阿拉伯芥Di19-2基因在開花時間及鹽分逆境下的功能性研究

Functional studies of Drought-Induced Protein 19-2 in flowering time and salt stress response in Arabidopsis thaliana

10.6342/NTU.2015.00553

吳典倫

Di19-2 ； IDD8/NUTCRACKER ； Di19蛋白結合位(DiBS) ； 鹽逆境 ； 乾旱逆境 ； 開花 ； Drought-induced protein 19 ； IDD8/NUTCRACKER ； Di19 binding site (DiBS) ； salt stress ； drought stress ； flowering

臺灣大學植物科學研究所學位論文

2015年

碩士

張英峯

繁體中文

在阿拉伯芥中Drought-Induced Protein 19 (Di19)基因家族中共有七個成員，這些基因會轉譯出非典型的鋅手指(zinc-finger)蛋白質，根據前人研究，阿拉伯芥乾旱誘導蛋白質 Di19 可能會參與不同的逆境訊息傳遞路徑。Di19-2 (At1G02750)基因表現會受到高鹽處理所誘導，且不會受到離層酸(ABA)所調控，但對於Di19-2基因和蛋白質功能還是未知的。前人利用酵母菌雙雜合方法(yeast two-hybrid)、pull-down 和雙分子互補螢光系統(bimolecular fluorescence complementation)分析，發現Di19-2會與IDD8/ NUTCRACKER (NUC)轉錄因子有交互作用。本論文利用阿拉伯芥原生質體系統進行轉錄活化分析，發現Di19-2蛋白質會與IDD8/NUC轉錄因子共同正向調節IDD8/NUC的目標基因SUS4之表現。本研究發現，Di19-2蛋白質具有轉錄因子活性，但是不會與其他Di19蛋白質結合之Di19 binding site (DiBS)結合。之後建立Di19-2大量表現株，發現在幼苗時期的主根生長較野生型對於鹽逆境較敏感，另外在土壤中Di19-2大量表現成株較野生型對於高鹽和乾旱逆境有不抗的外表型，暗示Di19-2基因在鹽和乾旱逆境反應中扮演重要角色。除了與逆境相關的研究結果，本研究也發現Di19-2大量表現株在開花時間的外表型較野生型提早，與開花調節相關基因在Di19-2大量表現株中表現量比野生型高，而在idd8/nuc突變株和Di19-2大量表現株的雜交株中，發現有回復idd8/nuc基因突變株造成延緩開花的外表型，暗示Di19-2很可能與IDD8 /NUC共同參與調控植物開花。

The Arabidopsis Drought-Induced Protein 19 (Di19) gene family encodes seven Cys/His-type zinc-finger proteins, most of which with unknown fuctions. The Di19s contain two atypical Cys/His zinc finger-like domains which may participate in different abiotic stress responses and signaling pathways. It was known that the transcripts of Di19-2 (At1G02750) increased in response to high-salt stress, but the protein expression pattern and functions remain unknown. Previous study showed that Di19-2 can interact with IDD8/NUC in yeast two-hybrid, pull-down and bimolecular fluorescence complementation (BiFC) assays. Here, it was found that Di19-2 interacts with IDD8/NUC and positively coregulate SUCROSE SYNTHASE 4 (SUS4) gene expression, but Di19-2 does not recognize Di19 binding site (DiBS, a known Di19-1/Di19-3 binding sequence). After generating the Di19-2 overexpression line, it was found the primary roots of Di19-2 overexprssion transgenic seedlings were shorter than wild type when encountered salt stress. Moreover, the adult of Di19-2 overexprssion transgenic plants showed lower salt and drought tolerance in the soil, which suggests that Di19-2 may play important role in salt and drought stress response. Interestingly, Di19-2 overexprssion transgenic plants showed early flowering phenotype. The flowering regulation genes were up-regulated in Di19-2 overexprssion transgenic plants. When idd8/nuc was crossed to Di19-2 overexprssion line, it showed the recovery of delay flowering phenotype in idd8/nuc mutant line. This suggests that Di19-2 may interact with IDD8/NUC to co-regulate flowering in Arabidopsis.