水稻白叶枯病菌无毒基因avrXa23的克隆及与AvrXa23互作的水稻蛋白的功能分析
发布时间：2018-09-06 13:50
【摘要】：由黄单胞菌水稻致病变种(Xanthomonas oryzae pv.oryzae,Xoo)引起的水稻白叶枯病是影响水稻生产的重要病害之一,严重制约水稻高产和稳产。水稻与Xoo的互作是研究植物与病原物互作的模式系统之一。寄主植物水稻(粳稻品种日本晴和籼稻品种93-11等)和病原菌Xoo(韩国菌株KACC10331、日本菌株MAFF311018和菲律宾菌株PXO99A等)的全基因组测序已经完成,为深入研究水稻与Xoo的互作奠定了生物信息基础。本实验室前期利用EZ-Tn5转座系统,构建了白叶枯病原菌PXO99A的突变体库,通过筛选PXO99A的突变体库,获得14个使CBB23感病的突变菌株。在此基础上,对这14个突变菌株的Tn5插入位点进行侧翼序列分析,发现有3个菌株Tn5插入在TALE(Transcription activator-like effectors)类基因的位置,这3个菌株分别命名为P99M2、P99M4和P99M5。本论文分为两部分:第一部分是克隆与水稻白叶枯病广谱抗病基因Xa23对应的无毒基因avr Xa23;第二部分是筛选水稻中与Avr Xa23互作的蛋白,并对相关蛋白及其编码基因进行功能分析。获得的主要结果如下:1.通过筛选PXO99A的Cosmid基因组文库,获得34个含有TALE类基因的Cosmid质粒(简称C1-C34)。将这34个Cosmid质粒转化到突变体菌P99M5中,发现C15转化进入P99M5后能够激发CBB23的抗病性,说明C15含有与Xa23基因对应的无毒基因。2.经过测序分析,发现C15中含有tal C9a和tal C9b两个候选的无毒基因。构建tal C9a和tal C9b的表达载体,分别命名为p HM1-Mi和p HM1-L。p HM1-L转化进入P99M5等突变体菌中能够使CBB23抗病,而p HM1-Mi无此功能。说明tal C9b是与Xa23相对应的Xoo无毒基因avr Xa23。3.avr Xa23是TALE类基因,全长4452bp,编码蛋白含1483氨基酸。通过Southern blot方法,证实avr Xa23存在于国内外Xoo代表性菌株中。Avr Xa23可以激活水稻中抗病基因Xa23的表达,avr Xa23的广泛存在可以解释Xa23对于白叶枯病菌的广谱抗病性。4.构建PXO99A诱导CBB23的c DNA文库。以Avr Xa23为诱饵,筛选该c DNA文库,初筛获得323个侯选克隆。通过测序后排除移码、复筛进一步验证获得43个候选阳性克隆。这43个克隆编码26种蛋白质,其中7个可能与抗病相关。将这7个基因的c DNA全长扩增并构建成prey载体进行酵母双杂交验证,然后进行GST pull-down体外验证,最终证明3个水稻蛋白质(Os IMPα、Os THIC和Os THI1)与Avr Xa23互作。5.生物信息学分析表明,Os IMPα为核转运蛋白,可能辅助Avr Xa23进入细胞核;Os THIC为嘧啶环前体合成酶,Os THI1为噻唑合成酶,两者都是维生素B1合成的关键酶。维生素B1作为植物抗病反应的激活剂,通过水杨酸和Ca2+相关的信号途径,使植物产生系统获得性抗性,对水稻抵抗Xoo的侵染具有重要作用。6.进一步的实验研究发现,Os THI1与Avr Xa23的N端互作;Os THI1定位在叶绿体上;利用CRISPR/Cas9技术敲除水稻近等基因系CBB4(含Xa4,抗白叶枯病原菌PXO61)中的Os THI1,获得的转基因植株叶片维生素B1含量下降,且对PXO61感病。总之,本研究成功克隆到对应于Xa23的无毒基因avr Xa23,该无毒基因广泛存在于检测的所有Xoo生理小种中,解释了Xa23对白叶枯病菌的广谱抗病机理。筛选到水稻中与Avr Xa23互作的3个蛋白:核转运蛋白Os IMPα以及参与维生素B1合成的Os THIC和Os THI1。维生素B1对水稻的获得性抗性很重要,我们推测在缺少Xa23基因的水稻中,Avr Xa23可能通过结合维生素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.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.111.47
本文编号：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.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.111.47
【参考文献】
相关期刊论文 前1条
1 高东迎,刘蔼民,周亦红,程艳军,向阳海,孙立华,翟文学;水稻抗白叶枯病基因Xa-25的分子定位[J];遗传学报;2005年02期
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