Cloning And Characterization Of MiRNAs And Their Targets,Including A Novel MiRNA-regulated Md-NBS Gene And Controlled Apple Altemaria Leaf Spot In Apple (Golden Delicious)

As an important plant non-coding small RNA, microRNA plays an important role in various stages of plant growth process. In plants, the precursors (primary RNA) transcription by RNA polymerase Ⅱ (RNA polymerase Ⅱ), and rely on their own sequence features and the secondary structure of the stem-loop to produce mature microRNA. Then cut by the DCL1enzyme to form the21-24nt length of small RNA, which regulates a variety of plant physiological metabolism, resistance and grow of plant (Chen,2004, Zhu et al2008). The miRBase database showed4053miRNA sequences which from52species registered (miRBaese Release18), they belong to different microRNA family, play an important role in plant growth and development or stresses. MiRNA functional diversity and can be passed between the rootstock and scion features make it save the species reproduction quality in grafted fruit trees obviously. However, at present the apple miRNA rarely reported, especially in the apple between miRNA and disease research is still blank. The research of relationship between miRNA and disease resistance has a very important value of theory and application in fruit. In this study, Apple’Golden Delicious’as test material, the miRNA and their precursors were cloned by various techniques. The function analyze showed that Md-miRLn11targeted an NBS class protein gene and regulated leaf spot disease. The mainly results are as follows:1. The high-throughput sequencing and bioinformatics technology was combined and used to prediction miRNAs in apple’Golden Delicious’. And than compared with the apples genomic and the miRBase and PMRB (plant miRNA database) database. Through the above two methods and in accordance to the RNA secondary structural characteristics, size, free energy and so on,146possible miRNA precursors were found. These pre-miRNA sequences were located on apple genome and it shows a focused bundle phenomenon, the same family miRNA often apperes at a2-3M position in the genome.2. According to the apple genomic information specific primers designed and than146miRNA precursors the cloned. The results showed that clone basically consistent with the predicted sequence (individual nucleotide sequences were different with the genome sequence, we revised them according to the several sequencing results). Than the Stem-loop primer method was used to clone mature miRNA of the146miRNA precursors. The resuled showed a total of11new miRNA,22conserved miRNA, and8non-conserved miRNA. Speculated that miRNA which can not be cloned may express in other periods of growth and development of apple or under stress conditions.3. Small RNA was extracted from apple’Golden Delicious’ leaf, phloem, flower, fruit pulp, peel for the real-time PCR assay to analyze those41mature miRNA expression in those tissues. The results showed that these miRNA expression in those tissues very differently. Comprehensive analysis, there is smaller amount of miRNA expression in the leaves and flowers, and larger amount of miRNA expression in the fruit. This result indicated that more miRNA involved in the process of apple fruit development and little miRNA related to the process of leaf and flower development.4. According to the apple genomic information, the SOPE Software was used to compare146mature miRNA formed from pri-miRNA to the apple genom, than their target sites were predicted at located in the genome. We found that almost all miRNA target not noly one target sites, individual is not found miRNA target site in the genome. And according to the miRNA prediction software of http://srna-tools.cmp.uea.ac.uk/to pridict the known conserved miRNA function, according to the miRNA prediction software of http://linux1.softberry.com/berry.phtml to pridict the new miRNA function. In able to predict functions for new miRNA, the method of5’RACE was used to identified the miRNAcutting position in the target gene. Ultimately proved Md-miRLn3, Md-miRLn4, Md-miRLn11can cut their respectively corresponding target genes, this indicat that each predicted miRNA function can be applied in future experiments. In one of them, Md-miRLn11, targeted a NBS class resistance gene, may be associated with plant disease related.5. According to the result of Md-miRLn11can cut Md-NBS gene, the Md-miRLn11and Md-NBS gene expression were analysised in40anti-susceptible apple varieties. Real-time PCR results showed that the Md-miRLn11expression was significantly higher in susceptible varieties than that in resistant varieties, and Md-NBS gene expression was significantly higher in resistant varieties than in the susceptible varieties. Thus these two preliminary experiments prove Md-miRLn11can control Md-NBS target gene expression.6. Further analysis of the Md-miRLn11and its target gene Md-NBS found that, when infiltrated Md-miRLn11in resistant varieties apple’JiGuan’then inoculated leaf spot pathogen,96.5%of the plants showed different degrees of leaf spot disease phenotype. When infiltrated Md-NBS in susceptible apple ’Fuji’ then inoculated leaf spot pathogen,53.9%of the plants showed resistance to leaf spot disease. There is29.5%of the plants still showed disease phenotype. Experimental results show that the apple Md-miRLn11can regulate Md-NBS gene expression and changes plant resistance against leaf spot disease, overexpression Md-miRLn11reduce Md-NBS gene expression causes leaf spot resistance reduced; Overexpressing Md-NBS gene expression can enhance rsistance to leaf spot resistance in a certain extent.In summary,146microRNA precursors were cloned from apple (Golden Delicious) by RNA sequencing and computational analysis, and found11to be novel.. Among them, Md-miRLnll targeted the Md-NBS gene (an NBS-LRR protein), and decreases resistance to Alternaria leaf spot.