Enhanced Resistance of Snowdrop Lectin (Galanthus nivalis L. Agglutinin)-Expressing Maize to Asian Corn Borer (Ostrinia furnacalis Guenée)

In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through the Agrobacterium tumefaciens-mediated also studied. Thirty-six independently derived plants were subjected to molecular analyses. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of three GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to ACB. These plants synthesized GNA at levels above 0.24% total soluble protein and enhanced resistance to ACB was demonstrated by exposing the plants to insects under greenhouse conditions. Semi-artificial diet bioassays also showed the toxic effect of GNA on ACB. Field evaluation of the transgenic plants supported the results from the artificial trial. In the present study, we have obtained new insect-resistant maize material for further breeding work and have found that GNA-expressing plants not only gained significant resistance to homopterans, but also showed toxicity to ACB, which is a type of Lepidoptera.     

Enhancement of resistance to aphids by introducing the snowdrop lectin genegna into maize plants

In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through theAgrobacterium tumefaciens- mediated method. The toxicity of GNA-expressing plants to aphids has also been studied. The independently derived plants were subjected to molecular analyses. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that thegna gene was integrated into maize genome and inherited to the following generations. The typical Mendelian patterns of inheritance occurred in most cases. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of nine GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to aphids. These plants synthesized GNA at levels above 0.22% total soluble protein, and enhanced resistance to aphids was demonstrated by exposing the plants to corn leaf aphid (Rhopalosiphum maidis Fitch) under greenhouse conditions. The nymph production was significantly reduced by 46.9% on GNA-expressing plants. Field evaluation of the transgenic plants supported the results from the inoculation trial. After a series of artificial self-crosses, some homozygous transgenic maize lines expressing GNA were obtained. In the present study, we have obtained new insect-resistant maize material for further breeding work.     

Resistance to green leafhopper (Nephotettix virescens) and brown planthopper (Nilaparvata lugens) in transgenic rice expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA)

Transgenic rice plants expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA) were screened for resistance to green leafhopper (Nephotettix virescens; GLH), a major homopteran pest of rice. Survival was reduced by 29% and 53% (P<0.05) respectively, on plants where GNA expression was tissue-specific (phloem and epidermal layer) or constitutive. Similar levels of resistance in GNA-expressing transgenic rice were previously reported for rice brown planthopper (Nilaparvata lugens; BPH). GNA binding to glycoproteins in gut tissues showed that BPH contained more "receptors" than GLH, and that the binding affinity was stronger, particularly in the midgut. Subsequent toxicity of GNA is thus unlikely to be directly related to the amount of lectin bound. GNA was not detected in the honeydew of either insect species when they were fed on GNA-expressing plants, in contrast to results from artificial diet studies. This result suggests that GNA is not being delivered to the insect efficiently. When offered a free choice vs control plants, BPH nymphs tended to avoid plants expressing GNA; avoidance was less pronounced and took longer to develop on plants where GNA expression was tissue-specific, In contrast to BPH, GLH nymphs were attracted to plants expressing GNA, whether constitutively or in a tissue-specific manner.     

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.     

Transgenic GNA Expressing Potato Plants Augment the Beneficial Biocontrol of Lacanobia Oleracea (Lepidoptera: Noctuidae) by the parasitoid Eulophus Pennicornis (Hymenoptera; Eulophidae)

The effect of expressing the gene encoding snowdrop lectin (Galanthus nivalis agglutinin, GNA) in transgenic potato plants, on parasitism of the phytophagous insect pest Lacanobia oleracea by the gregarious ectoparasitoid Eulophus pennicornis, was investigated in glasshouse trials. Expression of GNA (approx. 1.0% total soluble protein) by transgenic plants significantly reduced the level of pest damage, thus confirming previous studies. Furthermore, the presence of the parasitoid significantly reduced the levels of damage incurred either by the transgenic or control plants when compared to those plants grown in the absence of the parasitoid. For the GNA expressing plants the presence of the parasitoid resulted in further reductions (ca. 21%) in the level of damage caused by the pest species. The ability of the wasp to parasitise and subsequently develop on the pest larvae was not altered by the presence of GNA in the diet of the host. E. pennicornis progeny that developed on L. oleracea reared on GNA expressing plants showed no significant alteration in fecundity when compared with wasps that had developed on hosts fed on control potato plants, although mean size and longevity of female parasitoids was significantly reduced. The number of F ₂ progeny produced by parasitoids derived from hosts fed on GNA-expressing plants was not significantly different to those produced by parasitoids from hosts fed control plants. Results from the present study demonstrate that the use of transgenic plants expressing insecticidal proteins can be compatible with the deployment of beneficial insects and that the two factors may interact in a positive manner.     

农杆菌介导的雪花莲凝集素基因转入玉米骨干自交系

以农杆菌AGL0介导,将雪花莲凝集素基因转入玉米骨干自交系齐319和掖515胚性愈伤组织细胞,从筛选后的抗性愈伤组织获得再生植株。农杆菌浓度和共培养时间均能显著影响侵染后玉米愈伤组织的抗性频率。在农杆菌浓度OD600 0．2～0．3,共培养时间3d时,侵染后玉米愈伤组织的抗性频率最高,平均约4％。对再生植株及其子代基因组DNA的PCR及Southern杂交分析表明雪花莲凝集素基因已经整合到玉米基因组中,并遗传给后代。在蚜虫人工接种试验中,转基因植株上蚜虫的繁殖力为非转基因对照植株上的50％,这表明转基因植株抗蚜性显著增强。     

转修饰cry1Ac基因籼稻明恢81经花药培养获得抗虫DH系

对基因枪法获得的明恢81转修饰的cry1Ac基因当代植株进行花药培养,共接种花药2600枚,获得83份花培植株,其中双倍体植株43份,单倍体植株40份,PCR结果表明含有cry1Ac基因的植株55份,花培植株群体中转基因与非转基因植株的比值为2：1（55／28）,进一步结合Southern blot和ELISA分析,于花培植株当代筛选到转基因纯合株系36份,外源蛋白表达量上,花药来源于同一克隆的DH系的不同植株之间基本一致,最高的Cry1Ac含量达0．25％,田间抗虫性试验表明,经花药培养纯合获得的部分转基因纯合系植株对二化螟（chilo suppressalis)表现出高抗,而且主要农艺性状保持不变,以上结果表明水稻花药培养可以加速转基因的纯合与良种利用。     

Use of hi ii-elite inbred hybrids in Agrobacterium-based transformation of maize

Summary Hybrid embryos resulting from crosses between a highly regenerable maize germplasm (Hi II) and certain elite inbreds were treated with Agrobacterium tumefaciens containing the uidA (GUS) and pat genes under the control of different constitutive promoters. Six of the elite inbred lines were derived from a Lancaster background and three were derived from an Iowa Stiff Stalk background. Hybrid embryos from all three Stiff Stalk lines gave transgenic events at various frequencies, two of them at a comparable frequency to that observed with Hi II embryos. Embryos from only one Lancaster/Hi II hybrid were successfully transformed and the frequency was quite low. Additional Lancaster elite inbreds were then tested as a hybrid with Hi II and failed to produce a single transgenic event. The transgenic Hi II/elite events showed many characteristics of ‘hybrid vigor’ including more aggressive rooting, thicker stems, and taller stature than plants derived from Hi II events. The hybrid T₀ plants exhibited excellent tassel development in the greenhouse with abundant pollen shed. Seed set in the greenhouse was significantly (3–5-fold) higher than with Hi II transformats. Attempts to transform embryos derived from self or sibling crosses of the four inbred lines that were successful as hybrids with Hi II did not produce any transgenic events. T₀ plants having ∼50% elite genomic contribution perform nearly as well in the greenhouse as seed-derived elite inbred parents and offer a significantly reduced time line for recombinant protein product development from transgenic plants.     

雪花莲凝集素基因（gna）的改造及其抗蚜性

用定点突变方法对编码雪花莲凝集素（Galanthus nivalis agglutinin,GNA)前体蛋白的DNA序列进行了改造和转基因烟草9Nicotana tabacum L.）抗蚜性的研究。结果表明,将GNA编码序列中含有的稀有密码子改造后,GNA的表达水平从占总可溶性蛋白的0.17％增加到0.25％,转基因烟草的抗蚜性也随之增强,从平均抑制桃蚜（Myzus per-sicae(Sulzer））虫口密度63.7％显地提高到71.0％。     

转AtNHX1基因玉米的产生及其耐盐性分析

以玉米(ZeamaysL.)骨干自交系DH4866、齐319和鲁原16106的胚性愈伤组织为材料,采用农杆菌介导法将AtNHX1和hpt基因转入玉米培养细胞,经筛选获得了抗潮霉素的愈伤组织并再生植株。经PCR检测和Southernblot验证,确定了22.8%的再生植株为转基因植株。农杆菌液浓度、愈伤组织基因型及共培养时间对转化率均有明显影响。外源基因在转基因植株后代中的分离呈多样性,在部分株系中表现出孟德尔遗传规律。耐盐筛选表明,一些转基因植株及其后代具有很好的耐盐性,部分株系可在0.8%-1.0%NaCl溶液浇灌下萌发和生长。Northern杂交表明,植株耐盐性提高与AtNHX1基因的转录水平相一致。     

Influence of plant development and environment on transgene expression in potato and consequences for insect resistance

Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance.     

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.     

转AtNHX1基因玉米的产生及其耐盐性分析

以玉米(Zea mays L.)骨干自交系DH4866、齐319和鲁原16106的胚性愈伤组织为材料,采用农杆菌介导法将AtNHX1和hpt因转入玉米培养细胞,经筛选获得了抗潮霉素的愈伤组织并再生植株.经PCR检测和Southernblot验证,确定了22.8%的再生植株为转基因植株.农杆菌液浓度、愈伤组织基因型及共培养时间对转化率均有明显影响.外源基因在转基因植株后代中的分离呈多样性,在部分株系中表现出孟德尔遗传规律.耐盐筛选表明,一些转基因植株及其后代具有很好的耐盐性,部分株系可在0.8%-1.0%NaCl溶液浇灌下萌发和生长.Northern杂交表明,植株耐盐性提高与AtNHX1基因的转录水平相一致.     

Production of Transgenic Rice Homozygous Lines with Enhanced Resistance to the Rice Brown Planthopper

Mature seed‐derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was cotransformed with two plasmids, pWRG1515 and pRSSGNA1,containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β‐glucuronidase gene (gusA) and the snow‐drop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin‐containing medium, resistant callus was transferred to hygromycin‐containing regeneration medium for plant regeneration. Twenty‐six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy‐three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis. Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parentplants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes.Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding.This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis‐based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.     

影响农杆菌介导玉米优良自交系遗传转化的因素

以我国玉米骨干自交系9046,齐319,414,Mo17的幼胚为材料,在已经建立的农杆菌介导的玉米幼胚转化体系的基础上,研究了影响农杆菌介导玉米优良自交系遗传转化的因素,建立了优化的玉米优良自交系的遗传转化体系。研究结果表明,1．0—2．0mm的玉米幼胚是最适宜的转化受体;在感染液和共培养基中都加入乙酰丁香酮(200μmol／L)和抗坏血酸(50mg／L),能显著提高农杆菌对玉米的侵染能力;而感染前将幼胚高渗透压预处理未能提高转化率;延迟筛选有利于提高抗性愈伤组织的存活率。应用优化后的转化体系,获得了这4个玉米优良自交系的转基因植株,PCR阳性植株率为1．71％-4．09％。转化植株叶片总DNA的PCR和Southern杂交分析表明,T-DNA上的外源基因已经整合进了玉米基因组,并且在大多数转基因植株(71．4％)中为单位点插入。这一体系的建立,为进一步将有用基因导入玉米优良自交系奠定了基础。     

Effects of artificial diet containing GNA and GNA-expressing potatoes on the development of the aphid parasitoid Aphidius ervi Haliday (Hymenoptera: Aphidiidae)

Aphid parasitoids are important biological control agents. The possibility arises that whilst foraging on insect-resistant transgenic plants, they are themselves at risk from direct and indirect effects of the expression of a transgene used to control the pest species. A liquid artificial diet was successfully used to deliver the snowdrop lectin (Galanthus nivalis agglutinin; GNA) to the peach-potato aphid, Myzus persicae. Bioassays utilising artificial diet incorporating GNA, and excised leaves of the GNA-expressing transgenic potato line, GNA2#28, were performed to assess the potential effects of GNA on the development of the aphid parasitoid Aphidius ervi. The results indicate that GNA delivered via artificial diet to the aphids can be transferred through the trophic levels and has a dose-dependent effect on parasitoid development. Parasitoid larvae excreted most of the ingested GNA in the meconium but some of it was detected in the pupae. Although A. ervi development was not affected when developing within hosts feeding on transgenic potato leaves, this probably reflected sub-optimal expression of the toxin in the transgenic potato line used     

Engineering of enhanced glycine betaine synthesis improves drought tolerance in maize

Glycine betaine plays an important role in some plants, including maize, in conditions of abiotic stress, but different maize varieties vary in their capacity to accumulate glycine betaine. An elite maize inbred line DH4866 was transformed with the betA gene from Escherichia coli encoding choline dehydrogenase (EC 1.1.99.1), a key enzyme in the biosynthesis of glycine betaine from choline. The transgenic maize plants accumulated higher levels of glycine betaine and were more tolerant to drought stress than wild-type plants (non-transgenic) at germination and the young seedling stage. Most importantly, the grain yield of transgenic plants was significantly higher than that of wild-type plants after drought treatment. The enhanced glycine betaine accumulation in transgenic maize provides greater protection of the integrity of the cell membrane and greater activity of enzymes compared with wild-type plants in conditions of drought stress.     

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests

The adoption of pest‐resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.     

Development of transgenic rice pure lines with enhanced resistance to rice brown planthopper

Summary Mature seed-derived callus from an elite Chinese japonica rice cv. Ewan 5 was cotransformed with two plasmids, pWRG1515 and pRSSGNAl, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. Thirty-five independent transgenic rice plants were regenerated from 177 bombarded calluses. Eighty-three percent of the transgenic plants contained all three genes, as revealed by Southern blot analysis. Western blot analysis revealed that 23 out of 29 gna-containing transgenic plants expressed Galanthus nivalis agglutinin (GNA) (79%) at various levels, with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of all three transgenes (gna, hpt and gusA) in the R2 progeny. Amongst the R2 generation two independent homozygous lines were identified that expressed all three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing the survival, overall fecundity of BPH, retarding development, and decreasing the feeding of BPH. These BPH-resistant lines have been incorporated into a rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH.     

转苏云金杆菌毒蛋白基因玉米植株的获得及其抗虫性分析

玉米( Zea mays L.)转化成功与否与基因型密切相关.在转化过程中,除少数模式品种能够形成再生频率较高且易转化的Ⅱ型愈伤组织外,大多数栽培品种往往只能够形成再生频率较低且不易转化的Ⅰ型愈伤组织.因此探索Ⅰ型愈伤组织的诱导及其转化条件,提高转化效率,对直接改良玉米优良自交系具有重要意义.应用基因枪转化技术将苏云金杆菌( Bacillus thuringiensis ) cry1Ac3基因导入玉米优良自交系E28及340的Ⅰ型胚性愈伤组织中,经过膦丝菌素(PPT)或潮霉素(HygB)筛选,获得了再生植株.经PCR检测、Southern blot分析及Bt毒蛋白ELISA检测证实,外源基因已整合到玉米基因组中,并已获得表达.抗虫性分析结果表明,部分转基因玉米植株对玉米螟虫有较强的抗性.还比较了PPT和HygB两种筛选剂的筛选效果,表明PPT筛选的抗性愈伤组织的再生频率要高于HygB筛选的再生频率.