Screening Of BHLH Transcription Factors For Anthocyanin Biosynthesis Of Blue Honeysukle And Functional Verification Of LcTT8

Blue honeysuckle(Lonicera caerulea L.)are rich in anthocyanins.It is a good resource for extracting edible natural pigments,and it is also an economic and health fruit with great development potential.Pigmentation of blue honeysuckle fruits is a process of anthocyanin accumulation,and bHLH protein is an important transcription factor regulating anthoc yanin biosynthesis.However,the molecular regulation mechanism of anthocyanin biosynthesis in blue honeysuckle has not been reported.Studying the regulation mechanism of anthocyanin biosynthesis in blue honeysuckle can not only provide a theoretical basis for cultivating health-care plant varieties,but also provide a reference for studying the molecular mechanism of plant stress resistance.In this study,a blue honeysuckle variety named ‘Berel' was used as materials.The bHLH transcription factor Lc TT8 involved in anthocyanin biosynthesis in blue honeysuckle fruits was screened and its gene function was verified using transcriptome sequencing,bioinformatics analysis,construction of Lc TT8-EGFP green fluorescent vector and pBI121-Lc TT8 overexpression vector,gene transformation,subcellular localization,real-time fluorescence quantitative PCR,ELISA enzyme-linked immunosorbent assay,p H differential method,high performance liquid chromatography(HPLC)and other methods.The main results are shown as follows:(1)Identification and analysis of bHLH transcription factor family in blue honeysuckle fruitsThe conserved domains and conserved motifs of bHLH transcription factor in blue honeysuckle fruits were identified by NCBI CDD,MEME and phylogenetic tree methods.The results showed that 43 bHLH contained the conserved domains HLH(PF00010)or bHLH-MYC_N(PF14215).Almost all the 43 bHLH proteins of blue honeysuckle contain Motif 1 or Motif 2.Motif 1 and Motif 2 constitute the basic helix-loop-helix conserved domain.Furthermore,the results showed that there was a closer evolutionary relationship between bHLH transcription factors and the same conserved motifs.(2)Screening of bHLH related to anthocyanin biosynthesis in blue honeysuckle fruitsThe 43 bHLH sequences of blue honeysuckle were compared with protein databases Nr,Swiss-Prot,KEGG and COG(evalue < 0.00001)using blastx.The results showed that CL484.Contig3_All,Unigene 21245_All,Unigene 38564_All and Unigene 7373_All were related to anthocyanin biosynthesis.The 43 bHLHs of blue honeysuckle and 225 Arabidopsis bHLHs were combined to construct phylogenetic trees to predict the function of bHLHs.The results showed that bHLHs of blue honeysuckle could be divided into 14 subfamilies,while CL484.Contig3_All and Unigene 21245_All were clustered in the ? f subfamily which mainly regulated anthocyanin biosynthesis.The expression patterns of 43 bHLH genes from blue honeysuckle were analyzed by real-time fluorescence quantitative PCR(q RT-PCR).The results showed that the expression level of CL484.Contig3_All in fruits was significant higher than that in roots,stems,leaves and flowers.The expression level of CL484.Contig3_All increased sharply when fruits developed to the initial-veraison.Finally,we screened out the CL484.Contig3_All which regulated anthocyanin biosynthesis in blue honeysuckle fruits.(3)Cloning and bioinformatic analysis of the TT8 gene in blue honeysuckle fruitsThe CDs region of CL484.Contig3_All was obtained by assembling transcriptome data of blue honeysuckle fruits,named Lc TT8.The Lc TT8 gene was linked to the cloning vector and transferred to E.coli for sequencing.The length of Lc TT8 nucleotide sequence obtained was 2049 bp.Lc TT8 protein was acidic protein(p I=5.09),which contained 682 amino acids and its molecular weight was 75928.12.Lc TT8 was predicted to be located in the nucleus,which may contain two protein transmembrane helical domains and no signal peptide sites.In addition,we predicted the protein three-dimensional structure modeling of Lc TT8.Multiple sequence alignment between Lc TT8 and TT8 from other plants showed that they had the same conservative domain.Phylogenetic tree showed that TT8 s from blue honeysuckle and Prunus avium had the closest evolutionary relationship.(4)Subcellular localization and gene expression analysis of TT8 in blue honeysuckle fruitsSubcellular localization analysis was carried out using protoplast transformation method in Arabidopsis thaliana.Bright green fluorescence was observed in the nucleus,membrane and plasm of Arabidopsis thaliana protoplast transferred into empty vector,while the green fluorescence of Lc TT8-EGFP expression vector was only concentrated in the nucleus.The expression pattern of Lc TT8 in different tissues and fruit stages of blue honeysuckle was analyzed by q RT-PCR.The results showed that the expression of Lc TT8 was low in roots,stems,leaves and flowers,whilst the expression of Lc TT8 was high in fruits,which was 4-23 times higher than that in other tissues.During fruit development,the expression of Lc TT8 increased first and then decreased,reaching its peak at the fruit initial-veraison.(5)Functional validation of TT8 gene in blue honeysuckle fruitsThe pBI121-Lc TT8 was constructed and the function of Lc TT8 gene was verified by tobacco genetic transformation system.The results showed that the color of leaves and seed coats of Lc TT8 transgenic tobacco increased during varying degrees.The content of anthocyanins in transgenic tobacco leaves increased,which was determined by p H differential method.The results showed that the expression of F3 H,DFR and ANS genes in Lc TT8 transgenic tobacco leaves increased significantly compared with the gene expression in wild tobacco leaves using the q RT-PCR analysis of anthocyanin biosynthesis genes CHS,CHI,F3 H,DFR and ANS.The activity of related enzymes in anthocyanin biosynthesis of Lc TT8 transgenic tobacco leaves was detected.The results showed that the activities of DFR and ANS in Lc TT8 transgenic tobacco leaves were up-regulated obviously.The anthocyanin content in transgenic tobacco leaves was higher than that in wild tobacco leaves,and the cyanin-3-O-glucoside content in transgenic tobacco leaves was higher than that in wild tobacco leaves.