Heat stress induces heat shock proteins (HSPs) expression and HSP70 family is one of them that have been reported to involve in cellular protection against heat stress. But whether there is any association of genetic variation in the HSP70A1A gene with thermo tolerance is unknown. PCR-SSCP and DNA sequencing were used to detect possible SNPs in HSP70A1A gene in 890 Chinese Holstein cattle. Three fragments were amplified and five novel mutations were found in HSP70A1A gene in Chinese Holstein cattle. G/A mutation was found at nucleotide 1524 in coding region that resulting two genotypes of AA and AB. T/C mutation was found at nucleotide 3494 in 3'-UTR resulting three genotypes of CC, CD and DD. The other three point mutations, G/C at nucleotide 6400, C/T at nucleotide 6600 and G/A at nucleotide 6601 were also found in 3'-UTR resulting six genotypes of EE, EF, FF, EG, FG and GG. Linkage disequilibrium (LD) analysis showed that the nucleotide 6400, 6600 and 6601 were in strong LD (D>0.75). Association analysis indicated that AB, DD and FF genotype were the thermo tolerance genotype. SBYE Green I was used to quantify HSP70A1A mRNA expression in different tissues through quantitative real-time PCR assay. The results of the real-time PCR showed that the expression of HSP70A1A mRNA in the heart was significantly higher than that in the other tissues (P<0.05). We presume that these mutations could be used in marker assisted selection for anti-heat stress cows in our breeding program.

本实验采用PCR-SSCP和测序技术对荷斯坦奶牛和鲁西黄牛两个群体共673个个体热休克蛋白70基因(HSP70)的3'-侧翼区的遗传变异,及该突变对奶牛产奶性状和耐热性状的影响进行研究.研究结果表明设计引物扩增片段大小500 bp,扩增产物具有多态性.供试的72头鲁西黄牛和601头荷斯坦奶牛均发现3种基因型,为从、AB和BB型.测序分析发现热休克蛋白70基因(HSP701的3'-侧翼区存在1个新突变,T→C.关联分析结果表明,BB基因型荷斯坦奶牛个体,乳脂率、乳蛋白率和305 d产奶量,相对于AA基因型的个体都要高;红细胞钾含量和产奶量下降率比AA类型个体要低,且达显著水平(P<0.05).鲁两黄牛B基因基因频率为74.31%,而荷斯坦奶牛B基因基因频率却比较低,为14.89%.由于鲁西黄牛耐热性显著强于荷斯坦奶牛,且耐热性最高的BB型奶牛产奶量下降率显著低于耐热性差的AA型奶牛.等位基因B可作为选育抗热应激奶牛潜在的分子遗传标记.

Heat shock protein 70(HSP70) gene was regarded as a candidate gene for thermo resistance of cows.In this study,a 549 bp fragment of HSP70 gene of Chinese Holstein was amplified by polymerase chain reaction(PCR),the polymorphisms of three loci of HSP70 gene were detected by PCR-SSCP technique.By sequencing,three point mutations,G/C at nucleotide 6 400,C/T at nucleotide 6 600 and G/A at nucleotide 6 601,were identified in HSP70 gene sequence.χ2 test indicated that three polymorphisms in the population unfitted with Hardy-Weinberg equilibrium(P 0.01).Meanwhile,the relationship between milk performance and heat tolerance was analyzed.The results showed that in the three loci,the different genotypes of HSP70 gene had no significant influence on milk protein percent and fat percent,but had significant influence on 305-day milk yield.Individuals with genotype BB showed higher 305-day milk yield than those with genotypes AA and CC(P 0.05).And the cows with genotype BB showed lower potassium content in erythrocytes(PCE) and decrease rate of milk yield than those with genotypes AA and CC.It indicated that BB genotype was the advantageous genotype.These mutations could be used in MAS for anti-heat stress cows in our breeding program.

Karyotype analysis was carried out by means of culturing whole peripheral blood from 394 pigs,which were produced from three hybridization combinations of 13/17 Robertsonian translocation pig.According to karyotype all the pigs were divided into different groups.A RsaⅠrestriction enzyme polymorphic site(locus A) in a 1746 bp fragment of porcine POU1F1 gene was detected by using PCR-RFLP analysis and a mutation(locus E) in intron 5 of porcine POU1F1 gene was found by using PCR-SSCP analysis in three hybrid populations.At the same time,the effects on growth traits and carcass traits of POU1F1 gene were estimated according to their growth and slaughter data.The result of population genetics analyses demonstrated a significant effect on growth in locus A and a significant association with growth and loin eye area for locus E(P0.05).The results indicated that growth and loin eye area were correlated with polymorphisms of POU1F1 gene which could be used as an interpretation for genetic mechanism on heterozygous advantage of Robertsonian translocation at the molecular level.