本文選題：毛竹 + 赤霉素��； 參考：《浙江農(nóng)林大學(xué)》2016年碩士論文

【摘要】：赤霉素是調(diào)控植物生長(zhǎng)發(fā)育的五大激素之一,在如種子萌發(fā)、莖的伸長(zhǎng)以及果實(shí)發(fā)育等植物的多種生命活動(dòng)中發(fā)揮重要作用。通過(guò)分子遺傳學(xué)等方法,在水稻、擬南芥等植物中發(fā)現(xiàn)赤霉素傳導(dǎo)途徑的一些重要元件,包括赤霉素的受體蛋白GID1、赤霉素信號(hào)的負(fù)調(diào)控因子DELLA蛋白和DELLA蛋白降解相關(guān)的GID2蛋白。目前對(duì)赤霉素傳導(dǎo)途徑的研究表明,在赤霉素不存在或濃度較低的情況下,DELLA蛋白抑制植物的生長(zhǎng),而當(dāng)活性赤霉素存在時(shí),赤霉素與受體GID1結(jié)合后,導(dǎo)致后者構(gòu)象改變并與DELLA蛋白相互作用,誘導(dǎo)DELLA蛋白泛素化并通過(guò)蛋白酶途徑降解,激活下游信號(hào)轉(zhuǎn)導(dǎo),進(jìn)而調(diào)控植物生長(zhǎng)發(fā)育過(guò)程。毛竹我國(guó)分布最廣、經(jīng)濟(jì)價(jià)值最高的竹種,主要以無(wú)性系繁殖方式進(jìn)行更新生長(zhǎng),毛竹筍有著驚人的生長(zhǎng)速度,這種快速生長(zhǎng)現(xiàn)象具有重要的學(xué)術(shù)研究?jī)r(jià)值。有研究指出在竹筍的快速生長(zhǎng)期赤霉素的含量迅速升高,推測(cè)赤霉素在筍的快速生長(zhǎng)中會(huì)發(fā)揮關(guān)鍵作用,研究毛竹中的赤霉素合成和信號(hào)途徑具有重要意義。本研究以毛竹種子幼苗為材料,研究了赤霉素對(duì)毛竹種子萌發(fā)、幼苗生長(zhǎng)影響,并同源克隆獲得赤霉素信號(hào)轉(zhuǎn)導(dǎo)途徑中的三個(gè)關(guān)鍵基因,應(yīng)用生物信息學(xué)手段進(jìn)行基因序列分析。采用實(shí)時(shí)定量PCR方法研究這些基因在筍的快速生長(zhǎng)時(shí)期不用部位的表達(dá)情況,并利用酵母雙雜交系統(tǒng)驗(yàn)證了這些蛋白之間的互作關(guān)系,還通過(guò)農(nóng)桿菌介導(dǎo)花序侵染法獲得DELLA基因過(guò)表達(dá)擬南芥,初步探討了毛竹DELLA基因在植物中的功能,得到了以下結(jié)論:1.赤霉素促進(jìn)了毛竹種子的萌發(fā)和毛竹幼苗的生長(zhǎng),赤霉素濃度在100mg.L-1時(shí)作用最明顯,與對(duì)照組相比,種子發(fā)芽率提升了19.80%,莖長(zhǎng)提升了97.17%;2.通過(guò)同源克隆得到了毛竹中的三個(gè)基因PhGID1、PhGID2和PhSLR1全長(zhǎng),它們的ORF分別為1065bp、648bp和1866bp,分別編碼354、215和621個(gè)氨基酸。對(duì)氨基酸序列進(jìn)行生物信息學(xué)分析顯示,它們和其他植物中的同源蛋白質(zhì)氨基酸序列一致性較為保守。3.在快速生長(zhǎng)期的毛竹筍中,PhGID1、PhGID2和PhSLR1這三個(gè)基因在筍的頂部、中部、基部以及根部都有表達(dá),PhSLR1在筍的上、中部表達(dá)量較少,在根部較多,而PhGID1和PhGID2的表達(dá)情況相似,在筍的上中部表達(dá)量高,根部較少,這與PhSLR1基因的表達(dá)呈相反趨勢(shì)。4.原核表達(dá)實(shí)驗(yàn)表明,當(dāng)表達(dá)菌株為大腸桿菌BL21(DE3),表達(dá)載體為pET-32a時(shí),PhSLR1和PhGID2蛋白能夠表達(dá)且是可溶性的,PhGID1蛋白的表達(dá)以包涵體形式存在。5.酵母雙雜交結(jié)果顯示,PhGID2蛋白和PhSLR1有相互作用且作用于PhSLR1蛋白的C端GRAS結(jié)構(gòu)域,PhGID2蛋白和PhGID1蛋白也存在相互作用且不依賴于赤霉素的存在。6.將毛竹基因PhSLR1進(jìn)行轉(zhuǎn)基因?qū)嶒?yàn),觀察表型發(fā)現(xiàn)擬南芥轉(zhuǎn)基因植株比野生型要矮小,葉片和果莢也小、少。
[Abstract]:Gibberellin is one of the five hormones regulating plant growth and development, which plays an important role in many life activities of plants such as seed germination, stem elongation and fruit development. Some important elements of gibberellin transduction pathway were found in rice, Arabidopsis thaliana and other plants by means of molecular genetics, including gibberellin receptor protein GID1, the negative regulation factor Della of gibberellin signal and the GID2 protein associated with the degradation of della protein. Studies on the transmission pathway of gibberellin have shown that the Della protein inhibits the growth of plants without or at a low concentration of gibberellin, and when active gibberellin exists, gibberellin binds to the receptor GID1. The latter changes in conformation and interacts with Della protein, which induces the ubiquification and degradation of Della protein through protease pathway, activates downstream signal transduction, and then regulates plant growth and development. Phyllostachys pubescens are the most widely distributed species with the highest economic value in China. They mainly regenerate and grow by clonal reproduction. The bamboo shoots of Phyllostachys pubescens have an amazing growth rate, which has important academic research value. Some studies have pointed out that the content of gibberellin increases rapidly in the rapid growth period of bamboo shoots. It is suggested that gibberellin will play a key role in the rapid growth of bamboo shoots. It is of great significance to study gibberellin synthesis and signal pathway in Phyllostachys pubescens. In this study, the effects of gibberellin on germination and seedling growth of Phyllostachys pubescens were studied, and three key genes of gibberellin signal transduction pathway were obtained by homologous cloning. Gene sequence analysis was carried out by bioinformatics. Real-time quantitative PCR was used to study the expression of these genes during the rapid growth of bamboo shoots, and the interaction between these proteins was verified by yeast two-hybrid system. The overexpression of Della gene in Arabidopsis thaliana was also obtained by Agrobacterium tumefaciens mediated inflorescence infection. The function of Della gene in Phyllostachys pubescens was preliminarily studied, and the following conclusions were obtained: 1. Gibberellin promoted the germination of Phyllostachys pubescens seeds and the growth of Phyllostachys pubescens seedlings. The effect of gibberellin concentration was most obvious at 100 mg 路L ~ (-1). Compared with the control group, the germination rate of the seeds increased 19.80% and the stem length increased 97.17% ~ (2). Three genes, PhGID1, PhGID2 and PhSLR1, were cloned from Phyllostachys pubescens by homologous cloning. Their ORFs were 1065 BP, 648 BP and 1866 BP, respectively, encoding 354215 and 621 amino acids, respectively. Bioinformatics analysis of amino acid sequence showed that the amino acid sequence of homologous protein was conserved with that of other plants. The three genes, PhGID1, PhGID2 and PhSLR1, were expressed in the top, middle, base and root of bamboo shoots at the rapid growth stage, but the expression of PhGID1 and PhGID2 were similar in the middle part of shoot and the root, but the expression of PhGID1 and PhGID2 were similar. The expression of PhSLR1 gene in the upper and middle part of shoot was higher than that in the root, which was opposite to the expression of PhSLR1 gene. E. coli BL21 (DE3) and pET-32a were expressed in E. coli BL21 (DE3). The expression of PhSLR1 and PhGID2 proteins could be expressed in the form of inclusion body. The results of yeast two-hybrid showed that there was interaction between PhGID2 protein and PhSLR1, and the C-terminal GRAs domain of PhSLR1 protein, PhGID2 protein and PhGID1 protein, were also interacted and independent of the existence of gibberellin. 6. The transgenic experiment of Phyllostachys pubescens gene PhSLR1 showed that the transgenic plants of Arabidopsis thaliana were shorter than the wild type and the leaves and pods were smaller and less.
【學(xué)位授予單位】：浙江農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S795.7

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