山羊Zfy基因克隆分析及其敲除载体构建

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(Gen...

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

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 2 406 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 gene effectively and the knockout efficiency was 12.31%, 40.86%, 31.52%, and 26.37%, respectively. The targeting plasmid with the highest knockout efficiency was selected to transfect into GFFs cells, and a GFFs-positive cell line that could stably target the knockout of Zfy gene was sorted out by puromycin screening, and the mutation rate was 23.91% by PCR sequencing. In conclusion, this study successfully cloned the Zfy gene of Xinong Saanen dairy goats and revealed the physicochemical properties of the protein of Zfy gene, successfully constructed a CRISPR/Cas9 knockout vector for the Zfy gene of goats and screened the best knockout site, and also obtained subcloned cells for Zfy gene knockout, which provided the foundation for in-depth research on the function of Zfy gene of goats and the development of sex control technology.