The Research On MiRNA And Bioinformatics Analysis In Carica Papaya,Eucalyptus Grandis And Other Two Plant Species

MiRNAs are endogenous19-24nt small non-coding RNAs which can regulate their complementary target gene at the posttranscriptional levels. Recently, miRNAs are thought to play an essential role in plant growth, development and stress response. The purpose of this study is to identify the miRNA which are related to plant genetic quality. Deep sequencing analysis was carried out to identify the miRNA regulated in leaves,stem and roots of Carica papaya and plant infected by PRSV. At the same time, The new standard of miRNA bioinformatical identification was put forward after characteristics of all plant miRNA sequences were analyzed in miRBase database. The genome sequence were applied to bioinformatical identification of miRNA in Carica papaya, Eucalyptus grandis, Selaginella moellendorfii, Mimulus gutatus. MiRNA have been predicted in5species of Eucalyptus using EST in Cajanus cajan using EST and GSS by miRtour,78conserved miRNA precursor sequences and64conserved mature miRNA sequences were identified using high-throughput sequencing technology in Carica papaya. Comparative sequence analyses of mature miRNA sequences showed that uracil is the dominant base in the first position at the5'end, whereas cytosine is dominant at the19th position. Nucleotide bias has been found in miRNA flanking sequence. Purine is dominant base and the frequency of cytosine is very low compared with other bases in miRNA upstream sequences, whereas pyrimidine is dominant base in miRNA downstream sequences. The frequency of adenine is up to55.1%at the19th base from5'end, but its frequency is56.4%at the3rd from3'end in papaya miRNA*sequences.38high confidence miRNAs were found in Carica papaya. Cpa-miR169c, cpa-miR169d, cpa-miR408and cpa-miR482were expressed only in leaf of papaya.Cpa-miR164g and cpa-miR171h were expressed only in the root, but their expression level is relatively low. cpa-miR164g, cpa-miR167e, cpa-miR169c, cpa-miR169d, cpa-miR171h, cpa-miR172d, cpa-miR319c, cpa-miR390d, cpa-miR408, cpa-miR482were induced by virus infection in papaya.It was showed that31targets of miRNAs encoded transcription factors,3targets encoded proteins involved in translation,71encoded enzyme. In addition, there are7targets of miRNAs encoded disease resistance protein, the mainly regulated by cpa-miR171e, cpa-miR172c, cpa-miR396b and cpa-miR482.31specific miRNA gene was identified in papaya in which RTFL5was regulated by Cpa-miR-novel-7. But RTFL5could led to plant dwarf phenomenon, leaf spots and other symptoms infected by PRSV. The new rule of plant miRNA bioinformatics prediction was put forward through the analysis of the sequences and high confidence miRNA sequences in the miRBase database. The length of mature miRNA should be between19-24nt, whereas their base types should be4, their content of GC should be between30-70%. Average minimum free energy of single base should be less than-0.2kal/mol/nt. the content of GC is30-70%, the minimum free energy index should be greater than or equal to0.7in miRNA precursor sequences. The coding sequence should be removed by blast against the sequences in the PFAM database, and should not rely solely on the MFEI to distinguish with miRNA precursor sequences. The new standard is in accordance with78.9%miRNA sequences and91.1%high confidence miRNA sequences whereas66.9%miRNA sequences and76.4%high confidence miRNA sequences conform to the usual old standard.93.6%conserved papaya miRNA confirmed in experiments met the standards proposed in this study, whereas only78.2%conform to the old common standards. The new rule proposed in this study is obviously better than the old standard.594mature miRNAs were found using bioinformatics method in Carica papaya, which belong to99different miRNA familes.258miRNAs were predicted in whole genome sequences of Selaginella moellendorfii, which belong to78different miRNA familes.594miRNAs were identified in whole genome sequences of Mimulus gutatus, which belong to85different miRNA familes.1262miRNAs were found based on genome of Eucalyptus grandis, which belong to195different miRNA families. Nucleotide bias has been found in miRNA mature sequences and miRNA flanking sequences. Uracil and cytosine are the dominant bases in the first position and the19th position at the5'end respectively in every species of this paper. More importantly, C is rare base in miRNA upstream flanking sequences, whereas G is rare base in miRNA downstream sequences.Cajanus cajan microRNA has been predicted based on EST and GSS Sequences by miRtour, whereas miRNA in Eucalyptus has been predicted based on EST Sequences using the same method.26miRNA precursor sequences and19unique mature miRNAs were found in Eucalyptus. Nucleotide bias has been found in miRNA sequences that uracil and cytosine are the dominant bases in the first position and the19th position at the5'end respectively. Futhermore,3miRNAs were considered to regulate gene about wood formation.43unique mature miRNA sequences were found belonging to33different miRNA familes in Cajanus cajan.36coding RNAs were regulated by miRNA in which17targets were enzymes,2targets were transcription factors,7targets were protein involved in signaling transduction,5targets were protein participated in protein degradation pathway.