CAJ | 학술논문

谷氨酰胺合成酶(Glutamine synthetase,GS,E.C.6.3.1.2)是植物氨同化过程中的关键酶,对植物的氮素吸收和代谢起着至关重要的作用.谷氨酰胺合成酶还是除草剂草胺膦(Phosphinothricin(PPT)或Basta)的靶标酶.前期工作已从我国特有的豌豆(Pisum satium)品种中克隆了细胞质型谷氨酰胺合成酶(GS1)cDNA和叶绿体型谷氨酰胺合成酶(GS2)cDNA.为了验证谷氨酰胺合成酶的功能,构建了同时含有GSl cDNA和GS2 cDNA的植物表达载体p2GS.以该表达载体通过农杆菌介导法,转化小麦(Triticum aestivum)的未成熟胚愈伤组织,经PPT筛选及分化再生培养,获得了抗PPT的转基因小麦植株41株.PCR和基因组Southern杂交分析证实了GS1和GS2基因已经整合到转基因小麦的基因组.用除草剂草胺膦Basta溶液涂抹转p2GS小麦叶片,结果证明GS转基因植株可以抗高达0.3%的Basta溶液,而对照植株叶片逐渐变黄直至枯死.转基因小麦植株能正常结实.上述实验结果表明:1)GS基因在小麦植株中获得了有效表达,从而赋予小麦植株抗PPT特性;2)GS基因能够作为研究小麦遗传转化的筛选标记基因.
곡안선알합성매(Glutamine synthetase,GS,E.C.6.3.1.2)시식물안동화과정중적관건매,대식물적담소흡수화대사기착지관중요적작용.곡안선알합성매환시제초제초알련(Phosphinothricin(PPT)혹Basta)적파표매.전기공작이종아국특유적완두(Pisum satium)품충중극륭료세포질형곡안선알합성매(GS1)cDNA화협록체형곡안선알합성매(GS2)cDNA.위료험증곡안선알합성매적공능,구건료동시함유GSl cDNA화GS2 cDNA적식물표체재체p2GS.이해표체재체통과농간균개도법,전화소맥(Triticum aestivum)적미성숙배유상조직,경PPT사선급분화재생배양,획득료항PPT적전기인소맥식주41주.PCR화기인조Southern잡교분석증실료GS1화GS2기인이경정합도전기인소맥적기인조.용제초제초알련Basta용액도말전p2GS소맥협편,결과증명GS전기인식주가이항고체0.3%적Basta용액,이대조식주협편축점변황직지고사.전기인소맥식주능정상결실.상술실험결과표명:1)GS기인재소맥식주중획득료유효표체,종이부여소맥식주항PPT특성;2)GS기인능구작위연구소맥유전전화적사선표기기인.
Cloning of cytosolic (GS1) cDNA and chloroplast glutamine synthetase (GS2) cDNA from Pisum satium was carried out previously in our laboratory. To identify the functions of the GS genes, we first constructed a plant expression vector, p2GS, harboring two different isoenzymes, GS1 and GS2 cDNAs, under the control of two constitutive promoters of rice, Actin1 (Act1) and maize Ubiquitin (Ubi) genes. Then, using an Agrobacterium tumefaciens-mediated transformation method, we introduced GS1 and GS2 genes into wheat (Triticum aestivum) plants, producing 2GS-transgenic wheat plants using immature embryos as the explants.Presence of the transgenes GS1 and GS2 in wheat plants was confirmed by PCR and Southern blot hybridization analyses. Forty-one independent transgenic wheat plants with tolerance to an herbicide (Phosphinothricin,PPT) were generated. Results from Basta tests showed that the 2GS-transgenic wheat plants were endowed with herbicide-tolerant properties. Almost all of the transgenic plants were normal in morphology, and seed production was similar to that of the control wheat plants. Our study suggest that PPT resistance is conferred by effective expression of glutamine synthetases in transformed wheat plants, and glutamine synthetase genes can serve as a selective marker gene of wheat transformation system in our study.