Fish proteins not lipids are the major nutrients limiting the use of vegetable ingredients in catfish nutrition

The objective of this study was to determine the major nutrient limiting growth and lipid metabolism in African catfish fed diets composed of vegetable ingredients. Four diets were formulated from contrasted meal (fish meal: FM; vegetable meal: VM) and oil (fish oil: FO; vegetable oil: VO) sources. Replacement of FO by VO did not affect specific growth rate (SGR) and feed efficiency, whereas lower values were recorded in the case of FM replacement. LC‐PUFAs muscle contents were higher in fish fed control FMFO diet than in fish fed vegetable ingredients. However, the decrease in docosahexaenoic acid (DHA) concentration in FMVO group was limited compared to VM groups despite the same low DHA level in those three diets. These results may suggest an activation of LC‐PUFA biosynthesis from PUFA precursors brought with vegetable oils in FMVO group. This hypothesize is reinforced by the significant stimulation of elovl5 gene expression in liver and intestine from fish fed FMVO. Therefore, this study demonstrated that African catfish is able to bioconvert LC‐PUFAs at a significant biological level when FO is replaced by VO whereas the use of plant proteins has strong detrimental effects on growth performances.     

FADS2基因在奶牛乳腺细胞中的过表达和干扰研究

为了研究脂肪酸脱氢酶2(fatty acid desaturases 2,FADS2)基因在奶牛乳腺细胞脂肪酸代谢中的作用,本研究在奶牛乳腺上皮细胞中对FADS2基因进行过表达和干扰,研究FADS2基因表达对脂肪酸合成相关基因的调控及对奶牛乳腺上皮细胞中甘油三酯含量的影响。针对FADS2基因的CDS序列设计siRNA和过表达载体pcDNA3.1-FADS2-EGFP,转染奶牛乳腺细胞检测FADS2基因过表达和干扰对脂肪酸代谢相关基因表达的影响及细胞中甘油三酯含量的变化。结果显示,试验成功获得过表达载体pcDNA3.1-FADS2-EGFP和干扰片段,转染细胞后具有良好的过表达和干扰效果。FADS2基因过表达后,1-酰基甘油磷酸酰基转移酶(AGPAT1)、固醇调节元件结合蛋白裂解激活蛋白(SCAP)、3-磷酸甘油转移酶(GPAM)、脂肪酸延长链5(ELOVL5)、乙酰辅酶A酰基转移酶1(ACAA1)、脂肪酸脱氢酶1(FADS1)、二酰基甘油转酰基酶1(DGAT1)和过氧化物酶体增殖激活受体α(PPARα)基因显著下调(P<0.05),脂滴蛋白2(PLIN2)基因极显著上调(P<0.01)。FADS2基因干扰过后可引起AGPAT1、GPAM、ELOVL5、ACAA1、PLIN2和FADS1基因显著上调(P<0.05),脂肪酸合成胰岛素诱导基因1(INSIG1)极显著上调(P<0.01),DGAT1和PPARα基因显著下调(P<0.05)。甘油三酯检测结果显示,FADS2基因过表达和干扰均可降低奶牛乳腺上皮细胞中甘油三酯的含量。综上所述,在奶牛乳腺上皮细胞中,FADS2基因能调控脂质合成相关基因的表达,对乳腺脂质合成具有调控作用。     

Cloning and Bioinformatics Analysis of FADS2 Gene in Buffalo

This study was aimed to clone the buffalo Δ6-fatty acid desaturases (FADS2) gene using in-Silico cloning and analyze its genetic struction with bioinformatics, which provide a foundation for investigating the milk performance in buffaloes. Primers were designed according the sequence of FADS2 gene in dairy cow (GenBank accession No.:NM_001083444.1). The FADS2 gene was amplified by RT-PCR, and its sequence was analyzed by bioinformatics. Sequence analysis revealed that the buffalo FADS2 gene had 36 600 bp in length and consisted of 12 exons and 11 introns, containing an open reading frame (ORF) of 1 335 bp which encoding 444 amino acids. Sequence homology analysis indicated that the buffalo FADS2 protein gene showed 98.88%, 98.88%, 89.66%, 90.79%, 90.85%, 92.35% and 87.11% identity with that of Bos mutus, Bos taurus, Homo sapiens, Sus scrofa, Oryctolagus cuniculus, Orcinus orca and Rattus norvegicus, respectively. Protein prediction analysis showed that the molecular weight and isoelectric point (pI) of buffalo FADS2 were 52.51 ku and 8.75, respectively, and the FADS2 protein was weak alkali and the hydrophobicity protein without signal peptide. Phylogenetic tree analysis showed that FADS2 gene was highly conserved in different species and evolutionary processes,buffalo was close to Bos mutus and Bos taurus, and was far from Rattus norvegicus. This study suggested that FADS2 gene was successfully cloned in buffalo, which laid a foundation for clarifying the mechanism of milk performance in buffalo.     

FADS1基因对猪不饱和脂肪酸含量的影响研究

【目的】研究脂肪酸去饱和酶1(fatty acid desaturases 1,FADS1)基因对不饱和脂肪酸代谢的调控作用,为揭示其分子机制提供参考。【方法】利用PCR扩增猪FADS1基因的CDS区,将目的基因与pcDNA3.1(-)骨架载体连接获得重组表达载体pcDNA3.1-FLAG-FADS1,将重组表达载体瞬时转染宁乡猪肾成纤维细胞,转染后24、48 h分别收集细胞,利用实时荧光定量PCR和Western blotting检测FADS1基因的表达情况,经过遗传霉素(geneticin,G418)筛选之后获得稳定表达FADS1基因的细胞系,命名为1-4^#。利用甘油三酯检测试剂盒检测野生型和1-4^#细胞甘油三酯含量变化;利用气相色谱质谱法(gas chromatography-mass spectrometer,GC-MS)检测野生型和1-4^#细胞不饱和脂肪酸含量变化情况。【结果】成功获得过表达载体pcDNA3.1-FLAG-FADS1,在转录水平和蛋白水平检测均有过表达效果。通过G418筛选出1株稳定过表达FADS1基因的单克隆细胞。甘油三酯含量测定结果显示,与野生型宁乡猪肾成纤维细胞相比,过表达FADS1的宁乡猪肾成纤维细胞中甘油三酯的含量显著降低(P＜0.05)。不饱和脂肪酸检测结果显示,过表达FADS1后,总脂肪酸的含量极显著升高(P＜0.01),提高1.8倍,多不饱和脂肪酸(polyunsaturated fatty acid,PUFA)中ω-3 PUFA、ω-6 PUFA含量均极显著升高(P＜0.01),二十碳五烯酸(eicosapentaenoic acid,EPA)、二十二碳五烯酸(docosapentaenoic acid,DPA)、二十二碳六烯酸(docosahexaenoic acid,DHA)的相对含量极显著提高(P＜0.01),ω-3/ω-6值显著提高(P＜0.05)。【结论】成功构建超表达载体pcDNA3.1-FLAG-FADS1,并在宁乡猪肾成纤维细胞中成功筛选到了1株稳定超表达FADS1基因的单克隆细胞1-4^#,经检测,FADS1基因超表达能显著降低细胞中甘油三酯含量、提高ω-3 PUFA含量及占比,提高ω-3/ω-6值。     

中国荷斯坦牛FADS2基因3′端SNP突变对乳中脂肪酸组成的影响

【目的】FADS2是多不饱和脂肪酸合成中关键的限速酶之一,可催化食物中亚油酸(LNA,C18:2n6)合成γ-亚麻酸(GLA,C18:3n6)、二十碳五烯酸(EPA,C20:5n3)、二十二碳六烯酸(DHA,C22:6n3)等长链脂肪酸。试验旨在探讨中国荷斯坦牛FADS2基因3′端非编码区SNP突变对乳中脂肪酸含量的影响。【方法】随机选择20头无亲缘关系的中国荷斯坦牛样本,用直接测序列法检测FADS2基因3′端非编码区SNP突变位点。再以江苏某大型奶牛场551头中国荷斯坦牛为材料,用飞行时间质谱法对前期发现的2个SNP位点进行检测,同时利用最小二乘模型分析了SNP突变及其单倍型对乳中脂肪酸含量及其不饱和指数的影响。【结果】中国荷斯坦牛FADS2基因3′端非编码区存在3个SNP突变位点:c.1571 AG、c.2743 AG、c.2776 AG。c.1571 AG位点GG型为优势基因型,基因型频率为0.800,G为优势等位基因,基因频率为0.887。c.2776 AG位点AA为优势基因型,基因型频率为0.673,A为优势等位基因,基因频率为0.819。χ~2检验表明:c.2776 AG位点基因型分布均符合Hardy-Weinberg平衡(P0.05),而c.1571 AG位点基因型分布均偏离Hardy-Weinberg平衡(P0.05)。FADS2基因c.1571 AG位点与c.2776 AG位点间连锁不平衡系数r2为0.028,未达到显著水平(P0.05)。c.1571AG位点与c.2776 AG位点有3种单倍型,GA、GG和AA频率分别为0.705、0.181和0.114。多因素方差分析表明:FADS2-1571对C14:1含量、C14和C18不饱和指数的影响达到极显著水平(P0.01),对C18:0、SFA和MUFA含量的影响达到显著水平(P0.05)。GG型个体乳中C14:1含量、C14和C18不饱和指数显著高于AG型(P0.05)。FADS2-2776位点对C16:1含量、C16和C20不饱和指数的影响达到极显著水平(P0.01),对C14:1含量的影响达到显著水平(P0.05),GG型个体乳中C16:1含量、C16和C20不饱和指数显著高于AG型和AA型(P0.05)。同时,FDAS2-1571-2776单倍型对C16:1含量和C20不饱和指数的影响达到显著水平(P0.05),单倍型GG型个体乳中C16:1含量和C20不饱和指数显著高于GA型和AA型(P0.05)。【结论】FADS2基因3′端非编码区SNP突变对中国荷斯坦牛乳中脂肪酸组成有重要影响。在进一步验证其功能情况下,可作为影响中国荷斯坦奶牛乳脂肪酸组成的主效基因加以利用。