Identification Of Fruit Ripening-related MiRNAs And Their Target Genes In Blueberry And Genetic Transformation Of VcMIR156

Blueberry is an important small berry fruit crop which has a good effect on our health,with economical and nutritional value.Selecting the fine blueberry species and improving the quality of blueberry fruit are the most vital goals of blueberry breeding work,but the molecular mechanisms that control fruit development are little known in blueberry.MicroRNAs(miRNAs),a class of endogenous,20–24 nucleotides(nt)in length,single-stranded,non-coding RNAs,play vital roles in the regulation of fruit development and ripening.However,nothing is known about the miRNAs and their targets involved in blueberry fruit development and ripening.In this study,using high-throughput sequencing of small RNA,many miRNAs and their targets were identified in blueberry fruit.Integrative analysis of miRNAs and their targets revealed a complex miRNA-mRNA regulatory network involving a wide variety of biological processes.The findings will facilitate future investigations of the miRNA-mediated mechanisms that regulate fruit development and ripening in blueberry.In order to explore the function of miRNA in blueberry systemtically,VcMIR156 was cloned.To investigate the function of VcMIR156,overepression vector of VcMIR156 were constructed and then transformed into blueberry.Consequently,we obtain 5 positive transformants of VcMIR156.The main results are as follows.1.The libraries for miRNAome and degradome profiling were made from fruits at white and blue fruit stages,which represent fruit ripening onset and in progress,respectively.Among them,41 miRNAs were shown to be differentially expressed during fruit maturation,and 16 mi RNAs representing 16 families were further chosen to validate the sRNA sequencing data by stem-loop qRT-PCR.Meanwhile,178 targets were identified for 41 known and 7 novel miRNAs in WF and BF libraries using degradome sequencing,and targets of miR160 were validated using RLM-RACE(RNA Ligase-Mediated(RLM)-Rapid Amplification of cDNA Ends)approach?2.To systematically exploit the evolutionary conservation of the known miRNAs in plant species,a phylogeny tree was constructed using the sequences of mature miRNAs and pre-mi RNAs in blueberry and grape,all the members of the 9miRNA families were evolutionarily closer to each other.3.To provide clues for their roles during fruit maturation,the abundance of the miRNAs in the two libraries were analyzed,100 miRNAs were categorized into three groups based on their accumulation patterns.There were 68 miRNAs with up-regulated accumulation as fruit ripens,suggesting that they might play a role in fruit development and maturation4.The expression patterns of the target genes corresponding to the 6 miRNAs were also investigated by qRT-PCR at the same developmental stages as above.The apparently negative correlation between the miRNAs and their target genes indicates that the miRNAs exert considerable influence on the expression of their target genes.5.GO(Gene Ontology)functional analysis suggested that the targets were shown to be enriched biological processes,cellular components and molecular functions6.VcMIR156 was cloned and validated by using Northern blot and complementation test in Arabidopsis.The VcMIR156 gene was transformed into blueberry by using the Agrobacterium-mediate transformation method.Consequently we obtain 5 positive lines of VcMIR156.Moreover,polymerase chain reaction(PCR)and real-time quantitative PCR(qRT-PCR)of pre-miRNA and 6 SPLs all confirmed that the purpose gene had been successfully transferred into blueberry plants and expressed.These research results laid a foundaton for studying the functions of miRNAs in blueberry and improving cultivars by farther utilizing molecular genetics.