Characterization Of Potato Patatin Promoter And Signal Peptide In Transgenic Arabidopsis Thaliana

Patatin is a group of potato tuber-specific proteins with a molecular weight of about 40 k Da,which are usually glycosylated.Their content can account for about40% of the total soluble proteins in potato tubers.The Patatin promoter is a strong promoter and its tuber-specific expression confers on the coding gene.At high concentrations of sucrose,the Patatin promoter also initiates the expression of downstream genes in other parts of the potato plant.In the early stage,the 5?-end untranslated regions of the Patatin-encoding genes were classified into two major categories based on the presence or absence of an insertion sequence: Class I has no22 bp insertion sequence in its 5?-end untranslated region;Class II is in its 5?-The non-translated region has a 22 bp insertion sequence.The first 23 amino acid residues of Class I Patatin were presumed to be signal peptides,but this has not been demonstrated experimentally.Accordingly,previous graduate students in our laboratory constructed the GUS(p05 plasmid)and 23aa-GUS(p06 plasmid)vectors driven by the Class I Patatin promoter and genetically transformed the potato,but the GUS expression signal of the transgenic potato was weak.In this regard,we tested the above-mentioned expression characteristics in transgenic Arabidopsis.The results are as follows:1.Molecular identification of transgenic Arabidopsis plants and RT-PCR detection of transcriptional levels.In this experiment,two pro-Patatin:: GUS plants were obtained by transforming the p05 plasmid into Arabidopsis thaliana in the early stage of the experiment,named At G-1 and At G-2.The p06 plasmid was transformed into Arabidopsis obtained 6 strains of Pro Patatin:: 23aa-GUS transformants,named At SG-1~At SG-6.After PCR amplification and sequencing of the amplified products,the two p05 plasmid transformation lines were all transformed from the p06 plasmid and corrected strain nameas At G-1?At SG-7 and At G-2?At SG-8,respectively.The results of RT-PCR showed that,except for the wild-type non-banding,the At SG series of eight transformants showed a specific band,of which At SG-1 and At SG-3 were weak,At SG-2,At SG-4~At SG-8 are stronger.It showed that the 8 strains of At SG transgenic lines obtained by genetic transformation of p06 plasmid all had GUS gene expression.2.Reconstruction of Pro Patatin:: GUS,Pro Patatin:: 23aa-GUS and construction of Pro35s:: 23 aa.To verify whether Patatin's original 23 amino acid residues are the signal peptide sequence and localize the subcellular site of Patatin,we obtained twocopies of the Pro Patatin and Pro Patatin:: 23 aa GUS and Pro Patatin:: 23aa-GUS vectors.The fragment was then amplified with the Pro35s:: 23 aa DNA fragment and inserted into the plant genetic transformation vector p CAMBIA-1304 containing the GFP gene.Recombinant plasmids p1304-Gus,p1304-SGus,and p1304-SP were verified by the insertion of Pro Patatin,Pro Patatin:: 23 aa and Pro35s:: 23 aa,respectively,to form a new recombinant fragment Pro Patatin::GFP-GUS,Pro Patatin::23aa-GFP-GUS and Pro35s:: 23aa-GFP-GUS.3.Analysis of protein levels of GUS gene expression in transgenic Arabidopsis At SG plants.The wild-type,transgenic At SG-2 and At SG-7 strains of Columbia were individually selected for high-glucose-induced growth for 13 days,and each was weighed 70 mg.Protein was extracted by simple protein extraction.The primary antibody was anti-His-tag mouse monoclonal antibody(YB30401ES10)from Sigma,and the secondary antibody was goat anti-mouse Ig G(H+L)(A2016)monoclonal antibody labeled with horse-radish peroxidase from Biyuntian Biology.Western Blotting showed two clear protein bands with molecular weights between 65 and 70,and between 70 and 75 KDa,which were consistent with the histidine-tagged GUS protein molecular weights of 69.5 and 72.2 after theoretical cleavage/uncleaved signal peptides.It is thought that the signal peptide may be cleaved when it participates in transmembrane transport,resulting in two bands of different molecular weights,one is the molecular weight band before cleavage and the other is the molecular weight band that cuts the signal peptide.4.Analysis of the temporal and spatial characteristics of GUS gene expression in transgenic Arabidopsis At SG plants.GUS histochemical staining of the At SG-2 line of transgenic Arabidopsis plants was performed during the whole growth period.Seeds,germinated seeds,seedlings grown for 7 days,seedlings grown for 13 days,flowers,and fruit pods were used for staining.The results showed that the GUS gene was strongly expressed in the At SG-2 plant at the seed period and was no longer expressed during the seed germination period.It was also not expressed in seedlings grown 7days and 13 days,but was weakly expressed in the flower organ.The expression was mainly in the Style,petals,and stalks are not expressed in fruit pods.5.The effect of high sucrose concentration on GUS gene expression in At SG plants of transgenic Arabidopsis thaliana.It is known that the Patatin promoter is induced by high concentrations of sucrose in potatoes and the Patus promoter-driven GUS gene is transferred into Arabidopsis thaliana and induced using 8% sucrose.High sucrose induced At SG-2 strain seeds,germinated seeds,7-day-old seedlings,13-day-old seedlings,flowers,and fruit pods were stained.The results showed thatthe GUS gene was expressed in the cotyledon and radicle during the seed stage.The expression of the GUS gene was weakened during seed germination,and it was only expressed in the radicle but not in the endosperm.In the seedlings growing for 7 days,the cotyledon,suspensor,and root were all expressed,and the suspensor was most strongly expressed.Among the 13-day-old seedlings,the true leaves,suspensor and veins were most strongly expressed.Expression was found in flower organs,and it was most strongly expressed in style and petals.It is expressed in fruits.