水稻稻曲病菌遗传多样性及其抗DMI类杀菌剂分子机理的研究

发布时间：2018-03-03 18:32

本文选题：稻曲病　切入点：遗传多样性　出处：《华中农业大学》2015年博士论文　论文类型：学位论文

【摘要】：稻曲病是由Villosiclava virens引起的一种水稻穗部病害,在世界范围内造成严重的经济损失。本研究利用SNP和RAPD两种分子标记对湖北省不同水稻田的稻曲病菌进行群体遗传多样性分析,可明确稻曲菌株与水稻品种、地理环境的关系,从而更好的进行农业防治。目前稻曲病主要依靠化学防治,而DMI类杀菌剂是防治稻曲病的主要化学药剂,对稻曲病菌抗DMI类杀菌剂的分子机理进行了研究,可为稻曲病的有效防控提供理论依据。本研究取得的主要结果如下:1.分离自武汉华中农业大学试验田晚籼98和华粳952两个水稻品种的43个菌株,系统进化树显示相同水稻品种的菌株有聚在一起的趋势,证明了同一个田块相同水稻品种上的菌株有很大的相似性。另外,在来自湖北省宜昌望家、宜昌窑河、黄冈、阳新、荆州5块水稻田的110个稻曲菌株中,同一田块来源的不同菌株几乎来自不同水稻品种。遗传分析发现相同水稻田不同水稻品种上的菌株趋于聚在一起。Nei遗传多样性指数(H)和Shannon信息指数(I)都显示了所有菌株间的多态性比地理种群间的多态性要高。另外,MEGA5.2计算的种群间的平均遗传距离(0.006)高于种群内的平均遗传距离(0.0048)。FST值也显示了大部分种群间具有显著的遗传差异。同一田块中不同水稻品种上的菌株具有较高的相似性,说明了地理因素在稻曲病菌的进化过程中比水稻品种发挥了更重要的作用。2.克隆了稻曲病菌DMI类杀菌剂的靶标基因Vv CYP51。利用紫外诱变技术获得了对DMI类杀菌剂戊唑醇抗性的菌株,命名为UV10th。比较野生型菌株与UV10th的Vv CYP51基因的核苷酸序列和表达量,发现一个点突变,使得氨基酸137位由一个酪氨酸变成组氨酸(Y137H),并且在突变体中该基因过量表达。基因遗传转化分析发现含点突变Vv CYP51的转化子比野生型的转化子对戊唑醇具有较高的EC50值,而Vv CYP51表达量与EC50值没有相关性。分子对接与结合实验同时证明了野生型Vv CYP51蛋白比含点突变Y137H的Vv CYP51与戊唑醇有更高的亲和力。以上结果显示靶标基因上的点突变Y137H(而非靶标基因过量表达)是稻曲病菌抗DMI杀菌剂的原因。另外,含点突变的菌株在菌丝生长、产孢量、孢子萌发率等指标上相对野生型菌株没有明显差异,表现出对环境很好的适应性,预示在田间对DMI杀菌剂抗性的稻曲菌可能会出现和流行,需要实施监测。
[Abstract]:Rice smut is a rice panicle disease caused by Villosiclava virens, which causes serious economic losses worldwide. In this study, two molecular markers, SNP and RAPD, were used to analyze the population genetic diversity of Aspergillus oryzae in different paddy fields in Hubei Province. The relationship between the strain and the rice variety and geographical environment can be clarified, and the agricultural control can be carried out better. At present, chemical control is mainly relied on, and DMI fungicide is the main chemical agent to prevent and cure rice aspergillus. The molecular mechanism of resistance to DMI fungicides was studied. The main results obtained in this study are as follows: 1. 43 strains of late Indica 98 and Huaging952 were isolated from the experimental field of Wuhan Huazhong Agricultural University. The phylogenetic tree shows that the strains of the same rice varieties tend to gather together, which proves that the strains of the same rice varieties in the same field are very similar. In addition, the strains from Wangjia, Yichang, Yaohe, Huanggang and Yangxin in Hubei Province, Among 110 strains of rice koji from 5 paddy fields in Jingzhou, Genetic analysis showed that the strains on the same rice field tended to gather together. The genetic diversity index (H) and the Shannon information index (I) showed that. In addition, the average genetic distance between populations calculated by MEGA5.2 was 0.006) higher than that among populations within the population, indicating a significant genetic difference among most populations. Different strains of different rice varieties in the same field have high similarity. The results show that geographical factors play a more important role than rice varieties in the evolution of Aspergillus oryzae. 2. The target gene VvCYP51of DMI fungicides of Aspergillus oryzae was cloned. The DMI fungicide pentyl was obtained by ultraviolet mutagenesis. Azolol-resistant strains, Named UV10th. a point mutation was found by comparing the nucleotide sequence and expression of VV CYP51 gene between wild type strain and UV10th. The amino acid 137 changed from a tyrosine to a histidine Y137H, and the gene was overexpressed in the mutant. Genetic transformation analysis showed that the transformant containing point mutant Vv CYP51 had a higher EC50 value to tebuconazole than the wild-type transformant. However, there was no correlation between VV CYP51 expression and EC50 value. Molecular docking and binding experiments also demonstrated that wild-type Vv CYP51 protein had higher affinity to tebuconazole than that of point mutant Y137H Vv CYP51. The above results showed that the spot of target gene was higher than that of Vv CYP51 with point mutation Y137H. The mutant Y137H (instead of overexpression of non-target gene) is the cause of resistance to DMI fungicides in Aspergillus oryzae. There was no significant difference in hyphal growth, spore production and spore germination rate between wild type strains with point mutation, which showed good adaptability to the environment. It was suggested that Aspergillus ricoides resistant to DMI fungicides in the field might appear and be prevalent and need to be monitored.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S435.111.4

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