摘要
分析中国三种实验用小型猪线粒体DNA (mtDNA)D loop的多态性 ,建立各品种品系猪的遗传标记 ,为各品种品系猪的鉴别提供依据。应用PCR对西双版纳近交系小耳猪、广西巴马小型猪、贵州小型香猪和长白猪血液总DNA样品中mtDNAD loop进行扩增 ,用 2 3种限制性内切酶消化 ,观察其酶切多态。PCR扩增其mtDNAD loop 5′端 2 2 7bp高变区域 ,应用PCR SSCP和PCR直接测序分析 ,观察其单链构象多态和序列多态。结果显示 :3种小型猪之间未见酶切长度多态、单链构象多态和序列多态 ;与长白猪之间表现出单链构象多态和序列多态。本研究认为 :3种实验用小型猪之间mtDNA多态性贫乏 ,证明其在母系起源和进化上的一致性 ,亲源关系很近 ,应用PCR RFLP、PCR SSCP和PCR直接测序分析 ,尚不能作为 3种实验用小型猪品种品系鉴定的依据 ,但与长白猪等欧系猪比较有一定差异。
The present study is to analyze the polymorphism of the mitochondrial DNA (mtDNA) D-loop in Guizhou miniature Xiang pig, Guangxi Bama miniature pig and Xishuangbanna Small-ear inbred pig, all of those are used as laboratory miniature pigs in China, and Landrace, and to look for its cytoplasmic DNA markers to identify them. Pure and intact total DNA was obtained with leukocyte sediment added to the digestive solution STE (10 mmol/L Tris-HCl pH 8.0, 0.25 mmol/L EDTANa 2, 100 mmol/L NaCl), SDS(sodium dodecyl sulphate) and protease K, and was purified by Phenol/Chloroform extraction and ethanol precipitation. A pairs of primers were designed to amplify mtDNA D-loop of the pigs for PCR-RFLP(polymerase chain reaction-restriction fragment length polymorphism), the amplification products were digested by 23 restriction enzymes and detected with 2% agarose gel. Clear amplification band were obtained, and the fragment length was the same as that of early reports. Among the 36 pigs examined, there was no variation among or within each of the breeds showing monomorphism. Another pairs of primers were used to amplify mtDNA D-loop 5′-end 227 bp high variable regions, the amplification products were detected by PCR-SSCP(single string conformation polymorphism) and PCR-direct sequencing. There was no variation among or within three breeds of Chinese laboratory miniature pigs besides Landrace. By comparison of the sequences of the porcine mtDNA D-loop 5′-end, no difference was detected among the three breeds of Chinese laboratory miniature pigs, while 4 base variation with Landrace. It was concluded that the polymorphism of mtDNA D-loop within the three breeds of Chinese laboratory miniature pigs was poor, useful cytoplasmic DNA markers for distinguishing among them could not be obtained by the methods of PCR-RFLP,PCR-SSCP and PCR-directing sequence, also it could be used to distinguish them from Landrace, more attention should be paid to the study of the nDNA.
出处
《动物学报》
SCIE
CAS
CSCD
北大核心
2001年第4期425-430,共6页
ACTA ZOOLOGICA SINICA
基金
国家重点基础研究发展规划项目 (No .G2 0 0 0 0 16 10 6 )
国家"九五"重点科技攻关项目 (No .96 A2 3 0 6 0 1)
国家自然科学基
