水稻蛋白磷解酶2Ac-2負向調控滲透壓逆境抗性機制之探討

The Putative Role of Protein Phosphatase 2A Catalytic Subunit Isoform 2 as a Negative Regulator on Osmotic Stress Tolerance in Rice

邱靖雯(Ching-Wen Chiu) ； 林書增(Shu-Tseng Lin) ； 侯新龍(Shin-Lon Ho) ； 黃文理(Wen-Lii Huang)

《嘉大農林學報》 12卷1期 (2015/02) Pp. 27-45

摘要

本研究目的是藉由過量表現＂OsPP2Ac-2＂基因水稻轉殖細胞系，觀察在滲透壓逆境下細胞之生長情形，並以Nitroblue tetrazolium （NBT）與3,3' -diaminobenzidine （DAB）染色，探討＂OsPP2Ac-2＂基因與氧化逆境之相關性。結果顯示在水稻幼苗期＂OsPP2Ac-2＂基因表現有受滲透壓逆境抑制之趨勢，以農桿菌轉殖法於水稻中過量表現＂OsPP2Ac-2＂基因，在含hygromycin抗生素之篩選培養基中增生新的細胞團，以轉殖細胞系為計算標準期轉殖效率為13%。以PCR、南方墨點分析與即時定量PCR分析均證明為＂OsPP2Ac-2＂基因過量表現轉殖細胞系；Ox55及Ox89兩個轉殖細胞系培養於有或無添加0.3M sorbitol MSD_2培養基中，與未轉殖之TNG67一樣，均有隨培養天數增加細胞量逐漸增加的趨勢，唯轉殖細胞系於滲透壓處理下之細胞增殖與生長速率均有因對逆境更加敏感而減緩的趨勢。且逆境處理下Ox89轉殖細胞系NBT藍色淅色最深，而DAB黃褐色染色最淡，顯示轉殖細胞系有較多超氧陰離子累積與較少的過氧化氫形成，推測可能與超氧岐化酶活性降低有關。綜合上述結果顯示＂OsPP2Ac-2＂基因可能負向調控水稻於非生物逆境下之耐逆境相關下游基因之表現有關。

關鍵字

水稻 ； 蛋白磷解酶2Ac-2 ； 滲透壓逆境 ； 負向調控

並列摘要

The study aimed to understand the possible role of protein phosphatase 2A catalytic subunit isoform 2 (OsPP2Ac-2) on osmotic stress in rice by genetic transformation approach. The growth curve, nitroblue tetrazolium (NBT) and 3, 3'-diaminobenzidine (DAB) staining of transgenic cell lines under osmotic stress treatment were determined. The results showed that ＂OsPP2Ac＂ gene expression was inhibited under sorbitol treatment at seedling stage. The transformation efficiency is approximately up to 13%. All transgenic cell lines were confirmed by genomic PCR, Southern blot and quantitative real time PCR analysis. The growth curve of two transgenic cell lines, Ox55 and Ox89, either grown on MSD_2S_0 or MSD_2S_3 medium are similar to wild type, TNG67. But their growth efficiency are significantly decreased under sorbitol treatment owning to hypersensitive to osmotic stress. Besides, The NBT stain is stronger and DAB stain is lighter in ＂OxOsPP2Ac-2＂ overexpression line, Ox89, under sorbitol treatment for 20 days than in TNG67 which may derive from higher level of superoxide and low level of hydrogen peroxide accumulation. It may cause from lower enzyme activity of superoxide dismutase in Ox89 under sorbitol treatment. We concluded that ＂OsPP2Ac-2＂ gene may act as a negative regulator for osmotic stress tolerance in rice by affecting downstream stress responsive genes. In the future, more study is necessary to clarify the possible role of ＂OsPP2Ac-2＂ gene on stress tolerance in rice.

並列關鍵字

＂Oryza sativa＂ ； Protein phosphatase 2A catalytic subunit isoform 2 ； Osmotic stress ； Negative regulation