【摘要】：遺傳轉(zhuǎn)化系統(tǒng)是轉(zhuǎn)基因改良作物性狀和開展基因功能研究的重要手段。葡萄作為一種重要的園藝作物,其已有的轉(zhuǎn)化系統(tǒng)存在耗時長、效率低等諸多問題,極大的限制了品質(zhì)和抗性相關(guān)基因的功能研究。山葡萄(Vitis amurensis)具有較強的抗寒旱能力,其抗性相關(guān)基因功能的研究及信號通路的詮釋,將為葡萄抗逆品種的選育提供理論基礎(chǔ)。本研究以山葡萄組培苗為材料,分別從外植體的類型、抗生素濃度、農(nóng)桿菌菌株及菌液濃度、侵染時間和共培養(yǎng)時間幾個方面建立優(yōu)化葡萄的遺傳轉(zhuǎn)化體系。利用建立的遺傳轉(zhuǎn)化體系,將本實驗室前期獲得的抗寒相關(guān)轉(zhuǎn)錄因子VaERF057基因轉(zhuǎn)入山葡萄中,成功獲得過表達VaERF057基因的陽性愈傷。本研究為葡萄尤其是山葡萄中品質(zhì)和抗性相關(guān)基因的功能研究提供了一種高效、快速的方法。取得的主要結(jié)果如下:1、選用山葡萄葉柄、莖段和葉盤為外植體,比較了其形成愈傷組織的能力,發(fā)現(xiàn)與莖段和葉盤相比,葉柄形成的愈傷組織結(jié)構(gòu)疏松,生長迅速,更適合進行轉(zhuǎn)化試驗。因此,選用葉柄作為外植體開展后續(xù)轉(zhuǎn)化研究。2、以山葡萄葉柄為外植體,研究了不同濃度的潮霉素(hygromycin,Hyg)和卡那霉素(Kanamycin,Kan)對愈傷組織形成的影響。結(jié)果表明,僅4 mg/LHyg即可完全抑制葉柄愈傷分化,葉柄褐化死亡;添加Kan時,愈傷分化隨Kan濃度增加而降低,20 mg/L Kan即可完全抑制非轉(zhuǎn)化愈傷的分化,而葉柄未出現(xiàn)褐化壞死現(xiàn)象,因此選用20 mg/L的Kan進行轉(zhuǎn)化過程中陽性愈傷的篩選。3、以pSAK277-eGFP為轉(zhuǎn)化載體,以其含有的NPTII基因(新霉素磷酸轉(zhuǎn)移酶基因)和eGFP基因(綠色熒光蛋白基因)作為雙重篩選標(biāo)記,比較研究了農(nóng)桿菌菌株、菌液濃度、侵染時間和共培養(yǎng)時間對轉(zhuǎn)化效率的影響,建立了高效快速的山葡萄葉柄轉(zhuǎn)化體系。轉(zhuǎn)化的最適條件為:農(nóng)桿菌EHA105(OD600=0.5),侵染8min,共培養(yǎng)2天,在含20 mg/L Kan的培養(yǎng)基上篩選6-8周。一次轉(zhuǎn)化100個葉柄段可得到超過20個轉(zhuǎn)化愈傷。4、利用建立的轉(zhuǎn)化體系對山葡萄抗寒相關(guān)基因VaERF057進行遺傳轉(zhuǎn)化,并利用熒光定量PCR對得到的愈傷進行了驗證,.結(jié)果表明,抗性篩選的愈傷中VaERF057基因均上調(diào)表達。轉(zhuǎn)VaERF057陽性愈傷的獲得為進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.
【學(xué)位授予單位】：中國科學(xué)院武漢植物園
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S663.1

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