水稻根部低溫耐性分子機制之探討

Abstract

水稻經常因遭受非生物性之生理逆 境，如低溫而導致嚴重減產。因此極其重 要的是去瞭解水稻在低溫逆境下之耐冷機 制，尤其是其內部所有基因或蛋白質之表 現，找出與低溫反應相關之基因或蛋白 質。本計畫乃以水稻根部為系統，承續先 前已應用之基因差異顯示法，進一步以 PCR-selected cDNA substraction ， cDNA-AFLP 及蛋白質雙向電泳等方法來研 究在低溫下其內部所有基因與蛋白質之表 現。實驗結果顯示，由先前基因差異顯示 法對低溫具耐性(台農67 號, TNG67) 及對 低溫敏感(台中在來一號, TN1) 之水稻根 部所得之部份cDNA 片段逕行篩庫而獲致新 增基因， 如Reversibly glycosylated polypeptide protein (RGP), Dna-J related protein, NADPH-dependent aldose reductase, inorganic pyrophosphatase, Myb-like protein 與 ICT ， 將進一步分析其生理功能。在 PCR-selected cDNA substraction 之研究 上亦藉由反向北方墨氏法(revrse Northern blot) 初步獲得近20 個在低溫 下基因表現有差異之cDNA 片段, 目前正從 事定序之進一步方析。另外在以cDNA-AFLP 之方法來尋找早期與低溫訊息傳導有關之 基因，初步得到數個在低溫下可被誘導之 cDNA 片段，將繼續就其它不同引子之組合 進行篩選。最後本計畫亦針對水稻根部在 低溫或低溫處理後予以回溫其全部蛋白質 表現群相之差異利用蛋白質雙向電泳加以 分析，目前至少找到8 個受溫度影響其表 現的蛋白質。綜合言之，本計畫未來將就 上述cDNA 片段或蛋白質，進一步分析其與 低溫反應之關係，並檢測該基因如何在生 理功能上參與低溫耐性之調控。

Abiotic environmental stresses, such as low temperature frequently result in significant reduction of the potential yield of rice. Therefore, to understand the cold tolerance mechanism in rice, it is essential to evaluate and determine the genes/ or proteins expression profile in rice under chilling stress, and identify key transcripts or proteins that are associated with this process. To achieve this purpose, follows previous differential display (DDRT-PCR) approach, several methods including PCR-selected cDNA substraction, cDNA-AFLP and protein 2-D gel analysis have been applied to address this problem. The result showed that with partial cDNA fragments derived from DDRT-PCR using chilling tolerant (TNG 67) or chilling sensitive (TN1) rice cultivars as probes, several new genes have been found. These are reversibly glycosylated polypeptide protein (RGP), Dna-J related protein, NADPH-dependent aldose reductase, inorganic pyrophosphatase, Myb-like protein and ICT. The physiological function of these genes will be further characterized. Also about twenty cDNA fragments that expressed under chilling condition were identified by PCR-selected cDNA substraction and confirmed by reverse Northern blot. These cDNA fragments are now in sequencing. In addition, to look for genes involved in the early stage of cold signal transduction, another approach cDNA-AFLP was carried out in this study. So far a few cDNA fragments were obtained and other combination primer sets will be continuously used in searching for more genes. Finally, a protein 2-D gel analysis was used to reveal protein expression profiles difference in rice roots under chilling or re-warming condition. So far at least eight proteins were identified. In the future, genes or proteins identified above will be further analysis to dissect their relationship with cold tolerance in rice root.