香猪CYP3A29基因3'-UTR结构变异及其与肌肉雄激素水平的关联分析

doi:10.16431/j.cnki.1671-7236.2023.08.017

摘要/Abstract

摘要： 【目的】探究猪特有的细胞色素P450超家族3A亚族成员29(cytochrome P450 3A 29,CYP3A29)基因3'-UTR区核苷酸结构变异(structural variation,SV)对该基因表达的影响,解析该结构变异与香猪肉质性状形成的关系。【方法】以香猪、大白猪为研究对象,应用UCSC、miRBase、PITA、RBPsuite等软件预测CYP3A29基因3'-UTR区结构变异区间所包含的重复元件、miRNA和RBP结合位点;采用PCR方法对结构变异位点进行基因分型;计算群体基因型频率、基因频率及Hardy-Weinberg平衡状态;采用实时荧光定量PCR检测该变异对CYP3A29基因表达的影响;利用ELISA法检测香猪肌肉组织中CYP3A29蛋白含量和雄激素(T、DHT)水平,并使用SPSS 17.0软件对T、DHT含量与不同基因型进行Pearson相关性分析。【结果】生物信息学分析显示,CYP3A29基因3'-UTR的结构变异区全长303 bp,该变异区存在1个长为241 bp的短散在元件(short interspersed element,SINE),属于Pre0_SStRNA家族,位于Chr3:6 819 917―6 820 161,含有33个miRNAs和13个RBPs结合位点。基因分型显示,该结构变异在香猪、大白猪群体中呈现出丰富的多态性,表现为II基因型(插入型)、ID基因型(杂合型)、DD基因型(双缺失)。基因型群体分布频率显示,香猪中的D等位基因频率高于大白猪,该结构变异在香猪中处于Hardy-Weinberg平衡状态(P＞0.05),在大白猪中处于Hardy-Weinberg不平衡状态(P＜0.05)。实时荧光定量PCR和ELISA结果显示,CYP3A29基因ID基因型个体mRNA表达量和蛋白含量均显著高于II和DD基因型(P＜0.05)。相关性分析结果显示,香猪不同基因型个体CYP3A29基因含量与肌肉组织中T浓度呈显著相关(P＜0.05),与DHT浓度不相关(P＞0.05)。香猪ID基因型个体T浓度显著高于DD和II基因型(P＜0.05),DD基因型个体DHT浓度显著高于ID和II基因型(P＜0.05)。【结论】CYP3A29基因3'-UTR区长度为303 bp的结构变异在香猪群体中具有多态性,以缺失型D等位基因为主,即该结构变异在香猪中缺失SINE元件,导致CYP3A29基因表达上调,进而影响香猪肌肉组织中T浓度。CYP3A29基因3'-UTR结构变异与香猪肉质性状具有相关性。

关键词: 香猪; CYP3A29基因; 结构变异; 雄激素; 肉质

Abstract: 【Objective】 This study was conducted to investigate the effect of the structural variation (SV) in the pig-specific cytochrome P450 3A 29 (CYP3A29) gene 3'-UTR on its expression,and analyze the relationship between the SV and the formation of meat quality traits in Xiang pigs.【Method】 The repeated elements,miRNA,and RBPs binding sites in the SV of CYP3A29 gene 3'-UTR were predicted by UCSC,miRBase,PITA and RBPsuite softwares in Xiang pigs and Large White pigs.The SV site was genotyped by PCR,the genotype frequency,gene frequency and Hardy-Weinberg equilibrium were calculated.The mRNA expression of CYP3A29 gene was detected by Real-time quantitative PCR.The content of CYP3A29 protein and the level of androgen (T and DHT) in muscle were detected by ELISA.Pearson correlation analysis between the contents of T and DHT and different genotypes was performed by SPSS 17.0 software.【Result】 Bioinformatics analysis results showed that the SV length of CYP3A29 gene 3'-UTR was 303 bp,and there was a short interspersed element (SINE) with a length of 241 bp in this variation,belonging to Pre0_SStRNA family,located in Chr3:6 819 917―6 820 161,containing 33 miRNAs and 13 RBPs binding sites.The genotyping confirmed that the SV in Xiang pigs and Large White pigs showed rich polymorphism,which contained insertion (II),hybrid (ID) and deletion (DD).The frequency of D allele in Xiang pigs was higher than that in Large White pigs.The SV was in Hardy-Weinberg equilibrium in Xiang pigs (P＞0.05),but not in Large White pigs (P＜0.05).The results of Real-time quantitative PCR and ELISA showed that the mRNA expression and protein content of ID genotype of CYP3A29 gene in Xiang pigs were significantly higher than II and DD genotypes,respectively (P＜0.05).The correlation analysis results showed that the expression of CYP3A29 gene of different genotypes was significantly correlated with T concentration of muscle tissue in Xiang pigs (P＜0.05),but not correlated with DHT concentration (P＞0.05).The T concentration of ID genotype in Xiang pigs was significantly higher than that of DD and II genotypes (P＜0.05),and DHT concentration of DD genotype was significantly higher than that of ID and II genotypes (P＜0.05).【Conclusion】 The SV of 303 bp in CYP3A29 gene 3'-UTR was polymorphic in Xiang pigs,and the deletion D allele was the main one,that was the SV lacked the SINE element in Xiang pigs,which leaded to the up-regulation of CYP3A29 gene expression,thereby affecting the T concentration of muscle tissue in Xiang pigs.In summary,the SV of CYP3A29 gene 3'-UTR was correlated with the meat quality traits in Xiang pigs.

Key words: Xiang pigs; CYP3A29 gene; structural variation; androgen; meat quality

中图分类号:

S828

梁小芬, 冉雪琴, 牛熙, 李升, 黄世会, 王嘉福. 香猪CYP3A29基因3'-UTR结构变异及其与肌肉雄激素水平的关联分析[J]. 中国畜牧兽医, 2023, 50(8): 3199-3209.

LIANG Xiaofen, RAN Xueqin, NIU Xi, LI Sheng, HUANG Shihui, WANG Jiafu. Association Analysis Between Structural Variation of CYP3A29 Gene 3'-UTR and Androgen Level of Muscle Tissue in Xiang Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3199-3209.

参考文献

[1] BUȽA M,PRZYBYLSKI W,JAWORSKA D,et al.Formation of heterocyclic aromatic amines in relation to pork quality and heat treatment parameters[J].Food Chemistry,2018,276:511-519.
[2] LIYI W,SHU Z,YUQIN H,et al.CLA improves the lipo-nutritional quality of pork and regulates the gut microbiota in Heigai pigs[J].Food&Function,2022,13:12093-12104.
[3] 张婧.猪重要经济性状遗传参数估计[D].保定:河北农业大学,2020. ZHANG J.Estimation of genetic parameters of important economic traits in pigs[D].Baoding:Hebei Agricultural University,2020.(in Chinese)
[4] LIU F,BREWSTER C J,GILMOUR S L,et al.Relationship between energy intake and growth performance and body composition in pigs selected for low backfat thickness[J].Journal of Animal Science,2021,99(12):skab342.
[5] WEIDA W,JUNLIANG Z,XIAOYAN T,et al.Characterization and identification of pork flavor compounds and their precursors in Chinese indigenous pig breeds by volatile profiling and multivariate analysis[J].Food Chemistry,2022,385:132543.
[6] 李娜,张琪,刘洪亮,等.松辽黑猪与引入猪种肉品质性状的综合对比分析[J].黑龙江畜牧兽医,2022,16:55-59. LI N,ZHANG Q,LIU H L,et al.Comprehensive comparative analysis of meat quality traits between Songliao Black pigs and introduced pig breeds[J].Heilongjiang Animal Science and Veterinary Medicine,2022,16:55-59.(in Chinese)
[7] O'REILLY M W,KEMPEGOWDA P,WALSH M,et al.AKR1C3-mediated adipose androgen generation drives lipotoxicity in women with polycystic ovary syndrome[J].The Journal of Clinical Endocrinology and Metabolism,2017,102(9):3327-3339.
[8] TCHERNOF A,MANSOUR M F,PELLETIER M,et al.Updated survey of the steroid-converting enzymes in human adipose tissues[J].Journal of Steroid Biochemistry and Molecular Biology,2015,147:56-69.
[9] SCHIFFER L,ARLT W,O'REILLY M W.Understanding the role of androgen action in female adipose tissue[J].Frontiers of Hormone Research,2019,53:33-49.
[10] O'REILLY M W,HOUSE P J,TOMLINSON J W.Understanding androgen action in adipose tissue[J].Journal of Steroid Biochemistry and Molecular Biology,2014,143:277-284.
[11] ELFAKI I,MIR R,ALMUTAIRI F M,et al.Cytochrome P450:Polymorphisms and roles in cancer,diabetes and atherosclerosis[J].Asian Pacific Journal of Cancer Prevention,2018,19(8):2057-2070.
[12] MAKSYMCHUK O V,KASHUBA V I.Altered expression of cytochrome P450 enzymes involved in metabolism of androgens and vitamin D in the prostate as a risk factor for prostate cancer[J].Pharmacological Reports,2020,72(5):1161-1172.
[13] NISSEN P H,WINTERØ A K,FREDHOLM M.Mapping of porcine genes belonging to two different cytochrome P450 subfamilies[J].Animal Genetics,1998,29(1):7-11.
[14] 刘长存.猪CYP3A29羟化代谢T-2毒素的分子机制研究[D].武汉:华中农业大学,2013. LIU C C.Molecular mechanism study on the hydroxylation metabolism of T2 toxin by pig CYP3A29[D].Wuhan:Huazhong Agricultural University,2013.(in Chinese)
[15] YAO M,DAI M,LIU Z,et al.Comparison of the substrate kinetics of pig CYP3A29 with pig liver microsomes and human CYP3A4[J].Bioscience Reports,2011,31(3):211-220.
[16] LI X,HU X,JIN X,et al.IFN-γ regulates cytochrome 3A29 through pregnane X receptor in pigs[J].Xenobiotica,2014,45(5):373-379.
[17] 杨家大,吴声榕.利福平给药不改变从江香猪CYP3A29 mRNA的表达水平[J].凯里学院学报,2018,36(6):84-89. YANG J D,WU S R.Expression levels of CYP3A29 mRNA in Congjiang Xiang-pigs'tissues is not affected by rifampicin[J].Journal of Kaili University,2018,36(6):84-89.(in Chinese)
[18] SHUHAO F,CHENGCHENG K,YIGE C,et al.Copy number variation analysis revealed the evolutionary difference between Chinese indigenous pigs and Asian wild boars[J].Genes,2023,14(2):472.
[19] WRIGHT W C,CHENGE J,CHEN T.Structural perspectives of the CYP3A family and their small molecule modulators in drug metabolism[J].Liver Research,2019,3(3-4):132-142.
[20] 薛正楷,魏泓.重组肝CYP3A4与CYP3A29细胞微粒体药物代谢动力学比较[J].医药导报,2015,34(1):15-21. XUE Z K,WEI H.Comparison of pharmacokinetics between recombinant liver microsomes with CYP3A4 and CYP3A29[J].Herald of Medicine,2015,34(1):15-21.(in Chinese)
[21] JOACHIM W,ORSOLYA S,FRANÇOIS S,et al.Phenotypic impact of genomic structural variation:Insights from and for human disease[J].Nature Reviews Genetics,2013,14(2):125-138.
[22] PLATT R N 2 ND,VANDEWEGE M W,RAY D A.Mammalian transposable elements and their impacts on genome evolution[J].Chromosome Research,2018,26(1-2):25-43.
[23] VASSETZKY N S,BORODULINA O R,USTYANTSEV I G,et al.Analysis of SINE families B2,Dip,and Ves with special reference to polyadenylation signals and transcription terminators[J].International Journal of Molecular Sciences,2021,22(18):9897.
[24] MAQUAT L E.Short interspersed nuclear element (SINE)-mediated post-transcriptional effects on human and mouse gene expression:SINE-UP for active duty[J].Philosophical Transactions-Royal Society.Biological Sciences,2020,375(1795):20190344.
[25] YOUNG K M,JUNGYUN P,JOO L J,et al.Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3'-UTR[J].Nucleic Acids Research,2014,42(11):6999-7011.
[26] MENDELL J T.microRNAs:Critical regulators of development,cellular physiology and malignancy[J].Cell Cycle,2005,4(9):1179-1184.
[27] SCHIFFER L,ARLT W,STORBECK K.Intracrine androgen biosynthesis,metabolism and action revisited[J].Molecular and Cellular Endocrinology,2018,465:4-26.
[28] SEBO Z L,RODEHEFFER M S.Testosterone metabolites differentially regulate obesogenesis and fat distribution[J].Molecular Metabolism,2021,44:101141.
[29] ANDRÉ T,DANNICK B,INA M,et al.Androgens and the regulation of adiposity and body fat distribution in humans[J].Comprehensive Physiology,2018,8(4):1253-1290.
[30] 王亚兰.鸡雌雄胚蛋孵化期间甾体激素的变化及差异研究[D].武汉:华中农业大学,2019. WANG Y L.The chance rule and sex difference of steroid hormones in male and female hatched hen eggs[D].Wuhan:Huazhong Agricultural University,2019.(in Chinese)
[31] MANSOUR M F,PELLETIER M,TCHERNOF A.Characterization of 5α-reductase activity and isoenzymes in human abdominal adipose tissues[J].Journal of Steroid Biochemistry and Molecular Biology,2016,161:45-53.