藏猪和大约克猪<i>FABP</i>3基因多态性及差异表达分析

杨绍康; 薄素雪; 韩雨晴; 叶幼荣; 商鹏; 强巴央宗

doi:10.19303/j.issn.1008-0384.2023.06.001

藏猪和大约克猪FABP3基因多态性及差异表达分析

doi: 10.19303/j.issn.1008-0384.2023.06.001

杨绍康^{1, 2,},
薄素雪^{1, 2},
韩雨晴^{1, 2},
叶幼荣^{1, 2},
商鹏^{1, 2},
强巴央宗^{1, 2, ,}

1.
西藏农牧学院动物科学学院，西藏林芝 860000
2.
西藏特色农牧资源研发省部共建协同创新中心，西藏林芝 860000

基金项目: 西藏自治区重大科技专项（XZ202101ZD0005N）；国家自然科学基金地区科学基金项目（32160773）；中国农业大学基本科研业务费专项（2022TC002）

详细信息

作者简介:
杨绍康（1996 —），男，硕士，研究方向：动物遗传育种与繁殖（E-mail：yangshaokang0217@163.com）

通讯作者:
强巴央宗（1965 —），女，博士，教授，研究方向：高原遗传资源保护与利用（E-mail：qbyz628@126.com）

中图分类号: S813.1
计量
- 文章访问数: 559
- HTML全文浏览量: 191
- PDF下载量: 22
- 被引次数: 0
出版历程
- 收稿日期: 2022-09-22
- 修回日期: 2023-03-03
- 网络出版日期: 2023-06-02
- 刊出日期: 2023-06-28

Polymorphisms and Expressions of FABP3 in Tibetan and Yorkshire Pigs

YANG Shaokang^{1, 2
,},
BO Suxue^{1, 2},
HAN Yuqing^{1, 2},
YE Yourong^{1, 2},
SHANG Peng^{1, 2},
CHAMBA Yangzom^{1, 2
, ,}

1.
College of Animal Science, Tibet Agriculture and Animal Husbandry University, Linzhi, Tibet 860000, China
2.
Provincial and Ministerial Co-founded Collaborative Innovation Center for R&D on Tibet Characteristic Agricultural and Animal Husbandry Resources, Linzhi, Tibet 860000, China

摘要

摘要: 目的脂肪酸结合蛋白3（FABP3）参与长链脂肪酸的摄取及利用，在脂肪沉积中发挥重要作用。探究FABP3基因在藏猪和大约克猪间的基因多态性和表达差异可为藏猪品质改良提供分子机制参考。方法选取180日龄藏猪和大约克猪为研究对象，对两猪种FABP3基因5'侧翼区和CDS区进行单核苷酸多态性（SNPs）筛选，使用实时荧光定量检测FABP3基因在肝脏、背最长肌和背脂3个组织中的表达量。结果在FABP3基因5'侧翼区筛选到T-114C和C-635A等2个SNPs位点，且SNPs位点基因型频率在藏猪与大约克猪群体间均呈极显著差异（P ＜ 0.01）；经转录因子预测发现这2个SNPs位点与前脂肪细胞的更新、分化以及脂肪沉积相关，推测这两个多态性位点是参与调控FABP3基因表达的重要功能位点。FABP3基因在藏猪肝脏、背最长肌中的表达量极显著高于大约克猪（P＜0.01），在背脂中的表达量显著高于大约克猪（P ＜ 0.05）。结论推测FABP3基因可能为调控藏猪脂肪代谢的重要候选基因，在藏猪脂肪沉积中呈正向调控作用。
- 藏猪 /
- FABP3基因 /
- 脂肪沉积 /
- 多态性位点
Abstract: Objective Fatty acid binding protein 3 (FABP3) is involved in the uptake and utilization of long-chain fatty acids and plays an important role in fat deposition. To investigate the gene polymorphism and expression differences of FABP3 gene between Tibetan and York pigs could help improve the quality of Tibetan pigs for the genetic level. Methods In this study, single nucleotide polymorphisms (SNPs) in the 5' flanking region and CDS region of FABP3 in randomly selected 180-d-old Tibetan and Yorkshire pigs were tested. Expressions of FABP3 in the liver, longest dorsal muscle, and dorsal fat were detected using real-time fluorescence. Results Two SNPs, T-114C and C-635A, were found in FABP3 with significantly differentiated genotype frequencies between the two species of pigs (P＜0.01). Upon transcription factor prediction, these 2 SNPs loci were found to be associated with preadipocyte renewal, differentiation, and fat deposition, and it was hypothesized that they were important functional loci involved in the regulation of FABP3 gene expression. The expressions of FABP3 in the liver and longest dorsal muscle of Tibetan pigs were extremely significantly higher than those of Yorkshire pigs (P＜0.01), and that in the dorsal fat significantly higher than that of Yorkshire pigs (P＜0.05). Conclusion FABP3 might be closely related to the regulation of fat metabolism and deposition of Tibetan pigs which differs from Yorkshire variety.
- Tibetan pig /
- FABP3 /
- fat deposition /
- polymorphic loci

HTML全文

图 1 DNA琼脂糖凝胶电泳

M：Marker；1～3为藏猪；4～6为大约克猪。

Figure 1. DNA agarose gel electrophoresis

M: Marker; 1–3: Tibetan pigs; 4–6: Yorkshire pigs.

下载: 全尺寸图片幻灯片

图 2 FABP3基因SNPs位点测序峰

Figure 2. Sequence peak map of FABP3 mutation site

下载: 全尺寸图片幻灯片

图 3 组织总RNA琼脂糖凝胶电泳

M：Marker；1～3分别为藏猪肝脏、背脂、背最长肌组织；4～6分别为大约克猪肝脏、背脂、背最长肌组织。

Figure 3. Agarose gel electrophoresis of total RNA in tissue

M：Marker; 1–3: Liver, dorsal fat, and longest dorsal muscle tissues of Tibetan pig, respectively; 4–6: liver, dorsal fat, and longest dorsal muscle tissues of Yorkshire pig, respectively.

下载: 全尺寸图片幻灯片

图 4 FABP3基因在藏猪、大约克猪肝脏、背脂和背最长肌中的mRNA相对表达量

TP为藏猪，YY为大约克猪；*为显著差异（P ＜ 0.05），**为极显著差异（P ＜ 0.01）。

Figure 4. Relative expressions of FABP3 in liver, back fat, and longissimus dorsi muscle of TP and YY

TP: Tibetan pig; YY: Yorkshire pig; *: significant difference at P＜0.05;**: extremely significant difference at P＜0.01.

下载: 全尺寸图片幻灯片

表 1 FABP3基因5'侧翼区和CDS区引物序列

Table 1. Primer sequence of 5' flanking region and CDS region in FABP3

引物 Primer	扩增区域 Amplified region	引物序列（5′-3′） Primer sequences	退火温度 Annealing temperature/℃	产物大小 Product length/bp
5′−FABP3-1	356 bp至−460 bp	F: TCAGCCCAAGAGTGAGTTTC R: CCTTCTTCCTCGAAAGCG	56	817
5′−FABP3-2	−430 bp至−1365 bp	F: TCTGCTGGCTCAAGTTCAGT R: GAGAGGAGAAAGGAAACTCACT	58	953
5′−FABP3-3	−1342 bp至−2197 bp	F: TAGGAGTCAACTTTGGTGAGC R: CCAACTGAACTTGAGCCAGCA	59	856
5′−FABP3-4	−2195 bp至−3033 bp	F: CTGGGAACCTCCATATGTCG R: CTAAGCCACAATCTATCACCT	57	849
FABP3-CDS	34 bp至445 bp	F:CCTGTTCTGTCGTCTCTTTCTCA R:TGCCTCTTTCTCGTAAGTGCG	60	440

下载: 导出CSV

表 2 FABP3基因定量PCR引物序列

Table 2. Primer sequence of FABP3 for quantitative PCR

基因名称 Gene name	登录号 GeneBank number	引物序列（5′-3′） Primer sequences	退火温度 Annealing temperature/℃	产物大小 Product length/bp
FABP3	NM_001099931.1	F: ATGACCAAGCCTACCACAA R: AAGTTTGCCTCCATCCAGT	57	171
GAPDH	NM_001206359.1	F: CACCATCTTCCAGGAGCGAG R: CCCTTCAAGTGAGCCCCG	57	120

下载: 导出CSV

表 3 FABP3基因SNPs位点基因型频率及卡方检验

Table 3. Genotype frequency and chi-square test of SNPs on FABP3

位点 Loci	品种 Species	样本量 Sample size	基因型频率（个体数/频率） Genotype frequency (Individuals/Frequency)			基因频率 Gene frequency		χ²值 Cardinality	P值 P value
位点 Loci	品种 Species	样本量 Sample size	CC	CA	AA	C	A	χ²值 Cardinality	P值 P value
C-635A	大约克猪	39	16/0.410	16/0.410	7/0.179	0.615	0.385	0.693	0.707
	藏猪	28	15/0.520	12/0.410	28/1.000	0.000	1.000	0.000	1.000
	藏猪 vs 大约克猪	χ2=43.979；P＜0.01
T-114C			TT	TC	CC	T	C
	大约克猪	39	33/0.846	6/0.154	0/0	0.923	0.077	0.271	0.873
	藏猪	28	0/0.000	1/0.036	27/0.964	0.018	0.982	0.009	0.995
	藏猪 vs 大约克猪	χ2=63.476；P＜0.01

下载: 导出CSV

表 4 FABP3基因SNPs位点转录因子预测结果

Table 4. Predicted transcription factors in FABP3 SNPs

突变位点 Mutation Loci	突变前序列 Pre-mutation sequence	突变后序列 Post-mutation sequence	消失转录因子 Disappearance of transcription factors	新增转录因子 Addition of transcription factors
C-635A	TGGGGCGGGGG	TGGGGAGGGGG	ARNT2、 SUMO2、 RB1、 CTCF、SMAD4、LF11、CHD1、SUZ12、YY1	STAT5B、TCF7L2、TCF12、SREBP1、MYH11、SPI1、TP53、TBX21、HOXA9
T-114C	ACGCCTCGTCA	ACGCCCCGTCA	E4F1	HES5、CLOCK、WT1、EP300、THAP11、KLF5

下载: 导出CSV

参考文献(38)

[1]	SCHUMACHER M, DELCURTO-WYFFELS H, THOMSON J, et al. Fat deposition and fat effects on meat quality-a review [J]. Animals:an Open Access Journal from MDPI, 2022, 12(12): 1550.
[2]	VITALI M, DIMAURO C, SIRRI R, et al. Effect of dietary polyunsaturated fatty acid and antioxidant supplementation on the transcriptional level of genes involved in lipid and energy metabolism in swine [J]. PLoS One, 2018, 13(10): e0204869. doi: 10.1371/journal.pone.0204869
[3]	KIM B, MIN Y J, JEONG Y, et al. Comparison of growth performance and related gene expression of muscle and fat from Landrace, Yorkshire, and Duroc and Woori black pigs [J]. Journal of Animal Science and Technology, 2023, 65(1): 160−174. doi: 10.5187/jast.2022.e93
[4]	LI Q G, TAO Z, SHI L H, et al. Expression and genome polymorphism of ACSL1 gene in different pig breeds [J]. Molecular Biology Reports, 2012, 39(9): 8787−8792. doi: 10.1007/s11033-012-1741-6
[5]	GRZES M, SADKOWSKI S, RZEWUSKA K, et al. Pig fatness in relation to FASN and INSIG2 genes polymorphism and their transcript level [J]. Molecular Biology Reports, 2016, 43(5): 381−389. doi: 10.1007/s11033-016-3969-z
[6]	LI A N, WU L J, WANG X Y, et al. Tissue expression analysis, cloning and characterization of the 5’-regulatory region of the bovine FABP3 gene [J]. Molecular Biology Reports, 2016, 43(9): 991−998. doi: 10.1007/s11033-016-4026-7
[7]	CHMURZYŃSKA A. The multigene family of fatty acid-binding proteins (FABPs): Function, structure and polymorphism [J]. Journal of Applied Genetics, 2006, 47(1): 39−48. doi: 10.1007/BF03194597
[8]	SWEENEY T, O'HALLORAN A M, HAMILL R M, et al. Novel variation in the FABP3 promoter and its association with fatness traits in pigs [J]. Meat Science, 2015, 100: 32−40. doi: 10.1016/j.meatsci.2014.09.014
[9]	HONG J, KIM D, CHO K, et al. Effects of genetic variants for the swine FABP3, HMGA1, MC4R, IGF2, and FABP4 genes on fatty acid composition [J]. Meat Science, 2015, 110: 46−51. doi: 10.1016/j.meatsci.2015.06.011
[10]	WANG L J, LI L, JIANG J, et al. Molecular characterization and different expression patterns of the FABP gene family during goat skeletal muscle development [J]. Molecular Biology Reports, 2015, 42(1): 201−207. doi: 10.1007/s11033-014-3759-4
[11]	LI B, ZERBY H N, LEE K. Heart fatty acid binding protein is upregulated during porcine adipocyte development [J]. Journal of Animal Science, 2007, 85(7): 1651−1659. doi: 10.2527/jas.2006-755
[12]	GERBENS F, VAN ERP A J, HARDERS F L, et al. Effect of genetic variants of the heart fatty acid-binding protein gene on intramuscular fat and performance traits in pigs [J]. Journal of Animal Science, 1999, 77(4): 846−852. doi: 10.2527/1999.774846x
[13]	SERÃO N V L, VERONEZE R, RIBEIRO A M F, et al. Candidate gene expression and intramuscular fat content in pigs [J]. Journal of Animal Breeding and Genetics, 2011, 128(1): 28−34. doi: 10.1111/j.1439-0388.2010.00887.x
[14]	张敏. FABP3对LPS诱导的奶牛乳腺上皮细胞炎症反应的调控及机制研究[D]. 武汉: 华中农业大学, 2018. ZHANG M. Effects and mechanism of FABP3 gene on LPS-induced inflammation response in bovine mammary epithelial cells[D]. Wuhan: Huazhong Agricultural University, 2018. (in Chinese)
[15]	JIANG Y, LIU J L, LIU H T, et al. miR-381-3p inhibits intramuscular fat deposition through targeting FABP3 by ceRNA regulatory network [J]. Biology, 2022, 11(10): 1497. doi: 10.3390/biology11101497
[16]	CHMURZYNSKA A, SZYDLOWSKI M, STACHOWIAK M, et al. Association of a new SNP in promoter region of the porcine FABP3 gene with fatness traits in a Polish synthetic line [J]. Animal Biotechnology, 2007, 18(1): 37−44. doi: 10.1080/10495390600671560
[17]	BLECHA I M Z, SIQUEIRA F, FERREIRA A B R, et al. Identification and evaluation of polymorphisms in FABP3 and FABP4 in beef cattle [J]. Genetics and Molecular Research:GMR, 2015, 14(4): 16353−16363. doi: 10.4238/2015.December.9.3
[18]	LI X P, KIM S W, CHOI J S, et al. Investigation of porcine FABP3 and LEPR gene polymorphisms and mRNA expression for variation in intramuscular fat content [J]. Molecular Biology Reports, 2010, 37(8): 3931−3939. doi: 10.1007/s11033-010-0050-1
[19]	WANG B B, LI P H, ZHOU W D, et al. Association of twelve candidate gene polymorphisms with the intramuscular fat content and average backfat thickness of Chinese suhuai pigs [J]. Animals:an Open Access Journal from MDPI, 2019, 9(11): 858.
[20]	强巴央宗, 张浩, 纪素玲, 等. 藏猪屠宰性能和肉质测定与分析 [J]. 中国畜牧杂志, 2008, 44(21):10−11,48. QIANG B, ZHANG H, JI S L, et al. Determination and analysis of slaughter performance and meat quality of Tibetan pigs [J]. Chinese Journal of Animal Science, 2008, 44(21): 10−11,48.(in Chinese)
[21]	商鹏, 强巴央宗, 张博, 等. 藏猪选育群屠宰性能和肉质测定分析 [J]. 黑龙江畜牧兽医, 2015(2):30−32. doi: 10.13881/j.cnki.hljxmsy.2015.0092 SHANG P, Qiangbayangzong, ZHANG B, et al. Analysis of the slaughter performance and determination of meat quality in the selective breeding population of Tibetan pigs [J]. Heilongjiang Animal Science and Veterinary Medicine, 2015(2): 30−32.(in Chinese) doi: 10.13881/j.cnki.hljxmsy.2015.0092
[22]	张一君. 微卫星DNA标记与SNP芯片鉴定宁乡猪亲缘关系[D]. 长沙: 湖南农业大学, 2017. ZHANG Y J. Phylogenetic analysis of Ningxiang pig based on microsatellite DNA markers and SNP chip[D]. Changsha: Hunan Agricultural University, 2017. (in Chinese)
[23]	尹杭. 猪BMP7和BMP15基因3’-UTR多态性及其与繁殖性能的关系[D]. 南京: 南京农业大学, 2020. YIN H. Polymorphism of the 3’-UTR of the porcine BMP7 and BMP15 and their association with reproductive performance[D]. Nanjing: Nanjing Agricultural University, 2020. (in Chinese)
[24]	ARZATE-MEJÍA R G, RECILLAS-TARGA F, CORCES V G. Developing in 3D: The role of CTCF in cell differentiation [J]. Development, 2018, 145(6): 137729. doi: 10.1242/dev.137729
[25]	CARMONA-ALDANA F, ZAMPEDRI C, SUASTE-OLMOS F, et al. CTCF knockout reveals an essential role for this protein during the zebrafish development [J]. Mechanisms of Development, 2018, 154: 51−59. doi: 10.1016/j.mod.2018.04.006
[26]	肖成, 薛佳佳, 王晶, 等. 小尾寒羊S100A1基因编码区外显子克隆及表达分析 [J]. 中国畜牧杂志, 2021, 57(5):82−86. doi: 10.19556/j.0258-7033.20200622-01 XIAO C, XUE J J, WANG J, et al. Cloning and expression analysis of exon of S100A1 gene in small tail Han sheep [J]. Chinese Journal of Animal Science, 2021, 57(5): 82−86.(in Chinese) doi: 10.19556/j.0258-7033.20200622-01
[27]	ZENG Z, HUANG N N, ZHANG Y D, et al. CTCF inhibits endoplasmic reticulum stress and apoptosis in cardiomyocytes by upregulating RYR2 via inhibiting S100A1 [J]. Life Sciences, 2020, 242: 117158. doi: 10.1016/j.lfs.2019.117158
[28]	LUO D, XIAO H W, DONG J L, et al. B7-H3 regulates lipid metabolism of lung cancer through SREBP1-mediated expression of FASN [J]. Biochemical and Biophysical Research Communications, 2017, 482(4): 1246−1251. doi: 10.1016/j.bbrc.2016.12.021
[29]	XU W W, CHEN Q M, JIA Y H, et al. Isolation, characterization, and SREBP1 functional analysis of mammary epithelial cell in buffalo [J]. Journal of Food Biochemistry, 2019, 43(11): e12997.
[30]	BENGOECHEA-ALONSO M T, ERICSSON J. The phosphorylation-dependent regulation of nuclear SREBP1 during mitosis links lipid metabolism and cell growth [J]. Cell Cycle, 2016, 15(20): 2753−2765. doi: 10.1080/15384101.2016.1220456
[31]	MOISON C, CHAGRAOUI J, CARON M C, et al. Zinc finger protein E4F1 cooperates with PARP-1 and BRG1 to promote DNA double-strand break repair [J]. Proceedings of the National Academy of Sciences of the United States of America, 2021, 118(11): e2019408118. doi: 10.1073/pnas.2019408118
[32]	LIU Z Y, KRAUS W L. Catalytic-independent functions of PARP-1 determine Sox2 pioneer activity at intractable genomic loci [J]. Molecular Cell, 2017, 65(4): 589−603.e9. doi: 10.1016/j.molcel.2017.01.017
[33]	CERVANTES-CAMACHO C, BELTRÁN-LANGARICA A, OCHOA-URIBE A K, et al. The transient expression of Klf4 and Klf5 during adipogenesis depends on GSK3β activity [J]. Adipocyte, 2015, 4(4): 248−255. doi: 10.1080/21623945.2015.1007823
[34]	WU Q, FU C Y, LI M L, et al. CINP is a novel cofactor of KLF5 required for its role in the promotion of cell proliferation, survival and tumor growth [J]. International Journal of Cancer, 2019, 144(3): 582−594. doi: 10.1002/ijc.31908
[35]	WANG J, CHU Y F, XU M, et al. miR-21 promotes cell migration and invasion of hepatocellular carcinoma by targeting KLF5 [J]. Oncology Letters, 2019, 17(2): 2221−2227.
[36]	SCHAAP F G, BINAS B, DANNEBERG H, et al. Impaired long-chain fatty acid utilization by cardiac myocytes isolated from mice lacking the heart-type fatty acid binding protein gene [J]. Circulation Research, 1999, 85(4): 329−337. doi: 10.1161/01.RES.85.4.329
[37]	YI B, WANG J G, WANG S, et al. Overexpression of Banna mini-pig inbred line fatty acid binding protein 3 promotes adipogenesis in 3T3-L1 preadipocytes [J]. Cell Biology International, 2014, 38(8): 918−923. doi: 10.1002/cbin.10285
[38]	CHO K H, KIM M J, JEON G J, et al. Association of genetic variants for FABP3 gene with back fat thickness and intramuscular fat content in pig [J]. Molecular Biology Reports, 2011, 38(3): 2161−2166. doi: 10.1007/s11033-010-0344-3