Screening And Identification Of Anthocyanin-Related R2R3-MYB Family Genes Of Blue Honeysuckle

Blue honeysuckle(Lonicera caerulea L.)belongs to the family Caprifoliaceae and the genus Lonicera.It is well known for its rich nutrients,especially high content of anthocyanins.The color change of blue honeysuckle fruit during ripening is the result of the accumulation of anthocyanins.The regulatory role of R2R3-MYB transcription factors in the process of plant anthocyanin biosynthesis has been confirmed in many plants,but there is no research and report in blue honeysuckle.In this study,the excellent blue honeysuckle cultivar ‘Smurfs’ was used as test material.The transcription was performed in blue honeysuckle based on the previously transcriptome sequencing data,the bioinformatics was used to identify and analyze the R2R3-MYB transcription factors in blue honeysuckle.Cloned LcMYB90 and LcMYB5 gene and investigated the expression pattern and preliminary function of these two genes.The main findings are as follows:(1)Identification of R2R3-MYB family genes in blue honeysuckleBy using the HMM homology comparison method,59 R2R3-MYB transcription factors were screened from the blue honeysuckle fruit transcriptome database,and then the physical and chemical properties and conserved domain of the 59 genes were analyzed through online software such as Prot Param,MEME,NCBI-CDD,etc.The results showed that most of the proteins encoded by R2R3-MYB gene of blue honeysuckle were unstable and prone to degradation.And all these proteins had the typical structural characteristics of R2R3-MYB transcription factors,such as spiral-turn-helix and R2,R3 repeat.(2)Screening of R2R3-MYB transcription factors related to anthocyanin biosynthesis in blue honeysuckleAccording to the genetic similarity of the sequences,the bootstrap value and the classification of 126 R2R3-MYB reported in Arabidopsis thaliana and the 59 R2R3-MYB transcription factors in blue honeysuckle were divided into 20 subfamilies.Among them,15 LcR2R3-MYB genes may be related to anthocyanin biosynthesis.The expression patern of the 15 genes in different tissues and different fruit development periods were analyzed.The results showed that the expression patterns of these genes were significantly different.The expression level of CL6086 in the fruit is relatively higher than the other tissues.The expression in the different developmental stages of the fruit,The expression level of CL552 in the verasion stage was about twice as high as the expression level in the green fruit stage.Thus,CL6086,CL552 and Unigene12084 may play a positive role in regulating the anthocyanin biosynthesis of blue honeysuckle fruit,and were named LcMYB90,LcMYB5 and LcMYB4.In order to study the regulation of R2R3-MYB transcription factor on anthocyanin biosynthesis in blue honeysuckle,this study selected two genes LcMYB90 and LcMYB5 for functional verification.(3)Cloning and analysis of LcMYB90 and LcMYB5LcMYB90 and LcMYB5 were cloned from the mature fruits of blue honeysuckle.The LcMYB90 gene is 825 bp in length,encodes 274 complete amino acid sequences.The LcMYB5 gene is 963 bp in length,encodes 320 complete amino acids sequence.LcMYB90 and LcMYB5 contain the typical R2R3-MYB transcription factor characteristics.We also found that the R3 domains of LcMYB90 and LcMYB5 have the binding sites of b HLH family transcription factors,the two transcription factors may also interact with b HLH transcription factors family.In addition,the C-terminus of LcMYB90 also has an identified functional motif ANDV related to anthocyanin biosynthesis.(4)Functional identification of LcMYB90 and LcMYB5 geneThe overexpression vector p CAMBIA1300-LcMYB90-GFP,p CAMBIA1300-LcMYB5-GFP and the repression vector p FGC5941-LcMYB90,p FGC5941-LcMYB5 were constructed by double enzyme digestion method,A total of 3 LcMYB90 overexpression lines,3 LcMYB5 overexpression lines,4 LcMYB90 repression lines,4 LcMYB5 repression lines transgenic tobacco plants were obtained by Agrobacterium-mediated transformation of tobacco,and through PCR detection to determine the transgenic positive strains.Analysis of the expression of key structural genes in the anthocyanin biosynthesis pathway of LcMYB90 and LcMYB5 transgenic tobacco leaves,The results showed that compared with wild-type tobacco,the expression of CHS,CHI,F3 H,DFR,ANS in LcMYB90 overexpression and repression lines had no significant changes.The expression of CHI and F3 H in LcMYB5 overexpression transgenic tobacco leaves increased,and the expression of CHS and ANS increased significantly.The expression of CHS and ANS in LcMYB5 repression tobacco leaves decreased significantly.By measuring the color difference value of transgenic tobacco leaves,compared with wild-type tobacco seedlings LcMYB90 overexpression and repression lines had no significant changes in leaf color.LcMYB5 overexpression lines have higher a* values and darker leaf colors.LcMYB5 repression lines have higher b* values and lighter leaf colors,it is speculated that LcMYB5 is possible to increase the accumulation of anthocyanins by up-regulating the expression of CHS and ANS.