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