China Animal Husbandry and Veterinary Medicine ›› 2022, Vol. 49 ›› Issue (3): 837-844.doi: 10.16431/j.cnki.1671-7236.2022.03.005

• Special Focus: Genetic Breeding and Nutrition of Waterfowl • Previous Articles     Next Articles

Characteristic Analysis of circ-ZNF326 in Laying Ducks, Construction of Overexpression Vector and Its Effect on Proliferation of Small Intestinal Epithelial Cells

WEI Wenzhuo1,2, ZHENG Chao1,2, WU Yan1, LIANG Zhenhua1, PI Jingsong1, DU Jinping1, LI Chengfeng3, ZHANG Hao1   

  1. 1. Key Laboratory of Animal Embroyo Engineering and Molecular Breeding of Hubei Province, Institute of Animal Husbandry and Veterinary Sciences, Hubei Academy of Agricultural Sciences, Wuhan 430064, China;
    2. School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China;
    3. Hubei Shendan Healthy Food Co., Ltd., Anlu 432600, China
  • Revised:2021-10-31 Online:2022-03-05 Published:2022-03-03

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Abstract: 【Objective】 The study was aimed to clarify the basic characteristics of circ-ZNF326 and its effect on the proliferation of small intestinal epithelial cells in ducks.【Method】 Taking the Mallard duck as the research object, the duck embryo intestine was collected and cultured for small intestinal epithelial cells, RNA was extracted and reverse transcribed into cDNA, primers were designed with reference to the full-length sequence of circ-ZNF326, partial sequences of circ-ZNF326 were obtained by PCR reaction amplification, sequencing was performed to verify its loop formation mode using reverse splice sites.The nuclear and cytoplasmic RNAs were extracted using the PARISTM Kit to detect their nucleoplasmic distribution in the cells.The full-length sequence of circ-ZNF326 was synthesised and ligated onto the circ-ZNF326 eukaryotic expression vector using double digestion and T4 ligase, and then transformed into E.coli DH5α competent cells for expansion.The recombinant plasmid was detected by double enzyme digestion and agarose gel electrophoresis.The circ-ZNF326 overexpression vector was transfected into the small intestinal epithelial cells.The expression efficiency of circ-ZNF326 over expression was detected by Real-time quantitative PCR.The effect of over expression of circ-ZNF326 on the proliferation of small intestinal epithelial cells was detected by CCK-8.【Result】 circ-ZNF326 was formed by reverse splicing of exons 10, 11 and 12 of ZNF326 gene, and mainly distributed in the cytoplasm.circ-ZNF326 overexpression vector was successfully constructed and its expression efficiency was extremely significantly higher than that of no-load group (P<0.01).After transfection of circ-ZNF326 overexpression vector into small intestinal epithelial cells, the cell viability in circ-ZNF326 overload group was extremely significantly or significantly higher than that in no-load group within 24-72 h (P<0.01 or P<0.05).【Conclusion】 This experiment successfully verified the reverse splicing and annular structure of circ-ZNF326, proved that circ-ZNF326 was mainly enriched in the cytoplasm, constructed circ-ZNF326 overexpression vector, and confirmed that overexpression of circ-ZNF326 could improve the activity of small intestinal epithelial cells of laying ducks.The results provided a theoretical basis for further study on the biological functions of circ-ZNF326 and its regulation mechanism on the proliferation of small intestinal epithelial cells in laying ducks.

Key words: circRNA; laying duck; circ-ZNF326; nucleoplasmic distribution; overexpression vector

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