广灵驴DGAT2基因克隆、生物信息学分析及组织表达研究

doi:10.16431/j.cnki.1671-7236.2021.02.001

摘要/Abstract

摘要： 本研究旨在对广灵驴二酰基甘油酰基转移酶2（DGAT2）基因进行克隆，生物信息学分析和检测其在不同组织中的表达情况，为探究DGAT2基因在广灵驴脂肪沉积和提高乳脂率等方面的作用机制提供理论参考。根据GenBank上公布的马（登录号：XM_023645689.1）、双峰驼（登录号：XM_010973154.1）、绵羊（登录号：XM_027979550.1）等物种的DGAT2基因mRNA序列，利用Primer Premier 3.0在线工具设计同源引物，应用RT-PCR法扩增DGAT2基因序列，用生物信息学方法分析DGAT2基因编码序列，用实时荧光定量PCR技术检测DGAT2基因在广灵驴心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、肌间脂肪、皮下脂肪组织中的表达。结果显示，广灵驴DGAT2基因CDS序列1 086 bp，编码361个氨基酸，提交到GenBank，获得登录号：MT993643，其编码序列与马、牛、双峰驼、猪、绵羊、人、小鼠的同源性分别为99.0%、92.0%、93.5%、92.0%、92.7%、85.3%、84.1%。系统进化树分析表明，驴与马的亲缘关系最近，和小鼠的关系最远。DGAT2蛋白分子质量40.96 ku，脂肪系数92.85，等电点9.16，是一种具有跨膜区的稳定碱性疏水蛋白。DGAT2蛋白有28个磷酸化修饰位点，2个糖基化修饰位点，没有信号肽，主要定位在内质网，α-螺旋（39.89%）和无规则卷曲（36.01%）是主要的二级结构。DGAT2基因在检测的8个组织中都有表达，其中皮下脂肪中的表达量显著高于其余组织（P<0.05），其次是心脏、肝脏和肾脏，背最长肌中的表达量最低。本试验结果为探究DGAT2基因在广灵驴脂肪沉积和提高乳品质性状的作用奠定基础。

关键词: DGAT2基因; 广灵驴; 基因克隆; 生物信息学分析; 组织表达

Abstract: The study was aimed to clone,bioinformatics of diacylglycerol acyltransferase 2 (DGAT2) gene of Guangling donkey,detected its expression in different tissues,and provided theoretical reference for exploring the mechanism of DGAT2 gene in fat deposition and improving milk fat rate of Guangling donkey.Homologous primers was designed by Primer Premier 3.0 according to the mRNA sequences of DGAT2 gene of Equus caballus (accession No.:XM_023645689.1),Camelus bactrianus (accession No.:XM_010973154.1), Ovis aries(accession No.:XM_027979550.1),and other species published in GenBank.DGAT2 gene sequence of Guangling donkey was amplified with RT-PCR.The encoding sequence of DGAT2 gene was analyzed by bioinformatics methods,and the expression of DGAT2 gene in heart,liver,spleen,lung,kidney,longissimus dorsi muscle,intermuscular fat and subcutaneous fat of Guangling donkey was detected by Real-time quantitative PCR.The results showed that the CDS of Guangling donkey DGAT2 gene consisted of nucleotides of 1 086 bp encoding 361 amino acids.It was submitted to NCBI and the login number was MT993643.The coding sequence of DGAT2 gene showed 99.0%,92.0%,93.5%,92.0%,92.7%,85.3% and 84.1% identity with that of Equus caballus,Bos Taurus,Camelus bactrianus,Sus scrofa,Ovis aries,Homo sapiens and Mus musculus.Phylogenetic tree analysis revealed that Guangling donkey was most closely related to Equus caballus and farthest related to Mus musculus.The DGAT2 protein,with molecular weight of 40.96 ku,fat coefficient of 92.85 and isoelectric point of 9.16,was a stable basic hydrophobic protein with transmembrane region.There were 28 phosphorylation modification sites and 2 glycosylation modification sites in DGAT2 protein,with no signal peptide,it was mainly located in the endoplasmic reticulum.The secondary structure of DGAT2 protein was mainly of 39.89% alpha helix and 36.01% random coil.The DGAT2 gene was expressed in 8 tissues,among which the expression level in subcutaneous fat was significantly higher than that in other tissues (P<0.05),followed by heart,liver and kidney,and the lowest level was in the longissimus dorsi muscle.The experiment results provided a solid theoretical basis for further exploring the role of DGAT2 gene in fat deposition and improving milk quality traits in Guangling donkey.

Key words: DGAT2 gene; Guangling donkey; gene cloning; bioinformatics analysis; tissue expression

中图分类号:

S822

李武峰, 关家伟, 孙瑜彤, 邱丽霞, 杜敏. 广灵驴DGAT2基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2021, 48(2): 407-416.

LI Wufeng, GUAN Jiawei, SUN Yutong, QIU Lixia, DU Min. Cloning,Bioinformatics and Tissue Expression Analysis of DGAT2 Gene in Guangling Donkey[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 407-416.

参考文献

[1] GRISART B,COPPIETERS W,FARNIR F,et al.Positional candidate cloning of a QTL in dairy cattle:Identification of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition[J].Genome Research,2002,12(2):222-231.
[2] 吴东旺,孙丽媛,袁再美,等.动物DGAT基因的研究进展[J].中国畜牧兽医,2019,46(7):1945-1952. WU D W,SUN L Y,YUAN Z M,et al.Research progress on animal DGAT gene[J].China Animal Husbandry & Veterinary Medicine,2019,46(7):1945-1952.(in Chinese)
[3] TAKEMOTO K,FUKASAKA Y,YOSHIMOTO R,et al.Diacylglycerol acyltransferase 1/2 inhibition induces dysregulation of fatty acid metabolism and leads to intestinal barrier failure and diarrhea in mice[J].Physiological Reports,2020,8(15):e14542.
[4] AN X P,SONG S G,HOU J X,et al.Polymorphism identification in goat DGAT2 gene and association analysis with milk yield and fat percentage[J].Small Ruminant Research,2011,100(2-3):107-112.
[5] ZHOU F,ZHANG Y,TENG X,et al.Identification,molecular characteristics,and tissue differential expression of DGAT2 full-CDS cDNA sequence in Binglangjiang buffalo (Bubalus bubalis)[J].Archives Animal Breeding,2020,63(1):81-90.
[6] MUOZ M,GARCÍA-CASCO J M,CARABALLO C,et al.Identification of candidate genes and regulatory factors underlying intramuscular fat content through longissimus dorsi transcriptome analyses in heavy Iberian pigs[J].Frontiers in Genetics,2018,9:608.
[7] 王颜颜,托乎提·阿及德,肖海霞,等.新疆良种驴DGAT2基因第3内含子PCR-SSCP多态性与体尺性状的相关性分析[J].石河子大学学报(自然科学版),2011,29(1):40-44. WANG Y Y,TUOHU T,XIAO H X,et al.PCR-SSCP polymorphism of intron 3 of DGAT2 gene of fine-breed Xinjiang donkeys[J].Journal of Shihezi University(Natural Science),2011,29(1):40-44.(in Chinese)
[8] MAO H G,DONG X Y,CAO H Y,et al.Association of DGAT2 gene polymorphisms with carcass and meat quality traits in domestic pigeons (Columba livia)[J].British Poultry Science,2018,59(2):149-153.
[9] HU J,HI B,IE J,et al.Tissue expression and variation of the DGAT2 gene and its effect on carcass and meat quality traits in yak[J].Animals,2019,9(2):61.
[10] 田方圆,王台安,李艳敏,等.鸡MOGAT和DGAT基因的克隆及表达特性研究[J].畜牧兽医学报,2017,48(2):214-224. TIAN F Y,WANG T A,LI Y M,et al.Cloning and expression characteristics of MOGAT and DGAT genes in chicken(Gallus gallus)[J].Acta Veterinaria et Zootechnica Sinica,2017,48(2):214-224.(in Chinese)
[11] 邓章超,赵娟娟,夏琴,等.陆川猪DGAT2基因克隆、序列分析及表达水平研究[J].黑龙江畜牧兽医,2019,21:6-10. DENG Z C,ZHAO J J,XIA Q,et al.Cloning,sequence analysis and expression of DGAT2 gene in Luchuan pigs[J].Heilongjiang Animal Science and Veterinary Medicine,2019,21:6-10.(in Chinese)
[12] 麻延峰,方倩倩,王宏艳等.鹅Apo A1,CD36和DGAT2基因的cDNA克隆与序列分析[J].浙江农业学报,2015,27(10):1710-1719. MA Y F,FANG Q Q,WANG H Y,et al.cDNA cloning and sequence analysis of Apo A1,CD36 and DGAT2 genes from goose[J].Acta Agriculture Zhejiangensis,2015,27(10):1710-1719.(in Chinese)
[13] MCFIE P J,BANMAN S L,KARY S,et al.Murine diacylglycerol acyltransferase-2(DGAT2) can catalyze triacylglycerol synthesis and promote lipid droplet formation independent of its localization to the endoplasmic reticulum[J].Journal of Biological Chemistry,2011,286(32):28235-28246.
[14] 徐畅,庞喆,耿爽,等.牛二酰甘油酰基转移酶2基因(DGAT2)研究进展[J].畜牧与饲料科学,2016,37(Z1):4-7. XU C,PANG Z,GENG S,et al.Research progress on bovine diacylglycerol acyltransferase gene(DGAT2)[J].Animal Husbandry and Feed Science,2016,37(Z1):4-7.(in Chinese)
[15] HU S,GAO S,ZHU J,et al.Differential actions of diacylglycerol acyltransferase (DGAT) 1 and 2 in regula-ting lipid metabolism and progesterone secretion of goose granulosa cells[J].The Journal of Steroid Bioche-mistry and Molecular Biology,2020,202:105721.
[16] AL-SHUHAIB M B,AL-THUWAINI T M,AL-FIHAN R A,et al.Genotyping of diacylglycerol acyltransferase 2 gene in Holstein cattle popula-tion[J].Agriculturae Conspectus Scientificus,2019,84(2):211-218.
[17] 吕亚宁,贺琛昕,兰旅涛,等.猪肌内脂肪与肉品质的关系及其影响因素的研究进展[J].中国畜牧兽医,2020,47(2):554-563. LYU Y N,HE C X,LAN L T,et al.Research advances on the relationship between intramuscular fat and meat quality and influence factor of intramuscular fat in pigs[J].China Animal Husbandry & Veterinary Medicine,2020,47(2):554-563.(in Chinese)
[18] 姚怀兵,李玲,马强,等.哺乳动物DGAT基因与泌乳性状关联性研究进展[J].家畜生态学报,2020,41(4):1-7. YAO H B,LI L,MA Q,et al.Advances on the relationship between mammalian DGAT gene and lactation traits[J].Journal of Domestic Animal Ecology,2020,41(4):1-7.(in Chinese)
[19] 房兴堂,徐海霞,陈宏,等.徐淮白山羊DGAT2基因内含子3多态性与生长性状关联分析[J].生物技术通讯,2009,20(6):814-817. FANG X T,XU H X,CHEN H,et al.Association analysis on polymorphisms of DGAT2 gene intron 3 with growth traits in Xuhuai White goat[J].Letters in Biotechnology,2009,20(6):814-817.(in Chinese)
[20] 王国富,高树新,张立凡,等.金华猪DGAT1、DGAT2基因发育性表达特点及其与肌内脂肪的关联分析[J].华北农学报,2011,26(5):25-28. WANG G F,GAO S X,ZHANG L F,et al.Developmental expression characteristics of DGAT1 and DGAT2 gene and its association analysis with intramuscular fat in Jinhua pig[J].Acta Agriculturae Boreali-Sinica,2011,26(5):25-28.(in Chinese)
[21] ROE N D,HANDZLIK M K,LI T,et al.The role of diacylglycerol acyltransferase (DGAT) 1 and 2 in cardiac metabolism and function[J].Scientific Reports,2018,8(1):4983.
[22] AMIN N B,CARVAJAL-GONZALEZ S,PURKAL J,et al.Targeting diacylglycerol acyltransferase 2 for the treatment of nonalcoholic steatohepatitis[J].Science Translational Medicine,2019,11(520):v9701.
[23] VAZIRI N D,KIM C H,DANG B,et al.Downregulation of hepatic acyl-CoA:Diglycerol acyltransferase in chronic renal failure[J].American Journal of Physiology,2004,287(1):F90-F94.