本实验旨在通过探究SCD5基因缺失后对脂肪酸种类与组成的影响,为猪肉品质的改良提供新靶标.利用CRISPR/Cas9 技术对SCD5基因进行定点敲除,筛选获得纯合缺失单克隆细胞系,验证其敲除效率后,利用GC-FID对其脂肪酸含量进行检测分析.结果显示:SCD5基因缺失降低了其介导的限速反应产物C16:1和C18:1 的相对含量;在几种重要的多不饱和脂肪酸中,SCD5缺失在降低二十碳五烯酸(EPA)含量的同时引起了二十二碳五烯酸(DPA)升高;细胞中的单不饱和脂肪酸(MUFAs)含量也随着SCD5的缺失而降低,但多不饱和脂肪酸(PUFAs)含量上调.同时,SCD5基因缺失下调了对线粒体脂肪酸β-氧化具有重要影响的芥酸含量.综上,SCD5对脂肪酸的种类与组成具有重要的调控作用,也提示了其在脂质合成及代谢、脂肪沉积方面的重要作用.
为研究PLIN2基因对中国本土猪宁乡猪长链omega-3 多不饱和脂肪酸(ω-3 PUFA)的调控作用,利用CRISPR/Cas9 技术敲除宁乡猪原代肾成纤维细胞PLIN2基因,获得PLIN2纯合敲除细胞系,检测其脂肪酸含量及组成变化.结果显示:与野生型细胞相比,PLIN2-/-细胞的多不饱和脂肪酸总含量增加 55.92%,ω-3 PUFA含量增加 112.02%,多不饱和脂肪酸含量在总脂肪酸中的占比由 27.78%增加至 34.40%.因此,PLIN2基因对宁乡猪原代肾成纤维细胞多不饱和脂肪酸和ω-3 PUFA的形成具有正向调控作用.本研究为揭示PLIN2调控脂肪酸的分子机理提供了重要参考,并为猪肉品质改良的生物育种提供了候选靶点.
The macrophage CD163 surface glycoprotein is a member of the SRCR family class B, which has been identified as the key trigger in host–pathogen interactions, but its specific roles in sensing Glaesserella parasuis (G. parasuis) infection are largely unknown. Here, we investigated porcine CD163 in mediating the adhesion and immune response of G. parasuis using in vitro host–bacteria interaction models. CD163-overexpressing Chinese hamster ovary K1 cells (CHO-K1) showed obvious subcellular localization in the cytoplasm, especially in the cytomembrane. Although detection using scanning electron microscopy (SEM) confirmed the bacterial adhesion, there was no significant difference in the adhesion of G. parasuis to CHO-K1 cells between the presence and absence of CD163. In addition, similar results were observed in 3D4/21 cells. Meanwhile, bindings of G. parasuis to nine synthetic peptides, the bacterial binding motifs within SRCR domains of CD163, were weak based on a solid-phase adhesion assay and agglutination assay. Moreover, CD163 had no effect on the expression of G. parasuis-induced inflammatory cytokines (IL-6, INF-γ, IL-10, IL-4 and TGF-β) in CHO-K1 cells. In conclusion, these findings indicate that porcine CD163 plays a minor role in sensing G. parasuis infection.
随着分子生物学和动物遗传育种技术的快速发展,对于畜禽优良性状的研究已从对表型选育进入到对单个或多个重要性状关联基因的分子选育,如对猪肉质性状的相关研究,在影响猪背膘厚、肌内脂肪含量、火腿重量等方面筛选到许多关键基因.近年来,非洲猪瘟肆虐,对猪抗病的研究再次成为热点,也发现了众多关键基因,如CD163直接参与了猪呼吸与繁殖综合征病毒的侵入机制.除了疾病的影响外,繁殖性状对养猪业的发展也起着至关重要的作用.随着研究人员对各重要性状研究的不断深入,现已积累了大量重要育种价值基因的多维数据,且由此开发出了多种基因筛选技术,尤其是CRISPR-Cas9基因编辑技术的发现及其作为全基因组筛选工具的成功应用,以及在后基因组时代功能基因组筛选与多组学的联合应用等,进一步推动了遗传育种领域的发展.作者系统总结了猪肉质、抗病及繁殖性状相关的重要育种价值基因及其挖掘技术,以期为猪的遗传改良提供指导性建议和参考.
