Studies On MiRn51 And Target Genes And Establishing The Transient Expression System In Mulberry

Small RNA (miRNA, microRNA) are a group of endogenous single-strand non-coding small RNA containing 19-25 nucleotides, which regulated the expression level of target genes by the sequence complementary. Besides the negative regulation of target mRNA expression on degradation or translational inhibition, they play positive regulations by activation of miRNA. miRNAs are widely identified in animals, plants, microorganisms and viruses are involved in many biological processes of growth, develpoment, differentiation, cell apoptosis, and signal transduction.Mulberry (Morus L.) is a deciduous tree and always used as the forage for silkworm. It has an important economic and ecological value. With the completion of mulberry genome sequencing and establishment of small RNA library, we have a platform for the research of mulberry miRNA. At present, although the research about mulberry resistance, unique medicinal value and the relationship between silkworm have been reported, there has little information on the regulation ways between miRNA and its target genes.Based on M. notabilis small RNA library, we chose a novel miRNA (miRn51) as a candidate. We analyzed and predicted its precursor structure, upstream promoter sequence, and the target gene. We cloned the sequences of precursor and mature miRNA from M. notabilis genome DNA as well as its cDNA. Its precise site of cleavage of MnPP02 was verified by miRNA 5’-RACE. Two-month old mulberry seedlings were subjected to abiotic stresses (heat, drought, and high salt) and signal substance treatments (ABA, SA, H2O2, and MeJA). The expression profiles of miRn51 and its target genes before and after treatments were detected by qRT-PCR. Finally, we constructed an agrobacterium-mediated exogenous gene transient expression system in mulberry, which uesd to study the regulation relationships between miRn51 and its target gene. We detected the expression levels of target gene for the functional analysis of miRn51. The main findings of this study are as follows:1. Sequence and bioinformatics analysis of miRn51 from M. notabilis.Based on small RNA Illumina HiSeq-2000 sequencing for three M. notabilis tissues including leave, male flowers, and barks, we identified 85 conserved miRNAs and 262 tissue-specific miRNAs. According to their expression and annotation information of target genes, we obtained 10 miRNAs as candidates. Finally, we chose a mulberry miRNA (miRn51) discovered for the first time as object for further analysis. We analyzed its sequence, precursor secondary structure, promoter elements, and predicted target genes. The results showed that miRn51 has typical plant miRNA precursor secondary structural features. In addition to CAAT-box and TATA-box for transcription initiation, it also contains multiple stress response components such as drought (MBS), bacteria (TC-repeat), and necrosis (ARE). The predicted 8 potential target genes for miRn51 are belong to polyphenol oxidase gene families. The miRn51 regulated seven target mRNAs expression by degradation and the other target gene regulated by translational inhibition.The precursor sequence of miRn51 was cloned from the mulberry genome and the product is 87 nucleotides. Using stem-loop PCR, we obtained the mature miRn51 with the length of 71 nucleotides, suggesting the pre-miRn51 can be processed into mature miRn51.2. Verified the target gene of miRn51 and the expression profiles to various stress and signals treatments.QRT-PCR were carried out to confirm the expression levels of the miRn51 and seven target genes in five tissues of M. notabilis. The results showed that the expression pattern of miRn51 and target genes were quite different. For example, the morus012130 gene was expressed exclusively in male flowers, morus012131 and morus012128 genes were highly expressed in fruit and bark, respectively. Differences in the expression of target genes might reflect the functional differences and specialization of these genes. It is noteworthy that morus012121, morus012122 and morus012124 have little expression in root and bark. However, they are highly expressed in male flower, leave, and fruit. MiRn51 expression patterns were opposite compared with this three target genes in the same tissues, which is consistent with the regulation of mRNA and its target gene. We therefore predicted that the target genes for miRn51 would be morus012121, morus012122, and morus012124. The information analysis of morus012124 indicated that the gene encoded polyphenol oxidase (PPO2).The 5’-RACE assay indicated that the miRn51 was capable of cleavaging the morus012124 gene at the 10-11 and 7-8 sites with the cleavage ratio of 18/20 and 2/20, respectively. Two-month seedlings were subjected to high temperature, drought, cold, ABA, SA, H2O2 and MeJA treatments. We detected the expression levels of MnPPO2 and miRn51. The results showed that miRn51 and MnPPO2 gene had different expression patterns after stress treatments.3. Established an agrobacterium mediated transient expression system and expression analysis of miRn51 and MnPPO2.We constructed a recombinant plasmid PLGNL-35S-miRn51, which formed transient expression system mediated by agro-bacterium. We tested transfection effiicency under different injection buffer, different concentrations of bacteria, transformation time, and mulberry species using this transient expression system. The methods of GUS histochemical staining, polyphenol oxidase activity assay, and fluorescence quantitative PCR used to analysis transformation efficiency. The results indicated that the optimal mulberry transient expression system is transformed three days with the injection buffer 1 and concentrations of bacterial solution OD600=0.6. Based on these data, the relationship between miRn51 and MnPPO2 was disccused.