Studies on Transformation of N-acylamino Acid Racemase Gene (naaar) into the Rice (Oryza sativa L.)

Abstract

In this study, N-acylamino acid racemase gene (naaar) isolated from Deinococcus radiodurans was constructed into plant transformation vectors driven by CaMV35S, rbcS, globulin, or oleosin promoter, and transformed into the ‘TN67' rice (Oryza sativa L/cv/ Tainung 67) via Agrobacterium-mediated transformation. The frequencies of regeneration in the naaar transformed plantlets of four kinds of vectors were between 2.5% to 9.5%, the average of regenerated frequency is 5.8%. A 402 bp fragment of naaar gene could be amplified from 74% to 95% transformed rice plants among four kinds of transformants by PCR analysis. The results of Southern, Northern, and Western blot hybridization indicated that the naaar gene had been transformed and inserted into the chromosome of rice, expressed naaar mRNA and translated NAAAR protein, the data show the feasibility of using rice plants as bioreactor to produce high-value-protein to increase the economic value of rice.

本試驗將篩選自抗輻射奇異球菌(Deinococcus radiodurans)的胺基端醯化胺基酸消旋酵素(N-acylamino acid racemase, NAAAR)基因，構築至分別具有CaMV35S、rbcS、globulin 或 oleosin等啟動子的植物轉殖載體上，再以農桿菌法轉殖至’台農67號’水稻癒傷組織中，誘導再生植株。試驗結果顯示所有轉殖載體均有再生植株，4種質體之再生率介於2.5%~9.5%之間，平均再生率為5.8%。經PCR初步篩選，大部份的轉殖植株均具有naaar基因，4種質體之轉殖率介於74%~95%之間，平均轉殖率為85%。南方墨點分析之結果顯示四種質體之naaar基因已轉殖到再生水稻，並已插入其染色體組DNA中。北方墨點及西方墨點分析pKcnaaar及pKrnaaar之轉殖再生水稻顯示轉殖之naaar基因可在再生水稻葉片中轉譯出naaar mRNA，並表現NAAAR酵素(racemase)。試驗結果顯示利用naaar基因轉殖，以水稻來生產消旋酵素是可行的。