本文選題：稻曲病　切入點(diǎn)：遺傳多樣性　出處：《華中農(nóng)業(yè)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：稻曲病是由Villosiclava virens引起的一種水稻穗部病害,在世界范圍內(nèi)造成嚴(yán)重的經(jīng)濟(jì)損失。本研究利用SNP和RAPD兩種分子標(biāo)記對(duì)湖北省不同水稻田的稻曲病菌進(jìn)行群體遺傳多樣性分析,可明確稻曲菌株與水稻品種、地理環(huán)境的關(guān)系,從而更好的進(jìn)行農(nóng)業(yè)防治。目前稻曲病主要依靠化學(xué)防治,而DMI類殺菌劑是防治稻曲病的主要化學(xué)藥劑,對(duì)稻曲病菌抗DMI類殺菌劑的分子機(jī)理進(jìn)行了研究,可為稻曲病的有效防控提供理論依據(jù)。本研究取得的主要結(jié)果如下:1.分離自武漢華中農(nóng)業(yè)大學(xué)試驗(yàn)田晚秈98和華粳952兩個(gè)水稻品種的43個(gè)菌株,系統(tǒng)進(jìn)化樹(shù)顯示相同水稻品種的菌株有聚在一起的趨勢(shì),證明了同一個(gè)田塊相同水稻品種上的菌株有很大的相似性。另外,在來(lái)自湖北省宜昌望家、宜昌窯河、黃岡、陽(yáng)新、荊州5塊水稻田的110個(gè)稻曲菌株中,同一田塊來(lái)源的不同菌株幾乎來(lái)自不同水稻品種。遺傳分析發(fā)現(xiàn)相同水稻田不同水稻品種上的菌株趨于聚在一起。Nei遺傳多樣性指數(shù)(H)和Shannon信息指數(shù)(I)都顯示了所有菌株間的多態(tài)性比地理種群間的多態(tài)性要高。另外,MEGA5.2計(jì)算的種群間的平均遺傳距離(0.006)高于種群內(nèi)的平均遺傳距離(0.0048)。FST值也顯示了大部分種群間具有顯著的遺傳差異。同一田塊中不同水稻品種上的菌株具有較高的相似性,說(shuō)明了地理因素在稻曲病菌的進(jìn)化過(guò)程中比水稻品種發(fā)揮了更重要的作用。2.克隆了稻曲病菌DMI類殺菌劑的靶標(biāo)基因Vv CYP51。利用紫外誘變技術(shù)獲得了對(duì)DMI類殺菌劑戊唑醇抗性的菌株,命名為UV10th。比較野生型菌株與UV10th的Vv CYP51基因的核苷酸序列和表達(dá)量,發(fā)現(xiàn)一個(gè)點(diǎn)突變,使得氨基酸137位由一個(gè)酪氨酸變成組氨酸(Y137H),并且在突變體中該基因過(guò)量表達(dá)�；蜻z傳轉(zhuǎn)化分析發(fā)現(xiàn)含點(diǎn)突變Vv CYP51的轉(zhuǎn)化子比野生型的轉(zhuǎn)化子對(duì)戊唑醇具有較高的EC50值,而Vv CYP51表達(dá)量與EC50值沒(méi)有相關(guān)性。分子對(duì)接與結(jié)合實(shí)驗(yàn)同時(shí)證明了野生型Vv CYP51蛋白比含點(diǎn)突變Y137H的Vv CYP51與戊唑醇有更高的親和力。以上結(jié)果顯示靶標(biāo)基因上的點(diǎn)突變Y137H(而非靶標(biāo)基因過(guò)量表達(dá))是稻曲病菌抗DMI殺菌劑的原因。另外,含點(diǎn)突變的菌株在菌絲生長(zhǎng)、產(chǎn)孢量、孢子萌發(fā)率等指標(biāo)上相對(duì)野生型菌株沒(méi)有明顯差異,表現(xiàn)出對(duì)環(huán)境很好的適應(yīng)性,預(yù)示在田間對(duì)DMI殺菌劑抗性的稻曲菌可能會(huì)出現(xiàn)和流行,需要實(shí)施監(jiān)測(cè)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S435.111.4

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