Analysis of HHP1 Promoter Using Luciferase Reporter Assay System
Date Issued
2011
Date
2011
Author(s)
Lu, Yu-Jou
Abstract
HHP1 (heptahelical transmembrane protein 1), a protein with a predicted seven transmembrane domain structure homologous to mPRs (membrane progestin receptors), is determined to be a novel negative regulator in ABA and osmotic signaling. Sequence analysis showed that the fragment upstream to the coding region of HHP1 (5’ UTR + -1 ~ -1552 bp) contains many ABA-, dehydration-, salinity- and low temperature-responsive elements.
In this study, we tried to investigate the stress-responsive elements which are involved in HHP1 transcriptional regulation using Dual-Luciferase Reporter○R (DLRTM ) Assay System (Promega). Four different DNA fragments containing various length of the putative promoter region of HHP1 (U1, 5’ UTR + -1 ~ -1552 bp; U2, 5’ UTR + -1 ~ -1103 bp; U3, 5’ UTR + -1 ~ -740 bp and U4, 5’ UTR + -1 ~ -302 bp) were cloned into vectors harboring firefly luciferase (FLUC). These constructs (U1 ~ U4-pSP) were co-transfected into protoplasts isolated from Arabidopsis mesophyll with internal control plasmid 35S-pRL expressing Renilla luciferase (RLUC). Due to the methods used to isolate and transfect plasmid into protoplasts greatly affected the assay, a few modification were made to optimize the protoplasts isolation and transfection. Protoplasts isolated from 5 ~ 7 week-old Arabidopsis, 2 × 106 cells/MMG solution for transfection, and 100:1 mass ratio of U1 ~ U4-pSP to 35S-pRL gave the best transformation and normalized luciferase expression (FLUC/RLUC). From more than three independent experiments, higher luciferase expressions were observed in the U4-pSP construct which contains the shorter HHP1 upstream fragment.
The experiments were also performed when the protoplasts were treated with abscisic acid. A few interesting preliminary results were discussed.
Subjects
HHP1
Type
thesis
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