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
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
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
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
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
A plant expression vector containing a chimeric Bt29K gene coding for the activated Cry1Ac protein and the arrowhead proteinase inhibitior gene API B were introduced into the cotton cultivar Jihe321 mediated ...A plant expression vector containing a chimeric Bt29K gene coding for the activated Cry1Ac protein and the arrowhead proteinase inhibitior gene API B were introduced into the cotton cultivar Jihe321 mediated by Agrobactertium tumefaciens. Based on the results of kanamycin resistant testing, PCR detection for both foreign genes and insect bioassay using Heliethis armigera , nine transgenic homozygous cotton lines with insect resistance of more than 90% and better agronomic traits were bred through six generations from the original transgenic plants. Results from insect bioassay and sequence analysis of the PCR products of plants from some homozygous lines indicated that the chimeric Bt29K gene was stably inherited in these transgenic cotton lines. The main agronomic characters of these homozygous cotton lines, such as boll productivity and fibre strength, were better than that of the original cotton cv. Jihe321.展开更多
Bt5198, a new rice restorer line containing Bt gene, was developed from the cross and backcross of the elite restorer line Chenghui 177 with Bt Minghui 63, a transgenic Bt restorer line. The inbred lines were evaluate...Bt5198, a new rice restorer line containing Bt gene, was developed from the cross and backcross of the elite restorer line Chenghui 177 with Bt Minghui 63, a transgenic Bt restorer line. The inbred lines were evaluated using PCR amplification, test paper evaluation, insect resistance evaluation in both the laboratory and paddy fields, nursery evaluation of rice blast resistance and pedigree selection of agronomic traits. Larval mortalities on Bt5198 and Bt Minghui 63 were 100% when rice culms were inoculated with the eggs of the striped stem borer (SSB) in the laboratory. Bt5198 was highly resistant against SSB and the yellow stem borer (YSB) under field conditions. The F1 hybrids derived from Bt5198 and four cytoplasmic male sterile (CMS) lines were also highly resistant to SSB and YSB and had a significant heterosis. Two-year evaluation of rice blast resistance confirmed that the resistance levels of Bt5198 to leaf blast and neck blast were similar to those of Chenghui 177 and significantly better than those of Bt Minghui 63. Seed germination ability and pollen yield of Bt5198 were similar with Chenghui 177, suggesting that the introduction of the Bt gene into the new restorer line had no significant effects on seed vitality or the yield of seed production. To identify the presence of the Bt gene, it was effective to combine test paper examination with the evaluation of insect-resistance, both in the laboratory and under field conditions.展开更多
Rice production and quality are seriously affected by the lepidopteran pest,striped stem borer(SSB),in Northeast China.In this study,a synthetic cry1 C gene encoding Bacillus thuringiensis(Bt)δ-endotoxin,which is tox...Rice production and quality are seriously affected by the lepidopteran pest,striped stem borer(SSB),in Northeast China.In this study,a synthetic cry1 C gene encoding Bacillus thuringiensis(Bt)δ-endotoxin,which is toxic to lepidopteran pest,was transformed into a japonica rice variety(Jigeng 88)in Northeast China by Agrobacterium-mediated transformation.Through molecular detection and the Basta resistance germination assay,a total of 16 single-copy homozygous transgenic lines were obtained from 126 independent transformants expressing cry1 C.Finally,four cry1 C-transgenic lines(JL16,JL23,JL41,and JL42)were selected by evaluation of the Cry1 C protein level,insect-resistance and agronomic traits.The cry1 C-transgenic lines had higher resistance to SSB and higher yield compared with non-transgenic(NT)control plants.T-DNA flanking sequence analysis of the transgenic line JL42 showed that the cry1 C gene was inserted into the intergenic region of chromosome 11,indicating that its insertion may not interfere with the genes near insertion site.In summary,this study developed four cry1 C-transgenic japonica rice lines with high insect resistance and high yield.They can be used as insect-resistant germplasm materials to overcome the problem of rice yield reduction caused by SSB and reduce the use of pesticides in Northeast China.展开更多
A synthetic cry2A^* gene enco ding Bacillus thuringiensis(Bt) δ-endotoxi n that resi st ance to lepidopteran pest was transformed into japonica rice variety Jijing 88, which is the most widely cultivated variety i...A synthetic cry2A^* gene enco ding Bacillus thuringiensis(Bt) δ-endotoxi n that resi st ance to lepidopteran pest was transformed into japonica rice variety Jijing 88, which is the most widely cultivated variety in Jilin Province, Northeast China, by Agrobacterium-mediated transformation. A total of 106 independent transformants overexpressing cry2A^* gene driven by ubiquitin(Ubi) promoter was produced. Three single-copy homozygous transgenic lines were finally selected based on the results of PCR analysis, se gregation ratio of Bast a resistance, and Southern hybridiza tion analyse s. RT-PCR and enzyme linke dimmune sorbent assay(ELISA) revealed that cry2A^* transcripts and protein were highly expressed in these lines. The high level of Cry2A^* protein expression resulted in high resistance to rice striped stem borer as evidence d by insect feeding bioassays. Our results demonst rate that cry2A^* transgenic japonica rice confers resistance to the rice striped stem borer in the laboratory conditions.展开更多
Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacill...Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacillus thuringiensis(Bt)cotton with a single Cry1 Ac gene,growers are on the verge of adopting Bt cotton that carries the double gene(Cry1 Ac+Cry2 A)due to its better effectiveness against insect pests.Thus,the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.The expression levels of the Cry1 Ac and Cry2 A genes were evaluated in the leaves of 10 genotypes(2 parents and 8 Fhybrids)at 30 days after sowing(DAS),while samples of leaves,bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.The Fhybrids were developed through reciprocal crosses between two Bt(CKC-1,CKC-2)and two non-Bt(MNH-786,FH-942)parents.The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay(ELISA).The results showed that the MNH786 xCKC-1 hybrid had the highest concentrations of Cry1 Ac gene at30 DAS(3.08μg g^(-1))and 110 DAS(1.01μg g^(-1))in leaves.In contrast,the CKC-2 xMNH-786 hybrid showed the lowest concentrations of Cry1 Ac gene at 30 DAS(2.30μg g^(-1))and 110 DAS(0.86μg g^(-1)).The Fhybrid FH-942×CKC-2 showed the highest concentrations of Cry2 A gene at 30 DAS(8.39μg g^(-1))and 110 DAS(7.74μg g^(-1))in leaves,while the CKC-1 xMNH-786 hybrid expressed the lowest concentrations of Cry2 A gene at 30 DAS(7.10μg g^(-1))and 110 DAS(8.31μg g^(-1)).A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS,whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.When the expression pattern was compared between various plant parts in genotype CKC-2,it was found that leaves had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))at 70 DAS,followed by bolls(Cry1 Ac(1.66μg g^(-1))and Cry2 A(8.15μg g^(-1)))and flowers(Cry1 Ac(1.07μg g^(-1))and Cry2 A(7.99μg g^(-1))).The genotype CKC-2 had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))in the upper canopy but less accumulation(2.66μg g^(-1)of Cry1 Ac,8.09μg g^(-1)of Cry2 A)in the lower canopy at 70 DAS.Similarly,at 110 DAS,the expression levels of Cry1 Ac and Cry2 A in upper and lower canopy leaves were 1.52 and 7.92μg 9,and 0.99 and 7.54μg 9,respectively.Hence,the current study demonstrates that different genotypes showed variable expression for both of the Cry1 Ac and Cry2 A genes during plant growth due to different genetic backgrounds.The Cry2 A gene had three-fold higher expression than Cry1 Ac with significant differences in expression in different plant parts.The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1 Ac and Cry2 A for sustainable cotton production worldwide.展开更多
The Bt Cry IA (C) chloroplast expression cassette and OC chloroplast expression cassette were constructed. The Bt expression cassette contained the 3.5 kb wild type Bt Cry I4(C) gene under the control of the strong li...The Bt Cry IA (C) chloroplast expression cassette and OC chloroplast expression cassette were constructed. The Bt expression cassette contained the 3.5 kb wild type Bt Cry I4(C) gene under the control of the strong light-induced psbA promoter and terminator from rice (Oryza saliva . L) chloroplast, the gene: trnH-psbA-trnk from tobacco (Nicotiana tabacum. L) as the homologous fragment. The OC chloroplast expression cassette contained the OC gene under the control of 16S promoter and terminator from tobacco, the tobacco gene: psbA-ORF512 as homologous fragment. The two cassettes both had the aadA gene expression cassette as the selectable marker. Leaves of tobacco were cotransformed with the particle bombardment method. After selection by spectinomycin, the transformants were obtained. The integration of Bt and OC gene were confirmed by Southern-blotting analysis, and Western-blotting analysis. Proteinase inhibitor assays showed that the Bt and OC gene had expressed. Bioassays showed that the transgenic tobacco had a significant resistance to the larvae of cotton bollworm (helicoverpa zea).展开更多
Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish...Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB + 5 mg/L thidiazuron (TDZ) +7.5 mg/L AgNO3 + 0.1 mg/L NAA + 2 mg/L proline (L-pro) + 250 mg/L casein acid hydrolysate (CH) + 3% sucrose; the appropriate growth medium was 1/2 MSB + 1 mg/L IBA + 2 mg/L L-pro + 250 mg/L CH + 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB + 0.2 mg/L IAA + 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.展开更多
The damage status of L. invasa on different Eucalyptus varieties( clones) were surveyed in the field,and effects of Eucalyptus varieties on fitness,development and reproduction of this pest were studied in the study. ...The damage status of L. invasa on different Eucalyptus varieties( clones) were surveyed in the field,and effects of Eucalyptus varieties on fitness,development and reproduction of this pest were studied in the study. Results showed that:( 1) Most of the major Eucalyptus varieties( clones) could be attacked by L. invasa in Fujian Province,and galls formed on E. urophylla × E. camaldulensis DH201-2,E. gradis QG3,E. dunnii,E. urophylla cU6,E. grandis × E. urophylla GL9,E. robusta and E. exserta; L. invasa could develop and complete its life cycle on above varieties.( 2) The damage degree of different Eucalyptus varieties( clones) was different from fitness degree to L. invasa. L. invasa had different selection frequency,fecundity and number of larvae per gall on different Eucalyptus varieties( clones).( 3) In addition to little difference in female ratios,L. invasa feeding different Eucalyptus varieties( clones) had extremely significant differences in the indicators such as number of emerged L. invasa,body mass of female adult,fecundity,longevity,etc.( 4) According to the fitness index( FI),the host plants of L. invasa were divided into 4 types by cluster analysis: suitable host,moderately suitable host,less suitable host and unsuitable host; the fecundity of each female was more than 70 eggs in suitable host,which was adequate to lead to potential population outbreak of the pest. Host fitness index could be used as a comprehensive evaluation index for screening of resistant varieties( clones) in production.展开更多
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.
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.
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.
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.
文摘A plant expression vector containing a chimeric Bt29K gene coding for the activated Cry1Ac protein and the arrowhead proteinase inhibitior gene API B were introduced into the cotton cultivar Jihe321 mediated by Agrobactertium tumefaciens. Based on the results of kanamycin resistant testing, PCR detection for both foreign genes and insect bioassay using Heliethis armigera , nine transgenic homozygous cotton lines with insect resistance of more than 90% and better agronomic traits were bred through six generations from the original transgenic plants. Results from insect bioassay and sequence analysis of the PCR products of plants from some homozygous lines indicated that the chimeric Bt29K gene was stably inherited in these transgenic cotton lines. The main agronomic characters of these homozygous cotton lines, such as boll productivity and fibre strength, were better than that of the original cotton cv. Jihe321.
基金supported by the grant from the National Research and Development Project of Transgenic Crops of Ministry of Science and Technology of China (Grant No.JY03-B-11)
文摘Bt5198, a new rice restorer line containing Bt gene, was developed from the cross and backcross of the elite restorer line Chenghui 177 with Bt Minghui 63, a transgenic Bt restorer line. The inbred lines were evaluated using PCR amplification, test paper evaluation, insect resistance evaluation in both the laboratory and paddy fields, nursery evaluation of rice blast resistance and pedigree selection of agronomic traits. Larval mortalities on Bt5198 and Bt Minghui 63 were 100% when rice culms were inoculated with the eggs of the striped stem borer (SSB) in the laboratory. Bt5198 was highly resistant against SSB and the yellow stem borer (YSB) under field conditions. The F1 hybrids derived from Bt5198 and four cytoplasmic male sterile (CMS) lines were also highly resistant to SSB and YSB and had a significant heterosis. Two-year evaluation of rice blast resistance confirmed that the resistance levels of Bt5198 to leaf blast and neck blast were similar to those of Chenghui 177 and significantly better than those of Bt Minghui 63. Seed germination ability and pollen yield of Bt5198 were similar with Chenghui 177, suggesting that the introduction of the Bt gene into the new restorer line had no significant effects on seed vitality or the yield of seed production. To identify the presence of the Bt gene, it was effective to combine test paper examination with the evaluation of insect-resistance, both in the laboratory and under field conditions.
基金supported by grants from the Jilin Provincial Agricultural Science and Technology Innovation Project in China(CXGC2021TD014)the National Major Project of Breeding for Genetically Modified Organisms in China(2016ZX08001001-001-007)。
文摘Rice production and quality are seriously affected by the lepidopteran pest,striped stem borer(SSB),in Northeast China.In this study,a synthetic cry1 C gene encoding Bacillus thuringiensis(Bt)δ-endotoxin,which is toxic to lepidopteran pest,was transformed into a japonica rice variety(Jigeng 88)in Northeast China by Agrobacterium-mediated transformation.Through molecular detection and the Basta resistance germination assay,a total of 16 single-copy homozygous transgenic lines were obtained from 126 independent transformants expressing cry1 C.Finally,four cry1 C-transgenic lines(JL16,JL23,JL41,and JL42)were selected by evaluation of the Cry1 C protein level,insect-resistance and agronomic traits.The cry1 C-transgenic lines had higher resistance to SSB and higher yield compared with non-transgenic(NT)control plants.T-DNA flanking sequence analysis of the transgenic line JL42 showed that the cry1 C gene was inserted into the intergenic region of chromosome 11,indicating that its insertion may not interfere with the genes near insertion site.In summary,this study developed four cry1 C-transgenic japonica rice lines with high insect resistance and high yield.They can be used as insect-resistant germplasm materials to overcome the problem of rice yield reduction caused by SSB and reduce the use of pesticides in Northeast China.
基金funded by the National Major Project for Transgenic Organism Breeding, China (201408001001-009)
文摘A synthetic cry2A^* gene enco ding Bacillus thuringiensis(Bt) δ-endotoxi n that resi st ance to lepidopteran pest was transformed into japonica rice variety Jijing 88, which is the most widely cultivated variety in Jilin Province, Northeast China, by Agrobacterium-mediated transformation. A total of 106 independent transformants overexpressing cry2A^* gene driven by ubiquitin(Ubi) promoter was produced. Three single-copy homozygous transgenic lines were finally selected based on the results of PCR analysis, se gregation ratio of Bast a resistance, and Southern hybridiza tion analyse s. RT-PCR and enzyme linke dimmune sorbent assay(ELISA) revealed that cry2A^* transcripts and protein were highly expressed in these lines. The high level of Cry2A^* protein expression resulted in high resistance to rice striped stem borer as evidence d by insect feeding bioassays. Our results demonst rate that cry2A^* transgenic japonica rice confers resistance to the rice striped stem borer in the laboratory conditions.
基金Higher Education Commission,Pakistan for providing funds。
文摘Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacillus thuringiensis(Bt)cotton with a single Cry1 Ac gene,growers are on the verge of adopting Bt cotton that carries the double gene(Cry1 Ac+Cry2 A)due to its better effectiveness against insect pests.Thus,the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.The expression levels of the Cry1 Ac and Cry2 A genes were evaluated in the leaves of 10 genotypes(2 parents and 8 Fhybrids)at 30 days after sowing(DAS),while samples of leaves,bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.The Fhybrids were developed through reciprocal crosses between two Bt(CKC-1,CKC-2)and two non-Bt(MNH-786,FH-942)parents.The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay(ELISA).The results showed that the MNH786 xCKC-1 hybrid had the highest concentrations of Cry1 Ac gene at30 DAS(3.08μg g^(-1))and 110 DAS(1.01μg g^(-1))in leaves.In contrast,the CKC-2 xMNH-786 hybrid showed the lowest concentrations of Cry1 Ac gene at 30 DAS(2.30μg g^(-1))and 110 DAS(0.86μg g^(-1)).The Fhybrid FH-942×CKC-2 showed the highest concentrations of Cry2 A gene at 30 DAS(8.39μg g^(-1))and 110 DAS(7.74μg g^(-1))in leaves,while the CKC-1 xMNH-786 hybrid expressed the lowest concentrations of Cry2 A gene at 30 DAS(7.10μg g^(-1))and 110 DAS(8.31μg g^(-1)).A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS,whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.When the expression pattern was compared between various plant parts in genotype CKC-2,it was found that leaves had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))at 70 DAS,followed by bolls(Cry1 Ac(1.66μg g^(-1))and Cry2 A(8.15μg g^(-1)))and flowers(Cry1 Ac(1.07μg g^(-1))and Cry2 A(7.99μg g^(-1))).The genotype CKC-2 had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))in the upper canopy but less accumulation(2.66μg g^(-1)of Cry1 Ac,8.09μg g^(-1)of Cry2 A)in the lower canopy at 70 DAS.Similarly,at 110 DAS,the expression levels of Cry1 Ac and Cry2 A in upper and lower canopy leaves were 1.52 and 7.92μg 9,and 0.99 and 7.54μg 9,respectively.Hence,the current study demonstrates that different genotypes showed variable expression for both of the Cry1 Ac and Cry2 A genes during plant growth due to different genetic backgrounds.The Cry2 A gene had three-fold higher expression than Cry1 Ac with significant differences in expression in different plant parts.The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1 Ac and Cry2 A for sustainable cotton production worldwide.
基金supported by the National Natural Science Foundation of China(39570361).
文摘The Bt Cry IA (C) chloroplast expression cassette and OC chloroplast expression cassette were constructed. The Bt expression cassette contained the 3.5 kb wild type Bt Cry I4(C) gene under the control of the strong light-induced psbA promoter and terminator from rice (Oryza saliva . L) chloroplast, the gene: trnH-psbA-trnk from tobacco (Nicotiana tabacum. L) as the homologous fragment. The OC chloroplast expression cassette contained the OC gene under the control of 16S promoter and terminator from tobacco, the tobacco gene: psbA-ORF512 as homologous fragment. The two cassettes both had the aadA gene expression cassette as the selectable marker. Leaves of tobacco were cotransformed with the particle bombardment method. After selection by spectinomycin, the transformants were obtained. The integration of Bt and OC gene were confirmed by Southern-blotting analysis, and Western-blotting analysis. Proteinase inhibitor assays showed that the Bt and OC gene had expressed. Bioassays showed that the transgenic tobacco had a significant resistance to the larvae of cotton bollworm (helicoverpa zea).
基金Supported by National Natural Science Foundation of China(31301703)Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province[CX(14)5068]
文摘Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB + 5 mg/L thidiazuron (TDZ) +7.5 mg/L AgNO3 + 0.1 mg/L NAA + 2 mg/L proline (L-pro) + 250 mg/L casein acid hydrolysate (CH) + 3% sucrose; the appropriate growth medium was 1/2 MSB + 1 mg/L IBA + 2 mg/L L-pro + 250 mg/L CH + 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB + 0.2 mg/L IAA + 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.
基金Supported by Key Project of Fujian Department of Science and Technology(2012N0008)Science and Technology Project of Fujian Department of Forestry(MLK[2005])
文摘The damage status of L. invasa on different Eucalyptus varieties( clones) were surveyed in the field,and effects of Eucalyptus varieties on fitness,development and reproduction of this pest were studied in the study. Results showed that:( 1) Most of the major Eucalyptus varieties( clones) could be attacked by L. invasa in Fujian Province,and galls formed on E. urophylla × E. camaldulensis DH201-2,E. gradis QG3,E. dunnii,E. urophylla cU6,E. grandis × E. urophylla GL9,E. robusta and E. exserta; L. invasa could develop and complete its life cycle on above varieties.( 2) The damage degree of different Eucalyptus varieties( clones) was different from fitness degree to L. invasa. L. invasa had different selection frequency,fecundity and number of larvae per gall on different Eucalyptus varieties( clones).( 3) In addition to little difference in female ratios,L. invasa feeding different Eucalyptus varieties( clones) had extremely significant differences in the indicators such as number of emerged L. invasa,body mass of female adult,fecundity,longevity,etc.( 4) According to the fitness index( FI),the host plants of L. invasa were divided into 4 types by cluster analysis: suitable host,moderately suitable host,less suitable host and unsuitable host; the fecundity of each female was more than 70 eggs in suitable host,which was adequate to lead to potential population outbreak of the pest. Host fitness index could be used as a comprehensive evaluation index for screening of resistant varieties( clones) in production.