基于转录组测序的摘除卵巢藏姜母猪背最长肌脂代谢相关基因筛选与分析

Screening and analysis of genes related to lipid metabolism of longissimus dorsi muscle of ovary-removed Zangjiang sows based on transcriptome sequencing

【目的】探究摘除卵巢后藏姜母猪背最长肌脂肪沉积的动态规律,为揭示脂肪沉积的调控机制提供理论依据。【方法】于45日龄手术摘除藏姜母猪卵巢(试验组),对照组藏姜母猪在腹部相同部位进行创伤处理但不摘除卵巢。分别记录45、154和300日龄藏姜母猪的血清雌激素、甘油三酯和总胆固醇含量变化,300日龄屠宰采集背最长肌组织样品制作石蜡切片,并通过Illumina NovaSeq 6000测序平台完成测序。以|log2 Fold Change|>1且P<0.05为标准,采用DESeq2筛选出背最长肌组织中的差异表达基因(DEGs),通过GO和KEGG数据库分别进行GO功能注释分析及KEGG信号通路富集分析,找出可能参与脂代谢的基因,并以实时荧光定量PCR进行转录组测序结果验证。【结果】试验组藏姜母猪的血清甘油三酯含量显著高于对照组藏姜母猪(P<0.05,下同),总胆固醇含量极显著高于对照组藏姜母猪(P<0.01,下同),雌激素含量极显著低于对照组藏姜母猪,背最长肌脂肪含量显著低于对照组藏姜母猪。试验组与对照组藏姜母猪间存在278个DEGs,其中有33个DEGs与脂代谢相关。DEGs主要注释在胆汁酸分泌、磷脂酰肌醇3-激酶信号传导、蛋白激酶B信号传导、G蛋白偶联受体结合、糖胺聚糖结合、趋化因子活性及趋化因子受体结合等GO功能条目上,且主要富集在糖酵解/糖异生、淀粉和蔗糖代谢、碳水化合物消化和吸收、MAP激酶丝氨酸/苏氨酸磷酸酶活性、雌激素2-羟化酶活性及cGMP-PKG信号等KEGG信号通路中。实时荧光定量PCR检测到的目的基因表达趋势与转录组测序结果基本一致,表明转录组测序数据可靠。【结论】摘除卵巢后藏姜母猪血清雌激素水平降低,甘油三酯和总胆固醇水平升高,背最长肌的肌间脂肪含量降低,与CALML5、PLCZ1、PIK3C2G、IL6等33个参与脂代谢调控DEGs的差异表达有关,且涉及胆汁酸分泌、磷脂酰肌醇3-激酶信号传导调控等功能条目及糖酵解/糖异生、淀粉和蔗糖代谢等信号通路。

【Objective】This paper investigated the dynamic regulation of fat deposition in longissimus dorsi muscle of ovary-removed Zangjiang sows,to provide theoretical basis for revealing the regulatory mechanism of fat deposition.【Method】The ovaries of Zangjiang sows were surgically removed at 45 days of age(treatment group),with the control group of Zangjiang sows being traumatized in the same area of the abdomen without removing ovaries.The variation of serum estrogen,triglyceride and total cholesterol contents of Zangjiang sows were recorded at 45,154 and 300 days of age respectively.The longissimus dorsi muscle tissue samples were collected at 300 days of age for paraffin sectioning and sequencing by Illumina NovaSeq 6000 sequencing platform.The differentially expressed genes(DEGs)in the longissi-mus dorsi muscle tissues were screened by DESeq2 with|log2 Fold Change|>1 and P<0.05.The GO and KEGG databases were used for GO functional annotation analysis and KEGG signaling pathway enrichment analysis respectively,to identi-fy genes that might be involved in lipid metabolism.The transcriptome sequencing results were verified by real-time fluo-rescence quantitative PCR.【Result】The serum triglyceride content of Zangjiang sows in the treatment group was signifi-cantly higher than that of the control group(P<0.05,the same below),the total cholesterol content was extremely signifi-cantly higher than that of the control group(P<0.01,the same below),the estrogen content was extremely significantly lower than that of the control group,and the total fat content of longissimus dorsi muscle was significantly lower than that of the control group.There were 278 DEGs between the treatment group and control group of Zangjiang sows,of which 33 DEGs were related to lipid metabolism.The DEGs were mainly annotated on the GO function entries of bile acid secre-tion,phosphatidylinositol 3-kinase signaling,protein kinase B signaling,G protein-coupled receptor binding,glycosami-noglycan binding,chemokine activity and chemokine receptor binding.And they were mainly enriched in KEGG signa-ling pathways including glycolysis/gluconeogenesis,starch and sucrose metabolism,carbohydrate digestion and uptake,MAP kinase serine/threonine phosphatase activity,estrogen 2-hydroxylase activity and cGMP-PKG signaling.The expres-sion trend of the target genes detected by real-time fluorescence quantitative PCR was basically consistent with the tran-scriptome sequencing results,indicating that the transcriptome sequencing data were reliable.【Conclusion】Decreased se-rum estrogen levels,increased triglyceride and total cholesterol levels,and decreased intermuscular fat content of longis-simus dorsi muscle in ovary-removed Zangjiang sows are associated with differential expression of 33 DEGs involved in lipid metabolism regulation,including CALML5,PLCZ1,PIK3C2G,and IL6,and are involved in function terms such as bile acid secretion,phosphatidylinositol 3-kinase signaling regulation,and involved in signaling pathways such as gly-colysis/gluconeogenesis,starch and sucrose metabolism.