山羊FATP2基因的克隆及对前体脂肪细胞脂质沉积的影响

doi:10.11843/j.issn.0366-6964.2023.09.006

Abstract

Abstract: The aim of this study was to clone and analyze the fatty acid transport protein 2 (FATP2) gene by bioinformatics in goats, to examine the expression differences of FATP2 gene in different tissues of goats, and to further reveal the effect of interfering FATP2 gene on lipid metabolism in goat intramuscular precursor adipocytes. In this experiment, 10-month-old healthy Jianzhou goats (n=12) were used as experimental animals. The sequences of the FATP2 gene were amplified and cloned by RT-PCR, and sequence alignment, phylogenetic tree construction and bioinformatics analysis were performed. The relative expression levels of FATP2 gene in different tissues of goat and in different differentiation stages of goat intramuscular preadipocytes were detected by real-time fluorescence quantitative PCR. The SI-RNA interference sequence was synthesized and transfected into goat intramuscular preadipocytes. The FATP2 interference efficiency and the expression of the genes related to lipid metabolism were measured by real-time quantitative PCR; The effect of FATP2 interference on lipid droplet formation was observed by Bodipy staining and the triglyceride content was measured by GPO-Trinder enzymatic reaction. The results showed that the full length of the goat FATP2 gene sequence was 2 335 bp, CDS region was 1 863 bp, 5'UTR was 188 bp, 3'UTR was 284 bp, encoding 621 amino acids, and the highest expression of goat FATP2 gene was in the liver. RT-qPCR showed that the expression of FATP2 was interfered significantly in the cells. The Bodipy staining showed that the lipid droplets were significantly reduced after interfering with the FATP2 gene. The results of triglyceride assay showed that the triglyceride content in intramuscular adipocytes of goats was significantly reduced after interfering with FATP2. In this study, the CDS region sequence of goat FATP2 was successfully obtained. After interfering with FATP2, lipid deposition in goat intramuscular preadipocytes was significantly reduced and the expression of lipid metabolism related genes was significantly affected. These results provide important data to further elucidate the mechanism of FATP2's effect on the regulation of intramuscular formation in goats.

Key words: goat, FATP2 gene, RNA interference, intramuscular precursor adipocytes

CLC Number:

S827.2

TANG Yinmei, LI Qi, LI Haiyang, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Cloning of the Goat FATP2 Gene and Its Regulatory Effect on Lipid Deposition in Precursor Adipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3642-3652.

References

[1] 卢全晟,张晓莉.新疆肉羊养殖与羊肉生产消费发展现状分析[J].黑龙江畜牧兽医,2018(14):7-11.
LU Q S,ZHANG X L.Analysis of the current development of meat sheep breeding and mutton production and consumption in Xinjiang[J].Heilongjiang Animal Science and Veterinary Medicine,2018(14):7-11.(in Chinese)
[2] 朱江江,林亚秋,王永,等.山羊cc类趋化因子受体(CCR2)基因的克隆及组织表达分析[J].畜牧兽医学报,2016, 47(11):2202-2209.
ZHU J J,LIN Y Q,WANG Y,et al.Molecular cloning and tissue expression of goat chemokine (CC motif) receptor 2(CCR2) gene[J].Acta Veterinaria et Zootechnica Sinica,2016,47(11):2202-2209.(in Chinese)
[3] MELTON E M,CERNY R L,DIRUSSO C C,et al.Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1(FATP2/ACSVL1) reveals distinct patterns of trafficking of exogenous fatty acids[J].Biochem Biophys Res Commun,2013,440(4):743-748.
[4] SCHAFFER J E,LODISH H F.Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein[J].Cell,1994,79(3):427-436.
[5] 齐仁立,黄金秀,杨飞云,等.脂肪酸转运蛋白家族及其介导的脂肪酸跨膜转运[J].动物营养学报,2013,25(5):905-911.
QI R L,HUANG J X,YANG F Y,et al.Fatty acid transport protein family and their mediation in transmembrane transportation of fatty acids[J].Chinese Journal of Animal Nutrition,2013,25(5):905-911.(in Chinese)
[6] SCHAFFER J E.Fatty acid transport:the roads taken[J].Am J Physiol Endocrinol Metab,2002,282(2):E239-E246.
[7] ALICEA G M,REBECCA V W,GOLDMAN A R,et al.Changes in aged fibroblast lipid metabolism induce age-dependent melanoma cell resistance to targeted therapy via the fatty acid transporter FATP2[J].Cancer Discov,2020,10(9):1282-1295.
[8] FALCON A,DOEGE H,FLUITT A,et al.FATP2 is a hepatic fatty acid transporter and peroxisomal very long-chain acyl-CoA synthetase[J].Am J Physiol Endocrinol Metab,2010,299(3):E384-E393.
[9] CHEN Y T,YAN Q,LV M Y,et al.Involvement of FATP2-mediated tubular lipid metabolic reprogramming in renal fibrogenesis[J].Cell Death Dis,2020,11(11):994.
[10] VEGLIA F,TYURIN V A,BLASI M,et al.Fatty acid transport protein 2 reprograms neutrophils in cancer[J].Nature,2019, 569(7754):73-78.
[11] CHEN Y H,ZHAO Y,DENG Y,et al.FATP2 regulates non-small cell lung cancer by mediating lipid metabolism through ACSL1[J].Tissue Cell,2023,82:102105.
[12] ADESHAKIN A O,LIU W,ADESHAKIN F O,et al.Regulation of ROS in myeloid-derived suppressor cells through targeting fatty acid transport protein 2 enhanced anti-PD-L1 tumor immunotherapy[J].Cell Immunol,2021,362:104286.
[13] RAN L S,WU Y Z,GAN Y W,et al.Andrographolide ameliorates hepatic steatosis by suppressing FATP2-mediated fatty acid uptake in mice with nonalcoholic fatty liver disease[J].J Nat Med,2023,77(1):73-86.
[14] PEREZ V M,GABELL J,BEHRENS M,et al.Deletion of fatty acid transport protein 2(FATP2) in the mouse liver changes the metabolic landscape by increasing the expression of PPARα-regulated genes[J].J Biol Chem,2020,295(17):5737-5750.
[15] UCHIYAMA A,AOYAMA T,KAMIJO K,et al.Molecular cloning of cDNA encoding rat very long-chain acyl-CoA synthetase[J].J Biol Chem,1996,271(48):30360-30365.
[16] 王涛.猪候选基因SLC27A2和IFRD1的分离与鉴定[D].武汉:华中农业大学,2005.
WANG T.Isolation,identification of porcine candidate genes:SLC27A2 and IFRD1[D].Wuhan:Huazhong Agricultural University,2005.(in Chinese)
[17] HAUSMAN G J,NOVAKOFSKI J E,MARTIN R J,et al.The development of adipocytes in primary stromal-vascular culture of fetal pig adipose tissue[J].Cell Tissue Res,1984,236(2):459-464.
[18] XIONG Y,XU Q,LIN S,et al.Knockdown of LXRα inhibits goat intramuscular preadipocyte differentiation[J].Int J Mol Sci,2018, 19(10):3037.
[19] TIAN W,XIANG H,LI Q,et al.Oleic acid,independent of insulin,promotes differentiation of goat primary preadipocytes in vitro[J].Animal Prod Sci,2023,63(2):113-119.
[20] YANG C H,LI Q,LIN Y Q,et al.MCD inhibits lipid deposition in goat intramuscular preadipocytes[J].Genes,2023,14(2):440.
[21] 许晴,李倩,王永,等.过表达FGF10促进山羊皮下前体脂肪细胞分化[J].畜牧兽医学报,2019,50(10):1972-1984.
XU Q,LI Q,WANG Y,et al.Overexpression of FGF10 promotes the differentiation of subcutaneous preadipocyte in goat[J].Acta Veterinaria et Zootechnica Sinica,2019,50(10):1972-1984.(in Chinese)
[22] 许晴,林森,朱江江,等.山羊肌内前体脂肪细胞诱导分化过程中内参基因的表达稳定性分析[J].畜牧兽医学报,2018, 49(5):907-918.
XU Q,LIN S,ZHU J J,et al.The expression stability analysis of reference genes in the process of goat intramuscular preadipocytes differentiation in goat[J].Acta Veterinaria et Zootechnica Sinica,2018,49(5):907-918.(in Chinese)
[23] LOWE C E,O'RAHILLY S,ROCHFORD J J.Adipogenesis at a glance[J].J Cell Sci,2011,124(16):2681-2686.
[24] QIMUGE N,HE Z Z,QIN J,et al.Overexpression of DNMT3A promotes proliferation and inhibits differentiation of porcine intramuscular preadipocytes by methylating p21 and PPARg promoters[J].Gene,2019,696:54-62.
[25] WANG H T,LEE J H,TIAN Y.Critical genes in white adipose tissue based on gene expression profile following exercise[J].Int J Sports Med,2019,40(1):57-61.
[26] LOW K O,MAHADI N M,ILLIAS R M.Optimisation of signal peptide for recombinant protein secretion in bacterial hosts[J]. Appl Microbiol Biotechnol,2013,97(9):3811-3826.
[27] ARDITO F,GIULIANI M,PERRONE D,et al.The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review)[J].Int J Mol Med,2017,40(2):271-280.
[28] HIRSCH D,STAHL A,LODISH H F.A family of fatty acid transporters conserved from mycobacterium to man[J].Proc Natl Acad Sci U S A,1998,95(15):8625-8629.
[29] HEINZER A K,KEMP S,LU J F,et al.Mouse very long-chain acyl-CoA synthetase in X-linked adrenoleukodystrophy[J].J Biol Chem, 2002,277(32):28765-28773.
[30] QIU P S,WANG H Z,ZHANG M N,et al.FATP2-targeted therapies-A role beyond fatty liver disease[J].Pharmacol Res,2020, 161:105228.
[31] FENG K X,MA R S,LI H Q,et al.Upregulated SLC27A2/FATP2 in differentiated thyroid carcinoma promotes tumor proliferation and migration[J].J Clin Lab Anal,2022,36(1):e24148.
[32] 张鑫,周雪冰,高铭,等.虫草素通过激活AMPK和MAPK信号通路抑制脂代谢抑制人胃癌细胞的增殖和迁移[J].细胞与分子免疫学杂志,2022,38(6):513-521.
ZHANG X,ZHOU X B,GAO M,et al.Cordycepin inhibits the proliferation and migration of human gastric cancer cells by suppressing lipid metabolism via AMPK and MAPK activation[J].Chinese Journal of Cellular and Molecular Immunology,2022, 38(6):513-521.(in Chinese)
[33] GAO Q Z,ZHANG G J,ZHENG Y Q,et al.SLC27A5 deficiency activates NRF2/TXNRD1 pathway by increased lipid peroxidation in HCC[J].Cell Death Differ,2020,27(3):1086-1104.
[34] MOYA M,BENET M,GUZMÁN C,et al.Foxa1 reduces lipid accumulation in human hepatocytes and is down-regulated in nonalcoholic fatty liver[J].PLoS One,2012,7(1):e30014.
[35] KRAMMER J,DIGEL M,EHEHALT F,et al.Overexpression of CD36 and acyl-CoA synthetases FATP2,FATP4 and ACSL1 increases fatty acid uptake in human hepatoma cells[J].Int J Med Sci,2011,8(7):599-614.
[36] 骆金红,刘凤丹,谭晓山,等.贵州黑山羊不同肌肉组织FASN基因表达与肌内脂肪含量相关性分析[J].中国畜牧杂志,2023,59(5):162-165.
LUO J H,LIU F D,TAN X S,et al.Correlation between FASN gene expression and intramuscular fat content in different muscle tissues of black goats in Guizhou[J].Chinese Journal of Animal Science,2023,59(5):162-165.(in Chinese)
[37] 杨昌恒,李琪,黄维,等.山羊DGAT1基因的克隆及对前体脂肪细胞脂质沉积的调控作用研究[J].畜牧兽医学报,2022,53(1):76-87.
YANG C H,LI Q,HUANG W,et al.Cloning of goat DGAT1 gene and its regulation on lipid deposition in preadipocytes[J].Acta Veterinaria et Zootechnica Sinica,2022,53(1):76-87.(in Chinese)
[38] 庞至远,魏云涛,金泉秀,等.上调SCD1对乳腺癌细胞增殖凋亡的影响及与其相关通路的调控机制[J].现代肿瘤医学,2021,29(10):1668-1671.
PANG Z Y,WEI Y T,JIN Q X,et al.The effect of up-regulation of SCD1 on proliferation and apoptosis of breast cancer cells and the regulation mechanism of related pathways[J].Journal of Modern Oncology,2021,29(10):1668-1671.(in Chinese)
[39] BREJCHOVA K,RADNER F P W,BALAS L,et al.Distinct roles of adipose triglyceride lipase and hormone-sensitive lipase in the catabolism of triacylglycerol estolides[J].Proc Natl Acad Sci U S A,2021,118(2):e2020999118.
[40] 梁莎莎,庞春英,邓廷贤,等.河流型水牛LPL基因在不同组织及泌乳期的表达水平分析[J].中国奶牛,2022(7):12-16.
LIANG S S,PANG C Y,DENG T X,et al.Analysis of LPL gene expression in different tissues and lactation period of river buffalo[J].China Dairy Cattle,2022(7):12-16.(in Chinese)

Cloning of the Goat FATP2 Gene and Its Regulatory Effect on Lipid Deposition in Precursor Adipocytes

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	YAN Xiaochun, XI Haijiao, LI Jinquan, WANG Zhiying, SU Rui. Study on Estimates of Genomic Breeding Value of Fleece Traits in Inner Mongolia Cashmere Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 120-128.
[2]	ZHANG Chenjian, LI Yinxia, DING Qiang, LIU Weijia, WANG Huili, HE Nan, WU Jiashun, CAO Shaoxian. Efficient Preparation of CRISPR/Cas9-mediated Goat SOCS2 Gene Edited Embryos [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 129-141.
[3]	ZHANG De'an, YANG Ruozhu, LIU Jie, LIU Dewu, DENG Ming, LIU Guangbin, SUN Baoli, GUO Yongqing, LI Yaokun. Expression Analysis of Transcriptome in the Liver of Chuanzhong Black Goats Fed with Silage Neolamarckia Cadamba Substitute for Silage Corn [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 232-244.
[4]	SHAO Peng, TANG Yinmei, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Regulation Effect of PSMD9 on Lipid Deposition in Goat Precursor Adipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3653-3663.
[5]	LIU Xinhuan, YUN Jialei, MAO Li, LI Jizong, HAO Fei, HE Miaofeng, YANG Leilei, ZHANG Wenwen, CHENG Zilong, SUN Min, LIU Maojun, WANG Shaohui, BAI Juan, LI Wenliang. Isolation, Identification, Virulence Genes and Drug Resistance Analysis of Escherichia coli Isolated from Diarrheal Goat and Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3445-3454.
[6]	ZHANG Renbao, ZHOU Donghui, ZHOU Lisheng, GAO Xiaoxiao, LIU Nan, HE Jianning. Analysis of Genetic Structure of Conservation Population in Jining Gray Goats Based on 70 K SNP Chip [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2836-2847.
[7]	SHAO Yuexin, ZHANG Xinyu, GE Liyan, SHI Huaiping. Cloning and RNA Interference Analysis of ATF4 Gene in Xinong Saanen Dairy Goat [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2353-2364.
[8]	LIU Jie, XU Xiangping, DENG Ming, ZOU Xian, JIANG Shengwei, LIU Dewu, LIU Guangbin, SUN Baoli, GUO Yongqing, LI Yaokun. Effect of miR-144-5p Targeting WNT5a on the Proliferation and Apoptosis of Goat Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2421-2435.
[9]	XU Tiantian, ZHANG Tongtong, WANG Meng, WANG Xin. Transcription Factor Foxq1 Affects the Proliferation of Hair Follicle Stem Cells in Cashmere Goats via WNT/β-catenin Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2653-2661.
[10]	QUBI Wuqie, LI Yanyan, LI Xin, WANG Yong, WANG Youli, LIU Wei, ZHU Jiangjiang, LIN Yaqiu. APOA4 Gene Inhibits Intramuscular Adipocyte Differentiation in Goat [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 1927-1938.
[11]	BO Luqi, WANG Xueying, HOU Jiaming, ZHANG Li, ZHANG Jianhui, RUAN Rulin, ZHANG Bohan, WANG Shuang, SONG Mingxin, ZHANG Ziqun. Interference of Ts-ODC Gene of Trichinella spiralis by siRNA-757 Effect on Its Acid Resistance [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2114-2125.
[12]	ZHU Junru, WEI Wei, PEI Dangshuai, ZHANG Fenque, DUAN Yu, GUO Yongfeng, JIANG Yue, XIA Shuli, HAN Jing, HOU Jinxing, AN Xiaopeng. Effects of PDCD4 on the Apoptosis of Dairy Goat Mammary Epithelial Cells and the Synthesis of β-casein and TG [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1429-1440.
[13]	CHEN Cancan, JIANG Jing, SUN Xiaoyan, REN Hangxing, LI Jie. Expression of AGRP Gene in Goat Tissue and Its Action Mechanism on Melanin Production [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1441-1451.
[14]	DAN Yixin, YANG Lu, XIANG Hua, ZHANG Huanrong, REN Yupeng, YANG Falong, HE Honghong, ZHU Jiangjiang. Effects of BIRC5 on the Cycle and Apoptosis of Goat Testis Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1511-1524.
[15]	WANG Han, MENG Lijie, LIU Wenjiao, XU Yongjian, GONG Ting. Effect of TAS1R3 Gene Interference on Autophagy Related Factors in Leydig Cells of Xiang Pig [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1525-1534.