利用冷光報導載體系統分析含HHP1啟動子之基因片段

Abstract

阿拉伯芥 HHP1 (heptahelical transmembrane protein 1) 蛋白質是黃體激素之膜上接受體 mPR (membrane progestin receptor) 的同源蛋白質，具有7個穿膜區塊。目前認為HHP1在阿拉伯芥Abscisic acid (ABA) 與滲透壓訊息調控路徑中做為負調控因子，並可能連結低溫及滲透壓逆境間的訊息傳遞。進行序列分析後發現有許多與ABA、乾旱、高鹽與低溫逆境相關之可能的轉錄調控序列散佈於 HHP1的上游序列 (5’ UTR + -1 ~ -1552 bp) 中。 本研究希望藉由Dual-Luciferase Reporter○R (DLRTM ) Assay System了解這些與逆境相關的序列是否參與HHP1的轉錄調控。建構帶有四種不同長度 HHP1上游序列且具有firefly luciferase報導基因 (FLUC) 之pSP-luc+NF Fusion vector重組載體 (U1 ~ U4-pSP，U1：5’ UTR + -1 ~ -1552 bp；U2：5’ UTR + -1 ~ -1103 bp；U3：5’ UTR + -1 ~ -740 bp 與 U4：5’ UTR + -1 ~ -302 bp) 後，與做為內部對照組可表現 Renilla luciferase報導基因 (RLUC) 的35S-pRL共轉形至阿拉伯芥原生質體中短暫表現，觀察不同長度 HHP1上游序列經正規化 (FLUC/RLUC) 的相對冷光值，並分析這些啟動子片段驅動能力的變化。 由於純化出之原生質體健康程度與載體轉形過程對 DLRTM Assay System的實驗結果影響很大，本研究對原生質體之純化與短暫表現系統，進行部分修改後發現：純化5-7週大植株之真葉獲得之原生質體，使用2 × 106 cells/MMG solution之濃度，並以100：1之 U1 ~ U4-pSP與35S-pRL質量比進行轉形，可有效提高轉形效率並得到最佳的相對冷光值。使用上述之系統並配合 DLRTM Assay System，經過三重複以上之實驗，發現HHP1上游序列越短，其驅動FLUC之能力越強 (驅動能力 U4 > U3 > U2 >U1)。 此外本研究也對原生質體進行外加ABA處理，發現於該系統中，不同長度HHP1上游片段之驅動能力受ABA影響，似乎有提升之現象。

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.