细粒棘球绦虫锌指蛋白基因的克隆、序列分析及在不同发育阶段的表达分析

Cloning,Sequencing,and Expression Analysis at Different Development Stages of Zinc Finger Protein Gene of Echinococcus granulosus

旨在分析并预测细粒棘球绦虫锌指蛋白(Echinococcus granulosus zinc finger peotein,Eg-ZFP)结构、功能及其在细粒棘球绦虫发育过程中的表达模式,预测其在细粒棘球绦虫防治中潜在的作用。通过对前期细粒棘球绦虫原头蚴(protoscolex,PSC)转录组高通量测序结果中Unigene进行筛选,克隆Eg-ZFP基因并进行序列分析,实时荧光定量PCR(Realtime fluorescence quantitative PCR,qPCR)分析Eg-ZFP在原头蚴及成虫中的表达特征,利用全量组织原位杂交(Whole mount in situ hybridization,WISH)检测该蛋白mRNA在原头蚴和成虫组织中的分布情况。结果显示,细粒棘球绦虫Eg-ZFP基因的CDS序列长852bp,具有完整的开放阅读框(852bp),编码283个氨基酸,预测表明Eg-ZFP分子质量大小及等电点为31.6ku和8.89;qPCR检测发现,Eg-ZFP在原头蚴及成虫阶段均有表达,且在成虫阶段表达量高;全量组织原位杂交检测结果表明,Eg-ZFP mRNA在原头蚴及成虫中分布广泛,且在成虫的生殖系统中丰度较高,提示Eg-ZFP与虫体生殖发育有关。结果表明Eg-ZFP在细粒棘球绦虫发育过程中具有重要作用,为进一步研究Eg-ZFP在E.granulosus防治中的作用奠定基础。

The aim of this study is to analyze and predict the structure and function of the Echinococcus granulosus zinc finger protein(Eg-ZFP)and its expression pattern in the different development stages of Echinococcus granulosus(E.g),and predict its potential role in the prevention and control of E.g.Based on the results of the high-throughput sequencing of the protoscolex(PSC)transcriptome,Eg-ZFP gene was successfully cloned and to analyze its sequence characteristics by the bioinformatics software,quantitative real-time PCR(qPCR)was used to confirm the quantity of Eg-ZFP gene expression in different developmental stages of E.g,the distribution feature of Eg-ZFP mRNA was analyzed by whole mount in situ hybridization(WISH)in PSC and adult.The results suggested that the Eg-ZFP of E.g is at the length of852bp,containing an open reading frame of852bp which encoded283amino acids with a predicted molecular mass of31.6ku and PI of8.89.According to the analysis of qPCR,mRNA expression of Eg-ZFP was detected at PSC and adult,the Eg-ZFP mRNA expression levels was higher at adult.WISH detection shows that the Eg-ZFP mRNA is widely distributed in the PSC and adults and have higher abundance in the adult reproductive system,suggesting that Eg-ZFP may be related to the larval reproductive development.These indicated that Eg-ZFP could play an important role in the development of E.g.This will lay a foundation for further research on the role of Eg-ZFP in the prevention and treatment of E.g.