大白猪MYOD1、AKT3基因启动子区多态性及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2019.12.004

摘要/Abstract

摘要： 为探究MYOD1和AKT3基因启动子区的多态性及其可能存在的影响基因表达的分子调控机制，试验采用PCR直接测序的方法对大白猪MYOD1和AKT3基因启动子区的多态性进行检测，同时利用生物信息学分析方法预测了大白猪MYOD1和AKT3基因的核心启动子区、CpG岛和转录因子结合域。结果显示，MYOD1基因共预测到5个核心启动子区、1个CpG岛区域和10个转录因子结合域，且第5个核心启动子区位于CpG岛区域内；AKT3基因共预测到6个核心启动子区，未发现CpG岛的存在。通过直接测序的方法检测到MYOD1基因在G-361T处存在1个SNP突变，但在本试验群体中只发现1种基因型，同时该突变位点位于第1个核心启动子区内；AKT3基因在启动子区T-1709C处存在1个SNP突变，包括TT、TC和CC 3种基因型，其中TT基因型为优势基因型，T为优势等位基因。遗传多态性分析提示，该突变位点多态信息含量（PIC）介于0.25~0.5之间，表现为中度多态。本研究初步探究了大白猪MYOD1和AKT3基因启动子区的多态性并预测了启动子区可能的调控因子和调控元件，为进一步研究MYOD1、AKT3基因对肌肉生长发育的调控机制及将突变位点作为遗传标记用于分子选育提供指导和依据。

关键词: 猪; MYOD1基因; AKT3基因; 启动子; 多态性; 生物信息学

Abstract: To investigate the polymorphism of the promoter regions of MYOD1 and AKT3 genes and the possible molecular regulatory mechanism that affected gene expression,PCR direct sequencing was used to detect the polymorphism in the promoter regions of MYOD1 and AKT3 genes in Large White pigs.At the same time,the core promoter region,CpG island and transcription factor binding domain of MYOD1 and AKT3 genes were predicted by bioinformatics analysis.The results showed that there were 5 core promoter regions,1 CpG island region and 10 transcription factor binding domains of MYOD1 gene,and the fifth core promoter region was located in the CpG island region.A total of 6 core promoter regions were predicted for AKT3 gene,and no CpG island was found.1 SNP mutation was detected in MYOD1 gene at G-361T site by direct sequencing,but only 1 genotype was found in the experimental population,and the mutation site was located in the first core promoter region.There was 1 SNP-mutation in the promoter region of AKT3 gene T-1709C site,including TT,TC and CC genotypes,of which TT was the dominant genotype,and T was the dominant allele.Genetic polymorphism analysis indicated that the PIC value of this mutation site was 0.25 to 0.5,showing moderate polymorphism.This study preliminarily explored the polymorphism of MYOD1 and AKT3 genes promoter region and predicted the possible regulatory factors and regulatory elements in the promoter region,which would provide guidance and basis for the further study of the regulatory mechanisms of the MYOD1 and AKT3 genes for muscle growth and development and the use of mutation sites as genetic markers for molecular breeding.

Key words: pig; MYOD1 gene; AKT3 gene; promoter; polymorphism; bioinformatics analysis

中图分类号:

王飞, 宗秋芳, 慕京生, 吴圣龙, 包文斌. 大白猪MYOD1、AKT3基因启动子区多态性及生物信息学分析[J]. 中国畜牧兽医, 2019, 46(12): 3486-3494.

WANG Fei, ZONG Qiufang, MU Jingsheng, WU Shenglong, BAO Wenbin. Polymorphism and Bioinformatics Analysis of Promoter Regions of MYOD1 and AKT3 Genes in Large White Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3486-3494.

参考文献

[1] PAS M F W T,HARDERS F L,SOUMILLION A,et al.Genetic variation at the porcine MYF-5 gene locus.Lack of association with meat production traits[J].Mammalian Genome,1999,10(2):123-127.
[2] NAIDU P S,LUDOLPH D C,TO R Q,et al.Myogenin and MEF2 function synergistically to activate the MRF4 promoter during myogenesis[J].Molecular and Cellular Biology,1995,15(5):2707-2718.
[3] BERKES C A,TAPSCOTT S J.MyoD and the transcriptional control of myogenesis[J].Seminars in Cell & Developmental Biology,2005,16(4):585-595.
[4] WHITEMAN E L,CHO H,BIRNBAUM M J.Role of Akt/protein kinase B in metabolism[J].Trends in Endocrinology & Metabolism,2002,13(10):444-451.
[5] BELLACOSA A,TESTA,STAAL S,et al.A retroviral oncogene,AKT,encoding a serine-threonine kinase containing an SH2-like region[J].Science,1991,254(5029):274-277.
[6] HERS I,VINCENT E E,TAVARÉ J M.Akt signalling in health and disease[J].Cellular Signalling,2011,23(10):1515-1527.
[7] JONES P F,JAKUBOWICZ T,PITOSSI F J,et al.Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(10):4171-4175.
[8] LEE E A,KIM J M,LIM K S,et al.Effects of variation in porcine MYOD1 gene on muscle fiber characteristics,lean meat production,and meat quality traits[J].Meat Science,2012,92(1):36-43.
[9] URBAŃSKI P,PIERZCHAŁA M,KAMYCZEK M,et al.Relations between the polymorphism in the coding and 5'-flanking regions of the porcine MYOD1 and MYF5 genes and selected productive traits in line 990 gilts[J].Journal of Animal & Feed Sciences,2006,15(2):225-235.
[10] URBAŃSKI P,KURY J.Two new SNPs within exon 1 of the porcine MYOD1(MYF3) gene and their frequencies in chosen pig breeds and lines[J].Journal of Animal Breeding & Genetics,2004,121(3):204-208.
[11] VERNER J,HUMPOLICEK P,KNOLL A.Impact of MYOD family genes on pork traits in Large White and Landrace pigs[J].Journal of Animal Breeding & Genetics,2015,124(2):81-85.
[12] URBAŃSKI P,KURYŁ J.New SNPs in the coding and 5'-flanking regions of porcine MYOD1(MYF3) and MYF5 genes[J].Journal of Applied Genetics,2004,45(3):325-329.
[13] HAN X,JIANG T,YANG H,et al.Investigation of four porcine candidate genes (H-FABP,MYOD1,UCP3 and MASTR) for meat quality traits in Large White pigs[J].Molecular Biology Reports,2012,39(6):6599-6605.
[14] 曹婷,侯冠彧,施力光,等.五指山猪不同组织中Myf5与MyoD1基因的表达研究[J].家畜生态学报,2012,33(5):12-15. CAO T,HOU G Y,SHI L G,et al.Research on expression of Myf5 and MyoD1 genes in different tissues of Wuzhishan pig[J].Journal of Domestic Animal Ecology,2012,33(5):12-15.(in Chinese)
[15] CHO H,THORVALDSEN J L,CHU Q,et al.Akt1/PKBα is required for normal growth but dispensable for maintenance of glucose homeostasis in mice[J].Journal of Biological Chemistry,2001,276(42):38349-38352.
[16] WESSELINK J J.Molecular cloning,expression and characterization of the human serine/threonine kinase Akt-3[J].FEBS Journal,2010,265(1):353-360.
[17] 刘庆礼,李娟,王亚军.猪Akt3基因克隆序列分析及组织表达图谱研究[J].四川大学学报(自然科学版),2014,51(4):815-820. LIU Q L,LI J,WANG Y J.Molecular cloning sequence analysis and tissue expression of Akt3 gene in pigs (Sus scrofa)[J].Journal of Sichuan University (Natural Science Edition),2014,51(4):815-820.(in Chinese)
[18] LEE E A,KIM J M,LIM K S,et al.Effects of variation in porcine MYOD1 gene on muscle fiber characteristics,lean meat production,and meat quality traits[J].Meat Science,2012,92(1):36-43.
[19] 奉玲丽,张瑞门,黄叶,等.广西巴马小型猪MyoD1基因克隆及其真核表达载体的构建[J].中国畜牧兽医,2019,46(4):977-985. FENG L L,ZHANG R M,HUANG Y,et al.Cloning and eukaryotic expression vector construction of MyoD1 gene in Guangxi Bama mini-pig[J].China Animal Husbandry & Veterinary Medicine,2019,46(4):977-985.(in Chinese)
[20] 张雄,张勇,陈祥,等.猪肌分化因子1基因启动子区多态性及生物信息学研究[J].中国畜牧兽医,2016,43(5):1308-1315. ZHANG X,ZHANG Y,CHEN X,et al.Study on polymorphisms and bioinformatics of MYOD1 gene promoter region in pig[J].China Animal Husbandry & Veterinary Medicine,2016,43(5):1308-1315.(in Chinese)
[21] 陈亚楠.PDK1、FoxO3和Akt3基因多态性对大白猪生产性能的影响[D].石河子:石河子大学,2015. CHEN Y N.Effects of polymorphisms of PDK1,FoxO3 and Akt3 gene on production performances in Yorkshire[D].Shihezi:Shihezi University,2015.(in Chinese)
[22] 王利娜,陈仕毅,贾先波,等.家兔PIK3CA和AKT3基因多态性及其与生长性状的关联性分析[J].四川农业大学学报,2016,34(3):374-380. WANG L N,CHEN S Y,JIA X B,et al.Polymorphisms of PIK3CA and AKT3 genes and their association with growth traits of rabbits[J].Journal of Sichuan Agricultural University,2016,34(3):374-380.(in Chinese)
[23] 黄龙,吴本丽,何吉祥,等.中华鳖MyoD1基因SNP鉴定及其与生长性状的关联分析[J].生物技术通报,2019,35(4):76-81. HUANG L,WU B L,HE J X,et al.SNP identification of MyoD1 gene and its correlation with growth traits in Pelodiscus sinensis[J].Biotechnology Bulletin,2019,35(4):76-81.(in Chinese)
[24] ZHAO Z,FU Y X,HEWETT-EMMETT D,et al.Investigating single nucleotide polymorphism (SNP) density in the human genome and its implications for molecular evolution[J].Gene,2003,312:207-213.
[25] NIE Q,LEI M,OUYANG J,et al.Identification and characterization of single nucleotide polymorphisms in 12 chicken growth-correlated genes by denaturing high performance liquid chromatography[J].Genetics Selection Evolution,2005,37(3):339-360.
[26] 樊佳佳,刘小献,白俊杰,等.草鱼柠檬酸合酶基因SNP筛选及与生长性状的关联分析[J].华中农业大学学报,2014,33(3):84-89. FAN J J,LIU X X,BAI J J,et al.Detection of SNP incitrate synthase gene and association analysis with growth traits in grass carp (Ctenopharyngodon idellus)[J].Journal of Huazhong Agricultural University,2014,33(3):84-89.(in Chinese)
[27] DELAVAL K,FEIL R.Epigenetic regulation of mammalian genomic imprinting[J].Current Opinion in Genetics & Development,2004,14(2):188-195.
[28] PERCHARDE M,COLEY H M,WEBB A,et al.The context and potential of epigenetics in oncology[J].British Journal of Cancer,2009,100(4):571-577.
[29] 周迪,陈伟,赵佳福,等.地方黄牛MyoD1基因启动子多态性分析及其相关转录因子SF1验证[J].农业生物技术学报,2018,26(5):743-755. ZHOU D,CHEN W,ZHAO J F,et al.Polymorphism analysis of MyoD1 gene promoter and related SF1 transcription factors validation in local cattle (Bos taurus)[J].Journal of Agricultural Biotechnology,2018,26(5):743-755.(in Chinese)