Immature embryos of rice varieties "Xiushui11" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticid...Immature embryos of rice varieties "Xiushui11" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant cant were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56%-72% measured as numbers of Geneticin (G418)-resistant calli produced and 36%-60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38%-61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16%-75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.展开更多
Transgenic plants expressing foreign genes resulting in resistance against pests could make significant contribu-tion to sustainable agriculture.Considerable progress has been achieved to control the chewing insect pe...Transgenic plants expressing foreign genes resulting in resistance against pests could make significant contribu-tion to sustainable agriculture.Considerable progress has been achieved to control the chewing insect pests by the development of transgenic plants expressing Bacillus thuringiensis endotoxins(Barton and Miller,1993)or plant-derived proteins such as protease inhibitor(Hoffman et al.,1992;Hilder et al.,1993)or lectins(Boulter et al.,1990).However,there are fewer reports on the successful control of sap-sucking insects belonging to the order Homoptera by the use of genetic engineering technology.展开更多
Vegetative Insecticidal Proteins(VIPs), a large family of insecticidal proteins, are produced from Bacillus thuringiensis(Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticide...Vegetative Insecticidal Proteins(VIPs), a large family of insecticidal proteins, are produced from Bacillus thuringiensis(Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticides that confer a wider insecticidal spectrum and have stronger activity. This work compared the geographical distribution of Bt strains and their vip3 genes in different climatic zones in China, the tropical(Hainan Province), subtropical(Guangxi Province) and temperate zones(Heilongjiang Province). A total of 156 Bt strains were isolated from 841 soil samples in Hainan Province tropical region, 356 Bt strains from 1 420 soil samples in Guangxi Province and 167 Bt strains from 1 010 soil samples in different geographical regions in Heilongjiang Province. Twenty-two out of 156 strains from tropical Hainan Province and two out of 356 from subtropical Guangxi Province were found to express vip3 genes, while vip3 genes were not expressed from temperate zone in Heilongjiang Province. Restriction Fragment Length Polymorphism(RFLP) was used to identify different types of vip3 genes that were within the same family and three fulllength vip3 genes were isolated. The genes cloned from Bacillus thuringiensis strain SL3 expressed in the transformed E. coli BL21 strain. Through SDS-PAGE, 88.6 ku insecticidal protein was expressed. The bioassays used two-instar larva of Lepidoptera insects(Spodoptera exigua and Agrotis ipsilon) were performed. The results of the bioassays showed that the protein strongly inhibited the body weight increasement on Spodoptera exigua and Agrotis ipsilon in a standard bioassay. Taken together, the results indicated that the distribution of Bt strains and vip3 genes had regional preference. Tropical and subtropical regions were the rich resources of Bt strains and vip3 genes compared with temperate region. These results would undoubtedly facilitate the studies of insecticidal proteins and expand the list of the pest-killing candidates to make fully use of the extremely rich microbial resources. The new vip3 genes isolated in the current study might also help resolve the emerging insecticidal resistance problems.展开更多
A protocol is presented for genetically engineering loblolly pine (Pinus taeda L.) using particle bombardment. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to...A protocol is presented for genetically engineering loblolly pine (Pinus taeda L.) using particle bombardment. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Plasmid pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) cryIAc coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (Nos) terminator sequences, and the selectable marker gene, neomycin phosphotransferase II (nptII) controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected by kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was induced from the kanamycin-resistant callus, and transgenic plantlets were then produced. The presence of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern blot analysis, and insect feeding assays. The recovered transgenic plants were acclimatized and then established in soil.展开更多
In assessing an insect-resistant genetically engineered （IRGE） crop before its commercialization, researchers normally use so-called ＂Tier-1 assays＂ as the initial step to determine the effects of the crop on non-...In assessing an insect-resistant genetically engineered （IRGE） crop before its commercialization, researchers normally use so-called ＂Tier-1 assays＂ as the initial step to determine the effects of the crop on non-target organisms. In these tests, the insecticidal proteins （IPs） produced by the IRGEs are added to the diets of test organisms in the laboratory. Test organisms in such assays can be directly exposed to much higher concentrations of the test IPs than they would encounter in the field. The results of Tier-1 assays are thus more conservative than those generated in studies in which the organisms are exposed to the IPs by feeding on IRGE plant tissue or in the case of predators or parasites, by feeding on invertebrate prey or hosts that have fed on IRGE plant tissue. In this report, we consider three important factors that must be considered in Tier-1 assays： （i） methods for delivery of the IP to the test organisms; （ii） the need for and selection of compounds used as positive controls; and （iii） methods for monitoring the concentration, stability and bioactivity of the IP during the assay. We also analyze the existing data from Tier-1 assays regarding the toxicity of Bt Cry proteins to non-target arthropod species. The data indicate that the widely used Bt proteins have no direct toxicity to non-target organisms展开更多
Behavior and fitness are important ecological traits frequently measured in insect bioassays.A common method to measure them in soft-bodied herbivorous insects involves confining individuals to plant leaves using clip...Behavior and fitness are important ecological traits frequently measured in insect bioassays.A common method to measure them in soft-bodied herbivorous insects involves confining individuals to plant leaves using clip cages.Although studies have pre-viously highlighted the negative effects of clip cages on leaf physiology,little is known about the impact that using this confinement method has on insect fitness.The responses of different aphid genotypes/clones to different containment methods have not previously been investigated.Here we measured key fitness traits(intrinsic rate of natural increase,mean relative growth rate,time to reach reproductive adulthood and population doubling time)in the potato aphid,Macrosiphum euphorbiae Thomas(Hemiptera:Aphididae),when confined to plants using two methods:(1)clip cages to confine aphids to individual strawberry leaves and(2)a mesh bag to confine aphids to whole strawberry plants.Our study identified a strong negative impact on all the measured aphid fitness traits when using clip cages instead of mesh bags.We also identified genotype-specific differences in response to confinement method,where clip cage confinement differentially affected the fitness of a given aphid genotype compared to the same genotype on whole plants.These results suggest that clip cage use should be carefully considered when experiments seek to quantify insect fitness and that whole plants should be used wherever possible.Given the prevalence of clip cage use in insect bioassays,our results highlight the need for cau-tion when interpreting the existing literature as confinement method significantly impacts aphid fitness depending on their genotype.展开更多
文摘Immature embryos of rice varieties "Xiushui11" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant cant were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56%-72% measured as numbers of Geneticin (G418)-resistant calli produced and 36%-60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38%-61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16%-75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.
文摘Transgenic plants expressing foreign genes resulting in resistance against pests could make significant contribu-tion to sustainable agriculture.Considerable progress has been achieved to control the chewing insect pests by the development of transgenic plants expressing Bacillus thuringiensis endotoxins(Barton and Miller,1993)or plant-derived proteins such as protease inhibitor(Hoffman et al.,1992;Hilder et al.,1993)or lectins(Boulter et al.,1990).However,there are fewer reports on the successful control of sap-sucking insects belonging to the order Homoptera by the use of genetic engineering technology.
基金Supported by National Key R&D Projects(2017YFD0201200)Heilongjiang Provincial National Science Foundation(C2016025)
文摘Vegetative Insecticidal Proteins(VIPs), a large family of insecticidal proteins, are produced from Bacillus thuringiensis(Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticides that confer a wider insecticidal spectrum and have stronger activity. This work compared the geographical distribution of Bt strains and their vip3 genes in different climatic zones in China, the tropical(Hainan Province), subtropical(Guangxi Province) and temperate zones(Heilongjiang Province). A total of 156 Bt strains were isolated from 841 soil samples in Hainan Province tropical region, 356 Bt strains from 1 420 soil samples in Guangxi Province and 167 Bt strains from 1 010 soil samples in different geographical regions in Heilongjiang Province. Twenty-two out of 156 strains from tropical Hainan Province and two out of 356 from subtropical Guangxi Province were found to express vip3 genes, while vip3 genes were not expressed from temperate zone in Heilongjiang Province. Restriction Fragment Length Polymorphism(RFLP) was used to identify different types of vip3 genes that were within the same family and three fulllength vip3 genes were isolated. The genes cloned from Bacillus thuringiensis strain SL3 expressed in the transformed E. coli BL21 strain. Through SDS-PAGE, 88.6 ku insecticidal protein was expressed. The bioassays used two-instar larva of Lepidoptera insects(Spodoptera exigua and Agrotis ipsilon) were performed. The results of the bioassays showed that the protein strongly inhibited the body weight increasement on Spodoptera exigua and Agrotis ipsilon in a standard bioassay. Taken together, the results indicated that the distribution of Bt strains and vip3 genes had regional preference. Tropical and subtropical regions were the rich resources of Bt strains and vip3 genes compared with temperate region. These results would undoubtedly facilitate the studies of insecticidal proteins and expand the list of the pest-killing candidates to make fully use of the extremely rich microbial resources. The new vip3 genes isolated in the current study might also help resolve the emerging insecticidal resistance problems.
文摘A protocol is presented for genetically engineering loblolly pine (Pinus taeda L.) using particle bombardment. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Plasmid pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) cryIAc coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (Nos) terminator sequences, and the selectable marker gene, neomycin phosphotransferase II (nptII) controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected by kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was induced from the kanamycin-resistant callus, and transgenic plantlets were then produced. The presence of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern blot analysis, and insect feeding assays. The recovered transgenic plants were acclimatized and then established in soil.
文摘In assessing an insect-resistant genetically engineered （IRGE） crop before its commercialization, researchers normally use so-called ＂Tier-1 assays＂ as the initial step to determine the effects of the crop on non-target organisms. In these tests, the insecticidal proteins （IPs） produced by the IRGEs are added to the diets of test organisms in the laboratory. Test organisms in such assays can be directly exposed to much higher concentrations of the test IPs than they would encounter in the field. The results of Tier-1 assays are thus more conservative than those generated in studies in which the organisms are exposed to the IPs by feeding on IRGE plant tissue or in the case of predators or parasites, by feeding on invertebrate prey or hosts that have fed on IRGE plant tissue. In this report, we consider three important factors that must be considered in Tier-1 assays： （i） methods for delivery of the IP to the test organisms; （ii） the need for and selection of compounds used as positive controls; and （iii） methods for monitoring the concentration, stability and bioactivity of the IP during the assay. We also analyze the existing data from Tier-1 assays regarding the toxicity of Bt Cry proteins to non-target arthropod species. The data indicate that the widely used Bt proteins have no direct toxicity to non-target organisms
基金The authors would like to thank the Biotechnology and Biological Sciences Research Council and the Fruit Crop Science CTP for funding this projectthe Rural and Environment Science and Analytical Services(RESAS)Division of the Scottish Government through the Strategic Research Programme(2022-2027)+2 种基金the Underpinning National Capacity programme(Service 8.2 Maintenance of pest collections)for supporting the project through the James Hutton InstituteGaynor Malloch at the James Hutton Institute and Danielle Henderson-Holdings at Harper Adams University for their support with molecular workDr Edwin Harris at Harper Adams University for his support with data analysis.
文摘Behavior and fitness are important ecological traits frequently measured in insect bioassays.A common method to measure them in soft-bodied herbivorous insects involves confining individuals to plant leaves using clip cages.Although studies have pre-viously highlighted the negative effects of clip cages on leaf physiology,little is known about the impact that using this confinement method has on insect fitness.The responses of different aphid genotypes/clones to different containment methods have not previously been investigated.Here we measured key fitness traits(intrinsic rate of natural increase,mean relative growth rate,time to reach reproductive adulthood and population doubling time)in the potato aphid,Macrosiphum euphorbiae Thomas(Hemiptera:Aphididae),when confined to plants using two methods:(1)clip cages to confine aphids to individual strawberry leaves and(2)a mesh bag to confine aphids to whole strawberry plants.Our study identified a strong negative impact on all the measured aphid fitness traits when using clip cages instead of mesh bags.We also identified genotype-specific differences in response to confinement method,where clip cage confinement differentially affected the fitness of a given aphid genotype compared to the same genotype on whole plants.These results suggest that clip cage use should be carefully considered when experiments seek to quantify insect fitness and that whole plants should be used wherever possible.Given the prevalence of clip cage use in insect bioassays,our results highlight the need for cau-tion when interpreting the existing literature as confinement method significantly impacts aphid fitness depending on their genotype.
