论文部分内容阅读
谷氨酰胺合成酶 (Glutaminesynthetase,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 (Pisumsatium)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GS1cDNA和GS2cDNA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 (Triticumaestivum)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 4 1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。
Glutamine synthetase (GS, E.C. 6.3.1.2) is a key enzyme in plant ammonia assimilation and plays a crucial role in plant nitrogen uptake and metabolism. Glutamine synthetase is also a target enzyme for the herbicide Phosphinothricin (PPT) or Basta. Preliminary work Cytoplasmic glutamine synthetase (GS1) cDNA and chloroplast glutamine synthetase (GS2) cDNA have been cloned from the pea (Pisumsatium) species endemic to our country. In order to verify the function of glutamine synthetase, a plant expression vector p2GS containing GS1 cDNA and GS2 cDNA was constructed. The calli were transformed into immature embryos of Triticum aestivum by Agrobacterium tumefaciens-mediated method. After screening and differentiation and regeneration of PPT, 41 transgenic wheat plants resistant to PPT were obtained. PCR and genomic Southern hybridization analysis confirmed that the GS1 and GS2 genes have been integrated into the genome of transgenic wheat. Application of the herbicide phosphinothricin Basta solution to p2GS wheat leaves showed that the GS transgenic plants were resistant to up to 0.3% Basta solution and the leaves of control plants turned yellow until they died. Transgenic wheat plants are normally robust. The above experimental results show that: 1) GS gene is efficiently expressed in wheat plants, thereby giving the plant PPT resistance; 2) GS gene can be used as a screening marker gene for wheat genetic transformation.