The Study On Molecular Mechanism Of MYB Transcription Factor Regulating Anthocyanin Synthesis In Grape Berries

Grape is one of the most important fruit trees in the world,and it's the most processable fruit with the highest processing depth.Color plays vital role in defining the appearance quality of grape berries.It not only determines the market value of table grapes but also affects the processing applications and the quality of processed products.Anthocyanins are the main pigments which determine the appearance color of the grape berry skin.The proportion of various anthocyanins in the peel and the difference in their accumulation level make the grape skin appear red,purple or black.The MYB is one of the most extensive and critical transcription factors involved in anthocyanin biosynthesis.Several studies have shown that the coloration of grapes depends mainly on the genotypes of two closely linked MYB loci on chromosome 2.At present,the relationship between the genotypes and haplotype compositions of grape MYBA1 and MYBA2 loci and the color of the grape skin,and how the MYB alleles of MYBA1 and MYBA2 loci regulate the formation of grape fruit coloring traits,and how to use efficiently genotypes and haplotype compositions of MYB gene loci for molecular design breeding of fruit color traits is still lack of systematic and in-depth research.In this study,the genotypes and haplotype compositions of MYBA1 and MYBA2 loci of 213 grape germplasms were identified by specific primer sequences.The expression patterns of MYB regulatory genes and coloration-related structural genes were analysed in grape varieties from different haplotypes compositions,and the functions of VvmybA2r and VvmybA2w of MYB-regulated genes and VvPAL-like of structural genes in grapes were studied,which further provide the important foundations for better revealing the mechanism of grape fruit coloring.In this study,the genotypes and haplotype compositions of the MYBA1 and MYBA2 loci were identified by specific primer sequences based on the investigation of the color traits of 213 grape germplasms.The result showed that genotypes and haplotypes of 99.1%grape germplasms were correspond to their fruit color.At the same time,the haplotypes of‘Olarra Queen'and‘Yuanruihei'which are the 'phased phase" or 'repulsive phase' cultivar,were identified by anlysising the haplotypes and thier segregations from the self-fertilization population.The result showed that the haplotypes of 'Olarra Queen' and‘Yuanruihei'are both AC-N.The relationship analysis between the haplotype compositions of the MYBA1 and MYBA2 loci and the fruit peel color showed that the grape skins containing HapB or HapC-Rs was mostly red or purple,and grape skins containing HapE2 or HapC-N tend to be black.In addition,the greater the number of haplotypes containing the functional gene,the darker the color of the pericarp,closer to purple or black.The analysis of the genotypes and haplotypes of the two major populations(Vitis vinifera and the hybrids of V vinifera and V.Labrusca)revealed that the haplotype compositions were different,and the largest haplotype composition in colored grapes from Vitis vinifera is AC-Rs;while the largest haplotype composition of hybrids of V vinifera and V.Labrusca are AE1 and AE1E2.In this study,to verify the feasibility of 'based on the characteristics of grape MYB genotypes and haplotypes regulating fruit coloration and predicting the coloration pattern and the separation ratio of progeny populations of different parental combinations',the haplotypes of progeny of two hybridized combinations which are'Muscat Humburg' × 'Crimson seedless' and 'Qiuhongbao' × 'Cuibao seedless' in an unsuccessful state were identified,and the results revealed that the haplotype segregation ratios of the progeny of the two hybrid combinations are consistent with the expected results.In addition,the fruit coloring of hybridized progeny was investigated in the fruit matured.The survey results showed that the fruit coloring of the above-mentioned hybrid population was consistent with the genotypes and haplotypes of the MYBA1 and MYBA2 loci,which laid the foundation for the molecular design breeding of grape color traits.In this study,four main haplotype compositions(A,AC-Rs,AE2,and AE1E2)were selected as the research subjects,and two to four grape cultivars which were randomly selected from each of haplotypes composition types,were used as samples.The relationship between the expression patterns of regulatory genes and structural genes of grape cultivars of 4 haplotypes compositions during different developmental stages and the fruit colors was analyzed by using qRT-PCR.The results showed that PAL,4CL,CHS2,CHI1,and F3H1 showed similar expression trends in grape cultivars of different haplotypes compositions.F3 'H was expressed in both pigmented and non-pigmented grape varieties,while F3',5'H was expressed only in pigmented cultivars compared to non-pigmented cultivars,and F3',5'H/F3'H affects the shading of the pericarp.The DFR,LDOX,and UFGT showed similar expression trends in the pigmented grapevine cultivars.They all showed low expression before the initiation of fruit coloration process.The expression level are continuous increasing after the fruit coloration,and was consistent with the fruit coloring.The transcriptional expression of VvmybAl,VlmybA1-3,VlmybAl-2 and VlmybA2 was detected in the 'Kyoho' and 'Jumeigui' grapes from AE1E2 haplotype composition,and the similar expression patterns of VvmybAl,VlmybAl-3 and VlmybA2 and UFGT and GST were detected.The expression level was very low during the SGB to BGB period,and the expression level are increased rapidly during the VB period,and was highly expressed during the process of VB and MB period.The expression level of VlmybAl-2 is increased firstly and then decreased during fruit development,which was different from the expression patterns of VvmybA1,VlmybA1-3 and VlmybA2,indicating that the transcription factor had already started the regulation before fruit coloration.These results will help to further understand the functions of alleles of MYBA1 and MYBA2 locus and structural genes of anthocyanin synthesis.The full-length sequences of the VvmybA2r and VvmybA2w cDNAs were cloned based on the sequences of VvmybA2r and VvmybA2w from NCBI database.By analyzing the amino acid sequences,it was found that the N-terminus of VvmybA2r and VvmybA2w has a highly conserved R2R3 domain of the MYB transcription factor,and R3 domain contains a bHLH motif that interacts with the bHLH transcription factor,indicating that they may interact with a bHLH transcription factor in the grapevine.The expression patterns of VvmybA2r and VvmybA2w and anthocyanin synthesis related structural genes were analyzed in various organs and fruits of different developmental stages.It was found that the expression pattern of VvmybA2r was closely related to the fruit coloring process.The UFGT and LDOX expression trend is similar to that of VvmybA2r.Subcellular localization results indicate that the green fluorescence signals of VvmybA2r and VvmybA2w specifically appear in the cell nucleus.Transcriptional activation characteristics combined with bioinformatics analysis indicated that VvmybA2r has a significant transcriptional activation activity,whereas the loss of VvmybA2w transcriptional activation activity is the fact that the C-terminal transcriptional activation region is affected by the presence of its downstream SNP site(CA deletion).Both Yeast Two-Hybrid System and BiFC showed that UBE2A and SAP5 are the interaction proteins of VvmybA2w,and VvmybA2r and VvMYCAl and VvWDR1 have an interaction relationship.Yeast Two-Hybrid System also showed that VvmybA2r interacts with VvMYCAl and VvWDR1.Tobacco transient expression analysis indicated that VvmybA2w did not cause color change in tobacco leaves and was not directly involved in the pathway of anthocyanin biosynthesis.In addition,the results also showed that VvWDRl enhanced the effect of VvmybA2r in the regulation of anthocyanin biosynthesis.The above results suggested that MBW complexes from VvmybA2r,VvWDR1 and VvMYCA1 involved in the regulation of anthocyanin biosynthesis pathways,and VvmybA2w genes may play an important role in metabolic pathways of abiotic stress.A 2,000-bp 5'-flanking region of VvPAL-like was isolated from 'Summer Black'grapevine by PCR amplification,named pVvPAL-like.Sequence analysis of pVvPAL-like revealed several hormonal or stress-related cis-acting elements found in the VvPAL-like promoter sequence,indicating that VvPAL-like expression may be regulated by many factors such as auxin,ABA,ethylene,MeJA,salicylic acid,and light.To gain a better understanding of the expression and regulatory mechanism of VvPAL-like,a chimeric expression unit consisting of the ?-glucuronidase(GUS)reporter gene under the control of a 2,000 bp fragment of the VvPAL-like promoter was transformed into tobacco via Agrobacterium tumefaciens.Histochemical staining showed strong GUS staining in the cotyledon,hypocotyl and root of 3-day-old tobacco seedling.Furthermore,the histochemical staining of root,stem,fruit and leaf in transgenic tobacco indicated that strong GUS staining of xylem elements of the vascular tissues,vein,ovary and root in transgenic tobacco.The abiotic stress assay of transgenic tobacco showed that VvPAL-like promoter could be induced by Cu,ABA,IAA,NAA,IBA,2,4-D,MeJA,ETH and drought.Deletion analysis showed that three positive regulatory regions(-2000 to-1461 bp,-1461 to-930 bp,and-930 to-424 bp)and one negative regulatory region(-424 to-292 bp);the enthylene-response region located in the-1461/-930 fragment,while the element(s)for the MeJA-responsive expression may be present in the-424/-292 region in the VvPAL-like promoter.Furthermore,these findings will help us to better understand the expression pattern and regulation mechanism of VvPAL-like and the underlying molecular mechanisms by which VvPAL-like participates in biosynthesis of flavonoids and stress responses.