【摘要】：葡萄是世界重要的果樹資源,目前全世界栽培最廣泛的葡萄多屬于歐亞種群。這些栽培品種雖然品質(zhì)優(yōu)良,但是抗病性差,給葡萄生產(chǎn)帶來(lái)了困擾。中國(guó)作為葡萄起源地之一,有眾多在抗逆方面價(jià)值巨大的種質(zhì)資源,為葡萄分子育種提供了廣闊的基因資源。本研究從華東葡萄‘白河35-1’白粉菌誘導(dǎo)cDNA文庫(kù)中克隆得到VpPR10.1基因,構(gòu)建植物表達(dá)載體,以歐洲葡萄無(wú)核白胚性愈傷組織為受體進(jìn)行遺傳轉(zhuǎn)化。在葡萄遺傳轉(zhuǎn)化過(guò)程中,使用外源多胺以提高轉(zhuǎn)化效率,獲得轉(zhuǎn)基因無(wú)核白葡萄材料。主要取得以下結(jié)果:1.分別使用腐胺、精胺、亞精胺的不同濃度(10μM、100μM和1000μM)在遺傳轉(zhuǎn)化的不同時(shí)期(共培養(yǎng)階段、延遲篩選階段和篩選階段)對(duì)受體材料進(jìn)行處理。對(duì)于添加多胺處理的時(shí)期來(lái)說(shuō),在共培養(yǎng)階段添加效果優(yōu)于在延遲篩選階段添加,優(yōu)于篩選階段添加。對(duì)于不同種類濃度而言,腐胺1000μM和亞精胺100μM處理效果較好。研究結(jié)果表明:外源多胺優(yōu)化無(wú)核白體細(xì)胞胚遺傳轉(zhuǎn)化體系的最佳處理為:在共培養(yǎng)階段,培養(yǎng)基為1/2MS培養(yǎng)基+20g/L蔗糖+150μM乙酰丁香酮(AS)另添加1000μM腐胺或100μM亞精胺有助于提高抗性胚在篩選階段中的誘導(dǎo)率。誘導(dǎo)率分別達(dá)到了96.2%和88.8%,而對(duì)照組誘導(dǎo)率為44.4%。2.通過(guò)農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化方法得到華東葡萄基因VpPR10.1過(guò)表達(dá)無(wú)核白植株70個(gè),經(jīng)基因組PCR鑒定和蛋白表達(dá)水平鑒定出11個(gè)轉(zhuǎn)基因植株,陽(yáng)性率為15.7%。3.使用VpPR10.1過(guò)表達(dá)轉(zhuǎn)基因株系6904和野生型無(wú)核白離體葉片接種霜霉菌,苯胺藍(lán)染色結(jié)果顯示轉(zhuǎn)基因株系6904上霜霉菌生長(zhǎng)較野生型受到抑制,在接種后1-5天的觀察期內(nèi),接菌后2天菌絲上的吸器數(shù)量顯著減少;接種5天后統(tǒng)計(jì)單位面積產(chǎn)孢量,6904株系離體葉盤上的產(chǎn)孢量也顯著降低;DAB染色結(jié)果顯示,接種后76h、96h轉(zhuǎn)基因株系6904離體葉盤上細(xì)胞內(nèi)活性氧積累較野生型明顯,有明顯的超敏反應(yīng)。轉(zhuǎn)華東葡萄VpPR10.1基因能夠提高無(wú)核白葡萄對(duì)霜霉病的抗性。
[Abstract]:Grape is an important fruit resource in the world. At present, most of the most widely cultivated grapes in the world belong to Eurasian population. Although the quality of these cultivars is good, but the disease resistance is poor, which brings trouble to grape production. As one of the places of grape origin, China has many germplasm resources of great value in stress resistance, which provides broad genetic resources for grape molecular breeding. In this study, the VpPR10.1 gene was cloned from cDNA library of 'Baihe 35-1' Grape in East China, and the plant expression vector was constructed, and the non-white embryogenic callus of European grape was used as the receptor for genetic transformation. In the process of grape genetic transformation, exogenous polyamines were used to improve the efficiency of transformation and to obtain transgenic non-white grape materials. The main results are as follows: 1. The receptor materials were treated with putrescine, spermine and spermidine (10 渭 m 100 渭 M and 1000 渭 M) at different stages of genetic transformation (co-culture stage, delayed screening stage and screening stage) respectively. For the period of adding polyamines, the effect of adding polyamines in co-culture stage is better than that in delayed screening stage and in screening stage. For different kinds of concentration, putrescine 1000 渭 M and spermidine 100 渭 M had better effect. The results showed that the optimal treatment of exogenous polyamines to optimize the genetic transformation system of non-nuclear white somatic embryos was as follows: in co-culture stage, The addition of 1000 渭 M putrescine or 100 渭 M spermidine to the 1/2MS medium 20g/L sucrose 150 渭 M acetyl eugenone (AS) could increase the induction rate of resistant embryos in the screening stage. The induction rate was 96.2% and 88.8%, respectively, while that of the control group was 44.4% and 88.8%, respectively. 70 transgenic plants were obtained by Agrobacterium tumefaciens mediated transformation. Eleven transgenic plants were identified by genomic PCR identification and protein expression level, and the positive rate was 15.70.3. VpPR10.1 overexpression transgenic lines 6904 and wild type denuclearized leaves were inoculated in vitro. The results of aniline blue staining showed that the growth of the transgenic strain 6904 was more inhibited than that of wild type, and during the observation period of 1-5 days after inoculation. 2 days after inoculation, the number of hyphal absorbers was significantly reduced, and the spore production per unit area was counted after 5 days inoculation, and the spore production on leaves of isolated leaves of strain 6904 was also significantly decreased by DAB staining. The accumulation of reactive oxygen species (Ros) in the leaves of the transgenic strain 6904 at 76 h and 96 h after inoculation was significantly higher than that of the wild type and showed obvious hypersensitivity. Transgenic grape VpPR10.1 gene can improve the resistance to downy mildew in non-nuclear white grape.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S436.631

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 王辰;姚文孔;謝小青;童威霍;王磊;王杰;王躍進(jìn);;華東葡萄泛素連接酶基因VpUIRP2和VpUIRP3的抗白粉病特性分析[J];果樹學(xué)報(bào);2016年12期

2 劉長(zhǎng)命;楊瑞平;莫言玲;王永琦;鄭俊澅;張顯;;外源Spd預(yù)處理對(duì)甜瓜白粉病抗性及其內(nèi)源多胺的誘導(dǎo)分析[J];西北植物學(xué)報(bào);2016年01期

3 郭印山;郭修武;張海娥;;葡萄胚胎發(fā)育與敗育過(guò)程中胚珠的多胺含量變化[J];植物生理學(xué)通訊;2007年01期

4 段輝國(guó);黃作喜;林宏輝;;多胺在高等植物個(gè)體發(fā)育中的作用[J];西北農(nóng)業(yè)學(xué)報(bào);2006年02期

5 張文娥,王飛,潘學(xué)軍;葡萄屬植物(Vitis L.)再生系統(tǒng)的研究進(jìn)展[J];西北農(nóng)林科技大學(xué)學(xué)報(bào)(自然科學(xué)版);2003年S1期

相關(guān)博士學(xué)位論文 前3條

1 代玲敏;葡萄遺傳轉(zhuǎn)化體系與轉(zhuǎn)抗病相關(guān)基因研究[D];西北農(nóng)林科技大學(xué);2015年

2 周起;無(wú)核白葡萄體細(xì)胞胚再生體系建立與轉(zhuǎn)基因研究[D];西北農(nóng)林科技大學(xué);2014年

3 李桂榮;無(wú)核葡萄胚胎發(fā)育的生理特性和胚挽救育種技術(shù)的研究[D];西北農(nóng)林科技大學(xué);2013年

相關(guān)碩士學(xué)位論文 前1條

1 樊超紅;中國(guó)野生葡萄芪合酶基因轉(zhuǎn)化無(wú)核葡萄品種的研究[D];西北農(nóng)林科技大學(xué);2006年

，

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