摘要
【目的】鉴定山羊环状RNA(circular RNA,circRNA)铁氧还蛋白还原酶(circFDXR)的表达,建立其在山羊原代卵巢颗粒细胞中的高效表达方式。【方法】利用PCR扩增、DNA测序、RNase R酶消化、实时荧光定量PCR鉴定circFDXR的环状性质。通过核质分离、荧光原位杂交(FISH)检测circFDXR亚细胞定位。利用同源重组将circFDXR的线性序列无缝克隆至慢病毒载体pLC5-ciR,转染至293T细胞包装病毒后进行病毒滴度测定,并确定最佳感染复数(MOI),并以最佳MOI感染山羊原代卵巢颗粒细胞,通过实时荧光定量PCR检测过表达效率,并测序验证circFDXR是否正确环化。【结果】山羊circFDXR主要定位于卵巢颗粒细胞的细胞质中,耐受RNase R酶的消化。成功构建circFDXR慢病毒过表达载体,浓缩病毒滴度为5.49×10~9 TU/mL,感染山羊卵巢颗粒细胞的最佳MOI为100。实时荧光定量PCR结果显示,与对照组相比,过表达组circFDXR表达量极显著升高(P<0.01),测序结果显示过表达的circFDXR可以正确环化。【结论】circFDXR主要定位于卵巢颗粒细胞的细胞质中,相比线性RNA具有更高的稳定性。利用慢病毒包装的circFDXR在山羊原代卵巢颗粒细胞中可以正确环化,本试验结果为进一步研究circFDXR在山羊原代卵巢颗粒细胞中的功能奠定了理论基础。
【Objective】The purpose of this experiment was to identify the expression of circular RNA(circRNA)circFDXR in goat and establish its efficient expression mode in goat primary ovarian granulosa cells.【Method】The cyclic properties of circFDXR were identified by PCR amplification,DNA sequencing,RNase R digestion and Real-time quantitative PCR.Cytoplasmic separation and fluorescence in situ hybridization(FISH)were used to detect the subcellular localization of circFDXR.The linear sequence of circFDXR was seamlessly cloned into the Lentivirus vector pLC5-ciR by homologous recombination,and transfected into 293T cells to package the virus,then the virus titer was determined,and the optimal multiplicity of infection(MOI)was determined.The optimal MOI was used to infect goat primary ovarian granulosa cells.Real-time quantitative PCR was used to detect the overexpression efficiency,and sequencing verified whether circFDXR was cyclized correctly.【Result】Goat circFDXR was mainly localized in the cytoplasm of ovarian granular cells and tolerates digestion of RNase R enzymes.The circFDXR lentiviral overexpression vector was successfully constructed,the concentrated virus titer was 5.49×109 TU/mL,and the optimal MOI for infecting goat ovarian granulosa cells was 100.Real-time quantitative PCR results showed that the expression level of circFDXR in overexpressed group was extremely significantly higher than that in control group(P<0.01),and the sequencing results showed that the overexpressed circFDXR could be cyclized correctly.【Conclusion】circFDXR was mainly localized in the cytoplasm of granular cells and had higher stability than linear RNA.The circFDXR packaged by Lentivirus could be cyclized correctly in the primary ovarian granulosa cells of goats.The experimental results laid a theoretical basis for further study of the function of circFDXR in the primary ovarian granulosa cells of goats.
作者
刘杰
李之瀚
郭聪慧
冯光杭
薛文哲
刘德武
柳广斌
孙宝丽
郭勇庆
邓铭
邹娴
李耀坤
LIU Jie;LI Zhihan;GUO Conghui;FENG Guanghang;XUE Wenzhe;LIU Dewu;LIU Guangbin;SUN Baoli;GUO Yongqing;DENG Ming;ZOU Xian;LI Yaokun(College of Animal Science,South China Agricultural University,Guangzhou 510642,China;National Engineering Research Center for Livestock and Poultry Breeding,South China Agricultural University,Guangzhou 510642,China;Guangdong Key Laboratory of Agricultural Animal Genomics and Molecular Breeding,South China Agricultural University,Guangzhou 510642,China;Guangdong Key Laboratory of Animal Breeding and Nutrition,State Key Laboratory of Livestock and Poultry Breeding,Institute of Animal Science,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2024年第6期2330-2341,共12页
China Animal Husbandry & Veterinary Medicine
基金
广东省科技特派员项目(KTP20210344)
广东省现代农业产业技术体系项目(2023KJ127)。
