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。     

褐飞虱V-ATPase d亚基基因的克隆与mRNA表达分析

液泡型H+-ATPase(V-ATPase)在昆虫生长发育过程中具有重要作用。本文通过RT-PCR获得褐飞虱Nilaparvata lugens(Stl)V-ATPase d亚基基因NlVHA-d的cDNA序列,并通过实时荧光定量PCR对NlVHA-d基因的表达进行了分析。结果表明,NlVHA-d基因编码349个氨基酸,不同昆虫V-ATPase d亚基高度保守。NlVHA-d基因在褐飞虱2龄若虫中表达量最高,雌虫表达量显著高于雄虫表达量。LD10和LD30三唑磷处理的羽化3 d短翅雄虫NlVHA-d基因相对表达倍数分别是丙酮处理的2.15和2.46倍。亚致死剂量三唑磷处理褐飞虱短翅雄虫NlVHA-d基因的表达上调可能与褐飞虱再猖獗相关。     

大鼠电子传递黄素蛋白-泛醌氧化还原酶cDNA的克隆、功能表达和细胞定位

从大鼠肝线粒体纯化得到一个内膜结合蛋白, 经蛋白酶水解发现丰度最高的两个肽段含有相同的一个14肽序列. BLAST同源搜索找到了与这个14肽对应的大鼠基因组DNA. 利用RACE的方法得到一个完整开放阅读框的cDNA, 编码含616个氨基酸残基的蛋白质序列. 克隆得到的大鼠cDNA与人的ETF-QO具有很高的同源性. 通过序列比较发现克隆得到的cDNA对应大鼠ETF-QO的蛋白前体ETF-QOp, 而从大鼠肝线粒体纯化得到的内膜结合蛋白则是ETF-QO. 构建的pYES/ETF-QO在酵母中的表达产物富集于线粒体组分, 并具有ETF-QO的氧化-还原活性, 因此获得了ETF-QO在酵母中的功能表达, 表明ETF-QOp N端32肽是线粒体的引导肽, 且FAD及[4Fe-4S]被正确地组装. ETF-QO在大鼠的心、肝、肾表达较高, 而在脾与肺却很低. 在动物细胞中表达了ETF-QOp的GFP融合蛋白, 它在细胞内与线粒体的特异染料呈共定位.     

受褐飞虱取食的水稻幼苗的抑制消减cDNA文库的构建及筛选

采用抑制消减杂交方法,以褐飞虱取食32h的水稻幼苗及未受褐飞虱取食的水稻幼苗为作为对比材料构建了消减cDNA文库,以分离水稻幼苗中褐飞虱应答基因。随机从消减cDNA文库中挑选16个白色菌落提取质粒,进行PCR扩增,发现插入片段的长度位于100～900bp之间。以在受褐飞虱取食的水稻幼苗中特异表达的基因(BpHi008A)为探针,通过斑点印迹分析发现在抑制消减后的cDNA池中,目的基因得到有效富集。利用反向总RNA斑点印迹分析和Northern杂交验证,从消减cDNA文库中筛选到了25个基因受褐飞虱取食的诱导。其中有17个克隆与编码已知功能蛋白的基因有显著的同源性,它们分别参与蛋白质的折叠与降解、蛋白质与蛋白质的相互作用及信号传递、脂类代谢、胁迫反应、物质运输和细胞生长等。总体上,参与胁迫反应和衰老的基因在褐飞虱取食后表达增强。     

谷子肌动蛋白基因的克隆及序列分析

以谷子（Setaria italica）为材料,提取总RNA。根据植物肌动蛋白基因编码区的两端的保守序列设计了简并引物,用5'RACE方法扩增出了谷子肌动蛋白基因编码区序列。以豌豆肌动蛋白cDNA作探针进行的Southern杂交分析表明扩增出了目的基因。将所获得的片段克隆到T载体后进行测序,序列分析结果表明：谷子肌动蛋白基因的编码区长1131个核苷酸,编码了377个氨基酸;所得序列(命名为MIAc)与GenBank中注册的肌动蛋白基因序列的相似性均在60%以上,与其它肌动蛋白氨基酸序列的相似性达89%以上。根据高等植物肌动蛋白序列相似性重建了进化树,表明谷子肌动蛋白与水稻肌动蛋白异型体RAc2和RAc3之间的亲缘关系 最为密切,在进化过程中分化时间最为接近。     

谷子肌动蛋白基因的克隆及序列分析

以谷子 (Setariaitalica)为材料 ,提取总RNA。根据植物肌动蛋白基因编码区的两端的保守序列设计了简并引物 ,用 5’RACE方法扩增出了谷子肌动蛋白基因编码区序列。以豌豆肌动蛋白cDNA作探针进行的Southern杂交分析表明扩增出了目的基因。将所获得的片段克隆到T载体后进行测序 ,序列分析结果表明 :谷子肌动蛋白基因的编码区长 1 1 3 1个核苷酸 ,编码了 3 77个氨基酸 ;所得序列 (命名为MIAc)与GenBank中注册的肌动蛋白基因序列的相似性均在 6 0 %以上 ,与其它肌动蛋白氨基酸序列的相似性达 89%以上。根据高等植物肌动蛋白序列相似性重建了进化树 ,表明谷子肌动蛋白与水稻肌动蛋白异型体RAc2和RAc3之间的亲缘关系最为密切 ,在进化过程中分化时间最为接近     

褐飞虱遇险释放挥发性物质的提取及成分分析

利用Y型嗅觉仪测定不同试验条件下褐飞虱Nilaparvata lugens(Stl)对同类昆虫遭遇草间小黑蛛Erigonidium graminicolum Sundvall捕食时挥发性物质的行为反应,并利用固相微萃取、气相色谱与质谱技术分离鉴定挥发性物质。目的明确褐飞虱遇险释放挥发性物质的最佳提取条件,并分析该挥发性物质的成分。结果表明,用乙酸乙酯、甲醇和正己烷分别提取"褐飞虱2³龄若虫+草间小黑蛛成蛛"共存体释放的挥发性物质,发现乙酸乙酯提取物能引起褐飞虱极显著的逃避行为。用不同褐飞虱虫量(100、300、600头/500 mL瓶)、不同提取时间(1、3、6 h)和不同溶剂温度(15、25、35℃)分别提取"褐飞虱23龄若虫+草间小黑蛛成蛛"共存体释放的挥发性物质,发现乙酸乙酯提取物能引起褐飞虱极显著的逃避行为。用不同褐飞虱虫量(100、300、600头/500 mL瓶)、不同提取时间(1、3、6 h)和不同溶剂温度(15、25、35℃)分别提取"褐飞虱2³龄若虫+草间小黑蛛成蛛"共存体释放的挥发性物质,发现3003龄若虫+草间小黑蛛成蛛"共存体释放的挥发性物质,发现300⁶00头、3600头、3⁶ h和25℃下的提取物能引起褐飞虱极显著的逃避行为,明确了褐飞虱遇险释放挥发性物质的最佳提取条件。利用固相微萃取法分别萃取"褐飞虱26 h和25℃下的提取物能引起褐飞虱极显著的逃避行为,明确了褐飞虱遇险释放挥发性物质的最佳提取条件。利用固相微萃取法分别萃取"褐飞虱2³龄若虫"释放的挥发性物质和"褐飞虱23龄若虫"释放的挥发性物质和"褐飞虱2³龄若虫+草间小黑蛛成蛛"共存体释放的挥发性物质。气相色谱分析表明,保留时间16 min、21.5 min时,"褐飞虱"与"褐飞虱+草间小黑蛛"共存体释放的挥发物均出现新峰;保留时间10.94 min时,仅"褐飞虱+草间小黑蛛"共存体释放的挥发物出现新峰。推测保留时间10.94 min时出现的峰为褐飞虱被草间小黑蛛捕食时释放的示警挥发物。质谱分析进一步表明,这种挥发物具有与(E)-2-己烯醛相类似的化学结构,但其实际结构还需深入研究。     

褐飞虱核糖体小亚基蛋白S8基因NlRPS8的克隆及其表达分析

【目的】核糖体蛋白在生物体内具有重要作用,本研究旨在探明核糖体蛋白S8在褐飞虱Nilaparvata lugens生长发育过程中的表达规律和功能。【方法】本文根据褐飞虱基因表达谱提供的差异表达基因信息及转录组提供的基因核心序列,结合褐飞虱基因组比对分析,对褐飞虱核糖体S8基因进行了预测,并通过RT-PCR获得了褐飞虱核糖体小亚基蛋白S8的全长c DNA序列,命名为NlRPS8(Gen Bank登录号:KU341337)。【结果】NlRPS8基因全长627 bp,编码208个氨基酸。进化分析表明,褐飞虱与桑粉介壳虫Maconellicoccus hirsutus亲缘关系最为接近。应用荧光定量PCR分析了基因NlRPS8的表达规律,结果表明,NlRPS8基因在褐飞虱怀卵雌虫中表达量最高,而在雄成虫、初羽化雌成虫与1~5龄若虫表达量相对较低;在褐飞虱体内,NlRPS8基因在卵巢中表达量最高,中肠次之,在其他部位表达量较低。在抗性品种ASD7和RHT上,NlRPS8基因表达量分别为感性品种TN1上的1.99倍和2.14倍。NlRPS8基因在经过饥饿处理1 d的褐飞虱组中表达量为正常组的1.6倍。【结论】研究结果显示NlRPS8基因在褐飞虱生长、繁殖中发挥作用,为进一步研究NlRPS8基因在褐飞虱生长发育和对抗性品种适应中的功能提供了线索。     

褐飞虱2009年秋季回迁的雷达监测及轨迹分析

褐飞虱Nilaparvata lugens (Stål)是水稻生产上重要的迁飞性害虫,研究其迁飞、扩散规律,为早期预警和有效防治提供科学依据。2009年4月27日至10月11日, 在中国农业科学院植物保护研究所重大病虫害监测预警兴安雷达站内利用毫米波扫描昆虫雷达对褐飞虱的迁飞过程进行长期观测,高空探照灯诱虫器及佳多自动虫情测报灯诱虫器分别用来诱捕高空及地面的褐飞虱,对高空探照灯诱到的褐飞虱雌成虫随机挑选30头进行卵巢解剖, 并结合大区环流和利用Hysplit_4模型进行轨迹分析,研究了褐飞虱的秋季回迁过程和虫源。结果表明：褐飞虱秋季回迁高峰期出现在9月28日至10月7日,高峰日为10月1日, 高峰日内雷达回波主要在600~1 100 m范围内聚集成层,高空探照灯诱虫器内褐飞虱的数量达到了13 620头;卵巢发育级别以1~2级为主。轨迹分析显示：本次回迁的褐飞虱主要来自湖南衡阳和永州等地,随东北气流向广西柳州、南宁和崇左等方向迁飞。轨迹推断与褐飞虱实发虫情基本吻合,通过毫米波扫描昆虫雷达确定了褐飞虱秋季回迁的高度,为毫米波扫描昆虫雷达早期投入到预测预报的实践中奠定了基础,对我国褐飞虱早期预警体系的建立将提供必要的技术支持。     

谢氏宽漠王β-actin基因cDNA克隆、序列分析及表达量检测

β-actin是actin家族的一员,在维持细胞结构、运动和分裂等细胞生理活动方面发挥着重要作用,是基因定量实验中最常用的内参之一。本实验采用同源克隆和RACE技术扩增得到谢氏宽漠王Mantichorula semenowi β-actin基因。序列分析结果表明,该基因cDNA全长1 372 bp,开放阅读框长1 131 bp,编码376个氨基酸,5′和3′末端非翻译区域(UTR)分别为66 bp和175 bp;该序列与其他动物β-actin基因核苷酸序列具有96%～99%高度同源性。β-actin表达量检测结果显示热激后不同恢复时间其表达量无明显变化,且与未经热激处理的对照相比无显著差异。表明β-actin是研究受外界环境胁迫作用下昆虫体内不同基因表达水平的可靠内参基因。     

Molecular Cloning of ADP-Glucose Pyrophosphorylase Large Subunit cDNA from Oncidium

A full-length cDNA for ADP-glucose pyrophosphorylase large subunit (AGPL) was isolated from tropical epiphytic orchid Oncidium hybrid Goldiana. The cDNA was 1754 bp in length with an open reading frame of 1551 bp encoding 517 amino acids. The deduced amino acid sequence showed 73 % identity with those of potato isoform 3 (AGPL3) and Arabidopsis thaliana isoform 1 (AGPL1), 71 % identity with that of barley isoform BLPL. RT-PCR analysis showed that AGPL was expressed in mature leaf, immature leaf, developing inflorescence and flower of Oncidium. No expression was detected in roots.     

Bacterial Symbionts of the Brown Planthopper,Nilaparvata lugens (Homoptera: Delphacidae)

The brown planthopper (Nilaparvata lugens Stål), the most destructive pest of rice, has been identified, including biotypes with high virulence towards previously resistant rice varieties. There have also been many reports of a yeast-like symbiont of N. lugens, but little is known about the bacterial microbes. In this study, we examined the bacterial microbes in N. lugens and identified a total of 18 operational taxonomic units (OTUs) representing four phyla (Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes) by sequencing and analyzing 16S rRNA gene libraries obtained from three populations of N. lugens, which were maintained on the rice varieties TN1, Mudgo, and ASD7. Several of the OTUs were similar to previously reported secondary symbionts of other insects, including an endosymbiont of the psyllid Glycapsis brimblecombei, an Asaia sp. found in the mosquito Anopheles stephensi, and Wolbachia, found in the mite Metaseiulus occidentalis. However, the species and numbers of the detected OTUs differed substantially among the N. lugens populations. Further, in situ hybridization analysis using digoxigenin-labeled probes indicated that OTU 1 was located in hypogastrium tissues near the ovipositor and ovary in biotype 1 insects, while OTU 2 was located in the front of the ovipositor sheath in biotype 2 insects. In addition, masses of bacterium-like organisms were observed in the tubes of salivary sheaths in rice plant tissues that the insects had fed upon. The results provide indications of the diversity of the bacterial microbes harbored by the brown planthopper and of possible associations between specific bacterial microbes and biotypes of N. lugens.Close associations between insects and the microbes they harbor appear to be common. Symbionts have been found to contribute to the nutrition, development, reproduction, speciation, and defense against natural enemies of their host insects (1, 11, 18, 30, 39). The small brown planthopper (Laodelphax striatellus) and the white-backed planthopper (Sogatella furcifrea) also reportedly harbor an alphaproteobacterial Wolbachia symbiont (29) that can be transferred horizontally between different insect species and that affects its hosts'' sexual reproduction, cytoplasmic incompatibility, and immune responses (21, 38, 39).The brown planthopper, Nilaparvata lugens Stål (Homoptera: Delphacidae), is a monophagous insect herbivore of rice (13) that feeds on rice phloem and causes serious damage to rice crops. N. lugens reportedly harbors an intracellular, eukaryotic “yeast-like symbiont” (YLS) in the fat body, which plays a key role in recycling uric acid (3, 33). However, little is known about bacterial symbionts in N. lugens.It has been well recognized that diversity exists within insect species and that “biotypes” or populations that are adapted to or that prefer a particular host can frequently develop (10, 12). The behavioral and physiological responses during insect establishment on plants are feeding, metabolism of ingested food, growth, adult survival, egg production, and oviposition (34). In N. lugens, the biotype is assigned to a population with the ability to damage varieties of rice that carry resistance genes and that were previously resistant to it (5). It has been claimed that some biotypes of N. lugens differ in small morphological features, isozymes, and DNA polymorphisms (6, 25, 36). However, the precise nature of the virulence-conferring mechanisms in N. lugens biotypes (and their modes and stability of inheritance) is not clear. It is interesting to survey symbionts in different biotype populations of N. lugens.Generally, the 16S rRNA gene has been used as a molecular marker enabling the detection of as-yet-uncultured microbes, and it facilitates a profound investigation of microbial diversity (2, 22, 44). We initiated a study using molecular methods to investigate the bacterial symbionts of N. lugens. The major objective of this study was to identify bacterial microbes in N. lugens. The identified bacterial microbes appeared to be associated with different populations of N. lugens and in some cases were located in specific tissues, according to in situ hybridization (ISH) analyses.     

N-glycosylation Site Analysis Reveals Sex-related Differences in Protein N-glycosylation in the Rice Brown Planthopper (Nilaparvata lugens)

1. Download : Download high-res image (275KB)
2. Download : Download full-size image

Highlights

• •N-glycosylation site analysis of the hemipteran pest insect Nilaparvata lugens.
• •Differential N-glycosylation of proteins is observed between male and female adults.
• •Sex-specific glycoproteins are involved in insect reproduction.

     

A Simple Sequence Repeat- and Single-Nucleotide Polymorphism-Based Genetic Linkage Map of the Brown Planthopper,Nilaparvata lugens

In this study, we developed the first genetic linkage map for the major rice insect pest, the brown planthopper (BPH, Nilaparvata lugens). The linkage map was constructed by integrating linkage data from two backcross populations derived from three inbred BPH strains. The consensus map consists of 474 simple sequence repeats, 43 single-nucleotide polymorphisms, and 1 sequence-tagged site, for a total of 518 markers at 472 unique positions in 17 linkage groups. The linkage groups cover 1093.9 cM, with an average distance of 2.3 cM between loci. The average number of marker loci per linkage group was 27.8. The sex-linkage group was identified by exploiting X-linked and Y-specific markers. Our linkage map and the newly developed markers used to create it constitute an essential resource and a useful framework for future genetic analyses in BPH.     

水稻焦磷酸:D-果糖-6-磷酸1-磷酸转移酶α亚基的cDNA克隆及表达

F。。PZ（果糖一2,6一二磷酸）是真核生物中广泛存在的小分子代谢调节物,而PFP则是它的一个广泛存在于植物组织中的重要靶酶（Stilt1990）。该酶在80年代初被发现并为植物生化界所重视。它催化下列可逆反应：F。P＋PPi-Fl,。PZ＋Pi。此酶既可在酵解或生糖作用中催化形成净碳流（Hatzfeld等1989）,也可以与PFK或F;,6Pase形成循环催化PPi的产生和消除（Sung等1988）。许多植物的urn由a和P两种亚基组成（Botha等1988,Yan和Tao1984）。其中a亚基为调节亚基,与F。,。PZ对催化活性的调节有关;卢亚基为催化亚基,具有活性位…     

玉米21kD富硫种子贮存蛋白的cDNA克隆及其序列分析

利用逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）方法从玉米掖单－２０开花后１０ｄ的叶片中分离到２１ｋＤ玉米种子贮存蛋白ｃＤＮＡ（Ｎ２１ＫＺＹ）,并进行了序列分析．其编码蛋白包含２１１个氨基酸,极其富含甲硫氨酸,高达２７％;其Ｎ端有一个２１个氨基酸的信号肽．Ｎ２１ＫＺＹ及其编码蛋白和Ｃｈｕｉ等人分离的该基因的基因组克隆及其编码蛋白的同源性分别为９５．１％和９０．５％;两者的编码蛋白与玉米１０ｋＤ醇溶蛋白极其相似,其中间多出一个５４氨基酸的肽段和一个６氨基酸的肽段,这表明它们可能是来源于同一个祖先基因,后来通过基因重排、缺失或不均等交换等过程而形成的不同的蛋白质．     

水稻品种和稻虱缨小蜂对褐飞虱协同作用的模拟分析

利用我们先前的试验结果和文献资料,组建了水稻品种－褐飞虱－稻虱缨小蜂３者相互关系的模拟模型．模拟分析表明水稻品种能直接和间接地通过物理或化学特性影响稻虱缨小蜂的寄生作用,其中以影响褐飞虱卵垂直分布型、稻虱缨小蜂的迁入时间、未成熟期历期和怀印量等的水稻品种特性起主要作用．同时,模拟分析还表明中等抗性的水稻品种,配以增强稻虱缨小蜂寄生作用的品种特性,就能有效地控制褐飞虱的危害．文中还就协调水稻品种抗性与天敌协同控制褐飞虱的途径进行了讨论．     

Biological Studies on Agamermis unka (Nematoda: Mermithidae), a Parasite of the Brown Planthopper Nilaparvata lugens

The mermithid parasite, Agamermis unka, is the most important natural enemy of the brown planthopper (BPH), Nilaparvata lugens, in Korean rice fields. Very little is known about many aspects of the mermithid's life cycle and behavior, and a study was undertaken to close the data gap. The sex ratio of A. unka isolated from field-collected BPH showed a strong female bias. Even when several A. unka occurred within a BPH, the majority were females. Similarly, the sex ratio of field-collected A. unka adults that were in the soil was strongly biased towards females and, in many instances, the females were found in the absence of males. Females collected from the field from January to May and maintained in water at 25 C had a mean pre-oviposition period of 17-28 days and a mean oviposition period of 17-37 days, and averaged 543-1851 eggs/female. The eggs averaged 20, 17 and 36 days to hatch at 30, 25 and 20 C respectively, but none hatched at 15 C. Most of the eggs (96%) hatched at 25 and 20 C, but only 64% hatched at 30 C. Agamermis pre-parasites could be found on rice stems in the field and laboratory. In the field, BPH-susceptible and BPH-resistant rice cultivars showed no significant difference in the numbers of pre-parasites on the stem. In the laboratory, the number of pre-parasites recovered/rice stem was significantly higher in two out of three trials when BPH nymphs and adults were present. When BPH nymphs were exposed to the pre-parasites in the laboratory, 39% of the brachypterous females and 4.5% of the brachypterous males were parasitized, whereas 0.3% of the macropterous females and 0% of the macropterous males were parasitized. The parasitism data obtained under field conditions showed similar trends. The reason(s) for these differences in parasitism between the BPH sex and wing types that have been observed both in the laboratory and field is unknown. Because brachypterous males and macropterous females and males occur in lesser numbers than the brachypterous females, this may, in part, account for the differences in parasitism observed. However, BPH behavior cannot be discounted as a factor in the differential parasitism.