星豹蛛四个羧酸酯酶基因克隆及溴氰菊酯胁迫下的表达模式

Cloning of four carboxylesterase genes in Pardosa astrigera(Araneae:Lycosidae)and their expression profiles under deltamethrin stress

【目的】探究星豹蛛Pardosa astrigera羧酸酯酶基因PaCarE1-4是否与其代谢溴氰菊酯有关。【方法】采用RT-PCR技术克隆星豹蛛4个羧酸酯酶基因PaCarE1-4 cDNA序列,通过生物信息学软件分析其序列特性。采用RT-qPCR技术测定这4个羧酸酯酶基因在星豹蛛雌雄成蛛不同组织(头胸部、腹部和足)以及在不同浓度(LC_(10)=5.151 mg/L;LC_(30)=8.619 mg/L;LC_(50)=12.311 mg/L)溴氰菊酯胁迫12 h和LC_(30)浓度溴氰菊酯胁迫2,4,8,12,24和48 h雄成蛛中的相对表达水平。【结果】克隆获得星豹蛛羧酸酯酶基因PaCarE1-4(GenBank登录号分别为MZ643212,MZ643214,MZ643215和MZ643216)的全长cDNA序列,开放阅读框(ORF)分别长1653,1803,1827和1818 bp,分别编码550,600,608和605个氨基酸。组织表达谱结果表明,PaCarE1和PaCarE2在星豹蛛雌雄成蛛腹部中的表达量最高,且在雄成蛛腹部中的表达量高于雌成蛛中的;PaCarE3和PaCarE4在雌雄成蛛头胸部中的表达量最高,且PaCarE3在雌成蛛头胸部中的表达量高于雄成蛛中的,PaCarE4在雄成蛛头胸部中的表达量高于雌成蛛中的。LC_(30)浓度溴氰菊酯胁迫12 h诱导了星豹蛛雄成蛛中PaCarE1的表达,LC_(10)和LC_(30)浓度溴氰菊酯胁迫12 h诱导了PaCarE2的表达。LC_(30)浓度溴氰菊酯胁迫不同时间后,与对照组(丙酮处理组)相比,星豹蛛雄成蛛中PaCarE4的表达量与对照组均无显著差异,而PaCarE1的表达量在处理后2,8和12 h,PaCarE2的表达量在处理后12 h,以及PaCarE3的表达量在处理后24 h显著上调。【结论】羧酸酯酶基因PaCarE1,PaCarE2和PaCarE3可以被溴氰菊酯诱导表达,表明其可能参与星豹蛛对溴氰菊酯的代谢过程。本研究结果有助于了解星豹蛛对外源物质的代谢机理,为这一捕食性天敌的保护提供了新思路。

【Aim】To investigate whether carboxylesterase genes PaCarE1-4 of Pardosa astrigera are related to the metabolism of deltamethrin.【Methods】The cDNA sequences of four carboxylesterase genes PaCarE1-4 of P.astrigera were cloned by RT-PCR,and their sequence characteristics were analyzed by bioinformatics software.The relative expression levels of the four carboxylesterase genes in different tissues(cephalothorax,abdomen,and leg)of female and male adults of P.astrigera and in male adults of P.astrigera after exposed to different concentrations(LC_(10)=5.151 mg/L;LC_(30)=8.619 mg/L;and LC_(50)=12.311 mg/L)of deltamethrin for 12 h and LC_(30) of deltamethrin for 2,4,8,12,24,and 48 h were assayed by RT-qPCR.【Results】The full-length cDNA sequences of carboxylesterase genes PaCarE1-4(GenBank accession numbers:MZ643212,MZ643214,MZ643215 and MZ643216,respectively)were cloned.Their open reading frames(ORFs)are 1653,1803,1827 and 1818 bp,encoding 550,600,608 and 605 amino acids,respectively.Tissue expression profiles revealed that PaCarE1 and PaCarE2 were highly expressed in the abdomen of female and male adults of P.astrigera,with higher expression level in males than in females,while PaCarE3 and PaCarE4 were highly expressed in the cephalothorax of female and male adults of P.astrigera,with PaCarE3 showing higher expression level in females than in males and PaCarE4 showing higher expression level in males than in females.After treatment for 12 h,LC_(30) of deltamethrin induced the expression of PaCarE1 in male adults of P.astrigera,while LC_(10) and LC_(30) of deltamethrin induced the expression of PaCarE2.In male adults of P.astrigera exposed to LC_(30) of deltamethrin,the relative expression levels of PaCarE4 at various time points after treatment showed no significant difference from that in the control group(acetone treatment group),while the relative expression levels of PaCarE1 at 2,8 and 12 h after treatment,PaCarE2 at 12 h after treatment and PaCarE3 at 24 h after treatment were significantly upregulated as compared to that in the control group.【Conclusion】The carboxylesterase genes PaCarE1,PaCarE2 and PaCarE3 can be induced by deltamethrin,indicating that they may participate in the metabolism of deltamethrin in P.astrigera.The results of this study are helpful for understanding the metabolism mechanism of foreign substances in P.astrigera,providing new ideas for the protection of this predatory enemy.