Characterization Of The Inducible Promoter BjC-P

Transgenic crops are playing more and more important roles in agriculture. The best example is the use of the Bacillus thuringiensis (Bt) toxin to enhance insect resistance in several commercial transgenic crops. The expression patterns of the introduced genes have a great impact on crop improvement despite the properties of a given gene are definitely important to crop improvement. It is desirable to direct transgene expression only when the gene-encoded products are needed so as to minimize potential adverse effects on the growths of the plants and reduce yield drag. Thus, the characterization and use of inducible promoters can aid the development of genetically engineered crops.The BjCHI1 gene of Brassica juncea encodes a novel chitinase with two chitin-binding domains. The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fugal infection. These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses.Here, we report the detailed characterization of the BjCHI1 promoter. 5′-, 3′-ends and internal deletion analysis were performed in transgenic tobacco and Arabidopsis plants, and specific regions of the promoter that are response to wounding and MeJA inducibility were identified in a transient gene expression system of Nicotiana benthamiana leaves. The summary results are below:1. GUS expression under the driving of the BjCHI1 promoter (-1060/+17) was essentially undetectable in the young seedlings under normal growth conditions. GUS activity was first detected in the stigma of young flowers, peaked in the young siliques, and decreased when the siliques became older. No GUS expression was found in the mature siliques, seeds or root.2. The BjCHI1 promoter (-1060/+17) was inducible by NaCl, PEG, wounding and MeJA treatments. High levels of GUS expression were detected in the transgenic tobacco and Arabidopsis plants after wounding, NaCl, PEG, and MeJA treatment, indicating that the BjCHI1 promoter responses to both biotic and abiotic stresses.3. RT-PCR analysis confirmed that the expression of the BjCHI1 gene in B. juncea was inducible by PEG and NaCl.4. The transcription start site was determined by 5′-RACE, and was located at the 17th nucleotide (A) upstream of the translation initiation codon (ATG) of the BjCHI1 gene.5. A -805/+17 promoter fragment was enough to response to wounding and MeJA induction, which was proved in transgenic tobacco and Arabidopsis plants. The 397 bp region between -805 and -409 of the BjCHI1 promoter contains a cis-acting element that is essential for the wounding and MeJA inducibility.6. The -695/-620 region was necessary but not sufficient to confer MeJA-responsive expression. A T/G-box (AACGTG) locates in -353 play an important role in the expression of the BjCHI1 gene in response to MeJA treatment. The 76 bp region is coupled with the T/G-box (AACGTG) to confer full MeJA-inducible transcription of the BjCHI1 gene. This is the first T/G-box (AACGTG) element identified in a chitinase gene promoter.