Study On Cloning And Function Of Transcription Factor MYB For Anthaynin Biosynthesis In Mulberry Fruit

It is widely acknowledged that Mulberry not only has high nutritional value,but also contains a variety of health care functions.It has been listed by the Ministry of Health as "Affinal Drug and Diet" and has become a high-quality raw material for the development of functional foods.Mulberry is rich in anthocyanins,which is an important active ingredient in Mulberry and one of the significant sources of natural pigments.Anthocyanins are water-soluble natural pigments widely found in plants and it is focused on more researchers due to their important physiological and economical functions.Thus,the study of the regulation mechanism of anthocyanin synthesis has a critical role in scientific significance for the increase of anthocyanin content in plants and the innovation of germplasm.In this study,Dagu(black fruit)and Baiyuwang(white fruit)Mulberry cultivars were used as experimental materials;firstly,isolated the transcription factor MYB in anthocyanin synthesis and analyzing the transcription factor MYB in anthocyanin synthesis by bioinformatics analysis;secondly,defined the gene of anthocyanin synthesis structure and Ma MYBs expression patterns;third,researched the role of Ma MYB10 in the anthocyanin synthesis of mulberry.Finally,explored the new ways of regulating the anthocyanin synthesis of Mulberry,helping to improve the color of Mulberry and enhancing economic value of Mulberry.The main results are as follows: 1 Changes of Anthocyanin content in different Mulberry cultivars and fruit differentstages of growthUsed the CIRG chromatic aberration system evaluates the color change in appearance color of Mulberry quantitatively.There were significant differences in the CIRG values of the fruits of the two cultivars,the black 'Da Shi' and the white 'White Jade' Mulberry.The matured cultivars of ‘Da Shi' CIRG value is 7.22 and matured cultivars of Bai Yuwang CIRG value is 3.24,the CIRG of Bai Yuwang cultivars was significantly lower than Da Shi cultivar.With the growth of Mulberry fruit,the CIRG value of black Da Shi showed a rising trend and the color was significantly deepened,which was consistent with the change of Total Anthocyanin Content.However,the CIRG value of white Bai Yu Wang cultivar basically has little changed.Two types of anthocyanin ‘cyanidin-3-O-glucoside,cyanidin-3-O-rutin' were detected in the black 'Daishi' cultivar by HPLC,but were not detected in the white 'White Jade' cultivar.With the growth of fruit,the cyanidin-3-O-glucoside content of Da Shi cultivar has increasing alteration and the cyanidin-3-O-rutin content increased first and then decreased.The results showed that the difference in the Total Anthocyanin Content of mulberry may be the main reason for the difference in the color of different cultivars,which is related to the genetic characteristics of mulberry cultivars.2 Mulberry transcriptome sequencing and bioinformatics analysisConstructed Mulberry transcriptome sequencing data analytics platform ‘Illumina Hiseq4000' analyzed data of transcriptome sequencing in different mulberry cultivars.The results showed that a 53184 SNP markers were obtained,which sequencing depth were greater than 100;A total of 12,082 SSR sequences were obtained,including single nucleotides,dinucleotides and trinucleotides and they were accounted for 35.96%,37.61%,and 22.97%,respectively.Jointed sequencing data of ginseng-free transcriptome by software of Trinity,we obtained 32155 genes,which average length are 956 bp;and 55883 transcripts,which average length is 1161 bp.Predicted CDS by GENScan software,we obtained 19238 valid CDSs and the predicted rate was 59.83%.In the GO functional classification note,61 genes were obtained,which were related to synthesis of anthocyanin.In the KEGG metabolic pathway enrichment analysis,59 structural genes were found,which related to synthesis pathway in the anthocyanin of mulberry.In comparison with the NR database,42 MYB genes(34 named and 8 unnamed)were found in the transcriptome library of mulberry.3 Cloning of the full-length c DNA of transcription factor MYB in mulberryanthocyanin synthesisIn the transcriptome database of mature mulberry fruits,based on BLAST comparison of NR database,based on the MYB transcription factor associated with anthocyanin synthesis(compared with comp16540_c0 in the transcriptome),primers were designed using RACE technology to obtain MYB full-length c DNA sequences.It has a length of 1338 bp,a non-translated sequence of 245 bp at the 5' end,an untranslated sequence of 172 bp at the 3' end,305 amino acid were translated and named Ma MYB10,which has the typical domain of MYB transcription factor and belongs to R2R3 MYB transcription factor.The phylogenetic tree was constructed by NJ method and it was found that it has high homology participates in the regulation of anthocyanin synthesis in other species(Arabidopsis,strawberry,pear,etc.)MYB.Them have.4 Expression analysis of Ma MYBs and related genes in the synthesis of mulberryanthocyaninsBased on the transcriptome data of mature mulberry fruits,KEGG pathway was used to annotate 4 MYB fragments Ma MYB10,Ma MYB305,Ma MYB5 and Ma MYB48 family members were cloned.Real-time fluorescence quantitative quantification(q PCR)was used,and the expression of Ma MYB10 and Ma MYB305 was significantly different among the two cultivars.Compared with the Baiyuwang cultivar,the expression of Ma MYB10 and Ma MYB305 increased gradually with the development of fruit,which was consistent with the change of anthocyanin content.However,the expression of Ma MYB5 and Ma MYB48 in the Dashi cultivars was significantly lower than that of the Baiyuwang cultivars.Analysis the expression of structural gene in mulberry anthocyanin synthesis showed that the four structural genes(Ma CHS,Ma DFR,Ma ANS,and Ma UFGT)all had certain expression differences in Dashi and Baiyuwangang cultivars,among which Ma CHS,Ma DFR,and Ma UFGT.The expression was significantly higher than that of the Baiyuwang cultivar.With the development of the fruit,the expression of these three structural genes was increased,which was consistent with the change of anthocyanin content.However,the Ma ANS expression of the Dashi cultivars was significantly lower than that of the Baiyuwang cultivars.