蛋鸭circ-ZNF326的特征分析、过表达载体的构建及对小肠上皮细胞增殖的影响

【目的】 明确circ-ZNF326的基本特征及对蛋鸭小肠上皮细胞增殖的影响。【方法】 以绿头鸭为研究对象，采集鸭胚肠道并培养小肠上皮细胞，提取RNA并反转录为cDNA，参考circ-ZNF326的全长序列设计引物，通过PCR反应扩增获得circ-ZNF326的部分序列，测序后利用反向剪接位点验证其成环方式。利用PARISTM Kit提取细胞核与细胞质RNA检测其在细胞中的核质分布情况。合成circ-ZNF326全长序列并利用双酶切及T4连接酶将circ-ZNF326全长序列连接在PLCDH-ciR真核表达载体上，转化大肠杆菌DH5α感受态细胞扩大培养，提取重组质粒进行双酶切及琼脂糖凝胶电泳检测，将circ-ZNF326过表达载体转染小肠上皮细胞，利用实时荧光定量PCR技术检测circ-ZNF326过表达后的表达效率，利用CCK-8检测过表达circ-ZNF326对蛋鸭小肠上皮细胞增殖的影响。【结果】 circ-ZNF326是由ZNF326基因第10、11和12号外显子反向剪接而成，且主要分布在细胞质中。circ-ZNF326过表达载体构建成功，表达效率极显著高于空载组（P<0.01）。将circ-ZNF326过表达载体转染小肠上皮细胞，结果发现，circ-ZNF326过载组在24~72 h细胞活力极显著或显著高于空载组（P<0.01；P<0.05）。【结论】 本试验成功验证了circ-ZNF326的反向剪接与环状结构，证明了circ-ZNF326主要富集在细胞质中，并构建了circ-ZNF326过表达载体，证实了过表达circ-ZNF326可提高蛋鸭小肠上皮细胞的活力，为深入研究circ-ZNF326的生物学功能及其对蛋鸭小肠上皮细胞增殖的调控机制奠定了理论基础。

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

【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.