山葡萄高效遗传转化愈伤体系的建立

发布时间：2018-12-19 09:53

【摘要】：遗传转化系统是转基因改良作物性状和开展基因功能研究的重要手段。葡萄作为一种重要的园艺作物,其已有的转化系统存在耗时长、效率低等诸多问题,极大的限制了品质和抗性相关基因的功能研究。山葡萄(Vitis amurensis)具有较强的抗寒旱能力,其抗性相关基因功能的研究及信号通路的诠释,将为葡萄抗逆品种的选育提供理论基础。本研究以山葡萄组培苗为材料,分别从外植体的类型、抗生素浓度、农杆菌菌株及菌液浓度、侵染时间和共培养时间几个方面建立优化葡萄的遗传转化体系。利用建立的遗传转化体系,将本实验室前期获得的抗寒相关转录因子VaERF057基因转入山葡萄中,成功获得过表达VaERF057基因的阳性愈伤。本研究为葡萄尤其是山葡萄中品质和抗性相关基因的功能研究提供了一种高效、快速的方法。取得的主要结果如下:1、选用山葡萄叶柄、茎段和叶盘为外植体,比较了其形成愈伤组织的能力,发现与茎段和叶盘相比,叶柄形成的愈伤组织结构疏松,生长迅速,更适合进行转化试验。因此,选用叶柄作为外植体开展后续转化研究。2、以山葡萄叶柄为外植体,研究了不同浓度的潮霉素(hygromycin,Hyg)和卡那霉素(Kanamycin,Kan)对愈伤组织形成的影响。结果表明,仅4 mg/LHyg即可完全抑制叶柄愈伤分化,叶柄褐化死亡;添加Kan时,愈伤分化随Kan浓度增加而降低,20 mg/L Kan即可完全抑制非转化愈伤的分化,而叶柄未出现褐化坏死现象,因此选用20 mg/L的Kan进行转化过程中阳性愈伤的筛选。3、以pSAK277-eGFP为转化载体,以其含有的NPTII基因(新霉素磷酸转移酶基因)和eGFP基因(绿色荧光蛋白基因)作为双重筛选标记,比较研究了农杆菌菌株、菌液浓度、侵染时间和共培养时间对转化效率的影响,建立了高效快速的山葡萄叶柄转化体系。转化的最适条件为:农杆菌EHA105(OD600=0.5),侵染8min,共培养2天,在含20 mg/L Kan的培养基上筛选6-8周。一次转化100个叶柄段可得到超过20个转化愈伤。4、利用建立的转化体系对山葡萄抗寒相关基因VaERF057进行遗传转化,并利用荧光定量PCR对得到的愈伤进行了验证,.结果表明,抗性筛选的愈伤中VaERF057基因均上调表达。转VaERF057阳性愈伤的获得为进一步研究其基因的功能提供了重要材料。
[Abstract]:Genetic transformation system is an important means to improve the traits of transgenic crops and to study gene function. As an important horticultural crop, the existing transformation system of grape has many problems, such as long time consuming, low efficiency and so on, which greatly limits the research on the function of quality and resistance-related genes. (Vitis amurensis) has a strong ability to resist cold and drought. The study of gene function related to resistance and the interpretation of signal pathway will provide a theoretical basis for the breeding of grape varieties of resistance to stress. In this study, the optimal genetic transformation system of grape was established from explant type, antibiotic concentration, Agrobacterium tumefaciens strain and bacterial solution concentration, infection time and co-culture time. Using the established genetic transformation system, the cold-related transcription factor VaERF057 gene obtained in our laboratory was transferred into Vitis vinifera, and the positive callus expressing VaERF057 gene was successfully obtained. This study provides an efficient and rapid method for the functional study of quality and resistance-related genes in grape, especially in mountain grape. The main results are as follows: 1. The callus formation ability of petiole, stem segment and leaf disc of Vitis vinifera was compared with that of stem segment and leaf disk. The callus formed by petiole was looser and grew rapidly than that of stem segment and leaf disk. It is more suitable for transformation test. Therefore, the petiole was selected as explant to carry out subsequent transformation study. 2. The effects of hygromycin (hygromycin,Hyg) and kanamycin (Kanamycin,Kan) on callus formation were studied with petiole of Vitis vinifera as explant. The results showed that callus differentiation of petiole was inhibited completely and petiole browning died only for 4 mg/LHyg. With the addition of Kan, the callus differentiation decreased with the increase of Kan concentration, and the differentiation of non-transformed callus was completely inhibited at 20 mg/L Kan, but the petiole did not show browning and necrosis. Therefore, 20 mg/L Kan was selected to screen the positive callus in the transformation process. 3. PSAK277-eGFP was used as the transformation vector. The NPTII gene (neomycin phosphotransferase gene) and the eGFP gene (green fluorescent protein gene) were used as double screening markers to compare the concentration of Agrobacterium tumefaciens. The effect of infection time and co-culture time on the transformation efficiency was studied and an efficient and rapid transformation system of petiole of Vitis vinifera was established. The optimum conditions for transformation were as follows: Agrobacterium EHA105 (OD600=0.5), infected for 8 min, co-cultured for 2 days, and screened on the medium containing 20 mg/L Kan for 6-8 weeks. More than 20 transformed calli could be obtained from 100 petiole segments in a single transformation. 4. Genetic transformation of cold-resistant genes VaERF057 was carried out by using the established transformation system, and the calli were verified by fluorescence quantitative PCR. The results showed that the expression of VaERF057 gene was up-regulated in the resistant callus. The acquisition of transgenic VaERF057 positive callus provides important materials for further study of its gene function.
【学位授予单位】：中国科学院武汉植物园
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S663.1

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