Cloning And Functional Analysis Of Muscari Ameniacum Glycosyltransferase Gene MaGT1

At present,there are few understandings of the modified genes involved in the synthesis of anthocyanins and their modes of action.The molecular mechanism of the formation and modification of different blue anthocyanins is deeply understood,and the law is found to be an urgent problem to be solved.Based on the research of the research group,this study cloned a flavonoid glycosyltransferase gene MaGT1 from grape hyacinth for the first time.A series of experiments were carried out to explore the function of MaGT1 gene in anthocyanin coloring,and provide theoretical basis for flower color genetic engineering.The results are as follows:1.A flavonoid glycosidase gene MaGT1 was cloned from grape hyacinth.The full-length cDNA was 1338 bp,encoding 445 amino acids.The GenBank accession number was MK652470.The theoretical molecular weight is about 49.301 KD and the theoretical isoelectric point is 5.40.2.MaGT1 encoded amino acid sequence analysis shows that MaGT1 belongs to GT-B class,and its C-terminus contains a typical PSPG glycosyltransferase domain consisting of 44 amino acids..The similarity between MaGT1 and monocotyledons such as Elaeis guineensis,Phoenix dactylifera and Vitis vinifera is relatively high,ranging from 51% to 54%.3.The phylogenetic tree shows that MaGT1 is clustered in the clade FGGT(flavonoid glycosylglycosylase),which uses anthocyanin 3-glucoside as a substrate to attach glucose or rhamnose to the anthocyanin sugar moiety.Enzyme.4.The total amount of anthocyanins in the roots,bulbs,leaves and different flower development stages of grape hyacinth was determined.The results showed that the anthocyanin content increased with the development of flowers.The results of RT-PCR showed that the MaGT1 gene was predominantly expressed in flowers,but the expression level of MaGT1 was low in the roots and leaves with less anthocyanin content,and the expression of MaGT1 was regulated by development.When the grape hyacinth was in the S1 period,the expression of MaGT1 was almost absent,and it began to rise with the progress of flower development from the S2 period,and reached the highest peak during the S4 period.5.The prokaryotic expression vector pET-28a-MaGT1 was constructed.The optimal induction conditions of the recombinant egg were determined by adjusting the induction time,IPTG concentration and induction temperature: MaGT1 recombinant protein can be IPTG concentration 0.1 mM,28 ° C for 4 h.The best expression.In addition,recombinant proteins are mostly present in the form of inclusion bodies.6.The plant expression vectors pCambia1301-MaGT1,pART27-MaGT1 i and pFGC5941-MaGT1 i were constructed and transferred into Agrobacterium tumefaciens LBA4404.Agrobacterium tumefaciens LBA4404 containing recombinant plasmid was transformed into model plant tobacco by Agrobacterium-mediated leaf disc method.Nicotiana tabacum 'NC89'),selected for the selection pressure of pCambia1301,pART27 and pFGC5941,which can increase the mortality of non-resistant plants by more than 75% and the survival of resistant seedlings by 80%,respectively: pCambia1301(Hyg final concentration 250?g/L));pART27(Kana 0.2 g/L);pFGC5941(0.0003% glufosinate).7.By PCR,6 tobacco plants transfected with pART27-MaGT1 i were obtained.The phenotypic observation and the determination of anthocyanin content in the corolla showed that compared with the wild type,the tobacco color transferred to pART27-MaGT1 i was slightly deepened.The content of anthocyanins and flavonols in the corolla was higher than that in the wild type.8.The expression level of the anthocyanin synthesis pathway gene in transgenic tobacco was determined.The results showed that the expressions of the structural genes NtPAL,NtC4 H,Nt4CL,NtF3 H,NtF3'H and NtF3'5'H were similar to those of the wild type.The expression of key structural genes NtCHS,NtCHI,NtFLS,NtDFR,NtANS and NtUFGT increased,especially the NtCHS,NtCHI and NtDFR genes increased significantly;tobacco regulatory genes NtAN2 and NtAn1 a increased,but in There were differences in the performance of different lines,while the expression of NtAn1 b decreased.In summary,the grape hyacinth MaGT1 gene encodes a flavonoid glycosyltransferase(FGGT),which may be involved in the subsequent glycosylation modification of anthocyanin 3-O-glucoside in blue grape hyacinth.Interference with MaGT1 gene can cause changes in tobacco color,indicating that MaGT1 protein is an important modification step in the anthocyanin synthesis pathway,which can affect the color of other plants.