长角血蜱Flotillin-1基因的原核表达及生物信息学分析

Prokaryotic expression and bioinformatics analysis of Flotillin-1 gene fromHaemaphysalis longicornis

为了研究蜱源性Flotillin-1基因的结构、生物学特性及组织分布特征,以长角血蜱为研究对象,通过基因克隆、原核蛋白表达及生物信息学方法分析Flotillin-1蛋白的分子特性,同时结合RT-qPCR技术分析该蛋白在长角血蜱不同发育阶段和不同组织中的表达情况。结果显示,Flotillin-1基因的长度为1051 bp,约编码375个氨基酸残基,其蛋白质大小约41 ku,经超声破碎发现重组蛋白主要以包涵体的形式存在于沉淀中。生物信息学分析显示,该蛋白为亲水性蛋白,由α-螺旋(77.31%)、延伸链(9.80%)、β-转角(2.52%)和无规则卷曲(10.36%)4种结构组成,有44个磷酸化位点,其中丝氨酸位点19个、苏氨酸位点16个、酪氨酸位点9个,无信号肽区域和跨膜结构域。RT-qPCR分析结果显示,Flotillin-1在长角血蜱不同发育阶段中,在卵中表达丰度最高,在饱血若蜱体内表达丰度最低;在不同组织中,在卵巢中的表达丰度最高,在中肠中的表达丰度最低。本次研究结果不仅有助于了解该蛋白的基因结构和组织分布,还为后续研究该蛋白的功能提供了依据。

In order to study the structure,biological characteristics and tissue distribution of tick Flotillin-1 gene,the molecular characteristics of Flotillin-1 protein were analyzed by gene cloning,prokaryotic expression and bioinformatics methods,and at the same time,mRNA expression level of the Flotillin-1 gene in different developmental stages and tissue of Haemaphysalis longicornis were analyzed by RT-qPCR.The results showed that the length of Flotillin-1 gene was 1051 bp,encoding about 375 amino acids,and the protein size was about 41 ku.After ultrasonic fragmentation,it was found that the protein mainly existed in the form of inclusion.The biological information analysis showed that the protein was a hydrophilic protein,consisting ofα-helix(77.31%),extension chain(9.80%),β-turn(2.52%)and random coil(10.36%).There were 44 phosphorylation sites,including 19 serine sites,16 threonine sites,9 tyrosine sites,and no signal peptide region and transmembrane domain.RT-qPCR results showed that the expression abundance of Flotillin-1 at different developmental stages of H.longicornis was the highest in eggs but the lowest in nymphs,and the expression abundance in ovary was the highest but the expression abundance was lowest in the midgut in different developmental tissues.This study not only helped to understand the gene structure and tissue distribution of the protein,but also provided a basis for the subsequent study of the function of the protein.