Cold induced promoter from winter brassica napus

Details Cold induced promoter from winter brassica napus Cold induced promoter from winter brassica napus

C - Chemistry, Metallurgy

12

N

C12N 15/11 (2006.01) C07K 14/415 (2006.01) C12N 15/82 (2006.01)

Patent

CA 2146712

A 1.2 kb fragment of the 5' regulatory region (from bp -1107 to + 100) was fused to the GUS (.beta.-glucuronidase) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic Brassica napus plants only after incubation at 2°C. No expression was observed after incubation at 22°C, either in the presence or absence of abscisic acid. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and - 606 and a second positive regulatory region located between bp -302 and - 274. Deletion analyses and results from replacement with a truncated CaMV (cauliflower mosaic virus) 35S promoter suggest that the minimal size required for any maintenance of low temperature GUS expression is a -300 by fragment. Within this fragment are two 8 bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a bp core sequence in the low temperature- and dehydration-responsive elements (LTREs and DREs) identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes. Mutation of either one or both of the GGCAC core sequence of the putative LTRE's to AATTC resulted in loss of cold-inducible gene expression, providing for the first time, direct evidence that CCGAC is required for low temperature expression. Furthermore, replacement of an enhancer region (-605 to -1107) of the promoter with a more active enhancer from the 35S constitutive cauliflower mosaic virus (CaMV) promoter resulted in a "hybrid" promoter with increased low temperature induced activity several fold over that of the native promoter.

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