摘要
由小的干扰RNA(Small interfering RNA,siRNA)介导的RNA干扰(RNA interference,RNAi)是近年来快速发展的一种转录后基因沉默方法,已被广泛的应用于基因功能的研究,基因网络调控的探讨以及疾病的治疗等方面。多数的siRNA表达载体依赖RNA聚合酶Ⅲ启动子中的一种,操纵一段短发夹结构RNA(Small hairpin RNA,shRNA)在细胞或体内表达。这一类启动子主要包括人源和鼠源的U6启动子和人H1启动子等。为了探明鱼类自身的RNA聚合酶Ⅲ启动子是否能有效驱动shRNA在鱼体内表达,从而更好地利用RNAi进行鱼类基因功能和抗病毒研究,研究利用斑马鱼的H1和U6启动子以及草鱼的H1启动子,以草鱼呼肠孤病毒(Grass carp reovirus,GCRV)外衣壳蛋白VP7基因为靶基因,以增强型绿色荧光蛋白(eGFP)为报告基因,分别构建了三个shRNA表达载体:pZH1siGCRV-CMVeGFP、pZU6siGCRV-CMVeGFP和pCH1siGCRV-CMVeGFP。通过显微注射将三种表达载体分别导入稀有鮈鲫(Gobiocypris rarus)受精卵中。由于siRNA片段很短,其表达检测非常困难,研究采用stem-loop RT-PCR方法,对稀有鮈鲫胚胎发育不同时期的shRNA表达进行了检测。研究结果表明,采用的三种鱼类自身的RNA聚合酶Ⅲ启动子均能有效驱动GCRVsiRNA的表达;在取样的各个胚胎发育时期均能检测到GCRV siRNA的表达;stem-loop RT-PCR方法可以便捷检测siRNA的表达。研究构建的鱼类胚胎siRNA有效持续表达载体,建立的简易快捷siRNA检测方法,为进一步的抗GCRV转基因鱼研制以及siRNA的病毒复制干扰机制研究奠定了重要基础并提供有力的技术支撑。
RNA interference, mediated by small interfering RNA, is a fast developed post-transcriptional gene silencing method in resent years, which has been extensively used in the fields of gene function research, gene networks regulation and disease therapy, etc. Most siRNA expression vectors dependent on one of the RNA polymerase III promoters, operating small hairpin RNA expressing in cell or in vivo. These promoters mainly include human and mouse U6 promoters and human H1 promoter. In this study, in order to explore whether fish RNA polymerase III promoters could efficiently drive shRNA expression in vivo, and consequently make better use of RNAi to research gene function and virus resistance in fish, three shRNA expression vectorspZH1siGCRV-CMVeGFP, pZU6siGCRV-CMVeGFP and pCH1siGCRV-CMVeGFP were constructed by employing zebrafish H1/U6 promoters and grass carp H1 promoter respectively, using outer capsid protein VP7 gene of grass carp reovirus （GCRV） as target gene and eGFP as report gene. Then the three vectors were injected into rare minnow embryos respectively. Since siRNA was short and difficult to be detected, stem-loop RT-PCR technique has been introduced to detect shRNA expression in different development stages of rare minnow embryos. Experiment results have showed that all of the three fish RNA polymerase III promoters could efficiently drive shRNA expression. At the same time, GCRV siRNA can be detected in all of the sampling embryo development stages. And stem-loop RT-PCR technique was proved to be a simple and facile tool to detect siRNA. The construction of efficient and lasting siRNA expressing vector in fish embryo and establishment of easy and fast siRNA detection method have provided a solid foundation and forceful technique support for producing GCRV resistant transgenic fish and studying siRNA interfering mechanisms in virus replication.
出处
《水生生物学报》
CAS
CSCD
北大核心
2010年第5期1031-1036,共6页
Acta Hydrobiologica Sinica
基金
国家863项目(2007AA10Z186)资助
