转录组分析方法比较揭示不同生长阶段滩羊肝脏脂肪代谢的差异

Compare and Reveal Difference of Liver Fat Metabolism of Tan Sheep at Different Growth Stages Using Transcriptome Analysis Method

本试验旨在挖掘影响滩羊不同生长阶段肝脏脂肪代谢的差异基因。试验分别采集3、6、9月龄各3只公滩羊的肝脏左叶,然后分别以3月龄为对照组,6月龄为试验组;以6月龄为对照组,9月龄为试验组,采用Illumian软件进行转录组测序分析并进行实时荧光定量PCR(RTqPCR)验证。结果显示:与3月龄相比,6月龄筛选到265个差异表达基因(上调91个,下调174个);与6月龄相比,9月龄筛选到286个差异表达基因(上调195个,下调91个)。京都基因与基因组百科全书(KEGG)通路注释结果显示,与3月龄相比,6月龄主要富集在过氧化物酶体增殖物激活受体(PPAR)信号通路、不饱和脂肪酸的生物合成、胆固醇代谢等信号通路;与6月龄相比,9月龄主要富集在胰岛素抵抗、钙信号通路、腺苷酸活化蛋白激酶(AMPK)等信号通路。利用ClueGo将2组中的共有基因进行富集分析发现主要在胆固醇代谢、淀粉和蔗糖代谢、钙信号通路等信号通路。随机挑选的酰基辅酶A合成酶5(ACSL5)、载脂蛋白A4(APOA4)、血管生成素样蛋白8(ANGPTL8)、蛋白磷酸酶1调节亚基3C(PPP1R3C)和乙酰辅酶A羧化酶β(ACACB)5个基因的RT-qPCR验证结果与转录组测序结果一致。通过对不同月龄滩羊的肝脏进行转录组测序分析,筛选出ACSL5、APOA4可作为3、6月龄滩羊肝脏脂肪代谢调控的基因,PPP1R3C和ACACB作为6、9月龄滩羊肝脏脂肪代谢调控的候选基因,ANGPTL8、糖原蛋白2(GYG2)、5-羟色胺受体2A(HTR2A)和溶质载体家族8成员A3(SLC8A3)作为3、6、9月龄滩羊肝脏脂肪代谢调控的共有基因。

The purpose of this study was to explore the differential genes affecting liver fat metabolism at dif-ferent growth stages of Tan sheep.The left lobe of liver of 3 sheep at 3,6 and 9 months of age were collected.The control group was 3 months old,and the experimental group was 6 months old,and then 6 months old as the control group,9 months old as the experimental group.Finally,transcriptome sequencing was performed by Illumian software and real-time fluorescence quantitative PCR(RT-qPCR)verification was performed.The results showed that 265 differentially expressed genes(91 up-regulated and 174 down-regulated)were detected at 6 months of age compared with 3 months of age.Compared with 6 months of age,286 differentially ex-pressed genes(195 up-regulated and 91 down-regulated)were detected at 9 months of age.The Kyoto Ency-clopedia of genes and Genomes(KEGG)pathway annotation results showed that compared with 3 months of age,6 months of age were mainly enriched in peroxisome proliferator activated receptor(PPAR)signaling pathway,unsaturated fatty acid biosynthesis,cholesterol metabolism and other signaling pathways.Compared with 6 months of age,9 months of age was mainly enriched in insulin resistance,calcium signaling pathway,adenylate activated protein kinase(AMPK)and other signaling pathways.ClueGo was used to enrich the com-mon genes in the two groups and found that they were mainly in cholesterol metabolism,starch and sucrose metabolism,calcium signaling and other pathways.RT-qPCR results of 5 randomly selected acyl-CoA syn-thetase 5(ACSL5),apolipoprotein A4(APOA4),ANGPTL8(ANGPTL8),protein phosphatase 1 regulatory subunit 3C(PPP1R3C)and acetyl-CoA carboxylaseβ(ACACB)genes were consistent with the transcrip-tome sequencing results.Through transcriptomic sequencing analysis of liver of Tan sheep at different ages,ACSL5 and APOA4 are selected as the genes regulating liver fat metabolism of Tan sheep at 3 and 6 months of age,and PPP1R3C and ACACB are selected as the candidate genes regulating liver fat metabolism of Tan sheep at 6 and 9 months of age.ANGPTL8,glycogen 2(GYG2),serotonin receptor 2A(HTR2A),and sol-ute vector family 8 member A3(SLC8A3)are the common genes regulating liver lipid metabolism in Tan sheep aged 3,6 and 9 months of age.