湖南省怀化地区微小扇头蜱溴氰菊酯抗药性及与钠离子通道结构域Ⅲ基因突变位点的关联研究

Resistance to deltamethrin and its association with mutation sites in the sodium iron channel domain Ⅲ gene in Rhipicephalus microplus in Huaihua City of Hunan Province

目的检测湖南省怀化地区微小扇头蜱对溴氰菊酯的抗药性及蜱虫钠离子通道基因碱基突变位点,并探究两者间的关联。方法2022年6—9月,从湖南省怀化地区黄牛养殖场采集微小扇头蜱,采用成虫浸渍法检测所采集的蜱虫对溴氰菊酯的抗药性。采用PCR技术扩增抗药及非抗药微小扇头蜱钠离子通道结构域Ⅲ基因,经测序、比对,检测序列中的碱基突变位点。翻译微小扇头蜱钠离子通结构域Ⅲ基因,检测其氨基酸序列信号肽、跨膜结构域、磷酸化与糖基化位点,推导抗药及非抗药微小扇头蜱钠离子通道结构域Ⅲ蛋白三级结构,并对其差异进行比对分析。结合抗药性检测、序列与蛋白三级结构比对结果,分析碱基突变位点与抗药性间的关联。结果所采集微小扇头蜱对溴氰菊酯的半数致死浓度(LC_(50))为121.39 mg/L、95%致死浓度为(LC_(95))为952.61 mg/L;其对溴氰菊酯的抗性系数(RF)为9.24,抗药程度(RL)为Ⅱ级抗药。所获得的微小扇头蜱钠离子通道结构域Ⅲ基因序列长度为1010 bp,在抗药性微小扇头蜱钠离子通道结构域Ⅲ基因序列中检测出2个相邻碱基突变位点。抗药及非抗药微小扇头蜱钠离子通道结构域Ⅲ蛋白虽均无信号肽,但存在6个跨膜结构域、42个磷酸化与8个糖基化位点,且在其三级结构中存在明显的结构差异。结论湖南省怀化地区微小扇头蜱对溴氰菊酯表现出Ⅱ级抗药,且在抗药蜱株钠离子通道结构域Ⅲ基因序列中检出2个与其抗药性产生存在一定关联的突变位点。

Objective To investigate the level of deltamethrin resistance and mutation sites in the sodium iron channel gene in Rhipicephalus microplus in Huaihua City,Hunan Province,and to examine the correlation between deltamethrin resistance and mutation sites in the sodium iron channel gene in Rh.microplus.Methods Rh.microplus was sampled from multiple yellow cattle farms in Huaihua City,Hunan Province from June to September 2022,and the level of resistance to deltamethrin was determined in ticks using the adult immersion test.The sodium iron channel domain Ⅲ gene was amplified in deltamethrin-resistant and wild-type Rh.microplus using PCR assay.Following sequencing and sequence alignment,mutation sites were detected in bases.The sodium iron channel domain Ⅲ gene in Rh.microplus was translated,and the signal peptide,transmembrane domain,and phosphorylation and glycosylation sites were detected in amino acid sequences.The tertiary structures of the sodium iron channel domain Ⅲ protein of deltamethrin-resistant and wild-type Rh.microplus were deduced and compared,and the association between mutation sites in bases and resistance to deltamethrin was examined in Rh.microplus according the level of deltamethrin resistance,sequence alignment and protein tertiary structure.Results The median(LC_(50))and 95%lethal concentrations(LC_(95))of deltamethrin were 121.39 mg/L and 952.61 mg/L against Rh.microplus,with a resistance factor of 9.24 and level Ⅱ resistance.The sequence of the sodium ion channel domain Ⅲ gene was 1010 bp in size,and mutation sites were detected in two neighboring bases in the sequence of the sodium ion channel domain Ⅲ gene in deltamethrin-resistant Rh.microplus.Although no signal peptides were found in the sodium iron channel domain Ⅲ protein of deltamethrin-resistant or wild-type Rh.microplus,6 transmembrane domains,42 phosphorylation sites and 8 glycosylation sites were identified,with a significant difference in the tertiary structure of the sodium iron channel domain Ⅲ protein between deltamethrin-resistant and wild-type Rh.microplus.Conclusions Level II resistance to deltamethrin is detected in Rh.microplus in Huaihua City,Hunan Province,and two mutation sites that correlate with the emergence of deltamethrin resistance are identified in the sequence of the sodium iron channel domain Ⅲ gene in deltamethrin-resistant Rh.microplus.