鐮刀菌幾丁質合成酶Chs1和Chs2基因小片段RNA干擾功能分析
發(fā)布時間：2018-10-15 15:00
【摘要】：小麥赤霉病(Fusarium Head Blight,FHB)是由鐮刀菌(Fusarium)引起的一種世界性真菌病害。小麥作為世界第二大糧食作物,缺乏對赤霉病的天然抗性,易造成大規(guī)模的赤霉病害的爆發(fā),導致產量的大幅下降甚至顆粒無收。因此需要一種有效的方法來增強小麥對赤霉病的抵抗力。RNAi技術的出現(xiàn)對小麥抗性的提高提供了一個新思路。幾丁質是一種真菌細胞壁的重要組成成分,是由幾丁質合成酶(chitin synthases,Chs)催化合成的,病原真菌通常含有多個幾丁質合成酶基因,而植物中未發(fā)現(xiàn)幾丁質合成酶的存在,因此幾丁質合成酶是抗真菌藥物的理想靶標。本研究利用Gateway技術構架了不同的幾丁質合成酶(Chs1,Chs2)基因小片段的RNAi表達載體,通過RNAi技術篩選出對亞洲鐮刀菌生長發(fā)育和致病力影響較大的基因小片段,為小麥的抗病改良提供材料。1.將幾丁質合成酶(Chs1)基因分成5個基因片段,每個基因段都構建成具有單個莖環(huán)結構的RNAi表達載體,通過原生質體轉化法,將基因小片段導入野生型亞洲鐮刀菌5035,定點整合到PLS基因位點,在其體內表達產生干擾小片段。結果表明,與野生型5035相比,Chs1基因各段RNAi轉化子生長遲緩,分生孢子的產量有不同程度的降低,致病力下降了48.2%-68.6%,細胞壁幾丁質含量也下降了11%-23%。2.與Chs1構建方法相同,將Chs2分為5個基因片段,每個片段構建成單莖環(huán)的RNAi載體。通過原生質體轉化法,得到真菌轉化子,高滲透壓和酸性環(huán)境下,生長明顯比野生型5035緩慢;產孢量上也有不同程度的下降;在△Chs2-3,△Chs2-4,△Chs2-5這3個片段的轉化子中幾丁質含量下調了9%-25.3%;與野生型5035相比,Chs2基因RNAi真菌轉化子致病力降低了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.
【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S432.44
本文編號：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.
【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S432.44
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，本文編號：2272914
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