农杆菌介导的转谷氨酰胺合成酶基因小麦的抗除草剂特性研究(英文)

AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSGENIC WHEAT PLANTS WITH GLUTAMINE SYNTHETASES CONFER TOLERANCE TO HERBICIDE

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。

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.