大麥離層酸反應複合體ABRC1位於35S啟動子在菸草細胞表現之影響

Abstract

啟動子為基因表現與否的主要關鍵，故對可受離層酸Abscisic acid（ABA）誘導的啟動子之相關研究，將有利於瞭解植物的生理與對逆境之反應機制。 本實驗以大麥可受離層酸誘導的HVA22基因，其啟動子中之離層酸反應複合體ABRC1構築在花椰菜鑲嵌病毒35S啟動子中，接上β-glucuronidase（GUS）為報導基因，以含有一個到兩個套數不同方向性ABRC1的構築質體，利用電穿孔法將構築質體送到菸草原生質體中，做短暫表現分析（transient expression assay）。 結果顯示，不需離層酸的處理，各構築質體可以有基本的GUS表現活性，外加10-6M的離層酸可以使含有兩個套數正向的ABRC1的構築質體，受到離層酸的誘導最多達4.9倍，其次是同一構築質體經過啟動子缺失後，仍然可以受到3.8倍的誘導。另外，含有一個或兩個反向的ABRC1也可以分別在適當濃度的離層酸下受到誘導倍數分別為3和2倍，可見由單子葉植物而來的ABRC1可以在雙子葉中受到離層酸所誘導。 此外，在ABRC1之中外加一段ABRE類似的對稱性序列5'-CCCACGTGGG-3'，雖然會降低含有反向ABRC1構築質體的基本GUS表現活性，但並不會改變受離層酸誘導的程度，因此外加對稱式的ABRE類似序列，並不能幫助ABRC1對於離層酸的反應。 外加50μM茉莉酸（jasmonic acid, JA），並不會誘導含有兩個套數正向ABRC1的構築質體，反而會抑制含有兩個套數正向的構築質體和控制組質體pBI221SI的GUS表現活性，但以後者受到抑制的程度較高，若濃度低於50μM則只有控制組受到抑制。外加水楊酸（salicylic acid, SA）處理的實驗結果與JA處理相似，可見JA和SA對於35S啟動子和ABRC1都有影響。 若外加protein phosphatase的抑制劑Okadaic acid(OA)或protein kinase的抑制劑staurosporine(STA)，都會誘導含有兩個正向ABRC1質體的GUS表現，且誘導倍數與單獨離層酸處理時相當，但是當離層酸處理外加STA時，會降低質體可受誘導的倍數，證明protein kinase和phophatases參與了ABRC1被離層酸誘導的過程，外加NaCl，隨著濃度的提高，會使得質體的GUS表現活性降低。 因此，啟動子在不同環境下，會有不同程度的表現，雖然啟動子的操控因數複雜，但也代表植物為適應環境而必須有的生存方式。

The regulation of the promoter is a major key point for gene expression. To study ABA (abscisic acid) inducible promoter is a powerful tool for understanding how plants work in physiological processes and responses to stresses. The ABA responsive complex 1 (ABRC1) from barely HVA22 gene was constructed between 5' upstream CaMV 35S promoter fragment and the 35S promoter TATA box (from -90 to +8), which was fused to β-glucuronidase (GUS) reporter gene for promoter study. By transient expression assay, the construct carrying one or two copies of either direction ABRC 1 was introduced into tobacco protoplasts, and its expression of GUS was used as an indicator to the strength of promoter. The result indicate that all of the constructs carried ABRC1 could express GUS protein in tobacco protoplast without ABA treatment. ABA at a final concentration of 10-6 M induced GUS activity 4.9 times more than the one without ABA treatment in the chimeric 35S promoter with 2 copies of right orientation ABRC1. Deletion of 5'upstream 35S promoter fragment, however, didn't eliminate its response to ABA. In addition to right orientation of ABRC 1, the constructs carrying 1 or 2 copies of reverse orientaion ABRC1 could be induced by properly concentration ABA. This indicates that ABRC1 from monocot can be induced by ABA within dicot plant. The presence of a ABRE- like sequences (5'-CCCACGTGGG-3') of the proximal CE1 (coupling element 1), decreased the GUS expression of the constructs with the reverse orientation of ABRC1, but did not remove its response to ABA. Therefore, the insertion of a conserved ABRE like sequences into ABRC1 can not enhance the response of ABRC1 to ABA. The GUS activity of the construct carrying 2 copies of the right orientation ABRC1 could not be induced by jasmonic acid (JA) treatment. The GUS activity of this construct and the control pBI221SI were inhibited with 5 μM JA treatment. Below 50 μM JA treatment, only the GUS activity of control was inhibited. The result of salicylic acid (SA) was similar to that of JA treatment. Consequently, JA and SA affect both ABRC 1 and 35S promoter. Either Okadaic acid (OA), a protein phosphatase inhibitor, or staurosporine (STA), a protien kinase inhibitor, treatment could induce the GUS activity of the construct carrying 2 copies of the right orientation ABRC1. When both ABA and STA were present, the GUS activity was less than the one treated with only ABA or STA alone. This may indicate that both protein kinase and phosphatase are involved in the induction of ABA in ABRC1.