中国野生华东葡萄白河-35-1抗白粉病PR10基因的克隆与表达分析
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摘要
在前期的研究中,实验室以多年鉴定为高抗白粉病的中国野生华东葡萄白河-35-1为材料,构建了cDNA文库,并测序获得了与抗病有关的一些EST序列。本试验是在此基础上,对所获得的病程相关蛋白基因(PR10)的EST序列进行进一步地研究,以阐明该病程相关蛋白基因的结构,与其它物种中病程相关蛋白基因的同源关系,考察其拷贝数,表达特点及在葡萄抗白粉病过程中的表现,取得的主要研究结果有:
1.白河-35-1病程相关蛋白基因(VpPR10)cDNA及DNA序列的克隆和分析在已有PR10 EST序列的基础之上,设计特异引物,通过RACE技术,克隆获得了华东葡萄白河-35-1 PR10基因的完整cDNA序列,长827 bp组成,暂命名为VpPR10。根据该完整序列的开放阅读框区域设计引物,克隆获得了VpPR10的部分DNA序列,长602 bp。两条序列登录GenBank数据库,登录号分别为:DQ336289和DQ396808。序列分析表明,VpPR10基因序列除具有PR10基因普遍具有的Bet v1 family signature和P-loop模体外,还具有其它多个模体。序列比对表明,VpPR10蛋白序列与欧洲葡萄白玉霓的两个PR10蛋白分别具有79%和89%的相似性,和毛葡萄的一个PR10蛋白具有96%的序列相似性,和华东葡萄的另外一个PR10蛋白具有98%的相似性。
2.白河-35-1病程相关蛋白基因(VpPR10)基因的Southern blot分析将白河-35-1基因组DNA分别用EcoRⅠ,HindⅢ,XbaⅠ三种限制性内切酶进行单酶切,以获得的VpPR10 DNA序列为探针,进行Southern blot分析。结果表明,在每个泳道上,至少有4条杂交带,大小位于2.0至10.0 kb之间,因而VpPR10属于一个多基因家族。对白河-35-2、广西-2、湖南-1、玫瑰香、无核白、河岸葡萄和山平氏葡萄7个葡萄品种或株系的Southern blot分析表明,PR10基因在葡萄中均以多拷贝广泛存在。
3.白河-35-1病程相关蛋白基因(VpPR10)在mRNA水平的表达变化情况用实时荧光定量PCR技术,对对照及白粉菌接种后不同时间白河-35-1叶片中VpPR10在mRNA水平的表达变化情况进行分析。结果表明,在对照叶片中,VpPR10是组成性表达的,在接种过的叶片中,接种24 h后,VpPR10的表达量与对照相比稍微有所降低,在接种48和72 h后,VpPR10的表达量明显降低,并在72 h时达到最低值,。在接种96 h后,VpPR10的表达量恢复到对照的水平。说明在白河-35-1抗白粉病的过程中,VpPR10在转录水平即受到调节,参与抗病反应。
4.白河-35-1病程相关蛋白基因(VpPR10)的原核表达、重组蛋白的纯化及多克隆抗体的制备
将VpPR10的编码序列与原核表达载体pGEX-4T-1连接,构建重组表达载体。重组表达载体转入宿主菌E.coli BL21(DE3)后,用0.1 mM IPTG分别于30℃和37℃诱导3 h进行重组蛋白的表达,结果表明,重组蛋白在两种温度下都能够表达,但在30℃时大部分以可溶形式表达,在37℃时大部分以包涵体形式表达。
用0.1 mM的IPTG在30℃诱导3 h,使重组蛋白大部分以可溶形式表达。诱导后的菌体用细菌裂解液裂解后,收集可溶性蛋白,用GST亲和树脂进行纯化。将纯化后的重组蛋白免疫家兔制备了抗VpPR10融合蛋白的多克隆抗体。
5.白河-35-1叶片中病程相关蛋白(VpPR10)的表达变化情况
用制备的多克隆抗体对对照及白粉菌接种后不同时间白河-35-1叶片中VpPR10蛋白的表达变化情况进行分析。结果表明该抗体能够特异识别电泳迁移位置在17 kDa的蛋白,在未经白粉菌诱导的叶片中,VpPR10蛋白的表达量较高;在经白粉菌诱导后的叶片中,VpPR10蛋白的表达量在诱导24 h后仅有少量降低,但在诱导48 h和72 h后,VpPR10蛋白的表达量迅速降低,仅能够微弱地被检测到。在诱导96 h后,VpPR10蛋白的表达量迅速恢复到了诱导24 h后的水平,之后又有所提高。这说明,VpPR10在蛋白水平积极参与白河-35-1的抗白粉病的过程。
综上所述,我们研究获得的VpPR10基因全长序列、制备的探针和抗体、该基因在人工接种诱导后在华东葡萄中的一系列表现为进一步深入研究中国野生华东葡萄VpPR10基因提供了重要依据。
In the previous study, a PR10 EST sequence has been obtained from cDNA library prepared from Chinese wild Vitis pseudoreticulata clone Baihe-35-1 leaves,which is high resistant to Uncinula necator from years’investigation. This further study was to illuminate the PR10 sequence, homology with other PR10 proteins, copy number and expression pattern during the period of defending against Uncinula necator.
1. Cloning and analysis of PR10 cDNA and DNA sequence(VpPR10) from Baihe-35-1
The full length cDNA sequence of PR10 was cloned using RACE and specific primer and was temporarily designated as VpPR10. Genomic DNA fragment of VpPR10 was also cloned using one pair primers of VpPR10 open reading frame. Theses two sequences were submitted to GenBank under accession numbers DQ336289 and DQ396808, respectively. Besides the Bet v1 family signature and P-loop motifs which were common in PR10 proteins, there also existed some other motifs in VpPR10 protein sequence. Meanwhile, VpPR10 protein showed 79% and 89% sequence similarity with two PR10 proteins from Vitis vinifera Ugni Blanc, 96% with one PR10 protein from Vitis quinquangularis and 98% with another PR10 from Vitis pseudoreticulata.
2. Southern blot analysis of VpPR10 gene
Genomic DNA of Baihe-35-1 was digested with EcoRI, HindⅢand XbaI respectively and hybridised with the DNA sequence of VpPR10 as probe. More than four hybridised bands, ranging from 2.0 kb to 10.0 kb, were present in each lane, suggesting that VpPR10 belongs to a multi-gene family. Southern blot analysis of other 7 cultivars or accessions of Vitis indicated that PR10 genes generally existed with multiple copies.
3. Expression pattern of VpPR10 mRNA
Quantitative real-time RT-PCR was used to determine the relative mRNA expression levels of VpPR10. There was a constitutive expression of VpPR10 in control leaves. The transcript level of VpPR10 altered at different time after inoculation with Uncinula necator. In inoculated leaves VpPR10 expression began to decrease after 24 h and reached the lowest level after 72 h. Then it started to increase and obtained the normal level after 96 h compared with the control. These results showed that VpPR10 was regulated at transcript level and involved in resistance to U. necator of Baihe-35-1.
4. Expression of VpPR10 gene in E.coli, purification of VpPR10 fusion protein and antibody preparation
The coding sequence of VpPR10 was cloned into the pGEX-4T-1 vector, and then the pGEX-VpPR10 construct was transformed into E. coli BL21 for protein induction with 0.1 mM IPTG for 3 h at 30°C and 37°C, respectively. Fusion protein could be expressed at both temperatures, most proportion soluble at 30°C whilst insoluble at 37°C.
Expression of the VpPR10 fusion protein in solubility was induced with 0.1 mM IPTG at 30°C for 3 h. The bacterial cells were pelleted after incubation and suspended in lysis solution. Fusion protein was purified with Glutathione-Sepharose resin by affinity chromato- graphy. The polyclonal antibody against VpPR10 protein was raised using the purified fusion protein to immune a rabbit.
5. Expression pattern of VpPR10 protein
The polyclonal antibody was used to study VpPR10 protein change in U. necator inoculated leaves of Baihe-35-1 and the control. On Immunoblots the antibody recognized a protein migrating at about 17 kDa. The expression level of VpPR10 in control leaves was high and only slightly decreased at 24 h post-inoculation. The level was drastically decreased and could be just faintly detected at 48 and 72 h post-inoculation. Then VpPR10 gained a great increase to the normal level at 96 h post-inoculation. These results showed that VpPR10 positively involved in resistance to U. necator.
In conclusion, the full length sequence of VpPR10 gene, the probe and the antibody, and the expression pattern after induction by U. necator.from this study laid foundation for the further research of VpPR10 gene.
1.白河-35-1病程相关蛋白基因(VpPR10)cDNA及DNA序列的克隆和分析在已有PR10 EST序列的基础之上,设计特异引物,通过RACE技术,克隆获得了华东葡萄白河-35-1 PR10基因的完整cDNA序列,长827 bp组成,暂命名为VpPR10。根据该完整序列的开放阅读框区域设计引物,克隆获得了VpPR10的部分DNA序列,长602 bp。两条序列登录GenBank数据库,登录号分别为:DQ336289和DQ396808。序列分析表明,VpPR10基因序列除具有PR10基因普遍具有的Bet v1 family signature和P-loop模体外,还具有其它多个模体。序列比对表明,VpPR10蛋白序列与欧洲葡萄白玉霓的两个PR10蛋白分别具有79%和89%的相似性,和毛葡萄的一个PR10蛋白具有96%的序列相似性,和华东葡萄的另外一个PR10蛋白具有98%的相似性。
2.白河-35-1病程相关蛋白基因(VpPR10)基因的Southern blot分析将白河-35-1基因组DNA分别用EcoRⅠ,HindⅢ,XbaⅠ三种限制性内切酶进行单酶切,以获得的VpPR10 DNA序列为探针,进行Southern blot分析。结果表明,在每个泳道上,至少有4条杂交带,大小位于2.0至10.0 kb之间,因而VpPR10属于一个多基因家族。对白河-35-2、广西-2、湖南-1、玫瑰香、无核白、河岸葡萄和山平氏葡萄7个葡萄品种或株系的Southern blot分析表明,PR10基因在葡萄中均以多拷贝广泛存在。
3.白河-35-1病程相关蛋白基因(VpPR10)在mRNA水平的表达变化情况用实时荧光定量PCR技术,对对照及白粉菌接种后不同时间白河-35-1叶片中VpPR10在mRNA水平的表达变化情况进行分析。结果表明,在对照叶片中,VpPR10是组成性表达的,在接种过的叶片中,接种24 h后,VpPR10的表达量与对照相比稍微有所降低,在接种48和72 h后,VpPR10的表达量明显降低,并在72 h时达到最低值,。在接种96 h后,VpPR10的表达量恢复到对照的水平。说明在白河-35-1抗白粉病的过程中,VpPR10在转录水平即受到调节,参与抗病反应。
4.白河-35-1病程相关蛋白基因(VpPR10)的原核表达、重组蛋白的纯化及多克隆抗体的制备
将VpPR10的编码序列与原核表达载体pGEX-4T-1连接,构建重组表达载体。重组表达载体转入宿主菌E.coli BL21(DE3)后,用0.1 mM IPTG分别于30℃和37℃诱导3 h进行重组蛋白的表达,结果表明,重组蛋白在两种温度下都能够表达,但在30℃时大部分以可溶形式表达,在37℃时大部分以包涵体形式表达。
用0.1 mM的IPTG在30℃诱导3 h,使重组蛋白大部分以可溶形式表达。诱导后的菌体用细菌裂解液裂解后,收集可溶性蛋白,用GST亲和树脂进行纯化。将纯化后的重组蛋白免疫家兔制备了抗VpPR10融合蛋白的多克隆抗体。
5.白河-35-1叶片中病程相关蛋白(VpPR10)的表达变化情况
用制备的多克隆抗体对对照及白粉菌接种后不同时间白河-35-1叶片中VpPR10蛋白的表达变化情况进行分析。结果表明该抗体能够特异识别电泳迁移位置在17 kDa的蛋白,在未经白粉菌诱导的叶片中,VpPR10蛋白的表达量较高;在经白粉菌诱导后的叶片中,VpPR10蛋白的表达量在诱导24 h后仅有少量降低,但在诱导48 h和72 h后,VpPR10蛋白的表达量迅速降低,仅能够微弱地被检测到。在诱导96 h后,VpPR10蛋白的表达量迅速恢复到了诱导24 h后的水平,之后又有所提高。这说明,VpPR10在蛋白水平积极参与白河-35-1的抗白粉病的过程。
综上所述,我们研究获得的VpPR10基因全长序列、制备的探针和抗体、该基因在人工接种诱导后在华东葡萄中的一系列表现为进一步深入研究中国野生华东葡萄VpPR10基因提供了重要依据。
In the previous study, a PR10 EST sequence has been obtained from cDNA library prepared from Chinese wild Vitis pseudoreticulata clone Baihe-35-1 leaves,which is high resistant to Uncinula necator from years’investigation. This further study was to illuminate the PR10 sequence, homology with other PR10 proteins, copy number and expression pattern during the period of defending against Uncinula necator.
1. Cloning and analysis of PR10 cDNA and DNA sequence(VpPR10) from Baihe-35-1
The full length cDNA sequence of PR10 was cloned using RACE and specific primer and was temporarily designated as VpPR10. Genomic DNA fragment of VpPR10 was also cloned using one pair primers of VpPR10 open reading frame. Theses two sequences were submitted to GenBank under accession numbers DQ336289 and DQ396808, respectively. Besides the Bet v1 family signature and P-loop motifs which were common in PR10 proteins, there also existed some other motifs in VpPR10 protein sequence. Meanwhile, VpPR10 protein showed 79% and 89% sequence similarity with two PR10 proteins from Vitis vinifera Ugni Blanc, 96% with one PR10 protein from Vitis quinquangularis and 98% with another PR10 from Vitis pseudoreticulata.
2. Southern blot analysis of VpPR10 gene
Genomic DNA of Baihe-35-1 was digested with EcoRI, HindⅢand XbaI respectively and hybridised with the DNA sequence of VpPR10 as probe. More than four hybridised bands, ranging from 2.0 kb to 10.0 kb, were present in each lane, suggesting that VpPR10 belongs to a multi-gene family. Southern blot analysis of other 7 cultivars or accessions of Vitis indicated that PR10 genes generally existed with multiple copies.
3. Expression pattern of VpPR10 mRNA
Quantitative real-time RT-PCR was used to determine the relative mRNA expression levels of VpPR10. There was a constitutive expression of VpPR10 in control leaves. The transcript level of VpPR10 altered at different time after inoculation with Uncinula necator. In inoculated leaves VpPR10 expression began to decrease after 24 h and reached the lowest level after 72 h. Then it started to increase and obtained the normal level after 96 h compared with the control. These results showed that VpPR10 was regulated at transcript level and involved in resistance to U. necator of Baihe-35-1.
4. Expression of VpPR10 gene in E.coli, purification of VpPR10 fusion protein and antibody preparation
The coding sequence of VpPR10 was cloned into the pGEX-4T-1 vector, and then the pGEX-VpPR10 construct was transformed into E. coli BL21 for protein induction with 0.1 mM IPTG for 3 h at 30°C and 37°C, respectively. Fusion protein could be expressed at both temperatures, most proportion soluble at 30°C whilst insoluble at 37°C.
Expression of the VpPR10 fusion protein in solubility was induced with 0.1 mM IPTG at 30°C for 3 h. The bacterial cells were pelleted after incubation and suspended in lysis solution. Fusion protein was purified with Glutathione-Sepharose resin by affinity chromato- graphy. The polyclonal antibody against VpPR10 protein was raised using the purified fusion protein to immune a rabbit.
5. Expression pattern of VpPR10 protein
The polyclonal antibody was used to study VpPR10 protein change in U. necator inoculated leaves of Baihe-35-1 and the control. On Immunoblots the antibody recognized a protein migrating at about 17 kDa. The expression level of VpPR10 in control leaves was high and only slightly decreased at 24 h post-inoculation. The level was drastically decreased and could be just faintly detected at 48 and 72 h post-inoculation. Then VpPR10 gained a great increase to the normal level at 96 h post-inoculation. These results showed that VpPR10 positively involved in resistance to U. necator.
In conclusion, the full length sequence of VpPR10 gene, the probe and the antibody, and the expression pattern after induction by U. necator.from this study laid foundation for the further research of VpPR10 gene.
引文
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