阿拉伯芥Di19-2基因參與調控開花時間的角色

Abstract

Di19家族是一個與乾旱逆境調控有關的基因家族，被發現在土壤水分減少時表現量會上升，亦有研究指出部分Di19成員基因表現量受到鹽分逆境誘導，然而該家族蛋白質大部分的功能都未知，過去實驗室成員研究發現在酵母菌雙雜合系統 (yeast two-hybrid system)、試管內結合實驗 (in vitro pull-down assay)及雙分子螢光互補試驗(bimolecular fluorescence complementation, BiFC)中，阿拉伯芥Di19-2蛋白質與IDD8/NUC蛋白質有交互作用，IDD8/NUC屬於INDERTERMINATE DOMAIN轉錄因子家族，在阿拉伯芥中能夠調控開花轉變，而IDD8/NUC被發現能透過正向調節蔗糖合成酶四號基因 (Sucrose synthase4, SUS4)改變植物體內醣類代謝。本論文發現Di19-2大量表現株具有提早開花之現象，開花相關基因表現量也有提高，將Di19-2大量表現株與idd8突變株雜交後延遲開花性狀有回復現象，然而di19-2突變株並沒有出現明顯晚開花性狀，反而是di19-2 di19-5雙重突變株有比較明顯的延遲開花性狀。透過染色質免疫沉澱定量PCR (Chromatin Immunoprecipitation-qPCR, ChIP-qPCR)觀察到SUS4基因啟動子上部分片段有富集現象，IDD8/NUC轉錄因子透過共免疫沉澱分析，證實Di19-2蛋白質與IDD8/NUC蛋白質有交互作用，IDD8/NUC轉錄因子可能是與Di19蛋白質結合透過結合SUS4基因啟動子上特定區域去調控轉錄活性。

Drought-induced 19 (Di19) family is related to drought stress regulation. There are also studies indicated that some Di19 genes are induced by salt stress. However, most Di19 proteins are functionally unknown. Previous study found interaction of Arabidopsis Di19-2 protein with IDD8/NUC (Nutcracker) protein in yeast two-hybrid, in vitro pull-down assay, and bimolecular fluorescence complementation (BiFC). IDD8/NUC is an INDETERMINATE DOMAIN transcription factor, it regulates floral transition in Arabidopsis, and it affects sugar metabolism in plants by positively regulating Sucrose synthase 4 (SUS4). In this study, analysis of Di19-2 overexpression lines exhibit early flowering phenotype, and expression level of flowering related genes were up-regulated. The cross line of Di19-2 overexpression line and idd8 mutant line showed recovered flowering phenotype; however, di19-2 mutant lines did not show delayed flowering phenotype, and late flowering phenotype was observed in di19-2 di19-5 double mutant lines. Interaction between Di19-2 protein and NUC/IDD8 protein was confirmed by co-immunoprecipitation analysis. There was enrichment in promoter fragments of SUS4 gene in chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis, which suggests that IDD8/NUC transcription factor might interact with Di19-2 and regulate transcription activity by binding to specific fragment of SUS4 promoter.