The Isolation And Functional Analysis Of Brassica Juncea BjCHI1 Promoter

The Brassica juncea chitinase gene BjCHI1 is induced by wounding, treatment with jasmonate (JA), insect feeding or by fungal infection; indicating the promoter of BjCHI1 is an inducible promoter. To understand the regulation expression pattern of BjCHI1underling plant-pathogen interactions, it is important to clone and illuminate the function of the BjCHI1 promoter.Brassica juncea genomic DNA was digested with DraⅠ,EcoRⅤ and SmaⅠ respectively. A special adapter was ligated to the ends of the digested DNA fragments, and the ligated DNA was used as a template for PCR with adapter primer and gene special primer designed according to the sequence of the Brassica juncea BjCHI1 cDNA. One specific PCR product was generated when the DraⅠadapter ligated DNA used as template.The specific PCR product was isolated and cloned into pGEM-T Easy vector and then sequenced. The sequence was compared with the sequences in GenBank and found that a 120bp sequence at the 3′ end accorded with 5′ end of the BjCHI1 cDNA, demonstrated that this PCR amplified DNA fragment is the promoter of the BjCHI1gene and a novel promoter (1098bp) was obtained. A like TATA box was found in this promoter by PLACE analysis.Plant transformation vector pBI121 was digested completely with HindⅢ and BamHⅠ. The bigger fragment was isolated and ligated to the 1098bp BjCHI1 promoter fragment which had been cleaved with HindⅢ and BamHⅠto generate plant expression vector named pBICHP harboring the fusion gene CHP::GUS.pBICHP was electroporated into Agrobacterium tumefaciens strain EHA105. EHA105 containing pBICHP was activated and induced by Acetosyringone, bacterial suspension was then infiltrated into intercellular spaces of near fully expanded tobacco leaves using a 1mL plastic syringe by infiltrating 100μL of bacterial suspension into each spot. After agroinfiltration, the treated plants were maintained in a growth chamber at 28℃ under 16h light for 3 days before GUS assay of the infiltrated areas. Histochemically GUS activity detecting results demonstrated that the -1098bp BjCHI1 promoter fragment was active to drive the expression of the GUS gene.