[Objective] The aim was to learn the resistance of different tissues and organs of transgenic cotton to Spodoptera exigua （Hbner）. [Method] Flowers,the 1st,the 3rd,the 6th and the 14th leaves from the top of 33B,GK1...[Objective] The aim was to learn the resistance of different tissues and organs of transgenic cotton to Spodoptera exigua （Hbner）. [Method] Flowers,the 1st,the 3rd,the 6th and the 14th leaves from the top of 33B,GK12 and SGK321 were used to feed S. exigua neonates respectively. Survival larvae and dead ones were counted on the 3rd,the 7th,the 10th,the 16th and the 19th day; meanwhile,the pupae amount was recorded,and the pupae weight was measured at the 24th h after pupation. [Result] The survival curves,pupation rates and pupae weights of S. exigua feeding on different tissues of transgenic cotton were not significantly different from those of S. exigua feeding on the corresponding tissues of conventional cotton; pupation rate of S. exigua feeding on different leaves of the same cotton variety were not significantly different from each other,but all higher than that of S. exigua feeding on the flowers of that cotton; and there were no differences among pupation weights of S. exigua feeding on different leaves or flowers of the same cotton variety. [Conclusion] Transgenic cotton showed weak resistance to S. exigua. Hence,in the transgenic cotton fields,more attention should be paid to occurrence trend of S. exigua and its control.展开更多
Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimat...Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.展开更多
The high energy quota and versatility of use make willows (Salix spp.) attractive as bioenergy crops. Insect defoliation constitutes a threat to the profitability of willow growers. Hitherto, the breeding for resistan...The high energy quota and versatility of use make willows (Salix spp.) attractive as bioenergy crops. Insect defoliation constitutes a threat to the profitability of willow growers. Hitherto, the breeding for resistance against the main insect pests has been hampered by the fact that all known resistant willow clones are polyploids, and existing molecular breeding tools work most effectively for diploids. Here, we firstly report diploid willows highly resistant to the main insect defoliator, the leaf beetle (Phratora vulgatissima), offering new opportunities for breeding resistance. Leaf beetles exposed to three resistant clones (two S. purpurea one S. eriocephala) laid three to 27 times fewer eggs than females on a susceptible S. viminalis clone. Secondly, we show that beetles laid significantly more eggs on resistant clones if they were fed the susceptible clone prior to the oviposition monitoring test compared to when they prefed on resistant clones. Nevertheless, the differences observed between resistant and susceptible clones were pronounced in all cases. The food conditioning effect means that small differences in resistance among clones may be undetected.展开更多
PCR detection,quantitative real-time PCR(q-RTPCR),outdoor insect resistance,and disease resistance identification were carried out for the detection of genetic stability and disease resistance through generations(T2,T...PCR detection,quantitative real-time PCR(q-RTPCR),outdoor insect resistance,and disease resistance identification were carried out for the detection of genetic stability and disease resistance through generations(T2,T3,and T4)in transgenic maize germplasms(S3002 and 349)containing the bivalent genes(insect resistance gene Cry1Ab13-1 and disease resistance gene NPR1)and their corresponding wild type.Results indicated that the target genes Cry1Ab13-1 and NPR1 were successfully transferred into both germplasms through tested generations;q-PCR confirmed the expression of Cry1Ab13-1 and NPR1 genes in roots,stems,and leaves of tested maize plants.In addition,S3002 and 349 bivalent gene-transformed lines exhibited resistance to large leaf spots and corn borer in the field evaluation compared to the wild type.Our study confirmed that Cry1Ab13-1 and NPR1 bivalent genes enhanced the resistance against maize borer and large leaf spot disease and can stably inherit.These findings could be exploited for improving other cultivated maize varieties.展开更多
Both non-transgenic hybrid triploid poplars [ (Populus tomentosa×P.bolleana)×P.tomentosa ] and transgenic ones expressing cowpea trypsin inhibitor were cut at the base of the stem to produce auxoblasts, an...Both non-transgenic hybrid triploid poplars [ (Populus tomentosa×P.bolleana)×P.tomentosa ] and transgenic ones expressing cowpea trypsin inhibitor were cut at the base of the stem to produce auxoblasts, and used as source of leaves for insect feeding trials performed on 3 major insect species of poplar, the forest tent caterpillar ( Malacosoma disstria L.), gypsy moth ( Lymantria dispar L.) and willow moth ( Stilpnotia candida Staudinger). The height and basal diameter of trees were measured by the end of that year (2000). The results indicated that the growth elements of transgenic poplars were not interfered by the incorporation of the CpTI gene. Intriguingly, the height and basal diameter of the clone TG04 were much greater than that of the control. The transgenic foliage consumed by insects induced the increase of larval mortality, and decrease of larval wet weight gain, faecal output, pupal weight and egg deposition. Among them 3 transgenic clones, TG04, TG07 and TG71 received special attention for their outstanding insect resistance compared with other transgenic clones, which showed that the CpTI gene in them was expressed more actively and stably than in others.展开更多
A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis （Bt） fusion protein CrylAb/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） protein G10 was characterized and evaluated....A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis （Bt） fusion protein CrylAb/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein CrylAb/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer （Ostnnia furnacalis）, cotton boll worm （Helicoverpa armigera）, and armyworm （Mythimna separata）. This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.展开更多
Background:Sucking insect pests cause severe damage to cotton crop production.The development of insect resistant cotton cultivars is one of the most effective measures in curtailing the yield losses.Considering the r...Background:Sucking insect pests cause severe damage to cotton crop production.The development of insect resistant cotton cultivars is one of the most effective measures in curtailing the yield losses.Considering the role of morphological and biochemical host plant resista nee(HPR)traits in plant defense,12 cotton genotypes/varieties were evaluated for leaf area,leaf glanding,total soluble sugars,total soluble proteins,total phenolics,tannin and total flavonoids against fluctuating populations of whitefly,thrips and jassid under field conditions.Results:The population of these insects fluctuated during the growing seas on and remained above threshold level(whitefly>5,thrips>(8-10)f or jassid>1 per leaf)during late June and early July.Strong and negative association of whitefly(r=-0.825)and jassid(r=-0.929)with seed cotton yield was observed.Mean population of insects were the highest in Glandless-1 followed by NIA-82 and NIA-M30.NIAB-Kiran followed by NI AB-878 and Sadori were the most resistant,with the mean population of 1.41,1.60,1.66(whitefly);2.24,232,2.53(thrips)and 037,0.31,036(jassid),respectively.The resistant variety NIAB-Kiran showed less soluble sugars(8.54 mg.g^(-1)),soluble proteins(27.11 mg.g^(-1))and more phenolic(36.56 mg.g^(-1))and flavonoids(13.10mg.g^(-1))as compared with the susceptible check Glandless-1.Moreover,all insect populations were positively correlated with total soluble sugars and proteins.Whitefly populations exhibited negative response to leaf gossypol glands,total phenolics,tannins and flavonoids.The thrips and jassid populations had a significant and negative correlation with these four biochemical HPR traits.Conclusion:The ide ntified resistant resources and HPR traits can be deployed against sucking in sect pests'complex in future breeding programs of developing insect resistant cotton varieties.展开更多
The western flower thrips(WFT;Frankliniella occidentalis)is a mesophyll cell feeder that damages many crops.Management of WFT is complex due to factors such as high fecundity,short reproduction time,ability to feed on...The western flower thrips(WFT;Frankliniella occidentalis)is a mesophyll cell feeder that damages many crops.Management of WFT is complex due to factors such as high fecundity,short reproduction time,ability to feed on a broad range of host plants,and broad pesticide resistance.These challenges have driven research into developing alternative pest control approaches for WFT.This study analyzed the feasibility of a biological control-based strategy to manage WFT using RNA interference(RNAi)-mediated silencing of WFT endogenous genes.For the delivery of RNAi,we developed transgenic tomato lines expressing double-stranded RNA(dsRNA)of coatomer protein subunit epsilon(CopE)and Toll-like receptor 6(TLR6)from WFT.These genes are involved in critical biological processes of WFT,and their dsRNA can be lethal to these insects when ingested orally.Adult WFT that fed on the transgenic dsRNAexpressing tomato flower stalk showed increased mortality compared with insects that fed on wild-type samples.In addition,WFT that fed on TLR6 and CopE transgenic tomato RNAi lines showed reduced levels of endogenous CopE and TLR6 transcripts,suggesting that their mortality was likely due to RNAi-mediated silencing of these genes.Thus,our findings demonstrate that transgenic tomato plants expressing dsRNA of TLR6 and CopE can be lethal to F.occidentalis,suggesting that these genes may be deployed to control insecticide-resistant WFT.展开更多
Background To control the boll weevil Anthonomus grandis grandis(Coleoptera:Curculionidae),a key pest of cotton in the Americas,insecticides have been intensively used to manage their populations,increasing selection ...Background To control the boll weevil Anthonomus grandis grandis(Coleoptera:Curculionidae),a key pest of cotton in the Americas,insecticides have been intensively used to manage their populations,increasing selection pressure for resistant populations.Thus,this study aimed to detect insecticide resistance and assess insecticide control failure likelihood of boll weevil populations exposed to malathion,profenophos+cypermethrin,and fipronil insecticides.Results Twelve populations of the boll weevil were collected from commercial cotton fileds of the state of Bahia,northeastern Brazil.These populations were exposed to malathion,profenophos+cypermethrin mixture,and fipronil,at their respective maximum label dose for field applications.Three replicates of 10 adult beetles were exposed to the insecticides and mortality was recorded after 24 h treatment.The control failure likelihood was determined after 48 h.Highest median lethal times(LT_(50))were observed for malathion and the profenophos+cypermethrin mixture.Resistance to at least one insecticide was detected in 11 populations;three populations were resistant to malathion and profenophos+cypermethrin;seven were resistant to all insecticides tested.The resistance levels were low(<10-fold)for the three insecticides.Among 12 populations tested,58%of them exhibited significant risk of control failure for the insecticides malathion and profenophos+cypermethrin.The insecticide fipronil was efficient for the control of the boll weevil in 83%of the populations.Conclusions The results confirm the significant risk of insecticide control failure in the boll weevil populations to the main compounds used in the region.Thus,proper insecticide resistance management plans are necessary for the boll weevil in the region,particularly for malathion and profenophos+cypermethrin insecticides.展开更多
Expression vector pE14 with double resistance to virus and insect was constructed by inserting CW-cp gene and Bt-toxin gene one by into T-DNA of the same binary vector pE3. Tobacco was then transformed with Agrbacter...Expression vector pE14 with double resistance to virus and insect was constructed by inserting CW-cp gene and Bt-toxin gene one by into T-DNA of the same binary vector pE3. Tobacco was then transformed with Agrbacterium tumefaciens (At)GV311-SE carrying PE14. Nopaline assay and PCR amplification confirmed that both CW-cp gene and Bt-toxin gene had been introduced into tobacco genome by T-DNA of PE3. Test attack with virus and demonstrated, in some of the transgenic plants, double resistance to both infection by CMV and damage by Manduca sexta.展开更多
Calcium-dependent protein kinases(CDPKs)act as key signal transduction enzymes in plants,especially in response to diverse stresses,including herbivory.In this study,a comprehensive analysis of the CDPK gene family in...Calcium-dependent protein kinases(CDPKs)act as key signal transduction enzymes in plants,especially in response to diverse stresses,including herbivory.In this study,a comprehensive analysis of the CDPK gene family in upland cotton revealed that GhCPKs are widely expressed in multiple cotton tissues and respond positively to various biotic and abiotic stresses.We developed a strategy for screening insect-resistance genes from a CRISPR-Cas9 mutant library of GhCPKs.The library was created using 246 single-guide RNAs targeting the GhCPK gene family to generate 518 independent T0 plants.The average target-gene coverage was 86.18%,the genome editing rate was 89.49%,and the editing heritability was 82%.An insect bioassay in the field led to identification of 14 GhCPK mutants that are resistant or susceptible to insects.The mutant that showed the clearest insect resistance,cpk33/74(in which the homologous genes GhCPK33 and GhCPK74 were knocked out),was selected for further study.Oral secretions from Spodoptera litura induced a rapid influx of Ca2+in cpk33/74 leaves,resulting in a significant increase in jasmonic acid content.S-adenosylmethionine synthase is an important protein involved in plant stress response,and protein interaction experiments provided evidence for interactions of GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2.In addition,virus-induced gene silencing of GhSAMS1 and GhSAM2 in cotton impaired defense against S.litura.This study demonstrates an effective strategy for constructing a mutant library of a gene family in a polyploid plant species and offers valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.展开更多
Partially modified Bt Cry1Ac gene and the arrowhead proteinase inhibitor (API) gene were used to construct a plant transformation vector pBtiA and this construct was transferred into the genome of the hybrid popla...Partially modified Bt Cry1Ac gene and the arrowhead proteinase inhibitor (API) gene were used to construct a plant transformation vector pBtiA and this construct was transferred into the genome of the hybrid poplar 741 [ Populus alba L.×( P. davidiana Dode+ P. simonii Carr.)× P. tomentosa Carr.] by Agrobacterium _ mediated transformation. Ten kanamycin resistant plants have been regenerated. Upon insect bioassay using Clostera anachoreta (Fabricius), three of the examined plants were demonstrated to be highly resistant to the testing insects. The mortality of insect larvae on one plant was higher than 90% in 6 days after infestation and the growth of the survival larvae were seriously inhibited. Results of PCR and Southern blot analysis indicated that both Bt Cry1Ac gene and API gene were integrated as a single copy into the genomes of these three plants when Cry1Ac gene fragment was used as the probe. Protein dot blot immunoassay and ELISA analysis revealed that at least the Cry1Ac protein was produced in these three transgenic plants and the expression levels were estimated to be approximately 0.015% of the leaf total soluble protein. This is the first report on insect resistant transgenic hybrid poplar 741 that expresses two insecticidal protein genes.展开更多
Asian corn borer, Ostrinia furnacalis （Guen6e）, is the most important pest of maize in China and Southeast Asia. The objective of this study was to understand the genetic basis for Asian corn borer resistance. The s...Asian corn borer, Ostrinia furnacalis （Guen6e）, is the most important pest of maize in China and Southeast Asia. The objective of this study was to understand the genetic basis for Asian corn borer resistance. The study included 162 F2：3 populations derived from Mc37 （resistant） × Zi330 （susceptible） and field data were collected in 2004 and 2005. A linkage map was constructed from 118 SSR markers. Combined quantitative trait loci （QTL） analysis combined across environments was performed by composite interval mapping method （CIM）. Four QTLs with additive effects were detected for resistance to Asian corn borer leaf feeding damage in chromosome bins 1.08, 2.04/05, 4.01, and 10.04. Three putative QTLs were detected for Asian corn borer stalk damage in chromosome bins 1.01, 2.05, and 9.01. Maize resistance to Asian corn borer and European corn borer, O. nubilalis （Hubner）, may share a common mechanism. Genes responsible for DIMBOA biosynthesis are in chromosome bin 4.01 and may increase the observed resistance to Asian corn borer leaf feeding.展开更多
Grh2, a green rice leafhopper resistant gene from an indica cultivar DV85, was located on chromosome 11, and two RFLP markers C189 and G1465 were found to be linked to this gene. In order to transfer Grh2 into Taichun...Grh2, a green rice leafhopper resistant gene from an indica cultivar DV85, was located on chromosome 11, and two RFLP markers C189 and G1465 were found to be linked to this gene. In order to transfer Grh2 into Taichung65, a japonica cultivar with elite characters, backcross method with Taichung65 as the recurrent parent was used and the two RFLP markers were converted into CAPS markers for marker assisted selection (MAS). In the BC6F3 population, both phenotypic evaluation and MAS were conducted to screen the resistant plants with Taichung65 background. The linkage distance between CAPS markers and Grh2 was calculated and the efficiency of MAS was analyzed.展开更多
The research results of marker aided selection(MAS)for resistant varieties and lines against rice gall midge Orseolia oryzae Wood-Mason successfully in 1999 - 2002 were reported in the present paper. The molecular mar...The research results of marker aided selection(MAS)for resistant varieties and lines against rice gall midge Orseolia oryzae Wood-Mason successfully in 1999 - 2002 were reported in the present paper. The molecular markers linked to the gene Gm6 against rice gall midge were used to select and breed the resistant varieties and lines. The RAPD marker OPM06 was used to verify the existence actually of gene Gm6 in ten developed varieties resistant to gall midge such as Duokang1, Duokang2, Kangwen2, Kangwen3, Kang-wen5, Duokangzaozhan, Kangwenqinzhan, which were derived from Daqiuqi. For resistance breeding through PCRbased marker aided selection(MAS), the polymorphisms in the resistant and susceptible parents were i-dentified by RG476/Alu I and RG476/Sca I respectively. The RAPD marker OPM06(1.4 kb)was used to i-dentify 15 new resistance lines from F3 lines of Fengyinzhan1/Daqiuqi in 1999. 21 and 7 resistance lines were selected from F4 and F6 lines of KWQZ/Gui99(restored line of hybrid rice)using RG476/Alu I in 2000-2001 respectively. The Gm6 gene was transferred into the restored line of hybrid rice. In 2001 - 2002, RG214/ Hha I and G214/Sca I were used for selecting 11 and 5 resistance lines from F3 lines of KWQZ/IR56 and AXZ/KWQZ successfully. The application of the resistance gene through PCR-based marker aided selection is a new and effective approach in resistance breeding.展开更多
In agricultural production,a single insect-resistant and disease-resistant variety can no longer meet the demand.In this study,the expression vector pCAMBIA-3301-PR1 containing the disease-resistant gene PR1 was const...In agricultural production,a single insect-resistant and disease-resistant variety can no longer meet the demand.In this study,the expression vector pCAMBIA-3301-PR1 containing the disease-resistant gene PR1 was constructed by means of genetic engineering,and the PR1 gene was genetically transformed to contain the PR1 gene through the pollen tube method.In CryAb-8Like transgenic high-generation T7 receptor soybean,a new material that is resistant to insects and diseases is obtained.For T2 transformed plants,routine PCR detection,Southern Blot hybridization,fluorescence quantitative PCR detection,indoor and outdoor pest resistance identification and indoor disease resistance identification were performed.The results showed that there were 9 positive plants in the routine PCR test of T2 generation.In Southern Blot hybridization,both PR1 and CryAb-8Like genes are integrated in soybeans in the form of single copies.Fluorescence quantitative PCR showed that the expression levels of PR1 and CryAb-8Like genes are different in different tissues.The average expression levels of PR1 gene in plant roots,stems,and leaves are 2.88,1.54,and 5.26,respectively.CryAb-8Like genes are found in roots,stems,and leaves.The average expression levels were 1.36,1.39,and 4.25,respectively.The insectivorous rate of the CryAb-8Like gene in outdoor plants with positive insect resistance identification was 3.78%.The disc partition method was used indoors for pest resistance identification,and the bud length of transformed plants increased significantly.The average mortality rate of untransformed plants in indoor disease resistance identification was as high as 56.66%,and the average mortality rate of plants transformed with PR1 gene was 10.00%,and disease resistance was significantly improved.Therefore,a new material with resistance to diseases and insects is obtained.展开更多
Focusing on several commonly used insect resistance genes,we reviewed the advances in insect-resistant transgenic rice,and analyzed the problems and developing tendency in transgenic rice research in this paper.
This paper reports on a case study that involved the transfer of (1) methods for the analysis and (2) information on the fate and proper use of the agricultural chemicals, endosulphan, from Australia to Anhui Province...This paper reports on a case study that involved the transfer of (1) methods for the analysis and (2) information on the fate and proper use of the agricultural chemicals, endosulphan, from Australia to Anhui Province, China. A key outcome from the case study was that there was relatively little awareness of the potential environmental impacts from the use of endosulphan. Cross\|cultural constraints in the interaction were identified and areas which will require further effort in technology transfer were discussed.展开更多
The plasmid of pCDMARUBA-Hyg, which contained two insect-resistance genes, sbk （modified from CrylA（c）） and sck （modified from CpTI）, was transformed into an Agrobacterium EHA105 for infection of the calli of a ...The plasmid of pCDMARUBA-Hyg, which contained two insect-resistance genes, sbk （modified from CrylA（c）） and sck （modified from CpTI）, was transformed into an Agrobacterium EHA105 for infection of the calli of a super japonica rice Nanjing 45. Primarily, using polymerase chain reaction （PCR） detection with the primers of sbk and sck genes, 42 positive transgenic plants that were marker-free and contained the two target genes were selected from 97 regenerated plants. Results of southern-blotting indicated that 23, 11, 5, 2 and 1 plants had one, two, three, four and five copies of the transformed genes, respectively. Analysis of reverse transcription PCR （RT-PCR） and Bt gene testing paper showed that 28 T3 generation plants derived from four transgenic plants having a single copy were insect-resistant. Feeding experiment with rice stem borer revealed that the insect resistance was greatly increased with the larva mortality ranging from 94% to 100%. In addition, among the transgenic plants, three T3 transgenic plants possessed some desirable characteristics for breeding and production, such as plant height, seed-setting rate, 1000-grain weight and larva mortality. The mechanism of insect resistance of Bt .qene and its application in rice transgenic research were also briefly discussed.展开更多
A plant expression vector carrying both pea lectin gene and Soybean trypsin inhibitor gene has been constructed and transferred into tobacco via Agrobacterium mediated transformation. Transgenic plants are further co...A plant expression vector carrying both pea lectin gene and Soybean trypsin inhibitor gene has been constructed and transferred into tobacco via Agrobacterium mediated transformation. Transgenic plants are further confirmed by ELISA, PCR and PCR Southern assays. Results of bioassays show that transgenic plants display notably inhibitory effects to larvae development and survival of Heliothis armigera Hubner.展开更多
基金Supported by National Transgenic Major Project ( Safe Monitoring Technique of Transgenic Organism 2008ZX08012-004)~~
文摘[Objective] The aim was to learn the resistance of different tissues and organs of transgenic cotton to Spodoptera exigua （Hbner）. [Method] Flowers,the 1st,the 3rd,the 6th and the 14th leaves from the top of 33B,GK12 and SGK321 were used to feed S. exigua neonates respectively. Survival larvae and dead ones were counted on the 3rd,the 7th,the 10th,the 16th and the 19th day; meanwhile,the pupae amount was recorded,and the pupae weight was measured at the 24th h after pupation. [Result] The survival curves,pupation rates and pupae weights of S. exigua feeding on different tissues of transgenic cotton were not significantly different from those of S. exigua feeding on the corresponding tissues of conventional cotton; pupation rate of S. exigua feeding on different leaves of the same cotton variety were not significantly different from each other,but all higher than that of S. exigua feeding on the flowers of that cotton; and there were no differences among pupation weights of S. exigua feeding on different leaves or flowers of the same cotton variety. [Conclusion] Transgenic cotton showed weak resistance to S. exigua. Hence,in the transgenic cotton fields,more attention should be paid to occurrence trend of S. exigua and its control.
基金support of the National Natural Science Foundation of China(30771383)the Genetically Modified Organisms Breeding Major Projects, China(2013ZX08003-001)the National Basic Research Program of China (2009CB118902)
文摘Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.
文摘The high energy quota and versatility of use make willows (Salix spp.) attractive as bioenergy crops. Insect defoliation constitutes a threat to the profitability of willow growers. Hitherto, the breeding for resistance against the main insect pests has been hampered by the fact that all known resistant willow clones are polyploids, and existing molecular breeding tools work most effectively for diploids. Here, we firstly report diploid willows highly resistant to the main insect defoliator, the leaf beetle (Phratora vulgatissima), offering new opportunities for breeding resistance. Leaf beetles exposed to three resistant clones (two S. purpurea one S. eriocephala) laid three to 27 times fewer eggs than females on a susceptible S. viminalis clone. Secondly, we show that beetles laid significantly more eggs on resistant clones if they were fed the susceptible clone prior to the oviposition monitoring test compared to when they prefed on resistant clones. Nevertheless, the differences observed between resistant and susceptible clones were pronounced in all cases. The food conditioning effect means that small differences in resistance among clones may be undetected.
基金supported by the National Key Research and Development Program of China(2019YFD1002603-1)。
文摘PCR detection,quantitative real-time PCR(q-RTPCR),outdoor insect resistance,and disease resistance identification were carried out for the detection of genetic stability and disease resistance through generations(T2,T3,and T4)in transgenic maize germplasms(S3002 and 349)containing the bivalent genes(insect resistance gene Cry1Ab13-1 and disease resistance gene NPR1)and their corresponding wild type.Results indicated that the target genes Cry1Ab13-1 and NPR1 were successfully transferred into both germplasms through tested generations;q-PCR confirmed the expression of Cry1Ab13-1 and NPR1 genes in roots,stems,and leaves of tested maize plants.In addition,S3002 and 349 bivalent gene-transformed lines exhibited resistance to large leaf spots and corn borer in the field evaluation compared to the wild type.Our study confirmed that Cry1Ab13-1 and NPR1 bivalent genes enhanced the resistance against maize borer and large leaf spot disease and can stably inherit.These findings could be exploited for improving other cultivated maize varieties.
文摘Both non-transgenic hybrid triploid poplars [ (Populus tomentosa×P.bolleana)×P.tomentosa ] and transgenic ones expressing cowpea trypsin inhibitor were cut at the base of the stem to produce auxoblasts, and used as source of leaves for insect feeding trials performed on 3 major insect species of poplar, the forest tent caterpillar ( Malacosoma disstria L.), gypsy moth ( Lymantria dispar L.) and willow moth ( Stilpnotia candida Staudinger). The height and basal diameter of trees were measured by the end of that year (2000). The results indicated that the growth elements of transgenic poplars were not interfered by the incorporation of the CpTI gene. Intriguingly, the height and basal diameter of the clone TG04 were much greater than that of the control. The transgenic foliage consumed by insects induced the increase of larval mortality, and decrease of larval wet weight gain, faecal output, pupal weight and egg deposition. Among them 3 transgenic clones, TG04, TG07 and TG71 received special attention for their outstanding insect resistance compared with other transgenic clones, which showed that the CpTI gene in them was expressed more actively and stably than in others.
基金Project supported by the Fundamental Research Funds for the Central Universities(No.2017FZA6011)the National Key Transgenic Research Projects(No.2016ZX08010003)of China
文摘A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis （Bt） fusion protein CrylAb/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein CrylAb/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer （Ostnnia furnacalis）, cotton boll worm （Helicoverpa armigera）, and armyworm （Mythimna separata）. This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.
文摘Background:Sucking insect pests cause severe damage to cotton crop production.The development of insect resistant cotton cultivars is one of the most effective measures in curtailing the yield losses.Considering the role of morphological and biochemical host plant resista nee(HPR)traits in plant defense,12 cotton genotypes/varieties were evaluated for leaf area,leaf glanding,total soluble sugars,total soluble proteins,total phenolics,tannin and total flavonoids against fluctuating populations of whitefly,thrips and jassid under field conditions.Results:The population of these insects fluctuated during the growing seas on and remained above threshold level(whitefly>5,thrips>(8-10)f or jassid>1 per leaf)during late June and early July.Strong and negative association of whitefly(r=-0.825)and jassid(r=-0.929)with seed cotton yield was observed.Mean population of insects were the highest in Glandless-1 followed by NIA-82 and NIA-M30.NIAB-Kiran followed by NI AB-878 and Sadori were the most resistant,with the mean population of 1.41,1.60,1.66(whitefly);2.24,232,2.53(thrips)and 037,0.31,036(jassid),respectively.The resistant variety NIAB-Kiran showed less soluble sugars(8.54 mg.g^(-1)),soluble proteins(27.11 mg.g^(-1))and more phenolic(36.56 mg.g^(-1))and flavonoids(13.10mg.g^(-1))as compared with the susceptible check Glandless-1.Moreover,all insect populations were positively correlated with total soluble sugars and proteins.Whitefly populations exhibited negative response to leaf gossypol glands,total phenolics,tannins and flavonoids.The thrips and jassid populations had a significant and negative correlation with these four biochemical HPR traits.Conclusion:The ide ntified resistant resources and HPR traits can be deployed against sucking in sect pests'complex in future breeding programs of developing insect resistant cotton varieties.
基金supported by the Basic Science Research Program through the National Research Foundation(NRF),Ministry of Education,Korea(2021R1I1A1A01041938)a grant from the New Breeding Technologies Development Program,Rural Development Administration,Korea(PJ0165432022)supported in part by the BK21 Plus Program,Ministry of Education,Korea。
文摘The western flower thrips(WFT;Frankliniella occidentalis)is a mesophyll cell feeder that damages many crops.Management of WFT is complex due to factors such as high fecundity,short reproduction time,ability to feed on a broad range of host plants,and broad pesticide resistance.These challenges have driven research into developing alternative pest control approaches for WFT.This study analyzed the feasibility of a biological control-based strategy to manage WFT using RNA interference(RNAi)-mediated silencing of WFT endogenous genes.For the delivery of RNAi,we developed transgenic tomato lines expressing double-stranded RNA(dsRNA)of coatomer protein subunit epsilon(CopE)and Toll-like receptor 6(TLR6)from WFT.These genes are involved in critical biological processes of WFT,and their dsRNA can be lethal to these insects when ingested orally.Adult WFT that fed on the transgenic dsRNAexpressing tomato flower stalk showed increased mortality compared with insects that fed on wild-type samples.In addition,WFT that fed on TLR6 and CopE transgenic tomato RNAi lines showed reduced levels of endogenous CopE and TLR6 transcripts,suggesting that their mortality was likely due to RNAi-mediated silencing of these genes.Thus,our findings demonstrate that transgenic tomato plants expressing dsRNA of TLR6 and CopE can be lethal to F.occidentalis,suggesting that these genes may be deployed to control insecticide-resistant WFT.
基金supported by Foundation for Research Support of the State of Bahia(FAPESB)the CAPES Foundation(Brazilian Ministry of Education+1 种基金Finance Code 001)for financial supportBahia Association of Cotton Producers。
文摘Background To control the boll weevil Anthonomus grandis grandis(Coleoptera:Curculionidae),a key pest of cotton in the Americas,insecticides have been intensively used to manage their populations,increasing selection pressure for resistant populations.Thus,this study aimed to detect insecticide resistance and assess insecticide control failure likelihood of boll weevil populations exposed to malathion,profenophos+cypermethrin,and fipronil insecticides.Results Twelve populations of the boll weevil were collected from commercial cotton fileds of the state of Bahia,northeastern Brazil.These populations were exposed to malathion,profenophos+cypermethrin mixture,and fipronil,at their respective maximum label dose for field applications.Three replicates of 10 adult beetles were exposed to the insecticides and mortality was recorded after 24 h treatment.The control failure likelihood was determined after 48 h.Highest median lethal times(LT_(50))were observed for malathion and the profenophos+cypermethrin mixture.Resistance to at least one insecticide was detected in 11 populations;three populations were resistant to malathion and profenophos+cypermethrin;seven were resistant to all insecticides tested.The resistance levels were low(<10-fold)for the three insecticides.Among 12 populations tested,58%of them exhibited significant risk of control failure for the insecticides malathion and profenophos+cypermethrin.The insecticide fipronil was efficient for the control of the boll weevil in 83%of the populations.Conclusions The results confirm the significant risk of insecticide control failure in the boll weevil populations to the main compounds used in the region.Thus,proper insecticide resistance management plans are necessary for the boll weevil in the region,particularly for malathion and profenophos+cypermethrin insecticides.
文摘Expression vector pE14 with double resistance to virus and insect was constructed by inserting CW-cp gene and Bt-toxin gene one by into T-DNA of the same binary vector pE3. Tobacco was then transformed with Agrbacterium tumefaciens (At)GV311-SE carrying PE14. Nopaline assay and PCR amplification confirmed that both CW-cp gene and Bt-toxin gene had been introduced into tobacco genome by T-DNA of PE3. Test attack with virus and demonstrated, in some of the transgenic plants, double resistance to both infection by CMV and damage by Manduca sexta.
基金Biological Breeding of Stress Tolerant and High Yield Cotton Varieties(2023ZD04040)to L.M.National Natural Science Fund of China for Distinguished Young Scholars(32325039)+2 种基金National Natural Science Foundation of China(32272128)to S.J.,the National Natural Science Foundation of China(32401780)Key Scientific and Technological Project of Henan Province(222102110151)to S.L.,Major Science and Technology Project of Xinjiang Uygur Autonomous Region(2023A02003-2)to B.L.
文摘Calcium-dependent protein kinases(CDPKs)act as key signal transduction enzymes in plants,especially in response to diverse stresses,including herbivory.In this study,a comprehensive analysis of the CDPK gene family in upland cotton revealed that GhCPKs are widely expressed in multiple cotton tissues and respond positively to various biotic and abiotic stresses.We developed a strategy for screening insect-resistance genes from a CRISPR-Cas9 mutant library of GhCPKs.The library was created using 246 single-guide RNAs targeting the GhCPK gene family to generate 518 independent T0 plants.The average target-gene coverage was 86.18%,the genome editing rate was 89.49%,and the editing heritability was 82%.An insect bioassay in the field led to identification of 14 GhCPK mutants that are resistant or susceptible to insects.The mutant that showed the clearest insect resistance,cpk33/74(in which the homologous genes GhCPK33 and GhCPK74 were knocked out),was selected for further study.Oral secretions from Spodoptera litura induced a rapid influx of Ca2+in cpk33/74 leaves,resulting in a significant increase in jasmonic acid content.S-adenosylmethionine synthase is an important protein involved in plant stress response,and protein interaction experiments provided evidence for interactions of GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2.In addition,virus-induced gene silencing of GhSAMS1 and GhSAM2 in cotton impaired defense against S.litura.This study demonstrates an effective strategy for constructing a mutant library of a gene family in a polyploid plant species and offers valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.
基金ThePresidentialFoundationofTheChineseAcademyofSciences NaturalScienceFoundationofHebeiProvince China
文摘Partially modified Bt Cry1Ac gene and the arrowhead proteinase inhibitor (API) gene were used to construct a plant transformation vector pBtiA and this construct was transferred into the genome of the hybrid poplar 741 [ Populus alba L.×( P. davidiana Dode+ P. simonii Carr.)× P. tomentosa Carr.] by Agrobacterium _ mediated transformation. Ten kanamycin resistant plants have been regenerated. Upon insect bioassay using Clostera anachoreta (Fabricius), three of the examined plants were demonstrated to be highly resistant to the testing insects. The mortality of insect larvae on one plant was higher than 90% in 6 days after infestation and the growth of the survival larvae were seriously inhibited. Results of PCR and Southern blot analysis indicated that both Bt Cry1Ac gene and API gene were integrated as a single copy into the genomes of these three plants when Cry1Ac gene fragment was used as the probe. Protein dot blot immunoassay and ELISA analysis revealed that at least the Cry1Ac protein was produced in these three transgenic plants and the expression levels were estimated to be approximately 0.015% of the leaf total soluble protein. This is the first report on insect resistant transgenic hybrid poplar 741 that expresses two insecticidal protein genes.
基金funded by a grant of the National 973 Program of China (2006CB101701-7)the National Key Technologies R&D Program of China during the 11th Five-Year Plan period (2006BAD02A16-3,2006BAD08A06)
文摘Asian corn borer, Ostrinia furnacalis （Guen6e）, is the most important pest of maize in China and Southeast Asia. The objective of this study was to understand the genetic basis for Asian corn borer resistance. The study included 162 F2：3 populations derived from Mc37 （resistant） × Zi330 （susceptible） and field data were collected in 2004 and 2005. A linkage map was constructed from 118 SSR markers. Combined quantitative trait loci （QTL） analysis combined across environments was performed by composite interval mapping method （CIM）. Four QTLs with additive effects were detected for resistance to Asian corn borer leaf feeding damage in chromosome bins 1.08, 2.04/05, 4.01, and 10.04. Three putative QTLs were detected for Asian corn borer stalk damage in chromosome bins 1.01, 2.05, and 9.01. Maize resistance to Asian corn borer and European corn borer, O. nubilalis （Hubner）, may share a common mechanism. Genes responsible for DIMBOA biosynthesis are in chromosome bin 4.01 and may increase the observed resistance to Asian corn borer leaf feeding.
基金This work was conducted in Kyushu University,Japan by the first author during his visiting research supported by China Scholarship Counsel(CSC),the“948”Project of the Ministry of Agriculture of Chinathe Program for Outstanding Teachers by the Ministry of Education of China.
文摘Grh2, a green rice leafhopper resistant gene from an indica cultivar DV85, was located on chromosome 11, and two RFLP markers C189 and G1465 were found to be linked to this gene. In order to transfer Grh2 into Taichung65, a japonica cultivar with elite characters, backcross method with Taichung65 as the recurrent parent was used and the two RFLP markers were converted into CAPS markers for marker assisted selection (MAS). In the BC6F3 population, both phenotypic evaluation and MAS were conducted to screen the resistant plants with Taichung65 background. The linkage distance between CAPS markers and Grh2 was calculated and the efficiency of MAS was analyzed.
文摘The research results of marker aided selection(MAS)for resistant varieties and lines against rice gall midge Orseolia oryzae Wood-Mason successfully in 1999 - 2002 were reported in the present paper. The molecular markers linked to the gene Gm6 against rice gall midge were used to select and breed the resistant varieties and lines. The RAPD marker OPM06 was used to verify the existence actually of gene Gm6 in ten developed varieties resistant to gall midge such as Duokang1, Duokang2, Kangwen2, Kangwen3, Kang-wen5, Duokangzaozhan, Kangwenqinzhan, which were derived from Daqiuqi. For resistance breeding through PCRbased marker aided selection(MAS), the polymorphisms in the resistant and susceptible parents were i-dentified by RG476/Alu I and RG476/Sca I respectively. The RAPD marker OPM06(1.4 kb)was used to i-dentify 15 new resistance lines from F3 lines of Fengyinzhan1/Daqiuqi in 1999. 21 and 7 resistance lines were selected from F4 and F6 lines of KWQZ/Gui99(restored line of hybrid rice)using RG476/Alu I in 2000-2001 respectively. The Gm6 gene was transferred into the restored line of hybrid rice. In 2001 - 2002, RG214/ Hha I and G214/Sca I were used for selecting 11 and 5 resistance lines from F3 lines of KWQZ/IR56 and AXZ/KWQZ successfully. The application of the resistance gene through PCR-based marker aided selection is a new and effective approach in resistance breeding.
基金the National Major Special Project for Breeding New Varieties of Genetically Modified Organisms(2016ZX08004-004).
文摘In agricultural production,a single insect-resistant and disease-resistant variety can no longer meet the demand.In this study,the expression vector pCAMBIA-3301-PR1 containing the disease-resistant gene PR1 was constructed by means of genetic engineering,and the PR1 gene was genetically transformed to contain the PR1 gene through the pollen tube method.In CryAb-8Like transgenic high-generation T7 receptor soybean,a new material that is resistant to insects and diseases is obtained.For T2 transformed plants,routine PCR detection,Southern Blot hybridization,fluorescence quantitative PCR detection,indoor and outdoor pest resistance identification and indoor disease resistance identification were performed.The results showed that there were 9 positive plants in the routine PCR test of T2 generation.In Southern Blot hybridization,both PR1 and CryAb-8Like genes are integrated in soybeans in the form of single copies.Fluorescence quantitative PCR showed that the expression levels of PR1 and CryAb-8Like genes are different in different tissues.The average expression levels of PR1 gene in plant roots,stems,and leaves are 2.88,1.54,and 5.26,respectively.CryAb-8Like genes are found in roots,stems,and leaves.The average expression levels were 1.36,1.39,and 4.25,respectively.The insectivorous rate of the CryAb-8Like gene in outdoor plants with positive insect resistance identification was 3.78%.The disc partition method was used indoors for pest resistance identification,and the bud length of transformed plants increased significantly.The average mortality rate of untransformed plants in indoor disease resistance identification was as high as 56.66%,and the average mortality rate of plants transformed with PR1 gene was 10.00%,and disease resistance was significantly improved.Therefore,a new material with resistance to diseases and insects is obtained.
基金Supported by Grants from China National Major Special Project on New Varieties Cultivation for Transgenic Organisms(2008ZX08001-001)
文摘Focusing on several commonly used insect resistance genes,we reviewed the advances in insect-resistant transgenic rice,and analyzed the problems and developing tendency in transgenic rice research in this paper.
文摘This paper reports on a case study that involved the transfer of (1) methods for the analysis and (2) information on the fate and proper use of the agricultural chemicals, endosulphan, from Australia to Anhui Province, China. A key outcome from the case study was that there was relatively little awareness of the potential environmental impacts from the use of endosulphan. Cross\|cultural constraints in the interaction were identified and areas which will require further effort in technology transfer were discussed.
基金supported by the Natural Science Foundation of Jiangsu Province, China (Grant No. BK2008348)the China National Science & Technology Major Project for Breeding New Plant Varieties of Genetically Modified Organisms (GMOs) (Grant No. 2008ZX08001-004)
文摘The plasmid of pCDMARUBA-Hyg, which contained two insect-resistance genes, sbk （modified from CrylA（c）） and sck （modified from CpTI）, was transformed into an Agrobacterium EHA105 for infection of the calli of a super japonica rice Nanjing 45. Primarily, using polymerase chain reaction （PCR） detection with the primers of sbk and sck genes, 42 positive transgenic plants that were marker-free and contained the two target genes were selected from 97 regenerated plants. Results of southern-blotting indicated that 23, 11, 5, 2 and 1 plants had one, two, three, four and five copies of the transformed genes, respectively. Analysis of reverse transcription PCR （RT-PCR） and Bt gene testing paper showed that 28 T3 generation plants derived from four transgenic plants having a single copy were insect-resistant. Feeding experiment with rice stem borer revealed that the insect resistance was greatly increased with the larva mortality ranging from 94% to 100%. In addition, among the transgenic plants, three T3 transgenic plants possessed some desirable characteristics for breeding and production, such as plant height, seed-setting rate, 1000-grain weight and larva mortality. The mechanism of insect resistance of Bt .qene and its application in rice transgenic research were also briefly discussed.
文摘A plant expression vector carrying both pea lectin gene and Soybean trypsin inhibitor gene has been constructed and transferred into tobacco via Agrobacterium mediated transformation. Transgenic plants are further confirmed by ELISA, PCR and PCR Southern assays. Results of bioassays show that transgenic plants display notably inhibitory effects to larvae development and survival of Heliothis armigera Hubner.
