[目的]基于高叶酸小鼠睾丸转录组数据，筛选叶酸调控牛和小鼠繁殖力的同源基因，以期为通过叶酸改善雄性动物繁殖力奠定理论基础。[方法]选取24只健康、生长状况相近的11周龄C57BL/6雄性小鼠，随机分为3组(n=8):CON、A和B组，分别饲喂2、7和15 mg/kg叶酸，连续饲喂57 d。饲喂结束后分别测定3组小鼠左、右睾丸和精囊的重量，精子密度以及缓慢前进的精子比例，通过Illumina Novaseq™ 6000高通量测序进行小鼠睾丸转录组测序分析，对测序结果进行差异表达基因(DEGs)、GO功能注释和蛋白互作网络(PPI)分析，筛选叶酸调控精液品质和繁殖力的关键基因，并结合荷斯坦种公牛繁殖力相关同源基因和数量性状基因座(QTLs)数据，进一步筛选叶酸调控牛和小鼠雄性繁殖力的同源基因。[结果]相较于CON组，B组小鼠左、右精囊重量均极显著增加(P＜0。01)，A和B组小鼠精液中缓慢前进的精子比例均极显著降低(P＜0。01)。经RNA-Seq分析发现，在A vs CON组中筛选出423个DEGs，在B vs CON组中筛选出661个DEGs，A vs CON和B vs CON组中共有的上调DEGs 85个、下调DEGs 40个。通过GO功能注释分析发现，A vs CON组的DEGs显著富集在叶酸代谢和精液品质调控相关的GO条目，B vs CON组的DEGs显著富集在叶酸代谢和生殖器官发育相关的GO条目，A vs CON和BvsCON两个组共有的上调DEGs显著富集在叶酸代谢和精液品质调控相关等功能，共有的下调DEGs显著富集在一碳代谢过程和单精受精作用功能。通过DEGs的PPI网络分析发现，筛选出的核心节点(hub)基因可能通过影响精液品质而影响雄性动物繁殖力。同时本研究中筛选出的hub基因均为奶牛的同源基因，并找出了不同浓度叶酸影响荷斯坦种公牛繁殖力的同源基因及其附近的QTLs，包括每次妊娠的授精次数QTL、精子活力QTL等。将hub基因进一步与前人荷斯坦种公牛精子转录组差异表达基因进行比对，筛选出叶酸调控牛和小鼠繁殖力的同源基因:Serpinb1b基因(牛同源基因为SERPINB1)。[结论]饲喂7和15 mg/kg叶酸能够降低精液中缓慢前进的精子比例，通过转录组测序筛选出的Serpinb1b基因(牛同源基因为SERPINB1)是叶酸调控雄性动物繁殖力的关键基因之一，可为畜牧业实际生产中大型乳用动物荷斯坦牛的养殖及选育提供参考信息。
Screening of Key Homologous Genes for Bovine Fertility Based on High Folic Acid Mouse Testicular Transcriptome
[Objective]Based on transcriptome data of folic acid mice testis,the homologous genes of folic acid regulating bovine and mouse fertility were screened,which provided a theoretical foundation for improving male animal reproductive capacity through folic acid.[Method]Twenty four healthy and similarly growing 11 weeks old C57BL/6 male mice were randomly divided into three groups(n=8):CON,A,and B groups,fed with 2,7,and 15 mg/kg folic acid,respectively,for 57 consecutive days.After feeding,the weight of the left and right testes and seminal vesicles,sperm density,and the proportion of slow forward sperm were measured in three groups of mice.The transcriptome of mouse testes was sequenced using Illumina Novaseq™ 6000 high-throughput sequencing.The sequencing results were analyzed using differentially expressed genes(DEGs),GO functional annotation,and protein-protein interaction network(PPI)analysis to screen for key genes regulating semen quality and reproductive capacity by folic acid,and combined with homologous genes and quantitative trait genes related to reproductive capacity in Holstein bulls.Quantitative trait loci(QTLs)data were used to further screen for homologous genes that regulated male reproductive capacity in cattle and mice through folate regulation.[Result]Compared with CON group,the weight of the left and right seminal vesicles of mice in B group was extremely significantly increased(P＜0.01),and the unfavorable trait of the proportion of sperm slow foward in the semen of mice in both A and B groups were extremely significantly reduced(P＜0.01).Through RNA-Seq analysis,it was found that 423 DEGs were screened in A vs CON group,and 661 DEGs were screened in B vs CON group.A total of 85 upregulated DEGs and 40 downregulated DEGs were found in both A vs CON and B vs CON groups.Through GO functional annotation analysis,it was found that the DEGs in A vs CON group were significantly enriched in GO entries related to folate metabolism and semen quality regulation,while those in B vs CON group were significantly enriched in GO entries related to folate metabolism and reproductive organ development.The upregulated DEGs shared by both A vs CON and B vs CON groups were significantly enriched in folate metabolism and semen quality regulation,while the downregulated DEGs shared were significantly enriched in one-carbon metabolism and single sperm fertilization functions.Through PPI network analysis of DEGs,it was found that the selected hub genes might affect male animal fertility by affecting semen quality.At the same time,the hub genes screened in this study were all homologous genes of Holstein bulls,and homologous genes and their neighboring QTLs that affected the reproductive capacity of Holstein bulls at different concentrations of folic acid were identified,including inseminations per conception QTL and sperm motility QTL.Further comparing the hub gene with differentially expressed genes in the transcriptome of previous Holstein bull sperm,the homologous gene Serpinb1b(bovine homologous gene SERPINB1)was screened out,which regulated fertility in Holstein bulls and mice through folate regulation.[Conclusion]Feeding 7 and 15 mg/kg folic acid could reduce the proportion of slow forward sperm in semen.The Ser pinb1b gene(bovine homologous gene:SERPINB1)screened by RNA-Seq was one of the key genes for folic acid regulation of male animal fertility,which could provide reference information for the breeding and breeding of large dairy animal such as Holstein cows in the actual production of animal husbandry.
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