Study Of Nitrogen Assimilation Of Lactuca Dolichophylla Kitam.under Different Mixing Ratio Of Nitrogen Forms

With rich nutrient,strong adaptability,short growth period,lettuce（Lactuca dolichophylla Kitam.）is a hydroponic green leafy vegetables with large-scale production and usually used as a model plant of soil-free cultivation.To meet the need of large market demand and annual supply,alotof nitrogenous fertilizer is supplied.But the yield and quality does not keep pace with the application of nitrogenous fertilizer.Unreasonable fertilization not only reduces the yield and quality,but increases the cost of production and damages the ecological environment.Under the premise of good quality and high yield,improving nitrogen use efficiency is the effective way to increase profits,so the study of nitrogen nutrition regulation and nitrogen assimilation metabolism of lettuce is extremely important.This study adopted five treatments with different nitrogen forms of nutrient solution.Five treatments were conducted according to different nitrate/ammonium ratios:10:0（T₁）,7.5:2.5（T₂）,5:5（T₃）,2.5:7.5（T₄）,and 0:10（T₅）.The main research contents included the physiological characteristic,architecture and microstructure of roots,the difference and contact between roots and aerial parts on the growth and nutrition,the cloning and expression analysis of glutamine synthetase gene（LdGS）and glutamate synthase gene（LdGOGAT）in roots and aerial parts of lettuce.This study aimed at exploring the response of GS/GOGAT cycle to nitrogen forms from physiological and molecular levels,which could do benefit to the further study of nitrogen assimilation of lettuce.The main research results were as follows.1 Effects of different nitrogen forms on the roots of lettuceWith the decreasing of nitrate/ammonium ratio,the total length,surface area,volume,tips number,forks number and activity of roots increased first and declined later.The maximum was detected when nitrate/ammonium ratio was 7.5:2.5,but it declined to the minimum when nitrate/ammonium ratio was 0:10.A larger root length,surface area and activity of roots can provide more efficient assimilation ability.The tips number and forks number of roots showed the growth potential of root.Obviously mixed forms of nitrogen could improve the architecture and assimilation ability of roots.When nitrate/ammonium ratio was 7.5:2.5,the growth and architecture of the roots was optimal.With the decreasing of nitrate/ammonium ratio,roots average diameter showed increasing trend and reached the maximum when nitrate/ammonium ratio was 0:10.Observation of tip transverse section showed that different forms of nitrogen had remarkable effect on the microstructure of roots.The number of root cortex cell’s plies and the thickness of cell wall increased when the nitrate/ammonium ratio declined.2 Effects of different nitrogen forms on the growth and physiological characteristics of lettuceWith the decreasing of nitrate/ammonium ratio,the content of organic acid and soluble carbohydrate in roots exhibited an elevation in early stage and reduction in later stage.The maximum was detected when nitrate/ammonium ratio was 5:5 and 7.5:2.5,respectively.But soluble protein content in roots declined all along and had minimum level when nitrate/ammonium ratio was 0:10.On the contrary,the aerial parts showed reduced content of organic acid in the early stage but a great increase in later stage.Soluble carbohydrate content in aerial parts increased when nitrate/ammonium ratio declined.Soluble protein content,however,declined first and increased later.When nitrate/ammonium ratio was 5:5,content of organic acid in aerial parts was the minimum while soluble protein reached the maximum.The SOD activity of roots and aerial parts increased first and declined later when nitrate/ammonium ratio declined.The SOD activity was the highest when nitrate/ammonium ratio was 7.5:2.5 in roots and was 5:5 in aerial parts.On the contrary,the content of MDA and proline as well as the activity of POD and CAT exhibited the opposite trend both in aerial parts and in roots.With the decreasing of nitrate/ammonium ratio,root-shoot ratio declined while the dry weight of both roots and aerial parts increased first and declined later.When nitrate/ammonium ratio was 7.5:2.5,the dry weight of both roots and aerial parts were the highest,and root-shoot ratio was optimal.In summary,different forms of nitrogen had remarkable effect on the growth and nutrition of both roots and aerial parts,and roots physiological response was more sensitive.When nitrate/ammonium ratio was 7.5:2.5,the coordinated growth of the roots and aerial parts was optimal,as well as the growth and nutrition.3 Effects of different nitrogen forms on the activity of nitrogen assimilation enzymeWith the decreasing of nitrate/ammonium ratio,the nitrate content of roots and aerial parts showed a reducing trend and the nitrate content of aerial parts was higher.The maximum reached 285 μg/g and 326μg/g respectively when nitrate/ammonium ratio was 10:0,which were up to the standard of green vegetables（<432 μg/g）.The ammonium content of roots showed a reducing trend too.However,the ammonium content of roots increased first and declined later and was highest when nitrate/ammonium ratio was 7.5:2.5.With the decreasing of nitrate/ammonium ratio,the glutamine synthetase（GS）activity of roots and aerial parts increased first and declined later and was the highest when nitrate/ammonium ratio was 7.5:2.5.But it decreased significantly when ammonium ratio was more than 50%.The glutamate synthase（GOGAT）activity of roots and aerial parts increased first and declined later when nitrate/ammonium ratio declined,and the activity of NADH-GOGAT and FD-GOGAT had a same trend.The GOGAT activity was the highest when nitrate/ammonium ratio was 5:5 in roots and was 7.5:2.5 in aerial parts.4 Cloning and expression analysis of LdGS and LdGOGATThe full-length of cDNA of LdGS1 and LdGS2 was respectively 1077 bp and 1293 bp,and the coding sequence respectively encoded 358 amino acids and 430 amino acids.The encoding protein of LdGSl and LdGS2 all belonged to hydrophilic proteins and had no signal peptide and had a formula of C2095H3252N572O637S14 and C2095H3252N572O637S14,respectively.With the decreasing of nitrate/ammonium ratio,the expression of LdGS in roots and aerial parts increased first and declined later.The expression of LdGS2 was the highest when nitrate/ammonium ratio was 7.5:2.5 in roots and aerial parts.The expression of LdGS1 was the highest when nitrate/ammonium ratio was 7.5:2.5 in roots,but it was the highest when nitrate/ammonium ratio was 5:5 in aerial parts.The length of cDNA of LdFD-GOGAT gene fragment and LdNADH-GOGAT gene fragment was respectively 622 bp and 737 bp.The coding sequence encoded 207 and 245 amino acids,respectively.With the decreasing of nitrate/ammonium ratio,the expression of LdGOGAT in roots and aerial parts increased first and declined later.The expression of LdFd-GOGAT was the highest when nitrate/ammonium ratio was 7.5:2.5 in roots and aerial parts.The expression of LdNADH-GOGAT was the highest when nitrate/ammonium ratio was 5:5 in roots,but it was the highest when nitrate/ammonium ratio was 7.5:2.5 in aerial parts.5 Synergistic effects between GS and GOGAT in the process of nitrogen assimilationMixed forms of nitrogen effectively promoted the expression of LdGS and LdGOGAT of letture.Under the different nitrogen forms,the expression of LdGS2 and LdFd-GOGAT had a similar variation tendency,and their expression in aerial parts was higher than it in roots,the two isozyme activity as well.The expression and isoenzyme activity of LdGS1 had a similar variation tendency with LdNADH-GOGAT,which in roots was higher than it aerial parts.This showed that LdGS2 and LdFd-GOGAT played a leading role in nitrogen assimilation of aerial parts,but LdGSl and LdNADH-GOGAT did the key work in roots.The result was accorded with the localization of gene in different plant tissue.