Generation of CBF4::LUC transgenic plants and CBF4 promoter deletion assayeneration of CBF4::LUC transgenic plants and CBF4 promoter deletion assay
Date Issued
2007
Date
2007
Author(s)
Hsu, Po-Yen
Abstract
Plants suffered from many abiotic stresses in environment, including cold, heat, drought, or high salt. Drought stress, one of the most influential stresses to plants, and also to human, causes plants damage by dehydration. To resist drought stress, some physiological responses are induced in plants, such as stomata closure, decreased leaf area, leaf abscission and extension of roots to deeper, moist soil. The purpose of these responses is to reduce water loss or to increase water uptake. At the molecular level, plants also exhibited a complex signaling transduction under drought stress. DREB (DRE-binding protein) subfamily contains genes of transcriptional factors which bind to DRE (drought responsive element) cis-element of downstream genes and to induce gene expression. DREB subfamily is consisting of DREB1/CBF (C-repeat binding factor) and DREB2. DREB2 was induced by water deficient, while DREB1 /CBF was induced by low temperature. DREB1D (CBF4) was cloned in 2002. However, CBF4 was not induced by cold, but by drought. Although CBF4 is induced by water deficiency and is involved in ABA-dependent signaling transduction pathway, the upstream regulators of CBF4 are still unclear. To study the upstream regulator of CBF4, we used mutant screening method according to Ishitani (1997). We established a CBF4 promoter::LUC mutant pool, and will screen mutants which exhibit deregulated CBF4 expression by altered luminescence. Also, we would like to perform CBF4 promoter deletion assay to study which fragment of CBF4 promoter is most relevant to the induction of CBF4 genes, so that the responsive element will be used in the yeast one-hybrid assay. CBF4 5’ UTR sequence is still unclear in TAIR sequence database. To obtain precise CBF4 promoter length, 5’-RACE was performed to obtain CBF4 5’ UTR sequence. It was shown that CBF4 5’ UTR region is 84bp upstream of ATG. We establish a CBF4 promoter::LUC construct and transformed Arabidopsis by Argobacterium infection. LUC expression could be detected in the CBF4 promoter::LUC transgenic plants after drought treatment by Northern blot and RT-PCR. CBF4 promoter::LUC plants can also exhibit luminescence under drought stress. Southern blot was also performed to pick the CBF4 promoter::LUC transgenic plant that was T-DNA single-inserted. According to the promoter analysis by PLACE website, we constructed different length of CBF4 promoter fragments and acquired CBF4 promoter::GUS transgenic plants which contain various length of promoter sequence. In GUS staining test, CBF4 was expressed in cauline leaf under drought stress.
Subjects
CBF4
drought
transcriptional factor
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