Role Of Pitaya(Hylocereus Polyrhizus) MiR396b In Abiotic Stress Responses

Pitaya（Hylocereus）,belonging to Family Cactaceae,is a type of economical and nutritional fruits characterizing in high tolerance to drought stress,and cultivated in tropical and subtropical regions.Due to the influence of karstic topography,drought and other adverse environments are chief factors to constrain the growth and development of pitaya,thereby severely inhibit the yield and quality.miRNAs are endogenous single-strand non-coding small-molecular-weight RNAs with about 20nucleotides（nt）in length,and their precursors characterize in a stem loop structure,which are known to regulate gene expression at the post transcriptional level through cleavage of mRNAs and/or translational repression the corresponding target genes.Recently,the molecular response of pitaya to drought stress at transcriptomic level was documented,however,the roles of miRNAs in pitaya tolerance to drought stress had not yet been deciphered so far.Exploring the stress resistant miRNAs and their regulation patterns in pitaya is of great significance to development of pitaya industry,while it can optimiz pitaya species,improving their tolerance to stress.The main results are as follws:1.The precursor sequence of hpo-miR396b gene was cloned by PCR and named as hpo-MIR396b.The gene was 130 bp in length and its secondary structure can formed a typical stem-loop structure.The miR396b/miR396b*double-strand structural located on arms of the stem-loop,and the miR396b mature sequence（5’-UUCCACAGCUUUCUUGAACUU-3’）located on the 5’arm.Phylogenetic analysis showed that the plant MIR396b had a more recently evolutionary relationship in the family,but there were also a few species of intra-family plant aggregation,and the clustering of pitaya and the dicotyledonous species was more scattered,which further demonstrated that in addition to species differences,there were other factors that influence the formation of the MIR396b.2.Based on the RNA-seq results,psRNATarget and Targetfinder were used to predict the targets of hpo-miR396b.The functional annotation indicated that the target gene mainly encoded nuclear protein and participated in growth and development,most of which were homologous to Arabidopsis thaliana.Combined with the degradome sequencing results,four target genes of hpo-miR396b were obtained,i.e.GRF1（c56908.graph_c0）,GRF4（c52862.graph_c0）,GRF6（c39378.graph_c0）and GRF9（c54658.Graph_c0）,all of which were predicted to be members of the GRF family,and their cleavage sites confirmed that GRF was regulated by miR396b through specific cleavage at the site between the 10^thh and the 11^thh bases.3.A fragment of the target gene HpGRF6 of hpo-miR396b was obtained with PCR,which had an open reading frame（ORF）of 1,119bp（GeneBank:MK625692）,encoding 372 amino acids with two conserved domains,i.e.QLQ and WRC.The target interaction site located at the GRF sequence which owned the conserved“RSRKPVE”amino acid in Trp-Arg-Cys（WRC）region.Phylogenetic analysis indicated that HpGRF6 shared the highest homology with the protein of Chenopodium quinoa（XP₀21715016.1）.4.Transient expression of recombinant plasmid pBWA（V）HS-HpGRF6-GFP in tobacco leaf cells showed that HpGRF6 had nuclear localization signal（NLS）and distributed specifically in cell nucleus.5.To detect the expression of miRNA,a qRT-PCR system was established by comparing the methods between stem loop and poly A polymerase（PAP）,whose primers were designed based on the sequences of hpo-miR396b.Also,the stability of two reference genes,i.e.5S and U6,was assessed.The results demonstrated that the stem loop method showed superior sensitivity and specificity in comparison with PAP,and U6 was more stable as reference gene.Therefore,the stem loop method was more suitable for detecting of hpo-miRNA.6.Role of hpo-miR396b and its target HpGRF6 in abiotic stress responses was analyzed by stem-loop qRT-PCR.The results demonstrated that hpo-miR396b and target gene HpGRF6 were in response to adversity.Althouth diverse responses were investigated as exposure to the adversities,and the expressions of HpGRF6 showed exactly the opposite trends with hpo-miR396b,which substantially suggested that hpo-miR396b played a regulatary role in HpGRF6,leading to the involvement in abiotic stress response of pitaya.