Reference Gene Selection For Quantitative Real-time PCR Of MiRNA In Thellungiella Salsuginea And Preliminary Study On Tsa-miR319

Salt cress(Thellungiella salsuginea),a halophyte related to Arabidopsis thaliana,which has been developed as an important model plant for stress research because of its strong tolerance to abiotic stresses.The whole genome sequencing of salt cress has been completed,providing more reliable evidence.MicroRNAs(miRNAs)are a class of 20?24 nt in length,single-strand,endogenously initiated non-coding RNAs,which exist in eukaryotes extensively.Plant miRNAs play important roles in basic biological process such as plant growth,development,metabolism and immune response,they also response to various abiotic stresses.Quantitative real-time PCR(qRT-PCR)is an important technique to detect the expression pattern of plant miRNAs.Reference genes decrease the discrepancies caused by experimental samples,efficiency of reactions,and operations to make data normalized.In this study,we screened the best reference gene for miRNA expression normalization in salt cress,and analyzed the preliminary function of tsa-miR319.(1)We chose five housekeeping genes used in T.salsuginea,and fourteen tsa-miRNAs with uniform stability known from our Solexa sequencing data as candidate reference genes.We analyzed the expression stability of these genes in different tissues or under various stress conditions.After analysing by geNorm,tsa-miR162a and tsa-miR166a were the most stable genes for all experimental treatments;U6 snRNA,5S rRNA,tsa-miR162a and tsa-miR158 were the most stable genes in tissues of different developmental stages,while tsa-miR162a and U6 snRNA were the most stable genes under four abiotic stresses.NormFinder analysis showed that tsa-miR162a was the best reference gene for normalization of miRNA expression by qRT-PCR in T.salsuginea.(2)MEME analysis showed that miR319 in various plants was highly conserved.The precursors of tsa-miR319a,tsa-miR319b,tsa-miR319c obtained using bioinformatics analysis could be fold into stem-loop structures by Mfold software,with their mature sequences positioning on one arm.Then these three miRNA genes were successfully cloned.The expression of tsa-miR319a/b and tsa-miR319c changed in T.salsuginea seedlings under salt(300 mmol·L-1 NaCl)and cold(4?)treatments using qRT-PCR.The expression of tsa-miR319a/b was down-regulated after salt treatment,while tsa-miR319c changed in volatility.The expression of tsa-miR319a/b changed distinctly after cold treatment,while tsa-miR319c was stably expressed except strongly down-regulated at 5 hours of cold treatment.Moreover,both tsa-miR319a/b and tsa-miR319c expressed diversely in various tissues of different developmental stages,had sequential expression and tissue specificity.Thirty-five target genes of tsa-miR319 were predicted by psRNATarget.Annotated the target genes,we inferred that majorities encoded transcription factors and protein kinase,which might be involved in regulation of gene expression and energy metabolism.The over-expression vector of pre-tsa-miR319c was successfully constructed,and transformed into wild-type T.salsuginea using the floral dip method.As a result,31 transgenic seedlings with hygromycin resistance of T1 were screened out.