基于RNA-seq技术的雏鸡晚期离焦识别转录组学研究

Transcriptomic Study of Late Defocus Recognition in Chicks Based on RNA-seq Technology

目的:探讨晚期离焦识别的分子机制。方法:实验研究。将30只7 d龄白来航鸡随机分为负镜组、正镜组和对照组,分别给予雏鸡右眼-10 D/+10 D/0 D透镜诱导,干预时间为6 d。所有动物均在光学干预前后测量屈光度、眼轴和后极部各组织厚度等生物学参数。采用单因素方差分析对数据进行分析。眼球生物学参数测量完毕后分离眼后极部组织,提取RNA,应用RNA-seq技术筛选差异表达基因,并对其进行GO功能注释、KEGG通路分析及PPI网络分析,观察离焦干预对雏鸡后极部各组织转录组的影响。结果:负镜组、正镜组雏鸡的屈光度、眼轴长度以及脉络膜厚度,与对照组相比,差异具有统计学意义(P<0.05);以P<0.05和|log2FC|>1为筛选标准,负镜组与对照组相比,视网膜有203个差异表达基因,脉络膜有757个差异表达基因,巩膜有1509个差异表达基因;正镜组与对照组相比,视网膜有191个差异表达基因,脉络膜有378个差异表达基因,巩膜有1918个差异表达基因。与对照组比,负镜组与正镜组的重叠基因除LOC121112941外均表现出相同的差异表达趋势,GO分析通路存在50%以上重合,KEGG分析视网膜水平的重叠通路是花生四烯酸代谢、酪氨酸代谢和视黄醇代谢;脉络膜水平的重叠通路是内质网中的蛋白质加工,精氨酸和脯氨酸代谢和视黄醇代谢;巩膜水平的重叠通路则为细胞粘附分子、ECM-受体相互作用、粘着斑、细胞因子-细胞因子受体相互作用、硫代谢、糖胺聚糖生物合成-硫酸软骨素/硫酸皮肤素、Toll样受体信号通路、TGF-β信号通路和MAPK信号通路。通过PPI蛋白互作网络分析鉴定出离焦识别过程中各组织的关键基因。结论:本研究在转录组水平上筛选出晚期离焦识别过程的绝大多数重叠基因保持着相同的上调或下调表达趋势,不同方向的离焦信号可能诱导部分通路的相似变化。

Objective:To investigate the molecular mechanism of late defocus recognition.Methods:In this experimental study,307-day-old White Leghorn chicks were randomly divided into a minus-lens group,a plus lens group and a control group,and the chicks were induced with-10 D/+10 D/0 D lens in the right eye of the chicks respectively,with 6 days'intervention time.Biological parameters such as diopter,axial length,and thickness of each tissue in the posterior pole were measured in all animals before and after the optical intervention,and the data were analyzed by one-way ANOVA.After the measurement of the biological parameters of the eyeball,the posterior pole tissues of the eyes were isolated,RNA was extracted respectively,and differentially expressed genes(DEGs)were screened by RNA-seq technology.GO analysis,KEGG pathway analysis,and PPI network analysis was performed to observe the effect of defocus intervention on the transcriptome of each tissue in the posterior pole of chicks.Results:The diopter,axial length,and choroidal thickness of the chicks in the minus-lens group and the plus lens group were significantly different from those in the control group(P<0.05);with P<0.05 and|log2FC|>1 as the screening criteria,compared with the control group and the minus-lens group,there were 203 DEGs in the retina,757 DEGs in the choroid,and 1509 DEGs in the sclera;compared with the control group and plus-lens group,the retina had 191 DEGs,the choroid had 378 DEGs,and the sclera had 1918 DEGs.Compared with the control group,the overlapping genes of minus-lens group and plus-lens group all showed the same differential expression trend except for LOC121112941.There was more than 50%overlap in the GO analysis pathways.The common pathways in the KEGG analysis at the retinal level were arachidonic acid metabolism,tyrosine metabolism,and retinol metabolism;overlapping pathways at the choroidal level were protein processing in the endoplasmic reticulum,arginine and proline metabolism,and retinol metabolism;at the scleral level were cell adhesion molecules,ECM-receptor interactions,focal adhesions,cytokines-cytokine receptor interaction,sulfur metabolism,glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate,Toll-like receptor signaling,TGF-βsignaling,and MAPK signaling.Key genes in each tissue during out-of-focus recognition were identified by protein-protein interaction network analysis.Conclusion:This study screened out most of the overlapping genes in the late defocus recognition process at the transcriptome level,maintaining the same up-or down-regulation trend,and defocus signals in different directions may induce similar changes in some pathways.