陆川猪MYL2基因生物信息学分析、真核表达载体构建及组织表达研究

doi:10.16431/j.cnki.1671-7236.2023.05.007

Abstract

Abstract: 【Objective】 The pourse of this study was to investigate the structure and function of CDS region sequence of myosin light chain 2 (MYL2) gene and its encoded protein of Luchuan pigs,and to explore the effects of MYL2 gene on muscle growth and development,so as to provide molecular basis for the development and utilization of Landchuan pigs.【Method】 The CDS region of MYL2 gene in Luchuan pigs was amplified by RT-PCR.MegAlign software was used to compare MYL2 gene in Luchuan pigs with different species and construct phylogenetic tree.The physical and chemical properties,hydrophobicity and transmembrane structure of MYL2 protein were analyzed by bioinformatics software.The eukaryotic expression vector of MYL2 gene was constructure,the recombinant plasmid was transfected into C2C12 cells by the liposome method, and then the fluorescence was observed. Real-time quantitative PCR was used to detect the expression of MYL2 gene in different tissues of Luchuan pigs.【Result】 The results showed that the CDS region of MYL2 gene in Luchuan pigs was 501 bp length,encoded 166 amino acids,and the similarity was 99.6% with the wild boar MYL2 gene published in NCBI.There were two mutations,C mutates to T at 156 bp,which was synonymous mutation.Mutation from T to C at 404 bp caused amino acid mutation from isoleucine (I) to threonine (T).Bioinformatics analysis showed that the total number of MYL2 protein atoms was 2 633,the molecular mass was 18.879 ku,the theoretical isoelectric point (pI) was 4.83.There was no transmembrane structure,no signal peptide,and it was a non-secreted protein.The MYL2 protein had 16 sites that might be phosphorylated and 1 N-glycosylation site at the 78-position amino acid.The subcellular localization results showed that MYL2 protein was mainly found in cytoplasm (56.5%),cytoskeleton (21.7%), mitochondria (8.7%),nucleus (8.7%) and vacuoles (4.3%).The secondary structure of MYL2 protein was mainly composed of alpha helix,random coil,beta turn and extended chain,accounting for 57.83%,29.52%,9.04% and 3.61%,respectively.The results of cell tests showed that fluorescence was observed in both the experimental group and control group after 48 h.Compared with control group,the expression of MYL2 gene in experiment group was extremely significantly increased (P＜0.01).The expression of MYL2 gene in different tissues from high to low were heart,longissimus dorsi muscle,liver,subcutaneous fat,lung,spleen and kidney,and the expression in longissimus dorsi muscle was significantly higher than that in other tissues except heart (P＜0.05).【Conclusion】 In this study,the CDS region of MYL2 gene in Luchuan pigs was cloned successfully,the eukaryotic expression vector and tissue expression profile were constructed.The structure and function of the protein encoded by MYL2 gene were analyzed,which provided molecular basis for exploring the role of MYL2 gene in the growth and development of muscle and the development and utilization of Luchuan pigs.

Key words: Luchuan pigs; MYL2 gene; bioinformatics; eukaryotic expression vector

CLC Number:

PAN Pengcheng, LEI Zongquan, LI Xian, HU Xiangyun, QIN Qiantao, QIN Zhaoxian, GUAN Zhihui, CHEN Baojian, XIE Bingkun. Bioinformatics Analysis,Eukaryotic Expression Vector Construction and Tissue Expression of MYL2 Gene in Luchuan Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1796-1806.

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Bioinformatics Analysis,Eukaryotic Expression Vector Construction and Tissue Expression of MYL2 Gene in Luchuan Pigs

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