为了探索PID1(Phosphotyrosine interaction domain containing 1)基因的表达与脂肪沉积的关系, 文章利用兼并引物进行RT-PCR从猪脂肪和肌肉组织中克隆PID1基因CDS(Coding region)区全序列, 并采用荧光定量PCR方法对大白猪、鲁莱黑猪、莱芜猪3个猪品种的肝脏、脂肪和肌肉组织PID1基因mRNA表达进行了相对定量分析。结果表明: 经克隆、测序, 得到了猪PID1基因654 bp全编码区序列, 通过Blast比对, 与人、大鼠、牛有93.88%、66.94%、88.07%的同源性。PID1基因在同一个品种猪中mRNA表达水平总体表现为: 肝脏>脂肪>肌肉。在不同品种3种组织中PID1基因mRNA表达水平总体表现为: 莱芜猪>鲁莱黑猪>大白猪, 其中肝脏中差异显著(P<0.05), 但是在脂肪和肌肉组织中莱芜猪与鲁莱黑猪差异不显著(P>0.05)。对于高肌内脂肪(LWH)、中等肌内脂肪(LWI)和低肌内脂肪(LWL)沉积的3组莱芜猪, PID1基因在肝脏组织中的表达水平是LWH显著高于LWL(P<0.05), 在肌肉组织中则是LWH显著高于LWI和LWL(P<0.05)。PID1基因在莱芜猪品种内3个组织中mRNA表达量与IMF含量相关均不显著, 而在品种间3个组织中mRNA表达量与IMF含量呈显著正相关(P<0.05)。结果提示: PID1的表达可能与脂肪沉积性状存在一定的关系。
To explore the relationship between the expression of PID1 gene and fat deposition, we cloned CDS of PID1 from porcine fat and muscle tissues by RT-PCR using degenerate primers, and investigated expression of this gene in various tissues (i.e., liver, backfat, and muscle tissues) of different breeds (i.e., Yorkshire, Laiwu, and Lulai Black) by real-time fluorescence quantitative PCR. The results showed that 654 bp CDS of porcine PID1 was obtained by sequencing and was 93.88%, 66.94% and 88.07% identical to those of the human, rat, and Bos taurus, respectively. The expression of PID1 mRNA in various tissues and breeds, on the whole, tended to be liver > fat > muscle and Laiwu > Lulai Black > Yorkshire, respectively. For different breeds, PID1 mRNA abundance in liver had significant difference (P<0.05), but had no signifi-cant differences in fat and muscle tissues between Laiwu and Lulai Black (P>0.05). For the three groups of Laiwu pigs with high (LWH), intermediate (LWI), and low IMF content (LWL), PID1 mRNA level was higher in liver tissue of LWH than that of LWL significantly (P<0.05), and was higher in muscle tissue of LWH than that of LWI and LWL significantly (P<0.05). PID1 mRNA abundance was not correlated with IMF in these three tissues of Laiwu breed, but it was positively correlated with IMF in the tissues of these three breeds (P<0.05). These results implied that the expression of PID1 may be related to fat deposition.
[1] Qiu J, Ni YH, Gong HX, Fei L, Pan XQ, Guo M, Chen RH, Guo XR. Identification of differentially expressed genes in omental adipose tissues of obese patients by suppression subtractive hybridization. Biochem Biophys Res Commun, 2007, 352(2): 469-478.
[2] Liao YC, Si LZ, deVere White RW, Lo SH. The phospho-tyrosine-independent interaction of DLC-1 and the SH2 domain of cten regulates focal adhesion localization and growth suppression activity of DLC-1. Cell Biol, 2007, 176(1): 43-49.
[3] Wang B, Zhang M, Ni YH, Liu F, Fan HQ, Fei L, Pan XQ, Guo M, Chen RH, Guo XR. Identification and characterization of NYGGF4, a novel gene containing a phosphotyrosine-binding (PTB) domain that stimulates 3T3-L1 preadipocytes proliferation. Gene, 2006, 379: 132–140.
[4] Erkens T, van Poucke M, Vandesompele J, Goosens K, van Zeveren A, Peelman LJ. Development of a new set of ref-erence genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC/A. BMC Biotechnol, 2006, 6: 41.
[5] Yuan JS, Wang DL, Stewart Jr CN. Statistical methods for efficiency adjusted real-time PCR quantification. Biotechnol J, 2008, 3(1): 112–123.
[6] Zeng YQ, Wang GL, Wang CF, Wei SD, Wu Y, Wang LY, Wang H, Yang HL. Genetic variation of H-FABP gene and association with intramuscular fat content in Laiwu Black and four western pig breeds. Asian-Aust J Anim Sci, 2005, 18(1): 13–16.
[7] 庞卫军, 孙世铎, 李影, 陈国柱, 杨公社. 西部地区主要猪种和野猪H-FABP基因分子标记与IMF含量关系. 遗传, 2005, 27(3): 351–356.
[8] Gerbens F, van Erp AJ, Harders FL, Verburg FJ, Meuwissen TH, Veerkamp JH, te Pas MF. Effect of genetic variants of the heart fatty acid-binding protein gene on intramuscular fat and performance traits in pigs. J Anim Sci, 1999, 77(4): 846–852.
[9] Gerbens F, de Koning DJ, Harders FL, Meuwissen TH, Janss LL, Groenen MA, Veerkamp JH, Van Arendonk JA, te Pas MF. The effect of adipocyte and heart fatty acid-binding protein genes on intramuscular fat and backfat content in Meishan crossbred pigs. J Anim Sci, 2000, 78(3): 552–559.
[10] Houseknecht KL, Bidwell CA, Portocarrero CP, Spurlock ME. Expression and cDNA cloning of porcine peroxisome proliferator-activated receptor gamma (PPARγ). Gene, 1998, 225(1-2): 89–96.
[11] 陈杰, 赵茹茜, 杨晓静, 佟辉, 刘红林. ADD1基因PCR-SSCPs标记与猪肌内脂肪含量及背膘厚的关系. 南京农业大学学报, 2004, 27(3): 66–69.
[12] 王玢. 人类肥胖相关新基因NYGGF4、TNFAIP9的功能研究[学位论文]. 南京: 南京医科大学, 2007.
[13] Zhang CM, Chen XH, Wang B, Liu F, Chi X, Tong ML, Ni YH, Chen RH, Guo XR. Over-expression of NYGGF4 inhibits glucose transport in 3T3-L1 adipocytes via attenuated phosphorylation of IRS-1 and Akt. Acta Pharmacol Sin, 2009, 30(1): 120–124.
[14] 夏黎, 钱玲梅, 孔祥清, 倪毓辉, 张春梅, 童梅玲, 池霞, 吴伟玲, 郭锡熔. NYGGF4小鼠同源基因mRNA在遗传性ob/ob肥胖小鼠体内的表达. 实用儿科临床杂志, 2008, 23(19): 1488–1490.
[15] Dulor JP, Reyne Y, Nougues J. In vivo effects of a treatment with antibodies to adipocyte plasma membranes in the rabbit. Reprod Nutr Dev, 1990, 30(1): 49–58.
[16] 张春梅, 邱洁, 陈小慧, 王玢, 张敏, 郭锡熔. NYGGF4基因过表达对脂肪细胞胰岛素敏感性及分泌功能的影响. 中国当代儿科杂志, 2009, 11(10): 846–849.
[17] Satoh H. Lipid metabolism and obstructive liver disease. Nippon Rinsho, 2001, 59(Suppl.3): 186–189.
[18] 曾勇庆, 王根林, 魏述东, 王林云, 杨海玲, 曹洪防, 徐云华. 含不同比例莱芜猪血缘杂交猪胴体品质及肉质特性的研究. 遗传, 2005, 27(1): 65–69.
