Identification Of MiRNA Target Genes In Tea (Camellia Sinensis)and Their Expression Patterns Under Cold Stress

Low temperature is a common stress of tea plant that have seriously impact on high yield and high quality tea cultivation.MicroRNAs?miRNAs?are a kind of small noncoding RNAs that not only regulate plant growth and development,but also response to stress by targets genes in a post-transcriptional way.Therefore,studying the mechanism of miRNA and its target genes in response to cold stress play an important role in breeding cold-resistance tea varieties.In this study,we use degradome sequencing technology to identify the targets of miRNAs in tea?Camellia sinensis?.Among them,8 targets were validated by the RLM-5'RACE procedure.We studied the expression of some cold responding mi RNAs by quantitative real-time polymerase chain reaction?qRT-PCR?.By combining degradome data and the differentially expressed miRNAs,we analysed the expression profile of some miRNAs and their target genes.The main results were as follows:?1?we constructed two degradome libraries with and without 4? treatment.316 targets of 36 known miRNA families and 246 targets of 113 novel miRNAs were identified from the two libraries.GO analysis indicated that the mi RNA targets participate with many cellular and metabolism process.The cleavage sites of 8 targets corresponding to 7 miRNAs were verified by RLM-5'RACE procedure.The results were in line with the degradome sequencing data,showing the accuracy of degradome sequencing as a technology for miRNA target identification.?2?By screening the previous miRNA microarray data from our research group,we obtained 27 cold responding miRNAs,among them,9 were novel miRNAs.6 differentially expressed mi RNA were further analyzed by qRT-PCR.The results of qRT-PCR and microarray shared similar trends.85 targets of 13 miRNAs were identified by degrandome sequencing,most of them were transcription factors,some of them were enzymes and functional proteins.However,the other 14 mi RNAs were faied to find targets.?3?We studied the expression pattern of 5 differentially miRNA and its targets?miR156-SPL?miR164-NAC1?miR165-REV?miR396-GRF?miR6149-APX?under cold stress.qRT-PCR analysis revealed that miRNAs were down-regulation and its targets were up-regulation.We also used qRT-PCR to analyze the expression profile of 2 miR164 family members and their target gene NAC1 on two tea cultivars?LongJin 43 and YingHong 9?with opposite cold resistant.The expression of vvi-miR164 a showed no difference between the two cultivars,but vvi-miR164a₁ss21AG showed different expression.NAC1 up expression more in LongJin43 than in YingHong 9,indicating that NAC1 may account for the difference cold tolerance between tea cultivars.