›› 2018, Vol. 61 ›› Issue (9): 1010-1018.doi: 10.16380/j.kcxb.2018.09.002

• 研究论文 • 上一篇    下一篇

柑橘大实蝇滞育和非滞育蛹体内海藻糖含量的调控

 王佳, 樊欢, 熊克才, 刘映红*   

  1. (西南大学植物保护学院, 重庆400716)
  • 出版日期:2018-09-20 发布日期:2018-09-20

Regulation of the trehalose content in diapausing and non-diapausing pupae of the Chinese citrus fly, Bactrocera minax (Diptera: Tephritidae)

WANG Jia, FAN Huan, XIONG Ke-Cai, LIU Ying-Hong*   

  1. (College of Plant Protection, Southwest University, Chongqing 400716, China)
  • Online:2018-09-20 Published:2018-09-20

摘要: 【目的】通过比较柑橘大实蝇Bactrocera minax蛹滞育期与滞育前和滞育后以及滞育蛹与非滞育蛹体内海藻糖和葡萄糖含量的变化、海藻糖合成代谢途径中关键酶的活力变化以及关键酶基因的表达量变化,明确蛹滞育期间海藻糖合成代谢途径中关键酶对海藻糖含量的调控。【方法】利用分光光度法检测柑橘大实蝇滞育前(1日龄蛹)、滞育期(30, 60和90日龄蛹)以及滞育后(120和150日龄蛹)蛹体内海藻糖与葡萄糖含量的变化,以及海藻糖合成代谢途径中的海藻糖-6-磷酸合成酶(TPS)、海藻糖-6-磷酸磷酸酯酶(TPP)和海藻糖酶(Tre)活力的变化;利用实时定量荧光PCR(qPCR)检测TPS, TPPB, TPPC-1, TPPC-2和Tre-1基因表达量的变化。向1日龄蛹体内注射20-羟基蜕皮酮(20E)作为处理(以注射10%乙醇为对照),分别于注射后1和30 d比较处理组与对照组蛹体内海藻糖与葡萄糖含量、关键酶活力以及上述基因表达量的差异。【结果】柑橘大实蝇蛹进入滞育后,海藻糖含量显著升高,葡萄糖含量无显著变化;TPS和TPP的酶活力以及TPS, TPPC-1和TPPC-2表达量在化蛹后逐渐升高,于滞育期达到最高水平,维持至羽化前显著下降;TPPB表达量在整个蛹期无显著差异;Tre酶活力以及Tre-1表达量在化蛹后逐渐升高,于滞育早期达到最高水平,随后显著下降,羽化前再次显著上升。注射20E后1 d,与对照组相比,处理组蛹体内海藻糖与葡萄糖含量、关键酶(TPS, TPP和Tre)活力以及TPS, TPPC-2和Tre-1表达量无显著变化,TPPB表达量显著下降,TPPC-1表达量显著上升;注射后30 d,与对照组滞育蛹相比,处理组非滞育蛹海藻糖含量显著上升,葡萄糖含量、TPS和Tre酶活力、TPSTre-1表达量显著下降,TPP酶活力以及TPPBTPPC-2表达量无显著差异。【结论】柑橘大实蝇蛹体内海藻糖的含量在合成代谢途径中关键酶的调控下,随着滞育状态发生变化,表明海藻糖与滞育之间存在密切的关系,但其作用机理仍待进一步研究。

关键词: 柑橘大实蝇, 滞育, 海藻糖, 海藻糖-6-磷酸合成酶, 海藻糖-6-磷酸磷酸酯酶, 海藻糖酶, 20-羟基蜕皮酮

Abstract: 【Aim】 This study aims to compare the contents of trehalose and glucose, the activities of key enzymes in trehalose biosynthesis and catabolism pathway, and the expression levels of genes encoding these key enzymes among pupae at pre-diapause, diapause, and post-diapause stages, and between diapausing and non-diapausing pupae of the Chinese citrus fly, Bactrocera minax. 【Methods】 The contents of trehalose and glucose, and the activities of trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), and trehalase (Tre) in B. minax pupae in pupae at the pre-diapause stage (1 d-old pupae), diapause stage (30, 60 and 90 d-old pupae), and postdiapause stage (120 and 150 d-old pupae) were compared using spectrophotometry, and the expression levels of TPS, TPPB, TPPC-1, TPPC-2, and Tre-1 were determined using quantitative real-time PCR (qPCR). In addition, the neonate pupae were injected with 20-hydroxyecdysone (20E) as the treatment (injected with 10% ethanol as the control), and the contents of trehalose and glucose, the activities of key enzymes and the expression levels of the above genes at 1 and 30 d after injection were compared between the treatment and control groups. 【Results】 When B. minax pupae entered diapause, their trehalose content significantly increased, while their glucose content changed marginally as compared with those of pre- and post-diapause stages. The TPS and TPP activities and the expression levels of TPS, TPPC-1 and TPPC-2 increased gradually after pupation and reached the highest level during the diapause stage, then significantly decreased before adult emergence. The expression level of TPPB did not change significantly throughout the pupal stage. The Tre activity and the expression level of Tre-1 gradually increased after pupation and reached the highest level at the early diapause stage, then significantly decreased, followed by a significant increase before adult emergence. At 1 d after 20E injection, in pupae of the treatment group the contents of trehalose and glucose, the activities of TPS, TPP and Tre, and the expression levels of TPS, TPPC-2 and Tre-1 only changed slightly, while the expression level of TPPB significantly decreased and that of TPPC-1 significantly increased as compared to the control group. At 30 d after injection, in non-diapausing pupae of the treatment group the trehalose content increased significantly, the glucose content, the TPS and Tre activities, and the expression levels of TPS and Tre1 significantly decreased, while the TPP activity, and the expression levels of TPPB and TPPC-2 did not change significantly as compared to those in diapausing pupae of the control group. 【Conclusion】 The trehalose content in B. minax pupae varies with diapause status under the regulation of key enzymes in trehalose biosynthesis and catabolism pathway, displaying the close relationship between trehalose and diapause. The underlying mechanisms, however, need further investigation.

Key words: Bactrocera minax; diapause, trehalose, trehalose-6-phosphate synthase, trehalose-6-phosphate phosphatase, trehalase, 20-hydroxyecdysone