CSIRO Molecular and Health Technologies, and CSIRO Food and Nutritional Sciences, Sydney Laboratory, PO Box 184, North Ryde, NSW 1670, Australia.
Insect Biochem Mol Biol. 2011 Feb;41(2):77-89. doi: 10.1016/j.ibmb.2010.10.002. Epub 2010 Oct 28.
It has been suggested that Pentatomomorpha utilise the C(28) ecdysteroid, makisterone A (MakA), as the major moulting hormone rather than the more common C(27) hormone, 20-hydroxyecdsyone (20E). The present study is the first to examine this postulate at the level of the ecdysone receptor protein, a heterodimer of nuclear receptors EcR and USP. cDNAs encoding two alternatively spliced isoforms of EcR and a single USP were isolated from a high-quality cDNA library prepared from a representative pentatomomorphan, Nezara viridula (Nv). NvEcR and NvUSP were found to group phylogenetically with heteropteran and other insect EcRs and USP/RXRs, respectively. Sequence comparison and phylogenetic analysis of these proteins found them to be distinct from those belonging to other hemipteran ecdysone receptors characterised to date. Co-expression of the His(6)-tagged ligand binding regions (LBRs) of the two NvEcR variants with the FLAG-tagged LBR of NvUSP was achieved in insect cells employing appropriately constructed baculoviruses. The corresponding heterodimers, designated NvE10 and NvE11, were purified by affinity chromatography utilising the His(6) tags on their NvEcR subunits. The heterodimers displayed nanomolar affinity for [(3)H]ponasterone A (K(d) = 6.8-7.5 nM), characteristic of ecdysone receptors. MakA has a similar affinity to 20E for both NvE10 and NvE11, consistent with MakA being a major moulting hormone in N. viridula.
有人提出，半翅目昆虫可能利用 C(28) 蜕皮激素，即 makisterone A（MakA）作为主要蜕皮激素，而不是更常见的 C(27) 激素，即 20-羟蜕皮酮（20E）。本研究首次在蜕皮激素受体蛋白（EcR 和 USP 的核受体异二聚体）水平上检验了这一假设。从具有代表性的半翅目昆虫 Nezara viridula（Nv）的高质量 cDNA 文库中分离出编码两种替代剪接异构体的 EcR 和单个 USP 的 cDNA。NvEcR 和 NvUSP 在系统发育上分别与半翅目和其他昆虫的 EcRs 和 USP/RXRs 聚为一类。对这些蛋白质的序列比较和系统发育分析发现，它们与迄今为止表征的属于其他半翅目昆虫蜕皮激素受体的蛋白质明显不同。通过使用适当构建的杆状病毒，在昆虫细胞中实现了两种 NvEcR 变体的 His(6)-标记配体结合区（LBR）与 NvUSP 的 FLAG 标记 LBR 的共表达。相应的异二聚体，分别命名为 NvE10 和 NvE11，通过亲和层析利用其 NvEcR 亚基上的 His(6)标签进行纯化。这些异二聚体对 [(3)H]ponasterone A（K(d) = 6.8-7.5 nM）表现出纳摩尔亲和力，这是蜕皮激素受体的特征。MakA 对 NvE10 和 NvE11 的亲和力与 20E 相似，这与 MakA 是 N. viridula 的主要蜕皮激素一致。
