水稻根部低溫耐性分子機制之探討﹝3/3﹞

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。本計畫前二年已利用低溫下不同耐性 之水稻根部為材料，以cDNA-AFLP 及蛋 白質雙向電泳等方法找出諸多在低溫下表 現之基因與蛋白質。本年之計畫持別針對 其中3 個基因ADF, porin-like protein 及 β-glucosidase 等，釣取其cDNA 全長、決 定該基因於水稻基因體之考貝數及該基因 於不同非生物逆境，尤其是低温逆境之表 現。 結果發現ADF 屬於水稻多基因家族， 共有9 個isoforms.對不同生物性逆境 ADF-2 及ADF-3 基因具差異性表現。另外 則發現porin-like protein 基因為一具陰離子 通道特性之膜孔類似蛋白質。在三個水稻 品種TNG67、IR36 和PANGONIA 中可能 都僅有一個拷貝數。在台農67 號根部中經 過低溫處理後該基因之表現會立即被抑 制，而在24 小時後恢復。該基因表現直接 會受到高溫抑制。 此外本試驗以β-glucosidase 之基因專 一性引子進行該基因全長之釣取，意外因 引子之相似性，選殖到amine oxidase 基 因。經過南方墨點分析，發現此基因在水 稻基因體上可能僅有一個拷貝數。在北方 墨點分析中發現，此基因可受低溫處理所 誘導在一小時後大量表現，二小時後表現 降低，在RT-PCR 中也可觀察到相似之結 果。另外在不同逆境處理後，在地上部和 根部表現之情形也有所不同。此基因參與 多元胺之代謝路徑，將多元胺降解後，會 造成過氧化氫和胺的累積。其與水稻低溫 耐受性之關係仍待闡明。

Abiotic stresses, such as low temperature frequently result in significant reduction of the potential yield of rice. Therefore, in order 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. In past two years, we have identified several differentially expressed genes or proteins in response to low temperature by cDNA-AFLP and 2D gel methods. In this study, we especially focused on three genes, including ADf, porin-like protein and β-glucosidase to determine their corresponding gene copy numbers and expression patterns under various abiotic stress. After rice genome blast search, ADFs belong to a multiple gene families that consisted of 9 genes. RT-PCR analysis with 3’-specific primer pairs showed that ADF-2 and ADF-3 genes respond differentially to various abiotic stresses. In addition, the porin – like protein gene displays the property which similar to the anion channel of membrane protein. After southern blotting analysis, we found it’s a single copy gene in rice cultivars – TNG67, TN1 and Pangonia. In TNG67 root, the expression of this gene shut down when low temperature started after one hour, but it expressed again after 24 hours. Under differential abiotic stress treatment, it shows the porin-like protein gene expression repressed under heat treatment in both tissues. In order to get the full – length cDNA of beta – glucosidase gene, unexpectedly, due to the misannealing of primer pairs, we isolate another gene, which is amine oxidase. The southern blot analysis showed that there is only a single copy gene in the rice genome. After low temperature treatment, the expression of this gene was induced approximately two folds higher than the control. But after two hours, the expression reduced again. The gene may play an important role in polyamine degradation, that causes the H2O2 and NH3 accumulation. The coreelation between amine oxidase gene expression and rice cold tolerance would require further study.