Cloning and bioinformatics analysis of tae-miR399 andTaUBC24
The precursor and mature sequences of tae-miR399 were obtained according to the miRNA database of Dn1 and the wheat miRNA data of miRbase (http://www.mirbase.org/). According to the precursor and mature sequence of tae-miR399, the chromosome position of tae-miR399 was obtained in wheat open genome library wheat URGI (https://wheat-urgi.versailles.inra.fr/). The base conservation analysis of mature miRNA sequences was analyzed with WebLOGO (http://weblogo.berkeley.edu/logo.cgi). The online software RNAfoldWebSever was used to predict the stem-loop structure of miR399 precursors. The 2 kb promoter sequence upstream of the genes were downloaded from wheat genome (https://urgi.versailles.inra.fr/download/iwgsc/IWGSC_RefSeq_Assemblies/v1.0/ ) and analysed with PlantCARE software (http://bioinformatics. psb.ugent.be/webtools/plantcare/html/ ) for the active elements in its promoter. The potential target genes of tae-miR399 were predicted by psRNAtarget (http://plantgrn.noble.org/psRNATarget/analysis). The complete gene and protein sequences of TaUBC24 (Gene ID:TRIAE_CS42_1DL_TGACv1_062114_AA0208970.2) were obtained from wheat open genome library wheat URGI (https://wheat-urgi.versailles.inra.fr/). The physicochemical properties of TaUBC24 protein sequence was predicted by using ProtParam (https://web.expasy.org/protparam/). Plant UBC24 protein sequences were obtained from UniProt (https://www.uniprot.org/). Multiple sequence alignments were analyzed using DNAMAN. A phylogenetic tree was generated using neighbor joining (NJ) method with a 1000 bootstrap value by software MEGA 6.0. Subcellular localization prediction was performed by the online tool CELLO v.2.5 (http://cello.life.nctu.edu.tw). To obtain the full-length precursor sequences of tae-miR399 and CDS of TaUBC24 , total RNA was isolated from tillering nodes under -25°C treatment. The full-length precursor sequences of tae-miR399 was amplified using primerspre-tae-miR399 -F: 5’-TCGTGTGTGAATCACAGGG-3’ and primerspre-tae-miR399 -R: 5’-GGGCAATTCTCCTTTGGCA-3’, and Taq DNA Polymerase (CWBIO, Beijing, China). And the full-length CDS ofTaUBC24 was amplified using primers TaUBC24 -F: 5’-GCTTGGAGAGATGGATCTGTTTG-3’ and primers TaUBC24 -R: 5’-GTCACTGGCCACAGGCGA-3’, and Taq DNA Polymerase (CWBIO).