中国荷斯坦奶牛κ-酪蛋白可变剪切及蛋白突变类型的研究

Studies onκ-Casein Alternative Splicing and Protein Mutation Types in Chinese Holstein Cows

【目的】κ-酪蛋白蛋白突变与奶牛生产性能和乳品加工性能关系密切,监控中国荷斯坦奶牛κ-酪蛋白的蛋白突变,可以防止对生产不利的突变的发生。【方法】以50头不同泌乳期的中国荷斯坦奶牛为研究对象,获取每头奶牛乳中的体细胞,提取RNA,反转录为cDNA,设计不同引物,分别扩增κ-酪蛋白可变剪切体序列和CDS序列,并进行测序、蛋白突变分析、突变蛋白的磷酸化、糖基化分析。【结果】测序结果表明在不同泌乳期中国荷斯坦奶牛κ-酪蛋白只存在Ensemble数据库中公布的573 bp的剪切形式,不存在另一种剪切体。κ-酪蛋白的CDS区存在三处碱基突变,即c.536T>C、c.572C>A、c.633G>A,形成的蛋白突变型只有A型和B型两种。A型与B型相比,A型多了两个磷酸化位点(136位和142位)和一个O型糖基化位点(157位)。【结论】不同泌乳期的中国荷斯坦奶牛κ-酪蛋白只存在573 bp的剪切体,蛋白突变型只存在A型和B型,未检测到其它突变型,依据生产目的不同,可对A型奶牛与B型奶牛进行筛选。

[Objective]Protein mutation ofκ-casein was closely related to milk production performance and milk processing performance.Monitoringκ-casein mutation in Chinese Holstein dairy cows was to prevent the occurrence of harmful mutation.[Method]A total of 50 Chinese Holstein cows at different lactation stages were used as study subjects.Somatic cells were extracted from the milk of each cow,RNA was extracted and reverse transcribed into cDNA,and different primers were designed.Theκ-casein alternative spliceosome and CDS sequences were amplified and sequenced.Later,the protein mutation,phosphorylation and glycosylation of the mutant proteins were analyzed.[Result]The results of sequencing showed that theκ-casein of Chinese Holstein cows in different lactation stages only existed in the 573 bp splice form which was published in Ensemble database,and there was no other splice form.There are three mutations in the CDS region ofκ-casein.c.536T>C,c.572C>A,c.633G>A.Therefore,only two types of protein mutations.Type A and Type B,are formed.Compared with Type B,Type A has two more phosphorylation sites(positions 136 and 142)and one more O-glycosylation site(position 157).[Conclusion]In this study,κ-casein of Chinese Holstein cows was not found to have other alternative splices,only 573 bp splice form.And no other protein mutants ofκ-casein were detected,only Type A and Type B.Depending on the purpose of production,dairy cows producing Type A or Type Bκ-casein can be screened.