Studies on Transferring DAHP Synthase and Prephenate Dehydratase Genes into Rice (Oryza sativa L.)

Abstract

Abstract The 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DS) and prephenate dehydatase (PD) are the two imporptant enzymes in the biosynthesis of aromatic amino acids (trptophan, tyrosine and phenylalanine) in plants and microorganisms. In human, aromatic amino acids are enssential amino acids that depending on diet only. We attempted to increase the ratio of aromatic amino acids in rice, and raise its nutrition. In this study, the mutants of Corynebacterium glutamicum DS and PD gene which relieved their feedback inhibition and wild type (WDS and WPD) were constructed into plant transformation vectors driven by CaMV 35S, rubisco small subunit (rbc S) or globulin promoter. The constructed transformation vectors introduced into callus of rice (Oryza sativa L. cv. Tinung 67) via Agrobacterium-mediated transformation and double gene co-transformation. To establish the rice transformation system, and reveal the possibvility that DS, PD, WDS or WPD increase the aromatic amino acids in rice. The results of PCR, Sorthern and Northern hybridization analysis indicated DS, PD, WDS and WPD gene were present in the genome of transformed rice, and expressed in most of transformed rice. The regeneration rate of rice transferred with one gene was 4.6 %. The regeneration rate of co-transformation PD and DS genes were between 1.0~5.0 %. Increase 3 times and 2 times in DAHP synthase and prephenate dehydratase enzyme activity were found in transgenic rice, respectively. Transformed rice also found raise of aromatice amino acids between 2~5 times.

摘 要 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DS) 與 prephenate dehydratase (PD) 為在植物與細菌體內生合成芳香族胺基酸的兩個關鍵酵素。芳香族胺基酸中的苯丙胺酸 (phenylalanine) 及色胺酸(tryptophan)，為人體所必須的胺基酸，是依賴飲食攝取而來。所以若能在水稻中增加芳香族胺基酸的含量，將更有助於增加水稻的營養價值。 本試驗將分離自 Corynebacterium glutamicum 之解除回饋抑制型的 DS 與 PD 基因和野生型的 DS 與 PD 基因 (簡稱 WDS 與 WPD)，構築到有CaMV35S、rbcS 及 globulin啟動子的植物轉殖載體。並以農桿菌單獨或共同轉移DS 與 PD 基因到`台農 67 號´水稻(Oryza sativa L.)之成熟胚所誘導的癒傷組織，並誘導其再生。本研究同時也將GUS 報導基因轉移到`台農 67 號´水稻之成熟胚所誘導的癒傷組織。本研究之目的為建立水稻的基因轉殖系統，並探討轉移PD及DS基因到水稻，使其大量表現，以增加水稻芳香族胺基酸之可行性。 研究結果顯示，以農桿菌轉移單一基因到`台農 67 號´水稻之植株再生率，最高可達4.6%；共同基因轉移之植株再生率則介於 1.0 ~ 5.0 % 之間。以南方墨點法分析 PCR 正反應之轉殖植株，均可偵測到轉移基因的存在，以北方墨點分析亦大部分能偵測到其RNA。試驗結果顯示PD及DS基因有轉移到水稻染色體並表現其RNA，但沒有PD及DS基因共同轉移且有表現的情形。分析轉殖植株之DS 與 PD 之酵素活性，顯示在DS轉殖植株中之DS酵素活性，最高可提高三倍；在PD轉殖植株中之PD酵素活性，最高可提高二倍多。分析轉殖植株之胺基酸含量目前正在進行中。