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|標題:||Co-transformation of Corynebacterium glutamicum DAHP Synthase and Prephenate Dehydratase Genes into Rice (Oryza sativa L.) and Cloning of Rice DAHP Synthase Gene
共同轉殖Corynebacterium glutamicum DAHP Synthase和Prephenate Dehydratase 基因至水稻與選殖水稻DAHP Synthase基因之研究
|關鍵字:||芳香族胺基酸;3-deoxy-D-arabino-heptulosonate-7-phosphate synthase;提昇營養價值;農桿菌法共同轉移法;基因篩選;台農67號;G418;kanamycin抗生素篩選;prephenate dehydatase;aromatic amino acids;screen rice cDNA library;co-transformation;CaMV35S and globulin promoter||出版社:||分子生物學研究所||摘要:||
3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS)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。酪胺酸 (tyrosine)、苯丙胺酸 (phenylalanine)及色胺酸 (tryptophan) 屬於芳香族胺基酸，是人體所必須的胺基酸。但是，水稻之芳香族胺基酸的含量相對較其它種胺基酸為低，所以，提高水稻中此三種芳香族胺基酸的含量，將可提昇稻米的品質及營養價值。
本研究將由CaMV35S 及 globulin啟動子驅動分離自 Corynebacterium glutamicum 之解除回饋抑制型的 DAHPS 與 PD 基因和野生型的 DAHPS 與 PD 基因 (簡稱 WDS 與 WPD)，以農桿菌法共同轉殖到`台農67號´水稻(Oryza sativa L.)之成熟胚所誘導的癒傷組織，並誘導再生成植株。其目的為探討同時轉移兩個牽涉到芳香族胺基酸合成途徑中的關鍵酵素到同一株水稻，是否更能提高水稻芳香族胺基酸的含量。轉殖再生水稻以50 ppm之G418和150 ppm 之kanamycin進行初步篩選，經過聚合酵素連鎖反應 (PCR)、南方墨點及北方墨點分析之結果顯示，DAHPS與PD基因存在於轉殖植株的染色質，並可轉錄成RNA；且DAHPS與PD基因可共同轉殖到同一水稻植株，並可正常表現。本研究共獲得13株DAHPS與PD基因共同轉殖成功之轉殖植株。
DAHPS與PD共同轉殖植株之DAHPS酵素活性，在葉片較對照組增加1~2倍，在種子增加1~2.5倍； PD酵素活性在葉片較對照組增加1.5~2.5倍，在種子增加1~2.5。 DAHPS與PD共同轉殖植株之三種芳香族胺基酸(Phe、Tyr、Trp)含量均有增加的趨勢，葉片的胺基酸平均約增加2~3倍，種子胺基酸平均約增加1~2倍；其中有2株之葉片的三種胺基酸均較對照組分別增加8及5倍，有4株之種子的三種胺基酸均較對照組增加2倍。
本試驗以剪傷處理3天之`台農67號´水稻葉片polyA RNA為模版，以已發表之植物3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase核酸序列設計引子，進行RT-PCR，增幅出探針，選殖水稻cDNA庫，獲得一個擬似水稻DAHP synthase基因－rDS-15。rDS-15之核酸序列為1806 bp，可轉譯成具有一個502個胺基酸的open reading frame，分子量為55.8kDa。其不論是核酸序列或胺基酸序列都與菸草、阿拉伯芥及馬鈴薯等DAHP synthase基因有高達約75%的相似性，在第299~302 個胺基酸有DAHP synthase酵素之金屬離子結合位置的結構，在大腸桿菌表現載體系統，可表現出一個55 kDa蛋白，且具DAHP synthase活性。以rDS-15為探針與水稻葉片及根部之DNA與RNA均有雜交反應，證實所篩選之rDS-15為水稻DAHP synthase基因。
The 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DS) and prephenate dehydatase (PD) are the two key enzymes in the biosynthesis of aromatic amino acids in plants and microorganisms. Tyrosine, phenylalanine, and tryptophan are aromatic amino acids which are essential for human. Among 20 amino acids, the contents of aromatic amino acids in rice are relative lower than others. Therefore, attempts have been made to increase the amount of aromatic amino acids in rice via gene transformation and improve its nutrition.
In this study, the DS and PD gene of Corynebacterium glutamicum which were relieved their feedback inhibition and wild type (WDS and WPD) were constructed into plant transformation vectors driven by CaMV35S and globulin promoter. The constructed genes were transferred into mature callus of rice (Oryza sativa L. cv. Tainung 67) via Agrobacterium-mediated transformation.The purposes of this study are to explore the possibility for increase the contents of aromatic amino acids in rice via co-transformation of DS and PD genes.
Primary selection of regenerating plantlets was selected with 50 ppm of G418 and 150 ppm of kanamycin. The results of PCR, Southern and Northern hybridization analysis indicated that the DS, PD, WDS and WPD genes were present in the genome of transformed rice, and expressed in transformed rice. In this strudy, 13 co-transformed rice plants were obtained.
Increases in 1~2 times and 1~2.5 times of enzyme activities of DS were found in the leaves and seeds of co-transformed rice plants, respectively. Increases in 1.5~2.5 times and 1~2.5 times of enzyme activities of PD were found in the leaves and seeds of co-transformed rice plants, respectively. In general, increase in 2~3 times and 1~2 times of phenylalanine, tyrosine, and trptophan were found in the leaves and seeds of co-transformed rice plants, respectively. Among them, increases in 8 and 5 times of 3 aromatic amino acids in leaves were found in two transformants, and 2 times in seeds of 4 transformants.
A rice DS cDNA (rDS-15) was isolated from rice cDNA library probed with a RT-PCR fragment which was synthesized by using the designed primers from conserved nucleic acid sequences of published plant DS genes and rice mRNA as template. rDS-15 has 1806 bp and contains an open reading frame of 502 amino acids with predicted molecular weight of 55.8 kDa. Both of the nucleotide sequence and amino acid sequence of rDS-15 exhibits 75% homology with tobacco, Arabidopsis and potato DS genes. There is a metal binding site of plant DS at 299~302 amino acids. In E.coli expression system, rDS-15 can express a 55 kDa protein with DS activity. The results of Southern and Northern hybridization indicated that rDS-15 is presented in the rice leaves and expressed DS RNA.
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