镰刀菌几丁质合成酶Chs1和Chs2基因小片段RNA干扰功能分析
发布时间：2018-10-15 15:00
【摘要】：小麦赤霉病(Fusarium Head Blight,FHB)是由镰刀菌(Fusarium)引起的一种世界性真菌病害。小麦作为世界第二大粮食作物,缺乏对赤霉病的天然抗性,易造成大规模的赤霉病害的爆发,导致产量的大幅下降甚至颗粒无收。因此需要一种有效的方法来增强小麦对赤霉病的抵抗力。RNAi技术的出现对小麦抗性的提高提供了一个新思路。几丁质是一种真菌细胞壁的重要组成成分,是由几丁质合成酶(chitin synthases,Chs)催化合成的,病原真菌通常含有多个几丁质合成酶基因,而植物中未发现几丁质合成酶的存在,因此几丁质合成酶是抗真菌药物的理想靶标。本研究利用Gateway技术构架了不同的几丁质合成酶(Chs1,Chs2)基因小片段的RNAi表达载体,通过RNAi技术筛选出对亚洲镰刀菌生长发育和致病力影响较大的基因小片段,为小麦的抗病改良提供材料。1.将几丁质合成酶(Chs1)基因分成5个基因片段,每个基因段都构建成具有单个茎环结构的RNAi表达载体,通过原生质体转化法,将基因小片段导入野生型亚洲镰刀菌5035,定点整合到PLS基因位点,在其体内表达产生干扰小片段。结果表明,与野生型5035相比,Chs1基因各段RNAi转化子生长迟缓,分生孢子的产量有不同程度的降低,致病力下降了48.2%-68.6%,细胞壁几丁质含量也下降了11%-23%。2.与Chs1构建方法相同,将Chs2分为5个基因片段,每个片段构建成单茎环的RNAi载体。通过原生质体转化法,得到真菌转化子,高渗透压和酸性环境下,生长明显比野生型5035缓慢;产孢量上也有不同程度的下降;在△Chs2-3,△Chs2-4,△Chs2-5这3个片段的转化子中几丁质含量下调了9%-25.3%;与野生型5035相比,Chs2基因RNAi真菌转化子致病力降低了61.1%-73.6%。
[Abstract]:Wheat scab (Fusarium Head Blight,FHB) is a worldwide fungal disease caused by Fusarium (Fusarium). Wheat, as the second largest food crop in the world, lacks natural resistance to scab, which can easily cause a large-scale outbreak of scab, leading to a sharp decline in yield and even no harvest. Therefore, an effective method is needed to enhance the resistance of wheat to scab. The emergence of RNAi technique provides a new way to improve the resistance of wheat to scab. Chitin, an important component of the cell wall of fungi, is catalyzed by chitin synthase (chitin synthases,Chs). Pathogenic fungi usually contain multiple chitinase genes, but no chitinase is found in plants. Therefore, chitin synthase is an ideal target for antifungal drugs. In this study, the RNAi expression vectors of different chitinase (Chs1,Chs2) gene fragments were constructed by using Gateway technique, and the gene fragments which had great influence on the growth, development and pathogenicity of Fusarium asiatica were screened by RNAi technique. To provide materials for wheat disease resistance improvement. 1. Chitin synthase (Chs1) gene was divided into five gene fragments, each segment was constructed into a single stem loop structure of RNAi expression vector, through protoplast transformation method. The gene fragment was introduced into wild-type Fusarium asiatica 5035 and integrated into the PLS gene site. The results showed that compared with wild type 5035, the growth of RNAi transformants in each segment of Chs1 gene was slower, the yield of conidial spores was decreased in varying degrees, the pathogenicity was decreased by 48.2 to 68.6, and the content of chitin in cell wall was decreased by 11-23.2. The Chs2 was divided into five gene fragments, each of which was constructed into a single stem ring RNAi vector. The fungal transformants were obtained by protoplast transformation. Under high osmotic pressure and acidic environment, the growth rate of fungal transformants was slower than that of wild type 5035, and the spore yield decreased in varying degrees. The chitin content in the transformants of the three fragments of Chs2-3, Chs2-4, Chs2-5 was down-regulated 9- 25.3.The pathogenicity of RNAi transformants of Chs2 gene was decreased by 61.1% -73.6% compared with wild type 5035.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.44
本文编号：2272914
[Abstract]:Wheat scab (Fusarium Head Blight,FHB) is a worldwide fungal disease caused by Fusarium (Fusarium). Wheat, as the second largest food crop in the world, lacks natural resistance to scab, which can easily cause a large-scale outbreak of scab, leading to a sharp decline in yield and even no harvest. Therefore, an effective method is needed to enhance the resistance of wheat to scab. The emergence of RNAi technique provides a new way to improve the resistance of wheat to scab. Chitin, an important component of the cell wall of fungi, is catalyzed by chitin synthase (chitin synthases,Chs). Pathogenic fungi usually contain multiple chitinase genes, but no chitinase is found in plants. Therefore, chitin synthase is an ideal target for antifungal drugs. In this study, the RNAi expression vectors of different chitinase (Chs1,Chs2) gene fragments were constructed by using Gateway technique, and the gene fragments which had great influence on the growth, development and pathogenicity of Fusarium asiatica were screened by RNAi technique. To provide materials for wheat disease resistance improvement. 1. Chitin synthase (Chs1) gene was divided into five gene fragments, each segment was constructed into a single stem loop structure of RNAi expression vector, through protoplast transformation method. The gene fragment was introduced into wild-type Fusarium asiatica 5035 and integrated into the PLS gene site. The results showed that compared with wild type 5035, the growth of RNAi transformants in each segment of Chs1 gene was slower, the yield of conidial spores was decreased in varying degrees, the pathogenicity was decreased by 48.2 to 68.6, and the content of chitin in cell wall was decreased by 11-23.2. The Chs2 was divided into five gene fragments, each of which was constructed into a single stem ring RNAi vector. The fungal transformants were obtained by protoplast transformation. Under high osmotic pressure and acidic environment, the growth rate of fungal transformants was slower than that of wild type 5035, and the spore yield decreased in varying degrees. The chitin content in the transformants of the three fragments of Chs2-3, Chs2-4, Chs2-5 was down-regulated 9- 25.3.The pathogenicity of RNAi transformants of Chs2 gene was decreased by 61.1% -73.6% compared with wild type 5035.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.44
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