Molecular cloning and characterization of the promoter for the multiple stress-inducible gene BjCHI1 from Brassica juncea.
We have previously isolated a Brassica juncea cDNA encoding a novel chitinase BjCHI1 with two chitin-binding domains (Zhao and Chye in Plant Mol Biol 40:1009-1018, 1999). The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fungal infection. These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses. Here, we report the cloning and characterization of the BjCHI1 promoter. A 1,098 bp BjCHI1 genomic DNA fragment upstream of the ATG start codon was isolated by PCR walking and various constructs were made by fusing the BjCHI1 promoter or its derivatives to beta-glucuronidase reporter gene. The transgenic Arabidopsis plants showed that the BjCHI1 promoter responded to wounding and MeJA treatment, and to treatments with either NaCl or polyethyleneglycol (PEG 6000), indicating that the BjCHI1 promoter responses to both biotic and abiotic stresses. A transient gene expression system of Nicotiana benthamiana leaves was adopted for promoter deletion analysis, and the results showed that a 76 bp region from -695 to -620 in the BjCHI1 promoter was necessary for MeJA-responsive expression. Furthermore, removal of a conserved T/G-box (AACGTG) at -353 to -348 of the promoter greatly reduced the induction by MeJA. This is the first T/G-box element identified in a chitinase gene promoter. Gain-of-function analysis demonstrated that the cis-acting element present in the 76 bp region requires coupling with the T/G-box to confer full magnitude of BjCHI1 induction by MeJA.
Wu
Xue-Feng
XF
National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), Key Laboratory of Crop Genetics and Breeding, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
Wang
Chun-Lian
CL
Xie
En-Bei
EB
Gao
Ying
Y
Fan
Ying-Lun
YL
Liu
Pi-Qing
PQ
Zhao
Kai-Jun
KJ
D000085:Acetates / Q000494:pharmacology; D001483:Base Sequence; D001937:Brassica / Q000201:enzymology / Q000235:genetics*; D002688:Chitinases / Q000235:genetics*; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000494:pharmacology; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D054883:Oxylipins / Q000494:pharmacology; D010937:Plant Growth Regulators / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000494:pharmacology; D013314:Stress, Mechanical; D024363:Transcription Initiation Site
D000085:Acetates; D003517:Cyclopentanes; D018744:DNA, Plant; D054883:Oxylipins; D010937:Plant Growth Regulators; D010940:Plant Proteins; D011092:Polyethylene Glycols; D012965:Sodium Chloride; C072239:methyl jasmonate; D002688:Chitinases; D005966:Glucuronidase