cdna Molecular Cloning and Sequence Analysis of Porcine Perilipin ( PL IN) Gene

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1 2010,32 (1) : ,112 http :/ / xuebao. jlau. edu. cn Journal of Jilin Agricultural University E2mail : sina. com cdna Ξ,,,,, : PCR, PLIN,, PLIN cdna, 978 bp ( GenBank EU416277) 87 % 83 %, 325, 1 PAT21 2 A 1, 87 %,82 %,86 %,86 % : ; ; PCR ; ; : Q785 ; S82812 : A : (2009) Molecular Cloning and Sequence Analysis of Porcine Perilipin ( PL IN) Gene YANG Hong2wen, WANG Chun2feng, SU Chao2zhi, WEI Bi2fen, LI Hong2shu Guizhou Institue of Animal Husbandry and Veterinary Medicine, Guiyang , China Abstract : The partial cdna of porcine PLIN gene was amplified by RT - PCR with degenerate primers designed according to the conserved sequences of human, mouse, rat and cattle PLIN genes. The cdna ( GenBank accession No. EU416277) containing start codon was 978 bp in length, sharing 87 % and 83 % identity with those of human and mouse, respectively. The predicted peptide contained 325 amino acid, a PAT21 domain, two protein kinase A site and a Glu2rich acidic region. The comparison of the de2 duced amino acids sequence of porcine PLIN with those of human, mouse, cattle, and sheep showed that the amino acid homology was 87 %, 82 %, 86 % and 86 %, respectively. Cloning and sequencing of porcine PLIN gene would be helpful to study its function. Key words : porcine ; PLIN gene ; degenerate PCR ; cloning ; sequence analysis ( Perilipin,PLIN),, :, [122 ], PLIN, 2 PLIN,,, [324 ] Qi PLIN, [526 ], [729 ],PLIN Ξ : [ ( ) ] :,,,, : : :

2 : cdna 105 PLIN, PLIN PLIN cdna, PLIN RNA RNeasy Lipid Tissue Mini Kit (QIA2 GEN ) RNA,, , ( NM ) (NM ) (NM ) (NM ) PLIN, primer primier 510, LN1F : 5 2AGGATGKC (A/ C) (G/ A) T ( G/ C) AACAA ( G/ A) GG23 ; LN1R : 5 2AG GCGGGTRGAGATGGT23 ; LN2F : 5 2GACTTTGCTG GATGGAGACC23 ; LN2R : 5 2CAGGGCTG ( T/ C ) TACCTC(A/ T) CT PCR PLIN cdna RevertAid TM H Minus First Strand cdna Synthesis Kit ( Fermentas ) PCR : 215 L MgCl 2, 215 L dntp (2 mmol/ L),215 L 10, 1 L,1 L TaqE ( 1 U/ L ), 1 L cdna, 1315 L dh 2 O, 25 L PCR :94 4 min ; s, 50 s ( LN1 LN2 5816,5715 ),72 50 s, 35 ; min PCR 115 %,UNIQ210 DNA, p GEM R 2T, ( Escherichia coli) DH5, Amp + LB,, PCR, 114 DNAStar5101, (National Cen2 ter for Biotechnology Information, NCBI, http :/ / www. ncbi. nlm. nih. gov) BLAST(Basic Local Align2 ment Search Tool) ( Euro2 pean Bioinformatics Institute, EBI, http :/ / www. ebi. ac. uk) CLUSTAL W, http :/ / cn. expasy. org PLIN 2, PCR 2 ( 1), 374 bp ( LN1F, LN1R) 955 bp ( LN2F, LN2R), 978 bp cdna ( EU416277) 87 % 83 %, 325, ( 2) 212 cdna, PLIN ADRP(, GenBank NP ) TIP47 (47 kd, GenBank NP ) N 100 PAT21, 37 % 43 %( 2) PLIN 2 PLIN A (protein ki2 nase A, PKA) 1 [10211 ] ( 3) ( NP ) (NP ) (NP ) (NP ) 4 PLIN, 87 %,82 %,86 %,86 %, ( 4), Journal of Jilin Agricultural University

3 Sample ; A. LN1F LN1R The amplification results using primer LN1F and LN1R ; B. LN2F LN2R The amplification results using primer LN2F and LN2R ; M. DNA Marker DL PLIN RT - PCR Fig. 1. The amplification re sults of porcine PLIN gene by RT - PCR, A, The start codon is indicated by a short horizontal line, the protein kinase A sites are boxed, the Glu2rich acidic region is highlighted by underlining 2 PLIN cdna Fig. 2. Partial nucleotide and predicted amino acid sequence of porcine PLIN Journal of Jilin Agricultural University 2009, February

4 : cdna The asterisks indicate invariant amino acid ; : Means conserved substitutions ; 1 Means semi2conserved substitutions ; Blank indicate no conserved residues 3 N PAT21 Fig. 3. Amino acid sequence alignment of PAT21 domain of porcine PAT ( Perilipin,ADRP,TIP247) protein family 4 PLIN N Fig. 4. Hydrophobicity analysis of N2terminal of porcine perilipin protein 3 PCR, cdna,, PCR, ;, PLIN, PLIN PAT21, ADRP TIP47 PAT21 37 % 43 %, Lu [12 ] PLIN N 100 PAT21 ADRP TIP47 PAT21 40 % PLIN, PLIN PLIN,,, PLIN PLIN,,, [1 ], PLIN cdna, PLIN : [1 ] Tansey J T, Sztalryd C, Hlavin E M, et al. The central role of perilipin A in lipid metabolism and adipocyte lipolysis[j ]. IUBMB Life, 2004, 56 (7) : [2 ],,. (Perilipin) [J ]., 2006, 22 (12) : [3 ] Martinez2Botas J, Anderson J B, Tessier D, et al. Absence of per2 ilipin results in leanness and reverses obesity in Lepr (db/ db) mice [J ]. Nat Genet, 2000, 26 (4) : [4 ] Tansey J T, Sztalryd C, Gruia2Gray J, et al. Perilipin ablation re2 sults in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet2induced obesity[j ]. Proc Natl Acad Sci USA, 2001, 98 (11) : [5 ] Qi L, Corella D, Sorl J V, et al. Genetic variation at the perilipin ( PLIN) locus is associated with obesity2related phenotypes in White women[j ]. Clin Genet, 2004, 66 (4) : [6 ] Qi L, Shen H, Larson I, et al. Gender2specific association of a perilipin gene haplotype with obesity risk in a white population[j ]. Obes Res, 2004b, 12 (11) : ( 112 ) Journal of Jilin Agricultural University

5 GenBank GI : %, 609 ( G A) (C T),, Glu Asp, 2) nisrk bp, nisr ( GGAGG) TATAAAAT, nisr,1 699 bp nisr, bp nisk,, 1 3),,,, Nisin, ; nisrk, nisa, NICE, Nisin nisrk NICE, NICE, : [1 ] Suomalainen T, Lagstrgm H, MgttgJ, et al. Influence of whey2 based fruit juice containing Lactobacillus rhamnosus on intestinal well2being and humoral immune response in healthy adults [J ]. Food SC TEC,2006,7 (39) : [2 ] Pham M, Lemberg D A, Day A S. Probiotics : sorting the evidence from the myths [J ]. Med J Aust,2008, 188 (5) : [3 ]. [M]. :, 2008 : [4 ] Aires K A, Cianciarullo A M, Cameiro S M, et al. Production of human papillomavirus type 16 L1 virus2like particles by recombinant Lactobacillus casei cells [J ]. Appl Environ Microbiol, 2006, 72 (1) : [ 5 ] Johnson2Henry K C, Donato KA, Shen2Tu G, et al. Lactobacillus rhamnosus strain GG prevents enterohemorrhagic Escherichia coli O157 : H72induced changes in epithelial barrier function [J ]. Infect Immun,2008,76 (4) : [6 ] Van M J, Polman J, Beerthuyzen M, et al. Characterization of the Lactococcus lactis nisin A operon genes nisp, encoding a subtilisin2 like serine protease involved in precursor processing, and nisr, encoding a regulatory protein involved in nisin biosynthesis [J ]. J Bacteriol, 1993, 175 (9) : [7 ] Kuipers O P, Beerthuyzen M M, De Ruyter P G G A, et al. Au2 toregulation of nisin biosynthesis in Lactococcus lactis by signal transduction [J ]. J Biol Chem, 1995, 270 (45) : [8 ] De Ruyter P G G A, Kuipers O P, Beerthuyzen M, et al. Func2 tional analysis of promoters in the nisin gene cluster of Lactococcus lactis [J ]. J Bacteriol, 1996, 178 (12) : [ 9 ] Wu C M, Lin C F, Chang Y C, et al. Construction and characteri2 zation of nisin2controlled expression vectors for use in Lactobacillus reuteri [J ]. Biosci Biotechnol Biochem, 2006, 70 (4) : [10 ] Zhou X X, Li W F, Ma G X, et al. The nisin2controlled gene ex2 pression system : construction, application and improvements [J ]. Biotechnol Adv, 2006, 24 (3) : [11 ],. DNA [J ]., 2004, 16 (2) : [12 ] J, D W. [M]. 3 1,,,. :,2002 : 27230, ( 107 ) [7 ] Londos C, Brasaemle D L, Schultz C J, et al. Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells[j ]. Semin Cell Dev Biol, 1999a,10 (1) : [8 ] Londos C, Brasaemle D L, Schultz C J, et al. On the control of lipolysis in adipocytes[j ]. Ann N Y Acad Sci, 1999b, 892 : [9 ],,. (perilipin) [J ].,2006,37 (3) : [ 10 ] Jones B G, Thomas L, Molloy S S, et al. Intracellular trafficking of furin is modulated by the phosphorylation state of a casein ki2 nase II site in its cytoplasmic tail [J ]. EMBO J, 1995,14 (23) : [11 ] Ditti A S, Thomas L, Thomas G, et al. Interaction of furin in immature secretory granules from neuroendocrine cells with the AP21 adaptor complex is modulated by casein kinase II phosphory2 lation[j ]. EMBO J, 1997, 16 (16) : [12 ] Lu X, Gruia2Gray J, Copeland N G, et al. The murine perilipin gene: the lipid droplet2associated perilipins derive from tissue2 specific, mrna splice variants and define a gene family of ancient origin[j ]. Mamm Genome, 2001, 12 (9) : Journal of Jilin Agricultural University 2010, February