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Mechanism Of Phosphorylation Site And Adjacent Amino Acid Residues Regulates Nitrate Reductase Activity And Nitrogen Nutrition In Rice

Posted on:2020-05-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:R C HanFull Text:PDF
GTID:1483306302486414Subject:Crop Cultivation and Farming System
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Rice(Oryza sativa L.)is one of the most important grain crops in China and plays an important role in ensuring food security.Nitrogen is the main mineral element affecting rice yield.At present,the uptake rate of nitrogen fertilizer in Chinese rice fields is only about 40%.Compared with the major rice-producing countries,the nitrogen fertilizer application rate of rice in China is relatively high while the nitrogen utilization rate is lower.Nitrate reductase(NR)is a key Nitrate assimilating enzyme in plants,and its activity is regulated by post-translation phosphorylation modification.The activity of NR decreased rapidly from light to dark,which reduced the uptake and assimilation of nitrate nitrogen by plants.Therefore,it is of great practical significance to study the phosphorylation regulation mechanism of nitrate reductase activity in rice to improve the capacity of nitrate assimilation and nitrogen utilization in rice.In the study,according to the biochemical characteristics of rice nitrate reductase NIA1 phosphorylation site and adjacent amino acid residues,permanent phosphorylation,non-permanent phosphorylation,permanent oxidation and non-permanent oxidation were imitated by directed mutation of amino acid residues,and building NIA1 phosphorylation sites and neighboring amino acid residues of directed mutation into the transformation vecter,using japonica rice variety Kitaake as receptor material for genetic transformation,to study the effect of NIA1 directed mutation of phosphorylation site and adjacent amino acid residues on phosphorylation level and NR activity,and to provide theoretical foundation for the efficient utilization of nitrogen in rice.The main research results are as follows:(1)OsNia1 gene was cloned from japonica rice Kitaake.Through phosphorylation site prediction and homologous comparison analysis,it was speculated that the phosphorylation site that could regulate NR activity was Ser532.By gene and protein expression analysis,it was found that OsNia1 gene expression,protein expression and phosphorylation showed photoperiod,low temperature reduced the expression of OsNia1gene,and Nitrate nitrogen induced the expression of nitrate reductase gene.Constructed over-expression transgenic lines in which NIA1phosphorylation site S532and adjacent amino acid residues T531,M536and S627gene was mutated directly,through the phenotype and nitrogen utilization of transgenic lines were analyzed,found that the growth of NIA1phosphorylation sites and mutation of neighboring amino acid residues directed mutation lines and OsNia1 over-expression lines are suppressed,phosphorylation site S532and neighboring amino acids T531and S627directed mutation can improve the NR activity of transgenic lines,accelerated the nitrate nitrogen assimilation,and the directed mutation of the adjacent amino acid residue M536reduced the NR activity,and the nitrate assimilation ability was reduced.(2)The directed mutations of phosphorylation site of NIA1 can break the circadian rhythm of NR activity.NR activity of transgenic lines was significantly increased both in light and dark,and NR activity state is stable at about 80%.Directed mutation of adjacent amino acid residues T531and S627had positive influence on the total activity,actual activity and activity state of NR,but the circadian rhythm of NR was not affected.The NR activity of M536directed mutation transgenic lines were lower than that of wild type,and NR activity state was higher than that of wild type in the night.Studies have shown that the regulation of NR phosphorylation by directional mutation of phosphorylation site is an important way to improve NR activity and nitrate assimilation efficiency.(3)Compared with the wild type,empty vector lines,OsNia1 over-expression lines,and the NIA1 phosphorylation site transgenic lines had lower plant height,root length,dry matter weight,chlorophyll and soluble sugar content,and NR activity,ammonium nitrogen,free amino acid,nitrite,H2O2,and MDA content was higher at room temperature.Nitrate assimilation pathway was active of the NIA1 phosphorylation site directed mutation lines under low temperature stress,and nitrite content,H2O2content,MDA content,plant height,root length,dry weight,chlorophyll content,soluble sugar content,ammonium nitrogen and amino acid content were higher than in wild type,empty carrier lines and OsNia1over-expression lines.The content of ammonium nitrogen in all lines increased under low temperature stress.It indicated that the accumulation of nitrite was the reason for the weak growth of NIA1 phosphorylation site directed mutant transgenic lines under normal temperature,and the increase of NR activity enhanced the tolerance of rice to low temperature stress.(4)The plant height,aboground dry weight and chlorophyll content of the NIA1phosphorylation site transgenic lines were lower than those of the wild type,empty carrier lines and OsNia1 over-expression lines with ammonia nitrogen and nitrate nitrogen mixed nutrition as nitrogen source,while the NR activity,ammonium nitrogen,amino acids,nitrite,H2O2and MDA content were higher.The plant height,above ground dry weight,chlorophyll content,NR activity and ammonium nitrogen content,free amino acid content and nitrite content of NIA1 phosphorylation site directed mutation transgenic lines were higher than in wild type,empty carrier lines and OsNia1 over-expression lines with nitrate nitrogen as nitrogen source,and the H2O2and MDA content of leaves from all lines were no significant difference.There was no significant difference in the physiological indexes in all lines except NR activity with ammonia nitrogen as nitrogen source.When ammonia nitrogen and nitrate nitrogen mixed nutrition was used as nitrogen source,the content of ammonium nitrogen in the test material was the same as that of ammonia nitrogen,while the content of ammonium nitrogen was the lowest when potassium nitrate was used as nitrogen source.Compared with ammonium nitrate,the content of nitrite in the leaves of the NIA1 phosphorylation site transgenic lines cultured with nitrate nitrogen was slightly different from that of other test materials.It showed that the change of the NR activity and the rise of ammonium nitrogen content in rice leaves will not affect rice phenotype and physiology,nitrate assimilation rate increase lead to the excessive accumulation of nitrite is the reason for the weak growth of NIA1 phosphorylation site directed mutation transgenic lines with ammonium nitrate as nitrogen source,and the directed mutation of phosphorylation site improved NR activity,promoted the conversion efficiency of nitrate nitrogen,and enhanced the tolerance to ammonium deficiency.In conclusion,the directed mutation of NIA1 phosphorylation site can enhance the activity of NR and the assimilation rate of nitrate nitrogen under normal growth conditions,and the accumulation of nitrite nitrogen would cause adverse effects on the growth and development of rice.The directed mutation of phosphorylation site could improve the nitrogen nutrition of rice under low temperature stress and nitrogen deficiency,and reduce the accumulation of nitrite,and enhance the tolerance to low temperature and nitrogen hunger of rice.
Keywords/Search Tags:Rice, Nitrate reductase, Phosphorylation, NR activity, Nitrogen utilization, Nitrite
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