Articles
ISOLATION OF TWO GENES SIMILAR TO DREB1/CBF FROM THE SWEET CHERRY AND THEIR ANALYSIS BY TRANSFORMATION INTO ARABIDOPSIS
Article number
618_3
Pages
39 – 45
Language
English
Abstract
DREB1 (dehydration responsive element binding protein 1)/CBF (C-repeat binding factor) up-regulates the expression of a group of genes designated as COR (cold regulated)/RD (responsive to dehydration) genes in Arabidopsis at low temperature.
We isolated three DREB1/CBF-like genomic clones from the sweet cherry.
Two (D2A and D2B) were deduced to be complete ORFs.
Each putative protein had an EREBP/AP2 DNA binding domain motif, a potential nuclear localization signal, in the N-terminal.
The conserved EREBP/AP2 domain of the D2 proteins showed high identity (74 ~79%) with each DREB1/CBF protein.
In addition, TATA-box and G-box sequences, and MYB- and MYC-recognition sites were predicted in the 5-region of each gene.
Low temperatures induced expression of the D2 genes in sweet cherry.
Therefore, we renamed D2A and D2B as CIGA (cold induced gene A) and CIGB, respectively.
To investigate the effects of the CIG proteins at low temperatures, we transformed a binary vector carrying a fusion of the enhanced CaMV 35S promoter and CIGB into Arabidopsis using the floral dip method.
Both neomycin phosphotransferase and the CIGB gene were detected in only five transgenic plants by PCR. Some transgenic plants had a dwarf phenotype.
Although we are currently studying the expression of the CIG gene and its effects on freezing tolerance in the transgenic progeny, we postulate that this phenomenon is due to the transgene.
We isolated three DREB1/CBF-like genomic clones from the sweet cherry.
Two (D2A and D2B) were deduced to be complete ORFs.
Each putative protein had an EREBP/AP2 DNA binding domain motif, a potential nuclear localization signal, in the N-terminal.
The conserved EREBP/AP2 domain of the D2 proteins showed high identity (74 ~79%) with each DREB1/CBF protein.
In addition, TATA-box and G-box sequences, and MYB- and MYC-recognition sites were predicted in the 5-region of each gene.
Low temperatures induced expression of the D2 genes in sweet cherry.
Therefore, we renamed D2A and D2B as CIGA (cold induced gene A) and CIGB, respectively.
To investigate the effects of the CIG proteins at low temperatures, we transformed a binary vector carrying a fusion of the enhanced CaMV 35S promoter and CIGB into Arabidopsis using the floral dip method.
Both neomycin phosphotransferase and the CIGB gene were detected in only five transgenic plants by PCR. Some transgenic plants had a dwarf phenotype.
Although we are currently studying the expression of the CIG gene and its effects on freezing tolerance in the transgenic progeny, we postulate that this phenomenon is due to the transgene.
Authors
H. Kitashiba, T. Ishizaka, N. Matsuda, H. Nakano, T. Suzuki
Keywords
Prunus avium L., low temperature, cold regulated gene (COR), responsive to dehydration (RD)
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