一种免疫诱导型鲤启动子的克隆与功能分析

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

为发掘适用于基因工程抗病育种的鱼类启动子,通过实时荧光定量PCR实验对鲤Rab GTP酶(Ras-associated binding-GTPases 1a3,Rab1a3)基因的表达模式进行了分析,证实该基因在鳃、头肾等与机体免疫防御功能密切相关的组织内转录水平较高,且免疫激活后转录显著增强,符合基因工程抗病育种所需的外源免疫基因转录模式。从鲤细菌人工染色体文库中,使用Rab1a3基因特异引物筛选获得包含该基因区域的文库克隆,测序获得该基因完整序列,以及上下游调控序列。通过生物信息学手段,预测到长度为1014 bp的鲤Rab1a3基因启动子序列,该启动子不具有典型的TATA盒或CpG岛特征,存在多个免疫相关转录因子结合位点。在草鱼肾组织细胞系内验证该启动子活性,结果显示,绿色荧光蛋白基因和萤火虫荧光素酶基因都能够在该启动子驱动下表达,证实该片段具有启动子活性,且启动子活性在受到免疫诱导后增强,双荧光素酶报告基因检测结果显示,该启动子活性在免疫刺激后增强至免疫刺激前的8.67倍。研究表明,鲤Rab1a3基因启动子有望被开发成为免疫诱导型的基因工程元件,驱动外源免疫基因在鱼体内适时表达,抵御外界病原感染,同时避免非必要条件下的过度表达形成生长负担。

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.