不同萝卜抗源抗黑腐病相关基因差异表达比较研究
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摘要
黑腐病是严重影响十字花科蔬菜生产的世界性细菌病害。为了筛选萝卜黑腐病抗源和挖掘抗病优异基因,本文采用三种不同的鉴定方法对萝卜种质资源进行了鉴定,并采用DDRT-PCR和cDNA-AFLP对不同萝卜抗源抗黑腐病相关基因表达进行了研究,主要研究结果如下:
1.对40份初选萝卜种质分别接种Xcc8004和XccBJ两个菌株,进行黑腐病苗期抗性鉴定;对其中的8份代表性萝卜种质采用肉质根切片接种Xcc8004进行抗性鉴定;对其中的27份萝卜种质接种8个含有效应物基因的农杆菌,进行过敏反应鉴定。结果表明:不同萝卜种质苗期对黑腐病的抗性存在显著差异,筛选出对Xcc8004表现高抗的材料3份,对XccBJ表现高抗的材料1份。萝卜苗期不同材料对Xcc8004和XccBJ的抗性表现极显著相关,萝卜苗期和肉质根对Xcc8004的抗病性极显著相关。稳定可靠抗病资源的获得为萝卜抗病育种和抗病机理深入研究提供了基础材料。筛选出17份对不同效应物表现过敏反应的萝卜种质。不同抗源对不同效应物的过敏反应程度有所不同。
2.利用cDNA-AFLP技术,研究了4份萝卜抗病材料KB09-1、KB09-12、KB09-17、KB09-16和1份感病材料KB09-29接种和未接种黑腐菌Xcc8004的基因差异表达。256对引物组合在参试材料中扩增出790个差异表达条带。克隆得到的74个差异片段按其功能分为8类。在多份萝卜种质中都涉及信号转导、蛋白质代谢、光合作用、抗病防御及转运等序列的差异表达。不同萝卜抗源表达的抗性相关基因不完全相同。
3.对高抗黑腐病萝卜种质KB09-12分别接种效应物xopXccN和xopXccX2,利用cDNA-AFLP方法获得153个差异表达条带。25个测序条带分析表明,接种xopXccN和xopXccX2公共差异表达条带主要涉及信号转导和抗病防御,接种xopXccN的特异差异表达条带主要涉及光合作用,接种xopXccX2的特异表达差异条带主要涉及蛋白质合成、加工。比较KB09-12接种效应物与接种Xcc8004的基因差异表达发现:少部分差异条带同时出现在过敏反应和抗病反应中。
Black rot is a bacteria disease seriously influenced the production of cruciferous vegetable worldwide. In order to screen out the resistant radish germplasm and excellent disease resistance gene, three different methods were used to identify a set of radish germplasm, and DDRT-PCR and cDNA-AFLP were used to analyze the differentially expressed ESTs of five accessions of radish. The main results were as follows:
1. Forty radish germplasm were inoculated with strain Xcc8004 and XccBJ of Xanthomonas campestris pv. campestris (Xcc) at seedling stage, respectively, and the fleshy root of eight germplasm among them were sliced and inoculated with strain Xcc8004. Twenty-seven accessions of them were inoculated with eight Agrobacterium with different effectors. The result indicated that radish germplasm resistant to black rot was significantly different. Three highly resistant accessions of radish germplasm to Xcc8004 were selected; one highly resistant accession of radish germplasm to XccBJ was selected. The resistance of radish seedlings to Xcc8004 was significantly correlated to the resistance to XccBJ; radish resistance of seedlings and fleshy roots to Xcc8004 was also significantly correlated. Access to reliable resources provides the basic materials for the radish resistance breeding and resistance mechanism in-depth study. Seventeen radish germplasm exhibited hypersensitive response (HR) to different effectors. HR extent of different germplasm to different effectors was different.
2. cDNA-AFLP was employed to study the resistance mechanism of different radish germplasm to Xcc8004. Four resistant accessions of radish germplasm KB09-1, KB09-12, KB09-17, KB09-16 and one susceptible accession KB09-29 were used. 790 differentially expressed ESTs were obtained by 256 primers in all materials. 74 fragments were sequenced, and were divided into 8 categories according to their function. Genes related to signal transduction, protein metabolism, photosynthesis, defensive response and transport were differentially expressed in different accessions of radish germplasm. Gene differential expression of five accessions of radish germplasm against black rot disease was not identical.
3. Highly resistant accession of radish germplasm ?KB09-12? was inoculated with effectors xopXccN and xopXccX2, respectively, and 153 differentially expressed ESTs were obtained by cDNA-AFLP. Twenty-five fragments were sequenced. Common ESTs from the inoculation of two effectors were mostly involved in signal transduction and defensive responses. Specific ESTs in radish inoculated with xopXccN were mostly involved in photosynthesis, while specific ESTs in radish inoculated with xopXccX2 were mostly involved in protein synthesis and processing. In comparison with differentially expressed ESTs in KB09-12 inoculated with effectors and Xcc8004, a few ESTs appeared in both hypersensitive response and disease resistance response.
1.对40份初选萝卜种质分别接种Xcc8004和XccBJ两个菌株,进行黑腐病苗期抗性鉴定;对其中的8份代表性萝卜种质采用肉质根切片接种Xcc8004进行抗性鉴定;对其中的27份萝卜种质接种8个含有效应物基因的农杆菌,进行过敏反应鉴定。结果表明:不同萝卜种质苗期对黑腐病的抗性存在显著差异,筛选出对Xcc8004表现高抗的材料3份,对XccBJ表现高抗的材料1份。萝卜苗期不同材料对Xcc8004和XccBJ的抗性表现极显著相关,萝卜苗期和肉质根对Xcc8004的抗病性极显著相关。稳定可靠抗病资源的获得为萝卜抗病育种和抗病机理深入研究提供了基础材料。筛选出17份对不同效应物表现过敏反应的萝卜种质。不同抗源对不同效应物的过敏反应程度有所不同。
2.利用cDNA-AFLP技术,研究了4份萝卜抗病材料KB09-1、KB09-12、KB09-17、KB09-16和1份感病材料KB09-29接种和未接种黑腐菌Xcc8004的基因差异表达。256对引物组合在参试材料中扩增出790个差异表达条带。克隆得到的74个差异片段按其功能分为8类。在多份萝卜种质中都涉及信号转导、蛋白质代谢、光合作用、抗病防御及转运等序列的差异表达。不同萝卜抗源表达的抗性相关基因不完全相同。
3.对高抗黑腐病萝卜种质KB09-12分别接种效应物xopXccN和xopXccX2,利用cDNA-AFLP方法获得153个差异表达条带。25个测序条带分析表明,接种xopXccN和xopXccX2公共差异表达条带主要涉及信号转导和抗病防御,接种xopXccN的特异差异表达条带主要涉及光合作用,接种xopXccX2的特异表达差异条带主要涉及蛋白质合成、加工。比较KB09-12接种效应物与接种Xcc8004的基因差异表达发现:少部分差异条带同时出现在过敏反应和抗病反应中。
Black rot is a bacteria disease seriously influenced the production of cruciferous vegetable worldwide. In order to screen out the resistant radish germplasm and excellent disease resistance gene, three different methods were used to identify a set of radish germplasm, and DDRT-PCR and cDNA-AFLP were used to analyze the differentially expressed ESTs of five accessions of radish. The main results were as follows:
1. Forty radish germplasm were inoculated with strain Xcc8004 and XccBJ of Xanthomonas campestris pv. campestris (Xcc) at seedling stage, respectively, and the fleshy root of eight germplasm among them were sliced and inoculated with strain Xcc8004. Twenty-seven accessions of them were inoculated with eight Agrobacterium with different effectors. The result indicated that radish germplasm resistant to black rot was significantly different. Three highly resistant accessions of radish germplasm to Xcc8004 were selected; one highly resistant accession of radish germplasm to XccBJ was selected. The resistance of radish seedlings to Xcc8004 was significantly correlated to the resistance to XccBJ; radish resistance of seedlings and fleshy roots to Xcc8004 was also significantly correlated. Access to reliable resources provides the basic materials for the radish resistance breeding and resistance mechanism in-depth study. Seventeen radish germplasm exhibited hypersensitive response (HR) to different effectors. HR extent of different germplasm to different effectors was different.
2. cDNA-AFLP was employed to study the resistance mechanism of different radish germplasm to Xcc8004. Four resistant accessions of radish germplasm KB09-1, KB09-12, KB09-17, KB09-16 and one susceptible accession KB09-29 were used. 790 differentially expressed ESTs were obtained by 256 primers in all materials. 74 fragments were sequenced, and were divided into 8 categories according to their function. Genes related to signal transduction, protein metabolism, photosynthesis, defensive response and transport were differentially expressed in different accessions of radish germplasm. Gene differential expression of five accessions of radish germplasm against black rot disease was not identical.
3. Highly resistant accession of radish germplasm ?KB09-12? was inoculated with effectors xopXccN and xopXccX2, respectively, and 153 differentially expressed ESTs were obtained by cDNA-AFLP. Twenty-five fragments were sequenced. Common ESTs from the inoculation of two effectors were mostly involved in signal transduction and defensive responses. Specific ESTs in radish inoculated with xopXccN were mostly involved in photosynthesis, while specific ESTs in radish inoculated with xopXccX2 were mostly involved in protein synthesis and processing. In comparison with differentially expressed ESTs in KB09-12 inoculated with effectors and Xcc8004, a few ESTs appeared in both hypersensitive response and disease resistance response.
引文
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