Matsumoto Yukiko, Hattori Makoto
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
Ex. Insect-Plant Interaction Research Unit, National Institute Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
Arch Insect Biochem Physiol. 2019 Oct;102(2):e21602. doi: 10.1002/arch.21602. Epub 2019 Jul 22.
Multicopper oxidase (MCO) enzymes are present ubiquitously and act on diverse substrates. Recently, the presence of multiple MCO genes has been described in many insects. Based on sialotranscriptome data, we identified and comprehensively characterized six MCO genes: NcLac1S, 1G, and 2-5 in the green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae). NcLac1S and NcLac1G belong to the MCO1 ortholog of other insects. NcLac2 forms a clade with MCO2s involved in the sclerotization and pigmentation of the cuticle. NcLac3 and NcLac4 form a clade with NlMCO3 -5 of the hemipteran Nilaparvata luges. NcLac5 forms a clade with MCORPs (MCO-related proteins) that lack amino acid residues normally highly conserved in copper-coordinated MCOs. NcLac1S and NcLac3 were specifically expressed in the salivary glands; whereas NcLac5 was primarily expressed in the salivary glands. Only NcLac3 protein is considered to have laccase activity in the salivary glands and salivary sheaths ejected by the insect. NcLac1G expression was relatively high in the testis. NcLac2 and NcLac4 were specifically expressed in the integument and in Malpighian tubules, respectively. Knockdown by RNA interference (RNAi) of either NcLac2 and NcLac5 in nymphs caused high mortality. All NcLac2-knockdown nymphs showed depigmentation and soft cuticle, and eventually died, as did other MCO2-knockdown insects. DsNcLac5-injected nymphs (third, fourth, and fifth-instar) showed high mortality, but injection into adults had no effect on survival or number of eggs deposited, suggesting that NcLac5 is not essential for survival after molting (eclosion). NcLac5 could be a promising target gene for control of N. cincticeps.
多铜氧化酶（MCO）普遍存在并作用于多种底物。最近，在许多昆虫中都发现了多个MCO基因。基于唾液转录组数据，我们在褐飞虱（半翅目：叶蝉科）中鉴定并全面表征了六个MCO基因：NcLac1S、1G以及2 - 5。NcLac1S和NcLac1G属于其他昆虫的MCO1直系同源基因。NcLac2与参与表皮硬化和色素沉着的MCO2形成一个进化枝。NcLac3和NcLac4与半翅目褐飞虱的NlMCO3 - 5形成一个进化枝。NcLac5与MCO相关蛋白（MCORPs）形成一个进化枝，这些蛋白缺乏在铜配位MCO中通常高度保守的氨基酸残基。NcLac1S和NcLac3在唾液腺中特异性表达；而NcLac5主要在唾液腺中表达。仅NcLac3蛋白被认为在昆虫排出的唾液腺和唾液鞘中具有漆酶活性。NcLac1G在睾丸中的表达相对较高。NcLac2和NcLac4分别在体壁和马氏管中特异性表达。若虫中NcLac2和NcLac5的RNA干扰（RNAi）敲低导致高死亡率。所有敲低NcLac2的若虫都出现色素脱失和表皮变软，最终死亡，其他敲低MCO2的昆虫也是如此。注射dsNcLac5的若虫（三龄、四龄和五龄）死亡率很高，但注射到成虫体内对其存活或产卵数量没有影响，这表明NcLac5对于蜕皮（羽化）后的存活并非必需。NcLac5可能是控制褐飞虱的一个有前景的靶标基因。
