Abstract: The aim of this study was to obtain the coding sequence (CDS) of Occludin gene in Luchuan pig and carry out bioinformatics analysis of the sequence.A pair of specific primers were designed according to porcine Occludin gene sequence (accession No.:NM_001163647.2) published on GenBank,the jejunum tissue samples of Luchuan pig were collected,and RNA was extracted and then was reverse transcribed to synthesize cDNA.The target fragment was obtained by RT-PCR amplification,PCR products then were inserted into the pMD18-T vector,and then screened positive clones was sequenced.The obtained sequence was analyzed by bioinformatics software after sequencing.The results showed that the size of Occludin gene CDS in Luchuan pig was 1 569 bp,encoding 522 amino acids.The results of sequence alignment showed that the homology was 99.7% with reference sequence,and there were 3 different sites,the C→T was a missense mutation in the 16th position,which caused the leucine turns to phenylalanine at the 6th position,and the A→G at 1 059 bp position and the C→T at 1 218 bp position were synonymous mutations.This missense mutation site might be the reason of the intestinal barrier function in Luchuan pig was different from other pig breeds.The homology of Occludin gene in Luchuan pig were 83.8%,89.5%,88.1%,84.1%,88.3% and 86.6% with Mus musculus,Bos taurus,Homo sapiens,Mesocricetus auratus,Macaca nemestrina and Canis lupus,respectively.Phylogenetic tree analysis showed that the genetic distance between Luchuan pig and Bos taurus was the closest,and the genetic distance was the furthest with Canis lupus.The molecular weight of Occludin protein was 59.13 ku,the amino acid composition of the serine had a high content (9.2%).At the N-terminus of the peptide chain (Met),Occludin protein had an extinction coefficient of 96 415 in an aqueous solution and an instability index of 62.85,which was an unstable protein.Hydrophobic analysis results indicated that Occludin protein was a hydrophilic protein.There were five transmembrane helix regions of Occludin protein and two superfamily domains (MARVEL superfamily domain and Occludin-ELL superfamily domain) in Luchuan pig,and it had no signal peptide.The secondary structure prediction results showed that the alpha helix,extended chain and random coil of Occludin protein in Luchuan pig accounted for 41.00%,5.36% and 53.64%,respectively,and the tertiary structure was consistent with the secondary structure.This study successfully cloned and analyzed the sequence of Occludin gene CDS in Luchuan pig,which provided a reference for further study of the effect of Occludin gene on the intestinal barrier function of local pig breeds.

Key words: Luchuan pig; Occludin gene; cloning; bioinformatics analysis