Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper

Snowdrop lectin ( Galanthus nivalis agglutinin; GNA) has been shown previously to be toxic towards rice brown planthopper ( Nilaparvata lugens ; BPH) when administered in artificial diet. BPH feeds by phloem abstraction, and causes ‘hopper burn’, as well as being an important virus vector. To evaluate the potential of the gna gene to confer resistance towards BPH, transgenic rice ( Oryza sativa L.) plants were produced, containing the gna gene in constructs where its expression was driven by a phloem-specific promoter (from the rice sucrose synthase RSs1 gene) and by a constitutive promoter (from the maize ubiquitin ubi1 gene). PCR and Southern analyses on DNA from these plants confirmed their transgenic status, and that the transgenes were transmitted to progeny after self-fertilization. Western blot analyses revealed expression of GNA at levels of up to 2.0% of total protein in some of the transgenic plants. GNA expression driven by the RSs1 promoter was tissue-specific, as shown by immunohistochemical localization of the protein in the non-lignified vascular tissue of transgenic plants. Insect bioassays and feeding studies showed that GNA expressed in the transgenic rice plants decreased survival and overall fecundity (production of offspring) of the insects, retarded insect development, and had a deterrent effect on BPH feeding. gna is the first transgene to exhibit insecticidal activity towards sap-sucking insects in an important cereal crop plant.     

十六个水稻品种(系)对褐飞虱的抗虫性评价

本文通过苗期抗性鉴定、田间抗虫性鉴定、稻田节肢动物功能团优势度比较及产量测定的方法来评价16个水稻品种(系)对褐飞虱Nilaparvata lugens(Stl)的抗性。结果表明,有12个品种(系)对褐飞虱表现为抗级,2个品系表现为中抗。这14个抗虫-中抗的品种(系)在成株期也表现一定的抗虫性,且与对照TN1之间差异显著。本试验条件下,广占63-3S/华恢15、广两优106、Y58S/华15、广两优476、广占63-4S/华恢15这5个品种(系)不仅抗虫性好,而且产量也较高,分别比TN1和主栽品种扬两优6号高63.49%～66.57%和3.47%～11.57%,具有广泛的开发应用前景。     

Expression of the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: effects on predation by the grain aphid Sitobion avenae

Transgenic wheat plants containing the gene encoding snowdrop lectin (Galanthus nivalis agglutinin; GNA) under the control of constitutive and phloem-specific promoters were generated through the particle bombardment method. Thirty-two independently derived plants were subjected to molecular and biochemical analyses. Transgene integration varied from one to twelve estimated copies per haploid genome, and levels of GNA expression from 0 to ca. 0.2% of total soluble protein were observed in different transgenic plants. Seven transgenic plants were selected for further study. Progeny plants from these parental transformants were selected for transgene expression, and tested for enhanced resistance to the grain aphid (Sitobion avenae) by exposing the plants to nymphal insects under glasshouse conditions. Bioassay results show that transgenic wheat plants from lines expressing GNA at levels greater than ca. 0.04% of total soluble protein decrease the fecundity, but not the survival, of grain aphids. We propose that transgenic approaches using insecticidal genes such as gna in combination with integrated pest management present promising opportunities for the control of damaging wheat pests.     

Immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper,Nilaparvata lugens (Stal)

Rice brown planthoppers (Nilaparvata lugens) were fed on artificial diet containing snowdrop lectin (Galanthus nivalis agglutinin; GNA), which has been shown to be toxic towards this insect pest. In addition to decreasing survival, the lectin affected development, reducing the growth rate of nymphs by approximately 50% when present at a concentration of 5.3&mgr;M. Immunolocalisation studies showed that lectin binding was concentrated on the luminal surface of the midgut epithelial cells within the planthopper, suggesting that GNA binds to cell surface carbohydrate moieties in the gut. Immunolabelling at a lower level was also observed in the fat bodies, the ovarioles, and throughout the haemolymph. These observations suggest that GNA is able to cross the midgut epithelial barrier, and pass into the insect's circulatory system, resulting in a systemic toxic effect. Electron microscope studies showed morphological changes in the midgut region of planthoppers fed on a toxic dose of GNA, with disruption of the microvilli brush border region. No significant proteolytic degradation of GNA was observed either in the gut or honeydew of planthoppers fed on lectin-containing diet. The presence of glycoproteins which bind GNA in the gut of the brown planthopper was confirmed using digoxigen-labeled lectins to probe blots of extracted gut polypeptides.     

Expression of the Galanthus nivalis agglutinin (GNA) gene in transgenic potato plants confers resistance to aphids

Aphids, the largest group of sap-sucking pests, cause significant yield losses in agricultural crops worldwide every year. The massive use of pesticides to combat this pest causes severe damage to the environment, putting in risk the human health. In this study, transgenic potato plants expressing Galanthus nivalis agglutinin (GNA) gene were developed using CaMV 35S and ST-LS1 promoters generating six transgenic lines (35S1-35S3 and ST1-ST3 corresponding to the first and second promoter, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the GNA gene was expressed in leaves, stems and roots of transgenic plants under the control of the CaMV 35S promoter, while it was only expressed in leaves and stems under the control of the ST-LS1 promoter. The levels of aphid mortality after 5 days of the inoculation in the assessed transgenic lines ranged from 20 to 53.3%. The range of the aphid population in transgenic plants 15 days after inoculation was between 17.0 ± 1.43 (ST2) and 36.6 ± 0.99 (35S3) aphids per plant, which corresponds to 24.9–53.5% of the aphid population in non-transformed plants. The results of our study suggest that GNA expressed in transgenic potato plants confers a potential tolerance to aphid attack, which appears to be an alternative against the use of pesticides in the future.     

Anti-tumor and anti-viral activities of Galanthus nivalis agglutinin (GNA)-related lectins

Galanthus nivalis agglutinin (GNA)-related lectin family, a superfamily of strictly mannose-binding specific lectins widespread among monocotyledonous plants, is well-known to possess a broad range of biological functions such as anti-tumor, anti-viral and anti-fungal activities. Herein, we mainly focused on exploring the precise molecular mechanisms by which GNA-related lectins induce cancer cell apoptotic and autophagic death targeting mitochondria-mediated ROS-p38-p53 apoptotic or autophagic pathway, Ras-Raf and PI3K-Akt anti-apoptotic or anti-autophagic pathways. In addition, we further discussed the molecular mechanisms of GNA-related lectins exerting anti-viral activities by blocking the entry of the virus into its target cells, preventing transmission of the virus as well as forcing virus to delete glycan in its envelope protein and triggering neutralizing antibody. In conclusion, these findings may provide a new perspective of GNA-related lectins as potential drugs for cancer and virus therapeutics in the future.     

Crystallization and preliminary X-ray diffraction results of snowdrop (Galanthus nivalis) lectin

Well-ordered single crystals have been grown for a mannose-specific lectin from snowdrop (Galanthus nivalis) bulbs in the presence of methyl-alpha-D-mannoside. The space group symmetry is consistent with the orthorhombic space group P2(1)2(1)2(1). The unit cell parameters are a = 140.0 A, b = 64.7 A, c = 62.0 A. The asymmetric unit can accommodate a tetramer. The functional molecule (50,000 daltons) consists of four identical subunits and is highly specific for alpha 1,3-linked mannose oligosaccharides.     

The snowdrop lectin Galanthus nivalis agglutinin (GNA) and a fusion protein ButaIT/GNA have a differential affect on a pest noctuid Lacanobia oleracea and the ectoparasitoid Eulophus pennicornis

Fusion proteins have considerable potential as novel insect control agents because they enable the oral delivery of insecticidal peptides to the haemolymph of pests. Transport is achieved via fusion of the toxin to a carrier protein Galanthus nivalis agglutinin (GNA) that, after ingestion, binds to and crosses the insect gut epithelia. A fusion protein comprising a toxin from the South Indian red scorpion (Mesobuthus tamulus) that is fused to a GNA polypeptide (ButaIT/GNA) has a detrimental effect on the development of tomato moth Lacanobia oleracea (L.) (Lepidoptera: Noctuidae) larvae. The present study examines the effects of ButaIT/GNA and GNA, delivered orally or by injection, on the development of L. oleracea larvae, and the subsequent effects on the gregarious ectoparasitoid Eulophus pennicornis (Nees) (Hymenoptera: Eulophidae) developing on ButaIT/GNA‐ and GNA‐treated hosts. The fusion protein, but not GNA, reduces the growth of fifth stadium L. oleracea larvae. The development of E. pennicornis is not affected by the presence of ButaIT/GNA in hosts that ingest the protein, although it is affected when hosts are injected with the protein. This difference is considered to be a result of higher levels of fusion protein being present when the fusion protein is injected. Intact ButaIT/GNA is detected by immunoassay in the haemolymph of L. oleracea larvae after ingestion of the fusion protein. More unexpectedly, negative effects are observed for the growth of E. pennicornis larvae developing on hosts that have either ingested, or been injected with GNA.     

RFLP-facilitated investigation of the quantitative resistance of rice to brown planthopper ( Nilaparvata lugens)

Quantitative trait loci (QTLs), conferring quantitative resistance to rice brown planthopper (BPH), were investigated using 160 F₁₁ recombinant inbred lines (RILs) from the Lemont/Teqing cross, a complete RFLP map, and replicated phenotyping of seedbox inoculation. The paternal indica parent, Teqing, was more-resistant to BPH than the maternal japonica parent, Lemont. The RILs showed transgressive segregation for resistance to BPH. Seven main-effect QTLs and many epistatic QTL pairs were identified and mapped on the 12 rice chromosomes. Collectively, the main-effect and epistatic QTLs accounted for over 70% of the total variation in damage scores. Teqing has the resistance allele at four main-effect QTLs, and the Lemont allele resulted in resistance at the other three. Of the main-effect QTLs identified, QBphr5b was mapped to the vicinity of gl1, a major gene controlling leaf and stem pubescence. The Teqing allele controlling leaf and stem pubescence was associated with resistance, while the Lemont allele for glabrous stem and leaves was associated with susceptibility, indicating that this gene may have contributed to resistance through antixenosis. Similar to the reported BPH resistance genes, the other six detected main-effect QTLs were all mapped to regions where major disease resistance genes locate, suggesting they might have contributed either to antibiosis or tolerance. Our results indicated that marker-aided pyramiding of major resistance genes and QTLs should provide effective and stable control over this devastating pest. Received: 10 December 2000 / Accepted: 7 May 2001     

水稻麦黄酮对褐飞虱的抗性潜力

使用石油醚、乙酸乙酯、无水乙醇和水依次对抗性水稻品种IR36乙醇提取物进行萃取,并测试了4种萃取物对褐飞虱的活性。结果发现乙酸乙酯萃取物处理后3d的活性最强,褐飞虱1～2龄若虫和3～4龄若虫的死亡率分别是26．0％和48．0％。乙酸乙酯萃取物经柱层析分离得到麦黄酮。将麦黄酮定量加入人工饲料中饲养褐飞虱3龄若虫15d。结果表明,褐飞虱3、4龄若虫的排蜜露量随着麦黄酮在饲料中浓度（50～500μg／ml）的增加而减少,褐飞虱3、4龄若虫的死亡率却随着麦黄酮在饲料中浓度的增加而显著提高。当饲料中麦黄酮的浓度为500μg／ml时,褐飞虱3、4龄若虫的死亡率分别为58．21％和31．75％。麦黄酮50～500μg／ml浓度处理,褐飞虱3龄和4龄若虫的发育历期和相对生长量与对照之间没有明显的差异,表明麦黄酮有拒食作用。用500μg／ml的麦黄酮溶液涂抹到对褐飞虱敏感的水稻品种TN1植株上对褐飞虱雌成虫有明显的拒食作用和忌避产卵作用。本项研究结果表明水稻源的麦黄酮在水稻对褐飞虱的抗性中有重要的作用。     

Ferritin acts as the most abundant binding protein for snowdrop lectin in the midgut of rice brown planthoppers (Nilaparvata lugens)

The mannose-specific snowdrop lectin [Galanthus nivalis agglutinin (GNA)] displays toxicity to the rice brown planthopper Nilaparvata lugens. A 26kDa GNA-binding polypeptide from N. lugens midgut was identified by lectin blotting and affinity chromatography, and characterized by N-terminal sequencing. This polypeptide is the most abundant binding protein for GNA in the N. lugens midgut. A cDNA (fersub2) encoding this protein was isolated from an N. lugens cDNA library. The deduced amino acid sequence shows significant homology to ferritin subunits from Manduca sexta and other arthropods, plants and vertebrates, and contains a putative N-glycosylation site. Native ferritin was purified from whole insects as a protein of more than 400kDa in size and characterized biochemically. Three subunits of 20, 26 and 27kDa were released from the native complex. The 26kDa subunit binds GNA, and its N-terminal sequence was identical to that of fersub2. A second cDNA (fersub1), exhibiting strong homology with dipteran ferritin, was identified as an abundant cDNA in an N. lugens midgut-specific cDNA library, and could encode the larger ferritin subunit. The fersub1 cDNA carries a stem-loop structure (iron-responsive element) upstream from the start codon, similar to structures that have been shown to play a role in the control of ferritin synthesis in other insects.     

重庆秀山褐飞虱田间种群动态及其应用

分析23年(1984~2006)重庆市秀山县褐飞虱Nilaparvata lugens(Stal)观测圃与田间的系统调查资料,揭示出该虫年度间和年度内的田间种群发生动态规律。褐飞虱在观测圃条件下,2000年前常是大发生以上,2000年以来发生程度变轻,常是中等偏重以下,但近3年来又有回升的势头,各年均呈比大田发生偏重的趋势。年度间主害期呈推迟的趋势。年度内,稻苗移栽后即可见虫,一直到收获;前期虫量较小,中期增多,后期较大,主害期在后期。观测圃的褐飞虱发生高峰期比大田早5d左右。观测圃虫量在水稻前期与大田相当,中、后期有差异,特别是后期差异较大,是大田的2~10倍。观测圃的天敌种类主要是蜘蛛、黑肩绿肓蝽、螯蜂、隐翅虫和稻红瓢虫;天敌数量在水稻中期和后期分别比大田多30%和40%以上,对褐飞虱有一定的控制作用。还讨论褐飞虱田间种群动态研究在生产上的应用。     

雪花莲凝集素基因转化小麦及转基因小麦抗蚜性的研究

雪花莲凝集素对具有刺吸式口器的同翅目害虫具有毒杀作用。用基因枪法将1个新的雪花莲凝集素(GNA)基因转入普通春小麦品种中-60634和生产上正在推广的冬小麦高产品种——豫麦66中,分别获得了转基因小麦植株。抗蚜实验证明,转化gna基因的小麦植株对我国北方冬麦区的主要麦蚜——麦长管蚜和禾谷缢管蚜的抗性效果不尽相同。对禾谷缢管蚜,在接种当代即表现出明显的毒杀作用。对麦长管蚜,则表现为虫体发育减缓并且降低了其所生产的若蚜成活率。在自然放养条件下,gna基因则对这两种麦蚜的取食均起到了一定的抑制作用。     

惠州地区褐飞虱对几种药剂的抗药性监测

2009年采用稻茎浸渍法测定广东省惠州地区褐飞虱Nilaparvata lugens(Stl)种群对吡虫啉、噻嗪酮、异丙威、丁烯氟虫腈、烯定虫胺和毒死蜱等杀虫剂的敏感性,测定结果表明:当地褐飞虱种群对吡虫啉产生了极高水平抗性(抗性倍数为422.2倍),对噻嗪酮、异丙威产生了中等水平抗性(抗性倍数分别为11.0和14.0倍),对丁烯氟虫腈仍处于敏感性降低(抗性倍数为3.7倍),对烯定虫胺和毒死蜱敏感(抗性倍数<3倍)。基于褐飞虱对这6种药剂抗性的明显差异,对田间治理褐飞虱合理使用药剂进行了讨论。     

Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris

The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l^–1 at the 200 l scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.     

In vitro and in vivo binding of snowdrop (Galanthus nivalis agglutinin; GNA) and jackbean (Canavalia ensiformis; Con A) lectins within tomato moth (Lacanobia oleracea) larvae; mechanisms of insecticidal action

When fed in semi-artificial diet the lectins from snowdrop (Galanthus nivalis: GNA: mannose-specific) and jackbean (Canavalia ensiformis: Con A: specific for glucose and mannose) were shown to accumulate in vivo in the guts, malpighian tubules and haemolymph of Lacanobia oleracea (tomato moth) larvae. Con A, but not GNA, also accumulated in the fat bodies of lectin-fed larvae. The presence of glycoproteins which bind to both lectins in vitro was confirmed using labelled lectins to probe blots of polypeptides extracted from larval tissues. Immunolocalisation studies revealed a similar pattern of GNA and Con A binding along the digestive tract with binding concentrated in midgut sections. Binding of lectins to microvilli appeared to lead to transport of the proteins into cells of the gut and malpighian tubules. These results suggested that both lectins are able to exert systemic effects via transport from the gut contents to the haemolymph across the gut epithelium. The delivery of GNA and Con A to the haemolymph was shown to be dependent on their functional integrity by feeding larvae diets containing denatured lectins. Con A, but not GNA, was shown to persist in gut and fat body tissue of lectin-fed larvae chased with control diet for three days. Con A also shows more extensive binding to larval tissues in vitro than GNA, and these two factors are suggested to contribute to the higher levels of toxicity shown by Con A, relative to GNA, in previous long term bioassays.     

Consumption of snowdrop lectin (<Emphasis Type="Italic">Galanthus nivalis </Emphasis>agglutinin) causes direct effects on adult parasitic wasps

Honeydew is a common sugar-rich excretion of aphids and other phloem-feeding insects and represents the primary sugar in many agricultural systems. When honeydew-producing insects feed on genetically modified plants, the honeydew can contain amounts of the transgene product. Here we address whether this route of exposure poses a risk for non-target insects. Three species of parasitic wasps were selected: i.e. Aphidius colemani, Trichogramma brassicae and Cotesia glomerata, all of which are known to use honeydew as a carbohydrate source in the field. Wasps were fed sucrose solutions with varying concentrations of snowdrop lectin ( Galanthus nivalisagglutinin, GNA), a protein that has been engineered into crops to confer resistance against homopteran pests and that has been detected in honeydew. Parameters evaluated included gustatory response, longevity, fecundity, progeny emergence and sex ratio. While A. colemani and T. brassicae, but not C. glomerata, were able to detect GNA, this gustatory recognition had no effect on the acceptance of a GNA-sucrose solution. In all three species, GNA ingestion reduced parasitoid survival significantly. However, in respect to fecundity, negative effects were observed for T. brassicae but not for A. colemani. The results suggest that the effect of GNA consumption may depend on the specifics of a parasitoid's biology, especially its longevity and its mode of egg maturation.     

Repeated copulation in the brown planthopper, Nilaparvata lugens Stál (Homoptera; Delphacidae)

Abstract. 1. Mating of the brown planthopper, Nilaparvata lugens , was investigated in relation to oviposition. Females became unreceptive immediately after mating and showed various types of repelling behaviour to courting males. As a result, females usually did not mate repeatedly in quick succession, but after ceasing to lay fertilized eggs they behaved as virgins and mated again before producing more fertilized eggs.
2. Copulation, followed by deposition of fertilized eggs, occurred twice, or in a few cases three times, throughout the adult stage. Copulation lasted about 2 min at the first mating and about 1 min at the second or third mating.
3. When the number of fertilized eggs began to decrease rapidly, oviposition rate also decreased, but it increased again immediately after re-mating. Repeated copulation was also related to the potential rate of population increase. In the present experiments, the estimates of net reproductive rate ( R ₀) in brachypterous females was 287.7 (first mating), 88.3 (second mating) and 7.2 (third mating) and in macropterous females 286.8, 120.6 and 3.6 respectively.
4. In a patchy environment, repeated copulation following an invasion may contribute to the increase of a population.     

Identification of a rice gene (Bph 1) conferring resistance to brown planthopper (Nilaparvata lugens Stal) using STS markers

This study was carried out to identify a high-resolution marker for a gene conferring resistance to brown planthopper (BPH) biotype 1, using japonica type resistant lines. Bulked segregant analyses were conducted using 520 RAPD primers to identify RAPD fragments linked to the BPH resistance gene. Eleven RAPDs were shown to be polymorphic amplicons between resistant and susceptible progeny. One of these primers, OPE 18, which amplified a 923 bp band tightly linked to resistance, was converted into a sequence-tagged-site (STS) marker. The STS marker, BpE18-3, was easily detectable as a dominant band with tight linkage (3.9cM) to Bph1. It promises to be useful as a marker for assisted selection of resistant progeny in backcross breeding programs to introgress the resistance gene into elite japonica cultivars.     

地上部植食者褐飞虱对不同水稻品种土壤线虫群落的影响

地上和地下部生物群落的交互作用对于调控陆地生态过程具有重要作用。在盆栽条件下利用2×2析因设计研究了褐飞虱(Nilaparvata lugens)取食不同水稻品种后对土壤线虫群落的影响。结果表明, 褐飞虱侵害水稻9 d后, 感虫品种(广四和汕优63)的土壤线虫总数、属数及自生线虫(食细菌线虫、食真菌线虫和捕食性线虫)数量增加, 并且一般达到显著水平(P<0.05); 而上述指标在抗虫品种(汕优559和IR36)土壤中则呈现相反的趋势。植食性线虫数量在强感虫品种广四上显著增加(P<0.05), 而在强抗虫品种IR36上显著减少(P<0.05)。褐飞虱和水稻品种对土壤线虫的生态指数(线虫通道指数、Shannon-Wiener指数、成熟度指数、富集指数和结构指数)没有明显影响, 可能与供试土壤线虫群落组成单一及褐飞虱作用时间较短有关。总之, 褐飞虱强烈影响土壤线虫数量、群落组成和营养结构, 并且作用的方向(促进或抑制)和程度依赖于水稻的品种特性, 揭示出地上部植食者的短期侵害将对稻田土壤生态系统的结构和功能产生深远影响。