Cloning And Function Analysis Of Pigment-Related Transcription Factor BHLH In Pitaya

Recent years,as a typical tropical fruit,pitaya has become a new,special,excellent and high-developing project in agriculture,especially pitaya with red and purple flesh which are bright and nutritious.This is mainly because it contains special betaine,which has high value.It can be used not only as colorant in industrial production,but also as a drug for clinical treatment.At present,although the biosynthetic metabolic pathway of pitaya pigment has been basically clear and some key enzymes in the metabolic pathway have also been isolated and cloned,the regulation mechanism of pigment is not very clear.The full-length transcripts of variety with purple and red flesh?Zihonglong?and variety with white flesh?Jinghong long?were successfully constructed in the early stage of this experiment in laboratory,and the EST fragment of bHLH transcription factor differentially expressed with pigment was screened out,thus the molecular regulation mechanism of pigment synthesis was enriched and a theoretical basis for the cultivation of high-quality varieties of pitaya was provided.Based on the transcriptome data,six differentially expressing bHLH transcript sequences were screened.In this paper,the dragon fruits of different developmental stages were used as the material,the bHLH transcription factors related to pigment were screened by real-time PCR.The full-length sequence of the pigment-related bHLH transcription factor?HubHLH2?was cloned by RT-PCR,which suscessfully constructed a bait vector,established and screened a yeast two-hybrid library.The main findings are as follows:1.The expression of six bHLH candidate genes in different developmental stages of dragon fruit was analyzed by qRT-PCR.The results showed that the transcription factors of HubHLH11270,HubHLH3826,HubHLH49630 and HubHLH54630 did not show obvious changes in different developmental stages.The expression level of HubHLH1637 in red flesh showed a significant upward trend,while the expression level of HubHLH1637 in white flesh showed a trend of decreasing first and then increasing.However,the expression level of HubHLH2 in red flesh showed a trend of gradually decreasing,while the expression level of HubHLH2 in white flesh is higher than that in red flesh as a whole,and there is a difference between red and white flesh pitaya.2.According to the three generations of transcriptome data,the dragon fruit bHLH transcription factor was cloned by RT-PCR and named as HubHLH2.The complete open reading frame?ORF?of this gene is 1524 bp,encoding 508 amino acids,containing the bHLH-MYC_N domain and a typical HLH conserved domain,which is a hydrophobin and has no transmembrane region and signal peptide.Its predicted molecular weight is 124.55 KD,with the isoelectric point pI of the protein theory is 5.03.The amino acid sequence alignment result indicated that HubHLH2 has high homology with other higher plant bHLH proteins and is highly conserved in the HLH functional domain.expecially,the homology of HubHLH2 protein with saxifrage?Amaranthus hypochondriacus XP₀10679205.1?was the highest,reaching95.9%.Secondly,it has a higher homology with Kunote?Chenopodium quinoa XP₀21764267.1?and sugar beet?Beta vulgaris XP₀10679205.1?,which is 94.5%.The results of constructing phylogenetic tree showed that HubHLH2 was close to Arabidopsis JAMs protein and MYC protein,but the genetic distance from JAM3 was the closest.3.The tissue-specific analysis of HubHLH2 showed that HubHLH2 was mainly expressed in root,which is higher than the relative expression of stem and fruit.The result indicates that HubHLH2 may mainly play an important role in the root.The results of the treatment with meja showed that MeJA could promote the expression of HubHLH2,and the HubHLH2 expression increased after 1 h,but decreased after 2 h,fianlly,the expression of HubHLH2 started to pick up again and reached the maximum at 12 h.4.A three-frame yeast cDNA library was successfully constructed by using Invitrogen's Clone Miner 2 Library technology,including the total number of clones in the primary library was 1.44×10⁷,the recombination rate was 96%,and the average insert length was greater than 1 kb,and the total number of secondary libraries was9.6.×10⁶,the recombination rate was 100%,and the average insert length was greater than 1 kb;the library plasmid was transferred to the Y187 strain and the titer of the identified library was greater than 3×10⁷ cells/mL.5.The bait vector of pGBKT7-HubHLH2 was constructed and transformed into yeast Y2HOLD strain,which is tested by autoactivation and toxicity.The results showed that the bait protein?HubHLH2?was not self-activated and had no toxic effect on yeast strain.6.Mating method was used to screen the dragon fruit yeast library,and 13proteins which may interact with HubHLH2 were screened at early stage.The Blast alignment analysis revealed that the selected proteins were mainly enzymes related to glycolysis and gluconeogenesis and the Calvin cycle in photosynthesis,such as transmembraneATPase,cytochromeb6fcomplex,ironredoxenzyme glyceraldehyde-3-phosphate dehydrogenase etc.In addition,the interaction between HubHLH2 and SPL family genes was also found,so it is speculated that HubHLH2may not only be involved in dna repair,transcriptional and post-transcriptional regulation,cell signal transduction,apoptosis and other life processes,but also regulate pigment synthesis mediated by spl gene.