程包括細胞分裂和細胞伸長。對模式植物擬南芥(Arabidopsis thaliana)等的研究發(fā)現(xiàn):赤霉素(gibberellic acid,GA)和生長素(indole-3-acetic acid,IAA)在促進植物細胞分裂和伸長方面具有重要功能。IAA早期響應(yīng)基因SAUR在促進細胞伸長方面發(fā)揮了重要作用;GA途徑中的轉(zhuǎn)錄因子DELLA蛋白通過參與調(diào)控下游相關(guān)基因調(diào)節(jié)植物生長。本研究中:通過透射電鏡(transmission electron microscope,TEM)對毛竹竹筍顯微結(jié)構(gòu)進行分析;基于毛竹基因組數(shù)據(jù)庫進行SAUR和DELLA基因鑒定和生物信息分析;利用熒光實時定量(fluorescence quantification real-time PCR,qRT-PCR)、基因克隆、遺傳轉(zhuǎn)化、亞細胞定位、原核表達等分子生物學(xué)方法對SAUR和DELLA基因功能進行初步研究。主要研究結(jié)果如下:1.利用TEM對毛竹不同高度竹筍頂部細胞發(fā)育狀況進行觀察,在前期竹筍細胞較小,以細胞分裂為主;在發(fā)育中后期以細胞伸長為主,并伴隨著細胞的逐漸發(fā)育成熟。基于毛竹基因組數(shù)據(jù)庫對毛竹SAUR基因進行鑒定,得到38個毛竹SAUR(PheSAUR)基因,并對38個PheSAUR基因的編碼區(qū)長度、編碼蛋白氨基酸殘基數(shù)、啟動子元件分析及等電點等進行生物信息分析;多數(shù)PheSAUR基因沒有內(nèi)含子;對38個PheSAUR蛋白系統(tǒng)發(fā)育分析發(fā)現(xiàn)有多個姐妹對形成;對毛竹、水稻(Oryza sativa)及擬南芥的SAUR蛋白系統(tǒng)發(fā)育分析發(fā)現(xiàn)多數(shù)PheSAUR蛋白與水稻OsSAUR蛋白聚到一起,親緣關(guān)系更近;在PheSAUR、AtSAUR及Os SAUR蛋白中發(fā)現(xiàn)了3個保守性很高的基序。在毛竹基因組數(shù)據(jù)庫中鑒定得到1個毛竹DELLA基因,該基因沒有內(nèi)含子,并且丟失了部分保守結(jié)構(gòu)域。2.用qRT-PCR對不同PheSAUR基因的表達量進行分析,PheSAUR2、PheSAUR6、PheSAUR18、PheSAUR20、PheSAUR26、PheSAUR35等基因在實生苗中表現(xiàn)出組織特異性表達現(xiàn)象;多數(shù)PheSAUR基因在毛竹實生苗外施IAA后表達量上調(diào)。克隆得到6個毛竹PheSAUR基因,亞細胞定位研究發(fā)現(xiàn),PheSAUR4定位到細胞膜上,PheSAUR28定位到細胞質(zhì)中,PheSAUR29和PheSAUR34同時定位到細胞膜和細胞核中,毛竹PheSAUR蛋白可能參與到IAA信號接收和傳遞過程中;通過轉(zhuǎn)化擬南芥,35S:PheSAUR29-GFP在含有GA3的培養(yǎng)基上生長更快,PheSAUR29基因可能參與到GA途徑中;接著對毛竹實生苗進行外施GA3和烯效唑(S3307)處理并分析PheSAUR4和PheSAUR29基因的表達量,兩個基因均在外施GA3后上調(diào)表達,在外施S3307后下調(diào)表達,表明這兩個基因均可能參與到GA途徑中;同時發(fā)現(xiàn)了許多響應(yīng)GA3的基因(PheSAUR7、PheSAUR13、PheSAUR16、PheSAUR17、PheSAUR18等)。此外,構(gòu)建了PheSAUR4和PheSAUR29原核表達載體并探索得到兩個蛋白的原核表達條件。3.克隆得到毛竹DELLA基因(1 857 bp),沒有內(nèi)含子,GC含量為69.04%,編碼618個氨基酸,其蛋白含有DELLA蛋白全部典型的保守結(jié)構(gòu)域;與不同物種的DELLA蛋白進化分析發(fā)現(xiàn)該蛋白與水稻SLR1蛋白同源性最高,因此將克隆得到的該基因命名為PheSLR1;同時克隆得到PheSLR1基因上游1 933 bp的啟動子序列,并鑒定出GA和光響應(yīng)元件;經(jīng)qRT-PCR分析,PheSLR1在竹筍生長過程中表達量上升,在實生苗外施GA3后上調(diào)表達,外施S3307后下調(diào)表達;通過S3307處理的毛竹種子表現(xiàn)出發(fā)育緩慢的現(xiàn)象,PheSLR1在S3307處理的芽中的表達量較GA3和DMSO(對照)更低;亞細胞定位分析發(fā)現(xiàn)PheSLR1蛋白定位到細胞核中;用qRT-PCR方法對PheSLR1蛋白潛在下游基因(PheGA20ox、PheCesA1、PheCesA2、PheGASA6等)的表達量進行分析,這些基因多在竹筍發(fā)育過程中上調(diào)表達;此外,通過原核表達及純化得到PheSLR1蛋白。本研究基于毛竹基因組數(shù)據(jù)庫進行SAUR和DELLA基因的鑒定工作,并初步探索了PheSAUR29和PheSLR1等基因的功能,為這些基因參與毛竹竹筍發(fā)育機理研究提供了依據(jù)。
[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.
【學(xué)位授予單位】：中國林業(yè)科學(xué)研究院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S795.7;Q943.2

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