Cloning and tissue expression characteristics analysis of LADD 2 gene in Esox lucius

【Objective】 To clone LADD 2 gene of Esox lucius and analyze its tissue expression characteristics,which could provide theoretical basis for revealing the role of LADD 2 gene in the gonadal development of E.lucius.【Method】 Using the gonadal transcriptome data of E.lucius,the highly expressed gene LADD 2 in 126-day-old female E.lucius was screened.The coding region （CDS） sequence of LADD 2 gene was cloned,and bioinformatics analysis was performed using online tools such as DNAMAN 9.0,NCBI ORF finder,BLAST,ClustalX,ExPASy,ProtParam,PSORT II,SOMPA and SWISS-MODEL.Based on the amino acid sequences of fish ladder-type lectin homologs and C-type lectin re‐ceptors,a phylogenetic tree was constructed using the neighbor-joining （NJ） method in MEGA 5.1.The relative expres‐sion levels of LADD 2 gene in the gonads,intestines,muscles,liver,gills,brain,kidneys and head kidneys of E.lucius at 126,188,and 320 day-old were detected using real-time fluorescence quantitative PCR. 【Result】 The CDS sequence of LADD 2 gene in E.lucius was successfully cloned,with a total length of 609 bp,encoding 85 amino acids residues.The protein molecular weight was 9.78 kD,the theoretical isoelectric point （pI） was 7.30,and the molecular formula was C₈₆₃₅H₁₃₅₀₄N₂₃₃₄O₂₄₀₄S₁₁₅,indicating a hydrophilic protein.The LADD 2 protein had the highest content of leucine（11.86%）,and the lowest content of tryptophan （2.28%）.Phosphorylation site prediction showed that LADD 2 protein contained 147 serine sites,98 threonine sites and 44 tyrosine sites.Protein structure prediction revealed that LADD 2 pro‐tein mainly consisted of random coils β-turns,α-helices and β-folds,with 2 conserved PRT1 domains.Amino acid se‐quence homology analysis showed that the similarity between the LADD 2 protein of E.lucius and that of Dallia pectora‐lis was the highest at 99%.The phylogenetic tree indicated that LADD 2 protein of E.lucius clustered with fish laddertype lectin homologs and was closely related to Salmo salar,Oncorhynchus kisutch and Coregonus clupeaformis.Realtime fluorescence quantitative PCR results showed that the relative expression of LADD 2 gene in the ovaries of E.lucius gradually decreased with developmental time,and the difference in relative expression between the ovaries and testes be‐came smaller.Additionally,LADD 2 gene was expressed in the gills and brain.【Conclusion】 The function of LADD 2gene exhibits certain conservation and stability during evolution.LADD 2 protein may play an important role in the early ovarian development of E.lucius and may have immune activity in gills and brain during later developmental stages.LADD 2 protein may possess immune functions,thereby protecting the hepatopancreatic development of E.lucius.