昆虫激素对家蚕血淋巴和体壁酚氧化酶活性及其基因表达水平的影响

杨伟克 唐芬芬 刘增虎 董占鹏

摘要：【目的】研究外源昆蟲激素对家蚕酚氧化酶活性及其基因表达水平的影响，明确昆虫激素对酚氧化酶的调控作用，为深入解析家蚕酚氧化酶的功能及基因表达调控机制提供参考依据。【方法】对五龄第3 d发育良好的家蚕幼虫注射蜕皮激素20E和保幼激素JH，以注射等量0.1%二甲基亚砜（DMSO）为对照，分别在注射3、6、12和24 h后解剖家蚕幼虫采集血淋巴和体壁，检测分析家蚕酚氧化酶活性及其基因表达的变化情况。【结果】注射外源JH和20E均能促进家蚕血淋巴酚氧化酶原基因PPO1和PPO2的表达，有效增加酚氧化酶活性。家蚕血淋巴PPO1和PPO2基因对JH和20E的响应时间存在一定差异，其中，经JH处理后2个酚氧化酶原基因（PPO1和PPO2）仅在处理6和12 h时显著（P<0.05）或极显著（P<0.01）上调表达;而经20E处理后PPO1和PPO2基因从3 h开始一直持续到12 h均呈上调表达趋势。JH和20E对家蚕体壁PPO1和PPO2基因的影响恰好相反，具体表现为：JH能有效抑制家蚕体壁PPO1和PPO2基因的表达，降低酚氧化酶活性;20E则促进家蚕体壁PPO1基因上调表达，但不影响PPO2基因表达。【结论】家蚕酚氧化酶原基因PPO1和PPO2表达受JH和20E的调控，且激素对酚氧化酶原基因表达水平的影响与其酶活性变化规律一致，提示酚氧化酶不仅参与昆虫激素的代谢，还与激素协同调控家蚕的蜕皮变态。

关键词： 家蚕;昆虫激素;酚氧化酶;血淋巴;体壁;蜕皮变态

中图分类号： S883.89                          文献标志码： A 文章编号：2095-1191（2020）10-2573-07

Effects of insect hormone on activity of phenoloxidase and its gene expression inhemolymph and integument of Bombyx mori

YANG Wei-ke1， TANG Fen-fen2， LIU Zeng-hu1， DONG Zhan-peng1*

（1Sericulture and Apiculture Research Institute， Yunnan Academy of Agricultural Sciences， Mengzi， Yunnan  661101， China; 2College of Life Science and Technology， Honghe University， Mengzi， Yunnan  661101， China）

Abstract：【Objective】To clarify the regulation effect of insect hormones on phenoloxidase activity and gene expression of Bombyx mori， and study regulation effect of insect hormones on phenoloxidase， which could provide a theoretical reference for further research on the function of phenoloxidase and gene expression regulation mechanism. 【Method】Ecdysone 20E and juvenile hormone JH were injected into the silkworm at day 3 of 5th instar larva and the same amount of 0.1% dimethyl sulfoxide （DMSO） was injected as a control group. The phenoloxidase activity and gene expression level were determined in hemolymph and integument of silkworm at 3 h， 6 h， 12 h and 24 h after injection. 【Result】The results showed that the expression of prophenoloxidase genes PPO1 and PPO2 in hemolymph of silkworm were up regulated by 20E and JH， and the phenoloxidase activity was also higher than the control group. There was difference in response times of PPO1 and PPO2 in hemolymph of silkworm to 20E and JH. The expression of PPO1 and PPO2 were only significantly up-regulated at 6 h（P<0.05） and extremely up-regulated at 12 h（P<0.01） after JH treatment. While the expression of PPO1 and PPO2 were up-regulated from 3 h to 12 h after 20E treatment. The effects were opposite of JH and 20E on the expression of PPO1 and PPO2 in integument of silkworm. The expression of PPO1 and PPO2 were inhibited by JH and the phenoloxidase activity was decreased at the same time. 20E could induce the expression of PPO1，but did not affect the expression of PPO2 in integument of silkworm. 【Conclusion】The PPO1 and PPO2 expression in silkworm are regula-ted by JH and 20E. In addition， the effects of JH and 20E on the gene expression are consistent with the change rule of phenoloxidase activities. It is suggested that the phenoloxidase are involved in the metabolism of insect hormones and coordinately regulates the molting metamorphosis with JH and 20E in silkworm.

Key words： Bombyx mori; insect hormone; phenoloxidase; hemolymph; integument; molting metamorphosis

Foundation item： Modern Agricultural Sericulture Industrial Technology System Construction（Chief Scientist） Project of Yunnan（2019K-JTX006）; Applied Basic Research Project of Yunnan Academy of Agricultural Sciences（YJM 201710）

0 引言

【研究意義】酚氧化酶（Phenoloxidase，PO）是一类能将酚类氧化成邻苯醌或对苯醌的酶，广泛分布于动植物界，在生物体内发挥着极其重要的生理功能作用（陈晓宇等，2015;苑胜垒等，2016）。昆虫酚氧化酶通常以非活化状态的前体酚氧化酶原存在，主要分布在血淋巴和体壁等组织中（Yang et al.，2018）。酚氧化酶原通过酚氧化酶原激活系统转变为具有活性的酚氧化酶，参与调控昆虫的一系列生理过程，包括免疫防御、伤口愈合、表皮的鞣化和黑化等（Jearaphunt et al.，2014;Wu et al.，2017）。保幼激素和蜕皮激素是调控家蚕（Bombyx mori）生长发育与蜕皮变态的主要激素，研究家蚕酚氧化酶活性及其基因表达在外源激素处理后的变化规律，可为阐明激素对酚氧化酶基因的分子调控机制提供理论依据。……