稻曲病菌T-DNA插入突變體B2510的插入位點分析及致病相關(guān)基因Uvt3277功能的初步研究
發(fā)布時間：2019-06-19 15:52
【摘要】：水稻稻曲病是一種水稻穗部病害,現(xiàn)已由次要病害上升為主要病害。稻曲病菌(Ustilaginoideavirens)是稻曲病的病原真菌,在穗部形成的稻曲球嚴(yán)重影響水稻品質(zhì)和產(chǎn)量,稻曲球產(chǎn)生的毒素能對人畜健康產(chǎn)生危害。因此深入了解稻曲病的致病機理,可以為水稻稻曲病的防治提供理論依據(jù)。1、本文從稻曲病菌T-DNA插入突變體庫中篩選到一株致病力減弱的突變菌株B2510,通過分析突變菌株B2510的生物學(xué)性狀和T-DNA插入位點的側(cè)翼基因,為稻曲病菌的分子致病機理的研究提供方法和理論依據(jù)。與野生型P1相比,突變菌株B2510喪失了產(chǎn)孢能力,生長速率在MM培養(yǎng)基上明顯降低,而在PSA和TB3培養(yǎng)基中則沒有顯著差異。Southern雜交分析T-DNA的拷貝數(shù),結(jié)果顯示T-DNA以雙拷貝形式插入到突變菌株B2510的基因組中。運用hiTAIL-PCR技術(shù)擴增T-DNA插入位點的側(cè)翼序列,獲得兩個可能與致病性有關(guān)的基因UVC6TF和UVRASGAP。T-DNA分別插在基因UVC6TF的啟動子區(qū)域和UVRASGAP的下游3'端,且稻曲基因組和T-DNA序列均未丟失。半定量RT-PCR分別分析兩個基因在突變菌株B2510中的表達(dá)量,發(fā)現(xiàn)與P1相比均顯著下降。因此推測這兩個基因與稻曲病菌致病性相關(guān),可能在某一階段參與調(diào)控稻曲病菌在水稻上的致病過程。為進(jìn)一步了解基因的功能,構(gòu)建基因UVC6TF的沉默載體,并通過ATMT的方法導(dǎo)入稻曲病菌。獲得的2個沉默轉(zhuǎn)化子中UVC6TF的表達(dá)分別被抑制了 50%和90%,分析它們的產(chǎn)孢量、生長速率等生物學(xué)性狀和致病力檢測。結(jié)果發(fā)現(xiàn)該基因不影響菌株正常的生長發(fā)育,但是對分生孢子的產(chǎn)生過程有一定的調(diào)控作用。產(chǎn)孢能力降低,從而使其致病能力減弱。此外,T-DNA插入還破壞了基因UVRASGAP的表達(dá),下一步計劃則是對單個基因或是兩個基因進(jìn)行基因敲除、回補等方法進(jìn)一步研究基因功能,對稻曲病菌致病過程的解析具有重要意義。2、真菌對鐵離子的運輸有著特殊的機制。鐵轉(zhuǎn)運蛋白參與細(xì)胞內(nèi)鐵離子的跨膜運輸,有時也可作為二級調(diào)控影響基因的表達(dá)和機制,對保持體內(nèi)鐵離子的平衡和菌株致病因子的產(chǎn)生具有重要意義。Uvt3277是低親和力鐵轉(zhuǎn)運蛋白,且有研究闡明可能與稻曲病菌的致病過程相關(guān)。本研究則對基因Uvt3277的功能進(jìn)行進(jìn)一步地驗證。構(gòu)建發(fā)夾結(jié)構(gòu)沉默載體,用農(nóng)桿菌介導(dǎo)轉(zhuǎn)化的方法(ATMT)導(dǎo)入稻曲病菌中:分析沉默轉(zhuǎn)化子的表型,來驗證低鐵轉(zhuǎn)運蛋白在稻曲病菌的生長發(fā)育、致病性的關(guān)系。結(jié)果發(fā)現(xiàn)沉默菌株的生長速率與野生菌株P(guān)1相比有一定的降低,但基因沉默效率與生長速率之間沒有明顯的線性關(guān)系,說明基因Uvt3277可能協(xié)同其他調(diào)控因子影響稻曲病菌的生長發(fā)育。鐵脅迫性測定發(fā)現(xiàn)在一定的Fe2+濃度下,沉默菌株能夠利用Fe2+正常生長。另外,沉默菌株的致病能力都比P1強,表明鐵轉(zhuǎn)運蛋白在一定程度上影響到致病相關(guān)的負(fù)調(diào)控途徑。同時酵母雙雜交技術(shù)篩選出低鐵轉(zhuǎn)運蛋白的互作蛋白,從而進(jìn)一步為稻曲病菌致病機理的研究提供理論基礎(chǔ)。但是篩選到的候選蛋白太少,只得到兩個疑似蛋白:ATP/ADP carrier(AAC)protein和SH3 domain-containing protein,因此還需進(jìn)行驗證或重新酵母雙雜交篩選。
[Abstract]:Rice (Oryza sativa L.) is a disease of the ear part of rice, and is now the main disease of the secondary diseases. Utilaginoidea (Utilaginoidea) is a pathogenic fungus of rice-koji disease. The rice-koji balls formed in the ear part seriously affect the quality and yield of rice, and the toxin produced by rice-yeast can be harmful to the health of human and animal. Therefore, it is possible to provide a theoretical basis for the prevention and treatment of rice scab.1. A mutant strain B2510 with reduced pathogenicity is selected from T-DNA insertion mutant library. By analyzing the biological character of the mutant strain B2510 and the flanking gene of the T-DNA insertion site, the invention provides a method and a theoretical basis for the research on the molecular pathogenesis of the rice koji. The mutant strain B2510, compared to the wild type P1, lost the sporulation capacity and the growth rate was significantly reduced on the MM medium, while there was no significant difference in the PSA and TB3 medium. Southern blot analysis of the copy number of T-DNA results in the insertion of T-DNA into the genome of the mutant strain B2510 in a double-copy form. The flanking sequence of the T-DNA insertion site was amplified by using the hiTAIL-PCR technique to obtain two possible pathogenicity-related gene UVC6TF and UVRASGAP. T-DNA was inserted in the promoter region of the gene UVC6TF and the downstream 3 'end of the UVRASGAP, respectively, and both the rice-koji genome and the T-DNA sequence were not lost. The expression of the two genes in the mutant strain B2510 was analyzed by semi-quantitative RT-PCR. Therefore, it is presumed that the two genes are related to the pathogenicity of the rice-koji, and may be involved in the control of the pathogenic process of the rice-koji germs on the rice in a certain stage. In order to further understand the function of the gene, the silencing vector of the gene UVC6TF was constructed, and the rice koji was introduced by the method of ATMT. The expression of UVC6TF in the obtained 2 silent transformants was inhibited by 50% and 90%, respectively. The results show that the gene does not affect the normal growth and development of the strain, but has a certain regulation effect on the production process of the conidia. The ability to produce a spore is reduced, so that its pathogenic ability is reduced. In addition, that T-DNA insertion also destroy the expression of the gene UVRASGAP, and the next step is to further study the gene function of a single gene or two genes, such as gene knock-out and back-repair, which is of great significance to the analysis of the pathogenic process of the rice koji. The fungi have a special mechanism for the transport of iron ions. The iron transporter participates in the transmembrane transport of iron ions in the cell, and can also be used as the expression and mechanism of the secondary regulation and influence gene, which is of great significance for maintaining the balance of iron ions in the body and the generation of the pathogenic factors of the strain. Uvt3277 is a low-affinity iron transporter and has been shown to be related to the pathogenesis of rice-koji. This study further verifies the function of the gene Uvt3277. To construct the silencing vector of the hairpin structure, the method of Agrobacterium-mediated transformation (ATMT) was used to introduce the phenotype of the silencing transformant to verify the relationship between the growth and the pathogenicity of the low-iron transporter in the rice koji. The results showed that the growth rate of the silent strain is lower than that of the wild strain P1, but there is no obvious linear relation between the efficiency of the gene silencing and the growth rate. The determination of iron stress shows that the silent strain can grow with Fe2 + under a certain Fe2 + concentration. In addition, the pathogenic ability of the silent strain is higher than that of P1, indicating that the iron transporter has a certain degree of negative regulation of the disease. At the same time, the yeast two-hybrid technology is used to screen the interaction protein of the low iron transporter, thus further providing a theoretical basis for the research of the pathogenic mechanism of the rice koji. However, too few candidate proteins are screened, and only two suspected proteins are available: ATP/ ADP carrier (AAC) protein and SH3 domain-containing protein, and therefore, verification or re-yeast two-hybrid screening is required.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S435.111.4
本文編號：2502457
[Abstract]:Rice (Oryza sativa L.) is a disease of the ear part of rice, and is now the main disease of the secondary diseases. Utilaginoidea (Utilaginoidea) is a pathogenic fungus of rice-koji disease. The rice-koji balls formed in the ear part seriously affect the quality and yield of rice, and the toxin produced by rice-yeast can be harmful to the health of human and animal. Therefore, it is possible to provide a theoretical basis for the prevention and treatment of rice scab.1. A mutant strain B2510 with reduced pathogenicity is selected from T-DNA insertion mutant library. By analyzing the biological character of the mutant strain B2510 and the flanking gene of the T-DNA insertion site, the invention provides a method and a theoretical basis for the research on the molecular pathogenesis of the rice koji. The mutant strain B2510, compared to the wild type P1, lost the sporulation capacity and the growth rate was significantly reduced on the MM medium, while there was no significant difference in the PSA and TB3 medium. Southern blot analysis of the copy number of T-DNA results in the insertion of T-DNA into the genome of the mutant strain B2510 in a double-copy form. The flanking sequence of the T-DNA insertion site was amplified by using the hiTAIL-PCR technique to obtain two possible pathogenicity-related gene UVC6TF and UVRASGAP. T-DNA was inserted in the promoter region of the gene UVC6TF and the downstream 3 'end of the UVRASGAP, respectively, and both the rice-koji genome and the T-DNA sequence were not lost. The expression of the two genes in the mutant strain B2510 was analyzed by semi-quantitative RT-PCR. Therefore, it is presumed that the two genes are related to the pathogenicity of the rice-koji, and may be involved in the control of the pathogenic process of the rice-koji germs on the rice in a certain stage. In order to further understand the function of the gene, the silencing vector of the gene UVC6TF was constructed, and the rice koji was introduced by the method of ATMT. The expression of UVC6TF in the obtained 2 silent transformants was inhibited by 50% and 90%, respectively. The results show that the gene does not affect the normal growth and development of the strain, but has a certain regulation effect on the production process of the conidia. The ability to produce a spore is reduced, so that its pathogenic ability is reduced. In addition, that T-DNA insertion also destroy the expression of the gene UVRASGAP, and the next step is to further study the gene function of a single gene or two genes, such as gene knock-out and back-repair, which is of great significance to the analysis of the pathogenic process of the rice koji. The fungi have a special mechanism for the transport of iron ions. The iron transporter participates in the transmembrane transport of iron ions in the cell, and can also be used as the expression and mechanism of the secondary regulation and influence gene, which is of great significance for maintaining the balance of iron ions in the body and the generation of the pathogenic factors of the strain. Uvt3277 is a low-affinity iron transporter and has been shown to be related to the pathogenesis of rice-koji. This study further verifies the function of the gene Uvt3277. To construct the silencing vector of the hairpin structure, the method of Agrobacterium-mediated transformation (ATMT) was used to introduce the phenotype of the silencing transformant to verify the relationship between the growth and the pathogenicity of the low-iron transporter in the rice koji. The results showed that the growth rate of the silent strain is lower than that of the wild strain P1, but there is no obvious linear relation between the efficiency of the gene silencing and the growth rate. The determination of iron stress shows that the silent strain can grow with Fe2 + under a certain Fe2 + concentration. In addition, the pathogenic ability of the silent strain is higher than that of P1, indicating that the iron transporter has a certain degree of negative regulation of the disease. At the same time, the yeast two-hybrid technology is used to screen the interaction protein of the low iron transporter, thus further providing a theoretical basis for the research of the pathogenic mechanism of the rice koji. However, too few candidate proteins are screened, and only two suspected proteins are available: ATP/ ADP carrier (AAC) protein and SH3 domain-containing protein, and therefore, verification or re-yeast two-hybrid screening is required.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S435.111.4
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 卞英芳;于莎莎;牟仁祥;曹趙云;孫偉華;楊歡;林曉燕;陳銘學(xué);;高效液相色譜-高分辨質(zhì)譜法鑒定水稻稻曲病菌毒素[J];色譜;2015年10期
2 簡迎龍;;稻曲病的侵染規(guī)律與防治[J];江西農(nóng)業(yè);2015年08期
3 黃文藝;;淺談水稻稻曲病的發(fā)生規(guī)律及防治措施[J];農(nóng)業(yè)與技術(shù);2015年14期
4 王文斌;張榮勝;羅楚平;尹小樂;劉永鋒;陸凡;陳志誼;;中國主要稻區(qū)稻曲病菌的生物學(xué)特性及群體遺傳多樣性[J];中國農(nóng)業(yè)科學(xué);2014年14期
5 黃磊;胡建坤;俞咪娜;于俊杰;王亞會;鄭夢婷;鄭睿;劉永鋒;;稻曲病菌致病力喪失突變菌株B-1015的T-DNA標(biāo)記基因的克隆[J];中國農(nóng)業(yè)科學(xué);2014年13期
6 徐秋芳;陳晴晴;張金鳳;李碩;倪海平;周益軍;;酵母雙雜交篩選灰飛虱體內(nèi)水稻黑條矮縮病毒p10的互作蛋白[J];中國水稻科學(xué);2013年06期
7 俞咪娜;胡建坤;黃磊;于俊杰;尹小樂;聶亞鋒;陳志誼;劉永鋒;;稻曲病菌T-DNA插入突變體5062的插入位點分析[J];中國農(nóng)業(yè)科學(xué);2013年09期
8 李素貞;陳景堂;;植物鋅鐵轉(zhuǎn)運相關(guān)蛋白家族的研究進(jìn)展[J];生物技術(shù)通報;2013年02期
9 趙偉;黃杰;汪濤;戚仁德;;RNA干擾技術(shù)在真菌基因功能研究中的應(yīng)用[J];合肥師范學(xué)院學(xué)報;2012年06期
10 于俊杰;尹小樂;陳志誼;徐文秀;俞咪娜;胡建坤;劉永鋒;;稻曲菌交配型初探[J];植物病理學(xué)報;2012年06期
相關(guān)會議論文 前1條
1 劉永鋒;陳志誼;俞咪娜;于俊杰;胡建坤;聶亞鋒;;農(nóng)桿菌介導(dǎo)的水稻稻曲病菌突變體庫的構(gòu)建及其評價[A];中國植物病理學(xué)會2011年學(xué)術(shù)年會論文集[C];2011年
相關(guān)博士學(xué)位論文 前1條
1 陸孫杰;水稻OsMADS15基因功能研究及用酵母雙雜篩選互作蛋白[D];浙江大學(xué);2012年
相關(guān)碩士學(xué)位論文 前5條
1 鄭夢婷;稻曲病菌T-DNA插入突變體B1178、B3277側(cè)翼致病相關(guān)基因的克隆及功能初步分析[D];南京農(nóng)業(yè)大學(xué);2014年
2 張莉林;294個稻瘟病菌轉(zhuǎn)錄因子基因的敲除和功能分析[D];浙江大學(xué);2013年
3 王云艷;黑曲霉RNAi應(yīng)用的研究[D];華南理工大學(xué);2012年
4 丁正中;稻曲病菌突變體庫的構(gòu)建及其HOG1基因的克隆和功能初步分析[D];浙江師范大學(xué);2012年
5 張瑩;馬鈴薯StERF3轉(zhuǎn)錄因子的功能分析及其啟動子的克隆[D];華中農(nóng)業(yè)大學(xué);2009年
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