为了研究奶牛HSP70基因的mRNA表达规律和其紧密连锁的3个微卫星标记与耐热性状的相关性, 文中在日平均温湿度指数(THI)为86.2(高温期)、70.9(临界高温期)和56.8(适温期)时分别采集10头处于同一泌乳阶段的同龄健康奶牛的尾静脉血, 采用实时荧光定量RT-PCR技术分析不同条件下奶牛HSP70基因在外周血淋巴细胞的mRNA表达水平。结果表明: 随THI升高, HSP70 mRNA表达量呈上升趋势, 高温期奶牛的外周血淋巴细胞HSP70 mRNA表达丰度极显著高于临界高温期和适温期(P<0.01), 且与奶牛的耐热指标具有一定的遗传相关, 表明HSP70基因可以作为奶牛热应激反应的候选基因。选取23号染色体上与HSP70紧密连锁的3个微卫星标记BMS468、BM1258和BM1815, 用非变性聚丙烯酰胺凝胶电泳分析其在160头荷斯坦奶牛的遗传变异情况, 利用最小二乘法拟合线性模型初步探索了这3个微卫星座位与奶牛耐热指标的关系。相关分析表明: 耐热系数、红细胞钾含量及高温期日产奶量下降率的最有利基因型在BMS468基因座和BM1815基因座上, 分别为134 bp/ 128 bp(P<0.05)和186 bp /148 bp(P<0.05); BM1258基因座上, 最有利基因型101 bp /99 bp的高温期日产奶量下降率最低(P<0.05)。
The objective of this study was to investigate the variation of HSP70 mRNA level in dairy cows and relationships of its closely linked microsatellite loci with heat tolerance traits. Blood samples were collected from ten healthy Holstein cows with the same age and milking stage at different temperatures-humid-index (THI) (86.2, high temperature; 70.9, critical high temperature, and 56.8, optimum temperature). The mRNA levels of HSP70 of lymphocytes in peripheral blood were analyzed using real-time RT-PCR. The mRNA level of HSP70 was increased with the THI; the mRNA level of HSP70 at high temperature was higher than others (P<0.01). This indicated that the bovine HSP70 gene may act as a potential can-didate gene for response to heat shock. Genetic variation of three microsatellite loci BMS468, BM1258, and BM1815, which were closely linked to HSP70 gene on chromosome 23, was analyzed in 160 Holstein cows with non-denaturing poly-acrylamide gel electrophoresis. The association between these microsatellite loci and heat tolerance traits were analyzed by least square linear model. The results showed that 134 bp/128 bp at BMS468 and 186 bp/148 bp at BM1815 were the most favorable genotypes for HTC, red cell potassium, and decrement rate of milk yield in high temperature (P<0.05); 101 bp/99 bp at BM1258 was the most favorable genotype for decrement rate of milk yield in high temperature (P<0.05).
[1] Johnson HD. Bioclimate effects on growth, reproduction, and milk production. World Animal Science (Netherlands): Elsevier, 1987, 35–37.
[2] Du Preez JH, Hattingh PJ, Giesecke WH , Eisenberg BE. Heat stress in dairy cattle and other livestock under southern African conditions. III. Monthly tempera-ture-humidity index mean values and their significance in the performance of dairy cattle. Onderstepoort J Vet, 1990. 57(4): 243-248.
[3] Holter JB, West JW, McGilliard ML. Predicting ad libitum dry matter intake and yield of Holstein cows. J Dairy Sci, 1997, 80(9): 2188-2199.
[4] Valtorta SE, Gallardo MR. Evaporative cooling for Hol-stein dairy cows under grazing conditions. Int J Biomete-orol, 2004, 48(4): 213-217.
[5] Turner LW, Chastain JP, Hemken RW, Gates RS, Crist WL. Reducing heat stress in dairy cows through sprinkler and fan cooling. Appl Eng Agric, 1992, 48(4): 251-256.
[6] Smith TR, Chapa A, Willard S, Herndon Jr C, Williams RJ, Crouch J, Riley T, Pogue D. Evaporative tunnel cooling of dairy cows in the southeast. II: Impact on lactation per-formance. J Dairy Sci, 2006, 89(10): 3915-3923.
[7] Armstrong DV. Heat stress interaction with shade and cooling. J Dairy Sci, 1994, 77(7): 2044-2050.
[8] Silanikove N, Maltz E, Halevi A, Shinder D. Metabolism of water, sodium, potassium, and chlorine by high yielding dairy cows at the onset of lactation. J Dairy Sci, 1997, 80(5): 949-956.
[9] Ravagnolo O, Misztal I, Hoogenboom G. Genetic compo-nent of heat stress in dairy cattle, development of heat in-dex function. J Dairy Sci, 2000, 83(9): 2120-2125.
[10] 穆玉云. 乳牛耐热性的数量指标和遗传力. 中国畜牧杂志, 1990, 26(5): 46.
[11] Feige U, Morimoto R, Yahara I, Polla BS. Stress-inducible Cellular Responses. Birkhäuser Verlag Basel, Boston, Ber-lin, 1996, 239-254.
[12] Cai YF, Liu QH, Xing GD, Zhou L, Yang YY, Zhang LJ, Li J, Wang GL. Polymorphism of the promoter region of Hsp70 gene and its relationship with the expression of HSP70 mRNA, HSF1mRNA, Bcl-2 mRNA and Bax-A mRNA in lymphocytes in peripheral blood of heat shocked dairy cows. Asian-Aust J Anim Sci, 2005, 18(5): 734-740.
[13] Morimoto RI. Cells in stress: transcriptional activation of heat shock genes. Science, 1993, 259(5100): 1409-1410.
[14] Bruemmer-Smith S, Stüber F, Schroeder S. Protective functions of intracellular heat shock protein (HSP) 70 ex-pression in patients with severe sepsis. J Intensive Care Med, 2001, 27(12): 1835-1841.
[15] Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW. Quantitative reverse transcription- poly-merase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem, 2000, 285(2): 194-204.
[16] Bustin SA. Quantification of mRNA using real-time re-verse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol, 2002, 29(1): 23–39.
[17] 储明星, 周国利, 金海国, 石万海, 曹福存, 方丽, 叶素成, 朱颜. 7个微卫星座位与北京荷斯坦母牛体细胞评分关系的研究. 遗传学报, 2005, 32(5): 471-475.
[18] 李延璐, 储明星, 陈宏权, 方丽, 狄冉, 马月辉, 李奎. 绵羊微卫星BMS2508和FecB基因的多态及连锁分析. 遗传, 2009, 31(5): 500-507.
[19] 孙伟, 常洪, 金银, 王鹏, 钱建共, 吴文忠, 陈玲, 王伟. 湖羊产羔性状的微卫星标记与可能生产力的关联性分析. 畜牧兽医学报, 2009, 40(1): 7-14. [20] Rhoad AO. The Iberia heat tolerance test for cattle. Trop Agr, 1944, 21(9): 162-164.
[21] Livak KJ, Schmittgen TD. Analysis of relative gene ex-pression data using real-time quantitative PCR and the 2-△△Ct Method. Methods, 2001, 25(4): 402-408.
[22] Ahn SG, Thiele DJ. Redox regulation of mammalian heat shock factor 1 is essential for Hsp gene activation and protection from stress. Gene Dev, 2003, 17(4): 516-528.
[23] Zhang Y, Huang L, Zhang J. Targeted disruption of hsf1 leads to lack of thermotolerance and defines tis-sue-specific regulation for stress-induced Hsp molecular chaperones. Cell Biochem, 2002, 86(2): 376-393.
[24] Mosser DD, Caron AW, Bourget L. Role of the human h
