作 者：
何永彩;李增魁;陈炳华;张连生;李中余;何顺福;陈继勇;耿鹏程;胡晓宇;蒋硕;陈强;徐璐瑶;康明;李英
单 位：
果洛州动物疫病预防控制中心;西宁野生动物园;海东市乐都区芦花乡畜牧兽医站;青海省循化撒拉族自治县畜牧兽医站;玉树州动物疫病预防控制中心;青海大学农牧学院;海东市乐都区李家乡畜牧兽医站
关键词：
青海血蜱;Haemalin基因;克隆;生物信息学;转录水平
摘 要：
为构建青海血蜱Haemalin基因的cDNA全长序列,并分析该基因的生物信息学特征及其在青海血蜱不同发育阶段和雌蜱不同组织中的转录水平,本研究基于青海血蜱唾液腺转录组文库中检索到的一个功能注释为Haemalin的基因片段,采用cDNA末端快速扩增技术(RACE)引物设计软件设计Haemalin基因 3'RACE和5'RACE特异性引物,分别经RACE扩增青海血蜱Haemalin基因的3'端和5'端序列,测序后与其中间序列拼接获得该基因的cDNA全长序列.利用生物信息学软件分析该基因编码蛋白的信号肽、理化性质、跨膜结构域、结构域、蛋白质三级结构、B细胞抗原表位以及氨基酸序列比对,采用NCBI中的BLAST及MEGA 7.0软件分别分析Haemalin基因编码氨基酸序列的相似性及构建其系统进化树,采用qPCR检测Haemalin基因在不同发育阶段青海血蜱和雌蜱不同组织中的转录水平.结果显示,青海血蜱Haemalin基因cDNA全长583 bp,开放阅读框(ORF)423 bp,该基因编码蛋白的信号肽为N端的15个氨基酸,理论等电点4.71,属于亲水性蛋白,无跨膜结构域.该基因编码蛋白的三级结构分析结果显示,其存在两个典型的Kunitz/牛胰蛋白酶抑制剂(Kunitz/BPTI)结构域,两个结构域之间由单链连接,共包括3个α螺旋,2对反向平行的β折叠;在aa35~aa45、aa54~aa60、aa75~aa80、aa91~aa93、aa125~aa131等处可能存在潜在的B细胞抗原表位.相似性与进化树结果显示,青海血蜱Haemalin氨基酸序列与褐黄血蜱凝血酶抑制剂Haemalin氨基酸序列的相似最高达92%,并在进化树中二者处于同一进化分支.表明,青海血蜱Haemalin可能也为一种凝血酶抑制剂.qPCR结果显示,Haemalin基因在不同发育阶段青海血蜱中的转录水平为:卵>半饱血蜱>若蜱>饱血蜱>幼蜱>吸血96h的雌成蜱>游离雌成蜱,且其在青海血蜱雌蜱中肠中的转录水平极显著高于其他组织(P<0.0001).表明,Haemalin基因在青海血蜱成蜱吸血阶段及其在该蜱的血液消化中均发挥重要作用.本研究为进一步研究Haemalin基因的生物学功能及其在青海血蜱防控中的作用奠定基础.
译 名：
Cloning,bioinformatics and transcriptional analysis of Haemalin gene in Haemaphysalis qinghaiensis
关键词：
Haemaphysalis qinghaiensis%Haemalin gene%cloning%bioinformatics%transcription levels
摘 要：
To analyse the bioinformatics characteristics of the Haemalin gene of Haemaphysalis qinghaiensis and the transcriptional levels of the gene in different developmental stages and different tissues of female ticks,the full-length cDNA of the Haemalin gene was constructed.A gene fragment with a functional annotation of Haemalin was retrieved from the transcriptome library of the salivary gland of Haemaphysalis qinghaiensis.The 3'RACE and 5'RACE specific primers of Haemalin gene were designed using cDNA end rapid amplification(RACE)primer design software.The 3'and 5'end sequences of Haemalin gene amplified by RACE,respectively,and the full-length cDNA sequence of the gene was obtained by sequencing and splicing with its intermediate sequence.Bioinformatics software was used to analyze the signal peptide,physicochemical properties,transmembrane domains,structural domains,protein tertiary structure,B cell antigen epitope and amino acid sequence alignment of the protein coded by this gene.NCBI of BLAST and MEGA 7.0 software were respectively used to analyze the similarity of amino acid sequences encoded by the Haemalin gene and construct the phylogenetic tree of amino acid sequences encoded by the Haemalin gene,and fluorescence quantitative PCR(qPCR)was used to detect the transcription levels of the Haemalin gene at different developmental stages and different tissues of female ticks.The results showed that the cDNA of the Haemalin gene of Haemaphysalis qinghaiensis was 583bp in length with an open reading frame(ORF)of 423bp.The signal peptide of the protein encoded by the gene was 15 amino acids at the N-terminus.The theoretical isoelectric point of this protein was 4.71.It was a hydrophilic protein without a transmembrane domain.The tertiary structure of the protein showed the presence of two typical Kunitz/Bovine trypsin inhibitor(Kunitz/BPTI)domains,which were connected by a single chain and consisted of three alpha helices and two pairs of antiparallel beta folds.Potential B cell antigen epitopes may be located at aa35-aa45,aa54-aa60,aa75-aa80,aa91-aa93,aa125-aa131,etc.The similarity and phylogenetic tree results showed that the amino acid sequence of Haemalin from Haemaphysalis qinghaiensis was similar to that of Haemalin from Haemaphysalis flava,a thrombin inhibitor,by up to 92%,and they were in the same evolutionary branch in the phylogenetic tree,indicating that Haemalin from Haemaphysalis qinghaiensis may also be a thrombin inhibitor.The qPCR results showed that the transcription level of the Haemalin gene of Haemaphysalis qinghaiensis at different developmental stages was as follows:egg>half-engorged tick>nymph>engorged tick>larva>female adult ticks that feed blood for 96 hours>free female adult tick.The transcription level in the midgut of female Haemaphysalis qinghaiensis was significantly higher than that in other tissues(P<0.0001).This study indicated that the Haemalin gene played an important role in the blood-sucking stage of adult ticks in Haemaphysalis qinghaiensis,and it also played a crucial role in the blood digestion of Haemaphysalis qinghaiensis.This study laid the foundation for further investigating the biological function of the Haemalin gene and its role in the prevention and control of Haemaphysalis qinghaiensis.
