大麗輪枝菌腺苷酸激酶和寡糖基轉(zhuǎn)移酶STT3亞基毒力功能分析
發(fā)布時間：2018-09-09 18:57
【摘要】：棉花黃萎病(Verticillium Wilt of cotton)是一種土傳、維管束系統(tǒng)性病害,被稱為“棉花癌癥”,給棉花的產(chǎn)量和棉纖維的質(zhì)量都造成很大影響,其致病菌為大麗輪枝菌(Verticillium dahliae)。由于大麗輪枝菌極易產(chǎn)生新的生理小種,給研究帶來相當大的難度和很多的不確定性,因此對大麗輪枝菌的研究一直以來都是熱點。本實驗室利用寄主誘導(dǎo)的基因沉默技術(shù)(Host-induced gene silencing),以高致病力的大麗輪枝菌株V991為實驗材料,構(gòu)建一系列針對大麗輪枝菌靶標基因的煙草脆裂病毒(tobacco rattle virus, TRV)干擾載體,通過植物體內(nèi)dsRNA與靶標基因互作,并進行統(tǒng)計病情指數(shù)統(tǒng)計,從中篩選出20個與病原菌生長發(fā)育與致病性相關(guān)的基因。本文從中選擇腺苷酸激酶(adenylate kinase, AK)和寡糖基轉(zhuǎn)移酶STT3亞基(oligosaccharyl transferase STT3 subunit, OST STT3)進行深入研究,期望進一步研究病原菌基因生物學(xué)功能和拓寬植物抗逆基因數(shù)據(jù)庫。本研究的主要結(jié)果如下：1、利用實驗室構(gòu)建的HIGS體系,擴大本氏煙草培養(yǎng)規(guī)模,用含有AK和STT-3靶標基因片段的農(nóng)桿菌注射植株10天后,接種106個/mL大麗輪枝菌,統(tǒng)計植株病情指數(shù),并利用qRT-PCR進行植株真菌生物量檢測和靶標基因表達水平分析；2、利用融合PCR技術(shù),將靶標基因上、下游片段以及潮霉素抗性表達盒整合成一個完整的片段,通過BP反應(yīng)將其構(gòu)建至缺失突變體載體中；通過酶切反應(yīng),將新霉素抗性表達盒以及靶標基因表達盒構(gòu)建至pCAMBIA1302載體中,成為回補質(zhì)粒；然后將回補質(zhì)粒中的靶標基因開放閱讀框替換為GFP基因開放閱讀框,成為GFP表達質(zhì)粒。最后,通過農(nóng)桿菌介導(dǎo)的方法獲得陽性轉(zhuǎn)化子。3、將野生型、突變體以及回補體孢子接種本氏煙草,統(tǒng)計植株病情指數(shù)并進行生物量測定,分析不同菌株毒力水平。同時,用表達GFP的野生型以及缺失突變體孢子接種本氏煙草,然后通過共聚焦顯微鏡觀察兩者在植株根部侵染的區(qū)別,旨在進一步明確靶標基因在病原菌發(fā)病過程中所發(fā)揮的作用。4、將野生型、AK突變體以及回補體孢子滴加到察比克培養(yǎng)基上,分析在不同逆境情況下菌株生理指標(菌落直徑和產(chǎn)孢量)的差異。同時,將野生型、STT3突變體以及回補體孢子滴加到含有不同碳源的培養(yǎng)基上,分析菌株生理指標(菌落直徑和產(chǎn)孢量)的差異,從而進一步探索STT3基因在碳源利用率方面所發(fā)揮的作用。5、構(gòu)建含有靶標基因穩(wěn)定遺傳的Gateway干擾載體,轉(zhuǎn)化本氏煙草。對獲得的陽性植株接種病原菌,并對其進行抗病性分析以及分子水平檢測。本實驗利用突變體和回補體的構(gòu)建以及穩(wěn)定遺傳陽性植株的獲得,進一步探索AK和STT3基因在病原菌生長發(fā)育以及毒力方面發(fā)揮的作用,為農(nóng)作物病蟲害的防治提供了一定的實驗數(shù)據(jù)和理論基礎(chǔ)。
[Abstract]:Cotton Verticillium Wilt (Verticillium Wilt of cotton) is a systemic disease of vascular bundle, which is called "cotton cancer". It has a great influence on cotton yield and cotton fiber quality. The pathogenic bacteria is (Verticillium dahliae)., the pathogen of Verticillium dahliae. Because it is easy to produce new physiological species, it brings a great deal of difficulty and uncertainty to the research, so the research of Dendrotus dahliensis has been a hot spot all the time. The host induced gene silencing technique (Host-induced gene silencing),) was used to construct a series of (tobacco rattle virus, TRV) interference vectors of tobacco embrittlement virus (TBV) targeting the target gene of Verticillium dahliae (V991). Through the interaction of dsRNA and target genes in plants and the statistics of disease index, 20 genes related to the growth and pathogenicity of pathogenic bacteria were screened out. In this paper, adenylate kinase (adenylate kinase, AK) and oligosyltransferase (STT3) subunit (oligosaccharyl transferase STT3 subunit, OST STT3) were selected for further study in order to further study the gene biological function of pathogenic bacteria and broaden the database of plant stress resistance genes. The main results of this study were as follows: 1. Using the HIGS system constructed in the laboratory, we expanded the scale of tobacco cultivation. After 10 days of inoculation with Agrobacterium tumefaciens containing AK and STT-3 target gene fragments, we inoculated 106 / mL Rhizoctonia dahliensis. Plant disease index was counted, and plant fungal biomass and target gene expression level were analyzed by qRT-PCR. Using fusion PCR technique, the upstream and downstream fragments of target gene and hygromycin resistant expression box were integrated into a complete fragment. It was constructed into the deletion mutant vector by BP reaction, and the neomycin resistant expression box and target gene expression box were constructed into the pCAMBIA1302 vector by enzyme digestion. Then the open reading frame of the target gene in the complement plasmid was replaced by the open reading frame of the GFP gene to become the GFP expression plasmid. Finally, the positive transformant. 3 was obtained by Agrobacterium-mediated method. Spores of wild type, mutant and compensator were inoculated into Bentner's tobacco. The disease index and biomass of different strains were measured and the virulence levels of different strains were analyzed. At the same time, the wild type expressing GFP and the spores of the mutant were inoculated with Bentner's tobacco, and then the difference of infection in the root of the two plants was observed by confocal microscope. The purpose of this study was to further clarify the role of target gene in the pathogenesis of pathogenic bacteria, and to add spores of wild type AK mutant and compensator to Chabbik medium. The differences of physiological indexes (colony diameter and spore yield) under different stress conditions were analyzed. At the same time, the spores of wild type STT3 mutant and compensator were added to the medium containing different carbon sources, and the differences of physiological indexes (colony diameter and sporulation) were analyzed. In order to further explore the role of STT3 gene in the utilization of carbon source. 5, construct the Gateway interference vector with stable inheritance of target gene, and transform the tobacco. The positive plants were inoculated with pathogenic bacteria, and the disease resistance and molecular level were analyzed. The purpose of this study was to explore the role of AK and STT3 genes in the growth and virulence of pathogenic bacteria by constructing mutants and complement and obtaining stable genetically positive plants. It provides some experimental data and theoretical basis for the control of crop diseases and insect pests.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：博士后
【學(xué)位授予年份】：2016
【分類號】：S435.621.2
本文編號：2233278
[Abstract]:Cotton Verticillium Wilt (Verticillium Wilt of cotton) is a systemic disease of vascular bundle, which is called "cotton cancer". It has a great influence on cotton yield and cotton fiber quality. The pathogenic bacteria is (Verticillium dahliae)., the pathogen of Verticillium dahliae. Because it is easy to produce new physiological species, it brings a great deal of difficulty and uncertainty to the research, so the research of Dendrotus dahliensis has been a hot spot all the time. The host induced gene silencing technique (Host-induced gene silencing),) was used to construct a series of (tobacco rattle virus, TRV) interference vectors of tobacco embrittlement virus (TBV) targeting the target gene of Verticillium dahliae (V991). Through the interaction of dsRNA and target genes in plants and the statistics of disease index, 20 genes related to the growth and pathogenicity of pathogenic bacteria were screened out. In this paper, adenylate kinase (adenylate kinase, AK) and oligosyltransferase (STT3) subunit (oligosaccharyl transferase STT3 subunit, OST STT3) were selected for further study in order to further study the gene biological function of pathogenic bacteria and broaden the database of plant stress resistance genes. The main results of this study were as follows: 1. Using the HIGS system constructed in the laboratory, we expanded the scale of tobacco cultivation. After 10 days of inoculation with Agrobacterium tumefaciens containing AK and STT-3 target gene fragments, we inoculated 106 / mL Rhizoctonia dahliensis. Plant disease index was counted, and plant fungal biomass and target gene expression level were analyzed by qRT-PCR. Using fusion PCR technique, the upstream and downstream fragments of target gene and hygromycin resistant expression box were integrated into a complete fragment. It was constructed into the deletion mutant vector by BP reaction, and the neomycin resistant expression box and target gene expression box were constructed into the pCAMBIA1302 vector by enzyme digestion. Then the open reading frame of the target gene in the complement plasmid was replaced by the open reading frame of the GFP gene to become the GFP expression plasmid. Finally, the positive transformant. 3 was obtained by Agrobacterium-mediated method. Spores of wild type, mutant and compensator were inoculated into Bentner's tobacco. The disease index and biomass of different strains were measured and the virulence levels of different strains were analyzed. At the same time, the wild type expressing GFP and the spores of the mutant were inoculated with Bentner's tobacco, and then the difference of infection in the root of the two plants was observed by confocal microscope. The purpose of this study was to further clarify the role of target gene in the pathogenesis of pathogenic bacteria, and to add spores of wild type AK mutant and compensator to Chabbik medium. The differences of physiological indexes (colony diameter and spore yield) under different stress conditions were analyzed. At the same time, the spores of wild type STT3 mutant and compensator were added to the medium containing different carbon sources, and the differences of physiological indexes (colony diameter and sporulation) were analyzed. In order to further explore the role of STT3 gene in the utilization of carbon source. 5, construct the Gateway interference vector with stable inheritance of target gene, and transform the tobacco. The positive plants were inoculated with pathogenic bacteria, and the disease resistance and molecular level were analyzed. The purpose of this study was to explore the role of AK and STT3 genes in the growth and virulence of pathogenic bacteria by constructing mutants and complement and obtaining stable genetically positive plants. It provides some experimental data and theoretical basis for the control of crop diseases and insect pests.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：博士后
【學(xué)位授予年份】：2016
【分類號】：S435.621.2
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1 蘇曉峰;大麗輪枝菌腺苷酸激酶和寡糖基轉(zhuǎn)移酶STT3亞基毒力功能分析[D];中國農(nóng)業(yè)科學(xué)院;2016年
，本文編號：2233278
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