Identification Of Small RNAs In Salvia Miltiorrhiza Young Roots And Systematic Analysis Of Key Gene Families Involved In Small RNAs Pathways

Small RNAs play important and diverse roles in multiple developmental and physiological processes, antiviral defense, responding to biotic and abiotic stresse and so on. In recent years, the research of small RNA function and mechanism of action has become one of the hot spots in the life science. Salvia miltiorrhiza is a well-known traditional Chinese medicine (TCM) widely used for treating various human diseases, and it is also an emerging model plant for TCM studies because of its relatively small genome size, short life cycle and undemanding growth requirements. To our best knowledge, there is no report on the small RNA and mechanism in Salvia miltiorrhiza.In this study, the small RNA libraries and the degradome library were constructed from young root developing different stages. The miRNA, ta-siRNA and their targets were identified. Differential expression miRNA were analyzed. The three key gene families DCL, AGO and RDR involved in sRNA biogenesis pathway were cloned and function analyzed. Finally, RNAi vector were constructed to study the functions of DCL1and DCL2. The main conclusions obtained from this study are as follows:1. Five small RNA libraries were constructed from S. miltiorrhiza young roots at different developmental stages. We obtained22096287,24477247,22789461,23216990and21846663clean reads for each library. We identified176conserved miRNA belonging to39miRNA families and552novel miRNA. In addition,26miRNA mediated70TAS loci were found to generate ta-siRNAs.2. Based on the transcriptome data,196targets for conserved miRNA were predicted, with degradome library data,8targets of known miRNA have the right cleavage sites. The differential expression abundance of miRNAs in data sets was analyzed by counting the number of transcripts per million (TPM) clean tags in libraries. The results showed that miR156, miR164. miR166, miR167, miR168, miR169, miR172, miR390, miR396, miR408, miR4414, miR479, miR160, miR2118. miR1446and miR5225may be involved in young root development.3. DCL were the core components for small RNA biogenesis. We analyzed the SmDCL gene family in Salvia miltiorrhiza, using a comprehensive approach integrating genome-wide prediction, molecular cloning, gene expression profiling, and posttranscriptional regulation analysis. A total of five SmDCLs were identified.The absence of miR162in S. miltiorrhiza and the loss of miR162target site in SmDCL1were unexpectedly found. Further analysis showed that the miR162target site was not present in DCLl from ancient plants and was gained during plant evolution. Our results provide evidence for the gain and loss of miR162and its target sites in Dicer-like genes during evolution.4. AGO were the core components of RISC. In genome-wide, we identified ten SmAGO genes in S.miltiorrhiza. Full-length cDNAs of ten SmAGOs were subsequently cloned and sequenced. The results made S. miltiorrhiza become the first plant species with a full-set of experimentally validated full-length AGO cDNAs available. The newly identified SmAGOs were characterized using a comprehensive approach. Sequence features, gene structures, conserved domains and Phylogenetic relationships were analyzed.Using the modified5’-RACE method, we confirmed that SmAGO1and SmAGO2were targeted by S. miltiorrhiza miR168and miR403, respectively.5. RDR were the key components of siRNA biogenesis pathways. Through genome-wide predication and subsequent molecular cloning, five full-length S. miltiorrhiza RDR genes were identified. SmRDRs were responsive to methyl jasmonate treatment and cucumber mosaic virus infection. It provides a foundation for further studying the regulation and biological functions of SmRDRs and the biogenesis pathways of small RNAs in S. miltiorrhiza.6. The RNAi vector of DCL1and DCL2were constructed. The procedure of genetic transformation of Salvia miltiorrhiza was developed. The Optimization of genetic transformation was that the leaf fragments were infected by1m L OD600=0.5diluted20times Agrobacterium, the proper infection time was2minutes, the coculture time is60hours, the hygromycinB screening concentration is10mg/L.