| Betalains are water-soluble,nitrogen-containing plant pigment with various bioactive functions which are widely used in food,chemical industry,printing and dyeing,chemical industry,medicine,forage and other fields.Currently,structural genes involved in betalain biosynthetic pathway have been extensively elucidated in betalain-producing plants.However,regulatory factors involved in betalain biosynthesis are still not clear.MYB and WRKY transcription factors(TFs)play important roles in plant growth and development,physiological metabolism,stress resistance and hormone signal transduction.Pitaya(Hylocereus)is a kind of tropical fruits abundant with betalains.However,it is not clear how the MYB and WRK Y TFs regulate the betalain biosynthesis.In the present study,MYB and WRKY TFs involved in betalain biosynthesis of pitaya were screened from the transcriptome dadasets of ‘Guanhuahong’(red peel with red pulp)through bioinformatics,space-time expression,subcellular localization,yeast two hybrid,double luciferase report and virus-induced gene silencing(VIGS).The aim of the present study is to explore the roles of MYB and WRKY TFs in pitaya betalain biosynthesis.The main results are as follows:1.A total of 144 MYB and 73 WRK Y TFs were obtained from pitaya transcriptome datasets.Seven MYB and six WRKY TFs involved in betalain biosynthesis of pitaya were obtained based on FPKM values of the RNA-Seq.Two candidate MYB(Hp MYB39 and Hp MYB-like)and three candidate WRK Y(Hp WRKY40,Hp WRKY7 and Hp WRKY31)TFs related to betalain biosynthesis were obtained based on betalain contents and the results of real-time fluorescence quantitative PCR(q RT-PCR).2.Full-length c DNA sequences of Hp MYB39,Hp MYB-like,Hp WRKY40,Hp WRKY7,and Hp WRKY31 were cloned.Results from analyses of amino acid sequences and phylogenetic tree showed that Hp MYB39 was a R2R3 type MYB TF.Hp MYB-like had a single MYB domain and belonged to the MYB-related family.Hp WRKY7,Hp WRKY31 and Hp WRKY40 had only one WRK Y domain belonging to the WRKY family II class.Subcellular localization showed that Hp MYB39,Hp MYB-like,Hp WRKY40,Hp WRKY7,and Hp WRKY31 were nuclear proteins.3.Results from q RT-PCR showed that expression trends of Hp MYB39,Hp MYB-like,Hp WRKY40,Hp WRK Y7 and Hp WRKY31 were consistent with the key genes of Hp Cyt P450-like1,Hp Cyt P450-like2,Hp Cyt P450-like4 and Hp GT-like2 indicating that Hp MYB39,Hp MYB-like,Hp WRKY40,Hp WRKY7 and Hp WRKY31 are likely to play key roles in betalain biosynthesis.Silencing of Hp MYB39,Hp MYB-like,Hp WRKY40,Hp WRKY7 and Hp WRKY31 exhibited green and yellow patches in scales of dragon fruit and resulted in significant reduction of betacyanins and betaxanthins.Those results suggested that Hp MYB39,Hp MYB-like,Hp WRK Y40,Hp WRKY7 and Hp WRKY31 play key roles in the pitaya betalain biosynthesis.4.Results from yeast single-hybrid and double luciferase showed that Hp MYB39 could bind and activate betalain biosynthesis-related structural gene Hp Cyt P450-like1 promoter activity.Hp MYB-like could bind and activate promoter activity of Hp Cyt P450-like1 and Hp Cyt P450-like4.Hp WRKY40 could bind and activate the promoter activity of the structural gene Hp Cyt P450-like1.In summary,Hp MYB39,Hp MYB-like and Hp WRK Y40 are involved in betalain biosynthesis through selectively regulating expression of cytochrome P450-related structural genes. |
