简州大耳羊肌内脂肪细胞成脂分化差异表达基因的筛选与鉴定

旨在通过转录组测序技术获得简州大耳羊肌内前体脂肪细胞成脂分化前后的差异表达基因，并经生物信息学分析获得相关信号通路及可能发挥作用的关键功能候选基因。本研究以7日龄的健康简州大耳羊公羊为试验动物（n=3），采用胶原酶消化法分离获得其肌内前体脂肪细胞；利用Illumina平台对肌内前体脂肪细胞和诱导分化5 d的肌内脂肪细胞cDNA样品（n=3）进行高通量测序；以|log₂fold change|>0和P<0.05为阈值筛选获得差异表达基因，并利用clusterProfiler R包对其进行GO功能和KEGG通路富集；最后利用实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）技术检测功能候选基因在细胞分化前后的表达量变化。结果显示，共获得差异表达基因7 916个，其中4 143个为表达上调基因，主要富集到氧化磷酸化、核糖体合成和三羧酸循环通路等305条通路；3 773个为表达下调基因，且主要富集到甲状腺激素信号通路等303条通路；差异基因GO功能注释中52.8%为生物过程、13.4%是细胞组成和33.8%是分子功能。qRT-PCR结果表明，UCP3、ACACB、ACOT11、ACOX3、APOA1和WISP2在分化前后的山羊肌内脂肪细胞中的表达趋势与RNA-seq结果一致，提示这些基因适合作为下一步研究的功能候选基因。本研究筛选得到简州大耳羊肌内脂肪细胞成脂分化的差异基因，并确认了6个基因可作为功能候选基因。研究结果为阐明肉用山羊肌内脂肪细胞成脂分化的分子调控网络提供基础数据和系统资料。

Selection and Validation of the Differentially Expressed Genes during the Adipogenic Differentiation of Jianzhou Da'er Goat Intramuscular Adipocytes

This experiment aimed to obtain the differentially expressed genes before and after the adipogenic differentiation of Jianzhou Da'er goat intramuscular preadipocytes by the RNA-seq technology, and acquire the related signaling pathways and key candidate functional genes by bioinformatics analysis. The 7-day-old healthy male Jianzhou Da'er goat was used (n=3) and their intramuscular preadipocytes were isolated by collagenase digestion. Illumina platform was used to carry out the high-throughput sequencing to two groups of cDNA samples (n=3) of Jianzhou Da'er goat intramuscular preadipocytes and intramuscular adipocytes induced for 5 day, respectively. The differentially expressed genes were obtained according to the thresholds of|log₂fold change|>0 and P<0.05. The differentially expressed genes were enriched to GO terms and KEGG pathways using the clusterProfiler R. Ultimately, the qRT-PCR technology was performed to detect the change of expression levels of candidate functional genes before and after differentiation of preadipocytes. The results showed that there were 7 916 differentially expressed genes in the RNA-seq results during the adipogenic differentiation of Jianzhou Da'er goat intramuscular preadipocytes, including 4 143 up-regulated genes that were enriched to 305 KEGG pathways, including oxidative phosphorylation, ribosome synthesis, and citrate cycle pathways, etc., and 3 773 down-regulated genes that were enriched to 303 KEGG pathways, including thyroxine signaling pathways, and so on; In the results of GO function enrichment analysis, the biological process was 52.8% of GO terms, the cellular component was 13.4%, and the molecular function was 33.8%. The qRT-PCR results showed that the trends of UCP3, ACACB, ACOT11, ACOX3, APOA1, and WISP2 expression during the adipogenic differentiation of intramuscular preadipocytes were consistent with the results of RNA-seq, which indicated that these genes were the suitable candidate functional genes for further research. This study obtained the differentially expressed genes during the adipogenic differentiation of Jianzhou Da'er goat intramuscular adipocytes and verified 6 candidate functional genes. The results of this study provided the basic data and systematic information for elucidating the molecular regulatory networks during the adipogenic differentiation of meat goat intramuscular adipocytes.