Functional Analysis Of Transcription Factor PcMYB1 In Polygonum Cuspidatum

Many beneficial effects of resveratrol have been reported.Although stilbene synthases have been isolated and characterized,the transcriptional regulation underlying resveratrol biosynthesis is unknown.MYB have been well characterized in many plant species,however it is still not clear about the role of MYB involved in resveratrol biosynthetic pathway in Polygonum cuspidatum.In our previous study,transcriptional responses were investigated in Polygonum cuspidatum leaves using comparative RNA-seq based transcriptomic analysis and digital gene expression proling analysis.Our previous study found that a new MYB transcription factor,PcMYB1,could regulate resveratrol biosynthetic pathway in Polygonum cuspidatum.To assess the biological functions of PcMYB1 gene,this study will investigate tissue-specific expression profiles,subcellular localization,activity of PcMYB1 promoter,and overexpression of PcMYB1 in hairy roots.All together,these results will illustrate the biological functions of PcMYB1and provide new important information for the research of regulatory mechanism and the genetic improvement of Polygonum cuspidatum.The experimental results are as follows:Expression pattern analysis showed that PcMYB1 is widely expressed in all tissues examined,but predominantly in leaves.In contrast to PcMYB1 expression,the accumulation of resveratrol in root was the most.PcMYB1 expression was negative-correlated with accumulation of resveratrol implying that PcMYB1 may be a repressor of resveratrol biosynthesis in P.cuspidatum.Furthermore,we investigated the subcellular localization of PcMYB1 in Nicotiana benthamiana leaves by using the GFP fusion construct,35S::PcMYB1-GFP.PcMYB1-GFP was specifically localized in the nucleus,indicating that PcMYB1 functions as a transcription factor.hi TAIL-PCR was used to clone the PcMYB1 promoter from the leave genome DNA of P.cuspidatum,and bioinformatics analysis of it was conducted.The 2884 bp promoter sequence（Gen Bank accession number:MT811057）of the promoter was obtained.The promoter contained several core fragments（TATA-box and CAAT-box）essential for eukaryotic promoters,and also cis-elements that were responsive to Me JA and low-temperature and cis-acting regulatory elements that were associated with light and anaerobic responses.The recombinant expression vector pro PcMYB1::GUS were constructed.GUS histochemical staining was performed to analyze the activity of the promoter.All tissues of pro PcMYB1::GUS transgenic Athaliana were stained blue,that demonstrated that PcMYB1 promoter drove the GUS gene expression.PcMYB1 promoter is successfully cloned and it shows the activity of driving the expression of downstream GUS.An effective genetic transformation system for hairy roots of Polygonum cuspidatum was established.The leaves of P.cuspidatum was infected by Agrobacterium rhizogenes with the plasmid p CAMBIA1301 which contains GUS gene.The superior transformation system was as following.The leaves of the aseptic seedlings were used as explant materials,1/2MS medium was used as the induction medium.Ultrasound-assisted induction was used to infect for 5 s in suspension(OD₆₀₀=0.8).The positive transformed hairy roots were further identified by PCR technique and GUS histochemical staining,with the genetic transformation rate of about 90%.These results laid the foundation on gene function of P.cuspidatum.The negative regulation of PcMYB1 gene on resveratrol biosynthesis of Polygonum cuspidatum was confirmed by using transgenic hairy roots.When PcMYB1 was over-expressed in P.cuspidatum hairy roots,key genes（Pc RS,Pc PAL,Pc C4H）involved in the synthesis of resveratrol were down-regulated,and the content of resveratrol was reduced by 45%.Overexpression of PcMYB1 in P.cuspidatum hairy roots repressed resveratrol biosynthetic genes,leading to a reduction in the amounts of resveratrol.Taken together,these results further confirmed the negative role of PcMYB1 in the regulation of resveratrol biosynthesis.Previous reports have shown that the Sg2 subfamily R2R3-MYB transcription factor positively regulates the synthesis of resveratrol in grape.In this study,the R2R3-MYB transcription factor PcMYB1 of Sg4 subfamily negatively regulates the synthesis of resveratrol in P.cuspidatum,which helps to reveal a new regulatory mechanism of resveratrol synthesis.In addition,previously reported Sg4 subfamily R2R3-MYB transcription factors PcMYB1 mainly regulate the synthesis of lignin and flavonoids.In this study,the Sg4 subfamily R2R3-MYB transcription factor can also regulate the synthesis of resveratrol,further enriching the functional diversity of MYB gene.