鸡皮刺螨羧酸酯酶2基因克隆、序列分析及其表达特征研究  

Cloning,Sequence Analysis and Expression Characteristics of Carboxylesterase 2 Gene in Dermanyssus gallinae

作　　者：张学迪 徐楷 尹硕 刘晶[1] 王仲浩 秦建华[1] 吴鹏远 王传文 ZHANG Xuedi;XU Kai;YIN Shuo;LIU Jing;WANG Zhonghao;QIN Jianhua;WU Pengyuan;WANG Chuanwen(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Shijiazhuang Zhenghong Biotechnology Co.,Ltd.,Shijiazhuang 050000,China)

机构地区：[1]河北农业大学动物医学院,保定071001 [2]石家庄正鸿生物科技有限公司,石家庄050000

出　　处：《中国畜牧兽医》2024年第7期2973-2983,共11页China Animal Husbandry & Veterinary Medicine

基　　金：河北省自然科学基金(C2021204022);河北省教育厅青年基金(QN2021078);国家自然科学基金(32202830);河北省省属高等学校基本科研业务费研究项目(KY2022043)。

摘　　要：【目的】克隆鸡皮刺螨(Dermanyssus gallinae)羧酸酯酶2(CarE2)基因CDS区序列并进行生物信息学分析,同时检测其在鸡皮刺螨中的表达特征,以探究CarE2基因在鸡皮刺螨代谢抗性形成及发展中的作用。【方法】试验选择50只成年鸡皮刺螨雌螨,利用PCR扩增CarE2基因CDS区序列并测序,对其编码氨基酸序列进行多序列比对及系统进化树构建,通过生物信息学在线软件预测CarE2蛋白的理化性质及结构功能,并采用实时荧光定量PCR检测CarE2基因在鸡皮刺螨不同发育阶段及不同抗性品系中的表达模式,同时分析高效氯氰菊酯对鸡皮刺螨敏感品系和高效氯氰菊酯抗性品系中CarE2基因的诱导效应。【结果】鸡皮刺螨CarE2基因CDS区序列全长1665 bp,编码554个氨基酸。多序列比对结果显示,CarE2基因氨基酸序列具有高度保守的催化三联体(Ser-Glu-His)和五肽基序(Gly-X-Ser-X-Gly)。系统进化树结果表明,鸡皮刺螨CarE2与黑腹果蝇亲缘关系相近,共同划分为表皮酯酶分支。生物信息学分析结果显示,CarE2蛋白属于稳定的亲水蛋白,存在信号肽切割位点,且具有糖基化位点和多个磷酸化修饰位点,但不具有跨膜结构;CarE2蛋白二级结构以无规则卷曲为主,三级结构预测结果与二级结构一致。实时荧光定量PCR检测结果显示,CarE2基因在鸡皮刺螨若螨时期表达量最高,显著高于其他时期(P<0.05),成螨期表达量次之,在卵和幼螨时期的表达量相对较低;鸡皮刺螨高效氯氰菊酯抗性品系中CarE2基因的相对表达量是敏感品系的2.78倍;经高效氯氰菊酯胁迫处理后,鸡皮刺螨敏感品系中CarE2基因表达量与对照组相比差异不显著(P>0.05),而高效氯氰菊酯抗性品系中CarE2基因表达量与对照组相比显著升高(P<0.05)。【结论】本研究成功克隆鸡皮刺螨CarE2基因CDS区序列,其在鸡皮刺螨各发育阶段均有表达,且在高效氯氰菊酯抗性品系中过量表达�【Objective】The aim of this study was to clone the CDS region sequence of the carboxylesterase 2(CarE2)gene in Dermanyssus gallinae and conduct bioinformatic analysis,as well as detect its expression characteristics in Dermanyssus gallinae,so as to explore the role of CarE 2 gene in the formation and development of metabolic resistance in Dermanyssus gallinae.【Method】Fifty adult female Dermanyssus gallinae were selected,and the CDS region of CarE 2 gene was amplified by PCR and sequenced.Multiple sequence alignment and phylogenetic tree construction were carried out for its coding amino acid sequence.The physical and chemical properties and structural functions of CarE2 protein were predicted by bioinformatics online software.Real-time quantitative PCR was used to detect the expression patterns of CarE 2 gene in different developmental stages and different resistant strains of Dermanyssus gallinae,and to analyze the induction effect of cypermethrin on CarE 2 gene in sensitive and resistant strains of Dermanyssus gallinae.【Result】The CDS region of CarE 2 gene in Dermanyssus gallinae was 1665 bp,encoding 554 amino acids.Multiple sequence alignment results showed that the amino acid sequence of CarE 2 gene had highly conserved catalytic triad(Ser-Glu-His)and pentapeptide motifs(Gly-X-Ser-X-Gly).Phylogenetic tree analysis showed that CarE2 in Dermanyssus gallinae was closely related to Drosophila melanogaster,and was divided into epidermal esterase branch.Bioinformatics analysis results showed that CarE2 protein was a stable hydrophilic protein with signal peptide cleavage site,glycosylation site and multiple phosphorylation modification sites,but it did not have a transmembrane structure.The secondary structure of CarE2 protein was dominated by random coil,and the predicted tertiary structure was consistent with the secondary structure.The results of Real-time quantitative PCR showed that the expression of CarE 2 gene was the highest in the nymph stage,which was significantly higher than that in other sta

关 键 词：鸡皮刺螨 CarE2基因 克隆 序列分析 表达特征 

分 类 号：S858.31[农业科学—临床兽医学]