Expression,Characterization And Optimization Of Measurement Conditions Of Nitrate Reductase From Vitis Vinifera In Pichia Pastoris

Nitrate reductase?NR?can reduce nitrate into non-toxic N02-.Increasing the activity of nitrate reductase in fruits and vegetables,fruits and vegetables can make the nitrate content decreased,but also can improve the efficiency of nitrogen,increasing its production.In addition,there are reports that nitrate reductase are positively correlated in grain yield and protein content,so nitrate reductase can be biochemical indicators for breeding grain yield and protein content filtering.There are reports that NR and NO are related in plant resistance.This can provide ideas to ease the hurt of plant under stress conditions.In this study,we have optimized its promoter and the nucleotide sequence of the genetic code,which comes from the grapes of nitrate reductase VVNR1?Genbank NP₀01268049.1?amino acid sequence,then we synthesis new gene using PTDS and renamed VVNR1I.Then,we put the new gene into Pichia,and to secrete expressed in Pichia pastoris expression system.Finally,we optimized assay conditions of nitrate reductase from the concentration of KN03,the incubation time,the pH and the metal ions.The results showed that the optimal incubation time of the recombinant enzyme is 48 h.When the pH is about 6,the recombinant enzyme exhibited maximum activity.Within a certain range of concentrations,activity of grape nitrate reductase enhanced with increasing concentrations of KN03.When the concentrations of KN03 in medium greater than 1%,we continue to increase the concentration of KN03,having little effect on the activity of the recombinant enzyme.Mo6 + promote the activity of the recombinant enzyme obviously,it may be because molybdenum is an important component of nitrate reductase and molybdenum-containing enzymes.In this study,we constructed a grape nitrate reductase expression plasmid,so that it can carry on heterologous expression in Pichia pastoris,and then study the optimum conditions of the recombinant enzyme.This provides a basis for further research to explore some of the features of grapes nitrate reductase,such as nitrogen metabolism,the grain yield and protein content screening in breeding and reduce the nitrate content of fruit,plant resistance and so on.