HEREDITAS (Beijing) 2009 2, 31(2): 169 174 ISSN 0253-9772 www.chinagene.cn 研究报告 DOI: 10.3724/SP.J.1005.2009.00169 荷斯坦牛 HSP70-1 基因遗传多态性与乳腺炎抗性关系 1, 程维杰 2, 李秋玲 1, 王长法 1, 王洪梅 1, 李建斌 1, 孙延鸣 2, 仲跻峰 1., 250100; 2., 832000 1 以 253 头中国荷斯坦奶牛为研究对象, 检测 HSP70-1 基因的多态性, 并分析其多态性与中国荷斯坦牛体细胞评分 (Somatic cell score, SCS) 的相关性 首先以 PCR-SSCP 法寻找 HSP70-1 基因编码区的突变, 并通过测序确定突变的类型, 根据突变类型寻找合适的内切酶, 最终采用 PCR-RFLP 方法鉴定实验牛基因型 ; 然后分析基因多态性与中国荷斯坦牛 SCS 的相关性 结果表明 HSP70-1 基因的 1 623 bp 处产生 G A C 突变, 2 409 bp 处产生 G A 突变, 两位点都是沉默突变, 未引起氨基酸序列的改变 ; 经 χ 2 适合性检验, 中国荷斯坦牛在两个位点均未达到 Hardy-Weinberg 平衡状态 ; 同时, 群体基因座不同基因型与 SCS 相关分析的结果表明, 2409 位点基因型与 SCS 相关性不显著 (P>0.05), 1623 位点基因型与 SCS 相关性显著 (P<0.05), CC 型 SCS 显著低于 AG GG 型 (P<0.05), CC 基因型为乳腺炎抗性基因型 在中国荷斯坦奶牛群体中, HSP70-1 基因 CC 基因型可作为改良奶牛乳腺炎抗性性状的分子遗传标记 热休克蛋白 70; 限制性片段长度多态性 ; 单链构象多态性 ; 体细胞评分 ; 乳腺炎抗性 Genetic polymorphism of HSP70-1 gene and its correlation with resistance to mastitis in Chinese Holstein CHENG Wei-Jie 1, 2, LI Qiu-Ling 1, WANG Chang-Fa 1, WANG Hong-Mei 1, LI Jian-Bin 1, SUN Yan-Ming 2, ZHONG Ji-Feng 1 1. Dairy Cattle Rearch Center, Shandong Academy of Agricultural Science, Ji nan 250100, China; 2. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China Abstract: The polymorphisms of HSP70-1 gene in 253 Chinese Holstein dairy cows were studied, and the association between the polymorphisms and somatic cell score (SCS) were analyzed. PCR-SSCP, PCR-RFLP and DNA sequencing were used to investigate mutations in the coding region of HSP70-1 gene. The G A C mutation at 1 623 bp and G A mutation at 2 409 bp were found and both of them were silence mutations that caused no alteration in amino acid sequence. Chi-square test showed both loci weren t at Hardy-Weinberg disequilibrium in Chinese Holstein. In the meanwhile, the 收稿日期 : 2008 04 17; 修回日期 : 2008 06 27 基金项目 : (863 )( 2006AA10Z1D9, 2007AA10Z169), ( 2006YCX028) 作者简介 : (1979 ),,, E-mail: taijichi@126.com 通讯作者 : (1964 ),,,, E-mail: sdox2@163.com (1965 ),,,, E-mail: sym@shzu.edu.cn
170 HEREDITAS (Beijing) 2009 31 association of 2 409 locus and SCS was not significant. However, the polymorphism at 1623 locus affected SCS significantly (P<0.05). The SCS of genotype CC was significantly lower than that of genotype AG and GG (P<0.05), so genotype CC was mastitis resistant. These results suggest that genotype CC of HSP70-1 gene may be used as a molecular and genetic marker to improve the phenotype of anti-mastitis in Chinese Holstein dairy cows. Keywords: HSP70; restriction fragment length polymorphism; single-strand conformation polymorphism; somatic cell score; mastitis resistance,,,, [1],,,, (Somatic cell score, SCS) (Somaticcell count, SCC),, [2] 70(Heat shock protein 70, HSP70),,, HSP70, ; IL21 TGF2α, (SOD),, HSP70, HSP70 (HSP70-1 HSP70-2), 23q13,, 641 GenBank HSP70-1 HSP70-2 2, 3 HSP70-1, PCR-RFLP PCR-SSCP, SCS,, 1 1.1 1.1.1 实验样本 5, 253,, 3.8%, 20 1.1.2 主要试剂 Taq DNA ; EcoR Bgl Fok ( ) 1.2 1.2.1 基因组 DNA 的提取 - DNA 1.2.2 引物设计和 PCR 扩增 NCBI AY149618 HSP70-1 (19 526 bp) 9 HSP70-1 HSP70-2,, 3, HSP70-2, HSP70-1 3 PCR 2 000, 8 HSP70-1, PCR-SSCP, 200~450 bp 1 25 μl, : 50 ng 10 mmol/l dntp 1 μl l0 PCR buffer 2.5 μl
2 : HSP70-1 171 l0 μmol/l 1 μl 25 mmol/l Mg 2+ 1.5 μl Taq 2 U, 25 μl PCR : 94 2 min; 94 30 s, 52~58 30 s, 72 30 s, 30 ; 72 5 min 1.2.3 PCR-SSCP PCR 1%,,, 95 5 min, 5 min, 8% 1 TBE 150 V 12 h, 1.2.4 PCR-RFLP PCR-SSCP,,, SSCP PCR,, PCR-RFLP HSP70-1 12% PAGE : 1 TBE 120 V 5 h, 1.2.5 数据处理 SCC,, SCC SCS,, [3] : SCS = log 2 (SCC/10000)+3,, Hardy-Weinberg, SAS 8.2, Liu [4],, HSP70-1 : Yijklm=μ+Tj+Ck+Gl+Sm+eijklm : Yijklm ; μ ; Tj ; Ck ; Gl ; Sm ; eijklm 2 2.1 HSP70-1 PCR-SSCP P1 P2 P4 PCR, PCR P4 G A, 3 : AA GA GG HSP70-1 2409, EcoR Ⅱ, G, A,, 3 3 : AA(106 bp, 65 bp, 50 bp, 13 bp, 表 1 根据 HSP70-1 基因编码区设计的 9 对引物 (5 3 ) (bp) ( ) P0 P1 P2 P3 P4 P5 P6 P7 P8 F: GCGGCTTAGTCCGTGAGAACA R: ATCCCAACAGTCTCCATAACAAACA 1324~3440 2 117 59 F: GGGCCGTTTTCAGGA R: GGGTCTCCGAACTTGC 1414~1709 296 57 F: GAACCCGCAGAACACG R: CGAGTCGTTGAAGTAGGC 1650~1926 277 57 F: ATCAACGAGCCCACG R: GCTGGACGACAAGGTTC 1984~2298 315 57 F: TGTCGTCCAGCACCCA R: GCCCGTTGAAGAAGTCC 2288~2536 249 57 F: CGCATCCCCAAGGTG R: GGCTGGTTGTCCGGATA 2491~2774 284 57 F: CACCTACTCGGACAACC R: TCACCTCCTGGCACTT 2754~3199 446 57 F: CGGACAAGAAGAAGGTGC R: ATCCCAACAGTCTCCATAA 3161~3440 280 57 F: TACCGAGCGCCTCATC R: AACGCCTTGGTCTCCC 1605~1808 204 54
172 HEREDITAS (Beijing) 2009 31 9 bp, 6 bp); GG(65 bp, 60 bp, 50 bp, 46 bp, 13 bp, 9 bp, 6 bp); GA(106 bp, 65 bp, 60 bp, 50 bp, 46 bp, 13 bp, 9 bp, 6 bp) 13 bp, 65 bp 60 bp, 65 bp, 1 0.557,, χ 2, 1623 χ 2 179.843(P<0.01) 2409 χ 2 31.333(P<0.01), Hardy- Weinberg 图 1 2409 位点 EcoRⅡ 酶切图谱 (12% 聚丙烯酰胺凝胶电泳, 硝酸银染色 ) M: 50 bp DNA ladder marker P1 P2,, PCR, HSP70-1 1623, G A C 3, Fok G, Fok GG,, [5],,, 1614 G C, 1623 C Bgl, C, 189 bp 15 bp ; Fok 172 bp 32 bp 204 bp 15 bp 32 bp P8, 1 605~1 808 bp 204 bp ( 1) 1623, : AA( ), GG (Fok ), CC(Bgl ), AG(Fok, Bgl ), AC(Bgl, Fok ), GC( ) 2 2.2 253, 2~4, 2409 0.267, ; 1623 图 2 1623 位点酶切图谱 (12% ) A: Bgl, 1: CC ; 2: ; 3: B: Fok, 1: GG ; 2: ; 3: M: 50 bp DNA Ladder Marker 2.3 HSP70-1 SCS, SCS, 1~6, SCS (P<0.01), SCS (P<0.05), 1623 SCS (P<0.05), 2409 SCS (P>0.05)( 5) 1623 CC SCS GA GG,, 1623 CC ( 6) 3 3.1, : (1) :
2 : HSP70-1 173 表 2 中国荷斯坦牛 2409 突变位点基因频率和基因型频率 AA GA GG A G 253 0.095(24) 0.205(52) 0.70(177) 0.198 0.802 表 3 中国荷斯坦牛 1623 突变位点基因频率和基因型频率 253 AA CA CC GA GC GG A C G 0.032 (8) 0.047 (12) 0.403 (102) 0.277 (70) 0.043 (11) 0.198 (50) 0.194 0.448 0.358 表 4 基因座位在群体中的多态信息含量 杂合度 有效等位基因数及 χ 2 检验 (PIC) (He) (Ne) χ 2 1623 0.557 0.633 2.727 179.843 ** 2409 0.267 0.317 1.464 31.333 ** **: P 0.01 表 5 各因素对中国荷斯坦奶牛群的体细胞评分的影响 2409 1623 F (1~6 ) 9.20 ** 9.40 ** 6.06 * 0.76 2.47 * *: P 0.05; **: P 0.01 表 6 HSP70-1 基因 1623 位点不同基因型在群体中的最小二乘均值 (LSM) 及标准误 (SE) AA CA CC GA GC GG ± 6.1610±0.5305 ac 5.2696±0.5022 ac 5.1780±0.2552 a 5.8592±0.2875 bc 5.6349±0.4886 bc 5.8340±0.2969 ac : (P>0.05), ; (2)MHC:,, ; (3)β- : β- [6] β- TAP LAP BNBD1-13 [7], β- 3.2 HSP70-1 HSP70-1, HSP70, HSP70, IL- 10 Schmidt [8], IL-1β TNF-α, HSP IL-1β TNF-α Merz [9], HSP70, HSP70, IL-10 [10] ELISA HSP70, HSP70,, Liew [11], HSP70,, ; HSP70 ;, HSP70
174 HEREDITAS (Beijing) 2009 31,, HSP70, HSP70 3.3 HSP70-1,,,, HSP70-1, 1623 SCS, (2409, 1623 ), HSP70 2409 0.25 0.50, ; 1623 0.50,, 3.4 HSP70-1 5 6, 2409 SCS, 1623 SCS (P<0.05), CC GA GG CC,, AA 6, CA GC, CC CA GC SCS ; GG AA GA SCS ; C, C, C, mrna, 1623 HSP70, Pociot [12] HSP70-2(A/G), mrna, 1623,, 1623 HSP70, HSP70-1 mrna 参考文献 (References): [1] Heringstand B, Klemetsdal G, Ruane J. Selection for mastitis resistance in dairy cattle: a review with focus on the Nordic countries. Livest Prod Sci, 2000, 64(2): 95 106.[DOI] [2],,,,,.., 2001, (3): 39 40. [3] Shook GE. Genetic improvement of mastitis through selection on somatic cell count, veterinary clinics of North America. Food Anim Pract, 1993, 9(3): 563 581. [4] Liu BH. Statistical Genomics: Linkage, Mapping,and QTL Analysis. LLC: CRC Press, 1998, 404 409. [5],,.., 2003, 25(3): 327 329. [6] Goldammer T, Zerbe H, Molenaar A, Schuberth HJ, Brunner RM, Kata SR, Seyfert HM. Mastitis increases mammary mrna abundance of beta-defensin 5, toll-like-receptor 2 (TLR2), and TLR4 but not TLR9 in cattle. Clin Diagn Lab Immunol, 2004, 11(1): 174 185.[DOI] [7] Roosen S, Exner K, Paul S, Schroder JM, Kalm E, Looft C. Bovine β-defensins: Identification and characterization of novel bovine β-defensin genes and their expression in mammarygland tissue. Mamm Genome, 2004, 15(10): 834 842.[DOI] [8] Schmidt JA, Abdulla E. Down-regulation of IL-1 beta biosynthesis by inducers of the heat-shock response. Immunol, 1988, 141(6): 2027 2034. [9] Merz U, KellingHaus M, Hausler M. Partial liquid ventilation with surfactant: effects on gas exchange and lung pathology in surfactant depleted piglets. Intensive Care Med, 2000, 26(1): 109 116.[DOI] [10],,,,.., 2004, 25(6): 411 413. [11] Liew PK, Zulkifli I, Hair-Bejo M, Omar AR, Israf DA. Effects of early age feed restriction and heat conditioning on heat shock protein 70 expression, resistance to infectious bursal disease, and growth in male broiler chickens subjected to heat stress. Poultry Sci, 2003, 82(12): 1879 1885. [12] Pociot F, Ronningen KS, Nerup J. Polymorphic analysis of the human MHC-linked heat shock protein 70 (HSP70-2) and HSP70-hom genesin insulin-dependent diabetes mellitus (IDDM). Scand J Immunol, 1993, 8(5): 491 495.[DOI]