Cloning And Functional Study Of Grape Hyacinth MaFLs Gene Promoter

Grape hyacinth(Muscari spp.)is a perennial monocotyledonous bulb flower,with different degrees of blue as the main flower color,as well as white and pink varieties.It has a simple genetic background for flower color therefore it is an ideal material for breeding study of monocotyledon flower color.The previous research of the research group showed that the color of flower formation of grape hyacinth is mainly related to flavonoids,and put forward the hypothesis: flavonol synthase(Ma FLS)competes with dihydroflavonol-4-reductase(Ma DFR)for the same substrate,dihydroflavonol.The formation of flavonols and anthocyanins leads to two branches,both of which are involved in regulating the formation of blue and white grape hyacinth flower color.At present,cloning and functional verification of key genes for blue-white grape hyacinth flower formation have been studied,but how to regulate the flower formation mechanism in terms of transcriptional regulation is not clear.This study uses blue grape hyacinth 'M.aucheri'Dark Eyes 'and white grape hyacinth' M.aucheri 'White Beauty' as materials to clone and study the function of FLS gene and its promoter,an important catalyst in flavonol pathway.Main results in this study are as follows:1.A new FLS gene was cloned from M.aucheri 'Dark Eyes' and named Ma FLS1.The coding region of Ma FLS1 gene is 999 bp in length and encodes 332 amino acids.The accession number is MT198683,the protein molecular weight is 36.96 k Da,and the instability coefficient is 40.68.The prediction analysis belongs to unstable proteins.Ma FLS1 protein does not have a signal peptide and is not a secreted protein.2.Amino acid alignment analysis showed that the amino acid sequence homology between Ma FLS1 and Ma FLS was 93.43%,the similarities with onion Ac FLS-HRB and Ac FLS-H6 were 71.04% and 70.75%,and the similarity with Chinese narcissus Nta FLS was72.54%,the similarity with gentian Gt FLS was 65.07% and so on.Phylogenetic analysis showed that Ma FLS1 clustered in the monocotyledon family and had the closest evolutionary relationship with grape hyacinth Ma FLS and Chinese narcissus Nta FLS.3.Analysis of the spatial-temporal expression pattern of Ma FLS1 showed that the expression of Ma FLS1 was tissue-specific,hardly expressed in rhizomes and leaves of grape hyacinth.It was highly expressed during the S1(flower buds just formed)period in flowers,and then the expression level gradually decreased during the S2(flower buds start to color)period,and thereafter the gene was hardly expressed as the flower developed.The trend ofMa FLS gene expression was basically consistent with the previous research of the research group.4.Using the chromosome walking method to clone the blue and white Ma FLS gene promoter p HFLS(1257 bp),p BFLS(603 bp).Bioinformatics analysis showed that the sequence identity of the two promoters,p HFLS and p BFLS,was 26.08%.Except for the presence of 2 CAAT-box,1 TATA-box,and 1 G-box element at the same position,the two promoters differed greatly in the position and number of transcription factor binding site elements and other cis-acting elements: There are 4 CAAT-box,3 light-responsive elements G-box,1 I-box,1 ACE,1 cis-acting regulatory element A-box,4 TATA-box,1 transcription factor binding site element MYB,1 transcription factor binding site element MYb in p HFLS;p BFLS has 2 TATA-box,3 CAAT-box,2 transcription factor binding site elements MYC.5.According to the position of the cis-acting element on the promoter sequence,delete the promoter from the 5 'end segment by segment,construct the full-length p HFLS,p BFLS promoter and a series of deletion fusion reporter gene expression vectors,transform Agrobacterium GV3101 and transiently Transforming Tobacco spp.And analyzing GUS histochemical staining on tobacco leaves showed that both the p HFLS and p BFLS promoters had expression activity,and the p BFLS promoter activity was stronger.The key elements of p HFLS promoter activity are located in the region of-548 ?-224 bp;the key elements of p BFLS promoter activity are located in the region of-223 ?-86 bp.6.Construct the expression vector of the full length of the p HFLS and p BFLS promoters fused with the GUS gene,transform Agrobacterium GV3101 and genetically transform tobacco SR,and perform GUS histochemical staining on different tissues of the transgenic plants.The anther parts of the flowering stage are concentratedly expressed,and the p BFLS promoter activity is strong.Combined with the expression pattern of Ma FLS1 gene,it is speculated that the two promoters were expressed at the early stage of flowering,which has not been detected in this study.It is preliminarily presumed that p HFLS and p BFLS promoters are flower specific promoters.