葡萄无核相关基因表达、克隆及VvMADS46基因功能分析

发布时间：2018-06-24 06:10

  本文选题：葡萄 + 无核　； 参考：《西北农林科技大学》2017年硕士论文

【摘要】：葡萄(Vitis vinifera L.)是一种重要的经济果树,在国内外栽培面积很广。由于无核葡萄在鲜食水果中具有食用方便,口味极佳,营养丰富等优点,所以深受消费者青睐,在鲜食、制干方面具有很广阔的市场。近年来无核葡萄在世界鲜食葡萄中消费量有逐年上升的趋势,世界各葡萄生产大国对无核葡萄的研究与开发也逐渐加强,因此开展葡萄无核基因发掘及其功能研究具有重要的意义。本研究正是依据课题组前期通过有核和无核葡萄杂交后代进行的葡萄转录组分析,筛选出27个表达差异比较明显的基因,验证了这27个候选基因在胚珠败育前后三个关键时期的表达情况,并在无核葡萄‘无核白’和有核葡萄‘红地球’中克隆了5个表达差异比较大的基因,分析了这5个基因DNA序列的相似性,同时构建了VvMADS46基因的亚细胞定位载体和过表达载体,分别转化洋葱和番茄,并对转基因番茄进行功能研究,初步分析了VvMADS46的基因功能。获得了主要以下结果:1.采用半定量PCR技术分析了27个无核候选基因在多种有核和无核葡萄材料胚珠发育不同时期的表达情况,其中20个基因(HB1、GRAS、AP2-2、HB2、MYB1、MYB2、NAC26、GPDC8、SCD1、MADS46、TLP2、SCD2、KAN1、P450-1、NAC2、Knlike3、VVSTM、KAN1-2、GH3-1、TLP1)在无核葡萄材料胚珠中表达量较高,在有核葡萄材料胚珠中表达量低,而另外7个基因(NAC86、DDM、C2H2-1、C2H2-2、NAC41、GH3-9、GSLG)则在有核葡萄材料胚珠中表达量相对较高,这些基因的差异表达反应了它们与葡萄无核调控可能存在一定关系。2.在无核葡萄‘无核白’和有核葡萄‘红地球’中克隆了5个在无核葡萄及有核葡萄材料胚珠中表达差异比较大的基因MYB1、MYB2、NAC26、NAC41、HB1,基因全长大小分别为1686bp、1090bp、1054bp、1055bp和1657bp,测序结果显示在‘红地球’和‘无核白’中这些基因序列无明显差异。3.分析了VvMADS46基因在‘无核白’和‘红地球’的不同组织器官的表达情况,结果表明,VvMADS46在根、茎、叶、花序、花、卷须、果实等不同组织中均有表达,在花和花序中的表达量较高,在果实中的表达量次之,而在茎、叶和卷须中的表达量较低;同时在‘无核白’和‘红地球’两种不同材料中克隆了VvMADS46基因cDNA序列,比较了其序列差异,VvMADS46开放阅读框长度为618bp,编码206个氨基酸;并构建了VvMADS46基因的过量表达载体及亚细胞定位载体,分别侵染转化番茄子叶和洋葱表皮细胞,后期观察发现,过量表达VvMADS46基因的番茄果实及番茄种子都明显变小,表明VvMADS46与无核葡萄种子生长发育调控可能存在一定关系,荧光定位观察结果显示VvMADS46定位在植物细胞核上。另外在‘无核白’和‘红地球’两种不同材料中分别克隆了VvMADS46基因的启动子,分析了该基因的元件调控,序列比对发现在‘无核白’启动子中缺少了一个与胚乳发育相关的调控元件,该元件可能与VvMADS46参与的无核基因调控相关。
[Abstract]:Vitis vinifera L. It is an important economic fruit tree, which is widely cultivated at home and abroad. Because seedless grape has the advantages of convenient consumption, excellent taste and rich nutrition in fresh fruit, it is favored by consumers and has a broad market in fresh eating and drying. In recent years, the consumption of seedless grapes in the world fresh grapevine has been increasing year by year, and the research and development of seedless grapes have been gradually strengthened in the world. Therefore, it is of great significance to explore the seedless genes and their functions of grape. According to the transcriptome analysis carried out by the progeny of seedless and seedless grape in the early stage of the study group, 27 genes with obvious difference in expression were screened out. The expression of 27 candidate genes at three key stages before and after ovule abortion was verified, and five genes were cloned in seedless grape 'non-nuclear white' and nucleated grape 'red earth'. The similarity of the DNA sequences of the five genes was analyzed. The subcellular localization vector and over-expression vector of VvMADS46 gene were constructed and transformed into onion and tomato respectively. The function of transgenic tomato was studied, and the gene function of VvMADS46 was preliminarily analyzed. The main results are as follows: 1. The expression of 27 seedless candidate genes in various seedless and seedless grape ovules at different stages of ovule development was analyzed by semi-quantitative PCR technique. Twenty of them (HB1GRASA AP2-2Hb2mB1MYB2mB2mB26GPDC8 / MADS46-TLP2SCD2KN P450-1a NAC2SCD2SCD2KN450-1 NAC2SCD2KN like 3VSTMGHKAN1-2KAN3-1TLP1) were found to be highly expressed in the ovules of seedless grape. However, the other 7 genes (NAC86DDMU C2H2-1C2H2-2 + NAC41GH3-9 GSLG) were relatively high in the ovule of the nucleated grape. The differential expression of these genes reflected that there might be some relationship between these genes and the seedless regulation of grape. Five genes, MYB1, MYB2, MYB2, NAC26, NAC41, HB1, were cloned from seedless grape 'non-nuclear white' and seedless grape 'red earth'. The full length of the gene was 1686 BP, 1090 BP, 1054 BP, 1055 BP, and 1657 BP, respectively. There is no significant difference in the sequence of these genes between Red Earth and denuclearized Earth. The expression of VvMADS46 gene in different tissues and organs of 'non-nuclear white' and 'red earth' was analyzed. The results showed that VvMADS46 was expressed in roots, stems, leaves, inflorescences, flowers, tendrils, fruits and other tissues. The VvMADS46 gene cDNA sequence was cloned in two different materials of 'non-nuclear white' and 'Red Earth', while the expression level was lower in stems, leaves and tendrils, and the VvMADS46 gene cDNA sequence was cloned in two different materials: 'non-nuclear white' and 'Red Earth'. The length of VvMADS46 open reading frame was 618 BP, encoding 206 amino acids, and the overexpression vector and subcellular localization vector of VvMADS46 gene were constructed and infected into tomato cotyledon and onion epidermis cells respectively. The fruits and seeds of tomato which overexpressed VvMADS46 gene were obviously smaller, indicating that VvMADS46 might be related to the regulation of seed growth and development of seedless grape, and the fluorescence localization observation showed that VvMADS46 was located on the plant nucleus. In addition, the promoter of the VvMADS46 gene was cloned in two different materials, 'non-nuclear white' and 'Red Earth', and the element regulation of the gene was analyzed. Sequence alignment revealed that there was a lack of a regulatory element related to endosperm development in the 'non-nuclear white' promoter, which might be related to the regulation of the nucleoless gene involved in VvMADS46.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S663.1
，

本文编号：2060358