【作者】 李芳；

【导师】 丁洪浩；

【作者基本信息】 吉林大学 ， 动物营养与饲料科学， 2009， 硕士

【摘要】 本研究将采集荷斯坦奶牛血样,从全血中提取基因组DNA,以GenBank收录的牛HSF1全基因组核苷酸序列为模板设计11对特异性引物,通过PCR方法方法分段扩增了HSF1基因核苷酸序列。琼脂糖检测产物,出现特异性条带。运用SSCP方法,检测单核苷酸突变;应用测序手段确定突变碱基;酶切分型各SNP位点基因型。本研究采集血样,加抗凝剂用冰盒带回实验室,立即分离红细胞,测定红细胞钾离子浓度。应用SAS软件统计分析红细胞钾离子浓度与SNP基因型相关型。通过方差分析、最小二乘分析,得出红细胞钾离子浓度与SNP位点有显著相关性。红细胞钾离子浓度具有稳定性,不受外界环境、营养条件的影响,可以作为良好的耐热性能生理指标。本研究采集血样,分离红细胞膜,应用超微量ATP酶试剂盒测定红细胞膜上NKA活力。应用SPSS、SAS等软件分析红细胞膜NKA活力与SNP位点基因型的相关性。统计分析,发现NKA活力与HSF1基因的SNP位点没有显著相关性。本研究测量试验牛群体温,作者对所有实验牛体温进行准确测量,体温测量分成三个阶段:热应激临界期,热应激期,非热应激期。本实验运用直肠温度作为体温,每天测量三次取平均值,最后不同阶段的再取平均值,代表该时期的体温,比较三阶段体温变化情况。最后将不同阶段的体温平均,与HSF1基因的SNP位点进行相关性分析。结果发现,体温和SNP位点基因型没有显著相关性。综上该研究证明,证明HSF1基因组的两个SNP位点的基因型与红细胞钾离子浓度具有显著相关性,有望作为培育耐热性奶牛的遗传标记之一。更多还原

【Abstract】 Diry cattle are cold-resistent and nonhot-resistent livestock, appropriate to the external environment temperature of 8 23℃, when the critical temperature higher than 27℃, the eccrine sweat glands of cow does not finished developde ,so that slow perspiration causes the heat balance or imbalance, resulting in "Heat Stress Response".Heat stress refers to dairy cows is that when they encouter high temperature, role on the pituitary - adrenal cortex system the body caused the non-specific defense reactions and specific indications for systemic barriers.In summer, heat stress on lactating dairy cows causes great harm , in order to reduce the body heat the cow reduces feed intake. It has been determined, the temperature at 22 25℃, the feed intake began to decline; higher than 30℃, feed intake decrease more than 10%;hihger than 40℃,more than 50% decline in feed intake ,and the individual cows will cease to feed intake. Decline in feed intake of dairy cattle would cause malnutrition.Some studies showed that when the envierniment temperature higher than 21℃, increaseing temperature will cause to rise B T, body temperature rises 0.6℃, feed intake decreases 1.4 kg, 0.8 kg drop in milk production. Decline in milk production when the premises are another factor in the relative high humidity, if the temperature exceeds the premises for more than 28℃, heat stress causes inhibition of reproductive endocrine in dairy cattle , inhibiting the bubble of breast and breast duct formation and development , resulting in a decline in milk production . In addition, heat stress can reduce milk fat percentage ,due to high temperature ,cow find and feed preferred bait vessel and dislike rough material, so that the rumen acetic acid to propionic acid resulting from the scaled-down. Temperature between 4.4℃ 35℃, 15.5℃at the following virtually no change in milk fat percentage , 21.1℃and above the fat percentage will decline . Heat stress can significantly reduce the cow serum immunoglobulin concentration , leading to weakened immunity , increased multiple diseases such as mastitis, no placenta afterbirth, endometritis, hoof rot disease , al et. At the same time , because of reduceing feed intake, inadequate calcium , serum calcium concentration decreased and easy to cause a calcium deficiency disease, such as post-partum paralysis. Heat stress can also shorten the time of cow in empathema , prolong the estrus cycle, estrus or anestrus was not obvious , thus result of the timely breeding to conception rate and pregnancy rate declineing and the early embryonic mortality and abortion rate increaseing . There were reported that the average daily temperature increased from 33.3℃to 41.7℃, the pregnancy rate of dairy cows fell from 61.5% to 31%.Presently , there are three meas for the prevention and treatment of heat stress, domestic and abroad . First , environmental control, such as shading, ventilation, water cooling and other measures to promote the cow body heat elimination . Second , nutritional regulation , it is mainly addtion the concentration of various nutrients and variety additives to bait vessel which there is in favor of tolerance to heat stress of dairy cow, such as : sodium acetate , sodium bicarbonate and iso-butyric acid, isovaleric , potassium chloride , rumensin , digestive enzymes inhibitors , a variety of vitamins , minerals and so on. The third is to strengthen the feeding and management, such as adjusting work schedule , increase the feeding frequency, optimizing the feed composition, do a good job in environmental health, enhance disease prevention and control. Although the above measures up to some extent , reduce the symptoms of heat stress, it can not fundamentally eliminate the occurrence of heat stress, heat stress should not avoid dairy industry to a great loss. Therefore, the selection from the genetic point of cows on the heat resistance, in order to fundamentally solve the problem of heat stress in dairy cattle industry ,which caused huge losses , has become a study hot.of dairy cattle breeding at home and abroad.Studies have shown that the heat-resistant traits of cows are one of quantitative traits, different dairy breeds and individual cow response to heat stress exist differences , the heritability of the heat-resistant traits for Holstein cattle is 0.15-0.30. This shows can eliminate or reduce the heat stress on dairy cattle industry. by selecting the breeding of dairy heat resistance. Therefore, in this study ,we study candidate genes HSF1 polymorphism, statistical analysis of the HSF1 polymorphism and heat stress relevance, revealed the HSF1 gene and the relationship between the heat traits of cow dairy in the molecular level to explore genetic mechanism of heat shock respondent,by the genetic point of view to solve the problem of heat stress for reference, and then breeding in dairy cattle in the application of heat, which is important for dairy cows high-yielding, high efficiency, high-quality, and is significance for theoretical and practical .This study, we achieved the experiment in Dairy Cattle Research Centre, Shandong Academy of Agricultural Science, Jinan and some surrounding large-scale dairy farms.we analysis the cow’s DHI data to selecte cattles ,we collected all experiment sample from: Dairy Ltd. Jinan JIABAO first, second ranch, Ltd. Tianyuan dairy pastures, Animal Husbandry Research Institute, Bolameng, Jinlan, Qingdao, Linyi model dairy pastures, Dongying bumper harvest. We determined the temperature and humidity of barn; measured rectal temperature, a detailed record. we collected 718 blood samples of Holstein cow, collection vena cervicalis blood 15 ml, ACD anticoagulant, 3-packing, back to the laboratory with ice box. One template for the extraction of DNA, One template extracted erythrocyte used for the measurement of the potassium concentration, One template used for the measurement of activity of erythrocyte NKA.Erythrocyte potassium concentration determination: we measured the potassium content of erythrocyte by atomic absorption spectrophotometer in Center laboratory Dairy Cattle Research Centre, Shandong. Determination of NKA activity of erythrocyte membrane: inorganic phosphorus colorimetry: erythrocyte membrane Na +-K +-ATP activity measured in accordance with the amount of ATP AMD enzyme test kit instructions, the unit is the enzyme activity: U/mgprot, buy the kit from The first branch of the Institute.build bio-engineering, NanjingWe extracted and purificated DNA ,according to "Guide to molecular cloning experiments" .we measured the concentration and purityof DNA with the spectrophotometer, then diluted to 50 ng ?μl - 1, - 20℃cryopreservation standby. Genebank for landing in accordance with No. NC₀07312.2 （634454 639405） of the HSF1 gene sequence （4952bp）, we used Primer Premier5.0 to designe primer sequences, amplified different lengths fragments, the success of PCR detection agarose, Detected fragment by SSCP , if there is a mutation of nucleotide sequencing by SSCP or restriction enzyme electrophoresis to detected SNPs .we analysised all thermoresistance indicatrix with HSF1 gene polymorphism.We has found two new single nucleotide mutations of HSF1 by testing, respectively, 911 base pairs C→T and 4693 base pairs G→T. there are three genotypes of 911 points and 4693 by RLFP , respectively, CC, TC, TT and GG, GT, TT; their genotype frequencies are: 5.8%, 53.7% , 40.4%. And 28.1%, 28.7%, 43.2%. He and PIC results show that two mutations are moderate heterozygosity , chi-square test p <0.01 did not meet the Hardy-Weinberg balance. Pair linkage disequilibrium analysis, Two SNPs of HSF1 gene locus were in linkage disequilibrium by the matching statistic, D ’that （D’ = D / Dmax）. Between C911T and G4695T was weak linkage disequilibrium （weak linkage disequilibrium: D <0.5; strong linkage disequilibrium: D> 0.5; complete linkage disequilibrium: D = 1） （D = 0.45, r2 = 0.101）. D ’value is very low, indicated that the two sites once happen more possibility of reorganization. Carried out on the C911T and G4693T haplotype combinations are, respectively, are separately GGCC, GGTT, TTCC, TTTT, GGCT, GTCC, GTCT, GTTT, TTCT.In this study, we used the SAS and SPSS12.0 to test of significance and least squares analysis for genotype and erythrocyte potassium concentration, rectal temperature, and Na. K-ATP activity. The results show that the 4693-point erythrocyte potassium concentration ,TT genotype least squares mean concentrations of 488.11, less than GG genotype （890.83） and GT genotype （662.42）. Relative analysis for the three genotypes was significant （P <0.01）, rectal temperature was no significant difference .EPC 911 points ,CC genotype least squares mean concentrations of 780.63, more than CT genotype （626.44）, TT genotypes （626.34）. Relative analysis showed between CC and CT were significant difference （P <0.01）, three significant differences between genotypes （P <0.05）.Among the three genotypes NKA activity was no significant difference, but the trends and changes same as EPC, rectal temperature did not change significantly.In this study, PCR amplificated all exons and the vast majority of introns of thecow’s HSF1 gene , use SSCP, RFLP and sequencing to screen the PCR products for SNP, Two new SNPs were found, them genotyping analysis and relevance of their heat resistance and dairy correlation exist between the target, HSF1 gene is expected to become a candidate genes for breeding cow . In the future , this study was the accumulation of a large number of QTL data is important. for MAS更多还原