大黄鱼Rnd1基因表达载体构建及表达模式分析

为探究Rnd1基因在大黄鱼免疫应答过程中的作用，采用实时荧光定量PCR技术（qRT-PCR）对该基因的表达模式进行分析，同时构建原核表达载体pET-28a-Rnd1，并转化进大肠杆菌后诱导该蛋白的融合表达。研究结果表明:大黄鱼Rnd1基因ORF为699 bp，编码232个氨基酸；经序列多重比对和进化树构建发现，Rnd1的氨基酸序列高度保守；在健康大黄鱼的8个免疫组织中，Rnd1在肝脏中相对表达量最高，其次为脑，而在肠中表达量最低；变形假单胞菌攻毒后，Rnd1在肝脏中48 h时达到最高值，为对照组的25倍；在脾脏中则持续上调表达，尤其在48 h后升高更为明显。

Construction of Expression Vector and Expression Analysis of Rnd1 Gene in Larimichthys crocea

RNA sequencing was performed on the spleen samples of Larimichthys crocea challenged by Pseudomonas plecoglossicid in our previous study.We found that the Rnd1 gene was significantly up-regulated in the samples with relatively high bacterial load compared to those with relatively low bacterial load through differential gene expression analysis.The results suggested that this gene might play an important role in the infection of P.plecoglossicid to the L.crocea.In order to explore the function of Rnd1 in the immune response of L.crocea,the expression pattern of Rnd1gene was analyzed by quantitative real-time PCR (qRT-PCR)，and the prokaryotic expression vector of pET-28a-Rnd1was constructed to express the fusion protein in E.coli.The results showed that the full-length ORF of Rnd1 was 699 bp,encoding 232 amino acids.Multiple sequence alignment and evolutionary tree results showed that the amino acid sequence of Rnd1 was highly conserved.The expression level of Rnd1 gene was the highest in liver，followed by brain，and the lowest expression was observed in intestine among the eight examined tissues.The relative expression of Rnd1 gene peaked at 48h in liver after stimulated by P.plecoglossicid,which was 25 times as the control group.The gene expression of Rnd1 in spleen was continuously upregulated，especially after 48h of the infection.