Study On The Structure And Function Of 16 KDa Allergen Fag T 2 In Tartary Buckwheat

Buckwheat rich in amino acid-balanced protein and flavonoids with high biological activity,which is an important special crop for both medicine and food in western China.Buckwheat is one of the main food allergens.Many countries stipulate that food labels must be mandatory to indicate buckwheat ingredients.The allergy-causing substances in buckwheat food are allergic proteins in the grains.The existence of these allergic proteins greatly limits the addition scope of buckwheat as a functional food material.Therefore,reducing the content of buckwheat allergic protein and cultivating hypoallergenic buckwheat has become an urgent problem in the current buckwheat production.Modern biological technology has laid the technical foundation for the creation of hypoallergenic germplasm,but the first prerequisite is to clarify the biological functions of the main allergic proteins in buckwheat.At present,domestic and foreign researches on buckwheat allergens mainly included the isolation and identification of natural allergens,heterologous expression and immunological activity detection,the prediction and analysis of core epitope,allergen antibody preparation,and rapid detection of allergic reactions.Our laboratory previously identified the allergic protein with a molecular weight of 16 k Da,proving that it is the major allergen in tartary buckwheat.The allergic protein was reviewed by the World Health Organization’s Allergen Nomenclature Committee and named Fag t2.0101（Hereinafter referred to as Fag t 2）,and the immunological activity and core epitopes of Fag t 2 have explored.Based on the research results,this study uses“Jiujiang”tartary buckwheat as the material to explore the structure and function of the 16 k Da allergen Fag t 2 in tartary buckwheat through in vivo and in vitro experiments,in order to provide the basic information for the feasibility of hypoallergenic tartary buckwheat creation.The main research results obtained are as follows:（1）Fag t 2 was located in chromosome 7 of tartary buckwheat,and has no introns.Tissue expression analysis showed that the transcription level of Fag t 2 in seeds was significantly higher than that of other tissues and organs.Structure prediction analysis showed that the allergen Fag t 2 contains signal peptide with 19amino acids andα-amylase inhibitor domain.Natural Fag t 2 and Fag t 2 expressed in Pichia pastoris and Escherichia coli has heat stability and pepsin tolerance,but has no inhibitory activity onα-amylase extracted from germinated seeds of tartary buckwheat and porcine pancreatic amylase.Fag t 2 expressed in prokaryotic cells was purified,and a high titer rabbit anti-Fag t 2 polyclonal antibody was prepared.Non-radioactive isotope double-labeled(¹⁵N,¹³C)Fag t 2 expressed in prokaryotic cells was renatured,and NMR（Nuclear Magnetic Resonance）analysis did not obtain a resolvable protein structure.The possible reason is that the double-labeled Fag t 2with 10 Cys expressed in prokaryotic cell may have the incorrect disulfide bond formation,or the protein has a highly variable flexible region,resulting in the structure of Fag t 2 expressed in prokaryotic cell was different from natural.（2）Elements prediction of Fag t 2 promoter（1873bp upstream of coding region）showed that there were jasmonic acid,abscisic acid,drought and pathogenic bacteria and other stress regulatory elements,and 5 seed-specific expression elements at the 5’end of the promoter.The difference truncated promoters fused gus were stably transformed into Arabidopsis thaliana.GUS staining and activity determination showed that Fag t 2-P1（-1873bp-0）exhibited seed-specific expression.With the truncation of the 5’end of the promoter,the reduction of seed-specific elements,the expression of GUS in leaves,flowers,roots and other tissues gradually increases.Promoter activity analysis showed that the activity of truncated Fag t 2-P2（-1564 bp-0）was significantly higher than Fag t 2-P1,Fag t 2-P3（-1125 bp-0）and Fag t 2-P4（-302 bp-0）promoter.It is speculated that the truncated fragment of Fag t 2-P1 may contain the elements that inhibit promoter activity.Each transgenic Arabidopsis thaliana contained Fag t 2 promoters showed a certain difference in response to hormones.All truncated promoters responded to ABA and Me JA,but not to GA during the germination period;Both Fag t 2-P2 and Fag t 2-P3（seedlings）responded to ABA,Me JA and GA.Both Fag t 2-P1 and Fag t 2-P2 responded to mannitol during the germination period.The transcription factors obtained by yeast one-hybrid screening and the results of the promoter response to abiotic stress indicated that Fag t2 may play a biological function in physiological processes and stress tolerance.（3）Yeast hybrid library of tartary buckwheat was constructed based on SMART technique.Yeast two-hybrid screening showed that Fag t 2 can interact with functional proteins such as phenylalanine ammonia-lyase（PAL）,E3 ubiquitin protein ligase ARI2 and serine protein kinase.Ft PALB expressed in prokaryotic cells was purified,and high titer rabbit anti-PAL polyclonal antibody was prepared.In vitro,natural Fag t2 and Fag t 2 expressed in Pichia pastoris can activate the activity of two kinds of phenylalanine ammonia-lyases（Ft PALB and Ft PALN）in tartary buckwheat;Methods such as DOCKing prediction,Pull down,co-localization and Bi FC were used to further prove that Fag t 2 physically interacted with Ft PAL.（4）Recombinant plant binary expression vector was constructed,and a stable genetic Arabidopsis thaliana overexpressed Fag t 2（Fag t 2-OE）was obtained.Drought stress tolerance analysis showed that the survival rate,relative water content,total flavonoids content and CAT activity of Fag t 2-OE Arabidopsis thaliana were significantly higher than that of wild-type and negative control（Arabidopsis thaliana transformed with p CAMBIA3301）,and the contents of MDA,O^2-and H₂O₂ were lower than those of wild-type and negative control.The results show that Arabidopsis thaliana overexpressed Fag t 2 had increased the tolerance to drought stress.After drought stress,the transcription level of At PAL1 in the leaves of Fag t 2-OE Arabidopsis thaliana was significantly higher than that of wild-type and negative control;PAL activity in leaves at different stages showed that the PAL activity in Fag t 2-OE Arabidopsis thaliana leaves was significantly higher than that of the wild type.There was no PAL activity detected in the leaves of Fag t 2-OE and wild-type Arabidopsis thaliana with seedling age≥45 days.The ubiquitination of PAL was by western blotting.The degree of ubiquitination modification of PAL had a significant negative correlation with its activity（correlation coefficient r is-0.9392）.The post-translational ubiquitination modification of PAL in the leaves of adult Arabidopsis thaliana may be the main reason for the loss of PAL activity.The post-translational ubiquitination of PAL may not only lead to subsequent degradation,but also directly lead to inactivation.In vitro,ubiquitination analysis showed that Fag t 2 did not affect the ubiquitination of Ft PAL,and Fag t 2 increased the activity of Ft PAL independent of the inhibition of Ft PAL ubiquitination.（5）Fag t 2-OE Arabidopsis thaliana was resistant to Bacillus ginsengihumi.After inoculation with Bacillus ginsengihumi,the seeds germination rate and seedlings survival rate of Fag t 2-OE Arabidopsis thaliana were significantly higher than that of the wild type,and the number of bacteria in Fag t 2-OE Arabidopsis thaliana was significantly lower than that of the wild type.The transcription levels of At PAL1 gene and PR gene in SA signaling pathway were increased in Fag t 2-OE Arabidopsis thaliana,but significantly lower than the wild type,which indicated that the SA signaling pathway of Fag t 2-OE Arabidopsis thaliana in the resistance to Bacillus ginsengihum was inhibited.This result showed that the resistance of Fag t2-OE Arabidopsis thaliana to Bacillus ginsengihum was independent of SA.The above research results showed that the major allergen of tartary buckwheat,Fag t 2 can interact with Ft PAL and activate the activity of Ft PAL,which plays an important role in drought stress tolerance and pathogen infection resistance.Therefore,knocking out or mutating Fag t 2 in tartary buckwheat through genetic engineering methods is likely to result in the reduction or loss of basic resistance of tartary buckwheat.In-depth analysis of the relationship between Fag t 2 function and allergic epitopes is of great significance for rationally obtaining hypoallergenic tartary buckwheat through gene knockout or mutation.At the same time,the study found that the accumulation of flavonoids in leaves of Arabidopsis thaliana at the adult stage but no PAL activity was detected,which is likely to be related to the ubiquitination of PAL.This result is expected to reveal a new mechanism for rapid post-translational regulation of PAL.