Bioinformatics Prediction And Primary Functional Analysis Of MicroRNAs And Their Target Genes In Banana

MicroRNAs (miRNA) are a class of single-strand molecules RNA which is about19-25nt, non-protein-coding and endogenous founded in eukaryotes and some of DNA viruses, which have been reported to regulate gene expression negatively at the post-transcriptional levels by targeting mRNAs for degradation or inhibit protein translation. More and more studies showed that miRNA participate in the process of a series of important plant life processes, including self-regulation of growth and development; the differentiation regulation of cell, tissue; gene expression, etc. In addition, they played important roles in the response to environmental stress such as drought, salinity, barren and other pathogens.However, up to now there is no record of banana miRNA in the miRNA database. According to highly conserved miRNA and the secondary structure of precursors in plant, potential miRNA and its target genes in banana EST and GSS database were predicted by bioinformatic methods. And the study in-depth analysis the sequence features of miRNA, miRNA family diversity and its target gene function and so on.The difference expression of miRNA and their target mRNAs between root, leaf, flower and fruit of banana was studied by the RT-PCR and real-time RT-PCR method.Using the method described above, we could lay the foundation for the study of the banana microRNAs,explore the regulation mechanism of banana,s growth and development and provide theoretical basis for stress resistance. The main results are as follows:1. Some experiments were proceeded using the plant miRNA database available. Potential miRNA in banana EST and GSS database were predicted using bioinformatic approaches.33potential miRNA belonged to16miRNA families from18different precursors were searched through a very rigorous process of selection.The sequence analysis showed that the33potential miRNA pre-miRNA sequences varied from77-176bp in length; mature sequence length extents20to22bp.91possible target genes of25banana miRNA which encode proteins involved in transcription regulation, developmental processes, metabolism and stress response,were predicted by psRNATarget.2. Analysis and study on the species distribution of16miRNA families,14dicotyledoneae and5monocotyledonous were downloaded from the miRBase database. The results showed that11conserved miRNA belonged to16miRNA families and those conserved miRNA families have founded in dicotyledons and monocotyledons. MiR4995and miR5538considered as low degree conserved miRNA family. The study attempted to establish the phylogenetic tree of the typital miR156family, to study the molecular evolution of miRNA.3. On the base of extracting high quality small RNAs(less thanl50bases) with the modified CTAB method,14miRNA were checked in the young leaves of banana by the RT-PCR method. The results showed that, the amplified fragment of75bp contains the primer sequence and the complete sequence of the other detected miRNA, in addition to miR160a, miR319m and miR5538.4. Using total RNA extracted by the CTAB method as templates, the expression of12miRNA and5s rRNA in roots, leaves, flowers and fruits of banana was studied by real-time RT-PCR through stem-loop RT primers.The results showed that,8conserved miRNA belonged to12kinds of miRNA were detected75bp amplification products in different tissues of banana. In addition, significant tissue specificity was observed in other miRNA.Expression of miR166cb in the leaves, flowers and roots, but not expressed in fruit. miR319m was only detected in banana fruit, but not detected in other tissues. miR160was not detected the amplified products in the4different banana tissue.5. To verify the predicted miRNA and their target mRNAs,specific stem-loop RT primers were designed and roots, leaves, flowers and fruits of banana total RNA as template were used to profile the expression levels of target mRNAs(SPL,SRPK4,WRKY,GAMYB,DFR and F-box). The results showed that, each potential target mRNA exhibited a specific expression pattern in different tissues.4miRNA and their target mRNAs had effective expression levels in4tissues of banana, amplified bands varied from100-400bp in length. While GAMYB and DFR presented a different expression pattern.6. Through quantitative real-time RT-PCR expression, with5s rRNA gene as a reference gene for normalization, we have detacted the difference expression levels of9miRNA in young leaves of bananas.Data analysis shows that:When using the same amount of cDNA, the expression of housekeeping gene5s rRNA was0, the expression of miRNA respectively show miR156ga(0.973), miR156d(1.065), miR162(0.465),miR164e(0.868),miR166cb(0.255), miR167c(0.502), miR169h(0.611), miR399a(0.824) and miR4995(0.890). This shows these miRNA have a significant difference expression level in leaf.7. The difference expression of7miRNA between root, leaf, flower and fruit of bananas was studied by real-time RT-PCR.When the relative quantitation of7miRNA in bananas leaf was as l,the result showed that the highest expression in flower was miR167c, reaching11.290, obviously beyond the quantitation in root(0.300), leaf (1.00) and fruit (3.184). The highest expression of miR156d in flower (2.253), leaf (1.00) took the second place, fruit (0.336) for the3rd and a minimum of0.165for root.miR162expression in flower in an amount of (4.363), followed by root tissue (1.718), the leaf (1.00) and fruit (0.998) is almost the same as the expression; The expression of miR399a(2.684) was the highest in flowers, leaves (1.00) and roots (0.302) was more higher than fruit (0.067). Difference expression of miR169h, miR4995and miR164e was not significantly in4tissues.8. The difference expression of6target mRNA between root, leaf, flower and fruit of bananas was studied by real-time RT-PCR. When the relative quantitation of6target mRNAs in bananas leaf was as1, the result showed that each potential target mRNA exhibited a specific expression pattern in different tissues. The highest expression of WRKY in fruit(2.352), the lowest expression of DFR in flower, only0.015.The comparative expression level of each target mRNA was coherent with that been reported in miRNA, such as miR167c(11.290), which indicated that the relevant miRNA might have inverse proportion with target mRNA.The results showed that the targets SPL, SRPK4and DFR were also successfully amplified by real-time RT-PCR. Each potential target mRNA expression trend compared their miRNA have significant correlation.