圓葉菸草脯胺酸去氫酶及高粱穀胱甘肽還原酶基因的選殖及特性分析

Abstract

植物無法移動，因此必須有良好的防禦系統以避免遭受環境逆境的傷害。抗氧化系統以及脯胺酸在植物抵抗逆境均扮演重要的角色。穀胱甘肽還原酶 (Glutathione reductase, GR) 為植物重要的抗氧化酵素之一，負責將氧化態的穀胱甘肽 ( GSSG) 還原成還原態 GSH，避免細胞內氧化還原系統失衡；脯胺酸在植物中扮演滲透調節者的角色，會受到 Proline deydrogenase (ProDH) 代謝。為了了解 C4 型模式植物高粱 GR 基因以及圓葉菸草 ProDH 基因之特性，試驗中選殖並分析高粱的 3 個 GR 基因和圓葉菸草 ProDH 基因。結果顯示SbGR1 與 SbGR2 和水稻 OsGR1 及 OsGR2 胺基酸序列相似度分別達 93 % 與 95 %，SbGR3 與 OsGR3 則只有 49 %。與基因體組序列比對結果發現 SbGR3 基因中一段 Intron 未被移除，導致一個 stop codon 出現，讓此基因無法轉譯出正常的蛋白質。以大腸桿菌異源表現 SbGR，結果顯示 SbGR1 及 SbGR2 可表現出具有GR活性的蛋白質，而 SbGR3 蛋白質則不具 GR 活性；次細胞定位分析顯示 SbGR1 位於葉綠體的澱粉體，SbGR2 則位於細胞質。鹽逆境會誘導 SbGR1 及 SbGR2 表現，高溫 (45℃) 誘導 SbGR1 表現，但抑制 SbGR2 表現。這些結果顯示，水稻及高粱 GR 同源基因對高溫逆境的反應及蛋白質位置具有不同結果，此差異對抗氧化系統之影響仍有待研究。在 ProDH 基因部分，試驗中選殖出圓葉菸草的 ProDH 基因，稱為 NbProDH，基因全長為 1895 bp，ORF (Open reading frame) 為 1497bp，蛋白質分子量為 55 kDa。ProDH 表現量以葉片最高，其次是葉柄跟莖；ProDH 表現會受缺水抑制、受復水誘導，而脯胺酸會在缺水 12 小時後開始累積，試驗中亦建立大量表現 NbProDH 轉殖植株，以進行基因功能分析。

Plants couldn’t move, so they have developed a good defense system to avoid suffering from the damage of environmental stresse. An antioxidant defense system and proline play an important role in the resistance to stress in plants. Glutathione reductase (GR), one of the antioxidant defense systemic enzymes, mediates the reduction of oxidized glutathione (GSSG) to GSH to maintain cellular redox homeostasis. Proline is suggested to act as a compatible osmolyte and be degraded by proline dehydrogenase (ProDH). To understand thecharacteristics of GR gene in C4 model plant – sorghum and the ProDH gene in N. benthamiana, our study cloned and analized three sorghum GR genes and the N. benthamiana ProDH gene. The result showed that the amino acid sequence similarities of SbGR1 and rice OsGR1 were 93%, SbGR2 and OsGR2 were 95%, but only 49% for SbGR3 and OsGR3. Compared with the sequence of genomic DNA, we found that one fragment of intron wasn’t removed from the SbGR3 cDNA which resulted in a premature stop codon and let this gene can’t translate a normal protein. The expression of SbGR1 and SbGR2 in E. coli validated that it can be translated as a protein with GR activity. However, overexpression of SbGR3 in E. coli produced no GR activity. Subcellular localization of SbGR-GFP revealed that SbGR1 was localized to the chloroplast and SbGR2 to the cytosol. SbGR1 and SbGR2 were induced by salinity. A high temperature (45℃) induced the expression of SbGR1, but repressed the expression of SbGR2. These results show that the expression of homologous GR genes in rice and sorghum were different in response to the heat stress, and also different in the expressed protein location. Therefore, the differences of antioxidant defense systems between sorghum and rice have yet to be studied. In the other part, we isolated the proline dehydrogenase gene from Nicotiana benthamiana which called NbProDH. The full-length NbProDH gene is 1895 bp. The cloned cDNA contains open reading frames of 1497 bp and encodes protein of 499 amino acids with a molecular mass of 55 kDa. Under normal condition, the expression of NtProDH is higher in leaves than in petiole and stem. The expression of NbProDH was repressed by dehydration and induced by rehydration. And proline began to accumulate after dehydration for 12 hours. This studied also established the overexpression NbProDH transgenic plants for gene functional analysis.