BIRC5对山羊睾丸细胞周期、凋亡的影响  

Effects of BIRC 5 on the Cycle and Apoptosis of Goat Testis Cells

作　　者：但一昕 杨璐 向华 张焕容[1] 任玉鹏 杨发龙[1] 何翃闳 朱江江 DAN Yixin;YANG Lu;XIANG Hua;ZHANG Huanrong;REN Yupeng;YANG Falong;HE Honghong;ZHU Jiangjiang(College of Animal&Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China;Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province,Chengdu 610041,China)

机构地区：[1]西南民族大学畜牧兽医学院,成都610041 [2]青藏高原动物遗传资源保护与利用四川省重点实验室,成都610041

出　　处：《畜牧兽医学报》2023年第4期1511-1524,共14页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：四川省自然科学基金项目(2022NSFSC0082);西南民族大学优秀学生培养工程(2022NYXXS025);动物遗传育种四川省重点实验室开放基金(202202);四川省科技计划(2020JDJQ0010);四川省畜禽育种攻关项目(2021YFYZ0003);浙江省科技计划项目(2022C04017)。

摘　　要：旨在在克隆山羊BIRC 5基因的基础上,通过过表达或者敲低BIRC 5基因表达揭示BIRC 5基因对山羊睾丸细胞周期和凋亡的调控作用,为进一步完善BIRC5基因功能奠定基础。本研究以3只3日龄3 kg左右健康简州大耳羊公羊脾脏组织为模板,利用RT-PCR技术克隆山羊BIRC 5基因CDS区序列,并进行生物信息学分析。将克隆回收产物连接真核表达载体,从而构建pcDNA3.1(+)-BIRC 5。根据山羊BIRC 5基因序列设计并筛选有效siRNA。将pcDNA3.1(+)-BIRC 5和siRNA分别转染山羊睾丸细胞后,利用Western blot检测BIRC5蛋白表达,利用流式细胞仪检测细胞周期与凋亡,利用实时荧光定量PCR技术检测对凋亡相关基因Bax、Caspase 3、Caspase 7、Bcl-2、p 53、BCL 2L11和PARP 1的表达。结果,成功扩增山羊BIRC 5基因序列,长度为506 bp,包含5′UTR 28 bp,CDS区429 bp,3′UTR 49 bp,编码142个氨基酸残基,且与牛亲缘关系最近。过表达BIRC 5基因后抑制了细胞凋亡,且细胞被阻滞于G2/M+S期;同时可下调Caspase 7、p 53、BCL 2L11、Bcl-2和Bax基因mRNA的表达,但Caspase 3和PARP 1 mRNA表达无显著变化。而敲低BIRC 5基因表达后可显著促进细胞凋亡的发生,且细胞被阻滞于细胞被阻滞于G0/G1期,而Caspase 3、Caspase 7和PARP 1 mRNA表达显著上升,p 53和Bcl-2的表达显著下降,Bax和BCL 2L11表达无显著变化。BIRC 5基因可抑制山羊睾丸细胞的凋亡,并促进山羊睾丸细胞被阻滞于G2/M+S期。研究结果为进一步深入全面研究BIRC 5基因的功能提供重要的试验数据。The aim of the present study was to clone goat BIRC 5,and to reveal the role of BIRC 5 gene in regulating the cycle and apoptosis of goat testis cells through the overexpression or interference of BIRC 5.These data will be beneficial for exploring the role of BIRC 5 gene in goat.The BIRC 5 gene sequence was cloned by RT-PCR from spleen tissue of 3 three-day-old healthy Jianzhou male goats with 3 kg of body weight,and sequenced for further bioinformatics analysis using online softwares.The clone recovery product was ligated to a eukaryotic expression vector to construct pcDNA3.1(+)-BIRC 5.Effective siRNA was designed and screened based on goat BIRC 5 gene sequence.After pcDNA3.1(+)-BIRC 5 and siRNA were respectively transfected into goat testicular cells,the expression of BIRC5 protein was detected by Western blot,and cell cycle and apoptosis were detected by flow cytometry.Meanwhile,the expression of apoptosis-related genes Bax,Caspase 3,Caspase 7,Bcl-2,p 53,BCL 2L11 and PARP 1 were detected by RT-qPCR.A length of 506 bp BIRC 5 gene sequence was cloned successfully,including 28 bp of 5′UTR,429 bp of CDS region,and 49 bp of 3′UTR,encoding 142 amino acids.The phylogenetic tree showed that BIRC5 had the closest relative to Bos taurus.The overexpression of BIRC 5 gene inhibited the apoptosis of cells,and the cells were arrested in G2/M+S phase;it also down-regulated the expression of Caspase 7,p 53,BCL 2L11,Bcl-2 and Bax gene mRNA,but did not significantly change the mRNA expression of Caspase3 and PARP 1.siRNA interference of BIRC 5 gene promoted the apoptosis of cells,and the cells were arrested in G0/G1 phase;it also up-regulated the expression of Caspase 3,Caspase 7 and PARP 1 gene mRNA,and down-regulated the expression of p 53 and Bcl-2 gene mRNA,but did not significantly change the mRNA expression of Bax and BCL 2L11.Goat BIRC 5 gene could inhibit the apoptosis of testis cells and promote the arrest of goat testicular cells in G2/M+S phase.These data may lay a foundation for further study of BIRC 5 gene fun

关 键 词：山羊 BIRC 5基因 过表达 干扰 细胞周期 细胞凋亡 

分 类 号：S827.3[农业科学—畜牧学]