园艺学报 ›› 2019, Vol. 46 ›› Issue (4): 677-690.doi: 10.16420/j.issn.0513-353x.2018-0815

• 研究论文 • 上一篇    下一篇


王丽娟,龙俊宏,谢 竹,吴 柳,彭爱红,何永睿,龙 琴,陈善春,邹修平*   

  1. 西南大学/中国农业科学院柑桔研究所,国家柑桔品种改良中心,重庆 400712
  • 出版日期:2019-04-25 发布日期:2019-04-25
  • 基金资助:
    国家现代农业产业技术体系建设专项资金项目(CARS-27);重庆市社会事业与民生保障科技创新专项(cstc2016shms- ztzx80001);重庆市自然科学基金项目(cstc2017jcyjBX0020)

Cloning and Expression Characteristics of CsWRKY22 Promoters in Response to Xanthomonas citri subsp. citri

WANG Lijuan,LONG Junhong,XIE Zhu,WU Liu,PENG Aihong,HE Yongrui,LONG Qin,CHEN Shanchun,and ZOU Xiuping*   

  1. Citrus Research Institute,Southwest University/Chinese Academy of Agricultural Sciences,National Center for Citrus Varieties Improvement,Chongqing 400712,China
  • Online:2019-04-25 Published:2019-04-25

摘要: 由柑橘黄单胞杆菌致病变种(Xanthomonas citri subsp. citri,Xcc)引起的柑橘溃疡病严重危害着柑橘产业的健康可持续发展。前期研究表明,CsWRKY22可能是溃疡病感病基因。从‘晚锦橙’(Citrus sinensis Osbeck)中克隆了CsWRKY22的两个等位启动子pCsWRKY22-1和pCsWRKY22-2,长度分别为1 428和1 406 bp。序列分析表明两个等位启动子序列一致性为91.76%。两个等位启动子含有多种参与植物防御反应和生长发育的顺式作用元件,相同元件的数量和位置基本一致,不同的是,在TATA-box下游,pCsWRKY22-1含有2个22 bp长的TA-rich转录增强序列,而pCsWRKY22-2只含有1个。构建CsWRKY22启动子控制GUS报告基因的植物表达载体pCsWRKY22::GUS,并用农杆菌介导法转化‘晚锦橙’,经GFP活性检测和PCR鉴定获得pCsWRKY22-1::GUS和pCsWRKY22-2::GUS转基因植株各8株和4株。GUS组织化学染色、GUS酶活性及定量PCR分析表明,CsWRKY22启动子具有较强的机械伤诱导活性和柑橘溃疡病病原菌诱导活性,同时具有一定水平的基础表达特性。pCsWRKY22-1机械伤诱导活性高于pCsWRKY22-2,而pCsWRKY22-2溃疡病菌诱导活性高于pCsWRKY22-1。

关键词: 柑橘溃疡病, CsWRKY22, 启动子, 诱导, GUS, 不结球白菜, 分子生物学, 性状

Abstract: Citrus canker disease,induced by Xanthomonas citri subsp. citri(Xcc),is threating citrus industry in the world. Previous work has shown that the CsWRKY22 may be a susceptible gene for citrus canker. Two allelic promoters of the CsWRKY22 gene,named pCsWRKY22-1 and pCsWRKY22-2,were cloned from Wanjincheng orange(Citrus sinensis Osbeck). Sequencing showed that the lengths of pCsWRKY22-1 and pCsWRKY22-2 promoter obtained were 1 428 and 1 406 bp,respectively. Sequence analysis showed that the similarity of these two allelic promoters was 91.76%. PlantCare predication revealed that the two promoters contain multiple cis-acting elements,which were involved in plant defense response and development. Although some single nucleotide polymorphisms(SNPs)and indels(insert and deletion)were detected in their sequences,no obvious difference was found in numbers and locations of the elements between the two promoters. The obvious difference is that the pCsWRKY22-1 has two transcription enhancers“TA-rich”element just downstream of the“TATA-box”core promoter,while the pCsWRKY22-2 has only one. In order to study the function of CsWRKY22 promoters,the pCsWRKY22-1::GUS and pCsWRKY22-2::GUS vectors,in which GUS expression was controlled by pCsWRKY22-1 and pCsWRKY22-2,respectively,were constructed,and introduced into Wanjincheng orange by Agrobacterium-mediated transformation. Eight pCsWRKY22-1::GUS and four pCsWRKY22-2::GUS transgenic lines were obtained,and their transgenic nature was confirmed by GFP fluorescence and PCR analysis. GUS staining and enzyme activity and qPCR analysis showed that the two CsWRKY22 promoters possessed strong wounding- and Xcc-inducible activities. pCsWRKY22-1 displayed higher wounding- inducible expression level than pCsWRKY22-2,while pCsWRKY22-2 had higher Xcc-inducible expression level. The promoters also showed some levels of basic expression in leaf and stem tissues in transgenic plants before inoculation.

Key words: citrus canker, CsWRKY22, promoter, induction;GUS, Brassica campestris ssp. chinensis, molecular biology, trait