水稻 Ascorbate peroxidase 基因家族啟動子之選殖及分子特性分析

Abstract

Ascorbate peroxidase (APX) 是重要的抗氧化酵素，負責催化 H2O2 為 H2O 及 O2 。水稻APX由八個基因所組成，分別稱為OsAPX1~OsAPX8，其中 OsAPX1與OsAPX2位於細胞質，OsAPX3與OsAPX4位於過氧化體，而 OsAPX5~OsAPX8位於葉綠體，有關水稻APX基因調控及功能的相關研究目前仍然相當少。為了瞭解水稻各APX基因對環境逆境的反應，此試驗利用即時定量PCR (qPCR)分析基因表現，結果顯示多數APX基因受NaCl、缺水、重金屬及植物荷爾蒙誘導表現。分析不同發育時期葉部APX基因表現結果顯示，OsAPX2、OsAPX3 及OsAPX8在營養生長期表現量較高，而OsAPX1、OsAPX4、OsAPX5、OsAPX6及OsAPX7則在生殖生長期表現量較高。分析APX基因上游2 Kb啟動子的cis-acting element，結果顯示各啟動子含有許多與植物荷爾蒙(如JA、ABA及SA）以及非生物逆境(如乾旱、低溫及重金屬)相關的cis-acting element。此外，本試驗分析了OsAPXs基因的轉錄起始點，並選殖各基因上游1~1.5 kb啟動子，並以GUS做為報導基因，再利用農桿菌轉殖到水稻中，以探討OsAPX各啟動子之分子特性。PAPXs/GUS轉殖水稻經過組織化學染色分析後，結果顯示幼苗期OsAPX1、OsAPX4、OsAPX5、OsAPX7及 OsAPX8啟動子於根部及地上部都有相當高的活性，而 OsAPX2、OsAPX3 及 OsAPX6 啟動子主要則驅動GUS累積在地上部；孕穗期階段，幾乎全部OsAPX啟動子於葉鞘、莖部及葉身皆有驅動GUS表現，OsAPX2及OsAPX6在莖部則無GUS累積；穎花中，轉殖OsAPX1、OsAPX4、OsAPX5、OsAPX7及OsAPX8 啟動子的水稻在內外穎有較強的GUS表現，而 OsAPX2、OsAPX3及OsAPX6轉殖水稻的GUS則主要表現在花藥中；在種子中，OsAPX1、OsAPX3、OsAPX4、OsAPX5、OsAPX7及OsAPX8啟動子在成熟種子活性較高。這些結果顯示OsAPX基因家族各基因在不同的環境逆境下，受到不同程度的誘導表現，同時在水稻不同生長時期、不同組織也具有差異表現，顯示水稻在特定時期或組織可能有特定的APX基因參與抗氧化反應。本試驗對 OsAPX基因家族各基因表現及啟動子特性的綜合分析，除了讓我們更瞭解APX基因調控的模式之外，未來將可做為研究各APX基因功能的基礎。

Ascorbate peroxidase (APX) is a key antioxidative enzyme which catalyzes the conversion of H2O2 to H2O and O2 in plants. In rice, APX gene family is composed of eight genes, including two in the cytoplasm (OsAPX1and OsAPX2), two in the peroxisome (OsAPX3 and OsAPX4), and four in the chloroplast (OsAPX5, OsAPX6, OsAPX7 and OsAPX8), and yet their gene regulation and functions remain mostly uncharacterized. In order to understand their responses to environmental stresses, quantitative real-time PCR (qPCR) was conducted. The results showed that most OsAPX genes were induced by NaCl, dehydration, heavy metal, and plant hormones. Analysis of the gene expression patterns of the OsAPX genes at different developmental stages indicated that OsAPX2, OsAPX3, and OsAPX8 accumulated abundant transcripts in leaf tissues at the vegetative stage. In contrast, transcripts of OsAPX1, OsAPX4, OsAPX5, OsAPX6, and OsAPX7 highly increased at the reproductive stage. In silico prediction of the 2-kb promoter region upstream of OsAPX translational start codon revealed that lots of cis-elements are related to plant hormones and abiotic stress. In order to characterize the OsAPX promoters, transcriptional start sites (TSS) of OsAPX genes were identified. In addition, 1.0 to 1.5 kb promoter region was isolated to drive GUS expression, and PAPXs/GUS transgenic rice were generated. Histochemical analysis of PAPXs/GUS plants was conducted. High activity levels of OsAPX1, OsAPX4, OsAPX5, OsAPX7, and OsAPX8 promoters were observed in root and shoot tissue of rice seedlings, and the GUS expression driven by OsAPX2, OsAPX3 and OsAPX8 promoters was accumulated mainly in the shoot tissue of rice seedlings. Most of the PAPXs/GUS plants showed GUS activity in leaf sheath, stem, and blade, but GUS activity was not found in stem tissues of both PAPX2/GUS and PAPX6/GUS plants. Strong GUS expression driven by OsAPX1, OsAPX4, OsAPX5, OsAPX7, and OsAPX8 promoters was observed in the glumes of spikelet; meanwhile, OsAPX2, OSAPX3, and OsAPX6 promoters drove GUS expression mainly in anthers. Promoter activity in mature seeds was observed in OsAPX1, OsAPX3, OsAPX4, OsAPX5, OsAPX7, and OsAPX8 genes. In summary, the diverse expression patterns of OsAPX genes indicate that OsAPX genes are induced by different environmental stresses and developmentally regulated. The analysis of PAPXs/GUS transgenic plants further indicates that OsAPX genes have tissue- and stage- specificity. This study provides basic knowledge of the regulatory OsAPX promoters, which may contribute to further functional analysis of OsAPX genes.