Cold induced promoter from winter brassica napus

Abstract

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.degree. C. No expression was observed after incubation at 22.degree. 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 bp 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 5 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.