ABSTRACT
Objective:To clone swine leukocyte antigen,class II,DO alpha (SLA-DOA) gene from Banna mini-pig inbred line (BMI) and detect its mRNA expression level in 19 important tissues.Methods:The complete eDNA sequence of SLA-DOA gene was cloned by RT-PCR method from BMI and the mRNA expression pattern in BMI important tissues was examined by semi-quantative RT-PCR method.Nucleotide and protein sequences of SIA-DOA were used to carry out bioinformatics analysis and construct the phylogenetic tree.Results:The eDNA length of BMI SLA-DOA was 1 079 bp,which encoded a protein of 250 amino acids with molecular weight (Mw) 27.81 kD,and isoelectric point (pI) 6.48.Genome structure analysis showed it localized to Sus scrofa chromosomes 7 and consisted of four exons and three introns.Semi-quantitative expression analysis showed that SLA-DOA gene expressed highly in the lymph nodes and stomach;weakly in the heart,skin and duodenum and none in other 14 tissues.Functional bioinformatics analysis indicated that SLA-DOA protein contained one signal poptide,one transmembrane region,three conserved domains,four O-GlcNAc glycosylation sites,18 potential phosphorylation sites and to be located in the cytoplasm with 94.1％ certainty.Phylogenetic analysis demonstrated that BMI (pig) had the closest relationship with cattle.Conclusion:This study have successfully cloned the SLA-DOA gene of Banna mini-pig inbred line,performed bioinformatics analysis and tissue expression profile analysis.It will provide a basis for the studies of BMI xenotransplantation.
ABSTRACT
Objective To get TDRP1 gene of sterile and fertile boar of the Banna minipig inbred line (BMI), predict its function by bioinformatics analysis, and detect its expression patterns in the fertile boar.Methods Based on the NM_001198925 sequence, we designed specific primers and amplified BMI TDRP1 using RT-PCR method for sequen-cing and bioinformatics analysis.Meanwhile, the expression of TDRP1 in 17 organ tissues ( heart, liver, spleen, lung, kidney, thymus, lymph nodes, skin, duodenum, stomach, cerebrum, cerebellum, testis, epididymis, seminal vesicle, prostate, and bulbourethral gland) of fertile BMI boar and in the testis of sterile and fertile BMI boars was analyzed by semi-quantitative RT-PCR.Results The experiment obtained 680 bp cDNA sequence ( GenBank accession number:KJ186786) of BMI TDRP1, which encodes a protein of 186 amino acids with a predicted molecular weight (Mw) of 20.49 kDa and isoelectric point (pI) 5.86, and no signal peptide.It was a nuclear protein with a probability of 94.1%and had a leucine-rich nuclear export signals.Homology analysis of protein sequences revealed that BMI TDRP1 showed high identi-ty with that of humans, macaca mulatta, mouse and rat.The RT-PCR analysis showed that TDRP1 had a similar expression in the testes of sterile and fertile BMI boars.It was highly abundant in the seminal vesicle and prostate, moderately ex-pressed in cerebellum and testis and weakly expressed in cerebrum and kidney, while undetected in other 11 organ tissues. Conclusions We have cloned TDRP1 complete coding sequence, and found 2 SNPs,showing no difference in sequences and the testis mRNA expression levels between the fertile and sterile BMI boars.The multi-tissue transcription profile shows different expression levels in different organ tissues, being high in the seminal vesicle and prostate.The results of this study provide a foundation for further insight into the role of this gene in spermatogenesis.