Screening And Founctional Identification Of MYB Transcription Factor Family Genes Related To Flesh Color In Pitaya

In flesh of pitaya,the main pigment related to the color is betalain.It has been proved that the betalains has strong anti-oxidation.Through the study on the regulation of betalains synthesis,we can not only guide the breeding of new varieties from molecular level,but also explore the function of betalains more deeply.We collected flesh samples of H.polyrhizus and H.undatus,determined the contents of betacyains and betaxanthins respectively,analyzed the accumulation patterns of two pigments in pitaya.In order to further explore the mechanism of betalains’ synthesis,we studied the relationship between MYB transcription factors and betalains.MYB transcription factor genes are a large number and complex gene family,which participate in almost every stage of the growth and development of higher plants,in which R2R3 transcription factors play an important role in regulating plant secondary metabolism.In this study,flesh of 4 developmental stages(green fruit stage,pre-color conversion stage,color conversion stage and mature stage)in H.undatus and H.polyrhizus were used as experimental materials.Analyzing the defferences of expression between MYB family genes,the genes differentially expressed in H.polyrhizus were screened out.Analysing sequences by bioinformatics tools.Constructing yeast expression vector and plant expression vector to explore the mechanism of color conversion in pitaya at molecular level.The main results are as follows:1)The betacyanins and betaxanthins increased in "S" shapes in flesh,and increased rapidly from the color conversion stage,the content of betacyanins was higher than that of betaxanthins.2)A total of 88 MYB transcription factor genes were obtained by transcriptome data analysis.Removing 26 RPKM with zero or small differences genes,there were 63 left.Through heat map analysis,8 genes were significantly up-regulated during the colorconversion period and 10 genes were significantly down-regulated during the colorconversion period.qRT-PCR was used to analyze the DEGs of H.undatus and H.polyrhizus at four developmental stages,and six genes with more significant differences were obtained.Among them,Unigene0017577,Unigene0032771,Unigene0006295,Unigene0014228,Unigene0029017 were up-regulated in H.polyrhizu from the pre-color conversion stage to the color conversion stage,but on the contrary in H.undatus,and Unigene0017701 was significantly up-regulated in the pre-color conversion stage of H.polyrhizu.3)Based on the analysis of gene expression in flesh samples of H.polyrhizu at 10 stages,it was found that the expression of Unigene0032771 increased significantly on the 24th day after pollination,which was in the pre-color conversion stage;The expression of Unigene0014228 decreased significantly from 21 to 27 days after pollination,which was in the pre-color conversion stage,but increased significantly from 30 to 33 days,the color conversion stage.Unigene0006295 increased significantly from the 24th day to the 27th day after pollination,which was the pre-color conversions stage.Unigene0017577 increased significantly on the 30th and 33rd day after pollination,and the flesh in this period was in the stage of color conversion;Unigene0029017 was low at all developmental stages and significantly decreased on the 27th day after pollination.Unigene0017701 was relatively balanced and low in each period.To sum up,Unigene0032771 and Unigene0006295 had similar expression pattern and the expression level was the highest in the pre-color conversion stage,the form or was higher than the later.Unigene0014228 and Unigene0017577 had similar expression pattern,they all significantly up-regulated during the color conversion period,and the expression of Unigene0017577 was higher than that of Unigene0014228.Thus,Unigene0032771,Unigene0014228,Unigene0006295 and Unigene0017577 were picked as target genes and named HpMYB1,HpMYB2,HpMYB3,HpMYB4,respectively,to carry out deeper research.4)Four genes are all transcription factors,of which HpMYB1,HpMYB3,HpMYB4 are R2R3-MYB transcription factor with a R2 domin and a R3 domin.HpMYB2 contains a R2 domain and a Zinc finger domain which hidden at N treminal.5)In the gene silencing experiment,the results of HpMYB2 and HpMYB4 are more ideal.After silencing HpMYB2,the contents of betacyanins and betaxanthins were significantly decreased,and the gene expression was significantly down-regulated.After silencing HpMYB4,the content of betacyanins decreased significantly,and the gene expression was also significantly down-regulated.