关键词: 麦红吸浆虫, 滞育, 细胞色素c氧化酶, 酶活力, 基因表达

Abstract:  【Aim】 This study aims to explore the relationship between the changes in cytochrome c oxidase (COX) activity and gene expression of COX subunits SmCOX1 and SmCOX2 in Sitodiplosis mosellana and diapause development, so as to provide a theoretical basis for elucidating the energy metabolism mechanism of diapause of S. mosellana. 【Methods】 The SmCOX activities in S. mosellana larvae at different stages in the natural diapause process including pre-diapause, diapause, post-diapause quiescence and post-diapause development were measured using a COX assay kit. The full-length cDNA sequences of SmCOX1 and SmCOX2 were cloned using RT-PCR and RACE techniques, and analyzed by bioinformatics. qPCR technique was used to detect the expression pattern of SmCOX1 and SmCOX2 in the diapause process of S. mosellana. 【Results】 The SmCOX activity in larvae of S. mosellana in the diapause stage significantly decreased as compared to that in larvae in the pre-diapause stage, remained low level in larvae throughout the diapause stage, significantly increased in larvae in the post-diapause quiescent stage, and increased again in larvae in the post-diapause developmental stage which was significantly higher than those in larvae in the diapause and post-diapause quiescent stages. The open reading frames of SmCOX1 and SmCOX2 (GenBank accession number: PP466915 and PP466916, respectively) obtained were 1 536 and 660 bp in length, respectively, and contained more than 75% A+T base. SmCOX1 and SmCOX2 encoded the proteins of 511 and 220 amino acids with the predicted molecular weight of 57.70 and 25.76 kD, respectively. The amino acid sequence analysis indicated that SmCOX1 and SmCOX2 contained the classical REDOX copper coenzyme factors (CuA and CuB) and heme-containing metal centers heme a and heme a3 of mitochondria COX catalytic core. SmCOX1 and SmCOX2 displayed the highest amino acid sequence identity and the closest relationship to corresponding homologues from Orseolia oryzae (Cecidomyiidae). qPCR result showed that the expression levels of SmCOX1 and SmCOX2 exhibited significant difference in the diapause process of S. mosellana. The expression levels of SmCOX1 and SmCOX2 in larvae in the pre-diapause and post-diapause developmental stages were the highest, followed by those in larvae in the post-diapause quiescence stage, and those in larvae in the diapause stage were the lowest, showing the change trend basically consistent with the activity change of SmCOX. 【Conclusion】 The decrease of SmCOX activity and SmCOX1/2 expression during the diapause of S. mosellana were closely related to low oxygen consumption in diapause stage, while their increase was closely related to more energy demand in the diapause termination and post-diapause developmental stages.

Key words: Sitodiplosis mosellana, diapause, cytochrome c oxidase, enzyme activity, gene expression