發(fā)布時(shí)間：2018-06-24 06:10

  本文選題：葡萄 + 無核��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：葡萄(Vitis vinifera L.)是一種重要的經(jīng)濟(jì)果樹,在國內(nèi)外栽培面積很廣。由于無核葡萄在鮮食水果中具有食用方便,口味極佳,營養(yǎng)豐富等優(yōu)點(diǎn),所以深受消費(fèi)者青睞,在鮮食、制干方面具有很廣闊的市場。近年來無核葡萄在世界鮮食葡萄中消費(fèi)量有逐年上升的趨勢,世界各葡萄生產(chǎn)大國對無核葡萄的研究與開發(fā)也逐漸加強(qiáng),因此開展葡萄無核基因發(fā)掘及其功能研究具有重要的意義。本研究正是依據(jù)課題組前期通過有核和無核葡萄雜交后代進(jìn)行的葡萄轉(zhuǎn)錄組分析,篩選出27個(gè)表達(dá)差異比較明顯的基因,驗(yàn)證了這27個(gè)候選基因在胚珠敗育前后三個(gè)關(guān)鍵時(shí)期的表達(dá)情況,并在無核葡萄‘無核白’和有核葡萄‘紅地球’中克隆了5個(gè)表達(dá)差異比較大的基因,分析了這5個(gè)基因DNA序列的相似性,同時(shí)構(gòu)建了VvMADS46基因的亞細(xì)胞定位載體和過表達(dá)載體,分別轉(zhuǎn)化洋蔥和番茄,并對轉(zhuǎn)基因番茄進(jìn)行功能研究,初步分析了VvMADS46的基因功能。獲得了主要以下結(jié)果:1.采用半定量PCR技術(shù)分析了27個(gè)無核候選基因在多種有核和無核葡萄材料胚珠發(fā)育不同時(shí)期的表達(dá)情況,其中20個(gè)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)在無核葡萄材料胚珠中表達(dá)量較高,在有核葡萄材料胚珠中表達(dá)量低,而另外7個(gè)基因(NAC86、DDM、C2H2-1、C2H2-2、NAC41、GH3-9、GSLG)則在有核葡萄材料胚珠中表達(dá)量相對較高,這些基因的差異表達(dá)反應(yīng)了它們與葡萄無核調(diào)控可能存在一定關(guān)系。2.在無核葡萄‘無核白’和有核葡萄‘紅地球’中克隆了5個(gè)在無核葡萄及有核葡萄材料胚珠中表達(dá)差異比較大的基因MYB1、MYB2、NAC26、NAC41、HB1,基因全長大小分別為1686bp、1090bp、1054bp、1055bp和1657bp,測序結(jié)果顯示在‘紅地球’和‘無核白’中這些基因序列無明顯差異。3.分析了VvMADS46基因在‘無核白’和‘紅地球’的不同組織器官的表達(dá)情況,結(jié)果表明,VvMADS46在根、莖、葉、花序、花、卷須、果實(shí)等不同組織中均有表達(dá),在花和花序中的表達(dá)量較高,在果實(shí)中的表達(dá)量次之,而在莖、葉和卷須中的表達(dá)量較低;同時(shí)在‘無核白’和‘紅地球’兩種不同材料中克隆了VvMADS46基因cDNA序列,比較了其序列差異,VvMADS46開放閱讀框長度為618bp,編碼206個(gè)氨基酸;并構(gòu)建了VvMADS46基因的過量表達(dá)載體及亞細(xì)胞定位載體,分別侵染轉(zhuǎn)化番茄子葉和洋蔥表皮細(xì)胞,后期觀察發(fā)現(xiàn),過量表達(dá)VvMADS46基因的番茄果實(shí)及番茄種子都明顯變小,表明VvMADS46與無核葡萄種子生長發(fā)育調(diào)控可能存在一定關(guān)系,熒光定位觀察結(jié)果顯示VvMADS46定位在植物細(xì)胞核上。另外在‘無核白’和‘紅地球’兩種不同材料中分別克隆了VvMADS46基因的啟動(dòng)子,分析了該基因的元件調(diào)控,序列比對發(fā)現(xiàn)在‘無核白’啟動(dòng)子中缺少了一個(gè)與胚乳發(fā)育相關(guān)的調(diào)控元件,該元件可能與VvMADS46參與的無核基因調(diào)控相關(guān)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S663.1
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本文編號：2060358