桑螟(Glyphodes pyloalis)是蚕区桑园的主要害虫之一,鉴定其抵御各种环境胁迫相关的基因,是制定防治新策略的重要理论依据.对桑螟幼虫转录组数据的分析中,发现桑螟胞外铜锌超氧化物歧化酶(ecCu/ZnSOD)基因在应对高温胁迫时的表达量发生显著变化.为了明确ecCu/ZnSOD在桑螟应对不良环境中发挥的作用,克隆和鉴定了其编码基因GpecCu/ ZnSOD(GenBank登录号:MG566057).GpecCu/ ZnSOD基因的开放阅读框长726 bp,编码241个氨基酸,编码蛋白质序列与脐橙螟(Amyelois transitella)的一种SOD蛋白基因(GenBank登录号:XP_013197347.1)编码氨基酸序列的一致性高达83％.采用邻近相接法构建桑螟与其他昆虫的Cu/ZnSOD系统发生树,结果显示Cu/ZnSOD被分为ecCu/ZnSOD和icCu/ZnSOD两类,主要是随物种进化而进化.采用半定量RT-PCR检测不同温度处理桑螟5龄幼虫的GpecCu/ ZnSOD基因表达情况,结果显示幼虫体内的GpecCu/ZnSOD在高温(40℃)和低温(4℃)条件下表达量最高.构建pCold-GpecCu/ZnSOD重组表达质粒,并在大肠埃希菌中实现异源表达.利用镍柱亲和层析法分离纯化重组GpecCu/ZnSOD蛋白,采用WST-1法测定重组GpecCu/ZnSOD蛋白在40℃时的酶活性最高,并且经60～70℃处理后仍然具有较高的酶活性.采用牛津杯法分析重组GpecCu/ZnSOD的抗氧化活性表明:过表达GpecCu/ZnSOD的大肠埃希菌抗氧化的能力显著增强,显示GpecCu/ZnSOD具有较强的抗氧化功能.研究结果暗示GpecCu/ZnSOD在桑螟应对高温胁迫过程中发挥着重要作用.

桑螟(Glyphodes pyloalis)是桑树的主要害虫,因其对专用杀虫剂产生了一定抗性,导致防治效果下降.从分子水平研究桑螟对杀虫剂的抗性机制,有利于制定新的防治策略.通过对桑螟中肠转录组数据分析,鉴定了2个细胞色素P450基因CYP9A20和CYP324A 19.系统进化分析表明,桑螟CYP9A20和CYP324A19基因均属于CYP3家族,其中CYP324A 19基因编码氨基酸序列与同属鳞翅目的稻纵卷叶螟(Cnaphalocrocis medinalis)的同源序列亲缘关系比较近,序列相似度为63％;桑螟CYP9A20基因编码氨基酸序列同样与稻纵卷叶螟的同源序列亲缘关系较近,序列相似度高达80％.以浙江省杭州、湖州和海宁3个蚕区桑园采集的桑螟样本提取DNA,经qRT-PCR分析幼虫中肠的CYP9A20基因表达水平没有明显差异,而CYP324A19基因的表达水平存在显著差异,其中来自湖州、海宁的桑螟样本中该基因的表达量分别为来自杭州的桑螟样本的3.2倍、3.0倍.用15 μg/mL辛硫磷和15 μg/mL残杀威处理来自湖州的桑螟幼虫24 h后,qRT-PCR检测CYP9A20基因在幼虫中肠中的表达水平无明显变化,而CYP324A19基因的表达显著上调,辛硫磷、残杀威处理后该基因的表达量分别上调4.0倍、7.8倍.研究结果表明,桑螟的2个细胞色素P450基因在进化上出现了不同分歧,从而导致二者在功能上可能存在明显差异,CYP324A 19基因可能通过上调表达参与桑螟对杀虫剂胁迫的响应过程.

Heat tolerance is a key parameter that affects insect distribution and abundance. Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is a devastating pest of mulberry in the main mulberry-growing regions and can cause tremendous losses to sericulture by directly feeding on mulberry leaves and transmitting viruses to Bombyx mori. Moreover, G. pyloalis shows a prominent capacity for adaptation to daily and seasonal temperature fluctuations and can survive several hours under high temperature. To date, the molecular mechanism underlying the outstanding adaptability of this pest to high temperature remains unclear.

Recent studies highlighted that apolipoprotein D (ApoD) and its homologs exert neuroprotective and antioxidant functions in mammals and Drosophila. Unlike mammals and Drosophila, lepidopteran insects possess three distinct ApoD homologs. However, few information on their functions in lepidopteran insects are available. In this study, we investigated the protective potential of a novel ApoD homolog, BmApoD1, in Bombyx mori. Quantitative PCR analyses demonstrated that BmApoD1 is extensively expressed at low levels during the larval stage but abundantly expressed in the testis during the pupal and adult stages. Tryptophan fluorescence titration demonstrated that recombinant BmApoD1 protein can bind retinoic acid and ergosterol. In addition, we provided evidence that N-linked glycans of BmApoD1 are essential to BmApoD1 secretion, and three residues, namely, Asp69, Asp104, and Asp196, are the glycosylation sites of BmApoD1. Furthermore, we showed that BmApoD1 is significantly up-regulated in the larvae after oxidant or starvation treatment. The recombinant BmApoD1 protein can protect cells from oxidative stress induced by H2O2 and reduce actinomycin D-induced cell apoptosis. These observations, together with the transcriptional up-regulation of BmApoD1 in several tissues upon oxidative insult, identify BmApoD1 as a potent antioxidant. Our results demonstrate that BmApoD1 is critical for metabolic adaptation of B. mori to environmental challenges.