發(fā)布時(shí)間：2018-04-20 04:11

  本文選題：葡萄 + 體細(xì)胞胚發(fā)生途徑　； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：葡萄(Vitis spp.)是世界上重要的經(jīng)濟(jì)水果,尤其是歐洲葡萄(Vitis vinifera L.)品質(zhì)好、風(fēng)味純正、產(chǎn)量高,在世界葡萄栽培中占據(jù)著重要位置。但是,由真菌病害引起的白粉病給歐洲葡萄的生產(chǎn)和銷(xiāo)售帶來(lái)了巨大的經(jīng)濟(jì)損失,利用轉(zhuǎn)基因技術(shù)對(duì)歐洲葡萄進(jìn)行定向遺傳改良是解決這一問(wèn)題的有效途徑。中國(guó)野生華東葡萄白河-35-1對(duì)白粉病具有很強(qiáng)的抗性,挖掘中國(guó)野生葡萄種質(zhì)資源相關(guān)抗病基因和研究其功能對(duì)葡萄抗病育種具有重要的意義。課題組前期克隆到中國(guó)野生華東葡萄白河-35-1抗白粉病相關(guān)基因VpCN,轉(zhuǎn)化擬南芥表現(xiàn)出對(duì)白粉病抗性。本研究為進(jìn)一步驗(yàn)證該基因功能,建立5種葡萄體細(xì)胞胚胎再生途徑,并以葡萄原胚團(tuán)為受體材料,與農(nóng)桿菌共培養(yǎng),將VpCN基因轉(zhuǎn)入感白粉病的歐洲葡萄品種無(wú)核白,為改良?xì)W洲葡萄品種抗病性提供理論依據(jù)。具體內(nèi)容及取得的成果如下:1、誘導(dǎo)得到紅地球、無(wú)核白、赤霞珠、商-24、塘尾等5種葡萄的胚性愈傷組織以及體細(xì)胞胚。以紅地球、無(wú)核白、赤霞珠、商-24、塘尾的花藥、子房以及小花蕾為外植體,在MC、MS1、DM培養(yǎng)基上誘導(dǎo),得到胚性愈傷組織。無(wú)核白花藥在MC培養(yǎng)基(NN69+30 g/L蔗糖+2.5 u M 2,4-D+2.5 u M NOA+5.0 u M 4-CPPU+0.1 g/L肌醇+7 g/L TC瓊脂)上誘導(dǎo)效率最高,為24.00%。X6培養(yǎng)基(MS+60 g/L蔗糖+7 g/L TC瓊脂)比NB培養(yǎng)基更適合體胚的誘導(dǎo)。2、通過(guò)體細(xì)胞胚途徑獲得了無(wú)核白葡萄的再生株系,DM培養(yǎng)基(DKW+30 g/L蔗糖+2.5 u M 2,4-D+5.0 u M 6-BA+2.5 u M NOA+1.0 g/L肌醇+7 g/L TC瓊脂)更適合用于次生胚性愈傷的誘導(dǎo)。3、采用農(nóng)桿菌介導(dǎo)法,將攜帶VpCN基因的植物表達(dá)載體轉(zhuǎn)入歐洲葡萄無(wú)核白葡萄的原胚團(tuán)中,得到無(wú)核白葡萄的抗性胚。而且,原胚團(tuán)比體細(xì)胞胚更適用于無(wú)核白葡萄遺傳轉(zhuǎn)化的受體材料。4、構(gòu)建VpCN的亞細(xì)胞定位載體p BI221-VpCN-GFP,通過(guò)農(nóng)桿菌侵染將融合載體導(dǎo)入洋蔥表皮細(xì)胞,熒光顯微鏡觀察發(fā)現(xiàn)VpCN表達(dá)的蛋白定位于細(xì)胞核內(nèi)。
[Abstract]:Vitis spp..) Is one of the most important economic fruits in the world, especially the European grape vitis vinifera L. Good quality, pure flavor, high yield, in the world grape cultivation occupies an important position. However, the powdery mildew caused by fungal diseases has brought huge economic losses to the production and sale of European grapes. It is an effective way to solve this problem by using transgenic technology to improve the orientation of European grapes. The wild grape Baihe-35-1 has strong resistance to powdery mildew in the wild in China. It is very important to excavate the disease-resistant genes related to Chinese wild grape germplasm resources and to study its function for grape breeding. VpCN-1 gene associated with powdery mildew resistance of wild grape Baihe-35-1 was cloned and transformed into Arabidopsis thaliana to show resistance to powdery mildew. In order to further verify the function of this gene, five ways of somatic embryo regeneration were established, and grape proembryo mass was co-cultured with Agrobacterium tumefaciens to transfer VpCN gene into powdery mildew susceptible European grape varieties. To provide theoretical basis for improving the disease resistance of European grape varieties. The specific contents and results obtained are as follows: 1. The embryogenic calli and somatic embryos of red earth, non-nuclear white, Cabernet sauvignon, quotient-24 and Tangwei grape were obtained. Embryogenic calli were obtained by induction of red earth, non-nuclear white, Cabernet sauvignon, quotient 24, anthers, ovary and small flower buds on MCS 1DM medium. NN69 30 g / L sucrose 2.5 u M 2N 4-D 2.5 u M NOA 5.0 u M 4-CPPU 0.1 g / L inositol 7 g / L TC Agar was the most efficient in induction of non-nuclear white anthers. 24.00%.X6 medium MS 60 g / L sucrose 7 g / L TC Agar was more suitable for somatic embryogenesis than NB medium. The regenerated lines of non-nuclear white grape were obtained by somatic embryogenesis. DKW 30 g / L sucrose 2.5 u M 24-D 5.0 渭 M 6-BA was obtained by somatic embryogenesis. 2.5 UM NOA 1.0 g / L inositol 7 g / L TC Agar was more suitable for induction of secondary embryogenic callus by Agrobacterium tumefaciens. The plant expression vector carrying VpCN gene was transferred into the proembryo of non-white grape of European grape, and the resistant embryo of non-white grape was obtained. Moreover, proembryo cluster is more suitable than somatic embryo to construct the subcellular localization vector of VpCN, pBI221-VpCN-GFP. the fusion vector was introduced into onion epidermal cells by Agrobacterium tumefaciens. Fluorescence microscopy showed that the protein expressed by VpCN was located in the nucleus.
【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(lèi)號(hào)】：S663.1

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