南方中国荷斯坦牛耐热性状分子标记的研究
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摘要
奶业是畜牧业的重要组成部分,然而我国南方的高温、高湿的气候,严重影响着奶牛的生产性能、繁殖性能等,造成了巨大的经济损失。目前,人们对奶牛的耐热性状研究较少,且主要集中在生理指标方面。
本研究采用RAPD技术,从DNA水平对南方中国荷斯坦牛在5～9月的热应激反应作了研究,探索了与奶牛耐热性能相关的分子标记,并对耐热标记作了进一步的分析。具体内容如下:
1.RAPD标记的确定
以52头高产耐热组和41头高产不耐热组中国荷斯坦牛的DNA为材料,采用S441和S463两条RAPD引物,对荷斯坦牛的耐热性状进行研究。结果证实,S441和S463抗热应分子标记在耐热牛群中具有特异性。
2.RAPD标记在荷斯坦牛群中的检测结果
采用S441和S463引物一共测定了46头荷斯坦公牛,213头荷斯坦母牛。在公牛群中,具有S441和S463耐热候选标记的分别为8头和10头,同时具有两个耐热候选标记的5头;在母牛群中,具有S441和S463耐热候选标记的分别为25头和26头,同时具有两个耐热候选标记的12头。
3.RAPD标记与产奶量
S441、S463和无标记的母牛在热应激期间的产奶量下降率分别为3.49%、3.52%和22.67%,即有标记母牛的产奶量下降率显著低于无标记母牛。这种结果也验证了S441和S463都是耐热候选标记。
4.RAPD标记的生物信息学分析
根据两特异片段的测序信息,进行序列比较,结果显示S441序列与KCNN2基因的同源性达89%,其可能是KCNN2基因的内含子3;S463序列与NW_001508847.1的同源性达99%,NW_001508847.1的功能未知。根据已知KCNN2功能,初步判断其为耐热候选基因。
5.SCAR标记的转化
根据RAPD-S441、RAPD-S463标记的测序结果,利用Premier5.0软件设计SCAR引物。本研究成功地将RAPD-S441标记转化为耐热SCAR标记,并且能较好的区分高产耐热组和高产不耐热组。而RAPD-S463没有成功转换成SCAR标记。
6.SCAR-S441标记与乳成份的关系。
采用SCAR-S441分子标记,在5～9月测定了荷斯坦母牛的乳脂率、乳蛋白、乳糖和干物质。有SCAR标记的奶牛在6～9的乳脂率和乳蛋白均比5月高,无标记奶牛在6、7月的乳脂率和乳蛋白低于5月,而到8、9月恢复正常。6～9月乳糖的含量两组间没有差异(P＞0.05)。而在8月份,两组奶牛牛奶中的干物质均显著降低。
Dairy industry is an very important part of animal industry,but the weather in Southern China which is high temperature and humidity brings bad effect on diary's production and reproduction performance,resulting in vast economic loss.At present studies on diary hot stress is few and main on physiologic index.
For the reasons,molecular markers are used to study heat-tolerance in holstein.main work and conclusions are as follows:
1.Determination of the RAPD markers
In this paper,with DNA from 52 high-milk-yield,strong heat-tolerance and 41 high-milk-yield,weak heat-tolerance Chinese Holstein cattle as material,studies on heat-tolerance in Holstein has been performed by using the technology of RAPD-PCRwith selected S441 and S463 RAPD primers,The results indicated that S441 marker and S441 marker are RAPD marker of heat-tolerance in Holstein.
2.Detection of RAPD markers in Holstein cattle
46 heads of Holstein bulls and 213 heads of Holstein cows are measured by useing S441 and S463 markers.To bulls,8 heads of them had the S441 marker,10 heads had the S463 marker and 5 heads for two.To cows,25 heads of them had the S441 marker, 26 heads had the S463 marker and 12 heads for two.
3.The relationship between RAPD markers and the decline rate of milk yield By analyzing the decline rate of milk yield for different markers,the result shows that the cow which had S441 marker,S463 marke and none marker had the different decline rate of milk yield,which were 3.49%,3.52%and 22.67%respectively.For the decline rate of milk yield,the cows which had the heat-tolerance candidate marker were lower than those with on marker,and the difference was significantly(P＜0.05).
4.Bioinformatic analysis for RAPD markers
According to the sequences of S441 marker and S463 marker,it was found that the sequences of S441 and KCNN2 gene had 89%homology,it may be intron 3 in KCNN2 gene.The sequences S463 and NW_001508847.1 had 99%homology.The function of NW_001508847.1 were unkown.Based on known function of the KCNN2 gene,it may be a candidate gene for heat-tolerance.
5.Studis on SCAR markers of heat-tolerance in Holstein
Base on sequence of RAPD-S441 and RAPD-S463,use premeier5.0 software to design corresponding SCAR premier.The RAPD-S441 marker was converted to SCAR marker,but RAPD-S463 was not.
6.The relationship between SCAR-S441 and milk composition
Butterfat rate,milk protein,milk sugar and dry matter were detected during the monthes from May to Sep.In May,the butter fat rate and milk protein level of the cows which had the SCAR markers were lower than that in the other monthes,while the indexs of the cows which had not the SCAR marker was higher in May,Aug.,and Sep. than in june and july.The level of milk sugar of the cows which had different markers had no significant difference(P＞0.05).In August,the level of the dry matter of two types of the cows were both lower than in the other monthes,and the difference were significant(P＜0.05).
本研究采用RAPD技术,从DNA水平对南方中国荷斯坦牛在5～9月的热应激反应作了研究,探索了与奶牛耐热性能相关的分子标记,并对耐热标记作了进一步的分析。具体内容如下:
1.RAPD标记的确定
以52头高产耐热组和41头高产不耐热组中国荷斯坦牛的DNA为材料,采用S441和S463两条RAPD引物,对荷斯坦牛的耐热性状进行研究。结果证实,S441和S463抗热应分子标记在耐热牛群中具有特异性。
2.RAPD标记在荷斯坦牛群中的检测结果
采用S441和S463引物一共测定了46头荷斯坦公牛,213头荷斯坦母牛。在公牛群中,具有S441和S463耐热候选标记的分别为8头和10头,同时具有两个耐热候选标记的5头;在母牛群中,具有S441和S463耐热候选标记的分别为25头和26头,同时具有两个耐热候选标记的12头。
3.RAPD标记与产奶量
S441、S463和无标记的母牛在热应激期间的产奶量下降率分别为3.49%、3.52%和22.67%,即有标记母牛的产奶量下降率显著低于无标记母牛。这种结果也验证了S441和S463都是耐热候选标记。
4.RAPD标记的生物信息学分析
根据两特异片段的测序信息,进行序列比较,结果显示S441序列与KCNN2基因的同源性达89%,其可能是KCNN2基因的内含子3;S463序列与NW_001508847.1的同源性达99%,NW_001508847.1的功能未知。根据已知KCNN2功能,初步判断其为耐热候选基因。
5.SCAR标记的转化
根据RAPD-S441、RAPD-S463标记的测序结果,利用Premier5.0软件设计SCAR引物。本研究成功地将RAPD-S441标记转化为耐热SCAR标记,并且能较好的区分高产耐热组和高产不耐热组。而RAPD-S463没有成功转换成SCAR标记。
6.SCAR-S441标记与乳成份的关系。
采用SCAR-S441分子标记,在5～9月测定了荷斯坦母牛的乳脂率、乳蛋白、乳糖和干物质。有SCAR标记的奶牛在6～9的乳脂率和乳蛋白均比5月高,无标记奶牛在6、7月的乳脂率和乳蛋白低于5月,而到8、9月恢复正常。6～9月乳糖的含量两组间没有差异(P＞0.05)。而在8月份,两组奶牛牛奶中的干物质均显著降低。
Dairy industry is an very important part of animal industry,but the weather in Southern China which is high temperature and humidity brings bad effect on diary's production and reproduction performance,resulting in vast economic loss.At present studies on diary hot stress is few and main on physiologic index.
For the reasons,molecular markers are used to study heat-tolerance in holstein.main work and conclusions are as follows:
1.Determination of the RAPD markers
In this paper,with DNA from 52 high-milk-yield,strong heat-tolerance and 41 high-milk-yield,weak heat-tolerance Chinese Holstein cattle as material,studies on heat-tolerance in Holstein has been performed by using the technology of RAPD-PCRwith selected S441 and S463 RAPD primers,The results indicated that S441 marker and S441 marker are RAPD marker of heat-tolerance in Holstein.
2.Detection of RAPD markers in Holstein cattle
46 heads of Holstein bulls and 213 heads of Holstein cows are measured by useing S441 and S463 markers.To bulls,8 heads of them had the S441 marker,10 heads had the S463 marker and 5 heads for two.To cows,25 heads of them had the S441 marker, 26 heads had the S463 marker and 12 heads for two.
3.The relationship between RAPD markers and the decline rate of milk yield By analyzing the decline rate of milk yield for different markers,the result shows that the cow which had S441 marker,S463 marke and none marker had the different decline rate of milk yield,which were 3.49%,3.52%and 22.67%respectively.For the decline rate of milk yield,the cows which had the heat-tolerance candidate marker were lower than those with on marker,and the difference was significantly(P＜0.05).
4.Bioinformatic analysis for RAPD markers
According to the sequences of S441 marker and S463 marker,it was found that the sequences of S441 and KCNN2 gene had 89%homology,it may be intron 3 in KCNN2 gene.The sequences S463 and NW_001508847.1 had 99%homology.The function of NW_001508847.1 were unkown.Based on known function of the KCNN2 gene,it may be a candidate gene for heat-tolerance.
5.Studis on SCAR markers of heat-tolerance in Holstein
Base on sequence of RAPD-S441 and RAPD-S463,use premeier5.0 software to design corresponding SCAR premier.The RAPD-S441 marker was converted to SCAR marker,but RAPD-S463 was not.
6.The relationship between SCAR-S441 and milk composition
Butterfat rate,milk protein,milk sugar and dry matter were detected during the monthes from May to Sep.In May,the butter fat rate and milk protein level of the cows which had the SCAR markers were lower than that in the other monthes,while the indexs of the cows which had not the SCAR marker was higher in May,Aug.,and Sep. than in june and july.The level of milk sugar of the cows which had different markers had no significant difference(P＞0.05).In August,the level of the dry matter of two types of the cows were both lower than in the other monthes,and the difference were significant(P＜0.05).
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