扩展莫尼茨绦虫RNA结合蛋白基因的克隆及不同体节mRNA转录水平的分析

Molecular cloning of RNA binding protein gene and analysis of mRNA transcription in different segments of Moniezia expansa

为研究RNA结合蛋白在扩展莫尼茨绦虫生长发育中的功能,克隆了该基因的全长序列;同时以beta-Tubulin基因作为内参基因,采用SYBR Green real-time RT-PCR方法检测了该基因在扩展莫尼茨绦虫4个不同发育阶段,即头节、幼节、成节、孕节中的mRNA转录水平。结果显示,扩展莫尼茨绦虫RNA结合蛋白基因全长为1 905bp,编码562个氨基酸。进化分析表明,该基因与人类DAZ基因有一定的同源性;mRNA转录水平分析显示,目的基因和beta-Tubulin基因的Ct值与阳性质粒的浓度均呈良好的线性关系,相关系数均大于0.99。熔解曲线分析表明,产物为特异的单峰,具有较高的特异性。结果表明,此RNA结合蛋白在虫体各发育阶段中的表达丰度存在明显差异,在虫体成节的表达水平最高,其次为幼节、孕节和头节,初步推测该基因可能与人类DAZ基因有相似的功能,对扩展莫尼茨绦虫生殖细胞的发育具有一定的调控作用。

To explore the function of a RNA binding protein gene in the different development of Moniezia expansa ,the present study cloned the whole gene of the RNA binding protein gene and developed a SYBR Green real-time fluorescent quantitative PCR assay for detecting mRNA transcription using beta-Tubulin as an internal control. In result,the RNA binding protein contained 1 905 bp,which encoded 562 amino acids. Sequence analysis revealed that the gene had 45.75% similarity with human DAZ at the amino acids level. The mRNA transcription analysis demonstrated that a good linear relationships（〉0. 99） between the Ct value and the concentration of positive plasmid for each gene. The melting curve analysis showed the product was specific with a single peak. Real-time PCR results indicated that the expression abundance of the RNA binding protein gene was different in different segments. In conclusion,the expres- sion level of the gene was high in the mature segment, but low in the scolex. Based on these findings, we speculate that the function of the gene, like human DAZ may play a key role in the development of germ cells,which need a further research.