马铃薯赤霉素合成代谢途径关键酶基因GA2ox1、GA20ox1的克隆与功能分析

发布时间：2017-12-27 15:31

本文关键词：马铃薯赤霉素合成代谢途径关键酶基因GA2ox1、GA20ox1的克隆与功能分析　出处：《青海大学》2016年博士论文　论文类型：学位论文

【摘要】：赤霉素(GA)作为植物体内一种内源激素,在高等植物生长的整个生命周期,起着重要的调控作用。GA-20氧化酶和GA-2氧化酶是赤霉素合成途径中第三阶段的关键酶,它们均由多基因家族编码,参与着赤霉素水平的调控。GA-20氧化酶能促进活性赤霉素的合成,属于正向调控;而GA-2氧化酶则会将活性赤霉素转化为非活性的赤霉素,属负向调控。所以GA20ox基因和GA2ox基因的表达水平,将会影响到植物体内活性GA的合成,最终影响植株的生长发育。本研究首先以株高突变马铃薯植株为材料,建立了外源基因拷贝数的检测方法,并测定植株体内的赤霉素合成关键酶基因在不同时期不同组织内的表达量,分析表达差异,以明确赤霉素合成途径关键酶基因的表达水平与植株株高间的关系。以马铃薯青薯9号为材料,克隆了GA20ox1基因和GA2ox1基因,分析了序列特征,并通过农杆菌介导的遗传转化分析了过量表达目的基因对马铃薯植株的影响。从分子水平阐明了两基因的可能作用机理。本研究主要结果如下:1、利用实时荧光定量PCR方法估算出了转基因马铃薯中外源基因T-DNA的插入拷贝数,在所测的23株转基因株系中,12株为单拷贝插入,6株为双拷贝,5株为多拷贝,并通过对田间马铃薯农艺性状的分析。建立了快速检测外源基因拷贝数的方法。2、利用Real-time PCR方法,对赤霉素合成途径关键酶基因在马铃薯突变体材料M4P9中的表达量进行了检测分析,结果显示,在马铃薯生长的不同时期,以及各时期内的根、茎、叶中,各基因的表达量均发生了一定变化,CPS1基因在幼苗期的过量表达为植株的生长提供了充足的内源激素,GA20ox1基因在茎中变化明显,主要表现为在膨大期茎中的过量表达,可能与块茎淀粉累积有关,GA2ox1基因在块茎形成期茎中的过量表达,抑制了内源GA的合成,被认为是促使M4P9植株矮小的因素之一。外源喷施活性GA3能抑制植物体内自身活性GA的合成,主要表现为马铃薯植株体内赤霉素相关酶基因表达量的下降。3、克隆了马铃薯赤霉素合成途径中的StGA2ox1基因,测序得CDS全长1005bp,编码334个氨基酸。经对其编码的蛋白质二级结构预测,结果显示,α-螺旋为34.43%、无规则卷曲为34.13%、延伸主链为22.16%、β-转角为9.28%,为稳定的蛋白质。同源性比对结果显示,克隆得到的马铃薯GA2ox1基因编码的氨基酸序列与番茄GA2ox2基因、烟草GA2ox1、GA2ox2、GA2ox3基因的同源性较高,而与番茄GA2ox1基因的同源性最低。与NCBI上的GA2ox1(LOC102605134)基因序列相比,青薯9号中GA2ox1基因序列对应的其编码氨基酸也发生了一定的改变。4、克隆了马铃薯赤霉素合成途径中的StGA20ox1基因,测序得CDS全长1137bp,编码378个氨基酸,并对StGA20ox1基因编码的蛋白质进行了二级结构预测,结果显示,该蛋白质中α-螺旋为39.68%、无规则卷曲为33.33%、延伸主链为19.58%、β-转角为7.41%,为稳定的蛋白质。同源性比对结果显示,StGA20ox1与番茄GA20ox1基因、欧白英GA20ox1基因、烟草GA20ox基因的同源性较高,而与牵牛花的3类GA20ox基因的同源性都不高。与NCBI上的GA20ox1(AJ291453.1)基因序列相比,青薯9号中GA20ox1基因序列在多处发生了改变(共29处),经测序得青薯9号中的GA20ox1基因编码氨基酸较已公布的StGA20ox1基因编码的氨基酸,少了3个强碱性氨基酸,少了4个疏水氨基酸,多了4个极性氨基酸。5、改造并成功构建了一个新的质粒载体pCAEZ1383。构建了用于外源基因GA20ox1和GA2ox1遗传转化用的植物表达载体1383-GA20ox1和1383-GA2ox1。经遗传转化,获得含外源GA20ox1基因的闽薯1号转基因阳性苗7株;获得含外源GA2ox1基因的青薯9号转基因阳性苗1株。6、利用荧光定量方法,对获得的转基因阳性苗中,StGA2ox1基因和StGA20ox1基因的表达量进行了检测,结果表明,转基因植株中StGA2ox1基因和StGA20ox1基因的表达量均有不同程度的上调。经表型性状观察,过量表达达StGA20ox1基因可促进马铃薯植株株高生长和根的伸长。在青薯9号转基因植株中,StGA2ox1基因拷贝数为6;在闽薯1号转基因株系中,StGA20ox1基因拷贝数分别为3、2、4、2、2、4和2个。7、半定量PCR结果表明,过量表达StGA2ox1基因能不同程度的增加植株对非生物胁迫的抗逆性,在干旱胁迫下,StGA2ox1转基因株系较对照材料具有相对高的叶绿素含量、游离脯氨酸含量、相对叶片含水量。过量表达StGA20ox1基因则对马铃薯应对非生物胁迫影响不大。
[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.
【学位授予单位】：青海大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S532;Q943.2

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