| N-glycosylation plays an indispensable role in live organisms.In plants,N-glycosylation affects the biosynthesis and function of receptor proteins,cell wall biosynthesis and salt tolerance stress,plant hormone level,etc.However,the function of complex N-glycan in plants is relatively poorly understood.Therefore,in order to investigate the relationship between N-glycosylation modification and its function in plants,the first task is to release N-glycans from glycoconjugates.Compared with chemical method,enzymatic method is more often used for the release of N-glyans due to its advantages of strong specificity and mild working conditions.N-glycanase(PNGase)is a key enzyme for enzymatic release because it can release N-glycans from glycoprotein or glycopeptide.So far,different N-glycanases have been found to be deficient.For example,both PNGase F can be expressed in E.coli under recombinant conditions,and there is no self-glycosylation.However,it is unable to enzymatically digest the structure containing ?-1,3 fucose from plants or insects.PNGase A can cleaved structures containing ?-1,3 fucose,but it contains glycosylation which cannot be recombinant in Escherichia coli and can only act on glycopeptides.In order to meet the needs of rapid development of glycoomics,it is necessary to further develop new advantageous N-glycanase.At present,most researches on the role of N-glycosylation in plant growth and development focus on seed development,preservation and germination,as well as fruit ripening stage,but there is no research about the changes of N-glycosylation in different growth stages of plants.It is of great significance to further explore the role of N-glycosylation modification in plants' rooting development.A novel N-glycanase(PNGase Rz)was studied in this paper.To further understand the change of N-glycan in the growth of plant,this enzyme was used to release N-glycans in Arabidopsis thaliana glycoproteins at different growth stages.In order to further explore the role of N-glycosylation modification in rooting development,Arabidopsis seedlings were treated with different concentrations of PNGase Rz,and the changes in the root length and N-glycans composition of Arabidopsis seedlings were studied.The specific research content and results are as follows:1.Expression,purification and characterization of PNGase RzUsing BL21(DE3)system and expressing at 18? for overnight was selected to do the expression of PNGase Rz.A single strip was obtained by gradient elution through Nickel column.The results showed this enzyme had N-glycosase activity.Biochemical characterization results indicated that PNGase Rz's optimum reaction pH is 6.0;the optimum reaction temperature is 25?;PNGase Rz showed activity without the presence of metal ions,but Fe3+ will seriously inhibite the enzyme activity.PNGase Rz acts directly on both natural and denatured glycoproteins,and releases all types of N-glycans containing the core ?-1,3 fucose structure.2.Monosaccharide composition and glycosylation modification of N-glycans in Arabidopsis thaliana during different growth stagesThe monosaccharide composition,structure and relative content of N-glycans in Arabidopsis thaliana were studied by using N-glycanase PNGase Rz as the tool enzyme.The results showed that N-glycans were composed of G1cN,Gal,Man,Glc,Xyl and G1cA/Fuc;12 structures were found:4 of them belonged to high mannose type and 8 of them belonged to complex type.The N-glycan structural composition changed significantly during different growth stages of Arabidopsis.The relative peak ratio of high mannose type increased first,then decreased and increased again.The relative content of high mannose type increased from 11.40%in the seedling stage to 18.96%in the bolting stage,and then decreased to the lowest value of 1.66%in the mature stage.It finally rose to 6.63%in the aging stage.In contrast,the relative proportion of complex sugars decreased first and then increased and decreased again.The relative content decreased from 88.60%in the seedling stage to 81.04%in the bolting stage,and rose to the highest value of 98.34%in the mature stage,and it decreased to 93.3%during the aging period.The content of high mannose type in the late growth stages(the maturity stage and the aging stage)were significantly lower than that of other three growth stages,and the corresponding complex type was significantly higher than the three stages.Throughout the growth process,complex sugar is always the main component,and the structure containing xylose and fucose always accounts for the main part of complex sugar;Man3XylFucGlcNAc2,one of the typical structures in plant N-glycan,is always the highest structure.3.Effect of PNGase Rz on the Arabidopsis thaliana seedlingsThe seedlings of Arabidopsis were dealed with five treatments:Blank control(Blank),negative control ?(BSA0.05),negative control ?(BSA0.5),the experimental group I(E0.05)and experimental group ?(E0.5).Group E0.5 significantly inhibited the primary root length growth of Arabidopsis thaliana.The other groups grew normally.By comparing the N-glycans composition with different treatments,it was found that at the 8th hour of growth,there were significant differences between the complex type and high-mannose type in the control group and the two experimental groups.And this may results in the difference of the length of primary root of different groups.At the 5th day of growth,the difference decreased,but the difference between the E0.5 group and blank group was larger than that between the E0.05 group and blank group.By the 10th day of growth,most of the differences between groups were no longer significant.In general,in the 10-day growth cycle,the complex type content in Arabidopsis thaliana was always higher than the high mannose type;the structure containing xylose and fucose was always the main part of the N-glycan specteum of Arabidopsis thaliana.Among them,Man3XylFucGlcNAc2 has always been the most abundant structure;the difference between the N-glycan composition of the experimental group and the blank group gradually decreased and the difference finally disappeared. |
PDF Full Text Request