ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2018, Vol. 49 ›› Issue (9): 1818-1829.doi: 10.11843/j.issn.0366-6964.2018.09.003

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Construction and Expression of PHKG2 Gene Overexpression Vector and RNA Interference Vector in Congjiang Pig

WANG Yuan-yuan1,2,3, XU Hou-qiang1,2,3,4*, CHEN Wei1,2,4, ZHOU Di1,2,4, ZHANG Ming1,2,3, YANG Tao1,2,3   

  1. 1. Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region of Ministry of Education, Guizhou University, Guiyang 550025, China;
    2. Key Laboratory of Animal Genetics, Breeding and Reproduction in Guizhou, Guizhou University, Guiyang 550025, China;
    3. College of Animal Science, Guizhou University, Guiyang 550025, China;
    4. College of Life Science, Guizhou University, Guiyang 550025, China
  • Received:2018-02-02 Online:2018-09-23 Published:2018-09-26

Abstract:

The aim of this study was to clone the coding region of phosphorylase kinase gamma 2(PHKG2) gene in Congjiang pig and to explore the function of PHKG2 gene. In this study, the complete CDS region of PHKG2 gene in Congjiang pig was amplified by RT-PCR. By constructing the overexpression vector pEGFP-N3-PHKG2, four pairs of RNAi expression vectors targeting the Congjiang pig PHKG2 gene were designed and synthesized, and transiently transfected into C2C12 cell line and Congjiang pig kidney cells with normal growing cells as a blank control, the expression of green fluorescent protein of each recombinant vector was detected after 24 h. RNA was extracted from cells after 48 h. The expression levels of PHKG2 and glycogen metabolism related genes(glycogen phosphorylase (PYGM), glycogen synthase 1 (muscle)(GYS1), phosphoglyce rate mutase(PGAM2)) were detected by qRT-PCR, and the glycogen content of each group in cells was determined. The results showed that, after double enzyme digestion, sequencing detection and transient transfection of liposomes into C2C12 cells, it was verified that the overexpression vectors pEGFP-N3-PHKG2 and 4 RNAi expression vectors were successfully constructed. After transfecting into Congjiang pig kidney cells, compared with the blank and negative control (pEGFP-N3), the overexpression vector pEGFP-N3-PHKG2 transfected into cells extremely significantly increased the expression level of PHKG2 gene (P<0.01), and PGAM2 and PYGM genes expression were significantly up-regulated (P<0.05), and the glycogen content in cells was significantly reduced (P<0.05). In each RNAi vector, the interference efficiency of shRNA-1 was higher than that of the blank and negative control (NC). The expression levels of PHKG2 gene was extremely significantly down-regulated (P<0.01), and the expression levels of PGAM2, PYGM and GYS1 genes were significantly down-regulated (P<0.05), and significantly increased the glycogen content in the cells (P<0.05). shRNA-2 extremely significantly down-regulated the expression of PHKG2 gene (P<0.01); shRNA-3 also significantly interfered with the expression of PHKG2 (P<0.05); shRNA-4 did not interfere with the expression of the 4 genes detected obviously. The glycogen contents in cells didn't significantly increase after shRNA-2, shRNA-3, shRNA-4 transfected into the cells. In this study, the overexpression vectors and RNAi vectors of PHKG2 gene of Congjiang pig were successfully constructed. The expression of PHKG2, PYGM, PGAM2 and GYS1 genes and glycogen content were significantly affected. PHKG2 gene may be an important candidate gene affecting meat quality of Congjiang pig and to lay the foundation for further studying the role of PHKG2 gene in glycogen metabolism pathway.

Key words: Congjiang pig, PHKG2 gene, overexpression, RNAi

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