牦牛酪蛋白基因家族的克隆及组织表达分析

doi:10.16431/j.cnki.1671-7236.2019.01.007

摘要/Abstract

摘要：

本研究旨在克隆牦牛酪蛋白基因家族（CSN1S1、CSN1S2、CSN2和CSN3）的CDS区序列，鉴定其在牦牛不同组织中的表达水平。选取4岁龄左右处于泌乳期的健康类乌齐母牦牛3头，屠宰后分别采集乳腺、心脏、肝脏、骨骼肌组织，分别提取组织总RNA并反转录为cDNA，设计酪蛋白基因家族特异性引物扩增酪蛋白基因家族序列，进行生物信息学分析，并利用实时荧光定量PCR法分别检测酪蛋白家族基因mRNA水平。结果显示，克隆得到CSN1S1、CSN1S2、CSN2和CSN3基因cDNA序列分别为919、832、805和715 bp，其CDS区全长分别为645、669、690和585 bp，分别编码214、222、259和194个氨基酸残基。类乌齐牦牛酪蛋白基因家族与黄牛亲缘关系最近，其次是印度水牛，而与单胃动物猪的亲缘关系最远。组织表达结果显示，酪蛋白基因家族在组织中广泛表达，其中在乳腺组织中的表达量最高，其次是骨骼肌组织。在乳腺组织中CSN1S1、CSN1S2、CSN2基因之间表达量差异不显著（P > 0.05），但CSN2基因表达量显著高于CSN3基因（P < 0.05）。以上结果为酪蛋白基因家族在牦牛乳腺蛋白质代谢调控机制的研究提供了参考依据。

关键词: 牦牛; 酪蛋白基因家族; 克隆; 组织表达

Abstract:

The aim of this study was to clone the CDS of casein family genes (CSN1S1,CSN1S2,CSN2 and CSN3),and determine their expression in various tissues.Each of 3 healthy,about 4 years old Leiwuqi yak were selected.After slaughter,the tissues of mammary gland,heart,liver and skeletal muscle were collected for the total RNA extraction.The primers for casein family genes were designed and used for gene cloning,and then the sequences were analyzed using bioinformatics.The mRNA expression level of casein family genes were determined using Real-time PCR.The cDNA length of CSN1S1,CSN1S2,CSN2 and CSN3 genes were 919,832,805 and 715 bp,which containing 645,669,690 and 585 bp CDS and encoding 214,222,259 and 194 amino acids,respectively.The yak casein family was closest associated with that of Bos grunniens,followed by Bubalus bubalis,and distant with Sus scrofa.The tissue expression analysis showed that casein family genes were widely expressed in multiply tissues,which had the highest expression level in mammary gland,and followed in skeletal muscle.In addition,there was no difference among CSN1S1,CSN1S2 and CSN2 genes (P > 0.05),but the expression level of CSN2 gene was significantly higher than that of CSN3 gene in mammary gland (P < 0.05).These data provided basic information for the functional research of casein family genes in the regulation of protein metabolism in yak mammary gland,and laid a foundation for the development and utilization of yak genetic resources.

Key words: yak; casein family genes; cloning; tissue expression

中图分类号:

S823.85⁺5

王会, 柴志欣, 钟金城, 朱江江, 张成福. 牦牛酪蛋白基因家族的克隆及组织表达分析[J]. 《中国畜牧兽医》, 2019, 46(1): 54-61.

WANG Hui, CHAI Zhixin, ZHONG Jincheng, ZHU Jiangjiang, ZHANG Chengfu. Cloning and Tissue Expression of Casein Family Genes in Yak[J]. , 2019, 46(1): 54-61.

参考文献

[1] YU Q,HAN L,JIANG Y,et al.Analysis of the nutritional components and flavorous substances of White yak's milk[J].Acta Nutrimenta Sinica,2005,27(4):333-335.
[2] 李海梅,何胜华,刘天一,等.牦牛乳物理化学特性的研究进展[J].中国乳品工业,2009,37(8):35-40. LI H M,HE S H,LIU T Y,et al.Research developmentin physical and chemical characters of yak milk[J].China Dairy Industry,2009,37(8):35-40.(in Chinese)
[3] STERGIADIS S,LEIFERT C,SEAL C J,et al.Effect of feeding intensity and milking system on nutritionally relevant milk components in dairy farming systems in the North East of England[J].Journal of Agricultural and Food Chemistry,2012,60(29):7270-7281.
[4] 刘冬,黄玉军,赵海晴,等.牦牛乳蛋白质组成及特性[J].乳业科学与技术,2013,36(3):20-23. LIU D,HUANG Y J,ZHAO H Q,et al.Protein composition and characteristics of yak milk[J].Journal ofDairy Sciencce and Technology,2013,36(3):20-23.(in Chinese)
[5] LI H,MA Y,DONG A,et al.Protein composition of yak milk[J].Dairy Science and Technology,2010,90(1):111-117.
[6] SINGH L V,JAYAKUMAR S,SHARMA A,et al.Comparative screening of single nucleotide polymorphisms in β-casein and κ-casein gene in different livestock breeds of India[J].Meta Gene,2015,4(4):85-91.
[7] ZEPEDABATISTA J L,SAAVEDRAJIMÉNEZ L A,RUÍZFLORES A,et al.Potential influence of κ-casein and β-lactoglobulin genes in genetic association studies of milk quality traits[J].Asian-Australasian Journal of Animal Sciences,2017,30(12):1684-1688.
[8] ARDICLI S,SOYUDAL B,SAMLI H,et al.Effect of STAT1, OLR1,CSN1S1,CSN1S2 and DGAT1 genes on milk yield and quality traits in Holstein breed[A].International Scientific Agriculture Symposium[C].2017.
[9] BAI W L,YIN R H,DOU Q L,et al.A single nucleotide polymorphism and sequence analysis of CSN1S1gene promoter region in Chinese Bos grunniens(yak)[J].Animal Biotechnology,2010,21(1):36-41.
[10] CUI Y,YU T,QU X,et al.Genetic variation in the αS1-casein of Chinese yak (Bos grunniens)[J].European Food Research & Technology,2014,239(1):175-181.
[11] GROBLER R,VISSER C,CHESSA S,et al.Genetic polymorphism of CSN1S2 in South African dairy goat populations[J].South African Journal of Animal Science,2017,47(1):72-78.
[12] COSENZA G,IANNACCONE M,PICO B A,et al.The SNP g.1311T>C associated with the absence of β-casein in goat milk influences CSN2 promoter activity[J].Animal Genetics,2016,47(5):615-617.
[13] KUSZA S,ILIE D E,SAUER M,et al.Genetic polymorphism of CSN2 gene in Banat White and Carpatina goats[J].Acta Biochimica Polonica,2016,63(3):577-580.
[14] PATEL J B,CHAUHAN J B.Computational analysis of non-synonymous single nucleotide polymorphism in the bovine cattle kappa-casein (CSN3) gene[J].Meta Gene,2017,15:1-9.
[15] 胡言,桂林生,余横伟,等.中国荷斯坦牛CSN3基因多态性及其与泌乳性状的关联性[J].西北农林科技大学学报(自然科学版),2018,46(3):8-15. HU Y,GUI L S,YU H W,et al.Association between polymorphism of CSN3 gene and milk production traits of Chinese Holstein cattle[J].Journal of Northwest A&F University (Natural Science Edition),2018,46(3):8-15.(in Chinese)
[16] QU X,CUI Y,YU T,et al.Detection of αS2-casein variants in Chinese yak (Bos grunniens) by PCR-SSCP[J].European Food Research and Technology,2012,234(3):551-556.
[17] LI H,MA Y,DONG A,et al.Protein composition of yak milk[J].Dairy Science & Technology,2010,90(1):111-117.
[18] PANDYA G,RAMANI U,JANMEDA M,et al.Relative gene expression analysis of β-casein gene and its transcription regulatory genes in primary buffalo mammary epithelial cells of Surti and Jaffarabadi buffaloes[J].The Indian Journal of Animal Science,2018,88(3):319-321.
[19] HAYES J F,NG-KWAI-HANG K F,MOXLEY J E.Heritability of milk casein and genetic and phenotypic correlations with production traits[J].Journal of Dairy Science,1984,67(4):841-846.
[20] 李玲.CSN1S2、CSN2、CSN3基因在西农萨能奶山羊乳腺组织中的表达分析[D].杨凌:西北农林科技大学,2009. LI L.Expression analysis of CSN1S2,CSN2 and CSN3 genes in mammary gland of Xinong Saanen dairy goat[D].Yangling:Northwest A & F University,2009.(in Chinese)
[21] 蒲俊华,朱小瑞,绪欣,等.奶牛原代乳腺上皮细胞的培养及β酪蛋白mRNA的表达[J].中国畜牧兽医,2017,44(7):1975-1981. PU J H,ZHU X R,XU X,et al.Culture of dairy cow primary mammary epithelial cells and expression ofβ-casein mRNA[J]. China Animal Husbandry & Veterinary Medicine,2017,44(7):1975-1981.(in Chinese)
[22] WANG W,LUO J,ZHONG Y,et al.Goat liver X receptor α,molecular cloning,functional characterizationand regulating fatty acid synthesis in epithelial cells of goat mammary glands[J].Gene,2012,505(1):114-120.
[23] BEVILACQUA C,HELBLING J C,MIRANDA G,et al.Translational efficiency of casein transcripts in the mammary tissue of lactating ruminants[J].Reproduction Nutrition Development,2006,46(5):567-578.
[24] ROGERS J T.Ferritin translation by interleukin-1 and interleukin-6:The role of sequences upstream ofthe start codons of the heavy and light subunit genes[J].Blood,1996,87(6):2525-2537.
[25] DIAZ A D A,NODERER W L,WANG C L.Complete motif analysis of sequence requirements for translation initiation at non-AUG start codons[J]. Nucleic Acids Research,2018,46(2):985-994.
[26] BUGAUT A,BALASUBRAMANIAN S.5'-UTR RNA G-quadruplexes:Translation regulation and targeting[J].Nucleic Acids Research,2012,40(11):4727-4741.
[27] JOLIVET G,MEUSNIER C,CHAUMAZ G,et al.Extracellular matrix regulates alpha s1-casein gene expression in rabbit primary mammary cells and CCAAT enhancer binding protein (C/EBP) binding activity[J].Journal of Cellular Biochemistry,2001,82(3):371-386.
[28] LECHNER J,WELTE T,TOMASI J K,et al.Promoter-dependent synergy between glucocorticoid receptor and Stat5 in the activation of beta-casein gene transcription[J].Journal of Biological Chemistry,1997,272(33):20954-20960.
[29] GEYMAYER S,DOPPLER W.Activation of NF-kappa B p50/p65 is regulated in the developing mammary gland and inhibits STAT5-mediated beta-casein gene expression[J].The Faseb Journal,2000,14(9):1159-1170.
[30] LENASI T,KOKALJ-VOKAC N,NARAT M,et al.Functional study of the equine beta-casein and kappa-casein gene promoters[J].Journal of Dairy Research,2005,72(S1):34-43.