摘要
【目的】黑化反应在昆虫表皮骨化以及免疫防御过程中起着重要作用,酚氧化酶是黑化反应中的关键酶类,漆酶2(laccase2,LAC2)是酚氧化酶的一种,在昆虫变态发育和免疫系统中起着重要的作用。本研究旨在探讨LAC2在棉铃虫Helicoverpa armigera表皮骨化中表达模式及激素调控作用。【方法】采用PCR及RACE的方法,从棉铃虫5龄幼虫中得到了lac2 cDNA全序列。利用荧光定量PCR、激素处理及RNA干扰方法,对LAC2的表达模式差异和激素调控作用进行分析。【结果】序列分析表明,lac2 cDNA全长3 221 bp,编码框长度为2 268 bp,编码756个氨基酸残基。发育时序表达分析发现,lac2在幼虫各龄期表达规律相似,均在蜕皮期高水平表达,在5龄96 h转录水平达到最高峰。组织表达结果分析,lac2基因在幼虫表皮和成虫卵巢以及触角表达量较高。激素处理实验发现,保幼激素类似物(methoprene)对lac2基因转录有抑制作用;蜕皮激素(20-hydroxyecdysone)则促进其表达。进一步利用RNA干扰蜕皮激素受体EcR(ecdysone receptor)和USP(ultraspiracle isoform)基因发现,干扰后蜕皮激素受体的表达明显受到抑制,同时lac2基因的表达也显著受到抑制,表明蜕皮激素调控lac2基因转录。【结论】这些结果为进一步研究漆酶在昆虫表皮的骨化以及免疫防御等方面不同的生理功能提供理论依据。
[ Aim ] The melanization plays an important role in process of insect cuticle hardening and the immune defense, in which phenol oxidizing enzymes are the key enzymes. Laccase 2 （ LAC2）, as one of phenol oxidizing enzymes, plays an important role in metamorphosis and immune system of insects. The aim of this study was to explore its expression pattern and hormonal regulation during the cuticle hardening in the cotton bollworm, Helicoverpa armigera. [ Methods ] The full-length cDNA sequence of lac2 was amplified from the 5th instar larvae of the cotton bollworm by PCR and RACE methods. The expression patterns and hormonal regulation of lac2 were analyzed by using real-time quantitative PCR, hormone treatment and RNA interference methods. [ Results ] The sequence analysis showed that the full- length cDNA sequence of lac2 is 3 221 bp in length with an open reading frame of 2 268 bp encoding 756 amino acid residues. The developmental expression pattern showed that lac2 transcription was richly expressed in the molting stage of every instar and reached a peak in 96 h - 5th instar larvae. The spatial expression manifested that lac2 was richly expressed in the cuticle of larvae and in the ovary and antennae of adults. Further hormone treatment showed that juvenile hormone analogue （methoprene） inhibited lac2 transcription, while molting hormone （20-hydroxyecdysone） attenuated methoprene action and induced the expression of lac2. RNA interference of the molting hormone receptors EcR （ecdysone receptor） and USP （ ultraspiracle isoform） significantly inhibited the expression of ecr and usp, and injection of dsRNA of ecr and usp followed by injection of 20-hydroxyecdysone also inhibited the transcription of lac2. [ Conclusion ] Altogether, these results provide a foundation for further research of lac2 in insect cuticle hardening and immune defense.
出处
《昆虫学报》
CAS
CSCD
北大核心
2014年第2期150-160,共11页
Acta Entomologica Sinica
基金
国家自然科学基金项目(31172156)
关键词
棉铃虫
黑化
漆酶
免疫防御
RNA干扰
保幼激素类似物
蜕皮激素
Helicoverpa armigera
melanization
laccase
immune defense
RNA interference
juvenilehormone analogue
molting hormone
