从江香猪PDK4基因干扰载体的构建与检测

doi:10.16431/j.cnki.1671-7236.2018.04.005

Abstract

Abstract:

In order to further reveal the regulation function of PDK4 gene in intramuscular preadipocyte cells,this study collected the longissimus dorsi from the 5-day-old Congjiang Xiang pig.Type Ⅱ collagenase was adopted to digest the cells,and intramuscular preadipocyte cells were separated and cultured,and then the morphology were observed.According to the cloning PDK4 sequence,online software was used to design and synthesize three pairs of specific single siRNA and a pair of negative control sequences,annealing products were cloned into pLVX-shRNA2-Puro vector directly.The recombinant plasmids were verified by double enzyme digestion and sequencing.The constructed interference plasmids were transfected into the intramuscular preadipocyte cells of Congjiang Xiang pig.The most effective interference sequences were screened by Real-time PCR.The results showed that the primary culture of intramuscular preadipocyte cells started adherent in about 4 h,and the cell morphology was uniform after 24 h,the cells were firmly adherent.On the 5th day,the adherent cells were monolayer.The results of double enzyme digestion and sequencing showed that the four pairs of siRNA interference sequences designed and synthesized were correctly connected with the vector plasmid,indicating that the PDK4 gene interference vectors were constructed successfully,and the transfection of the intramuscular preadipocyte cells was successful.The results of Real-time PCR showed that the interfering vector could effectively reduce the mRNA expression of PDK4 gene in intramuscular preadipocyte cells with the best interference efficiency of 81.90%.In this study,we successfully cultured the intramuscular preadipocyte cells and constructed the interference vector of PDK4 gene,which laid the foundation for further research on the regulation mechanism of PDK4 gene on lipid metabolism.

Key words: Congjiang Xiang pig; PDK4 gene; intramuscular preadipocyte cell; interference vector; interference efficiency

CLC Number:

S828

YANG Yang, XU Houqiang, CHEN Wei, XU Min, CHEN Yinglian. Construction and Detection of the Interference Vector of PDK4 Gene in Congjiang Xiang Pig[J]. , 2018, 45(4): 873-880.

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Construction and Detection of the Interference Vector of PDK4 Gene in Congjiang Xiang Pig

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