布莱凯特黑牛CB1基因克隆测序及表达分析

Cloning,sequencing and expression of CB1 gene in Black cattle

本试验旨在研究布莱凯特黑牛CB1基因CDS区序列和表达情况,探讨CB1基因与脂肪沉积的关系。运用RT-PCR技术克隆CB1基因CDS区序列,并进行生物学分析;运用qRT-PCR技术检测不同组织的mRNA相对表达量;以及运用免疫组化技术对蛋白进行定位分析,运用Western blot技术检测CB1蛋白在不同组织的表达量。结果显示,克隆获得的布莱凯特黑牛CB1基因CDS为1495 bp,编码477个氨基酸。进化树显示布莱凯特黑牛CB1基因与牛、山羊、绵羊同源性最高(>98%)。qRT-PCR结果显示CB1基因在不同组织均有表达,在脂肪组织中相对表达量最高;免疫组化显示CB1基因主要在脂肪细胞膜上表达;Western blot结果显示CB1蛋白在脂肪组织和肌肉组织均有表达,在脂肪组织表达量显著高于肌肉组织。综上推测,CB1基因参与脂质代谢相关调节过程,为肉牛肉质性状的分子选育提供参考。

This experiment was to study the sequence and expression of the CDS region of the CB1 gene in Black cattle,and to explore the relationship between the CB1 gene and fat deposition.In this test,the sequence of the CDS region of the CB1 gene was cloned using RT-PCR technology,and biological analysis was performed.The relative expression of mRNA in different tissues was detected using qRT-PCR;and the protein was analyzed immunohistochemically using Western blot.The expression of CB1 protein in different tissues was detected.The results showed that the CDS of CB1 obtained by cloning was 1495 bp,encoding 477 amino acids.The phylogenetic tree showed that the CB1 gene of Black cattle had the highest homology with that of Bos taurus,goat,and sheep(>98%).qRT-PCR results showed that CB1 was expressed in different tissues,and the relative expression was highest in the adipose tissue.Immunohistochemistry showed that CB1 was mainly expressed on the cell membrane in the adipose tissue.CB1 protein was expressed in the adipose tissue and muscle tissue.The expression level in the adipose tissue was significantly higher than that in the muscle tissue.In summary,the present research suggested that the CB1 gene was involved in lipid metabolism-related processes,providing a reference for molecular selection of meat and beef quality traits.