山羊Zfy基因克隆分析及其敲除载体构建  

Cloning Analysis and Construction of Knockout Vector of Zfy Gene in Goat

作　　者：黄敏 谢晓刚 何琪富 曹旭阳 董翔宸 康健[1] 刘军[1] 权富生[1] HUANG Min;XIE Xiaogang;HE Qifu;CAO Xuyang;DONG Xiangchen;KANG Jian;LIU Jun;QUAN Fusheng(College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China;Department of Animal Engineering, Yangling Vocational & Technical College, Yangling 712100, China)

机构地区：[1]西北农林科技大学动物医学院,杨凌712100 [2]杨凌职业技术学院动物工程分院,杨凌712100

出　　处：《畜牧兽医学报》2022年第3期711-721,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：陕西省农业科技创新重点项目(NYKJ-2015-081);陕西省重点研发计划项目(2020NY-019)。

摘　　要：旨在对西农萨能奶山羊Zfy基因进行克隆和生物信息学分析,并利用CRISPR/Cas9技术成功获得敲除Zfy基因的奶山羊成纤维细胞株,为进一步探索Zfy基因功能及性别调控提供基础数据。本研究以健康的西农萨能奶山羊为对象,采集3只3月龄公山羊睾丸组织并提取总RNA。根据NCBI中预测的山羊Zfy基因mRNA序列信息(GenBank登录号:XM_018044893)设计引物,利用RT-PCR技术分段克隆获得山羊Zfy基因序列,使用相关生物信息学软件分析Zfy基因CDS区核苷酸序列并预测其蛋白质的结构和功能。针对山羊Zfy基因CDS区共设计了4条sgRNA,构建4个打靶载体并转染山羊胎儿成纤维细胞(GFFs),利用T7E1酶切检验打靶载体的切割效率,通过嘌呤霉素筛选敲除Zfy基因的GFFs阳性单克隆细胞,经PCR扩增、测序检测突变率。结果显示,成功克隆得到了长度为2406 bp的西农萨能奶山羊Zfy基因CDS区序列。同源性以及系统进化树分析表明,山羊Zfy基因序列与马鹿、牛、绵羊的同源性高,亲缘关系较近,与小鼠的同源性最低,亲缘关系最远。生物信息学软件分析显示,山羊Zfy基因共编码801个氨基酸,分子质量为90.37 ku,Zfy蛋白含66个磷酸化位点,等电点为5.66,亲水性较高,无信号肽,是一个结构不稳定的非分泌蛋白。T7E1酶切发现,4个打靶载体均可发挥敲除活性,切割效率分别为12.31%、40.86%、31.52%、26.37%。选择切割效率最高的打靶质粒转染山羊胎儿成纤维细胞(GFFs),经嘌呤霉素筛选获得能稳定靶向敲除Zfy基因的GFFs阳性细胞株,PCR测序检测突变率为23.91%。综上所述,本研究成功克隆了西农萨能奶山羊Zfy基因并揭示了该基因所编码蛋白的理化性质;成功构建了山羊Zfy基因CRISPR/Cas9敲除载体,并筛选出最佳敲除位点,获得了Zfy基因敲除的亚克隆细胞,为深入研究山羊Zfy基因功能及开发性别控制技术奠定了基础。The aim of this study was to clone and analyze the Zfy gene by bioinformatics in Xinong Saanen dairy goats,and to successfully establish goat fibroblasts cell lines with Zfy gene knockout by CRISPR/Cas9 technique,and provide basic data for further exploring the function and sex regulation of Zfy gene.In this study,the samples of testicles were collected from 3 healthy Xinong Saanen male goats during 3-month-old and total RNA were extracted,respectively.Primers were designed according to the predicted mRNA sequence(GenBank accession No.:XM_018044893)of Capra hircus Zfy gene published in NCBI,and the Zfy gene was segmental amplified and cloned by RT-PCR.The nucleotide sequence and protein structure and function of the CDS region of the Zfy gene were analyzed by related bioinformatics softwares.Four pairs of sgRNAs were designed according to the CDS region of the Zfy gene to construct 4 targeting vectors later transfected into goat fetal fibroblasts(GFFs).The knockout efficiency of the targeting vectors was confirmed by T7E1 digestion method.Then the Zfy gene knockout positive monoclonal GFFs cells were isolated through puromycin screening,and subsequently,PCR amplification and sequencing were used to detect the mutation rate.The results showed that the sequence of the CDS region of the Zfy gene of Xinong Saanen dairy goats with a length of 2406 bp was successfully cloned.The homology and phylogenetic tree analysis showed that the Zfy gene sequence of goat had high homology with Cervus elaphus,Bos taurus and Ovis aries,and their genetic distance was closer,the homology with Mus musculus was the lowest and the genetic distance was the farthest.Bioinformatics softwares analysis showed that the goat Zfy gene encoded 801 amino acids,and the molecular mass was 90.37 ku.The Zfy protein contained 66 phosphorylation sites,had an isoelectric point of 5.66,was highly hydrophilic,had no signal peptide,and was a structurally unstable non-secretory protein.T7E1 digestion revealed that all 4 targeting vectors could knock out the

关 键 词：Zfy基因克隆 西农萨能奶山羊 生物信息学分析 基因敲除 

分 类 号：S827.2[农业科学—畜牧学]