Cloning And Functional Analysis Of The Transcription Factor BrWRKY33 And Its Up-stream Regulatory Sequence From Chinese Cabbage

WRKY transcription factor is a plant-specific transcription factor,and WRKY33 belongs to WRKY protein group I,which WRKY domain highly conserved.Most of WRKY protein are located in the nucleus,and can form a hetero or homologous dimers by the interactions between proteins to regulate the expression of target genes.The regulation of transcription factor WRKY are complex,and how to regulate its downstream target gene becomes the hotspot in current study,the function of transcription factor gene BrWRKY33 and its upstream regulatory sequence are analyzed and discussed.The SSH cDNA library of Chinese cabbage(Brassica rapa subp.pekinensis cv.longbaiⅡ) inoculating Ecc(Erwinia carotovora subsp,carotovora,Ecc) is obtained,and select highly homologous,then cloned the coding region of BrWRKY33 from the Chinese cabbage genome by PCR,which have a length of 1443bp,encoding 480 amino acids.RT-PCR analysis shows that the BrWRKY33 begins to express in 6h after Chinese cabbage inoculating Ecc,and the gene has a highest accumulation in 24hpi as the infection time grows.Analyse the resistance identification of Arabidopsis mutant,wild-type and BrWRKY33 over-expression plants respectively,and get the disease statistics after inoculate Ecc 6hpi,12hpi, 24hpi.Disease index mainly lies in 53.6%～65.3%after inoculate with Ecc 6hpi,63.4%～74.8% after 12hpi,90.9%～98.7%after 24hpi.The statistics show that the difference obvious between transgenic plants and mutant ones in Ecc disease-resistant,and has not significant difference between transgenic plants and wild-type.The expression of BrWRKY33 in Arabidopsis is detected through RT-PCR,PR1 and BGL-2 are defense-regulated genes which associated with SA-regulated defense responses,PR1 has an enhanced expression in mutant-1 compared to wild-type,which suggest that the WRKY33 may have a negetive effect on SA;while the expression of JA-regulated defense genes AOS,have on difference in basically expression;Interestingly PDF1.2 which associated with JA/ET-regulated defense responses,have a lower expression in mutant compared to wild-type,but have a higher expression in over-expression lines 1,3,5,6 compared to wild-type.These results show that BrWRKY33 may have a positive regulation in the Arabidopsis JA/ET-mediated defense response to Ecc.A 1755bp up-stream regulatory sequence of transcription factor gene BrWRKY33 was amplified by PCR from Chinese cabbage(Brassica rapa subp.pekinensis cv.longbaiⅡ). Deletion mutant with a length of 315bp of the up-stream regulatory sequence was then constructed and transformed into Arabidopsis thaliana.The results indicated that the presence of some W box sequences in -1755～315bp region upstream the start codon was related to the down regulation of the gene expression,and the -315bp region contains the essential sequence for gene transcription.The difference in GUS staining in transgenic plants after and before inoculating with soft rot bacteria suggested that WRKY33 gene play a role in the resistance against soft rot disease.