Cloning And Target Vlidation Of Drought Responsive MiRNAs/isomirs In Pitaya (Hylocereus Polyrhizus)

As the most worldwide stress plants confronted with,drought was considered as the primary reason that caused crop failure.Getting better understanding of how plants responded to drought could direct us to improve the tolerance of crops against drought,tranditionally by screening out the better germplasms or creating new ones.microRNA(miRNA)was a class of small non-coding RNAs whose length were usually between 18 nt to 20 nt.It played very essential roles in development and other progresses of plant species,thus helped plants in stress response by reprogramming their development.Pitaya(Hylocereus),a local plant in tropical areas,was introduced and promoted in provinces in southern China on account of appealing taste and rich nutrition.As a cactus species,pitaya exhibited great tolerance to drought stress.Therefore,full understanding of the molecular mechanism and identification of the key genes of pitaya in response to drought stress would help us to find the better germplasm with best behavior in response to drought and the target genes which would improve drought tolerance of susceptible plants.In previous works,the key protein-coding genes in response to drought stress was identified in pitaya by suppression subtractive hybridization(SSH).However,no effort was levered to find key miRNAs helping pitaya to adapt to drought condition.In addition,no report was published about miRNAs in cactaceae.To understand molecular mechanism of pitaya in response to drought at miRNA level,elite local cultiver ‘Zihonglong’(Hylocereus polyrhizus ‘Zihonglong’)was chosen as object,subcultured clones were used as materials.15% polyethylene glycol(PEG)was used to apply drought stress,cytophysiological traits of materials were measured.In combination of bioinformatics and molecular cloning,hairpin and mature miRNAs expressed in pitaya was cloned and annotated.Their conservation among Plantae was investigated.Subsequently,targets of miRNAs were predicted by bioinformatics tools and validated by parallel analysis of RNA ends(PARE)and partially by RNA ligase mediated 5’ rapid amplification of cDNA end(RLM-5-RACE).The conservation of targeting relation between miRNA and mRNA in Plantae was investigated and discussed.Using stemloop reverse transcription-quantification PCR(RT-qPCR),expression of pitaya miRNAs under drought condition was quantified and profiled.By original Practical Extraction and Reporting Language(PERL)script,pitaya miRNA isoforms(isomiRs)were identified,classified and quantified.Their targets were also predicted and validated.In addition,their roles in miRNA mediated drought response were discussed and the first plant isomiR database was provided.The main contents and results in present study were as follows: 1.Cytophysiological traits under drought stress were measearedDrought treatment was applied by PEG for subcultured clones of pitaya seedlings for 72 hours and materials were sampled every 12 hours.It was observed that contents of osmotic adjustment substances,i.g.soluble protein(SP),soluble sugar(SS)and free proline(Pro),activities of protective enzyme,i.g.catalase(CAT),superoxide dismutase(SOD),peroxidase(POD),increased while cellular water potential(WP)decreased.Although relative electrical conductivity(REC)increased and malondialdehyde(MDA)accumulated,they were still at a very low level indicating robust tolerance of pitaya seedlings to drought.2.Hairpin and mature sequence of 36 pitaya miRNAs were clonedSamples growth under different conditions were collected for construction of one transcriptome and two small RNA(sRNA)libraries.The raw data were submitted to SRA(Sequence Read Archive)database of NCBI(https://www.ncbi.nlm.nih.gov/sra).Accession number were SRR8767412,SRR8767414,SRR8767415.In combination of bioinformatics and molecular cloning,34 and 30 mature sequences accounting for 39 hairpins were identified,among which six miRNAs were identified by both methods.Among identified hairpins,36 of them were experimentally cloned which were consisted of 21 known and three novel families.Most of the mature miRNAs was 21 nt in length(81.94%)and started with uridine(44.44%).Conservaion analysis discovered that some miRNA families were lineage-specific conserved,e.g.MIR403 was only found in dicotyledons.In addition,sequences of mature miRNAs in the same family were highly conserved,e.g.only in a few plants the eighth and twentieth nucleotides of miR162 were substituted with guanosine.3.Cleavage events on 48 Unigenes directed by 32 miRNAs were validatedTo find target genes of miRNAs,binding sites of miRNAs on pitaya Unigenes were predicted by psRNATarget,TargetFinder,GSTAr,and 337,1,064,50,119 sites were obtained,respectively.Drought treatment was carried out for subcultured clones of pitaya seedlings.Samples were collected for construction of one degradome library.The raw data were submitted to SRA database of NCBI.Accession number was SRR8767413.Degradome data in combination with predicted miRNA binding sites were used for PARE by original PERL script.Significant cleavage events(P value ≤ 0.05)were discovered from 50 sites on 48 Unigenes directed by 32 miRNAs.About half of the target genes(45.83%)coded transcription factors,e.g.squamosa promoterbinding-like protein(SPL),transcription factor GAMYB(MYB),auxin response factor(ARF),NAC domain-containing protein(NAC),homeobox-leucine zipper protein(HD-ZIP).RLM-5-RACE was subsequently carried out for 15 of them and 11 were successfully validated.Investigation on targeting relation between miRNA and mRNA showed that most targeting relation were conserved in Plantae,e.g.miR156 was found targeting SPL in all reference plants,while some were conserved in only several plants,e.g.miR396 was found targeting 1-aminocyclopropane-1-carboxylate synthase(ACS)in six reference species.4.Differential expression of 30 miRNAs under drought treatment was profiledThe suitable reference genes,thioredoxin-like protein YLS8(YLS8)and 40 S ribosomal protein(RP40S),for RT-qPCR in pitaya under abiotic stress were screened out by NormFinder,geNorm,BestKeeper and validated by calibration of eight stress responsive genes.Whereafter,expression of 33 miRNAs whose targets were validated was profiled.In results,30 were significantly differentially expressed at at least single time point indicating a temporal involvement of pitaya miRNAs in drought response.5.Roles of pitaya isomiR in response to drought treatment were exploredFrom sRNA data,395 and 2,543 templated and non-templated pitaya isomiRs were identified and annotated by original PERL script.For terminal position,3’ addition or truncation was found in most templated isomiRs,while NO DRIFT type greatly increased the number of non-templated isomiRs.In the rest non-templated isomiRs,difference was also mostly found at 3’ terminus.For substitution,most of them were SS(single SNP)following by 3V(3’ terminal variation).For length,templated isomiRs didn’t show any preference while non-templated ones were mostly 21 nt,the same as miRNA.Comparison with cloned sRNAs,55,accounting for 40 templated and 15 nontemplated isomiRs,were found.Considerable poly uridine was observed at 3’ termini of pitaya non-templated isomiRs and upregulation was proved by RT-qPCR.To check whether isomiRs could slice mRNA as miRNAs do,criteria of functional isomiR were hypothesized.Targets of them were predicted by GSTAr and validated by PARE.It was evident that some isomiRs assisted miRNAs to slice mRNA at the same or nearby sites,while the others might target totally different targets because of variations at their 5’ termini which thus increased the targets of the miRNAs.Aforementioned results indicated that,isomiRs were not byproducts during miRNA maturation.It involved in drought response in pitaya and diversified the miRNA mediated gene regulation.6.Plant isomiRs databases were constructedA total of 677 sRNA-Seq datasets for 23 plant species growing under normal condition were obtained by retrieving public database.Large-scale identification and annotation of isomiRs were carried out.From 6,167 hairpins,98,734 plant isomiRs were found,among which most of them were from Oryza sativa(24,503),Arabidopsis thaliana(17,516),Glycine max(13,478),Medicago truncatula(7,584).Targets of identified isomiRs were predicted by TargetFinder and psRNATarget.Bioinformatics results in combination with computer languages,was used to provide the first plant isomiR database,Plant isomiR Atlas.It has been developed using the Structured Query Language(MySQL)as a backend database and uses Professional Hypertext Preprocessor(PHP)as a frontend.Plant isomiR Atlas is hosted on a 16 core Intel Xeon machine with Ubuntu as an operating system and allows for the rapid searches and easy to browse interface is equipped with several search options and is available at www.mcr.org.in/isomir.A total of 433 sRNA-Seq datasets for 16 plant species growing under 21 different abiotic stresses were retrieved from published papers.By different methods,16,157 and 2,028 differentially expressed isomiRs were identified,respectively,among which 1,991 were called by both.Target genes of differentially expressed isomiRs were predicted by psRNATarget.Bioinformatics results in combination with computer languages,was used to provide the first database for plant differentially expressed isomiRs under abiotic stresses,Diff isomiRs.It has been developed using the MySQL as a backend database and uses PHP as a frontend.Plant isomiR Atlas is hosted on a 16 core Intel Xeon machine with Ubuntu as an operating system and allows for the rapid searches and easy to browse interface is equipped with several search options and is available at www.mcr.org.in/diffisomirs.Conclusively,this work reported miRNAs/isomiRs in cactus species for the first time,and the mechanic model of miRNA/isomiR mediated drought response in pitaya was initially revealed in aspect of conservation,targeting relation and differential expression,etc.