中国野生华东葡萄VpCN基因转化欧洲葡萄无核白的研究

发布时间：2018-04-20 04:11

本文选题：葡萄 + 体细胞胚发生途径　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：葡萄(Vitis spp.)是世界上重要的经济水果,尤其是欧洲葡萄(Vitis vinifera L.)品质好、风味纯正、产量高,在世界葡萄栽培中占据着重要位置。但是,由真菌病害引起的白粉病给欧洲葡萄的生产和销售带来了巨大的经济损失,利用转基因技术对欧洲葡萄进行定向遗传改良是解决这一问题的有效途径。中国野生华东葡萄白河-35-1对白粉病具有很强的抗性,挖掘中国野生葡萄种质资源相关抗病基因和研究其功能对葡萄抗病育种具有重要的意义。课题组前期克隆到中国野生华东葡萄白河-35-1抗白粉病相关基因VpCN,转化拟南芥表现出对白粉病抗性。本研究为进一步验证该基因功能,建立5种葡萄体细胞胚胎再生途径,并以葡萄原胚团为受体材料,与农杆菌共培养,将VpCN基因转入感白粉病的欧洲葡萄品种无核白,为改良欧洲葡萄品种抗病性提供理论依据。具体内容及取得的成果如下:1、诱导得到红地球、无核白、赤霞珠、商-24、塘尾等5种葡萄的胚性愈伤组织以及体细胞胚。以红地球、无核白、赤霞珠、商-24、塘尾的花药、子房以及小花蕾为外植体,在MC、MS1、DM培养基上诱导,得到胚性愈伤组织。无核白花药在MC培养基(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琼脂)上诱导效率最高,为24.00%。X6培养基(MS+60 g/L蔗糖+7 g/L TC琼脂)比NB培养基更适合体胚的诱导。2、通过体细胞胚途径获得了无核白葡萄的再生株系,DM培养基(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琼脂)更适合用于次生胚性愈伤的诱导。3、采用农杆菌介导法,将携带VpCN基因的植物表达载体转入欧洲葡萄无核白葡萄的原胚团中,得到无核白葡萄的抗性胚。而且,原胚团比体细胞胚更适用于无核白葡萄遗传转化的受体材料。4、构建VpCN的亚细胞定位载体p BI221-VpCN-GFP,通过农杆菌侵染将融合载体导入洋葱表皮细胞,荧光显微镜观察发现VpCN表达的蛋白定位于细胞核内。
[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.
【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

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