Molecular cloning and bioinformatic analysis of TRIM9-1 gene

The total RNA was extracted by TRIzol reagent (Takara, Tokyo, Japan), and the cDNA template was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocols. Two specific primers LvTRIM9-1-1F and LvTRIM9-1-1R (Table S1) were designed to amplify and validate the sequence of LvTRIM9-1 from the genome and transcriptome data (31 (link)). PrimeStar GXL DNA Polymerase (Takara, Japan) was used to amplify the gene. After the quality was assessed by electrophoresis on 1% agarose gel, the specific product was purified using Gel Extraction Kit (Omega, Norcross, GA, USA), cloned into the pMD19-T vector (TaKaRa, Japan), and transformed into DH5α competent cells (TransGen, China) for sequencing.
The complete ORF and amino acid sequence of LvTRIM9-1 was deduced using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). Conserved protein domains were predicted with SMART (http://smart.embl-heidelberg.de/). Different TRIM protein sequences (Table S2) were obtained from the UniProtKB/Swiss-prot and NCBI database. Multiple-sequence alignment and phylogenic analysis were performed using the neighbor-joining (NJ) method by ClustalW and MEGA 6.