禾谷缢管蚜阳离子通道基因RSC1的克隆、分子特性及诱导表达分析

Molecular cloning,characterization and induced expression analysis of ion channel gene RSC1 in Rhopalosiphum padi( Hemiptera: Aphididae)

【目的】SC1通道(sodium channel 1)是昆虫体内一种重要的离子通道,被认为是一种开发新型杀虫剂的神经靶标。本研究拟克隆禾谷缢管蚜Rhopalosiphum padi的SC1通道基因,并初步分析其生理功能及其与SC1类通道、电压门控钠离子通道、电压门控钙离子通道的进化关系。【方法】采用RT-PCR技术,克隆了禾谷缢管蚜SC1基因完整的开放阅读框;利用实时荧光定量PCR技术,分析禾谷缢管蚜成蚜在不同浓度的高效氯氟氰菊酯诱导下SC1基因表达变化。【结果】获得了禾谷缢管蚜SC1基因(命名为RSC1)完整的开放阅读框(Gen Bank登录号为KU640190),其长度6 687bp,编码2 228个氨基酸。RSC1具有SC1通道的结构特征,有一个不同于电压门控钠离子通道和电压门控钙离子通道的特殊DEEA模体(motif)。系统进化分析结果显示,RSC1与电压门控钠离子通道组成一个进化枝,电压门控钙离子通道组成另外一个进化枝,SC1与电压门控钠离子通道在进化上有更近的起源关系。实时荧光定量PCR分析结果表明,LC15,LC35和LC503种剂量的高效氯氟氰菊酯处理6 h后,禾谷缢管蚜RSC1基因表达量相对于清水对照显著下调,表达量分别为对照的0.57,0.82和0.78倍;3种剂量的高效氯氟氰菊酯处理24 h后,禾谷缢管蚜RSC1基因表达量分别为对照的2.19,1.33和1.19倍,其中LC15(0.1484 mg/L)胁迫下RSC1基因的表达量显著上调。【结论】SC1类通道与电压门控钠离子通道在进化起源上有更近的关系。RSC1通道可能是高效氯氟氰菊酯的次级靶标。由于RSC1和其同源基因只存在于节肢动物中,脊椎动物尚未发现该类基因,因此这类通道可能作为开发新型杀虫剂的神经靶标。

【Aim】SC1（ sodium channel 1） is an important ion channel in arthropods including insects.It is considered as a possible new neuro target. This study aims to investigate the SC1 gene from Rhopalosiphum padi,and to analyze the primary biological functions of the gene and the evolutionary relationship of SC1 channels with voltage-gated sodium channel and voltage-gated calcium channel.【Methods】RT-PCR technique was used to clone the full-length ORF of RC1 gene from R. padi. After R. padi adults were exposed to different concentrations of λ-cyhalothrin for 6 and 24 h,respectively,the expression levels of SC1 were analyzed by real-time quantitative PCR. 【Results】The ORF of RSC1（ Gen Bank accession no： KU640190） is 6 687 bp in length with the deduced amino acid sequence of2 228 residues. Structural analysis showed that the RSC1 has the typical structural feature of SC1 channel,and contains a unique DEEA motif,which is different from the motif of sodium channel and calcium channel. Phylogenetic analysis indicated that RSC1 and its orthologs from other animals consist of a sub-group,which is more similar to the sodium channel family. In R. padi adults exposed to λ-cyhalothrin for 6 h,the expression level of RSC1 gene significantly decreased to 0. 57-,0. 82- and 0. 78-fold that of the control,respectively,while increased to 2. 19-,1. 33- and 1. 19-fold that of the control,respectively,after 24 h insecticide exposure. The expression level of RSC1 significantly increased in R.padi adults exposed to λ-cyhalothrin at the dosage of LC15（ 0. 1484 mg /L）. 【Conclusion】The SC1 channel family are evolutionarily closer to the voltage-gated sodium channel family. RSC1 is the possible secondary target of λ-cyhalothrin. Since RSC1 gene and its orthologs have been found in invertebrates but not in vertebrates,these channels can be used as neurotarget to develop novel insecticides.