【作者】 刘爽；

【导师】 姜晶；

【作者基本信息】 沈阳农业大学 ， 蔬菜学， 2020， 硕士

【摘要】 植物通过转化酶将蔗糖分解为葡萄糖和果糖,来为植物提供生长发育的能源物质,以及作为与外界环境反应的信号因子,以此来调控植物的生长发育周期。转化酶分为酸性转化酶和碱性/中性转化酶(alkaline/neutral invertase,cytoplasmic invertases,CINs)。目前对于酸性转化酶的功能及其作用机制已有深入地研究,但CINs由于该家族蛋白的非糖基化特征造成其不稳定性,对其功能知之甚少。近来研究表明其在拟南芥、水稻等模式植物的器官发育和产品品质形成具有重要作用。但在以果实作为产品器官的园艺作物-番茄中的功能尚不清楚。本研究以Micro-Tom番茄为试材,对果实中表达量较高的SlCIN7基因进行转化酶活性、组织细胞定位及基因的沉默与过表达研究,对其功能进行了初步探究。主要研究结果如下:1)番茄SlCIN7开放阅读框长1996bp,它编码一个等电点为5.84,具有655个氨基酸残基的蛋白质,其分子量是73.51kDa。通过构建系统进化树发现SlCIN7被分类为α亚族,亚细胞定位预测表明其定位于线粒体中。2)构建pET30-SlCIN7原核表达载体,在大肠杆菌中诱导表达,提取总蛋白进行体外酶活性分析,结果表明SlCIN7具有转化酶活性,可以在体外催化蔗糖分解,并且其最适酶活pH值为7.0。3)通过PCR扩增获得SlCIN7基因的启动子全长,利用Gateway技术构建了含SlCIN7启动子的GUS表达载体,转化农杆菌GV3101后,在番茄果实中进行了瞬时表达,结果表明SlCIN7主要在果肉、维管束和胶质胎座中有表达活性。进一步构建了SlCIN7-GFP融合的植物表达载体,在烟草叶肉细胞中进行了瞬时表达,结果表明SlCIN7蛋白定位于线粒体中。4)构建SlCIN7的RNA干扰载体及过表达载体,利用农杆菌介导转化番茄Micro-Tom,经选择标记的PCR鉴定及目的基因的表达分析,获得过表达株系50株,沉默植株40株。5)过表达SlCIN7基因的转基因株系表型与未转基因植株无明显变化。但沉默SlCIN7基因后,转基因番茄果实中没有种子。进一步对其花粉生活力检测结果发现花粉活力明显降低,活性氧染色结果表明转基因花粉粒中活性氧含量明显升高。以上结果表明SlCIN7基因具有转化酶活性,在番茄中沉默该基因影响果实发育并导致单性结实。这为进一步研究SlCIN7基因在番茄果实成熟过程中的功能奠定了基础。更多还原

【Abstract】 Plants use invertase to decompose sucrose into glucose and fructose to provide plants with energy materials for growth and development,as well as signal factors that respond to the external environment to regulate plant growth and development cycles.Invertases are divided into acid invertases and alkaline / neutral invertases,cytoplasmic invertases(CINs).At present,the function of acid invertase and its mechanism of action have been intensively studied,but CINs are not stable due to the non-glycosylation characteristics of this family of proteins,and little is known about their functions.Recent studies have shown that it plays an important role in organ development and product quality formation of idopsis,rice and other model plants.However,its function in tomato,a hortiural crop with fruits as product organs,is unclear.In this study,Micro-Tom tomato was used as a test material to study the invertase activity,tissue cell localization,gene silencing and overexpression of SlCIN7 gene with high expression in fruits,and its function was preliminary explored.The main findings are as follows:1)Bioinformatics analysis of SlCIN7,we found that the open reading frame is 1996 bp long,it encodes a protein with an isoelectric point of 5.84,655 amino acid residues,and a molecular weight of 73.51 k Da.By constructing a phylogenetic tree,SlCIN7 was classified as an alpha subfamily.Subcellular localization prediction showed that it was localized in mitochondria.2)The pET-SlCIN7 prokaryotic expression vector was constructed and induced to express in E.coli,and the purified protein SlCIN7 was obtained.In vitro enzyme activity analysis showed that SlCIN7 has invertase activity and can catalyze the decomposition of sucrose in vitro,and its optimum enzyme activity p H is 7.0.Sucrose is the only catalytic substrate of SlCIN7.3)The full length of the promoter of the SlCIN7 gene was obtained by amplification.A GUS expression vector containing the SlCIN7 promoter was constructed using the Gateway technology.After transforming Agrobacterium GV3101,it was transiently expressed in tomato fruits.Expressive activity in tube bundles and glia.A SlCIN7-GFP fused plant expression vector was further constructed and transiently expressed in tobacco mesophyll cells.The results showed that the SlCIN7 protein was localized in mitochondria.4)Constructing the RNA interference vector and overexpression vector of SlCIN7,using Agrobacterium-mediated transformation of tomato Micro-Tom,through PCR identification of selectable markers and expression analysis of target genes,50 overexpression lines and 40 silent plants were obtained.5)There was no significant change in the phenotype of transgenic lines overexpressing the SlCIN7 gene compared with non-transgenic plants.However,after silencing the SlCIN7 gene,there was almost no seed in the transgenic tomato fruit.Furthermore,the pollen viability test results showed that the vitality of pollen was significantly reduced,and the reactive oxygen species staining results showed that the content of reactive oxygen species in transgenic pollen grain 9 was significantly increased.In this study,we have preliminarily regulated the expression of SlCIN7 gene in tomato,laying a foundation for further research on the function of SlCIN7 gene in tomato fruit maturation.更多还原