斯氏狸殖吸虫线粒体Cytb基因在不同虫期的转录水平分析

Transcription level of mitochondria gene Cyt b in different stages of Pagumogonimus skrjabini

目的研究斯氏狸殖吸虫线粒体Cytb(cytochrome B)基因在不同虫期的转录水平以更深入了解该吸虫的物质代谢途径,探索药物作用的药靶。方法分别提取斯氏狸殖吸虫5个虫期的总RNA,并反转录为cDNA。将目的基因质粒作为标准品制作标准曲线,以5个虫期的cDNA为模板,特异引物为实验组引物,卫氏并殖吸虫的18SrDNA基因引物作为内参引物做实时荧光定量PCR,分析斯氏狸殖吸虫线粒体Cytb基因分别在5个虫期的转录水平。结果标准曲线线性关系良好,回归系数γ=0.994。产物熔解曲线分析结果均显示为单一波峰。定量分析结果显示,斯氏狸殖吸虫线粒体Cytb转录主要在囊蚴期、30d幼虫期及60d童虫期,并且是逐步升高趋势,但成虫期转录较少,虫卵期没有转录。结论斯氏狸殖吸虫线粒体Cytb基因在不同虫期的转录水平有差别,在60d童虫期高转录,提示Cytb基因在幼虫的发育和移行中起着重要作用。

In this study, we aimed to analyze the transcription level of mitochondria gene Cytb in metacercariae, larva- 30d, larva-60d, adult, and egg of Pagurnogonirnus skrjabini. The mRNA of metacercariae, larva-30d, larva-60d, adult and egg of P. skrjabini were extracted with genomic extraction kit, and transcripted reversely into cDNA. With 18SrDNA of Par- agonimus westerrnani as an internal standard primer, Cytb genes were amplified by real-time PCR for establishing standard curves to evaluate the copy number of genes. Those quantitative analyses were reliable because the R value of standard curves was 0. 994 （〉0.98）, and the melting curve showed a single peak. There were increasing trend of transcription of Cytb gene at metacercariae stage, larva-30d stage, and larva-60d stage. There were less transcription of Cytb gene at adult stage and no transcription at egg stage. There were differences about the transcriptional level of mitochondria Cytb gene at different stages of Pagumogonirnus skrjabini, and peaked at larva-60d stage, suggesting that Cytb gene may play a role in the development and migrating of larva.