Cloning And Expression Analysis Of Pigment Synthesis -Related Transcription Factor HuMYB In Pitaya

MYB transcription factor is a member of a large super-family with a diversity of functions,which widely involved in plant growth and development,secondary metabolic regulation and stress response.Recently,a great deal of research has focused on the mechanism of MYB transcription factors involved in the synthesis of plant pigments.The red flesh pitaya is rich in abundant pigment,which plays an important role in anti-oxidation and anti-cancer.However,the involvement of transcription factor MYB in the regulation of pigment synthesis had not yet been extensively elaborated in pitaya so far.Thus,it is essential to excavate the key genes related to the pigmentation synthesis and therefore lay the foundation for explicating the molecular mechanisms of the regulation of pigment synthesis in pitaya fruit.Based on the previous work,different developmental stages of pitaya fruit were used as materials in the current work,The pigment-related MYB transcription factors were screened according to the gene expression trend by qRT-PCR and the full-length sequence of the pigment-associated MYB transcription factor(HuMYB1841)was cloned by RACE method.In addition,in order to further explore the function of the gene,a vector for yeast one-hybrid expression was constructed.This study is of great significance for further exploring the key genes involved in the metabolism and synthesis of pigment and the creation of new germplasm containing rich pigments by genetic engineering.The main findings are as follows:1.Using the stability evaluation software geNorm and NormFind and combining with qRT-PCR,the internal reference genes TBP2 and YLS8,which are most suitable for different developmental stages of pitaya fruit,were screened out from 17 candidate internal reference genes.2.The temporal and spatial expression of four MYB transcription factors in different developmental stages of pitaya fruit was elucidated from the gene expression level by the combination of qRT-PCR and transcriptome data analysis.The expression level of MYB1841 was up-regulated and then down-regulated during the whole fruit development of Hylocereus polyrhizus,and reached the highest value in the color-breaking period(H2).The expression level,however,was extremely low or not expressed in the Hylocereus undatus fruit,which is similar to the trend of CYP450,a key gene in sugar beet pigmentation pathway.Moreover,the expression trend of MYB1222 and MYB1841 was consistent,but the expression abundance was low.The overall expression of MYB636 and MYB1082 in the fruit development stage of pitaya was unstable and showed a fluctuating trend.Therefore,it was speculated that the MYB1841 gene is closely related to the synthesis of pitaya fruit pigment.3.The full-length MYB1841 sequence was cloned using RACE methodology,and named as HuMYB1841,whose characterizes in 911 bp total length,and 639 bp ORF,could encoding 212 amino acids.Moreover,the result of bioinformatics analysis showed that the protein encoded by HuMYB1841 contains the conserved structural regions of MYB family,which belongs to R2R3 transcription factors.4.The successful fusion protein expression vector(pBWA(V)HS-HuMYB1841-GFP)was transformed into protoplast co-culture of Arabidopsis by PEG-mediated electrochemical method with(pBWA(V)HS-GFP as control.The results of laser confocal microscopy showed that HuMYB1841 protein have nuclear localization signal,which was consistent with the prediction result of bioinformatics software,Hence,the result provided new information for further understanding of the function of HuMYB1841 gene.5.The key gene CYP450 promoter sequence involved in the beet pigment synthesis pathway was cloned by conventional PCR.The sequence length was 1547 bp,and the main regulatory elements in the promoter were predicted by onlinesoftware.The binding sites of multiple MYB transcription factors were found.6.In order to further explore the function of gene HuMYB1841,bait vector(pAbAi-CYP450)and prey vector(pGADT7-HuMYB1841)were constructed for yeast one-hybrid experiment.In addition,bait yeast strain was successfully constructed.