水稻Ascorbate peroxidase基因家族功能分析 1. 水稻Ascorbate peroxidase基因家族之特性及表現 2. 水稻Ascorbate peroxidase 8 (OsAPx8)的功能分析

Abstract

在植物中Ascorbate peroxidase是一個關鍵的抗氧化酵素，可將細胞中H2O2轉變成水。水稻中共有8個APx基因，其中兩個位於細胞質、兩個位於peroxisome，四個表現在葉綠體中，然而水稻APx基因對環境外在刺激的反應仍未清楚研究。為廣泛了解APx基因表現模式，以即時定量PCR來分析APx基因表現，我們的結果顯示水稻多數的APx基因大量累積在幼苗地上部組織，在重金屬處理可誘導水稻根部APx1與APx3誘導10倍以上表現量，另外幾乎所有的APx基因都被缺水、鹽分及ABA處理誘導而大量在根部及地上部表現。植物荷爾蒙處理顯示除了APx7外，水楊酸顯著的在根部誘導APx基因表現。以次細胞蛋白定位分析OsAPx1及OsAPx2位置，結果顯示OsAPx1與OsAPx2皆在細胞質和細胞核中表現。我們過去的研究顯示，水稻APx8會受鹽分誘導表現，為進一步了解其在鹽分逆境下之扮演的角色，試驗中分析水稻APx8啟動子驅動GUS報導基因之轉殖水稻，結果顯示GUS在葉片、葉鞘、莖部、內外穎、種子胚乳及胚中累積，刻傷及鹽分處理也提高GUS在葉片中的累積。分析APx8基因嵌入突變株osapx8的活性，結果顯示地上部減少40%的APx活性，鹽分處理下也呈現較低的誘導性。在鹽分耐受性測試中，osapx8表現對鹽分敏感之外表型，以上結果顯示OsAPx8是一個在鹽分逆境下具有保護植物功能的重要基因。

In plants, ascorbate peroxidase is a key antioxidative enzyme which catalyze the conversion of H2O2 to H2O. In rice, APx gene family is composed of eight genes, including two cytosolic isofoms, two putative peroxisomal isoforms, and four chloroplastic isoforms, and yet their gene expression patterns response to external stimuli remain mostly uncharacterized. To comprehensively understand the specific gene expression patterns of each individual of the APx gene, quantitative real-time PCR was conducted. Our results showed that most transcripts of APx genes are highly accumulated in the shoot tissue. The heavy metal response of APx indicated that more than 10 folds of APx1 and APx3 transcripts were induced by copper in root. Furthermore, almost all APx gene were significantly enhanced by dehydration, salt, and ABA either in root or shoot. Phytohormones treatments revealed that salicyclic acid notably increased the expression of all APx genes, except APx7, in root. Subcellular localization analysis demonstrated that both cytosolic isoform OsAPx1 and OsAPx2 are localized at cytosol and nucleus. Our previous study showed that OsAPx8 is highly induced by salt stress in root. To further understand its roles in salt stress, PAPx8::GUS transgenic rice was generated. PAPx8::GUS transgenic plants showed that GUS accumulated in blade, sheath, stem, lemma, palea, endosperm and embryo. Wounding and salt stress enhanced the accumulation of GUS protein in leaf. Analysis of APx8 knockout mutant, osapx8, showed that APx activity decreased 40% in leaf, while lower inducibility could be observed upon salt treatment. Salt tolerant assay indicated that osapx8 showed a salt sensitive phenotype. All these data let us conclude that OsAPx8 is a crucial gene in protecting rice from salt stress.