【摘要】：禾谷鐮刀菌是導(dǎo)致多種作物赤霉病的主要致病菌。赤霉病不僅導(dǎo)致農(nóng)作物減產(chǎn),給世界造成巨大的經(jīng)濟損失;其還產(chǎn)生多種毒素,包括單端孢霉烯B類毒素DON和NIV,這些毒素對人和家畜的健康造成巨大威脅。目前,使用多菌靈等咪唑類農(nóng)藥制劑是防治赤霉病的主要方法,該方法在過去一段時間內(nèi)上取得了一定的效果;但是近年來,此類防治方法弊端日益突出。首先,農(nóng)藥污染問題日益嚴重;其次,多年頻繁的使用農(nóng)藥,造成抗藥突變菌株的不斷涌現(xiàn),防治效果越來越差。因此,建立新的赤霉病防治方法和防治體系迫在眉睫。本文利用RNA干擾技術(shù),研究亞洲鐮刀菌關(guān)鍵基因Fg Pkc不同片段的干擾效果。將該基因CDS序列分成8個片段,每個片段大小約為500bp,分別構(gòu)建RNA干擾載體,并通過原生質(zhì)體轉(zhuǎn)化的方法,轉(zhuǎn)入到亞洲鐮刀菌5035菌株中。經(jīng)過通用及特異PCR鑒定、Southern鑒定共得到10個單拷貝轉(zhuǎn)化子。除片段3和5分別得到2個單拷貝陽性轉(zhuǎn)化子外,其余片段均得到1個單拷貝陽性轉(zhuǎn)化子。為進一步研究所得轉(zhuǎn)化子的特性,進行了一系列實驗,包括表型鑒定、田間花期接種、胚芽鞘接種以及分生孢子形態(tài)鑒定。主要結(jié)果如下:1.在SNA培養(yǎng)基上,培養(yǎng)4天后,轉(zhuǎn)化子生長速率與野生型菌株具有明顯差異,其中Pkc5-2和Pkc5-12生長最為緩慢,與野生型相比生長速率分別下降62.01%、63.67%;Pkc3-3生長速率下降幅度最小,為4.88%,Pkc8-16生長速率與野生型無明顯差異。2.田間花期接種結(jié)果顯示,除Pkc7-15外和野生型致病率無顯著性差異外,轉(zhuǎn)化子Pkc4-7,Pkc5-2,Pkc5-12、Pkc6-9與野生型菌株相比,致病率均有顯著性降低。其中Pkc4-7,Pkc5-2,Pkc5-12田間致病率下降更為明顯,與野生型菌株相比,分別下降72.10%、57.12%、54.96%(14天)。3.胚芽鞘接種顯示,所有轉(zhuǎn)化子致病率上與野生型相比均有極顯著性(P0.01)下降。Pkc5-2,Pkc5-12致病率下降程度尤為顯著,致病率分別下降60.75%、52.91%,與大田接種接種結(jié)果一致。4.轉(zhuǎn)化子分生孢子形態(tài)發(fā)生變化,轉(zhuǎn)化子產(chǎn)生的分生孢子均比野生型短,在寬度上與野生型無明顯差別。綜合上述實驗結(jié)果,Fg Pkc基因為禾谷鐮刀菌生長、致病相關(guān)的關(guān)鍵基因,將各基因片段沉默后,轉(zhuǎn)化子表型在生長速率、田間致病率、胚芽鞘致病率與野生型相比均有極顯著性的差異。尤其是第5個片段,其干擾效果更為明顯,不論是其生長速率、田間致病率,還是胚芽鞘致病率與野生型相比均有極顯著性差異,且比其他片段在干擾效果上更為明顯。且第5個片段得到兩個相互獨立的單拷貝轉(zhuǎn)化子,兩個轉(zhuǎn)化子的實驗結(jié)果基本一致,進一步證明了Pkc第5個片段對亞洲鐮刀菌的致病率起關(guān)鍵作用。可以將該片段構(gòu)建植物表達載體中,其產(chǎn)生的si RNA抑制亞洲鐮刀菌的生長,從而達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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S432.44

【共引文獻】

相關(guān)期刊論文 前10條

1 徐曉輝;劉延吉;王淼;田曉艷;;干旱脅迫對楊樹幼苗葉片內(nèi)ABA和CaM含量的影響[J];安徽農(nóng)業(yè)科學(xué);2006年11期

2 郝立華;王偉霞;王靈敏;尚忠林;;植物異三聚體G蛋白在細胞信號轉(zhuǎn)導(dǎo)中的作用機制研究進展[J];安徽農(nóng)業(yè)科學(xué);2009年04期

3 張媛華;王超;;G蛋白在植物中的研究進展[J];安徽農(nóng)業(yè)科學(xué);2012年05期

4 崔洪宇;吳波;吳東凱;吳菊;;蔬菜嫁接抗病增產(chǎn)機理的探討[J];北方園藝;2007年10期

5 曹慧;王孝威;付循成;;高等植物的鉀營養(yǎng)[J];北方園藝;2008年02期

6 姜躍麗;;鈣處理對切花采后壽命影響及機理研究[J];北方園藝;2008年09期

7 周錦琳,田野;酸性刺激對大鼠骨骼肌細胞胞漿Ca~(2+)的影響[J];北京體育大學(xué)學(xué)報;2005年09期

8 王艷輝;賈慧;司賀龍;馬繼芳;郝會芳;董金皋;;不同基因型玉米受HT-毒素脅迫后細胞內(nèi)CaM的動態(tài)變化[J];河北農(nóng)業(yè)大學(xué)學(xué)報;2007年05期

9 孫艷秋;李寶聚;陳捷;石延霞;;葡聚六糖誘導(dǎo)后對黃瓜細胞內(nèi)鈣信使的影響[J];河北農(nóng)業(yè)大學(xué)學(xué)報;2008年05期

10 葉楠;李永剛;文景芝;;利用mRNA差異顯示技術(shù)(DDRT-PCR)研究大豆抗疫霉根腐病的分子機制[J];東北農(nóng)業(yè)大學(xué)學(xué)報;2008年09期

相關(guān)會議論文 前3條

1 錢永強;孫振元;韓蕾;巨關(guān)升;;野牛草相連分株對異質(zhì)水分環(huán)境的生理響應(yīng)及其調(diào)控[A];中國觀賞園藝研究進展（2010）[C];2010年

2 王欣;劉持年;呂征;王育虎;;酸棗仁湯含藥血清對皮質(zhì)酮損傷的PC12細胞[Ca~(2+)]i、cAMP的影響[A];中華中醫(yī)藥學(xué)會方劑學(xué)分會2007年年會論文集[C];2007年

3 忻耀杰;滕磊;;喉源性咳嗽時間節(jié)律性的臨床觀察與探討[A];第六次全國中西醫(yī)結(jié)合變態(tài)反應(yīng)學(xué)術(shù)大會論文匯編[C];2013年

相關(guān)博士學(xué)位論文 前10條

1 劉巖;開竅藥對血腦屏障與腦細胞功能及其機制的影響[D];成都中醫(yī)藥大學(xué);2010年

2 王瑾;抗β1-腎上腺素受體自身抗體對T淋巴細胞增殖和分泌功能的影響[D];山西醫(yī)科大學(xué);2011年

3 楊朝暉;鈉離子通道阻斷劑利多卡因體內(nèi)外干預(yù)關(guān)節(jié)軟骨的生物學(xué)效應(yīng)[D];山西醫(yī)科大學(xué);2011年

4 孔祥培;玉米促分裂原活化蛋白激酶C族MAPKK基因ZmMKK4的分離及功能分析[D];山東農(nóng)業(yè)大學(xué);2011年

5 劉玉鯤;玉米MAPK基因家族的鑒定及ZmMPK4基因?qū)Φ墓δ芊治鯷D];山東農(nóng)業(yè)大學(xué);2011年

6 戴海英;鈣信號轉(zhuǎn)導(dǎo)途徑在自體移植皮片過度色素沉著中的作用[D];第二軍醫(yī)大學(xué);2011年

7 吳君;基于β_3-AR探討縮泉丸補腎縮尿的作用機制[D];廣州中醫(yī)藥大學(xué);2011年

8 孫濤;川貝母止嗽顆粒治療急性支氣管炎的作用機理研究[D];成都中醫(yī)藥大學(xué);2011年

9 張?zhí)m;Akt2是Akt家族中抗氧化應(yīng)激的主要激酶并通過多條信號途徑抑制過氧化氫誘導(dǎo)的細胞凋亡[D];湖南師范大學(xué);2011年

10 龍白;槲皮素、地錢黃酮、DADS對人肝癌HepG2細胞增殖和凋亡的影響及相關(guān)作用機理的研究[D];中南大學(xué);2010年

相關(guān)碩士學(xué)位論文 前10條

1 劉媛媛;番茄農(nóng)桿菌介導(dǎo)基因轉(zhuǎn)化技術(shù)體系的優(yōu)化與番茄LeJA2基因的功能研究[D];山東農(nóng)業(yè)大學(xué);2009年

2 季彥林;馬鈴薯塊莖糖苷生物堿積累的光誘導(dǎo)效應(yīng)研究[D];甘肅農(nóng)業(yè)大學(xué);2010年

3 姚順平;無機銅試劑誘導(dǎo)棉花抗黃萎病的研究[D];新疆農(nóng)業(yè)大學(xué);2010年

4 蘭敏娟;擬南芥NPCL基因調(diào)節(jié)花粉萌發(fā)和生長的機制分析[D];河北農(nóng)業(yè)大學(xué);2011年

5 李洋洋;AtCNGC2在茉莉酸信號通路中的作用[D];山東師范大學(xué);2011年

6 王蕾;煙草磷脂酶C基因的表達模式和過表達NtPLC4轉(zhuǎn)基因煙草提高抗病性研究[D];山東農(nóng)業(yè)大學(xué);2011年

7 劉婧;設(shè)施栽培對甜櫻桃花芽形態(tài)分化和生理特性的影響[D];山東農(nóng)業(yè)大學(xué);2011年

8 朱倩蓉;調(diào)控劑的電子傳遞性質(zhì)對骨骼肌型鈣釋放通道調(diào)控作用的影響[D];華東師范大學(xué);2011年

9 張娜;eATP調(diào)節(jié)擬南芥根系生長的信號轉(zhuǎn)導(dǎo)機制[D];河北師范大學(xué);2011年

10 喻向前;金魚PP2A-B"家族調(diào)節(jié)亞基β基因在胚胎發(fā)育中的功能研究[D];湖南師范大學(xué);2011年

，

本文編號：2119353