大型迪庆藏猪不同生长阶段肌肉生长差异基因及其调控通路分析

Screening of differential genes for muscle growth and regulatory pathways analysis in large Diqing Tibetan pig at different growth stages

关键词:  大型迪庆藏猪  RNA-seq  肌肉生长  差异表达基因  调控途径

Key words:  large Diqing Tibetan pig  RNA-seq  muscle growth  differential expression gene  regulatory pathway

摘要:

为筛选大型迪庆藏猪不同生长阶段肌肉生长差异的关键基因并分析其调控途径，以40、80和120 kg的大型迪庆藏猪为对象，基于RNA-seq技术对背最长肌组织进行转录组测序，对获得的测序数据进行拼接、比对、注释和差异分析，筛选与肌肉生长相关的差异基因进行STEM趋势分析、功能分析并构建差异基因互作网络。结果表明:1)10~40 kg阶段与40~80 kg阶段、40~80 kg阶段与80~120 kg阶段比较共检测到730 个、981 个基因显著差异表达；2)差异表达基因STEM趋势分析显示，共有4 个差异显著模块，模块11和14的差异基因先上调表达后轻微下调；模块9和10的差异基因先上调后下调表达；3)差异基因互作网络显示，10~40 kg vs 40~80 kg，FOS基因位于调控网络核心，与EGRs、CCL2和NOR-1等基因有互作关系，NOR-1基因上调导致肌肉生长速度加快，FOS基因下调抑制肌源性分化，EGRs基因下调导致胶原纤维束减少，CCL2基因下调导致肌肉再生受损；40~80 kg vs 80~120 kg，FOXO1、PDK4和PPARD等基因位于网络中心，SFRPs基因上调阻止成肌细胞终末分化，FZD7基因下调减缓肌纤维肥大速度，FOXO1下调导致肌生长抑制素mRNA下降减缓肌肉萎缩进程，PPARD与FOXO1均降低PDK4含量从而使肌肉丙酮酸脱氢酶复合物活性增强，对碳水化合物氧化和葡萄糖摄取增加，肌纤维变粗，与大型迪庆藏猪40至80 kg生长速度显著快于10至40 kg阶段，而80 kg之后缓慢下降的规律吻合；4)挑选了12 个差异基因进行qPCR验证，EGR1、SFRP1和FOXO1等12 个基因定量验证结果与转录组测序结果一致。综上，本研究利用RNA-seq技术筛选出12 个影响大型迪庆藏猪不同生长阶段肌肉生长的差异基因，可为解析地方猪肌肉生长的遗传调控机制、应用分子标记辅助选择提高迪庆藏猪瘦肉产量提供参考。

Abstract:

In order to screen the key genes of muscle growth in different growth stages of large Diqing Tibetan pig(TP)and analyze their regulatory pathways, the transcriptome of longissimus dorsi(LD)of TPs of 40, 80 and 120 kg was sequenced based on RNA-seq, and the sequencing data were spliced, compared, annotated and analyzed. The differential genes related to muscle growth were screened for STEM analysis and function analysis, and the differential gene interaction network was constructed. The results showed that: 1)There were respectively 730 and 981 genes significantly differentially expressed in 10-40 kg stage and 40-80 kg stage and 40-80 kg stage and 80-120 kg stage. 2)STEM analysis of differential expression genes(DEGs)showed that there were four modules with significant differences. The DEGs in modules 11 and 14 were up-regulated first and then slightly down-regulated. The DEGs of modules 9 and 10 were up-regulated first and down-regulated later. 3)The differential gene interaction network analysis showed that at 10-40 kg vs 40-80 kg, FOS gene was located in the center of regulatory network and interacted with EGRs, CCL2 and NOR-1. The up-regulation of NOR-1 leads to the acceleration of muscle growth, the down-regulation of FOS inhibits myogenic differentiation, the down-regulation of EGRs lead to the decrease of collagen fiber bundles, and the down-regulation of CCL2 leads to the damage of muscle regeneration. At 40-80 kg vs 80-120 kg, FOXO1, PPARD, PDK4 and other genes were located in the center of the network. The up-regulation of SFRPs genes prevent myoblasts from entering the terminal differentiation. The down-regulation of FZD7 slows down muscle fiber hypertrophy, down-regulation of FOXO1 leads to the decrease of myostatin mRNA and slows down the process of muscle atrophy. PPARD and FOXO1 both reduce the content of PDK4, so as to enhance the activity of muscle pyruvate dehydrogenase complex, increase carbohydrate oxidation and glucose uptake, and thicken muscle fibers, which is consistent with the law that the growth rate of TPs is faster at 40-80 kg, significantly faster than that at 10-40 kg, and slowly decreases after 80 kg. 4)A total of twelve DEGs were selected for qPCR verification. EGR1, SFRP1, FOXO1 were confirmed by qPCR and RNA-seq. In conclusion, this study identified twelve DEGs affecting muscle growth of TPs at different growth stages by RNA-seq. This study can provide a reference for analyzing the genetic regulation mechanism of muscle growth of local pigs and improving the lean meat yield of TP by molecular marker assisted selection.