水稻外分泌蛋白與白葉枯病相關性的研究
發(fā)布時間：2018-07-14 18:17
【摘要】：水稻白葉枯病(Bacterial Blight, BB)是一種由革蘭氏陰性細菌黃單胞桿菌水稻變種(Xanthomonas Oryzae pv. Oryzae, Xoo)引起的水稻細菌性維管束病害,疣粒野生稻(Oryza meyeriana L.)對白葉枯病表現(xiàn)為免疫。本研究以疣粒野生稻和感病水稻品種日本晴為研究對象,分析了外分泌蛋白質(zhì)在抗/感水稻與白葉枯病菌互作中的作用,具體的研究結果如下：1.利用Semi-Quantitative PCR和Quantitative Real Time PCR分析14個受Xoo誘導的疣粒野生稻差異蛋白的轉錄水平,結果表明其中8個差異蛋白的轉錄水平與其蛋白表達水平一致。另外,疣粒野生稻中過氧化酶活性的本底水平約是日本晴中的3.1倍,而且二者葉片中該酶活性均受Xoo侵染的誘導而上調(diào)。2.從疣粒野生稻Xoo應答蛋白中選取兩個信號蛋白(OmCS1、OmSCP8)及兩個病程相關蛋白(OmPOP11、OmR40c1)進行基因功能的初步分析。OmCS1蛋白被預測帶有一個CHORD-SGT1 (CS)結構域。過量表達OmCS1的水稻能夠顯著提升對白葉枯病的耐病性,而且該過表達株系中參與SA合成與積累的關鍵基因OsPAL和OsNPR1的表達均上調(diào),我們推測OmCS1的耐病性可能與SA抗病信號途徑關。OmPOP11被預測是一個帶有Prolyl OligoPeptidase (POP)活性的蛋白。過量表達OsPOP11的水稻也能夠顯著提升對白葉枯病的耐病性,其具體的耐病機制尚待進一步研究。OmSCP8是一個帶有Serine CarboxyPeptidases (SC)結構域的蛋白,與Brassinosteroids (BR)信號途徑中的關鍵蛋白BRS1屬于同源蛋白。OmSCP8轉基因水稻也在一定程度上提高了對白葉枯病的耐病性,而且在該轉基因株系中,參與SA合成與積累的關鍵基因OsPAL也上調(diào)表達。3. OmR40c1被預測是一個帶有RicinB(蓖麻凝集素)結構域的蛋白,該蛋白定位于細胞質(zhì)膜上,且能與血紅細胞發(fā)生凝集作用。部分PR基因、SA和JA合成與積累的關鍵基因在OmR40c1轉基因水稻中均下調(diào)表達。對OmR40c1和5’UTR-OmR40c1轉基因水稻分別接種白葉枯病菌生理小種P10(PXO124)和P8(PXO280),二者的病斑長度與野生型相似,該結果暗示OmR40c1可能不參與水稻對白葉枯病的抗性。然而在鹽脅迫下,OmR40c1轉基因水稻種子的發(fā)芽率、株高、Na+及K+含量均高于野生型,該結果暗示OmR40c1在一定程度上提高了水稻的耐鹽性,且實驗表明這種耐性的強度在苗期高于成株期。4.采用2D-DIGE (Two-Dimensional Difference In Gel Electrophoresis)分析白葉枯病脅迫下感病水稻日本晴懸浮細胞的外分泌蛋白質(zhì)組的變化,結果發(fā)現(xiàn)30個蛋白點的表達量上調(diào),2個下調(diào)。質(zhì)譜鑒定為7個水稻蛋白和4個白葉枯病菌蛋白,生物信息學分析預測其中7個水稻蛋白分別參與水稻防御、氧化還原及細胞壁修飾等生理過程,亞細胞定位實驗表明其中3個白葉枯病菌蛋白均分布在細胞膜上。在白葉枯病菌P10(PXO124)中過量表達其中一個蛋白點Xoo3654對應的基因,一定程度上弱化了白葉枯病菌的致病性,推測該基因可能作為一個負調(diào)控因子參與了病原菌的致病性。5.殼聚糖酶OsCHIT16是響應白葉枯病菌侵染的日本晴差異蛋白,來源于疣粒野生稻和日本晴的CHIT16等位基因在編碼區(qū)域存在差異,但二者均定位于細胞質(zhì)膜上。OsCHIT16和OmCHIT16轉基因水稻均增強了對白葉枯病菌的耐受性,部分PR (Pathogenesis Related Protein)基因、參與JA(Jasmonic Acid)和SA(Salicylic Acid)合成與積累的關鍵基因在轉基因水稻中均下調(diào)表達。根據(jù)以上結果推測栽培稻和疣粒野生稻CHIT16等位基因增強水稻對白葉枯病菌的耐病性可能不依賴于JA和SA途徑,且二者間序列的差異并不影響CHIT16基因功能。綜上所述,本研究利用2D-DIGE分析了白葉枯病菌脅迫下感病水稻日本晴懸浮細胞的外分泌蛋白質(zhì)組的變化,發(fā)現(xiàn)其中一個蛋白點Xoo3654可能是一個致病負調(diào)控因子。并且,本研究發(fā)現(xiàn)過量表達OmCS1、OsPOP11、OmSCP8、 OsCH1T16及OmCHIT16等基因均能增強水稻的耐病性。本研究為分析疣粒野生稻的抗病機理提供了新的理論依據(jù),為研究其它植物與病原細菌的互作提供了新參考。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S435.111.47
，
本文編號：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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S435.111.47
，
本文編號：2122554
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