Profiling Of Chilling-stress-responsive MiRNAs And Preliminary Study On Tsa-miR396in Thellungiella Salsuginea

MicroRNAs (miRNAs) are a class of endogenous, single-strand, non-coding RNAs with about20-24nt in length. MiRNAs are widespread regulators of gene expression in animals, plants and microorganisms. Plant miRNAs play an important regulatory role in plant growth, development, hormone regulation and immune response, and response to a variety of biotic and abiotic stresses (such as nutrient stress, drought stress, salt stress, cold stress, etc.).Salt cress(Thellungiella salsuginea) is a close-relative of Arabidopsis thaliana, which have been developed as a new salt tolerance model plant. Salt cress lacked endogenous ice nucleation, indicating a potential for supercooling. Freezing tolerance increased from-13to-18.5℃after cold acclimation in salt cress. In this study, we analyzed the profile of miRNAs in salt cress after chilling treatment, and preliminary function of tsa-miR396.(1) We used the real-time quantitative PCR method to analyze the expression patterns of members of the9miRNA families (tsa-MIR160, tsa-MIR167, tsa-MIR169, tsa-MIR171, tsa-MIR172, tsa-MIR319, tsa-MIR396, tsa-MIR403and tsa-MIR408) in salt cress after exposure to4℃for0,2,5,8,12and24hours. Our results show that the expression levels of tsa-miR160abc, tsa-miR171a and tsa-miR171bcde first decreased at5hours and then increased at8hours and decreased again at12or24hours after4℃treated. The expression levels of tsa-miR167abc, tsa-miR169a, tsa-miR169bc, tsa-miR169deg, tsa-miR172abc, tsa-miR319a, tsa-miR319b and tsa-miR408decreased after4℃treated. The expression levels of tsa-miR169f increased at8,12,24hours after4℃treated. The expression levels of tsa-miR396and tsa-miR403first increased at2,5hours respectively and then decreased at8hours.(2) The study of miRNA function depends on the identification of its target genes. We predicted target genes of tsa-miR396which responded to chilling stress rapidly by psRNATarget. As a result,25target genes were predicted. Then we identified Thhalv10024915m as a target gene of tsa-miR396by5’RACE (rapid amplification of cDNA ends). Thhalv10024915m was found to be cleaved at the8-9-nt position and the10-11-nt position. The target site was identified in the coding region of Thhalv10024915m.(3) We successfully cloned pre-tsa-miR396, and constructed over-expression vector of pre-tsa-miR396. This work laid a foundation for in-depth study of the regulatory mechanisms of chilling-stress-responsive miRNAs in salt cress.