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【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。
【Objective】 Blackening reaction plays an important role in ossification and immune defense of insect epidermis. Phenoloxidase is a key enzyme in melanization. Laccase 2 (LAC2) is a kind of phenoloxidase, In insect metamorphosis and immune system plays an important role. This study aimed to investigate the expression pattern and hormone regulation of LAC2 in the epidermal ossification of Helicoverpa armigera. 【Method】 The complete cDNA sequence of lac2 was obtained from the 5th instar larvae of cotton bollworm by PCR and RACE. Fluorescent quantitative PCR, hormone treatment and RNA interference methods were used to analyze the differences of LAC2 expression patterns and hormone regulation. 【Result】 Sequence analysis showed that the full-length cDNA of lac2 was 3 221 bp in length with a coding sequence of 2 268 bp encoding 756 amino acid residues. Developmental timing analysis showed that lac2 expressed at different stages of larvae at a high level and reached the peak at 96 hours of the 5th instar larvae. Tissue expression analysis, lac2 gene expression in larvae epidermis and adult ovary and antennae higher. Hormone treatment experiments showed that methoprene inhibited the transcription of lac2 gene and 20-hydroxyecdysone promoted its expression. Further use of RNA interference ecd (ecdysone receptor) and USP (ultraspiracle isoform) gene found that the expression of ecdysone receptor was significantly inhibited after interference, and lac2 gene expression was also significantly inhibited, indicating that ecdysone regulatory lac2 gene Transcription. 【Conclusion】 These results provide the theoretical basis for further studying the different physiological functions of laccase in the ossification and immune defense of insect epidermis.