摘要
【目的】克隆鸡过氧化还原酶1(peroxiredoxin 1,PRDX1)基因,探究其对鸡前脂肪细胞分化的作用,为进一步分析PRDX1功能奠定基础。【方法】采用RT-qPCR检测PRDX1在AA白羽肉鸡不同组织中的表达;采用ELISA法检测鸡前脂肪细胞系(immortalized chicken preadipocyte cell line,ICP1)成脂分化0和48 h的PRDX1酶活;采用PCR克隆鸡PRDX1基因的编码序列,构建真核表达载体并将其转染到ICP1中,24 h后采用油酸钠诱导ICP1分化,通过油红O染色检测ICP1脂滴积累情况;利用RT-qPCR和Western-blot检测过表达PRDX1后成脂相关基因的mRNA和蛋白表达水平,并对PRDX1进行功能生物信息学分析。【结果】PRDX1在AA白羽肉鸡卵巢中的表达量最高,在肌胃周围脂肪中的表达量最低。ICP1分化48 h后,PRDX1的酶活极显著降低。成功构建了pCMV-HA-PRDX1真核表达载体。过表达PRDX1降低了ICP1细胞中脂滴的积累,且成脂相关基因C/EBPα、PPARγ、FABP4和FAS的表达水平均呈下降趋势。PRDX1基因编码199个氨基酸,相对分子质量为22.31 ku,PRDX1可以与TXN、SRXN1、SOD2等蛋白相互作用,不同物种间PRDX1具有较高的相似性。【结论】成功克隆并构建了鸡PRDX1真核表达载体,PRDX1过表达可抑制鸡前脂肪细胞分化。
[Purpose]To clone the chicken peroxiredoxin 1(PRDX1)gene and investigate its ef-fect on the differentiation of chicken preadipocytes,laying the foundation for further analysis ofPRDX1 function.[Methods]RT-qPCR was used to detect the expression of PRDX1 in different tissues of AA white feathered broiler chickens;ELISA was used to detect the PRDX1 enzyme activ-ity at 0 and 48 hours of adipogenic differentiation in an immunized chicken preadipocyte cell line(ICP1);PCR was used to clone the coding sequence of the chicken PRDX1 gene,and then,the euka-ryotic expression vector was construct and transfect into ICP1,after 24 hours,ICP1 differentiationwas induced by sodium oleate,and the accumulation of lipid droplets in ICP1 was detected by oil redO staining;RT-qPCR and Western-blot were used to detect the mRNA and protein expression levelsof lipid related genes overexpressing PRDX1.The functional bioinformatics analysis of PRDX1 wasperformed.[Results]The expression level of PRDX1 was the highest in ovaries of AA white feath-er broilers,and the lowest in perigastric fat.After ICP1 differentiation 48 hours,the enzyme activityof PRDX1 decreased extremely significantly.The pCMV-HA-PRDX1 eukaryotic expression vectorwas successfully constructed.Overexpression of PRDX1 decreased the accumulation of lipid dropletsin ICP1 cells,and the expression levels of lipid-related genes C/EBPα,PPARγ,FABP4 and FASshowed a downward trend.PRDX1 gene encoded 199 amino acids,the relative molecular weight was22.31 ku,PRDX1 could interact with TXN,SRXN1,SOD2 and other proteins,PRDX1 had high sim-ilarity among different species.[Conclusion]The eukaryotic expression vector of PRDX1 is suc-cessfully cloned and constructed.Overexpression of PRDX1 can inhibit the differentiation of chickenpreadipocytes.
作者
李美琦
王园园
崔金妍
杜金铭
王维禹
刘殿辉
孙婴宁
LI Meiqi;WANG Yuanyuan;CUI Jinyan;DU Jinming;WANG Weiyu;LIU Dianhui;SUN Yingning(College of Life Science and Agriculture Forestry,Qiqihar University,Qiqihar 161000,China)
出处
《云南农业大学学报(自然科学版)》
北大核心
2025年第2期42-49,共8页
Journal of Yunnan Agricultural University:Natural Science
基金
黑龙江省自然科学基金项目(LH2024C124)
黑龙江省省属本科高校优秀青年教师基础研究支持计划(YQJH2023103)
齐齐哈尔大学2023—2024年度研究生创新科研项目(QUZLTS_CX20-23026)。
