Cloning and functional analysis of an immune-induced promoter in common carp　(Cyprinus carpio)

The study aimed to explore a teleost promoter applicable to genetic engineering breeding for disease resistance. The expression pattern of Ras-associated binding-GTPases 1a3 (Rab1a3) gene in common carp (Cyprinus carpio) was analyzed by quantitative real time PCR experiments. The transcription level of Rab1a3 gene was high in tissues closely related to immune defense, including gill and head kidney, while the transcription was enhanced after immune stimulation. This transcription pattern well fitted the conceived perfect expression pattern of heterologous immune gene in transgenic fish. Bacterial Artificial Chromosome (BAC) library of common carp was screened by PCR, using Rab1a3 specific primers. A BAC clone containing Rab1a3 gene was found and then sequenced, to obtain the complete genomic sequence of Rab1a3 gene, including its upstream and downstream regulatory sequences. A putative 1014 bp promoter of Rab1a3 gene, with multiple binding sites of immune related transcription factors, was predicted using several bioinformatics tools, while TATA box and CpG islands of typical promoters were absent. The promoter activity was verified in Ctenopharyngodon idella kidney cell lines, indicating that transcription of both green fluorescent protein (GFP) and firefly luciferase genes was able to be initiated by the 1014 bp fragment. After immune stimulation, the promoter activity reached 8.67 times as compared with before, determined by dual luciferase reporter assay. These results suggested that C. carpio Rab1a3 promoter could be a potential transgenic element with immune inducible characteristics, which initiates transcription of heterologous immune gene against exogenous infection in proper expression pattern, avoiding excessive transcription in unnecessary conditions.