水稻外分泌蛋白与白叶枯病相关性的研究
发布时间：2018-07-14 18:17
【摘要】：水稻白叶枯病(Bacterial Blight, BB)是一种由革兰氏阴性细菌黄单胞杆菌水稻变种(Xanthomonas Oryzae pv. Oryzae, Xoo)引起的水稻细菌性维管束病害,疣粒野生稻(Oryza meyeriana L.)对白叶枯病表现为免疫。本研究以疣粒野生稻和感病水稻品种日本晴为研究对象,分析了外分泌蛋白质在抗/感水稻与白叶枯病菌互作中的作用,具体的研究结果如下：1.利用Semi-Quantitative PCR和Quantitative Real Time PCR分析14个受Xoo诱导的疣粒野生稻差异蛋白的转录水平,结果表明其中8个差异蛋白的转录水平与其蛋白表达水平一致。另外,疣粒野生稻中过氧化酶活性的本底水平约是日本晴中的3.1倍,而且二者叶片中该酶活性均受Xoo侵染的诱导而上调。2.从疣粒野生稻Xoo应答蛋白中选取两个信号蛋白(OmCS1、OmSCP8)及两个病程相关蛋白(OmPOP11、OmR40c1)进行基因功能的初步分析。OmCS1蛋白被预测带有一个CHORD-SGT1 (CS)结构域。过量表达OmCS1的水稻能够显著提升对白叶枯病的耐病性,而且该过表达株系中参与SA合成与积累的关键基因OsPAL和OsNPR1的表达均上调,我们推测OmCS1的耐病性可能与SA抗病信号途径关。OmPOP11被预测是一个带有Prolyl OligoPeptidase (POP)活性的蛋白。过量表达OsPOP11的水稻也能够显著提升对白叶枯病的耐病性,其具体的耐病机制尚待进一步研究。OmSCP8是一个带有Serine CarboxyPeptidases (SC)结构域的蛋白,与Brassinosteroids (BR)信号途径中的关键蛋白BRS1属于同源蛋白。OmSCP8转基因水稻也在一定程度上提高了对白叶枯病的耐病性,而且在该转基因株系中,参与SA合成与积累的关键基因OsPAL也上调表达。3. OmR40c1被预测是一个带有RicinB(蓖麻凝集素)结构域的蛋白,该蛋白定位于细胞质膜上,且能与血红细胞发生凝集作用。部分PR基因、SA和JA合成与积累的关键基因在OmR40c1转基因水稻中均下调表达。对OmR40c1和5’UTR-OmR40c1转基因水稻分别接种白叶枯病菌生理小种P10(PXO124)和P8(PXO280),二者的病斑长度与野生型相似,该结果暗示OmR40c1可能不参与水稻对白叶枯病的抗性。然而在盐胁迫下,OmR40c1转基因水稻种子的发芽率、株高、Na+及K+含量均高于野生型,该结果暗示OmR40c1在一定程度上提高了水稻的耐盐性,且实验表明这种耐性的强度在苗期高于成株期。4.采用2D-DIGE (Two-Dimensional Difference In Gel Electrophoresis)分析白叶枯病胁迫下感病水稻日本晴悬浮细胞的外分泌蛋白质组的变化,结果发现30个蛋白点的表达量上调,2个下调。质谱鉴定为7个水稻蛋白和4个白叶枯病菌蛋白,生物信息学分析预测其中7个水稻蛋白分别参与水稻防御、氧化还原及细胞壁修饰等生理过程,亚细胞定位实验表明其中3个白叶枯病菌蛋白均分布在细胞膜上。在白叶枯病菌P10(PXO124)中过量表达其中一个蛋白点Xoo3654对应的基因,一定程度上弱化了白叶枯病菌的致病性,推测该基因可能作为一个负调控因子参与了病原菌的致病性。5.壳聚糖酶OsCHIT16是响应白叶枯病菌侵染的日本晴差异蛋白,来源于疣粒野生稻和日本晴的CHIT16等位基因在编码区域存在差异,但二者均定位于细胞质膜上。OsCHIT16和OmCHIT16转基因水稻均增强了对白叶枯病菌的耐受性,部分PR (Pathogenesis Related Protein)基因、参与JA(Jasmonic Acid)和SA(Salicylic Acid)合成与积累的关键基因在转基因水稻中均下调表达。根据以上结果推测栽培稻和疣粒野生稻CHIT16等位基因增强水稻对白叶枯病菌的耐病性可能不依赖于JA和SA途径,且二者间序列的差异并不影响CHIT16基因功能。综上所述,本研究利用2D-DIGE分析了白叶枯病菌胁迫下感病水稻日本晴悬浮细胞的外分泌蛋白质组的变化,发现其中一个蛋白点Xoo3654可能是一个致病负调控因子。并且,本研究发现过量表达OmCS1、OsPOP11、OmSCP8、 OsCH1T16及OmCHIT16等基因均能增强水稻的耐病性。本研究为分析疣粒野生稻的抗病机理提供了新的理论依据,为研究其它植物与病原细菌的互作提供了新参考。
[Abstract]:Rice bacterial blight (Bacterial Blight, BB) is a bacterial vascular disease of rice caused by Gram negative bacilli (Xanthomonas Oryzae pv. Oryzae, Xoo), and verruca wild rice (Oryza meyeriana L.) is immune to white leaf blight. This study was made of wart wild rice and susceptible rice varieties in Japan. The effects of exocrine protein on the interaction of resistant / susceptible rice and bacterial leaf blight were analyzed. The specific results were as follows: 1. the transcriptional water level of 14 Xoo induced verruca wild rice was analyzed by Semi-Quantitative PCR and Quantitative Real Time PCR, and the results showed that 8 of the different proteins were transcribed. In addition, the level of peroxidase activity in verruca wild rice was about 3.1 times more than that in Japan, and the activity of this enzyme in the two leaves was all induced by Xoo infection and up regulated by.2., two signal proteins (OmCS1, OmSCP8) and two course related proteins (OmPOP11, OmSCP8) were selected from the Xoo response protein of verruca wild rice. OmR40c1) preliminary analysis of gene function.OmCS1 protein was predicted to have a CHORD-SGT1 (CS) domain. Excessive expression of OmCS1 in rice could significantly increase the resistance to blight of white leaf blight, and the expression of OsPAL and OsNPR1, the key gene involved in SA synthesis and accumulation in the overexpressed strain, was up-regulated, and we speculated the disease resistance of OmCS1. .OmPOP11 may be predicted to be a protein with Prolyl OligoPeptidase (POP) activity with the SA disease resistance signal pathway. Excessive expression of OsPOP11 in rice can also significantly increase the resistance to blight of white leaf blight. Its specific tolerance mechanism remains to be further studied by.OmSCP8, a protein with Serine CarboxyPeptidases (SC) domain. The key protein BRS1 in the Brassinosteroids (BR) signal pathway belongs to the homologous protein.OmSCP8 transgenic rice, which also improves the resistance to blight of white leaf blight to some extent, and in the transgenic line, the key gene OsPAL that participates in SA synthesis and accumulation is also up to up expression.3. OmR40c1 is predicted to be a RicinB (Castor agglutination). Protein in the domain of the domain, which locates on the cytoplasmic membrane and can agglutinate with blood red cells. Some of the key genes for the synthesis and accumulation of PR, SA and JA are downregulated in OmR40c1 transgenic rice. P10 (PXO124) and P8 (PXO280), respectively, for OmR40c1 and 5 'UTR-OmR40c1 transgenic rice, respectively. The length of the two was similar to the wild type. The results suggested that OmR40c1 might not be involved in the resistance to rice blight. However, under salt stress, the germination rate, plant height, Na+ and K+ content of OmR40c1 transgenic rice seeds were higher than those of the wild type. The results suggested that OmR40c1 increased the salt tolerance of rice to a certain extent, and the experiment showed this The strength of seed tolerance in seedling stage was higher than that of adult plant stage.4. using 2D-DIGE (Two-Dimensional Difference In Gel Electrophoresis) to analyze the changes in exocrine protein group of Japanese clear suspension cells under bacterial blight under the stress of white leaf blight. The results showed that the expression of 30 protein spots was up and 2 down regulated. The mass spectrum was identified as 7 rice proteins and 4 The protein, bioinformatics analysis and bioinformatics analysis predicted that 7 of the rice proteins were involved in the physiological processes of rice defense, redox and cell wall modification. The subcellular localization experiment showed that 3 of them were distributed on the cell membrane. One protein point Xoo3654 was overexpressed in the P10 (PXO124) of the bacterial leaf blight pathogen. The corresponding gene weakens the pathogenicity of the bacterial leaf blight to some extent. It is presumed that the gene may participate in the pathogenic.5. chitosan enzyme OsCHIT16 as a negative regulator, which is the Japanese clear difference protein in response to the infection of the pathogen of the bacterial leaf blight, which is derived from the CHIT16 alleles of the wild rice and the Japanese clear region in the coding region. In the difference, both.OsCHIT16 and OmCHIT16 transgenic rice were both located on the cytoplasmic membrane, and both.OsCHIT16 and OmCHIT16 transgenic rice enhanced the tolerance to the bacterial leaf blight. Some PR (Pathogenesis Related Protein) genes, the key genes involved in the synthesis and accumulation of JA (Jasmonic Acid) and SA (Salicylic Acid) were down regulated in transgenic rice. It is presumed that the resistance of rice to Rice Leaf Blight by CHIT16 allele of cultivated rice and verruca wild rice may not depend on the JA and SA pathway, and the difference of the sequence between the two does not affect the function of the CHIT16 gene. In this study, the exocrine egg of the Japanese clear suspension cell of the infected rice under the stress of the bacterial leaf blight was analyzed by 2D-DIGE. The changes in the white matter group found that one of the protein points Xoo3654 may be a pathogenic negative regulator. Moreover, this study found that overexpression of OmCS1, OsPOP11, OmSCP8, OsCH1T16 and OmCHIT16 could enhance the tolerance to rice. This study provides a new theoretical basis for the analysis of the resistance mechanism of wart wild rice, and for the study of other plants. The interaction of pathogens and pathogenic bacteria provides a new reference.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.111.47
，
本文编号：2122554
[Abstract]:Rice bacterial blight (Bacterial Blight, BB) is a bacterial vascular disease of rice caused by Gram negative bacilli (Xanthomonas Oryzae pv. Oryzae, Xoo), and verruca wild rice (Oryza meyeriana L.) is immune to white leaf blight. This study was made of wart wild rice and susceptible rice varieties in Japan. The effects of exocrine protein on the interaction of resistant / susceptible rice and bacterial leaf blight were analyzed. The specific results were as follows: 1. the transcriptional water level of 14 Xoo induced verruca wild rice was analyzed by Semi-Quantitative PCR and Quantitative Real Time PCR, and the results showed that 8 of the different proteins were transcribed. In addition, the level of peroxidase activity in verruca wild rice was about 3.1 times more than that in Japan, and the activity of this enzyme in the two leaves was all induced by Xoo infection and up regulated by.2., two signal proteins (OmCS1, OmSCP8) and two course related proteins (OmPOP11, OmSCP8) were selected from the Xoo response protein of verruca wild rice. OmR40c1) preliminary analysis of gene function.OmCS1 protein was predicted to have a CHORD-SGT1 (CS) domain. Excessive expression of OmCS1 in rice could significantly increase the resistance to blight of white leaf blight, and the expression of OsPAL and OsNPR1, the key gene involved in SA synthesis and accumulation in the overexpressed strain, was up-regulated, and we speculated the disease resistance of OmCS1. .OmPOP11 may be predicted to be a protein with Prolyl OligoPeptidase (POP) activity with the SA disease resistance signal pathway. Excessive expression of OsPOP11 in rice can also significantly increase the resistance to blight of white leaf blight. Its specific tolerance mechanism remains to be further studied by.OmSCP8, a protein with Serine CarboxyPeptidases (SC) domain. The key protein BRS1 in the Brassinosteroids (BR) signal pathway belongs to the homologous protein.OmSCP8 transgenic rice, which also improves the resistance to blight of white leaf blight to some extent, and in the transgenic line, the key gene OsPAL that participates in SA synthesis and accumulation is also up to up expression.3. OmR40c1 is predicted to be a RicinB (Castor agglutination). Protein in the domain of the domain, which locates on the cytoplasmic membrane and can agglutinate with blood red cells. Some of the key genes for the synthesis and accumulation of PR, SA and JA are downregulated in OmR40c1 transgenic rice. P10 (PXO124) and P8 (PXO280), respectively, for OmR40c1 and 5 'UTR-OmR40c1 transgenic rice, respectively. The length of the two was similar to the wild type. The results suggested that OmR40c1 might not be involved in the resistance to rice blight. However, under salt stress, the germination rate, plant height, Na+ and K+ content of OmR40c1 transgenic rice seeds were higher than those of the wild type. The results suggested that OmR40c1 increased the salt tolerance of rice to a certain extent, and the experiment showed this The strength of seed tolerance in seedling stage was higher than that of adult plant stage.4. using 2D-DIGE (Two-Dimensional Difference In Gel Electrophoresis) to analyze the changes in exocrine protein group of Japanese clear suspension cells under bacterial blight under the stress of white leaf blight. The results showed that the expression of 30 protein spots was up and 2 down regulated. The mass spectrum was identified as 7 rice proteins and 4 The protein, bioinformatics analysis and bioinformatics analysis predicted that 7 of the rice proteins were involved in the physiological processes of rice defense, redox and cell wall modification. The subcellular localization experiment showed that 3 of them were distributed on the cell membrane. One protein point Xoo3654 was overexpressed in the P10 (PXO124) of the bacterial leaf blight pathogen. The corresponding gene weakens the pathogenicity of the bacterial leaf blight to some extent. It is presumed that the gene may participate in the pathogenic.5. chitosan enzyme OsCHIT16 as a negative regulator, which is the Japanese clear difference protein in response to the infection of the pathogen of the bacterial leaf blight, which is derived from the CHIT16 alleles of the wild rice and the Japanese clear region in the coding region. In the difference, both.OsCHIT16 and OmCHIT16 transgenic rice were both located on the cytoplasmic membrane, and both.OsCHIT16 and OmCHIT16 transgenic rice enhanced the tolerance to the bacterial leaf blight. Some PR (Pathogenesis Related Protein) genes, the key genes involved in the synthesis and accumulation of JA (Jasmonic Acid) and SA (Salicylic Acid) were down regulated in transgenic rice. It is presumed that the resistance of rice to Rice Leaf Blight by CHIT16 allele of cultivated rice and verruca wild rice may not depend on the JA and SA pathway, and the difference of the sequence between the two does not affect the function of the CHIT16 gene. In this study, the exocrine egg of the Japanese clear suspension cell of the infected rice under the stress of the bacterial leaf blight was analyzed by 2D-DIGE. The changes in the white matter group found that one of the protein points Xoo3654 may be a pathogenic negative regulator. Moreover, this study found that overexpression of OmCS1, OsPOP11, OmSCP8, OsCH1T16 and OmCHIT16 could enhance the tolerance to rice. This study provides a new theoretical basis for the analysis of the resistance mechanism of wart wild rice, and for the study of other plants. The interaction of pathogens and pathogenic bacteria provides a new reference.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.111.47
，
本文编号：2122554
本文链接：https://www.wllwen.com/shoufeilunwen/nykjbs/2122554.html
