Identification Of Mi RNAs Related To Betalain Biosynthesis In Pitaya

Pitaya is a new fruit,which is developing rapidly in recent years.Red pulp pitaya（H.monacanthus）is the only fruit containing abundant betalains.However,studies on betalain biosynthesis in pitaya are poorly documented.Compared with transcription factors involved in the regulation of betalain biosynthesis,negatively regulate target gene expression by miRNAs in betalain biosynthesis is not clear yet.miRNAs regulate its target genes mRNA at post-transcriptional level via the RISC（RNA-induced silencing complex）by binding with the AGO protein to cleavage target mRNA or repress target mRNA translation.miRNA play important roles in plant development,abiotic stress and biotic stress response as well as pigment regulation.In this study,candidate miRNAs and its target genes related to betalain biosynthesis regulation were identified based on small RNA and transcriptome database from pitaya flesh.The mechanisms of these miRNAs regulating betalain biosynthesis in pitaya flesh could provide the theory for pitaya molecular breeding work.The main results are as follows:1.Six small RNA libraries i.e.Hp19d₁,Hp19d₂,Hp25d₁,Hp25d₂,Hp29d₁ and Hp29d₂ were constructed using three different developmental pulp stages（19 d,25 d and 29 d after artificial pollination）of ‘Guanhuahong’ pitay.128 conserved miRNAs and 41 novel miRNAs were identified,among which 95 conserved miRNAs belonging to 53 miRNA families.Differential expression levels of 18 conservative miRNA and 1 novel miRNA（PC-5p-2975₃03）were detected in 19 d（before color conversion）,25 d（after color conversion）and 29 d（mature period）of three periods suggeting those 19 candidate miRNAs were involved in betalains biosynthesis in pitaya.2.Three transcriptome libraries were constructed from three stages（19 d,25 d and 29 d after artificial pollination）of ‘Guanhuahong’ pitaya pulp.A total of 7.46 Gb,9.10 Gb and 10.23 Gb of raw RNA-Seq data were generated in three libraries,respectively.68,505 transcripts were de novo assembled.These transcripts were further assembled into 39,737 genes with a average length of 879 bp.The 39,737 genes were functionally annotated based on public protein databases and,of these,3,988 genes were significantly differentially expressed between any two coloration stages,while 124 genes were differentially expressed among all three stages.A RNA-Seq library was constructed and sequenced by Target Finder software,31 genes were identified as targets of the novel and differential expression miRNAs in three stages（19 d,25 d and 29 d after artificial pollination）（P≤0.05）.3.Identification of 35 candidate miRNAs related to the accumulation of betalains in pitaya pulp by real-time quantitative PCR（Figure 16 and 17）.It was found that 12 candidate miRNAs were highly expressed in the turn color period（23 d）of ‘Guanhuahong’ pitaya flesh,of which 7 pitaya miRNAs including ata-miR172b-3p₁ss1AC,PC-5p-1835₄91,ssp-miR156_L+1R-1₁ss4AC,aly-miR157d-5p_L+1₁ss4AC,cpa-miR159 a,PC-3p-4296098₁,and PC-3p-53338₁8 in the different developmental stages of ‘Guanhuahong’ pitaya pulp were higher than that of ‘Guanhuabai’ pitaya pulp.This means that they are likely to play a regulatory role in the conversion of pitaya pulp.4.Expression pattern of key miRNAs and their target genes are then further investigated on 7 stages of pitaya pulp development and ripening.The expression level of these key miRNAs and their target genes displays a complementary pattern.For example,nta-miR6020b-comp234190_c0 in ‘Guanhuahong’ pitaya pulp.But,nta-miR6020b-comp234190_c0 showing upward and downward fluctuating trends in ‘Guanhuabai’ pitaya pulp,the regularity is unapparent.nta-miR6020 b shows significantly higher expression level at the conversion stage in ‘Guanhuahong’ pitaya,with a steady increase at expression level during fruit ripening in ‘Guanhuabai’ pitaya,but the total was lower than that of ‘Guanhuahong’ pitaya.These experimental evidences indicate that nta-miR6020 b may have particular function at betalains biosynthesis.5.Subsequently,5′RACE and transient expression system in tobacco are used to verify the target genes of miRNAs.A total of 4 miRNAs and 5 corresponding target genes were verified to be interacting in 5′RACE and tobacco transient expression system experiments.They are mdm-miR858-comp15143_c0,mdm-miR858-comp24362_c0,PC-5p-2975₃03-comp23458_c0,PC-5p-192₇269-comp29967_c0 and PC-5p-23845₃9-comp28219_c0,respectively.