番茄材料PI128216抗疮痂病T3小种基因Rx4启动子的克隆与分析  

Cloning and Analysis of Promoter Sequence of Rx4 Gene for Resistance to Bacterial Spot Race T3 in Solanum pimpinellifolium Accession PI128216

作　　者：曹海鹏[1] 裴成成[1] 张晓飞[1] 杨文才[1] 

机构地区：[1]中国农业大学农学与生物技术学院蔬菜学系/设施蔬菜生长发育调控北京市重点实验室,北京100193

出　　处：《农业生物技术学报》2015年第7期851-862,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31372073);现代农业产业技术体系北京市创新团队果类蔬菜项目(No.GCTDZJ2014033001)

摘　　要：抗病基因的启动子在黄单胞杆菌属(Xanthomonas)病原菌与寄主植物互作中起着重要的作用。番茄疮痂病是由黄单胞杆菌引起的一种严重威胁番茄生产的病害,已有研究表明,病原菌T3小种(X.perforans)与抗病基因Rx4的识别可能与该基因的启动子有关。因此,本研究采用PCR技术从醋栗番茄(Solanum pimpinellifolium)材料PI128216基因组DNA中克隆了Rx4上游2 149 bp的核苷酸序列。经与Plant Cis-acting Regulatory DNA Elements(PLACE)数据库对比发现,该序列中含有启动子基本核心作用元件,赤霉素、脱落酸、乙烯等激素诱导相关的顺式作用元件,干旱、盐胁迫、高低温等非生物胁迫相关的顺式作用元件,大量与病原菌诱发因子表达调节相关的转录因子WRKY和MYB结合的元件,以及抗性元件W-box、G-box等。将该启动子4个不同长度的缺失片段分别与GUS报告基因融合构成表达载体,利用农杆菌介导瞬时表达体系在番茄子叶与烟草(Nicotiana benthamiana)叶片中进行表达。GUS活性定量分析结果显示,仅有-338^-41之间297 bp片段活性很弱,其余3个片段-552^-41之间511 bp片段、-1285^-41之间1 244 bp片段和-1 285^-552之间733 bp片段的活性依次增强,但差异不明显。因此,-1285^-338之间的区域对Rx4基因的启动子活性有重要作用。这一发现为进一步研究该启动子类型以及Rx4与番茄疮痂病病原菌T3小种的互作提供了参考。Promoters of resistance genes play important roles on interactions between the pathogen of Xanthomonas and the host plant. Bacterial spot caused by Xanthomonas is a disease that severely threats tomato production. Current data suggest that the recognition of the pathogen race T3 to the resistance gene Rx4 might be associated with the promoter of the gene. Therefore, a 2 149 bp upstream sequence of the resistance gene Rx4 was obtained from the genomic DNA of Solanum pimpinellifolium accession PI128216 by PCR amplification in this study. Comparing to the database of Plant Cis- acting Regulatory DNA Elements（PLACE） revealed that the sequence contained the basic core promoter elements, gibberellin, abscisic acid,and ethylene related cis- elements, dehydration, salt, and cold response- related cis- elements, large number of pathogen- related transcription factors such as WRKY and MYB binding elements, and resistance- related elements W-box, G-box. A series deletion fragments of the promoter sequence were fused with the GUS gene and transiently expressed in tomato（Nicotiana benthamiana） cotyledon and tobacco leaves. Quantitative analysis of GUS activity indicated that the activity was very weak using a 297 bp fragment from-338 to-41.The remaining three fragments of 511 bp from-552 to-41, 1 244 bp from-1 285 to-41, and 733 bp from-1 285 to-552 showed increased activities but the differences were not remarkably. These results suggested that the region from-1 285 to-338 had an important role on promoter activity of the Rx4 gene.This finding provides useful information for further investigating the promoter types as well as the interaction between the resistance gene Rx4 and the race T3 pathogen of bacterial spot in tomato.

关 键 词：番茄 抗性基因Rx4 启动子活性 瞬时表达 

分 类 号：S668.108[农业科学—果树学]