Cloning And Functional Analysis Of Promoter Of 16kD Allergen From Tartary Buckwheat

Buckwheat,with high nutritional and medicinal value,is rich in flavonoids and protein.However,more and more attention has been paid to the allergic reaction caused by tartary buckwheatb and 16 kD allergen of tartary buckwheat(TBW16)is one of the causes of allergic symptoms.The TBW16 promoter sequence was cloned and analyzed.In order to determine its activity,the promoter was truncated from the 5 'end and the promoter fragments with luciferase were transformed into buckwheat protoplast to drive luciferase transient expression.According to the corresponding elements on the promoter,the response of the promoter to hormone regulation was studied.In addition,the tissue specificity of the TBW16 gene expression was analysed.Interference vectors of TBW16 were transformed into tartary buckwheat and arabidopsis thaliana to carry out silencing and overexpression of TBW16 gene with molecular biology methods.That lays a foundation for further study of TBW16 function and eliminating allergens.Conclusions are as follows :(1)Quantitative analysis of the expression of TBW16 gene in stems,roots,leaves and seeds of buckwheat was carried out.The results showed that the gene was expressed abundantly in tartary buckwheat seeds,and its expression in buckwheat roots,leaves and stems was much lower.The promoter of this gene is a seed specific promoter.(2)The 1876 bp TBW16 promoter sequence was obtained by genomic walking method and the promoter has the following basic components: transcriptional start site(-38 bp),TATA-box(-72 bp),CAAT-box(-50 bp),MeJA-responsive elements(TGACGmotif,-1584bp;CGTCA-motif,-1407bp),GA-responsive element(GARE-motif,-198bp?-1815bp)?ABA-responsive element(ABRE-motif,-156bp)andseed-specific element(RY repeat,-92bp?-134bp).(3)The full-length TBW16 promoter was constructed into a recombinant vector TBW16P-pCAPMBIA1301,which was transferred into the explants roots and stems,leaves and seeds of Tartary Buckwheat by microprojectile bombardment and activate the ?-glucuronidase(GUS)gene transient expression in the explants.The explants after staining showed that the expression of GUS in seeds was the highest and there are no visible GUS trace in roots and stems.(4)The full-length promoter was truncated from the 5 'end and obtained 4 promoter fragments,which were recombinded into double fluorescent reporter vectors respectively.The 4 recombinant vectors were transferred into the leaf protoplasts of tartary buckwheat for transient expression.The results showed that there was a region of regulating transcription in the TBW16 promoter,which was located from 1564 bp to-1876 bp.The increased expression of the reporter gene with GA,MeJA and ABA hormone treatment showed that the TBW16 promoter was responsive to three hormone stress components.(5)The TBW16 interference vector(TBW16i-pTCK303)was constructed by inserting TBW16 reverse and forward interference plates into the pTCK303 vector.TBW16 overexpression fragment was inserted into pTCK303 vector to construct TBW16 overexpression vector(TBW16m-pTCK303).It is important for the establishment of tartary buckwheat low sensitive germplasm.