摘要
【目的】明确FABP1基因对猪肌内脂肪细胞脂肪酸和脂肪合成的影响,为进一步解析FABP1基因对猪肌内脂肪沉积和脂质代谢的调控作用提供依据。【方法】设计香苏杂交猪FABP1基因干扰载体的靶序列并合成,通过载体连接构建shRNA-FABP1干扰载体并测序验证;将正确的干扰靶序列转染猪肌内脂肪细胞,通过转染效果和实时荧光定量PCR筛选干扰效率最高的靶序列进行后续试验。采用TRIzol®Reagent总RNA提取试剂盒提取转染shRNA-FABP1干扰载体后的猪肌内脂肪细胞的总RNA,以cDNA为模板进行实时荧光定量PCR检测脂肪沉积相关基因的相对表达量。运用气相色谱—质谱联用仪靶向检测猪肌内脂肪细胞内中长链脂肪酸(LCFA)的含量;采用GPO-PAP酶法检测猪肌内脂肪细胞甘油三酯(Triglyceride,TG)和胆固醇的含量。【结果】成功构建了shRNA-FABP1干扰载体并筛选出最佳干扰效率片段。FABP1基因干扰显著(P<0.05,下同)或极显著(P<0.01,下同)降低了脂肪沉积相关基因的相对表达量,包括LPL、FABP3、IGFBP4和OLR1。在FABP1基因干扰后的猪肌内脂肪细胞中共检测出38种脂肪酸,29种脂肪酸有差异;其中,FABP1基因干扰上调包括棕榈酸(C16:0)、硬脂酸(C18:0)和油酸(C18:1N9)等17种脂肪酸的比例,下调包括二十碳五烯(C20:5N3)、十七碳烯酸(C17:1N7)和十三烷酸(C13:0)等12种脂肪酸的比例。FABP1基因干扰显著或极显著降低了香苏杂交猪肌内脂肪细胞内TG和胆固醇的含量。【结论】干扰FABP1基因通过抑制与脂肪酸转运和TG合成相关基因的表达,从而改变脂肪酸的组成比例,降低TG和胆固醇的含量。
【Objective】To clarify the effect of FABP1 gene on fatty acid and lipid synthesis in porcine intramuscular adipocytes and to provide a basis for further analysis of the regulatory role of FABP1gene on intramuscular fat deposition and lipid metabolism in pigs.【Method】Target sequences of FABP1 gene interference vector of Xiangsu crossed pigs were designed and synthesized,and then shRNA-FABP1 interference vector was constructed by vector ligation and validated by sequencing.The correct interfering target sequence was transfected into porcine intramuscular adipocytes,and the target sequence with the highest interference efficiency was screened by transfection effect and real-time fluorescence quantitative PCR(RT-qPCR)for next experiments.Total RNA from porcine intramuscular adipocytes transfected with shRNAFABP1 interference vector was extracted by the TRIzol®Reagent total RNA extraction kit.RT-qPCR was used to detect the expression of genes related to fat deposition with cDNA being a template.Then the content of medium and long chain fatty acids(LCFA)in porcine intramuscular adipocytes was detected by targeted detection of gas chromatography-mass spectrometry(GC-MS),and the contents of triglycerides(TG)and cholesterol in porcine intramuscular adipocytes were detected by the GPO-PAP enzymatic method.【Result】The shRNA-FABP1 interference vector was successfully constructed and the fragment with the best interference efficiency was screened.FABP1 gene interference significantly(P<0.05,the same below)or extremely significantly(P<0.01,the same below)reduced the relative expression of fat deposition-related genes,including LPL,FABP3,IGFBP4 and OLR1.A total of 38 fatty acids were also detected in porcine intramuscular adipocytes withFABP1 gene interference,among which 29 had difference.FABP1 gene interference up-regulated the proportion of 17 fatty acids including methyl palmitate(C16:0),stearic acid(C18:0)and methyl oleate(C18:1N9),and down-regulated the proportion of 12 fatty acids including eicosapentaenoic(C
作者
熊讯
李永
阮涌
许厚强
XIONG Xun;LI Yong;RUAN Yong;XU Hou-qiang(College of Animal Sciences,Guizhou University/Key Laboratory of Ministry of Education for Animal Genetics,Breeding and Reproduction in the Plateau Mountains Region,Guiyang,Guizhou 550025,China;College of Life Sciences,Guizhou University,Guiyang,Guizhou 550025,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2023年第8期2454-2464,共11页
Journal of Southern Agriculture
基金
贵州省科技计划项目[黔科合服企〔2018〕4007(002)号]
贵州省农业重大产业科学研究攻关项目(黔教合KY字〔2019〕011号)。
