水稻白葉枯病菌無毒基因avrXa23的克隆及與AvrXa23互作的水稻蛋白的功能分析
發(fā)布時(shí)間：2018-09-06 13:50
【摘要】：由黃單胞菌水稻致病變種(Xanthomonas oryzae pv.oryzae,Xoo)引起的水稻白葉枯病是影響水稻生產(chǎn)的重要病害之一,嚴(yán)重制約水稻高產(chǎn)和穩(wěn)產(chǎn)。水稻與Xoo的互作是研究植物與病原物互作的模式系統(tǒng)之一。寄主植物水稻(粳稻品種日本晴和秈稻品種93-11等)和病原菌Xoo(韓國菌株KACC10331、日本菌株MAFF311018和菲律賓菌株P(guān)XO99A等)的全基因組測序已經(jīng)完成,為深入研究水稻與Xoo的互作奠定了生物信息基礎(chǔ)。本實(shí)驗(yàn)室前期利用EZ-Tn5轉(zhuǎn)座系統(tǒng),構(gòu)建了白葉枯病原菌PXO99A的突變體庫,通過篩選PXO99A的突變體庫,獲得14個(gè)使CBB23感病的突變菌株。在此基礎(chǔ)上,對這14個(gè)突變菌株的Tn5插入位點(diǎn)進(jìn)行側(cè)翼序列分析,發(fā)現(xiàn)有3個(gè)菌株Tn5插入在TALE(Transcription activator-like effectors)類基因的位置,這3個(gè)菌株分別命名為P99M2、P99M4和P99M5。本論文分為兩部分:第一部分是克隆與水稻白葉枯病廣譜抗病基因Xa23對應(yīng)的無毒基因avr Xa23;第二部分是篩選水稻中與Avr Xa23互作的蛋白,并對相關(guān)蛋白及其編碼基因進(jìn)行功能分析。獲得的主要結(jié)果如下:1.通過篩選PXO99A的Cosmid基因組文庫,獲得34個(gè)含有TALE類基因的Cosmid質(zhì)粒(簡稱C1-C34)。將這34個(gè)Cosmid質(zhì)粒轉(zhuǎn)化到突變體菌P99M5中,發(fā)現(xiàn)C15轉(zhuǎn)化進(jìn)入P99M5后能夠激發(fā)CBB23的抗病性,說明C15含有與Xa23基因?qū)?yīng)的無毒基因。2.經(jīng)過測序分析,發(fā)現(xiàn)C15中含有tal C9a和tal C9b兩個(gè)候選的無毒基因。構(gòu)建tal C9a和tal C9b的表達(dá)載體,分別命名為p HM1-Mi和p HM1-L。p HM1-L轉(zhuǎn)化進(jìn)入P99M5等突變體菌中能夠使CBB23抗病,而p HM1-Mi無此功能。說明tal C9b是與Xa23相對應(yīng)的Xoo無毒基因avr Xa23。3.avr Xa23是TALE類基因,全長4452bp,編碼蛋白含1483氨基酸。通過Southern blot方法,證實(shí)avr Xa23存在于國內(nèi)外Xoo代表性菌株中。Avr Xa23可以激活水稻中抗病基因Xa23的表達(dá),avr Xa23的廣泛存在可以解釋Xa23對于白葉枯病菌的廣譜抗病性。4.構(gòu)建PXO99A誘導(dǎo)CBB23的c DNA文庫。以Avr Xa23為誘餌,篩選該c DNA文庫,初篩獲得323個(gè)侯選克隆。通過測序后排除移碼、復(fù)篩進(jìn)一步驗(yàn)證獲得43個(gè)候選陽性克隆。這43個(gè)克隆編碼26種蛋白質(zhì),其中7個(gè)可能與抗病相關(guān)。將這7個(gè)基因的c DNA全長擴(kuò)增并構(gòu)建成prey載體進(jìn)行酵母雙雜交驗(yàn)證,然后進(jìn)行GST pull-down體外驗(yàn)證,最終證明3個(gè)水稻蛋白質(zhì)(Os IMPα、Os THIC和Os THI1)與Avr Xa23互作。5.生物信息學(xué)分析表明,Os IMPα為核轉(zhuǎn)運(yùn)蛋白,可能輔助Avr Xa23進(jìn)入細(xì)胞核;Os THIC為嘧啶環(huán)前體合成酶,Os THI1為噻唑合成酶,兩者都是維生素B1合成的關(guān)鍵酶。維生素B1作為植物抗病反應(yīng)的激活劑,通過水楊酸和Ca2+相關(guān)的信號途徑,使植物產(chǎn)生系統(tǒng)獲得性抗性,對水稻抵抗Xoo的侵染具有重要作用。6.進(jìn)一步的實(shí)驗(yàn)研究發(fā)現(xiàn),Os THI1與Avr Xa23的N端互作;Os THI1定位在葉綠體上;利用CRISPR/Cas9技術(shù)敲除水稻近等基因系CBB4(含Xa4,抗白葉枯病原菌PXO61)中的Os THI1,獲得的轉(zhuǎn)基因植株葉片維生素B1含量下降,且對PXO61感病。總之,本研究成功克隆到對應(yīng)于Xa23的無毒基因avr Xa23,該無毒基因廣泛存在于檢測的所有Xoo生理小種中,解釋了Xa23對白葉枯病菌的廣譜抗病機(jī)理。篩選到水稻中與Avr Xa23互作的3個(gè)蛋白:核轉(zhuǎn)運(yùn)蛋白Os IMPα以及參與維生素B1合成的Os THIC和Os THI1。維生素B1對水稻的獲得性抗性很重要,我們推測在缺少Xa23基因的水稻中,Avr Xa23可能通過結(jié)合維生素B1合成酶Os THIC和Os THI1,抑制水稻的抗性。
[Abstract]:Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important diseases affecting rice production, which seriously restricts the high and stable yield of rice. Genome sequencing of species 93-11 and pathogenic bacteria Xoo (Korean strain KACC10331, Japanese strain MAFF311018 and Philippine strain PXO99A etc.) has been completed, which lays a bioinformatics foundation for further study of the interaction between rice and Xoo. Fourteen mutant strains susceptible to CBB23 were obtained from the mutant library of PXO99A. On this basis, the flanking sequences of the insertion sites of Tn5 in 14 mutant strains were analyzed. Three strains of Tn5 were found to be inserted into the positions of TALE (Transcription activator-like effectors) genes. These three strains were named P99M2, P99M4 and P99M5, respectively. The first part is to clone AVR Xa23, a non-toxic gene corresponding to the broad-spectrum resistance gene Xa23 of rice bacterial blight. The second part is to screen the protein interacting with Avr Xa23 in rice and analyze the function of the related protein and its coding gene. The main results are as follows: 1. The Cosmid genomic library of PXO99A was screened and obtained. Thirty-four Cosmid plasmids containing TALE-like genes (C1-C34) were obtained. These 34 Cosmid plasmids were transformed into mutant strain P99M5. It was found that C15 transformed into P99M5 could stimulate the disease resistance of CBB23, suggesting that C15 contained a non-toxic gene corresponding to Xa23 gene. 2. Sequencing analysis showed that C15 contained two candidate non-toxic bases, tal C9a and tal C9b. The expression vectors of Tal C9a and tal C9b, named P HM1-Mi and P HM1-L.p HM1-L, were constructed and transformed into mutant strains such as P99M5 to make CBB23 resistant, but p HM1-Mi had no such function. The results showed that tal C9b was a TALE-like gene with a length of 4452 BP and a protein containing 1483 amino acids. Avr Xa23 can activate the expression of Xa23 gene in rice. The widespread existence of AVR Xa23 can explain the broad-spectrum resistance of Xa23 to bacterial blight. 4. The C DNA Library of CBB23 induced by PXO99A was constructed. Avr Xa23 was used as bait to screen the C DNA library. 323 candidate clones were obtained. 43 candidate positive clones were identified by exclusion of code-shifting after sequencing. These 43 clones encoded 26 proteins, 7 of which may be related to disease resistance. The full-length C DNA of the seven genes were amplified and constructed into a prey vector for yeast two-hybrid verification. GST pull-down in vitro validation was carried out. Bioinformatics analysis showed that Os IMP alpha was a nuclear transporter and might assist Avr Xa23 to enter the nucleus; Os THIC was a pyrimidine ring precursor synthase; Os THI 1 was a thiazole synthase, both of which were key enzymes in the synthesis of vitamin B1. Vitamin B1 acted as a plant disease resistance transporter. Corresponding activators, through salicylic acid and Ca2+ related signaling pathways, make plants produce systemic acquired resistance and play an important role in rice resistance to Xoo infection. 6. Further experimental studies have shown that OsTHI 1 interacts with the N-terminal of Avr Xa23; OsTHI 1 is located in chloroplasts; and the Near-isogenic Line CBB4 (containing Xa4) is knocked out by CRISPR/Cas9 technology. Os THI 1 in the transgenic plants of resistance to bacterial leaf blight pathogen PXO61 decreased the content of vitamin B1 in the leaves and was susceptible to PXO61. In conclusion, AVR Xa23, a non-toxic gene, was cloned successfully. The non-toxic gene was widely found in all the Xoo physiological races tested and explained the broad-spectrum resistance mechanism of Xa23 to bacterial leaf blight. Three proteins interacting with Avr Xa23, Os IMP alpha and Os THIC and Os THI 1. Vitamin B1, which are involved in the synthesis of vitamin B1, were screened out to be important for rice acquired resistance. We speculate that Avr Xa23 may inhibit rice resistance by binding vitamin B1 synthase Os THIC and Os THI 1 in rice lacking Xa23 gene.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S435.111.47
本文編號：2226541
[Abstract]:Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important diseases affecting rice production, which seriously restricts the high and stable yield of rice. Genome sequencing of species 93-11 and pathogenic bacteria Xoo (Korean strain KACC10331, Japanese strain MAFF311018 and Philippine strain PXO99A etc.) has been completed, which lays a bioinformatics foundation for further study of the interaction between rice and Xoo. Fourteen mutant strains susceptible to CBB23 were obtained from the mutant library of PXO99A. On this basis, the flanking sequences of the insertion sites of Tn5 in 14 mutant strains were analyzed. Three strains of Tn5 were found to be inserted into the positions of TALE (Transcription activator-like effectors) genes. These three strains were named P99M2, P99M4 and P99M5, respectively. The first part is to clone AVR Xa23, a non-toxic gene corresponding to the broad-spectrum resistance gene Xa23 of rice bacterial blight. The second part is to screen the protein interacting with Avr Xa23 in rice and analyze the function of the related protein and its coding gene. The main results are as follows: 1. The Cosmid genomic library of PXO99A was screened and obtained. Thirty-four Cosmid plasmids containing TALE-like genes (C1-C34) were obtained. These 34 Cosmid plasmids were transformed into mutant strain P99M5. It was found that C15 transformed into P99M5 could stimulate the disease resistance of CBB23, suggesting that C15 contained a non-toxic gene corresponding to Xa23 gene. 2. Sequencing analysis showed that C15 contained two candidate non-toxic bases, tal C9a and tal C9b. The expression vectors of Tal C9a and tal C9b, named P HM1-Mi and P HM1-L.p HM1-L, were constructed and transformed into mutant strains such as P99M5 to make CBB23 resistant, but p HM1-Mi had no such function. The results showed that tal C9b was a TALE-like gene with a length of 4452 BP and a protein containing 1483 amino acids. Avr Xa23 can activate the expression of Xa23 gene in rice. The widespread existence of AVR Xa23 can explain the broad-spectrum resistance of Xa23 to bacterial blight. 4. The C DNA Library of CBB23 induced by PXO99A was constructed. Avr Xa23 was used as bait to screen the C DNA library. 323 candidate clones were obtained. 43 candidate positive clones were identified by exclusion of code-shifting after sequencing. These 43 clones encoded 26 proteins, 7 of which may be related to disease resistance. The full-length C DNA of the seven genes were amplified and constructed into a prey vector for yeast two-hybrid verification. GST pull-down in vitro validation was carried out. Bioinformatics analysis showed that Os IMP alpha was a nuclear transporter and might assist Avr Xa23 to enter the nucleus; Os THIC was a pyrimidine ring precursor synthase; Os THI 1 was a thiazole synthase, both of which were key enzymes in the synthesis of vitamin B1. Vitamin B1 acted as a plant disease resistance transporter. Corresponding activators, through salicylic acid and Ca2+ related signaling pathways, make plants produce systemic acquired resistance and play an important role in rice resistance to Xoo infection. 6. Further experimental studies have shown that OsTHI 1 interacts with the N-terminal of Avr Xa23; OsTHI 1 is located in chloroplasts; and the Near-isogenic Line CBB4 (containing Xa4) is knocked out by CRISPR/Cas9 technology. Os THI 1 in the transgenic plants of resistance to bacterial leaf blight pathogen PXO61 decreased the content of vitamin B1 in the leaves and was susceptible to PXO61. In conclusion, AVR Xa23, a non-toxic gene, was cloned successfully. The non-toxic gene was widely found in all the Xoo physiological races tested and explained the broad-spectrum resistance mechanism of Xa23 to bacterial leaf blight. Three proteins interacting with Avr Xa23, Os IMP alpha and Os THIC and Os THI 1. Vitamin B1, which are involved in the synthesis of vitamin B1, were screened out to be important for rice acquired resistance. We speculate that Avr Xa23 may inhibit rice resistance by binding vitamin B1 synthase Os THIC and Os THI 1 in rice lacking Xa23 gene.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S435.111.47
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 高東迎,劉藹民,周亦紅,程艷軍,向陽海,孫立華,翟文學(xué);水稻抗白葉枯病基因Xa-25的分子定位[J];遺傳學(xué)報(bào);2005年02期
，本文編號：2226541
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