Aspartic proteases（APs）是一種蛋白水解酶，其廣泛存在於所有生物中。數種AP已被證明對於應對生物和非生物逆境至關重要。熱逆境是影響植物生長發育的主要非生物逆境。透過蛋白質體學，我們發現水稻AP的Oryzasin 1與水稻第一族群小分子量熱休克蛋白質Oshsp16.9A在高溫下有相互作用。為了探討Oryzasin 1在水稻耐熱性中的作用，我們將OsAP基因轉入水稻基因組中，以建立過量表現Oryzasin 1的水稻轉植株並以PCR確認是否成功，以供進一步研究。獲得Oryzasin 1過量表現轉植株(OsAP-OEs)後，我們比較了轉植株與野生型(WT)在高溫下的種子發芽率及幼苗存活率。研究成果表明，在熱處理條件下，OsAP-OE轉殖株的種子發芽率和幼苗存活率更高，與野生型相比，在耐熱性方面有顯著提升。此外，我們測量OsAP-OE轉殖株與WT種子在熱處理後的TTC還原活性(TTC reduction activity)和電解質滲漏率(electrolyte leakage)。TTC還原活性實驗中，WT經熱處理後喪失還原能力，而OsAP-OE轉植株仍然能將TTC還原。在電解質滲漏實驗中，熱處理對OsAP-OE轉植株的影響比WT小，代表OsAP-OE的細胞膜受損較少。最後，我們比較了OsAP-OE轉植株與WT的花粉在高溫處理後的存活率，而OsAP-OE轉植株亦取得較好的表現。綜合上述研究，我們證實OsAP能夠正向調控水稻的耐熱性，使OsAP-OE轉植株擁有較好的耐熱表現;Aspartic proteases (APs) are a group of proteolytic enzymes that are broadly distributed in all organisms. Several APs have been demonstrated to be essential for responses to biotic and abiotic stresses. Heat stress is the major abiotic stress seriously influencing plant growth and development. Using proteomic approaches, we found the interplay between oryzasin 1, a rice AP, and Oshsp16.9A, a rice class I small heat shock protein, in rice seeds during high temperature. Here, we are trying to characterize the role of oryzasin 1 in thermotolerance of rice seeds. We inserted oryzasin 1 gene into the rice genome to establish oryzasin 1-overexpressing rice plants, named as OsAP-OEs for further study. The PCR results showed that we first successfully insert oryzasin 1 into the rice genome. We compared the germination rate of seeds and survival rates of seedling between the transgenic plants and the wild-type (WT) plants exposed to heat treatment. The results showed that OsAP-OE transgenic lines have better performance in the germination and the survival rates under heat stress conditions. The germination rate of seeds and survival rate of seedlings of OsAP-OE transgenic lines were higher in comparison with the WT. In addition, we compared TTC reduction activity and electrolyte leakage of the WT and OsAP-OE seeds after heat treatment. Seedlings were viable among each transgenic line, whereas WT seedlings were sensitive to heat stress condition in the result of the TTC reduction. Besides that, the conductivity of the ion leakage also showed that oryzasin 1-overexpression line less effect in the membrane leakage than the WT line. The percentages of pollen viability of transgenic lines were higher in comparison with the WT under high temperature. These results confirm that the OsAP-OE seeds have higher thermotolerance than the WT. Our data revealed that oryzasin 1 positively regulates heat stress tolerance in rice.
