褐飞虱表皮蛋白基因NlICP的克隆及功能研究

表皮蛋白与几丁质结合构成抵御外界不良环境的昆虫角质层, 在昆虫的生长发育及蜕皮硬化中具有重要的作用。为了探讨表皮蛋白基因在褐飞虱Nilaparvata lugens生长发育中的功能, 本研究根据褐飞虱转录组测序信息, 对其中1个预测为编码表皮蛋白的Unigene36450序列进行了克隆, 并应用荧光定量PCR和RNA干扰（RNAi）技术分别对该基因的表达规律和功能进行了研究。结果表明： 克隆的 Unigene36450全长cDNA开放阅读框长585 bp, 编码的蛋白含194个氨基酸, 具有典型的表皮蛋白R&R保守结构域, 命名为NlICP。转录水平的时序表达分析发现, NlICP仅在褐飞虱若虫期表达, 且在3龄若虫体内表达量最高, 提示该基因编码的蛋白属于幼虫表皮蛋白。RNA干扰结果显示, 取食dsNlICP的1龄末2龄初（孵化后第3 天）若虫在干扰6 d和8 d时, NlICP基因的表达量分别较取食dsGFP的对照组下降58.8%和45.6%, 差异极显著(P<0.01); 干扰后部分褐飞虱若虫因蜕皮不完全死亡, 干扰5 d的存活率较对照下降26.7%。本研究结果提示, NlICP与褐飞虱若虫的生长发育及蜕皮相关, 可以作为褐飞虱防治的潜在靶标基因。     

Silencing pyruvate kinase (NlPYK) leads to reduced fecundity in brown planthoppers,Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Pyruvate kinase (PYK) operates in the glycolytic pathway, responsible for regulating the balance between glycolysis and gluconeogenesis. The previous work indicates PYK acts in development of Drosophila embryos and in embryonic muscle growth, from which it may be inferred that PYK acts in insect fecundity. More to the point, as a central enzyme in the glycolytic pathway, PYK acts in many energy‐spending functions in most organisms. On the background findings that triazophos (TZP) stimulates fecundity via increase activities of several genes in brown planthoppers, Nilaparvata lugens, we investigated the combined influence of TZP and silencing a N. lugens PYK (NlPYK) on reproduction‐linked biological performance parameters. Here, we report that TZP+dsNlPYK treatments led to reduced (by 26%) ovarian, but not fat body, protein content relative to controls. Ovarian (35%) and fat body (54%) soluble sugar contents were reduced. TZP+dsNlPYK treatments also led to reduced (by about 24%) fecundity, expressed as numbers of eggs laid. These data show directly that NlPYK acts in insect fecundity, probably via increases in glucose metabolism.     

褐飞虱过氧化物酶基因NlPOD1的克隆、鉴定及功能分析

【目的】探明褐飞虱Nilaparvata lugens过氧化物酶基因NlPOD1的分子特性、表达模式和生物学功能。【方法】基于褐飞虱转录组数据,利用PCR技术克隆获得NlPOD1基因全长cDNA序列;利用生物信息学手段分析其序列特征;通过qRT-PCR技术检测NlPOD1在褐飞虱不同发育时期(卵、1-5龄若虫和初羽化雌雄成虫)和5龄若虫不同组织(头部、脂肪体、血淋巴和肠道)中的表达水平,以及不同微生物(大肠杆菌Escherichia coli、金黄色葡萄球菌Staphylococcus aureus和金龟子绿僵菌Metarhizium anisopliae)（1×10⁷/mL）注射感染褐飞虱5龄若虫不同时间后的诱导表达模式;利用RNAi技术沉默褐飞虱 5 龄若虫NlPOD1基因,并通过生物测定分析NlPOD1基因沉默和接种金龟子绿僵菌(1×108/mL)后褐飞虱的存活率及致死中时(LT₅₀)。【结果】获得褐飞虱NlPOD1全长 cDNA 序列(GenBank登录号: MZ682107),其开放阅读框长2 049 bp,编码682个氨基酸,含有一个典型的动物亚铁血红素过氧化物酶结构域(An_peroxidasedomain),且N端包含一段由29个氨基酸残基组成的信号肽。系统发育分析表明,NlPOD1与半翅目其他昆虫的POD亲缘关系较近,其中与茶翅蝽Halyomorpha halys POD亲缘关系最近。发育表达谱结果表明,NlPOD1在褐飞虱不同发育阶段均有表达,其中卵期表达量最低,5龄若虫中表达量最高;组织表达谱结果表明,NlPOD1在褐飞虱5龄若虫不同组织中均有表达,且肠道和血淋巴中的表达量显著高于头部和脂肪体中的;微生物诱导表达模式表明,与注射PBS的对照组相比,大肠杆菌诱导48 h内各时间点NlPOD1在褐飞虱5龄若虫中的表达量显著上调,而在金黄色葡萄球菌和金龟子绿僵菌诱导下,NlPOD1表达量均呈现先上调后回稳的态势。RNAi分析结果显示,显微注射dsNlPOD1可显著抑制NlPOD1的表达水平。通过RNAi抑制NlPOD1表达后褐飞虱5龄若虫存活率不变,但注射dsNlPOD1联合金龟子绿僵菌感染对褐飞虱5龄若虫的LT₅₀(4.5 d)显著低于注射dsGFP联合金龟子绿僵菌感染的对照组的LT₅₀(5.4 d),说明沉默NlPOD1后褐飞虱对金龟子绿僵菌侵染的抵御能力显著降低。【结论】NlPOD1在褐飞虱病原防御过程中发挥重要作用,可作为开发RNAi和病原真菌联合介导的褐飞虱防治技术中的一个潜在靶标。     

The OST‐complex as target for RNAi‐based pest control in Nilaparvata lugens

RNAi‐based pest control strategies are emerging as environment friendly and species‐specific alternatives for the use of conventional pesticides. Because N‐glycosylation is important for many biological processes, such as growth and development, the early steps of protein N‐glycosylation are promising targets for an RNAi‐based pest control strategy. Through injection of dsRNAs, the expression of the catalytic subunits of the oligosaccharyl transferase complex was efficiently silenced in nymphs of the notorious rice pest insect Nilaparvata lugens. Silencing of both STT3 isoforms resulted in a high mortality of the N. lugens nymphs. However, our data reveals the occurrence of a functional redundancy between the two isoforms when silencing only one of the isoforms. These observations confirm the potential to use the early genes in the N‐glycosylation pathway as targets for an RNAi‐based pest control strategy. In addition, the existence of a functional redundancy between the two STT3 isoforms presents a factor which one must take into account when designing RNAi‐based approaches.     

Effects of temperature on fitness costs in chlorpyrifos‐resistant brown planthopper,Nilaparvata lugens (Hemiptera: Delphacidae)

Insecticide resistance is inevitable if an insecticide is widely used to control insect pests. Fortunately, the resistance‐associated fitness costs often give chances to manage resistances. In most cases, the fitness cost in resistant insects is often evaluated under laboratory conditions for insect development, which limits its practical application in pest control in the field. In a laboratory population R9 with 253‐fold resistance to chlorpyrifos after nine‐generation selection with chlorpyrifos, the relative fitness was only 0.206 under laboratory conditions (25°C, humidity 70%–80% and 16 h light/8 h dark photoperiod), when compared to S9, a susceptible counterpart (resistance ratio = 2.25‐fold) from the same origin as R9 but without any selection with insecticides. Temperatures varied the resistance‐associated fitness costs, with enhanced costs at high temperatures and reduced costs at low temperatures, such as 0.174 at 32°C and 0.527 at 18°C. The copulation rate and fecundity were two key factors for the reduced costs at low temperatures. Another finding was that R9 individuals needed much more time to recover from heat shock than that of S9, but R9 and S9 individuals were similarly sensitive to cold shock. The low fitness cost at low temperatures would increase the overwintering population, which might further increase risks of rapid development and widespread distribution of chlorpyrifos resistance in Nilaparvata lugens.     

褐飞虱卵黄蛋白的分离及其生化特性

电泳结合不同染色方法证实, 褐飞虱Nilaparvata lugens (Stål)卵黄蛋白为一种糖脂结合蛋白,其分子量约为314 kD,由148 kD、124.5 kD和39.6 kD 3个亚基组成。免疫反应证明,卵黄蛋白只存在于生育期的雌性褐飞虱成虫体内。褐飞虱卵黄蛋白具有种的特异性,其免疫血清与白背飞虱的卵黄蛋白无交叉反应。     

褐飞虱乙酰胆碱酯酶基因全长cDNA的克隆及序列分析

利用反转录聚合酶链式反应(RT_PCR)结合快速扩增cDNA末端(RACE)技术克隆了褐飞虱Nilaparvata lugens 乙酰胆碱酯酶基因cDNA。该cDNA全长2 467 bp,包含一个1 938 bp的开放阅读框(GenBank登录号AJ852420); 在推导出的646个氨基酸残基的前体蛋白中, N端的前30个氨基酸残基为信号肽,随后的616个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为69 418 D。在一级结构中,形成催化活性中心的3个氨基酸残基(Ser242,Glu371和His485),以及在亚基内形成二硫键的6个半胱氨酸完全保守; 位于催化功能域的14个芳香族氨基酸中有10 个完全保守。该酶的氨基酸序列与黑尾叶蝉的同源性最高,达83%。对来自23种昆虫（包括褐飞虱）的30个乙酰胆碱酯酶的聚类分析显示,褐飞虱的乙酰胆碱酯酶与其中6个Ⅱ型乙酰胆碱酯酶（AChE2）同属一个支系; 此外,只存在于昆虫AChE2中的超变区及特异的氨基酸残基,也存在于褐飞虱的乙酰胆碱酯酶中。以上结果表明,所克隆的褐飞虱的乙酰胆碱酯酶基因是一个与黑腹果蝇的orthologous型基因同源的AChE2基因。     

Imidacloprid‐induced transference effect on some elements in rice plants and the brown planthopper Nilaparvata lugens (Hemiptera: Delphacidae)

Abstract The widespread use of imidacloprid against insect pests has not only increased the rate of the development of target pest resistance but has also resulted in various negative effects on rice plants and Nilaparvata lugens resurgence. However, the effect of imidacloprid on elements in rice plants and the transference of these element changes between rice and N. lugens are currently poorly understood. The present study investigated changes of Cu, Fe, Mn, Zn, Ca, K, Mg and Na contents in rice plants following imidacloprid foliar sprays in the adult female of N. lugens that develops from nymphs that feed on treated plants and honeydew produced by females. The results indicated that imidacloprid foliar spray significantly increased Fe and K contents in leaf sheaths. Generally, Fe, Mn, K and Na contents in leaf blades were noticeably decreased, but Ca contents in leaf blades for 10 and 30 mg/kg imidacloprid treatments were significantly increased. The contents of most elements except K and Mg in the adult females and honeydew were significantly elevated. Multivariate statistical analysis showed that Fe, Mn and Na in leaf blades and Fe and Mn in leaf sheaths could be proportionally transferred to N. lugens. The relationship between most elements in adult female bodies and in the honeydew showed a positive correlation coefficient. There were significant differences in the contents of some elements in rice plants and N. lugens from different regions.     

Molecular characterization,spatial‐temporal expression and magnetic response patterns of iron‐sulfur cluster assembly1 (IscA1) in the rice planthopper,Nilaparvata lugens

The mechanisms of magnetoreception have been proposed as the magnetitebased, the chemical radical-pair and biocompass model, in which magnetite particles, the cryptochrome (Cry) or iron-sulfur cluster assembly 1 (IscA1) may be involved. However, little is known about the association among the molecules. Here we investigated the molecular characterization and the mRNA expression of IscA1 in different developmental stages, tissues and magnetic fields in the migratory brown planthopper (BPH), Nilaparvata lugens. NlIscA1 contains an open reading frame of 390 bp, encoding amino acids of 129, with the predicted molecular weight of 14.0 kDa and the isoelectric point of 9.10. Well-conserved Fe-S cluster binding sites were observed in the predicted protein. Phylogenetic analysis demonstrated NlIscA1 to be clustered into the insect's IscA1. NlIscA1 showed up-regulated mRNA expression during the period of migration. The mRNA expression of NlIscA1 could be detected in all the three tissues of head, thorax and abdomen, with the highest expression level in the abdomen. For the macropterous migratory Nilaparvata lugens, mRNA expression of NlIscA1 and N. lugens cryptochromel (Nlcry1) were up-regulated under the magnetic fields of 5 Gauss and 10 Gauss in strength (vs. local geomagnetic field), while N. lugens cryptochrome 2 (Nlcry2) remained stable. For the brachyterous non-migratory Nilaparvata lugens, no significant changes were found in mRNA expression of NlIscA1, Nlcry1 and Nlcry2 among different magnetic fields. These findings preliminarily reveal that the expression of NlIscA1 and Nlcry1 exhibited coordinated responses to the magnetic field. It suggests some potential associations among the putative magneto-sensitive molecules of cryptochrome and iron-sulfur cluster assembly.     

Characterization of a Nilaparvata lugens (Stål) brummer gene and analysis of its role in lipid metabolism

The brummer (bmm) genes encode the lipid storage droplet‐associated triacylglycerols (TAG) lipases, which belong to the Brummer/Nutrin subfamily. These enzymes hydrolyze the ester bonds in TAG in lipid metabolism and act in insect energy homeostasis. Exposure to some agricultural chemicals leads to increased fecundity, which necessarily involves lipid metabolism, in some planthopper species. However, the biological roles of bmm in planthopper lipid storage and mobilization have not been investigated. Here, the open reading frame (ORF) of bmm (Nlbmm) was cloned and sequenced from the brown planthopper (BPH; Nilaparvata lugens). The ORF is 1014 bp encoding 338 amino acid residues. Nlbmm contained patatin domains and shared considerable evolutionary conservation with other insect bmms. Nlbmm is highly expressed in the fat body, consistent with its roles in lipid metabolism. Injection with Nlbmm double‐stranded RNA (dsNlbmm) led to reduced Nlbmm mRNA accumulation, but did not influence expression of several genes related to lipid synthesis including acyl‐CoA‐binding protein (ACBP), acetyl‐CoA carboxylase (ACC), and a lipophorin receptor (LpR). Nlbmm knockdown led to increased TAG contents in whole bodies, accumulation of total fat body lipid, and decreased hemolymph lipid content. Nlbmm knockdown did not influence the synthesis and distribution of glycerol. We infer that Nlbmm acts in TAG breakdown and fat metabolism in N. lugens.     

AChE在水稻抗性诱导的褐飞虱凋亡中肠细胞中的定位及表达

【目的】褐飞虱Nilaparvata lugens是水稻的重要害虫之一。本研究旨在了解水稻品种抗性诱导的褐飞虱中肠细胞凋亡与细胞中乙酰胆碱酯酶(acetylcholinesterase,ACh E)的关系。【方法】从4龄Ⅰ型褐飞虱若虫中肠组织分离原代细胞,用不同抗性的水稻幼苗汁液处理第一代继代细胞,利用TUNEL染色法检测细胞的凋亡情况,再分别利用免疫组织化学和荧光定量PCR技术对ACh E进行亚细胞定位和检测其表达水平的变化。【结果】在对照组(未处理)细胞中,检测不到代表凋亡细胞核的绿色荧光,而免疫组化后阳性反应颜色很浅,表明细胞中存在ACh E的本底水平表达。感虫水稻品种TN1幼苗汁液处理细胞中,细胞的凋亡率为8%,抗性水稻B5幼苗汁液处理的细胞中有65%的细胞发生凋亡;而抗性水稻TKM-6幼苗汁液处理的细胞中则有85%的细胞发生凋亡。免疫组化的检测结果表明,所有凋亡的细胞中都存在ACh E的积累,且主要分布在细胞质中;ACh E在凋亡后期并不迁入凋亡小体中,而是集中在细胞核附近。荧光定量PCR检测表明,TKM-6,B5和TN1幼苗汁液处理的细胞中ACh E的表达量分别为对照细胞的29.9,18.4和8倍,该结果与细胞水平上免疫组化的检测结果一致。【结论】本研究结果证实了水稻品种抗性与其诱导的中肠细胞凋亡率呈正相关,且凋亡细胞的细胞质中存在ACh E的积累。这些发现为揭示ACh E在褐飞虱与水稻的互作中的功能、促进抗性水稻新品种的选育及开发新的褐飞虱防治措施提供了一定的参考信息。     

褐飞虱细胞色素P450基因CYP4C62的原核表达及多克隆抗体的制备

【目的】细胞色素P450单加氧酶在昆虫生长发育和适应环境过程中发挥着重要功能。【方法】本研究克隆了褐飞虱Nilaparvata lugens细胞色素P450基因CYP4C62的开放阅读框（不含信号肽编码序列部分）,在大肠杆菌Escherichia coli中实现了高效表达,经Ni-NTA琼脂糖凝胶亲和层析柱纯化得到了重组的CYP4C62蛋白。将该蛋白免疫日本大耳白兔Oryctolagus cuniculus雄兔,制备了兔抗CYP4C62血清抗体。采用间接ELISA方法检测了血清抗体的效价;并通过Western印迹杂交检测了该抗体的免疫学特异性。【结果】结果表明,通过大肠杆菌表达出的CYP4C62蛋白相对分子量为56 kD。间接ELISA法检测表明,制备的兔抗CYP4C62抗体的效价达到1∶100 000。Western印迹杂交证实,该抗体既可与异源表达的CYP4C62蛋白特异性结合,也可以与褐飞虱总蛋白中内源的CYP4C62特异性结合,表明具有较好的免疫反应特异性。【结论】CYP4C62多克隆抗体的成功制备,为后续分析CYP4C62在褐飞虱各组织中的时空表达水平,并通过免疫组织化学法定位分析该蛋白的组织、细胞及亚细胞分布规律,及最终解析CYP4C62的生物学功能奠定了基础。     

褐飞虱成虫体内磁性物质检测

地磁定向是昆虫远距离迁飞定向的重要机制之一.本研究以褐飞虱Nilaparvata lugens长翅型和短翅型成虫为研究对象,利用MPMS-7型号超导量子干涉磁强计(磁场范围为±4.8 mA/m,温度范围为1.9 ～ 400 K)检测虫体内的磁性物质,明确其体内的分布状况.结果表明:褐飞虱长翅型雄成虫整个虫体的温度退磁曲...     

Genome‐wide analysis of Nilaparvata lugens nymphal responses to high‐density and low‐quality rice hosts

The brown planthopper (BPH) Nilaparvata lugens is an economically important pest on rice plants. In this study, the higher population density and yellow‐ripe stage of rice plants were used to construct adverse survival conditions (ASC) against BPH nymphs. Simultaneously, the low population density and tillering stage of rice plants were used to establish a suitable survival condition (SSC) as a control. Solexa/Illumina sequencing was used to identify genes of BPH nymphs responding to ASC. Significantly longer duration development of BPH nymphs and significantly lower brachypterous ratio of BPH adults were observed by ASC compared with SSC. A total of 2 544 differentially expressed genes (DEGs) were obtained and analyzed by BLASTx, Gene Ontology and KEGG Orthology. Gene ontology analysis revealed that the DEGs were mainly involved in categories of cell, cell part, cellular process, binding, catalytic, organelle and metabolic processes. 1 138 DEGs having enzyme commission numbers were assigned to different metabolic pathways. The largest clusters were neurodegenerative diseases (137, 12.0%), followed by carbohydrate metabolism (113, 9.9%), amino acid metabolism (94, 8.3%), nucleotide metabolism (76, 6.7%), energy metabolism (64, 5.6%), translation (60, 5.3%), lipid metabolism (58, 5.1%), and folding, sorting and degradation (52, 4.6%). Expressing profile of 11 DEGs during eight nymphal developmental stages of BPH were analyzed by quantitative real‐time polymerase chain reaction. The 11 genes exhibited differential expression between ASC and SSC during at least one developmental stage. The DEGs identified in this study provide molecular proof of how BPH reconfigures its gene expression profile to adapt to overcrowding and low‐quality hosts.     

不同浓度氯化钙浸种处理对水稻防御酶活性和抗褐飞虱的影响

[目的]研究不同浓度氯化钙(calcium chloride,CaCl2)浸种处理对水稻防御酶活性和抗褐飞虱Nilaparvata lugens的影响.[方法]分别用10,20,30,40和50 mmol/L CaCl2溶液浸泡水稻种子48 h,以蒸馏水浸种为对照,待水稻长至分蘖期时,检测褐飞虱3龄若虫取食胁迫下各浓度...     

吡虫啉对褐飞虱DNA甲基化多态性的影响

为了探知基因组甲基化是否参与了昆虫抗药性, 本研究在室内对褐飞虱 Nilaparvata lugens连续9个世代的3龄若虫施用吡虫啉, 用AFLP检测褐飞虱抗性产生过程中DNA甲基化多态性的变化。利用25对AFLP引物共获得120个位点, 其中15个位点呈现甲基化多态性, 共获得78条多态性条带。根据多态性条带在不同世代样本中出现的多少计算多态性条带比例, 其中最高比例出现在G5代（10.26%）, 最低比例出现在G6代（1.28%）。多态性条带在不同世代间比例的变化趋势表明, 褐飞虱对吡虫啉的筛选产生快速应答。在筛选早期（G1, G2和G3）世代间DNA甲基化多态性比例差异相对较小, 变化范围在3.85%～6.41%之间; 在筛选中期（G4, G5和G6）世代间比例差异较大, 变化范围在1.28%～10.26%之间; 在筛选后期（G7, G8和G9）世代间比例差异相对较小, 变化范围在5.13%～7.69%之间。结果说明, 吡虫啉的连续施用能够诱导褐飞虱基因组产生甲基化变异, 初步揭示甲基化在褐飞虱抗药性产生过程中参与了基因组的表达调控。     

Analysis of differential proteins in two different wing‐types of female Nilaparvata lugens

Nilaparvata lugens (stal) is a rice pest and contains long‐winged and short‐winged varieties, called the wing differentiation. This study compared the protein profiles of the two wing‐types in females and two wing‐disc types 5th‐instar females by two‐dimensional electrophoresis analysis. We detected 172 and 174 protein spots in adults and 5th‐instar nymphs, respectively. The number of proteins with higher content in the long‐winged (disc) individuals is much more than that in the short‐winged (disc) individuals. A total of 32 differential protein spots were found, of which 20 were successfully identified. Their main function is about catabolic process, fiber and nucleoside binding, and they constitute 52 protein–protein interactions, which is around the glycolysis as the core. These results enrich the research on the protein Level in wing development, and provide more references for future studies.