不同品种猪PBD-119基因的克隆与差异表达研究

Cloning and different expression of PBD-119 gene in different breeds of pigs

为获得猪β防御素-119(PBD-119)基因mRNA序列以及在不同品种猪体内组织表达量模式,该试验以民猪、大白猪和野杂猪为试验动物,采用RT-PCR方法扩增PBD-119基因mRNA并进行生物信息学分析,利用qRT-PCR的方法检测该基因在不同品种猪组织中的差异表达情况。RT-PCR扩增结果表明:在3个群体猪3种类型组织(肝脏,脾脏和血液)中均能扩增出特异性片段,测序结果显示片段长度为408bp,通过对PBD-119基因序列的比对分析,该基因在第263位碱基上存在多肽位点的改变(C263T),并在第53氨基酸位点发生改变,由脯氨酸变为丝氨酸(P-S),但并不影响二硫键结构;进化树分析表明,猪与人、倭黑猩猩和马的亲缘关系较近,与羊驼和双峰驼亲缘关系较远。qRT-PCR结果显示,PBD-119基因在3个品种猪的各组织中均有表达,且表达存在差异,以品种为研究对象比较相同组织的差异表达,PBD-119基因在肝脏中的表达为野杂猪>民猪>大白猪,在脾脏中表达为大白猪>民猪>野杂猪,而在血液中的表达为民猪>野杂猪>大白猪;以相同品种为目标,考察不同组织间PBD-119基因差异表达,结果发现大白猪不同于民猪和野杂猪在肝脏、脾脏和血液中的表达,表现为脾脏最高,其次为肝脏、血液,而民猪和野杂猪的表达为肝脏和血液中较高,脾脏最低。该试验结果为进一步探究PBD-119基因的生物学功能及免疫防御相关的候选基因提供基础依据。

In order to explore the differences in the expression levels,the structure and function of porcine beta defensin-119(PBD-119)genes in different tissues of different breeds of pigs,PBD-119 gene of Min pig,Large White pig and Wild boars was amplified by RT-PCR and analyzed by bioinformatics,the difference expression level of PBD-119 gene in the tissues of different breeds of pigs was detected by qRT-PCR method.RT-PCR amplification results showed that the specific fragment was amplofied in the three types of tissues(liver,spleen and blood)of three different breeds of pigs,sequencing results showed that the fragment length was 408 bp,the gene on the 263 th base existed the change of the polypeptide loci(C263 T),and the 53 th amino acid happened change from proline to serine(P-S)through comparative analysis the sequence of PBD-119,but had no effect on the disulfide bond structure;the evolutionary tree showed that the nucleotide sequence of PBD-119 gene was closely to Homo sapiens,Pan paniscus and Equus caballus,but it was far from the Camelus ferus and Vicugna pacos.The results of qRT-PCR showed that PBD-119 gene was expressed in the liver,spleen and blood of Min pigs,Large White pigs and Wild boars,and the expression was different.In the different breeds,the expression of PBD-119 in liver was Wild pigs>Min pigs>Large white pigs,the expression in spleen was Large white pigs>Min pigs>Wild pigs,the expression in blood was Min pigs>Wild pigs>Large white pigs.The PBD-119 expression was different between different groups,the results found that the expression in Large white pigs was different from Min pigs and Wild pigs,the expression in spleen was the highest,followed by liver and blood,while the expression of Min pigs and Wild pigs was higher in liver and blood,and the lowest in spleen.The results of this experiment provide a basis for further exploring the biological function of PBD-119 gene in pigs and the shape candidate genes related to meat quality.