Study On Differential Expression Of Mirnas In Response To Ectropis Obliqua Feeding Stress In Tea Plant (Camellia Sinensis)

Tea plant(Camellia sinensis)is one of the most important economic plants in the world,and also is one of the most popular nonalcoholic beverages in the world.Tea Ectropis obliqua(E.oblique),a major pest of tea plant,cause great damage to growth and development of tea plant.MicroRNAs(miRNAs)are a class of 17~25 nucleotides,endogenous,small noncoding RNAs that play important roles in responses to biotic and abiotic stresses.Understanding expression patterns of miRNA in response to E.oblique feeding stress is necessary to gain insight into the mi RNA-dependent post-transcriptional gene regulation in responses to insect herbivory?In this study,we used high-throughput sequencing combined with microarray hybridization to identify differentially expressed miRNAs in tea plant in response to E.oblique feeding stress and mechanic wound stress,respectively.Degradome sequencing was used to analyse the target of the differentially expressed miRNAs,then RLM-5'RACE was used to validate the result of degradome sequencing.The results are as follows:(1)Three groups of samples were used to construct small RNA(smRNA)libraries for high-throughput sequencing: tea plant with insect feeding treatments as group STL,with mechanical damage treatment as group SML and without treatment as group SCKL,After removing the low-quality reads,adapter,and redundant sequences,about 2363062 ?2207927 and 3775038 unique sequences were obtained in group STL?SML and SCKL libraries for searching tea miRNAs,respectively(2)In group STL ? SML and SCKL,101 ? 106 and 112 conserved miRNA were identified,and 462 ? 406 and 478 species-specific tea miRNAs were also detected.Compared with control sample,56 miRNAs were differentially expressed under E.oblique feeding stress,of which,32 miRNAs were up regulated and 24 were down regulated;50miRNAs were differentially expressed under mechanic wound stress of which,28 miRNAs were up regulated and 22 were down regulated.While the result of microarray indicated that compared with control sample,37 miRNAs were differentially expressed under E.oblique feeding stress,of which,13 miRNAs were up regulated and 24 were downregulated;38 miRNAs were differentially expressed under mechanic wound stress of which,10 miRNAs were up regulated and 28 miRNAs were down regulated.(3)4 up regulated miRNAs and 5 down regulated miRNAs out of the differentially expressed miRNAs under E.oblique feeding stress identified by high-throughput sequencing,showed consistent expression profile compared with the result of microarray,the 9 miRNAs are most likely to be responsive to E.oblique feeding;4 up regulated miRNAs and 5 down regulated miRNAs out of the differentially expressed miRNAs under mechanic wound stress identified by high-throughput sequencing,showed consistent expression profile compared with the result of microarray,the 9 miRNAs are most likely to be responsive to mechanic wound.After we deducted 5 mi RNAs that have consist expression profile under E.oblique feeding stress and under mechanic wound stress,4differentially expressed miRNAs are likely to be influnced by oral secretions of E.oblique.(4)Based on degradome sequencing of C.sinensis done by our research team before,6 of the 9 different expressed mi RNAs in response to E.oblique feeding were indentified to target TCP family transcript factors?ARF response factors and SCL protein,etc.The RLM-5?RACE experiment was successfully used to map the cleavage sites in 3target genes of C.sinensis.