虹鳟免疫诱导型基因Cathelicidin2启动子功能分析

Functional Analysis of the Immune-Induced Promoter of the Rainbow Trout Cathelicidin2 Gene

本研究通过实时荧光定量PCR实验对虹鳟(Oncorhynchus mykiss)Cathelicidin2(Cath2)基因的转录模式进行了分析。结果显示,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,在细菌和病毒感染后,转录水平均显著升高。对基因上游调控序列进行启动子和转录因子结合位点预测,发现该启动子具有真核生物典型的TATA盒和CAAT盒结构,基因上游直至第一内含子区域内,密集存在多个免疫相关转录因子结合位点,其中,2个核因子κB(Nuclear factor kappa B,NFκB)预测结合位点均位于核心启动子正链区域。在草鱼(Ctenopharyngodon idellus)肾组织细胞系内,绿色荧光蛋白和萤火虫荧光素酶基因都能够在该启动子驱动下表达,表明其具有启动子活性,且启动子活性在受到免疫诱导后增强,包括细菌脂多糖(Lipopolysaccharides,LPS)模拟的细菌感染和聚肌胞苷酸(Polyinosinic polycytidylic acid,Poly I:C)模拟的病毒感染。双荧光素酶报告基因检测显示,启动子活性在与NFκB转录因子表达时,增强至4.39倍,证明Cath2基因受NFκB通路调控。研究表明,虹鳟Cath2基因能够在多种免疫刺激诱导下表达,其启动子可以应用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。

This study aimed to investigate the transcriptional regulation of a teleost antimicrobial peptide. The expression pattern of the rainbow trout Cathelicidin2（Cath2） gene was analyzed by quantitative real-time PCR（q RT-PCR）. Cath2 was expressed in tissues closely related to immune defense, including the gill and head kidney, and expression significantly increased after both bacterial and viral infections. Promoter and transcription factor binding sites were analyzed for the upstream regulatory sequence of Cath2 gene. The predicted promoter contained characteristic eukaryotic TATA and CAAT boxes, as well as multiple binding sites for immune-related transcription factors, including two candidate binding sites for nuclear factor kappa B（NFκB） on the positive strand of the core promoter. In Ctenopharyngodon idella kidney cell lines, transcription of both green fluorescent protein and firefly luciferase genes was initiated by the predicted Cath2 promoter. Both the bacterial mimetic lipopolysaccharide and the viral mimetic polyinosinic polycytidylic acid up-regulated promoter activity. As shown in the dual luciferase reporter assay, promoter activity was enhanced by co-expression of the transcription factor NFκB, indicating that Cath2 is regulated by the NFκB pathway. These results suggest that expression of the rainbow trout Cath2 gene could be induced by different immune stimuli, and its promoter might serve as an immune-inducible transgenic element. The Cath2 promoter may initiate the transcription of heterologous immune genes against exogenous infection in a proper expression pattern, avoiding excessive transcription under unnecessary conditions; therefore, it has potential for genetic engineering approaches in the breeding of disease-resistant fish.