柞蚕免疫相关基因Apdorsal的克隆鉴定与表达分析

Cloning,Identification and Expressional Analysis of Immunity Related Genes Apdorsal from Antheraea pernyi

ReL／NF—κB蛋白家族是一种重要的转录因子，在机体免疫应答、炎症反应、细胞凋亡与生长发育过程中起着重要调控作用。采用RACE技术克隆了2个柞蚕Rel／NF—κB相关蛋白基因彻dorsalA和Af）dorsalB的全长cDNA序列（GenBank登录号：JF488068，JF488069）。经BLAST比对表明2紊序列编码的蛋白质都含有Rel／NF—κB家族的典型氨基酸序列RHD；系统进化分析显示ApdorsalA蛋白与其它物种的ReL／NF—κB蛋白的序列相似度在32％-78％之间，与家蚕的RelA、烟草天蛾的Dorsal有较近的亲缘关系，说明Apdorsal属于Rel／NF—κB家族的Dorsal类。利用real—timePCR技术检测Apdorsal基因mRNA的组织分布以及经不同微生物诱导处理后的转录变化，结果表明Apdorsal基因mRNA在柞蚕蛹各组织中均有转录；柞蚕蛹脂肪体中的apdorsal基因mRNA经ApNPV诱导处理后的转录水平最高，经毕赤酵母和枯草芽孢杆菌诱导处理后的转录水平次之，经E．coli诱导处理后的转录水平最低，说明柞蚕蛹脂肪体中的Apdorsal可能与对病毒、革兰阳性菌和真菌的免疫有关。

Rel/NF-κB family proteins are important transcriptional factors which play crucial roles in immune response, inflammation, ceil apoptosis, growth and development. The full length cDNAs of two Rel/NF-κB related genes were cloned from Antheraea pernyi using RACE technology and were designated as ApdorsalA and ApdorsalB （GenBank accession No. JF488068 and JF488069）. BLAST search in GenBank indicated that both ApdorsalA and ApdorsaIB contain typical amino acids RHD of Rel/NF-κB family. Phylogenic analysis displayed that the sequence similarity of ApdorsalA with that of the other species is between 32%-78%. Especially, Apdorsal has a closer evolutionary relationship with that of Bombyx mori RelA and Maduca sexta Dorsal, showing that Apdorsal belongs to Dorsal class proteins of Rel/NF-KB family. The quantitative real-time RT-PCR was used to analyze the distribution of Apdorsal in different tissues and after challenge with different pathogens. The results showed that mRNA of Apdorsal gene was expressed in all tissues. The transcription level of Apdorsal gene mRNA was the highest in fat body after injection with Antheraea pemyi nucleopolyhedrovirus. The transcription level of Apdorsal gene mRNA ranked the second after injection with Pchia pastoris and Bacillus subtillis. The transcription level of Apdorsal gene mRNA was the Lowest after injection with E. coil, indicating that Apdorsal is probably involved in immune response against virus, fungus and Gram-positive bacterium in fat body of Antheraea pernyi.