藏猪肌肉生长抑制素（MSTN）基因的多态性研究及其序列分析
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摘要
本文通过自行设计引物，利用PCR-SSCP技术分析了藏猪肌肉生长抑制素(MSTN)基因3个外显子的多态性；利用计算机网络技术和生物信息学方法，比较分析了藏猪与登录在GenBank中的其它猪种以及其它物种MSTN基因编码区序列特点。本研究为藏猪遗传资源的全面分析积累了资料，为合理利用藏猪优良的遗传资源提供了理论依据。
研究结果表明，藏猪MSTN基因外显子1表现多态性，存在A、C两种等位基因和AA、AC、CC三种基因型，三种基因型频率分别为20.54％、52.68％、26.78％，A、C两种等位基因的频率分别为46.88％和53.12％。通过序列测定，将所获序列和GenBank中登录的猪MSTN基因全序列(AY208121)相应片段进行比对，首次拼接出了藏猪MSTN基因完整的编码区序列，共1128bp(GenBank登录号为EF612791)，A、T、G、C四种碱基的含量分别为32.8％、25.3％、21.1％、20.8％，其中G＋C的含量(41.9％)低于A＋T的含量(58.1％)。藏猪MSTN基因编码区71bp处存在A→C单碱基突变，该突变导致编码的第24位氨基酸由天冬酰胺改变为苏氨酸。
藏猪和其它猪种MSTN基因编码区序列比较结果显示，品种间MSTN基因编码区序列碱基组成差异不大，品种间核苷酸多样度Pi＝0.00197，平均核苷酸差异数K＝2.222；MSTN基因编码区71bp处的突变仅在藏猪中发现；藏猪MSTN基因编码的蛋白质20种氨基酸含量不均衡，以亮氨酸含量最为丰富，组氨酸和色氨酸含量最少，按极性分析，极性氨基酸的含量高于非极性氨基酸；藏猪MSTN基因对同义密码子的使用具有强烈的偏倚性，其它猪种也表现类似现象。
藏猪和其它物种MSTN基因编码区序列比较结果显示，藏猪与人、猩猩、牛、羊、狗、鼠的相似度都在90％以上，藏猪与鸡和大马哈鱼的相似度分别为83.0％和56.2％；不同物种对同义密码子使用的偏倚程度和偏爱类型各不相同，具有种属特异性；用邻接法构建MSTN基因NJ系统发育树，藏猪与其它哺乳纲动物聚在一个分支上，而属于鸟纲的鸡和鱼纲的大马哈鱼聚在另两个分支上，这与传统的生物学分类法结果一致。
In this thesis, the polymorphisms of myostatin gene three complete exons in Tibetan pig were analyzed by PCR-SSCP technology. Utilizing computer network technique and biological message means, the characteristics of myostatin gene coding region sequences were analyzed comparatively between Tibetan pig and other breeds or species from GenBank. The results accumulated datas for analysing entirely Tibetan pig's heredity resource, and provided theoretic gists for utilizing reasonly Tibetan pig's excellent heredity resource.
The research results showed that there was a polymorphism site in exonl in Tibetan pig, and the frequence of AA, AC and CC genotypes were 20.54%, 52.68%, 26.78%, respectively. The frequency of allele A and B were 46.88% and 53.12%. Through comparing the sequencing results with pig myostatin gene relevant sequence (GenBank accession number, AY208121), the Tibetan pig myostatin gene complete coding region sequence (1128bp) was abtained firstly. The sequence was submitted to GenBank under accession number EF612791. The content of A, T, G and C were 32.8%, 25.3%, 21.1%, 20.8%, respectively. GC base (41.9%) was lower than AT (58.1%). One single nucleotide mutation (A→C) in 71bp of coding region in Tibetan pig was found, and this mutation resulted in 24th amino acid change (Asn→Thr).
The analysis of myostatin gene coding region sequences between Tibetan pig and other breeds indicated that the difference of base contents were unconspicuous. The nucleotide diversity was 0.00197, and the average number of nucleotide differences was 2.222 in breeds. The mutation of myostatin gene in 71bp was exclusive for Tibetan pig. The contents of twenty kinds of amino acid coding myostatin gene in Tibetan pig were unbalanced. Leucine was the most; histidine and tryptophan were the least. According to the polarity, the content of polarity amino acid was higher than nopolarity amino acid. The usage of synonymous codon was very biased in Tibetan pig, and the similar phenomenons were represented in other breeds.
The analysis of myostatin gene coding region sequences between Tibetan pig and other species showed that the similarity between Tibetan pig and other lactation animals were above 90% all. The similarity between Tibetan pig and chicken, salmon were 83.0%, 56.2%, respectively. The analysis of codon usage indicated that the usage of synonymous codon was very biased, and the biased sorts were different among species. Phylogenetic tree of myostatin gene established by neighbor-joining method showed that Tibetan pig and other lactation animals assembled together, moreover, chicken and samlon assembled on other two branches, so the results were in accordance with traditional taxonomy in biology.
研究结果表明，藏猪MSTN基因外显子1表现多态性，存在A、C两种等位基因和AA、AC、CC三种基因型，三种基因型频率分别为20.54％、52.68％、26.78％，A、C两种等位基因的频率分别为46.88％和53.12％。通过序列测定，将所获序列和GenBank中登录的猪MSTN基因全序列(AY208121)相应片段进行比对，首次拼接出了藏猪MSTN基因完整的编码区序列，共1128bp(GenBank登录号为EF612791)，A、T、G、C四种碱基的含量分别为32.8％、25.3％、21.1％、20.8％，其中G＋C的含量(41.9％)低于A＋T的含量(58.1％)。藏猪MSTN基因编码区71bp处存在A→C单碱基突变，该突变导致编码的第24位氨基酸由天冬酰胺改变为苏氨酸。
藏猪和其它猪种MSTN基因编码区序列比较结果显示，品种间MSTN基因编码区序列碱基组成差异不大，品种间核苷酸多样度Pi＝0.00197，平均核苷酸差异数K＝2.222；MSTN基因编码区71bp处的突变仅在藏猪中发现；藏猪MSTN基因编码的蛋白质20种氨基酸含量不均衡，以亮氨酸含量最为丰富，组氨酸和色氨酸含量最少，按极性分析，极性氨基酸的含量高于非极性氨基酸；藏猪MSTN基因对同义密码子的使用具有强烈的偏倚性，其它猪种也表现类似现象。
藏猪和其它物种MSTN基因编码区序列比较结果显示，藏猪与人、猩猩、牛、羊、狗、鼠的相似度都在90％以上，藏猪与鸡和大马哈鱼的相似度分别为83.0％和56.2％；不同物种对同义密码子使用的偏倚程度和偏爱类型各不相同，具有种属特异性；用邻接法构建MSTN基因NJ系统发育树，藏猪与其它哺乳纲动物聚在一个分支上，而属于鸟纲的鸡和鱼纲的大马哈鱼聚在另两个分支上，这与传统的生物学分类法结果一致。
In this thesis, the polymorphisms of myostatin gene three complete exons in Tibetan pig were analyzed by PCR-SSCP technology. Utilizing computer network technique and biological message means, the characteristics of myostatin gene coding region sequences were analyzed comparatively between Tibetan pig and other breeds or species from GenBank. The results accumulated datas for analysing entirely Tibetan pig's heredity resource, and provided theoretic gists for utilizing reasonly Tibetan pig's excellent heredity resource.
The research results showed that there was a polymorphism site in exonl in Tibetan pig, and the frequence of AA, AC and CC genotypes were 20.54%, 52.68%, 26.78%, respectively. The frequency of allele A and B were 46.88% and 53.12%. Through comparing the sequencing results with pig myostatin gene relevant sequence (GenBank accession number, AY208121), the Tibetan pig myostatin gene complete coding region sequence (1128bp) was abtained firstly. The sequence was submitted to GenBank under accession number EF612791. The content of A, T, G and C were 32.8%, 25.3%, 21.1%, 20.8%, respectively. GC base (41.9%) was lower than AT (58.1%). One single nucleotide mutation (A→C) in 71bp of coding region in Tibetan pig was found, and this mutation resulted in 24th amino acid change (Asn→Thr).
The analysis of myostatin gene coding region sequences between Tibetan pig and other breeds indicated that the difference of base contents were unconspicuous. The nucleotide diversity was 0.00197, and the average number of nucleotide differences was 2.222 in breeds. The mutation of myostatin gene in 71bp was exclusive for Tibetan pig. The contents of twenty kinds of amino acid coding myostatin gene in Tibetan pig were unbalanced. Leucine was the most; histidine and tryptophan were the least. According to the polarity, the content of polarity amino acid was higher than nopolarity amino acid. The usage of synonymous codon was very biased in Tibetan pig, and the similar phenomenons were represented in other breeds.
The analysis of myostatin gene coding region sequences between Tibetan pig and other species showed that the similarity between Tibetan pig and other lactation animals were above 90% all. The similarity between Tibetan pig and chicken, salmon were 83.0%, 56.2%, respectively. The analysis of codon usage indicated that the usage of synonymous codon was very biased, and the biased sorts were different among species. Phylogenetic tree of myostatin gene established by neighbor-joining method showed that Tibetan pig and other lactation animals assembled together, moreover, chicken and samlon assembled on other two branches, so the results were in accordance with traditional taxonomy in biology.
引文
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