版纳微型猪近交系精子发生相关基因PHF7的分子特征及转录调控研究

Study of Molecular Characteristics and Transcriptional Regulation of Spermatogenesis-Related Gene PHF7 in Banna Mini-Pig Inbred Line

【目的】获得版纳微型猪近交系(Banna mini-piginbredline,BMI)精子发生相关基因PHF7的分子特征,构建其转录调控网络。【方法】通过RNA-seq技术对BMI睾丸进行全转录组测序;利用RT-PCR技术从BMI睾丸分离PHF7全长编码序列并分析其分子特征和蛋白功能;进一步基于睾丸全转录组测序数据,借助相关数据库对PHF7基因进行功能注释,分析其与非编码RNA(miRNA和lnRNA)间的调控关系,并构建其转录调控网络。【结果】成功获得了BMI睾丸转录组数据;通过RT-PCR获得了BMI PHF7 CDS区全长1155 bp,编码384个氨基酸;生物信息分析表明该基因位于猪13号染色体,含11个外显子,PHF7蛋白包含由54个氨基酸组成的PHD锌指结构域和112个氨基酸组成的扩展PHD锌指结构域,无规则卷曲在二级结构中占比最高;进化分析表明,BMI PHF7氨基酸序列在哺乳动物中较为保守,与双峰驼和羊驼的亲缘关系最为接近;蛋白互作网络分析发现,BMI PHF7与10个蛋白存在相互作用;基因功能分析发现,PHF7主要参与金属离子结合过程;基因靶向作用分析发现,猪PHF7受ssc-miR-149、sscmiR-769-3p、ssc-miR-324、ssc-miR-296-3p、ssc-miR-133b、ssc-miR-7142-3p、ssc-miR-27b-3p、ssc-miR-193a-5p等8个miRNA的靶向调控。【结论】阐明了BMI PHF7基因的分子特征、蛋白质结构并构建了转录调控网络,为深入研究PHF7在BMI精子发生过程的功能奠定基础。

【Objective】The purpose of this study is to obtain the molecular characteristics of PHF7 gene related to spermatogenesis in the Banna mini-pig inbred line(BMI)and construct its transcriptional regulatory networks.【Method】The whole transcriptome sequencing was carried out using testis tissues from adult BMI.The full-length coding sequence of PHF7 was isolated using RT-PCR technology and its molecular characteristics and corresponding protein function were analyzed.Further,the function of PHF7 gene was annotated by combining transcriptome data with related databases.Finally,the transcrip⁃tional regulatory networks between PHF7 and non-coding RNAs were constructed.【Result】The results indicated that the transcriptome data of BMI testis were successfully obtained.1155 bp CDS region of PHF7,which encoded 384 amino acids,was obtained by RT-PCR.Bioinformatics analysis implied that PHF7 gene was located on chromosome 13 of the pig genome containing 11 exons,and the PHF7 protein contained a PHD zinc finger domain composed of 54 amino acids and an extended PHD zinc finger do⁃main composed of 112 amino acids.The secondary structure of PHF7 contained mainly random coils.Phy⁃logenetic analysis in multi mammals suggested that the amino acid sequence of BMI PHF7 was relatively conserved and had the closest genetic relationship with Bactrian camel and alpaca.The analysis of inter⁃acted networks of proteins-protein indicated that BMI PHF7 had interaction with 10 proteins.The func⁃tion analysis of PHF7 indicated that it was mainly involved in the metal ion binding process.The analysis of miRNA-gene targeting showed that pig PHF7 was regulated by 8 miRNAs including ssc-miR-149,ssc-miR-769-3p,ssc-miR-324,ssc-miR-296-3p,ssc-miR-133b,ssc-miR-7142-3p,ssc-miR 27b-3p and ssc-miR-193a-5p.【Conclusion】This study obtained BMI testicular whole transcriptome data,clarified the molecular characteristics and protein structure of the BMI PHF7 gene,and con⁃structed a transcriptional regulatory network,which may lay a foundation for in-depth study of the func⁃tion of PHF7 in the process of BMI spermatogenesis.