鸡Apob基因组织表达与CRISPR/Cas9敲除系统的构建

Tissue Expression and Construction of CRISPR/Cas9 Knockout System of Apob in Chicken

旨在探究Apob基因在鸡肝脂质代谢过程中的功能。本研究对鸡Apob蛋白进行理化性质分析;利用RT-qPCR检测Apob基因在4周龄黄羽肉鸡组织中的表达情况,每组设置3个重复,进行3次平行试验。根据鸡Apob蛋白关键结构域,在Apob基因外显子设计3对sgRNA,构建Cas/gRNA载体;将重组质粒转染DF-1细胞后,利用T7核酸内切酶I(T7 endonuclease I,T7EI)酶切法和TA克隆测序法筛选敲除活性位点并计算敲除效率。利用RT-qPCR检测基因敲除后亚克隆细胞中Apob基因mRNA表达情况。结果表明,鸡Apob的相对分子质量为523.356 ku,平均亲水性为-0.300,为稳定的蛋白质,并且该基因主要在鸡的肝、肾和小肠组织表达。敲除载体转染至DF-1细胞后,T7EI酶切发现,Cas/gRNA6、Cas/gRNA7和Cas/gRNA8三个位点均可发挥敲除活性,TA克隆测序结果表明,三者的敲除效率分别为33.3%、65%和80%。同时,RT-qPCR结果显示,转染Cas9/gRNA7、Cas9/gRNA6、Cas9/gRNA8的细胞中Apob基因mRNA表达水平约分别下调99.96%(P<0.01)、85%(P<0.01)、47%(P<0.05)。综上所述,本研究揭示了鸡Apob基因在组织中的表达特点和蛋白的理化性质;成功构建了鸡Apob基因CRISPR/Cas9敲除载体,并筛选出最佳敲除位点,获得了Apob基因敲除的亚克隆细胞,为进一步探索Apob基因在鸡肝中的功能奠定了基础。

The study aimed to explore the function of Apob gene in the lipid metabolism of chicken liver.The physical and chemical properties of chicken Apob protein were analyzed;RT-qPCR was used to detect the expression of Apob gene in the tissues of 4-week-old yellow broiler chickens,3 replicates were set up in each group and 3 parallel experiments were carried out.Three pairs of sgRNA were designed according to the sequence of key domains of chicken Apob protein to construct Cas/gRNA vectors;T7 endonuclease I(T7EI)digestion method and TA cloning sequencing method were used to screen knockout active sites and calculate gene knockout efficiency in cells after the Cas/gRNA vector was transfected into DF-1 cells;The mRNA expression of Apob gene was detected in subcloned cells using RT-qPCR after gene knockout.The results showed that the relative molecular mass of chicken Apob was 523.356 ku and the average hydrophilicity was-0.300,which was a stable protein.And the Apob gene mainly expressed in chicken liver,kidney and small intestine tissues.T7EI digestion result showed that Cas/gRNA vectors could knock out the gene effectively.TA clone sequencing results showed that knockout efficiency of 3 active sites(Cas/gRNA6,Cas/gRNA7 and Cas/gRNA8)were 33.3%,65%and 80%,respectively.At the same time,RT-qPCR results showed that the expression level of Apob genes in transfected Cas9/gRNA7,Cas9/gRNA6,Cas9/gRNA8 cells were down-regulated by about 99.96%(P<0.01),85%(P<0.01),47%(P<0.05),respectively.In summary,we revealed the expression characteristics of Apob gene in chicken tissues and the physicochemical properties of the protein;Then we contructed Cas/gRNA knockout vectors,screened the optimal knock sites,and established Apob knockout subclonal cells successfully,which laid a foundation for exploring the function of Apob gene in chicken liver.