In Silico Cloning,RT-PCR Verification and Tissues Expression of Coding Region Sequence of Novel Swine Gene KCNJ12

doi:10.7668/hbnxb.2014.01.009

Abstract

Abstract: The KCNJ12 sequences of allied species of pig were downloaded from NCBI and compared by online BLAST to obtain the EST sequences,the referenced genomic sequences and high-throughput genomic sequences of pig. Subsequently we used silicon cloning strategy to gain the coding sequence and a few flanking sequence of KCNJ12 through Lasergene software. According to the coding regions,we designed specific primers and conducted experimental verification using Banna mini-pig inbred line(BMI). Meanwhile,the expression of KCNJ12 in 31 important tissues of BMI was analyzed by semi-quantitative RT-PCR. The research obtained 1 290 bp coding sequence(GenBank accession number: KC505155,the corresponding amino acid accession number: AGK36093) of swine KCNJ12. Bioinformatics analysis showed that the KCNJ12 gene encoded a protein of 429 amino acids with a predicted molecular weight(Mw) of 48. 63 kDa and isoelectric point(pI) 5. 66. Protein structural analysis suggested that it contained two conserved domains,three transmembrane regions and no signal peptide; hydrophobicity analysis indicated that both N-terminal and C-terminal of the protein were hydrophilic; and it was a cytosolic protein with a probability of 94. 1%. Phylogenetic analysis demonstrated that pig had the closest relationship with cattle andsheep. The analysis of tissue expression showed that the expression of KCNJ12 examined in 31 tissues had obvious differences. It was highly abundant in the cerebellum; moderately expressed in muscle,colon,cecum,esophagus, skin,brain,hypothalamus and spinal cord; and weakly expressed in thymus,liver,rectum and brain stem; while undetected in submandibular gland,testis,thyroid,epididymis,lung,heart,spleen,adrenal gland,lymph nodes,kidneys,pancreas,duodenum,jejunum,ileum,stomach,pituitary,pineal gland and sublingual gland. These results will provide a basis for the study of KCNJ12 function in swine.

Key words: Potassium inwardly-rectifying channel, subfamily J, member 12(KCNJ12), In silico cloning, B an-na mini-pig inbred line(BMI), Bioinformatics, Tissue expression

CLC Number:

QIN Cai-yan, HUO Jin-long, WANG Pei, WANG Shu-yan, MIAO Yong-wang, PAN Wei-rong, ZHA Xing-qin, ZENG Yang-zhi. In Silico Cloning,RT-PCR Verification and Tissues Expression of Coding Region Sequence of Novel Swine Gene KCNJ12[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(1): 41-49. doi: 10.7668/hbnxb.2014.01.009.

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