亚洲镰刀菌FgPkc基因小片段相关功能的鉴定

发布时间：2018-07-13 12:21

【摘要】：禾谷镰刀菌是导致多种作物赤霉病的主要致病菌。赤霉病不仅导致农作物减产,给世界造成巨大的经济损失;其还产生多种毒素,包括单端孢霉烯B类毒素DON和NIV,这些毒素对人和家畜的健康造成巨大威胁。目前,使用多菌灵等咪唑类农药制剂是防治赤霉病的主要方法,该方法在过去一段时间内上取得了一定的效果;但是近年来,此类防治方法弊端日益突出。首先,农药污染问题日益严重;其次,多年频繁的使用农药,造成抗药突变菌株的不断涌现,防治效果越来越差。因此,建立新的赤霉病防治方法和防治体系迫在眉睫。本文利用RNA干扰技术,研究亚洲镰刀菌关键基因Fg Pkc不同片段的干扰效果。将该基因CDS序列分成8个片段,每个片段大小约为500bp,分别构建RNA干扰载体,并通过原生质体转化的方法,转入到亚洲镰刀菌5035菌株中。经过通用及特异PCR鉴定、Southern鉴定共得到10个单拷贝转化子。除片段3和5分别得到2个单拷贝阳性转化子外,其余片段均得到1个单拷贝阳性转化子。为进一步研究所得转化子的特性,进行了一系列实验,包括表型鉴定、田间花期接种、胚芽鞘接种以及分生孢子形态鉴定。主要结果如下:1.在SNA培养基上,培养4天后,转化子生长速率与野生型菌株具有明显差异,其中Pkc5-2和Pkc5-12生长最为缓慢,与野生型相比生长速率分别下降62.01%、63.67%;Pkc3-3生长速率下降幅度最小,为4.88%,Pkc8-16生长速率与野生型无明显差异。2.田间花期接种结果显示,除Pkc7-15外和野生型致病率无显著性差异外,转化子Pkc4-7,Pkc5-2,Pkc5-12、Pkc6-9与野生型菌株相比,致病率均有显著性降低。其中Pkc4-7,Pkc5-2,Pkc5-12田间致病率下降更为明显,与野生型菌株相比,分别下降72.10%、57.12%、54.96%(14天)。3.胚芽鞘接种显示,所有转化子致病率上与野生型相比均有极显著性(P0.01)下降。Pkc5-2,Pkc5-12致病率下降程度尤为显著,致病率分别下降60.75%、52.91%,与大田接种接种结果一致。4.转化子分生孢子形态发生变化,转化子产生的分生孢子均比野生型短,在宽度上与野生型无明显差别。综合上述实验结果,Fg Pkc基因为禾谷镰刀菌生长、致病相关的关键基因,将各基因片段沉默后,转化子表型在生长速率、田间致病率、胚芽鞘致病率与野生型相比均有极显著性的差异。尤其是第5个片段,其干扰效果更为明显,不论是其生长速率、田间致病率,还是胚芽鞘致病率与野生型相比均有极显著性差异,且比其他片段在干扰效果上更为明显。且第5个片段得到两个相互独立的单拷贝转化子,两个转化子的实验结果基本一致,进一步证明了Pkc第5个片段对亚洲镰刀菌的致病率起关键作用。可以将该片段构建植物表达载体中,其产生的si RNA抑制亚洲镰刀菌的生长,从而达到防治赤霉病的目的。
[Abstract]:Fusarium graminearum is the main pathogen causing scab in many crops. Scab not only leads to the reduction of crop yield and causes great economic losses in the world, but also produces a variety of toxins, including Monosporin B toxoid don and NIV, which pose a great threat to the health of human beings and domestic animals. At present, the use of carbendazoles and other imidazole pesticide formulations is the main method to control scab, which has achieved some results in the past period of time, but in recent years, the disadvantages of this method are increasingly prominent. First, the problem of pesticide pollution is becoming more and more serious; secondly, the frequent use of pesticides for many years, resulting in the emergence of drug-resistant mutant strains, the control effect is getting worse and worse. Therefore, it is urgent to establish a new control method and control system of scab. In this paper, RNA interference technique was used to study the interference effect of different fragments of FG Pkc gene in Fusarium asiatica. The CDS sequence of the gene was divided into eight fragments, each of which was about 500 BP in size. The RNAi vector was constructed and transferred into Fusarium asiatica strain 5035 by protoplast transformation. A total of 10 single copy transformants were identified by general and specific PCR. With the exception of fragments 3 and 5, two single-copy positive transformants were obtained, and one single-copy positive transformant was obtained in all the other fragments. In order to further study the characteristics of transformants, a series of experiments were carried out, including phenotypic identification, field anthesis inoculation, coleoptile inoculation and conidial morphology identification. The main results are as follows: 1. On SNA medium, the growth rate of transformants was significantly different from that of wild type strains after 4 days of culture. The growth rate of Pkc5-2 and Pkc5-12 was the slowest, and the growth rate of Pkc5-2 and Pkc5-12 decreased 62.01% and 63.67%, respectively. The growth rate of Pkc8-16 was not significantly different from that of wild type. The results of field inoculation at flowering stage showed that the pathogenicity of the transformant Pkc4-7Pkc5-2Pkc5-12Pkc6-9 was significantly lower than that of wild-type strains except for Pkc7-15 and wild type. The pathogenicity of Pkc4-7Pkc5-2Pkc5-12 decreased more obviously than that of wild-type strains, and decreased by 54.96% (14 days). Coleoptile inoculation showed that the pathogenicity of all transformants was significantly lower than that of wild type (P0.01). The decrease of pathogenicity of Pkc5-2Pkc5-12 was more significant than that of wild type. The pathogenicity of all transformants was decreased by 60.75% and 52.91%, respectively, which was consistent with the results of field inoculation. The conidial morphology of the transformants changed. The conidiospores produced by the transformants were shorter than those of the wild type, and there was no significant difference in width between the transformants and the wild type. Based on the above results, the key genes related to the growth and pathogenicity of Fusarium graminearum were obtained. After silencing the gene fragments, the phenotypes of transformants were expressed at the growth rate and the pathogenicity in the field. The pathogenicity of coleoptile was significantly different from that of wild type. The interference effect of the fifth fragment was more obvious than that of the wild type, and it was more obvious than that of the other fragments in terms of growth rate, field pathogenicity and coleoptile pathogenicity. Two independent single-copy transformants were obtained from the fifth fragment, and the experimental results of the two transformants were basically consistent, which further proved that the fifth fragment of Pkc played a key role in the pathogenicity of Fusarium asiatica. The siRNA produced by the fragment can inhibit the growth of Fusarium asiatica and control scab.
【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

【共引文献】