奶牛HSP和NOS3基因多态性与血清生化指标的相关性研究
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摘要
以荷斯坦和荷-娟F_1牛作为实验动物,采用比色法对不同群体奶牛在热应激和非热应激期血清中的丙二醛(MDA)含量、总超氧化物歧化酶(T-SOD)活力、总抗氧化能力(T-AOC)和一氧化氮合酶活力(NOS)四个指标进行检测分析,并比较了群体间抗氧化能力的差异;采用PCR-SSCP技术对与耐热性状密切相关的热应激基因(HSP)和一氧化氮合酶基因(NOS3)进行了SNP分析,对部分基因型个体的PCR产物进行测序和序列比较,并分析了标记基因型与不同奶牛血清生化指标间的相关。结果表明:
(1)热应激期,两个群体奶牛血清MDA含量、T-SOD活力差异显著(P＜0.05),NOS活力差异极显著(P＜0.01)。非热应激期,四个指标均无显著差异(P＞0.05)。不同温度下,荷-娟F_1和荷斯坦牛血清MDA含量、T-SOD活力差异均极显著(P＜0.01),NOS活力差异显著(P＜0.05),荷斯坦牛血清中T-AOC差异显著(P＜0.05),而荷-娟F_1 T-AOC差异不显著(P＞0.05)。
(2)HSP基因发现两个多态位点(HSPA8-intron7、HSPA3-exon1),NOS3基因发现五个多态位点(NOS3-exon3、NOS3-exon4、NOS3-exon8、NOS3-exon13和NOS3-exon16)。测序发现HSPA8-intron7座位存在T缺失突变以及C→G转换和T→C颠换突变;HSPA8-exon4座位发现A缺失突变;HSPA3-exon1座位发现C→A颠换突变;NOS3-exon8座位发现G→A颠换突变。
(3)NOS3-exon4座位,荷斯坦牛与荷-娟F_1牛基因型分布差异显著(P＜0.05),其它座位基因型分布均无显著差异(P＞0.05)。
(4)HSPA3-exon1和NOS3-exon13扩增片段,两个奶牛群体都属中度多态,HSPA8-intron7扩增片段,均为低度多态;NOS3-exon3扩增片段,荷斯坦牛群体为高度多态,而荷-娟F_1群体为中度多态;NOS3-exon8和NOS3-exon16扩增片段,荷斯坦牛群体为低度多态,荷-娟F_1群体为中度多态。
(5)NOS3基因五个位点不同基因型与NOS活力的相关性分析表明:荷斯坦牛,NOS3-exon3座位AC基因型与NOS活力呈正相关,A基因为优势等位基因;NOS3-exon13座位AB基因型与NOS活力呈正相关,B基因为优势等位基因。荷-娟F_1牛NOS3-exon3座位AA和AB基因型与NOS活力呈正相关,A基因为优势等位基因;NOS3-exon4座位CC基因型与NOS活力呈正相关,C基因为优势等位基因。NOS3-exon8座位AC基因型与NOS活力呈正相关,C基因为优势等位基因。
(6)HSP基因两个位点不同基因型与血清抗氧化指标的相关性分析表明:荷斯坦牛,HSPA3-exon1座位AB和BB基因型与T-SOD活力呈正相关,B等位基因为优势等位基因。荷-娟F_1牛HSPA3-exon1座位AA基因型与MDA含量呈正相关,AB和BB基因型与MDA含量呈负相关;AB和BB基因型与T-SOD活力呈正相关,AA基因型与T-SOD活力呈负相关,B等位基因为优势等位基因。HSPA8-intron7座位AB基因型与T-SOD活力呈正相关,B等位基因为优势等位基因。
Holstein and Holstein×JerseyF_1 dairy cows were used to study heat tolerant traits.The effects and changes of heat stress in the MDA contents,T-SOD,T-AOC and NOS activities were evaluated by colorimetric method in two groups of 40 healthy Holstein and Holstein×JerseyF_1;SNPs of HSP and NOS3 gene were analyzed by PCR-SSCP technique.Gene and genotype frequencies were calculated for the polymorphic loci in HSP gene and NOS3 gene. Variance analysis was completed for distribution of genotypes frequency between different groups.Effects of genotypes in different locus on heat tolerant traits were estimated.The results indicated that:
(1)During heat stress period,the MDA contents and T-SOD activities of two groups dairy cows were differ significantly(P＜0.05)and NOS activities had extremely significant differences(P＜0.01),the T-AOC had no significant differences.However,during non-heat stress period,there were no significant differences on the MDA contents,the T-SOD,T-AOC and NOS activities between two groups.The MDA contents decreased(P＜0.01)and the T-SOD、NOS activities increased(P＜0.01)of both Holstein×Jersey F_1 and Holstein;the T-AOC of Holstein serum increased evidently(P＜0.05)between hot and cold season.
(2)HSP gene and NOS3 were amplified to identify the nucleotide diversity. Polymorphisms were detected in the loci of HSPA8-intron7,HSPA3-exon1,NOS3-exon3, NOS3-exon4,NOS3-exon8,NOS3-exon13 and NOS3-exon16.The analysis results of polymorphisms were HSPA8-intron7 T deletion mutation and C→G、T→C,HSPA8-exon4 A deletion mutation,HSPA3-exon1 C→A,NOS3-exon8 G→A.
(3)The distribution of genotype frequency was differ significantly between Holstein and Holstein×JerseyF_1 in NOS3-exon4(P＜0.05),others had no differences(P＞0.05).
(4)Both Holstein and Holstein×JerseyF_1 were midrange polymorphisms on the amplification fragments of HSPA3-exon1 and NOS3-exon13 and both were low polymorphisms on the amplification fragments of HSPA8-intron7;Holstein was high polymorphisms,however,Holstein×JerseyF_1 was midrange polymorphisms on the amplification fragments of NOS3-exon3;Holstein was low polymorphisms and Holstein×JerseyF_1 was midrange polymorphisms on the amplification fragments of NOS3-exon8 and NOS3-exon16.
(5)The correlation between NOS3 gene and NOS activity were analyzed by using the data of the two groups respectively.To Holstein,the results showed genotype BC of the HSPA3-exon1 and genotype AB of NOS3-exon13were positively related to NOS activity,the allele B was dominant allele.To Holstein×JerseyF_1,genotype AA and AB of NOS3-exon3 were positively related to NOS activity,the allele A was dominant allele.Genotype CC of NOS3-exon4 was positively related to NOS activity,the allele C of this site was dominant allele.The allele C of this site was dominant allele.
(6)In the analysis of the correlation between HSP gene and anti-oxidative capability using data of the two groups of cows respectively.To Holstein,genotype AB and BB of HSPA3-exon1 were positively related to T-SOD activity,the allele B of this site might was dominant allele.To Holstein×JerseyF_1,genotype AA of HSPA3-exon1 was positively related to MDA content,genotype AB and BB was negatively related to MDA content;AB and BB were positively related to T-SOD activity,the allele B of this site might was dominant allele. Genotype AB was positively related to T-SOD activity,the allele B of this site was dominant allele.
(1)热应激期,两个群体奶牛血清MDA含量、T-SOD活力差异显著(P＜0.05),NOS活力差异极显著(P＜0.01)。非热应激期,四个指标均无显著差异(P＞0.05)。不同温度下,荷-娟F_1和荷斯坦牛血清MDA含量、T-SOD活力差异均极显著(P＜0.01),NOS活力差异显著(P＜0.05),荷斯坦牛血清中T-AOC差异显著(P＜0.05),而荷-娟F_1 T-AOC差异不显著(P＞0.05)。
(2)HSP基因发现两个多态位点(HSPA8-intron7、HSPA3-exon1),NOS3基因发现五个多态位点(NOS3-exon3、NOS3-exon4、NOS3-exon8、NOS3-exon13和NOS3-exon16)。测序发现HSPA8-intron7座位存在T缺失突变以及C→G转换和T→C颠换突变;HSPA8-exon4座位发现A缺失突变;HSPA3-exon1座位发现C→A颠换突变;NOS3-exon8座位发现G→A颠换突变。
(3)NOS3-exon4座位,荷斯坦牛与荷-娟F_1牛基因型分布差异显著(P＜0.05),其它座位基因型分布均无显著差异(P＞0.05)。
(4)HSPA3-exon1和NOS3-exon13扩增片段,两个奶牛群体都属中度多态,HSPA8-intron7扩增片段,均为低度多态;NOS3-exon3扩增片段,荷斯坦牛群体为高度多态,而荷-娟F_1群体为中度多态;NOS3-exon8和NOS3-exon16扩增片段,荷斯坦牛群体为低度多态,荷-娟F_1群体为中度多态。
(5)NOS3基因五个位点不同基因型与NOS活力的相关性分析表明:荷斯坦牛,NOS3-exon3座位AC基因型与NOS活力呈正相关,A基因为优势等位基因;NOS3-exon13座位AB基因型与NOS活力呈正相关,B基因为优势等位基因。荷-娟F_1牛NOS3-exon3座位AA和AB基因型与NOS活力呈正相关,A基因为优势等位基因;NOS3-exon4座位CC基因型与NOS活力呈正相关,C基因为优势等位基因。NOS3-exon8座位AC基因型与NOS活力呈正相关,C基因为优势等位基因。
(6)HSP基因两个位点不同基因型与血清抗氧化指标的相关性分析表明:荷斯坦牛,HSPA3-exon1座位AB和BB基因型与T-SOD活力呈正相关,B等位基因为优势等位基因。荷-娟F_1牛HSPA3-exon1座位AA基因型与MDA含量呈正相关,AB和BB基因型与MDA含量呈负相关;AB和BB基因型与T-SOD活力呈正相关,AA基因型与T-SOD活力呈负相关,B等位基因为优势等位基因。HSPA8-intron7座位AB基因型与T-SOD活力呈正相关,B等位基因为优势等位基因。
Holstein and Holstein×JerseyF_1 dairy cows were used to study heat tolerant traits.The effects and changes of heat stress in the MDA contents,T-SOD,T-AOC and NOS activities were evaluated by colorimetric method in two groups of 40 healthy Holstein and Holstein×JerseyF_1;SNPs of HSP and NOS3 gene were analyzed by PCR-SSCP technique.Gene and genotype frequencies were calculated for the polymorphic loci in HSP gene and NOS3 gene. Variance analysis was completed for distribution of genotypes frequency between different groups.Effects of genotypes in different locus on heat tolerant traits were estimated.The results indicated that:
(1)During heat stress period,the MDA contents and T-SOD activities of two groups dairy cows were differ significantly(P＜0.05)and NOS activities had extremely significant differences(P＜0.01),the T-AOC had no significant differences.However,during non-heat stress period,there were no significant differences on the MDA contents,the T-SOD,T-AOC and NOS activities between two groups.The MDA contents decreased(P＜0.01)and the T-SOD、NOS activities increased(P＜0.01)of both Holstein×Jersey F_1 and Holstein;the T-AOC of Holstein serum increased evidently(P＜0.05)between hot and cold season.
(2)HSP gene and NOS3 were amplified to identify the nucleotide diversity. Polymorphisms were detected in the loci of HSPA8-intron7,HSPA3-exon1,NOS3-exon3, NOS3-exon4,NOS3-exon8,NOS3-exon13 and NOS3-exon16.The analysis results of polymorphisms were HSPA8-intron7 T deletion mutation and C→G、T→C,HSPA8-exon4 A deletion mutation,HSPA3-exon1 C→A,NOS3-exon8 G→A.
(3)The distribution of genotype frequency was differ significantly between Holstein and Holstein×JerseyF_1 in NOS3-exon4(P＜0.05),others had no differences(P＞0.05).
(4)Both Holstein and Holstein×JerseyF_1 were midrange polymorphisms on the amplification fragments of HSPA3-exon1 and NOS3-exon13 and both were low polymorphisms on the amplification fragments of HSPA8-intron7;Holstein was high polymorphisms,however,Holstein×JerseyF_1 was midrange polymorphisms on the amplification fragments of NOS3-exon3;Holstein was low polymorphisms and Holstein×JerseyF_1 was midrange polymorphisms on the amplification fragments of NOS3-exon8 and NOS3-exon16.
(5)The correlation between NOS3 gene and NOS activity were analyzed by using the data of the two groups respectively.To Holstein,the results showed genotype BC of the HSPA3-exon1 and genotype AB of NOS3-exon13were positively related to NOS activity,the allele B was dominant allele.To Holstein×JerseyF_1,genotype AA and AB of NOS3-exon3 were positively related to NOS activity,the allele A was dominant allele.Genotype CC of NOS3-exon4 was positively related to NOS activity,the allele C of this site was dominant allele.The allele C of this site was dominant allele.
(6)In the analysis of the correlation between HSP gene and anti-oxidative capability using data of the two groups of cows respectively.To Holstein,genotype AB and BB of HSPA3-exon1 were positively related to T-SOD activity,the allele B of this site might was dominant allele.To Holstein×JerseyF_1,genotype AA of HSPA3-exon1 was positively related to MDA content,genotype AB and BB was negatively related to MDA content;AB and BB were positively related to T-SOD activity,the allele B of this site might was dominant allele. Genotype AB was positively related to T-SOD activity,the allele B of this site was dominant allele.
引文
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