真菌gfp表达载体构建及农杆菌介导转化胶孢炭疽菌研究

发布时间：2018-04-18 07:28

  本文选题：胶孢炭疽菌 + 重组PCR技术　； 参考：《山东农业大学》2017年硕士论文

【摘要】：核桃(Juglans regia L.)是重要的“木本油料”战略树种,但随着核桃集中栽培面积的扩大和抗病品种的缺乏,核桃病害日渐严重。核桃炭疽病(anthracnose)是由胶孢炭疽菌(Colletotrichum gloeosporioides)引起的,可致果实坏疽、叶片焦枯,同时还危害嫩梢,导致产量锐减,是目前核桃生产中的灾难性病害。本论文构建了适于真菌的绿色荧光蛋白(Green fluorescent protein,gfp)表达载体,农杆菌介导法(Agrobacterium tumefaciens-mediated transformation,ATMT)对胶孢炭疽菌进行了遗传转化得到了带有绿色荧光蛋白标记的胶孢炭疽菌菌株,为研究胶孢炭疽菌对核桃的侵染过程,了解核桃与胶孢炭疽菌的互作机制提供了技术支撑。研究结果如下:1.真菌gfp独立表达载体(DL-gfp)的构建。以真菌trp C基因的启动子Ptrp C和终止子Ttrp C作为gfp和潮霉素磷酸转移酶基因(Hygromycin B phosphortransferase gene,hph)的启动子和终止子。通过PCR重组技术(Overlap PCR)分别构建了hph的表达元件(Ptrp C+hph+Ttrp C)和gfp表达元件(Ptrp C+gfp+Ttrp C);通过In-fusion酶连接技术,将两个表达元件与带有T-DNA区间的线性化Vector(p Green II 62-SK)相连,构建独立表达载体DL-gfp。2.真菌gfp融合表达载体(RH-gfp)的构建。以真菌trp C基因的启动子Ptrp C和终止子Ttrp C作为gfp和hph基因的启动子和终止子进行构建。通过Overlap PCR将启动子Ptrp C和hph基因重组到一起,且hph基因敲除终止密码;将Overlap PCR获得片段(Ptrp C+hph)与gfp、终止子Ttrp C和线性化后的Ti质粒(p Green II 62-SK)通过In-fusion酶连接重组,构建融合表达载体RH-gfp。3.重新分离纯化致病力较强的m9胶孢炭疽菌。实验室4℃斜面保存的胶孢炭疽菌m9菌株,直接在马铃薯葡萄糖琼脂培养基(PDA)培养,菌丝徒长,产孢量少,菌株产孢能力下降,通过将m9菌株重新侵染核桃叶片,获得大量的m9的分生孢子,在PDA平板上纯化后使用。4.ATMT转化胶孢炭疽菌。优化转化条件:胶孢炭疽菌分生孢子浓度为107个/m L,农杆菌GV3101(含DL-gfp载体)浓度OD620≈0.5;乙酰丁香酮(AS)诱导浓度为200μM,转化菌株的潮霉素B筛选浓度为100μg/m L,用于农杆菌菌株抑制的氨噻肟头孢霉素100μg/m L;诱导转化温度为22℃,时间为2天。5.获得了带有荧光蛋白标记的胶孢炭疽菌m9转化子。经过多次继代培养,筛选出能够稳定表达gfp标记基因和hph筛选基因的转化子,为后续研究转化子和野生型菌株生物学特性的差异以及观察胶孢炭疽菌转化子和核桃之间病原互作的活体动态过程奠定了基础。
[Abstract]:Juglans regia L.It is an important "woody oil" strategic tree species, but with the expansion of concentrated cultivation area and the lack of disease-resistant varieties, walnut disease is becoming more and more serious.Anthracnoseis is caused by Colletotrichum gloeosporioides. it can cause fruit gangrene, scorched leaves, and damage young shoots, which results in sharp decrease of yield, which is a catastrophic disease in walnut production.In this paper, the expression vector of green fluorescent protein green fluorescent protein (GFP) suitable for fungi was constructed. Agrobacterium tumefaciens-mediated transformation by Agrobacterium tumefaciens was used for genetic transformation of Bacillus anthracis to obtain Bacillus anthracis strain labeled with green fluorescent protein.It provides technical support for studying the infection process of anthrax on walnut and understanding the interaction mechanism between walnut and anthracis.The results are as follows: 1.Construction of fungal gfp independent expression vector DL-gfp.The promoter Ptrp C and the Terminator Ttrp C of trp C gene were used as promoter and Terminator of gfp and hygromycin B phosphortransferase gene.Ptrp C hph Ttrp C) and Ptrp C gfp Ttrp C of hph were constructed by PCR recombination technique, and two expression elements were connected with linearized Vector(p Green II 62-SKK with T-DNA interval by In-fusion enzyme ligation technique.The independent expression vector DL-gfp.2. was constructed.Construction of fungal gfp fusion expression vector RH-gfp.The promoters Ptrp C and Ttrp C of trp C gene were used as promoters and Terminators of gfp and hph genes.The promoter Ptrp C and hph gene were recombined together by Overlap PCR, and the hph gene knockout stop codon was removed, and the Overlap PCR fragment was ligated with GFP, Ttrp C and the linearized Ti plasmid p II 62-SKK were ligated by In-fusion enzyme.Construction of fusion expression vector RH-gfp.3.To reisolate and purify M. 9 anthracis with strong pathogenicity.The strain m9 was cultured directly in the potato glucose Agar medium (PDAs). The mycelium was long, the sporulation quantity was low, and the sporulation ability of the strain was decreased. The strain m9 was infected with the leaves of walnut again.A large number of conidia of m9 were obtained and purified on PDA plate. 4. ATMT was used to transform Bacillus anthracis.The optimum transformation conditions were as follows: the conidial concentration of Bacillus anthracis was 107 / mL, the concentration of Agrobacterium tumefaciens GV3101 (containing DL-gfp carrier) was OD620 鈮，

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