Identification And Isolation Of MiRNA In Tea(Camellia Sinensis)and Their Expression Characteristics Study On Response To Ectropis Obliqua Feeding Stress

MicroRNAs are a class of endogenous,non-coding single-stranded small RNAs that regulate numerous genes by guiding target mRNA cleavage in plant, and miRNAs play an important role in various aspects of plant growth and development process and environment stress responses. So it is becoming concerned focus about the regulation role of miRNA.. However, little attetion has been focused on the function of miRNA in plant defense mechanisms against pests, and a few tea miRNA sequences have been reported. So the paper performed the small RNA deep sequencing, miRNA microarray, qRT-PCR method etc and combined with bioinformatics analysis, isolated and identified miRNAs in tea(Camellia sinensis) by using these technologies, on this basis we found differently expressed miRNAs in tea plants after E. oblique attack to further confirm their characteristics and functions. The main results of paper were as follows:(1) Solexa high-throughput sequencing enabled the exploration of small RNA populations in tea plant,8,796,700effective sequences were obtained, accounting for81.30%of all reads,27%of all miRNA sequences were more than100times by sequences analyzing. We used qRT-PCR approach to detect the miRNAs of different expressed levels, the results were consistent with the high throughput sequencing results.(2) In all obtained miRNA sequences,253were coserved miRNA belonging to30miRNA families and574tea-specific sequences. The number of16conserved miRNA family members were hiher than other plant species (Arabidopsis etc). Bioinformatics analysis showed target genes of many tea miRNAs involved in plant growth and development, secondary metabolism, stress response process and signal transduction etc.(3) Microarray validated the sequencing results and obtained169miRNA sequences, included130conserved miRNAs and39tea-specific miRNAs. Further analysis revealed that35miRNAs were significantly with higher abundance in leaf, while60miRNAs were higher in root.(4) Based on miRNA microarray screening demonstrated a total of29different expressed miRNAs in tea plants after E. oblique attack, included27up-regulated expressed miRNAs and2down-regulated expressed miRNAs. In the different expressed miRNAs, miR159, miR319, miR396associated tagert genes had a role of regulating defense response, GA, ABA etc hormone synthesis and metabolism, and so on.(5) In significant different expressed miRNAs in tea plants after E.oblique attack, we random selected seven miRNAs to verify their expression patterns by using qRT-PCR approach. The results showed that their expressed levels all lower than the respective control after E.oblique attack and E.oblique oral secretion treatments. While five of seven miRNAs’ expressed levels also obviously lower than their respective control after MeJA treatment.