陆川猪PDK4基因序列分析、真核表达载体构建及组织表达分析

doi:10.16431/j.cnki.1671-7236.2020.08.001

Abstract

Abstract: This study was aimed to understand the sequence information of pyruvate dehydrogenase kinase 4 (PDK4) gene CDS region and the structure and function of the encoded protein in Luchuan pigs,construct a eukaryotic expression vector of PDK4 gene,and analyze the expression of PDK4 gene in different tissues,which laid a foundation for elucidating the molecular mechanism of PDK4 gene in the growth and development of Luchuan pigs.The CDS region of PDK4 gene in subcutaneous fat of Luchuan pigs was amplified by RT-PCR technology,the structure and function of PDK4 gene were predicted and analyzed using bioinformatics software.It was inserted into a eukaryotic expression vector to obtain pEGFP-N1-PDK4,the recombinant plasmid was transfected into 3T3-L1 cells by the liposome method,and then the fluorescence was observed.The expression of PDK4 gene mRNA in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and subcutaneous fat in Luchuan pigs were detected by Real-time quantitative PCR.The results showed that the CDS region of PDK4 gene in Luchuan pigs was 1 224 bp in length,encoding 407 amino acids,and had 99.8% homology with the CDS region of PDK4 gene in Sus scrafa published on NCBI.Bioinformatics analysis of the protein encoded by PDK4 gene in Luchuan pigs revealed that its molecular mass was 46.144 ku,the total number of atoms was 6 509,the theoretical isoelectric point (pI) was 7.21,and the number of positively and negatively charged amino acids was 42,respectively.There were 2 N-glycosylation sites and 33 phosphorylation sites of PDK4 protein.Subcellular localization results showed that 34.8% of PDK4 protein was present in mitochondria,30.4% was present in cytoplasm,26.1% was present in nucleus,and plasma membrane and vacuole membrane each accounted for 4.3%.Compared with control group,the expression of PDK4 gene in experimental group extremely significantly increased (P<0.01).The expression of PDK4 gene was most abundantly expressed in subcutaneous fat,followed by liver,lung,heart,spleen and kidney,it had the lowest expression in longissimus dorsi muscle,and the expression of subcutaneous fat was extremely significantly higher than that of longissimus dorsi muscle (P<0.01).This experiment successfully amplified the CDS region of PDK4 gene and constructed a eukaryotic expression vector containing it,and successfully predicted its structure and function,providing a theoretical basis for studying the genetic improvement of subcutaneous fat deposition in Luchuan pigs.

Key words: Luchuan pigs; PDK4 gene; cloning; sequence analysis; eukaryotic expression vector; tissue expression

CLC Number:

PAN Pengcheng, WEN Binhua, XIE Wan, JIANG Changjin, JIAO Di, CHEN Baojian, GUAN Zhihui, XIE Bingkun. Sequence Analysis,Eukaryotic Expression Vector Construction and Tissue Expression Analysis of PDK4 Gene in Luchuan Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(8): 2337-2347.

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Sequence Analysis,Eukaryotic Expression Vector Construction and Tissue Expression Analysis of PDK4 Gene in Luchuan Pigs

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