北京油鸡purH基因结构与表达特征研究

Characterization of Expression of purH gene in Beijing Fatty Chicken ( Gallus gallus)

提取北京油鸡8种组织的总RNA,利用RT-PCR检测purH基因mRNA的差异表达。将purH基因完整开放阅读框定向插入pEGFP-N3,构建带有GFP报告基因重组表达载体pEGFP-purH,利用脂质体介导法将其导入北京油鸡体外培养细胞中,进行G418药物筛选和克隆化培养。结果表明:北京油鸡purH基因(GenBank登录号:EU334506)编码区为1782 bp,编码593 aa的多肽,转染后24 h、48 h和72 h,pEGFP-purH转染率在10.3%～53.2%之间,绿色荧光均匀分布于细胞质与细胞核中,随表达量的增加,绿色荧光在细胞核中聚集成团块状或颗粒状。经药物筛选和克隆化培养,获得表达pEGFp-purH融合蛋白的克隆细胞株,RT-PCR与Western blotting检测确认pEGFP-purH已整合到北京油鸡成纤维细胞基因组中,在优化的条件下,阳性细胞凋亡率、生长与增殖状况与对照组比较差异不显著(P>0.05)。

The specific expression of purH gene in eight different tissues of Beijing fatty chicken was investigated by RT-PCR in this study. The full-length cDNA of purH was inserted into pEGFP-N3 multi-cloning sites to construct recombinant vector pEGFP-purH. The lipofectin method was used to transfect the pEGFP-purH into fibroblast cells. Bioinformatic analysis showed CDS region of the Beijing fatty chicken purH gene was 1782bp, which encoded a 483-amino-acids-long peptide. The recombinant pEGFP-purH was constructed with GFP as reporter gene to transfect into Beijing fatty chicken fibroblasts. The expression efficiency ranged from 10.3% to 53.2% in 24, 48, and 72 h after transformation. RT-PCR and Western blotting analyses showed that pEGFP-purH had been integrated into the genome of Beijing chicken fibroblast cell with normal expression level. In optimized condition, there was no significant effect （P〉0.05） on apoptosis ratio, growth and reduplication state comparing with the control group.