Molecular cloning of heat shock protein gene HSP90 and effects of abamectin and double-stranded RNA on its expression in Panonychus citri (Trombidiformes: Tetranychidae)
Keywords:
citrus red mite, abamectin, HSP90, double-stranded RNA, resistance mechanismAbstract
Panonychus citri McGregor (Trombidiformes: Tetranychidae), the widely distributed citrus red mite, has developed resistance to most registered acaricides. Adaptation of arthropods to extreme environmental conditions has been related to increased expression of their heat shock proteins (HSPs). The objectives of this study were to explore the relationship between HSPs and resistance of Panonychus citri to the acaricide abamectin and the adaptation of Panonychus citri to high temperatures. The full-length cDNA of the HSP90 gene was cloned from an abamectin-sensitive strain of Panonychus citri. This gene consisted of 2,495 nucleotides with a complete open reading frame (ORF) of 2,169 nucleotides. This gene encoded a polypeptide of 721 amino acids with a predicted molecular weight of 83.44 kDa, a theoretical isoelectric point of 5.06, a 3' untranslated region (UTR) of 228 bp, and a 5' UTR of 98 bp. The results of real-time PCR analyses indicated that the expression of the HSP90 gene in P. citri was markedly affected by the concentration of abamectin, the duration of exposure to it and the temperature, suggesting that the up-regulation of the HSP90 gene may play an important role in abamectin resistance and adaptation to high temperatures in Panonychus citri. The results of RNA interference experiments indicated that the HSP90 gene from adult female Panonychus citri was sensitive to down-regulation by double-stranded RNA (0.1–0.2 μg/μL). This study provides a molecular basis for further analysis of the relationships between the HSP90 gene and the resistance of Panonychus citri to abamectin and to high temperatures.
Resumen
Panonychus citri McGregor (Trombidiformes: Tetranychidae), el ácaro rojo de los cítricos ampliamente distribuido, ha desarrollado resistencia a la mayoria de los acaricidas registrados. La adaptación de los artrópodos a condiciones ambientales extremas se ha relacionado con el aumento de expresión de sus proteínas de choque térmico (HSPs, en inglés). El objetivo de este estudio fue explorar la relación entre el HSP y la resistencia de Panonychus citri al acaricida, abamectina, y la adaptación de Panonychus citri a altas temperaturas. El ADNc de longitud completa del gen HSP90 fue clonado a partir de una cepa de Panonychus citri sensible de abamectina. Este gen consistió en 2,495 nucleótidos con un marco de lectura abierto completo (ORF) de 2169 nucleótidos. Este gen codifica un polipéptido de 721 aminoácidos con un peso molecular predicho de 83.44 kDa, un punto isoeléctrico teórico de 5.06, una región 3' no traducida (UTR) de 228 pb y una 5' UTR de 98 pb. Los resultados del análisis de PCR en tiempo real indicaron que la expresión del gen HSP90 en P. citri fue marcadamente afectada por la concentración de abamectina, la duración de la exposición a la misma y la temperatura, lo que sugiere que la sobre regulación del gen HSP90 puede jugar un papel importante en la resistencia a la abamectina y a la adaptación a las altas temperaturas en P. citri. Los resultados de los experimentos de interferencia de ARN indicaron que el gen HSP90 de la hembra adulta de Panonychus citri fue sensible a la baja regulación de ARN de doble cadena (0.1-0.2 g/l). Este estudio provee una base molecular para el análisis adicional de las relaciones entre el gen HSP90 y la resistencia de Panonychus citri a la abamectina y a altas temperaturas.
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