从江香猪TPM3基因的克隆与序列分析
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摘要
[目的]对从江香猪TPM3基因进行扩增、克隆和序列分析。[方法]通过RT-PCR从猪背最长肌中扩增TPM3基因CDS区并进行克隆,利用DNAMAN和BioEdit等软件对克隆的序列进行分析。[结果]成功克隆了从江香猪TPM3基因并构建了pUCm-T-TPM3载体;获得了TPM3-1和TPM3-2两个序列,TPM3-1有两个碱基的差异,相似度达99.73%;TPM3-2有一个碱基的变化,同时插入了一段76 bp的片段,相似度达99.87%;密码子偏好性分析显示,UUG编码亮氨酸的频率为0.49%,而CUG编码亮氨酸的频率为2.89%,说明第49位碱基的转换可能提高蛋白的合成效率;RNA二级结构分析显示,碱基突变会影响TPM3基因RNA二级结构和最小自由能的变化;蛋白理化性质分析显示,氨基酸的改变对α螺旋、β折叠及转角等二级结构影响不明显。[结论]从江香猪TPM3基因的碱基突变可能影响原肌球蛋白的合成,为探究其对从江香猪肉质及种资源的开发利用提供试验依据。
[Objective]It was to clone and sequence analysis of Congjiang xiang pig TPM3 gene. [Methods]The CDS region of TPM3 gene in Congjiang xiang pig was cloned by RT-PCR and its sequence was analyzed using software such as DNAMAN and Bio Edit. [Results]The CDS region of TPM3 gene from Congjiang xiang pig was successfully cloned and the p UCm-T-TPM3 vector was constructed. We had also acquired two different sequences involving in TPM3-1 and TPM3-2. Sequence analysis results had showed that TPM3-1 had two different sites comparing original sequence and its similarity was 99. 73%.On the other sequence,TPM3-2 had a changed base and inserted a DNA fragment of 76 bp length as well as similarity is99. 87% comparing with the original sequence. The codon bias analysis showed that the UUG-encoded leucine frequency was0. 49%,while the CUG-encoded leucine frequency was 2. 89%,indicating that the 49 th base conversion may increase protein synthesis efficiency. RNA secondary structure analysis showed that base mutations affected the changes of TPM3 RNA secondary structure and minimal free energy. Analysis of protein physicochemical properties showed that amino acid changes had no significant effect on secondary structures such as α-helix,β-sheet,and turn. [Conclusion]The base mutation of TPG3 gene in Jiangxiang pig may affect the synthesis of tropomyosin. It provides experimental basis for exploring the development and utilization of Jiangjing pork meat and its resources.
[Objective]It was to clone and sequence analysis of Congjiang xiang pig TPM3 gene. [Methods]The CDS region of TPM3 gene in Congjiang xiang pig was cloned by RT-PCR and its sequence was analyzed using software such as DNAMAN and Bio Edit. [Results]The CDS region of TPM3 gene from Congjiang xiang pig was successfully cloned and the p UCm-T-TPM3 vector was constructed. We had also acquired two different sequences involving in TPM3-1 and TPM3-2. Sequence analysis results had showed that TPM3-1 had two different sites comparing original sequence and its similarity was 99. 73%.On the other sequence,TPM3-2 had a changed base and inserted a DNA fragment of 76 bp length as well as similarity is99. 87% comparing with the original sequence. The codon bias analysis showed that the UUG-encoded leucine frequency was0. 49%,while the CUG-encoded leucine frequency was 2. 89%,indicating that the 49 th base conversion may increase protein synthesis efficiency. RNA secondary structure analysis showed that base mutations affected the changes of TPM3 RNA secondary structure and minimal free energy. Analysis of protein physicochemical properties showed that amino acid changes had no significant effect on secondary structures such as α-helix,β-sheet,and turn. [Conclusion]The base mutation of TPG3 gene in Jiangxiang pig may affect the synthesis of tropomyosin. It provides experimental basis for exploring the development and utilization of Jiangjing pork meat and its resources.
引文
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[2]杨静娴.原肌球蛋白的分子生物学研究进展[J].大连医科大学学报,2004,26(2):144-147.
[3]Perry S V.Vertebrate tropomyosin:Distribution,properties and function[J].Journal of Muscle Research&Cell Motility,2001,(22):5-49.
[4]田兴国.猪肌肉组织EST的基因克隆与表达[D].广州:华南农业大学,2004:25-29.
[5]周硕,鲁正,吴华,等.原肌球蛋白的功能与相关疾病的研究进展[J].中华临床医师杂志,2016,(11):1601-1604.
[6]叶保国,张小辉,徐铁山.北京鸭和淮南麻鸭胸肌发育过程TPM1和TPM3基因的表达变化研究[J].黑龙江畜牧兽医,2014,(19):67-70.
[7]孙成娟,许厚强,陈伟,等.贵州从江香猪Fox O4、PRKAA1基因在不同组织中的表达分析[J].基因组学与应用生物学,2017,36(3):841-846.
[8]邱阳,陈晓雅,孙峥嵘.原肌球蛋白3与雄激素受体配体结合域之间的相互作用[J].中国医科大学学报,2016,45(11):975-976.
[9]孙岩岩,蔡惠芬,罗卫星,等.贵州黑山羊、贵州白山羊TFB1M基因第7外显子SNP研究[J].生物技术,2014,24(2):32-36.
[10]郑芳霞,王嘉福,冉雪琴.贵州地方猪品种对氧磷脂酶1基因的多态性研究[J].遗传繁育,2013,40(5):144-148.
[11]Hal B.Zabin,Martin P Horvath,Thomas Charles Terwilliger.Approaches to predicting effects of single amino acid substitutions on the function of a protein[J].Biochemistry,1991,30(25):6230-6240.
[12]Jiqiang Ling,Patrick O’Donoghue,Dieter S9ll.Genetic code flexibility in microorganisms:novel mechanisms and impact on physiology[J].Nature Reviews Microbiology,2015,13(11):707.
[13]白优,张勇,陈祥,等.从江香猪DRD1基因多态及其生物信息学分析[J].基因组学与应用生物学,2017,(02):570-574.