Expression Characteristics Of Glutamine Synthetase Of Wheat Cultivars With Different Nitrogen Use Efficiency

Glutamine synthetase(GS)is the key enzyme of plant nitrogen metabolism,to clarify the relationship of nitrogen use efficiency of wheat and the expression characteristics of GS isoenzyme,we cloned the GS isozyme genes,the preparation of the specific antibody of GS isoenzyme,expression characteristics of glutamine synthetase of different nitrogen use efficiency of wheat genotypes in different tissues and organs were investigated after anthesis.The results showed that the peak of GS activity appeared at anthesis to 8 days after anthesis in kernel,function leaf,outer glume,peduncle,rachis and root of N efficient cultivars Ym49 and N inefficient cultivars XN509,but the peak of main product of nitrogen assimilation(free amino acid,soluble protein)later than the peak of GS activity,appeared at 8 days to 16 days after anthesis.The GS activity in functional leaf of N efficient cultivars always fell sharply at 24 days after anthesis,while N inefficient cultivars always fell sharply at 8 days after anthesis,showed that the high assimilation ability of N efficient genotypes keep longer.The results of Native-PAGE showed that the cytosolic isoform glutamine synthetase isoenzyme(GS1)activity was generally found in different organs,but the chloroplastic isoform glutamine synthetase isoenzyme(GS2)was only present in green organs.GS2 m isozyme activity was found in green organs,and its migration rate was different from GS1 and GS2.GS2 m isozyme activity was only present in anthesis to 8 days after anthesis,and the highest activity appeared at anthesis in function leaf.GS2 is mainly involved in nitrogen assimilation,GS2 activity in different organs was as follows: functional leaf > outer glume > peduncle > rachis;GS1 is mainly involved in the transfer of nitrogen recycling,GS1 activity at 8 days after anthesis was as follows: kernel ≈ rachis > function leaf > outer glume > peduncle,showed that functional leaf and outer glume is given priority to with function of nitrogen assimilation,peduncle both assimilation and transfer function.GS1 remains a highly active after anthesis in rachis,and N efficient cultivars higher than N inefficient cultivars,showed that rachis is given priority to with function of nitrogen transport and distribution function,and the nitrogen transport ability of N efficient cultivars is stronger.The results of Western blot and q PCR showed that Ta GS2 protein subunits and gene expression level were higher in green organs,especially in the functional leaf,and the expression level up to the highest at anthesis,and N efficient cultivars higher than N inefficient cultivars.Ta GS1.1 protein subunit and gene expression level were higher in different organs,mainly expressed in rachis,and the expression level up to the highest at anthesis and 8 days after anthesis.Ta GS1.2 protein subunit and gene mainly expressed in rachis,and the expression level up to the highest at 8 days after anthesis and 16 days after anthesis,and N efficient cultivars was significantly higher than that of N inefficient cultivars,which suggests Ta GS1.2 mainly assist Ta GS1.1 in nitrogen transportation and assimilation in the grain-filling stage when nitrogen transport and the assimilation of larger demand,which is important to the grain-filling;higher the expression level of Ta GS1.2 in rachis,the more nitrogen transport and assimilation,so as to improve the utilization rate of nitrogen.Ta GS1.3 protein subunit and gene mainly expressed in kernel and outer glume,and the expression level up to the highest at anthesis in outer glume,but the expression level up to the highest at 24 days after anthesis in kernel,and N efficient cultivars was significantly higher than that of N inefficient cultivars,which suggests Ta GS1.3 mainly involved in nitrogen assimilation in the grain-filling,higher the expression level of Ta GS1.3,the more nitrogen stored in grain,and improve the grain filling rate in the grain-filling stage,so as to improve the utilization rate of nitrogen.Based on the above experiment,GS isozymes are tissue specific expression and has the characteristics of non overlapping functions,and the influencing mechanism of nitrogen use efficiency is different.