The Functional Analysis Of Glutamine Synthetase In Nitrogen Metabolism In Wheat

The assimilation, absortion and translocation of nitrogen and the formation of amino acid, protein and other nitrogenous organic compounds are crucial for crops, which are associated with the growth and development of crop and are closely related to the nitrogen utilization.Through the methods of gene mutation with excessive expression, QTL location and plant physiology, the pervious studies have pointed that GS and its activities have an obvious positive correlation with nitrogen utilization and the crops’ harvest and its appearance;they also turned out that GS2locates in both chloroplast, mitochondria and is related to nitrogen assimilation by using immunchemistry, chromatography and Western-bltting techniques and other cell-biochemistry technologies; GS1locates in cytochylema of vascular tissue and is associated with nitrogen translocation and recycle. However,the specific function of diverse GS isoenzymes isn’t clarified in the enzyme level.Through Native-PAGE combining with activity staining method, We are the first one that isolate four diffrerent activity GS isoenzyme from wheat:GS2, GSxl, GSx2and GS1,according to mobility, we need futher identify subunits of GS and isoenzyme subcellular location, from the view of growth and development of wheat,confirming their precise function in the process of nitrogen assimilazation, absorbtion and translocation.we purify chloroplast and mitochondria of wheat’s leaf by using differential centrifugation and sucrose density-gradient centrifugation technique, and testify that mobility and activity of plasmid GS2which only exists in chloroplast is maximum in results of Native-PAGE combining with activity staining and western-blot, and is constituted only with45KD subunit, and belongs to plasmid GS. we preliminary judge that GSx1,GSx2and GS1exist in cytochylema. we recover GS isoenzyme from Native-PAGE and identify the subunits of GS isoenzymes through western-blot, the results display that GS1consists of38KD subunit and exist in cytochylema; subunits of GSxl composes of45KD subunit, subunits of GSx2is constituted with38KD and45KD subunit.According to results of western-blot, we identify protein spot of45KD of2-DE gel in the scope of PH4.7-5.9through MALDI-TOF-MS/MS.Results show that its plasmid GS,we not detect cytoplasmic GS near38KD, and possible cause is that relative abundance of GS1is too low. We isolate and purifiy subunit of38KD by Native-PAGE combining with SDS-PAGE and authenticate GS protein again by adopting LC-MS/MS, results reveal that protein sequence of GSxl consistent with plasmid GS2, results of identification of GS1arise three types of protein: GS1,GSr1and GSr2.The study of the spatio-temporal expression of wheat GS isoenzyme have shown that, in the stem, GS1activities play leading roles in different growth periods of the wheat, and GS2, GSx1、 GSx2only have weak expressions in the green stem. Stem contains abundant vascular bundle which play a signficantly part in transportation, making a further result that GS1have related to transference of nitrogen. In leaves, GS2activities play leading roles in the functional leaves, and gradually weakened and disappeared with the senescence of plant. The photosynthesis of functional leaves are strong, the chloroplast is the place of photosynthesis processes and the reduction of NO3-, which provide raw materials and energy for nitrogen assimilation, this also proved that GS2is involved in nitrogen assimilation. GS1have a weak activity in the entire growth period of leaves, this perhaps due to photosynthetic tissue is more thriving than conducting tissue. GS1transport nitrogen which are assimilated by the functional leaves to the developmental organs, it also assimilate ammonia which is degraded by the nitrogenous compounds of senescence leaves to glutamine and then transfer to sink organs such as seeds. GSx1exists only in thriving function leaves but its activity is high, for example, the flag leaves from anthesis to14days after anthesis, the photorespiration is vigorous, we guess it participate in the assimilation of ammonia which are released by photorespiration. GSx2exists only in the leaves of7days after anthesis and its activity is very weak, we still cannot speculate its function. In the grain there only have GS1activity and the activity is very high in the formation of the grain, then cutting down; but the composition of the subunit have great differences with stems and leaves. The GS1of grain at7days after anthesis only have subunit of39KD, in the14days after anthesis we have detected the subunit of39kD、43kD45kD and49kD. In the21and28days after anthesis, there have extra subunit of33KD. Grains don’t have microtubules organizations, the function of GS1has not been determined.