本文選題：薯塊 + 遺傳轉(zhuǎn)化�。� 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：自1983年首次獲得轉(zhuǎn)基因株系以來,農(nóng)桿菌介導(dǎo)的馬鈴薯遺傳轉(zhuǎn)化體系在不斷的改進(jìn)和完善,目前已成功建立不同基因型和外植體類型的轉(zhuǎn)化體系,但仍存在基因型依賴和轉(zhuǎn)化效率低等問題。遺傳轉(zhuǎn)化技術(shù)的發(fā)展與成熟為馬鈴薯晚疫病抗性機制的研究提供了新的契機。StNTP是調(diào)控馬鈴薯晚疫病抗性機制的NAC轉(zhuǎn)錄因子,前期研究發(fā)現(xiàn)在本式煙中沉默Nb NTP基因?qū)е峦硪卟≈虏×υ鰪?本研究利用優(yōu)化的轉(zhuǎn)化體系進(jìn)一步探究StNTP在馬鈴薯晚疫病抗性中的作用。選取馬鈴薯晚疫病感病材料304413.19、304413.89和Désirée及抗病材料301071.3、304413.40,通過觀察試管薯形成特點選取幼嫩試管薯為外植體,探索篩選壓、分化培養(yǎng)基組成對轉(zhuǎn)化效率的影響,建立相應(yīng)的轉(zhuǎn)化體系。利用優(yōu)化的體系獲得StNTP轉(zhuǎn)基因陽性株系,對StNTP基因進(jìn)行晚疫病抗性功能驗證。取得研究結(jié)果如下:1.對304413.19、304413.40和Désirée試管薯形成特點觀測,結(jié)果表明304413.19初始結(jié)薯時間比Désirée和304413.40提前一周,初步確定自接種起培養(yǎng)55d形成的304413.19試管薯和接種60d的304413.40和Désirée試管薯為外植體優(yōu)化遺傳轉(zhuǎn)化體系。2.基于組織培養(yǎng)條件下的試管薯形成表型,對301071.3和304413.89兩種基因型試管薯形成進(jìn)行兩種光周期處理和對301071.3基因型進(jìn)行蔗糖處理實驗,結(jié)果表明全黑暗誘導(dǎo)試管薯的提前形成,短日照能增加單株結(jié)薯數(shù)和單薯塊重。60g/L蔗糖濃度有利于301071.3基因型試管薯的形成。3.以304413.19、304413.40和Désirée為受體對卡那(Kan)濃度和分化培養(yǎng)基組成兩個因素進(jìn)行優(yōu)化。304413.19、304413.40和Désirée最適Kan濃度分別為50mg/L,50mg/L和60mg/L。三個基因型中只有Désirée成功獲得轉(zhuǎn)基因株系,最優(yōu)分化培養(yǎng)基組合為(MS+0.1mg/L IAA+2mg/L ZT+60mg/L Kan+400mg/L Cef),植株再生率和外植體轉(zhuǎn)化率分別為52.27%和11.36%。4.成功構(gòu)建StNTP超量表達(dá)載體,利用優(yōu)化的Désirée轉(zhuǎn)化體系轉(zhuǎn)入馬鈴薯受體材料Désirée中,獲得StNTP1轉(zhuǎn)基因株系5個,StNTP2轉(zhuǎn)基因株系3個,經(jīng)晚疫病抗性鑒定,病斑面積分析顯示,與對照相比超量株系晚疫病抗性顯著增強,說明StNTP增強馬鈴薯晚疫病抗性。
[Abstract]:Since the first transgenic lines were obtained in 1983, the Agrobacterium tumefaciens mediated transformation system of potato has been continuously improved and improved. At present, the transformation system of different genotypes and explants has been successfully established. However, there are still some problems such as genotype dependence and low efficiency of transformation. The development and maturity of genetic transformation technology provide a new opportunity for the study of resistance mechanism of potato late blight. StNTP is the NAC transcription factor to regulate the resistance mechanism of potato late blight disease. Previous studies showed that silencing NbNTP gene in tobacco could enhance the pathogenicity of late blight disease. This study further explored the role of StNTP in potato late blight resistance by using the optimized transformation system. Potato late blight susceptible materials 304413.89 and D 茅 sir 茅 e and disease-resistant material 301071.3A30444.13.40 were selected. The young tuber was selected as explant by observing the formation characteristics of test-tube tuber, and the effects of selection pressure and the composition of differentiation medium on the transformation efficiency were explored, and the corresponding transformation system was established. StNTP transgenic positive lines were obtained by using the optimized system, and the function of late blight resistance of StNTP gene was verified. The results are as follows: 1. The characteristics of tuber formation in vitro were observed. The results showed that the initial tuber setting time of 304413.19 was one week earlier than that of D 茅 sir 茅 e and D 茅 sir 茅 e. 304413.19 tubers formed from 55 days after inoculation and 304413.40 and D 茅 sir 茅 e from 60 days were identified as the optimal genetic transformation system of explants. Based on the phenotype of tuber formation under tissue culture conditions, two photoperiod treatments and sucrose treatments were carried out for the formation of 301071.3 and 304413.89 genotypes, respectively. The results showed that the formation of tube-tuber was induced in full darkness. Short sunshine could increase the number of tubers per plant and the sucrose concentration of 60 g / L sucrose per plant, which was beneficial to the formation of 301071.3 genotype tube-tuber. The optimum concentration of Kan and the composition of differentiation medium were optimized by using 304413.19 ~ 304413.40 and D 茅 sir 茅 e as receptors. The optimum concentration of Kan was 50 mg / L 50 mg / L and 60 mg / L respectively. Among the three genotypes, only D 茅 sir 茅 e successfully obtained transgenic lines. The optimal differentiation medium was (MS 0.1 mg / L IAA 2 mg / L ZT 60 mg / L Kan 400 mg / L ef). The regeneration rate and explant transformation rate were 52.27% and 11.36% respectively. The overexpression vector of StNTP was successfully constructed and transformed into potato receptor D 茅 sir 茅 e by using the optimized D 茅 sir 茅 e transformation system. Three transgenic lines of StNTP1 were obtained. The results showed that the transgenic lines were resistant to late blight, and the spot area was analyzed. Compared with the control, the resistance to late blight was significantly enhanced by StNTP, which indicated that StNTP enhanced the resistance to late blight in potato.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S532

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