Identification And Regulatory Mechanism Of MYB Transcription Factors On Anthocyanin Biosynthesis In Potato Tuber

Potato(Solanum tuberosum L.)is the third most important food crop worldwide.The germplasm of potato is highly diverse,colorful potatoes have development potential in market for they are not only rich in nutrition,but also have good processing quality and high added value.The pigmented potatoes are a rich source of anthocyanins.As the most important flavonoids in plants,anthocyanins are the main pigments responsible for the red-blue colour of potatoes,they have potential health benefits in humans,such as super strong oxidation resistant ability,suppression of inflammation,safeguard against cardiovascular diseases and improvement of human immunity etc.,its medical value has attracted more attention.Therefore,the study on anthocyanin regulatory mechanism in potato tuber has important scientific significance and applicable value for in-depth analysis of anthocyanin metabolism pathway and cultivation of colorful potato varieties.It’s been reported that anthocyanin biosynthesis is mainly controlled by three transcription factors(TFs)including MYB,basic helix-loop-helix(b HLH)and WD40,among them,the MYB is the most important TF.In this thesis,an integrative analysis of the transcriptome and metabolome of potato tubers from three colored potato clones(Y: white-pale yellow-yellow,R: white-pale yellow-red,P: pale purple-purple-dark purple)at three developmental stages(tuber formation stage-S1,tuber developmental stage-S2 and tuber maturation stage-S3)was performed using high-throughput RNA sequencing,furthermore,combined with the RNA-seq data of the purple varieties "Heimeiren",white varieties "Xindaping",and red varieties "Lingtian Hongmei",the differentially expressed genes and TFs involved in anthocyanin biosynthesis were discovered,the function of anthocyanin-related MYB transcription factors and regulatory mechanism on anthocyanin biosynthesis in potato were further investigated.The main results were summarized as follows:1.Transcriptome analysis on three different pigmented clones(CIP 302281.17-Y,CIP 302281.25-R and CIP 302281.15-P)at three growth stages(S1,tuber expansion stage-S2 and tuber maturity stage-S3)was performed to screen out 15 pathway genes and 13 candidate TFs involved in anthocyanin biosynthesis of potato tuber;eight TFs were up-regulated in red and purple tubers at three growth stages,including one R2R3-MYB TF and one MYB-related TF,the rest TFs were b ZIP,WRKY,HB-other,AUX/IAA,b HLH,and GNAT;five transcription factors were down-regulated in colored flesh at the three growth stages,which were RWP-RK,MADS-MIKC,NAC,b HLH and C2H2,respectively.These TFs may be involved in anthocyanin biosynthesis in potato tuber,which requires further investigation.2.In order to identify and investigate the function of MYB TFs in potato,a total of 111 StR2R3-MYB TFs,138 MYB-related TFs,three 3R-MYB and one 4R-MYB were distinguished.The characteristics of chromosomal allocation,gene structure,protein conserved domains,and genetic relationships of the 111 R2R3-MYB TF family members were comprehensively analyzed.111 StR2R3-MYB TFs were phylogenetically classified into 31 subfamilies based on highly-conserved gene structural compositions and motifs.StR2R3-MYB genes might be related to biosynthesis of anthocyanin were further analyzed based on RNA-seq data and eight candidate R2R3-MYB TFs were screened.3.In order to investigate the flavonoid content and composition changes in white and colored potato flesh,metabolome sequencing of the flesh of Y,R and P at S1,S2 and S3 stages was carried out as well.A total of 72 flavonoids were identified.The accumulation of delphinidin(Dp),petunidin(Pt),malvidin(Mv),cyanidin(Cy)and peonidin(Pn)led to the dark purple appearance of the P clone during tuber develop ment;the high accumulation of Pg derivatives and with Cy and Pn derivatives in red flesh of R led to the red coloration at the S3 stage.4.The WGCNA analysis was used by combining transcriptome and metabolome data to study the function of the candidate MYB TFs and to understand the regulatory mechanism on anthocyanin accumulation in potato tuber.Three core modules positively correlated with anthocyanin content and four core modules negatively correlated with anthocyanin accumulation were determined,and two core MYB TFs-StMYB3 and StMYBATV,respectively.It was found that StMYB3 and StMYBATV can negatively regulate anthocyanin biosynthesis via transient assay and stable transformation,furthermore,StMYB3 and StMYBATV can bind to the anthocyanin transcription regulator Stb HLH1 to inhibit StDFR promoter activity by the bimolecular fluorescence complementation(BiFC)assay,which was a key structural anthocyanin pathway gene.During the color accumulation of potato tubers,the transcriptional activators StAN1 and Stb HLH1 positively regulate anthocyanin biosynthesis,whereas StMYB3 and StMYBATV were involved in feedback regulatory mechanisms underlying the biosynthesis of flavonoid accumulation in colored potatoes during tuber development.