本文關(guān)鍵詞：馬鈴薯赤霉素合成代謝途徑關(guān)鍵酶基因GA2ox1、GA20ox1的克隆與功能分析　出處：《青海大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 馬鈴薯 轉(zhuǎn)基因 赤霉素 GA20-氧化酶 GA2-氧化酶 遺傳轉(zhuǎn)化 非生物脅迫

【摘要】：赤霉素(GA)作為植物體內(nèi)一種內(nèi)源激素,在高等植物生長的整個(gè)生命周期,起著重要的調(diào)控作用。GA-20氧化酶和GA-2氧化酶是赤霉素合成途徑中第三階段的關(guān)鍵酶,它們均由多基因家族編碼,參與著赤霉素水平的調(diào)控。GA-20氧化酶能促進(jìn)活性赤霉素的合成,屬于正向調(diào)控;而GA-2氧化酶則會(huì)將活性赤霉素轉(zhuǎn)化為非活性的赤霉素,屬負(fù)向調(diào)控。所以GA20ox基因和GA2ox基因的表達(dá)水平,將會(huì)影響到植物體內(nèi)活性GA的合成,最終影響植株的生長發(fā)育。本研究首先以株高突變馬鈴薯植株為材料,建立了外源基因拷貝數(shù)的檢測方法,并測定植株體內(nèi)的赤霉素合成關(guān)鍵酶基因在不同時(shí)期不同組織內(nèi)的表達(dá)量,分析表達(dá)差異,以明確赤霉素合成途徑關(guān)鍵酶基因的表達(dá)水平與植株株高間的關(guān)系。以馬鈴薯青薯9號(hào)為材料,克隆了GA20ox1基因和GA2ox1基因,分析了序列特征,并通過農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化分析了過量表達(dá)目的基因?qū)︸R鈴薯植株的影響。從分子水平闡明了兩基因的可能作用機(jī)理。本研究主要結(jié)果如下:1、利用實(shí)時(shí)熒光定量PCR方法估算出了轉(zhuǎn)基因馬鈴薯中外源基因T-DNA的插入拷貝數(shù),在所測的23株轉(zhuǎn)基因株系中,12株為單拷貝插入,6株為雙拷貝,5株為多拷貝,并通過對(duì)田間馬鈴薯農(nóng)藝性狀的分析。建立了快速檢測外源基因拷貝數(shù)的方法。2、利用Real-time PCR方法,對(duì)赤霉素合成途徑關(guān)鍵酶基因在馬鈴薯突變體材料M4P9中的表達(dá)量進(jìn)行了檢測分析,結(jié)果顯示,在馬鈴薯生長的不同時(shí)期,以及各時(shí)期內(nèi)的根、莖、葉中,各基因的表達(dá)量均發(fā)生了一定變化,CPS1基因在幼苗期的過量表達(dá)為植株的生長提供了充足的內(nèi)源激素,GA20ox1基因在莖中變化明顯,主要表現(xiàn)為在膨大期莖中的過量表達(dá),可能與塊莖淀粉累積有關(guān),GA2ox1基因在塊莖形成期莖中的過量表達(dá),抑制了內(nèi)源GA的合成,被認(rèn)為是促使M4P9植株矮小的因素之一。外源噴施活性GA3能抑制植物體內(nèi)自身活性GA的合成,主要表現(xiàn)為馬鈴薯植株體內(nèi)赤霉素相關(guān)酶基因表達(dá)量的下降。3、克隆了馬鈴薯赤霉素合成途徑中的StGA2ox1基因,測序得CDS全長1005bp,編碼334個(gè)氨基酸。經(jīng)對(duì)其編碼的蛋白質(zhì)二級(jí)結(jié)構(gòu)預(yù)測,結(jié)果顯示,α-螺旋為34.43%、無規(guī)則卷曲為34.13%、延伸主鏈為22.16%、β-轉(zhuǎn)角為9.28%,為穩(wěn)定的蛋白質(zhì)。同源性比對(duì)結(jié)果顯示,克隆得到的馬鈴薯GA2ox1基因編碼的氨基酸序列與番茄GA2ox2基因、煙草GA2ox1、GA2ox2、GA2ox3基因的同源性較高,而與番茄GA2ox1基因的同源性最低。與NCBI上的GA2ox1(LOC102605134)基因序列相比,青薯9號(hào)中GA2ox1基因序列對(duì)應(yīng)的其編碼氨基酸也發(fā)生了一定的改變。4、克隆了馬鈴薯赤霉素合成途徑中的StGA20ox1基因,測序得CDS全長1137bp,編碼378個(gè)氨基酸,并對(duì)StGA20ox1基因編碼的蛋白質(zhì)進(jìn)行了二級(jí)結(jié)構(gòu)預(yù)測,結(jié)果顯示,該蛋白質(zhì)中α-螺旋為39.68%、無規(guī)則卷曲為33.33%、延伸主鏈為19.58%、β-轉(zhuǎn)角為7.41%,為穩(wěn)定的蛋白質(zhì)。同源性比對(duì)結(jié)果顯示,StGA20ox1與番茄GA20ox1基因、歐白英GA20ox1基因、煙草GA20ox基因的同源性較高,而與牽�；ǖ�3類GA20ox基因的同源性都不高。與NCBI上的GA20ox1(AJ291453.1)基因序列相比,青薯9號(hào)中GA20ox1基因序列在多處發(fā)生了改變(共29處),經(jīng)測序得青薯9號(hào)中的GA20ox1基因編碼氨基酸較已公布的StGA20ox1基因編碼的氨基酸,少了3個(gè)強(qiáng)堿性氨基酸,少了4個(gè)疏水氨基酸,多了4個(gè)極性氨基酸。5、改造并成功構(gòu)建了一個(gè)新的質(zhì)粒載體pCAEZ1383。構(gòu)建了用于外源基因GA20ox1和GA2ox1遺傳轉(zhuǎn)化用的植物表達(dá)載體1383-GA20ox1和1383-GA2ox1。經(jīng)遺傳轉(zhuǎn)化,獲得含外源GA20ox1基因的閩薯1號(hào)轉(zhuǎn)基因陽性苗7株;獲得含外源GA2ox1基因的青薯9號(hào)轉(zhuǎn)基因陽性苗1株。6、利用熒光定量方法,對(duì)獲得的轉(zhuǎn)基因陽性苗中,StGA2ox1基因和StGA20ox1基因的表達(dá)量進(jìn)行了檢測,結(jié)果表明,轉(zhuǎn)基因植株中StGA2ox1基因和StGA20ox1基因的表達(dá)量均有不同程度的上調(diào)。經(jīng)表型性狀觀察,過量表達(dá)達(dá)StGA20ox1基因可促進(jìn)馬鈴薯植株株高生長和根的伸長。在青薯9號(hào)轉(zhuǎn)基因植株中,StGA2ox1基因拷貝數(shù)為6;在閩薯1號(hào)轉(zhuǎn)基因株系中,StGA20ox1基因拷貝數(shù)分別為3、2、4、2、2、4和2個(gè)。7、半定量PCR結(jié)果表明,過量表達(dá)StGA2ox1基因能不同程度的增加植株對(duì)非生物脅迫的抗逆性,在干旱脅迫下,StGA2ox1轉(zhuǎn)基因株系較對(duì)照材料具有相對(duì)高的葉綠素含量、游離脯氨酸含量、相對(duì)葉片含水量。過量表達(dá)StGA20ox1基因則對(duì)馬鈴薯應(yīng)對(duì)非生物脅迫影響不大。
[Abstract]:Gibberellin (GA), as an endogenous hormone in plants, plays an important role in regulating the whole life cycle of higher plants. GA-20 oxidase and GA-2 oxidase are key enzymes in the third stage of gibberellin biosynthesis. They are encoded by multiple gene families and participate in the regulation of gibberellin level. GA-20 oxidase can promote the synthesis of gibberellin. It belongs to the positive regulation. GA-2 oxidase will transform the active gibberellin into the inactive gibberellin, which is a negative regulation. Therefore, the expression level of GA20ox gene and GA2ox gene will affect the synthesis of active GA in the plant and ultimately affect the growth and development of the plant. The plant height mutant of potato plants as materials, the method of exogenous gene copy number, and determination of plant gibberellin synthesis key enzyme gene expression in different tissues, differential expression analysis, the relationship between the expression of key enzymes and plant gibberellin biosynthetic genes clear height between. GA20ox1 and GA2ox1 genes were cloned from potato 9, and their sequence characteristics were analyzed. The effect of over expressed target genes on potato plants was analyzed by Agrobacterium mediated transformation. The possible mechanism of the two gene was elucidated at the molecular level. The main results of this study are as follows: 1, using real-time fluorescence quantitative PCR method to estimate the exogenous transgenic potato T-DNA gene insert copy number, in the 23 transgenic strains, 12 strains were single copy insertion, 6 were double copies, 5 were multiple copies, and through the analysis on potato agronomic the characters of the field. A method for rapid detection of the copy number of foreign genes was established. 2, using the Real-time PCR method, the expression of key enzymes in GA biosynthesis gene in potato mutants in M4P9 content were detected and analyzed, results showed that the growth of potato in different periods, and each period of roots, stems and leaves, the expression of each gene are changed, CPS1 gene in seedling stage overexpression for plant growth provides plenty of endogenous hormones, GA20ox1 gene changes in the stem, mainly for over expression in the expansion period of the stem, may be related to tuber starch accumulation, the formation of GA2ox1 gene overexpression in the stem tuber, inhibit the synthesis of endogenous GA, is considered is one of the driving factors of M4P9 small plants. Exogenous GA3 can inhibit the synthesis of GA in the plant, which is mainly manifested in the decrease of the gene expression of gibberellin related enzymes in the potato plants. 3, the StGA2ox1 gene in the synthesis pathway of gibberellin was cloned, and the total length of CDS was 1005bp, and 334 amino acids were encoded. According to the prediction of the two level structure of the encoded protein, the results showed that the alpha helix was 34.43%, the irregular coil was 34.13%, the extended main chain was 22.16%, the beta corner was 9.28%, and it was a stable protein. Homology comparison results showed that the amino acid sequence encoded by potato GA2ox1 gene was highly homologous with tomato GA2ox2 gene, tobacco GA2ox1, GA2ox2 and GA2ox3 gene, but the lowest homology with tomato GA2ox1 gene. Compared with the GA2ox1 (LOC102605134) gene sequence on NCBI, the encoded amino acid corresponding to the GA2ox1 gene sequence in green potato No. 9 also changed a certain amount. 4, cloning the StGA20ox1 gene of potato gibberellin biosynthesis pathway, sequencing of CDS is 1137bp, encoding 378 amino acids, and the StGA20ox1 gene encoding protein was predicted, the two stage structure showed that the protein in alpha helix 39.68%, random coil 33.33%, extension 19.58%, p-turns backbone 7.41%, stable protein. Homology comparison showed that StGA20ox1 had high homology with tomato GA20ox1 gene, Ou Baiying GA20ox1 gene and tobacco GA20ox gene, but not homologous with 3 kinds of GA20ox genes of morning glory. NCBI and GA20ox1 (AJ291453.1) gene sequences compared to sequences of GA20ox1 gene in 9 Qingshu changed in many places (29 in total), the StGA20ox1 gene encoding GA20ox1 gene encoding amino acid amino acid sequencing to Qingshu 9 in less than published, 3 basic amino acids, less the 4 hydrophobic amino acids, more than 4 polar amino acids. 5. A new plasmid vector pCAEZ1383 was constructed and successfully constructed. A plant expression vector, 1383-GA20ox1 and 1383-GA2ox1, used for genetic transformation of exogenous gene GA20ox1 and GA2ox1, was constructed. Through genetic transformation, 7 transgenic GA20ox1 positive seedlings of Min tuber 1 were obtained, and 1 transgenic positive seedlings of green potato No. 9 containing exogenous GA2ox1 gene were obtained. 6, fluorescence quantitative method was used to detect the expression level of StGA2ox1 gene and StGA20ox1 gene in transgenic positive seedlings. The results showed that the expression level of StGA2ox1 gene and StGA20ox1 gene in transgenic plants increased to varying degrees. Through the observation of phenotypic traits, overexpression of StGA20ox1 gene could promote the growth of potato plants and the elongation of root. In the transgenic plants of Qingshu 9, the copy number of StGA2ox1 gene was 6. In StGA20ox1 1 transgenic lines, the copy number of StGA20ox1 gene was 3, 2, 4, 2, 2, 4 and 2, respectively. 7, semi quantitative PCR results showed that overexpression of StGA2ox1 can be increased in different degree in plant abiotic stress tolerance under drought stress, StGA2ox1 transgenic lines compared with control material has relatively high chlorophyll content, free proline content, relative leaf water content. The overexpression of StGA20ox1 gene had little effect on the response to abiotic stress in potato.
【學(xué)位授予單位】：青海大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S532;Q943.2

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