Seed priming is an effective seed pretreatment technology that enhances germination and overall crop performance by optimizing seed hydration and metabolic processes before planting.Seed quality is a critical determin...Seed priming is an effective seed pretreatment technology that enhances germination and overall crop performance by optimizing seed hydration and metabolic processes before planting.Seed quality is a critical determinant of cotton(Gossypium hirsutum)crop performance,influencing germination,plant vigor,and yield.This study evaluates the effects of seed priming with potassium salts(1%and 2%KCl and K2SO4)on germination,morphological traits,and Cry1Ac gene expression in three Bt cotton cultivars(IUB-2013,NIAB-878B,FH-142)as Cry1Ac enhance the pest resistance in Bt cotton and reduce the plant’s dependence on chemical insecticides.Seeds were primed for six hours,air-dried,and sown in the field.Germination rates,plant height,number of bolls per plant,boll weight,seed cotton yield,and ginning outturn(GOT)were assessed at crop maturity.Cry1Ac gene expression was quantified to explore the influence of priming treatments on transgene activity.Results demonstrated that 1%K2SO4 priming significantly enhanced germination and yield-related traits,with Cry1Ac expression peaking in the IUB-2013 cultivar under 1%K2SO4 treatment.These findings suggest that potassium-based halopriming improves cotton seedling establishment and Bt gene expression.This study addresses the critical gaps in understanding the effects of seed halopriming on morphological traits,germination,and expression of the Cry1Ac gene in Bt cotton while providing a novel eco-friendly and cost-effective halopriming approach,offering the potential to improve cotton production.展开更多
Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis(Bt)have proven to be highly effective in managing some key pests.However,the evolution of resistance by the target pests threa...Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis(Bt)have proven to be highly effective in managing some key pests.However,the evolution of resistance by the target pests threatens the sustainability of Bt crops.The L31S mutation in a tetraspanin encoded by Harm TspC5(previously known as Ha TSPAN1)has been shown to confer dominant resistance to the Bt protein Cry1Ac in Helicoverpa armigera,a globally damaging lepidopteran pest.However,the broader implications of the L31S mutation in the tetraspanins of other lepidopteran species remain unclear.The evolutionary analyses in this study indicate that TspC5s have evolved in a species-specific manner among the lepidopteran insects.To investigate the role of TspC5s in conferring dominant resistance to Cry1Ac,we used the piggyBac-based transformation system to generate four transgenic H.armigera strains that express exogenous TspC5 variants from three phylogenetically close species(Helicoverpa zea,Helicoverpa assulta and Heliothis virescens)and one phylogenetically distant species(Plutella xylostella).In comparison with the background SCD strain of H.armigera,the transgenic strains expressing HzeaTspC5-L31S,HassTspC5-L31S,or HvirTspC5-L31S exhibited significant resistance to Cry1Ac(10.0-,21.4-,and 81.1-fold,respectively),whereas the strain expressing PxylTspC5-L27S remained susceptible.Furthermore,the Cry1Ac resistant phenotypes followed an autosomal dominant inheritance pattern and were closely linked to the introduced mutant TspC5s.These findings reveal the conserved role of TspC5s from Helicoverpa and Heliothis species in mediating the dominant resistance to Cry1Ac,and they provide crucial insights for assessing resistance risks related to mutant tetraspanins and devising adaptive resistance management strategies for these major lepidopteran pests.展开更多
The effects of mepiquat chloride(DPC)on the Cry1Ac protein content in Bacillus thuringiensis(Bt)cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt ...The effects of mepiquat chloride(DPC)on the Cry1Ac protein content in Bacillus thuringiensis(Bt)cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and-efficiency cotton cultivation.This study was conducted using Bt cotton cultivar‘Sikang 3'during the 2020 and 2021 growing seasons at Yangzhou University Farm,Yangzhou,Jiangsu Province,China.Potted cotton plants were exposed to high temperature and drought stress,and sprayed with either 20 mg L^(-1)DPC or water(CK).Seven days after treatment,the Cry1Ac protein content,α-ketoglutarate content,pyruvic acid content,glutamate synthase activity,glutamic oxaloacetic transaminase activity,soluble protein content,and amino acid content were measured,and transcriptome sequencing was performed.DESeq was used for differential gene analysis.Under the DPC treatment,the Cry1Ac protein content increased by 4.7-11.9% compared to CK.Theα-ketoglutarate content,pyruvic acid content,glutamate synthase activity,glutamic oxaloacetic transaminase activity,soluble protein content,and amino acid content all increased.Transcriptome analysis revealed 7,542 upregulated genes and 10,449 downregulated genes for DPC vs.CK.Gene ontology(GO)and Kyoto Encyclopedia of Gene and Genomes(KEGG)analyses showed that the differentially expressed genes were mainly involved in biological processes,such as carbon and amino acid metabolism.For example,genes encoding 6-phosphofructokinase,pyruvate kinase,glutamic pyruvate transaminase,pyruvate dehydrogenase,citrate synthase,isocitrate dehydrogenase,2-oxoglutarate dehydrogenase,glutamate synthase,1-pyrroline-5-carboxylate dehydrogenase,glutamic oxaloacetic transaminase,amino-acid N-acetyltransferase,and acetylornithine deacetylase were all significantly upregulated.The DPC treatment increased pyruvate,α-ketoglutarate,and oxaloacetate by increasing the operational rate of the glycolytic pathway of the citric acid cycle.It also significantly upregulated the genes encoding glutamate synthase,pyrrolidine-5-carboxylic acid dehydrogenase,glutamate oxaloacetate transaminase,and N-acetylglutamate synthetase,while it downregulated the genes encoding glutamine synthetase.Therefore,the synthesis of aspartic acid,glutamic acid,pyruvate,and arginine increased after treatment with DPC,and the Cry1Ac protein content was increased by regulating carbon and amino acid metabolism.展开更多
A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression c...A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API-B genes was constructed. These two insect-resistant genes were transferred into two cotton ( Gossypium hirsutum L.) varieties ( or lines) via Agrobacterium-mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0% - 99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Ac and API-B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect-resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API-B with that expressing Cry1Ac only revealed that the insect-resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect-resistant genes reported here is reasonable.展开更多
基金National Natural Science Foundation of China(3216045632360474+2 种基金32360486)grants from the Provincial Basic Research Program(Natural Science)([2020]1Z018)Provincial Key Technology R&D Program([2021]YiBan272).
文摘Seed priming is an effective seed pretreatment technology that enhances germination and overall crop performance by optimizing seed hydration and metabolic processes before planting.Seed quality is a critical determinant of cotton(Gossypium hirsutum)crop performance,influencing germination,plant vigor,and yield.This study evaluates the effects of seed priming with potassium salts(1%and 2%KCl and K2SO4)on germination,morphological traits,and Cry1Ac gene expression in three Bt cotton cultivars(IUB-2013,NIAB-878B,FH-142)as Cry1Ac enhance the pest resistance in Bt cotton and reduce the plant’s dependence on chemical insecticides.Seeds were primed for six hours,air-dried,and sown in the field.Germination rates,plant height,number of bolls per plant,boll weight,seed cotton yield,and ginning outturn(GOT)were assessed at crop maturity.Cry1Ac gene expression was quantified to explore the influence of priming treatments on transgene activity.Results demonstrated that 1%K2SO4 priming significantly enhanced germination and yield-related traits,with Cry1Ac expression peaking in the IUB-2013 cultivar under 1%K2SO4 treatment.These findings suggest that potassium-based halopriming improves cotton seedling establishment and Bt gene expression.This study addresses the critical gaps in understanding the effects of seed halopriming on morphological traits,germination,and expression of the Cry1Ac gene in Bt cotton while providing a novel eco-friendly and cost-effective halopriming approach,offering the potential to improve cotton production.
基金primarily funded by a grant from the National Natural Science Foundation of China(31930093)Additional support was provided by the Natural Science Foundation of Jiangsu Province,China(BK20230983)the Project of Fund for Stable Support to Agricultural Sci-Tech Renovation,China(xjnkywdzc-2022004)。
文摘Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis(Bt)have proven to be highly effective in managing some key pests.However,the evolution of resistance by the target pests threatens the sustainability of Bt crops.The L31S mutation in a tetraspanin encoded by Harm TspC5(previously known as Ha TSPAN1)has been shown to confer dominant resistance to the Bt protein Cry1Ac in Helicoverpa armigera,a globally damaging lepidopteran pest.However,the broader implications of the L31S mutation in the tetraspanins of other lepidopteran species remain unclear.The evolutionary analyses in this study indicate that TspC5s have evolved in a species-specific manner among the lepidopteran insects.To investigate the role of TspC5s in conferring dominant resistance to Cry1Ac,we used the piggyBac-based transformation system to generate four transgenic H.armigera strains that express exogenous TspC5 variants from three phylogenetically close species(Helicoverpa zea,Helicoverpa assulta and Heliothis virescens)and one phylogenetically distant species(Plutella xylostella).In comparison with the background SCD strain of H.armigera,the transgenic strains expressing HzeaTspC5-L31S,HassTspC5-L31S,or HvirTspC5-L31S exhibited significant resistance to Cry1Ac(10.0-,21.4-,and 81.1-fold,respectively),whereas the strain expressing PxylTspC5-L27S remained susceptible.Furthermore,the Cry1Ac resistant phenotypes followed an autosomal dominant inheritance pattern and were closely linked to the introduced mutant TspC5s.These findings reveal the conserved role of TspC5s from Helicoverpa and Heliothis species in mediating the dominant resistance to Cry1Ac,and they provide crucial insights for assessing resistance risks related to mutant tetraspanins and devising adaptive resistance management strategies for these major lepidopteran pests.
基金supported by the National Natural Science Foundation of China(31901462)the Natural Science Foundation of the Jiangsu Higher Education Institutions,China(22KJA210005)+1 种基金the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD)the Brand Professional Construction Program of Jiangsu Higher Education Institutions,China。
文摘The effects of mepiquat chloride(DPC)on the Cry1Ac protein content in Bacillus thuringiensis(Bt)cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and-efficiency cotton cultivation.This study was conducted using Bt cotton cultivar‘Sikang 3'during the 2020 and 2021 growing seasons at Yangzhou University Farm,Yangzhou,Jiangsu Province,China.Potted cotton plants were exposed to high temperature and drought stress,and sprayed with either 20 mg L^(-1)DPC or water(CK).Seven days after treatment,the Cry1Ac protein content,α-ketoglutarate content,pyruvic acid content,glutamate synthase activity,glutamic oxaloacetic transaminase activity,soluble protein content,and amino acid content were measured,and transcriptome sequencing was performed.DESeq was used for differential gene analysis.Under the DPC treatment,the Cry1Ac protein content increased by 4.7-11.9% compared to CK.Theα-ketoglutarate content,pyruvic acid content,glutamate synthase activity,glutamic oxaloacetic transaminase activity,soluble protein content,and amino acid content all increased.Transcriptome analysis revealed 7,542 upregulated genes and 10,449 downregulated genes for DPC vs.CK.Gene ontology(GO)and Kyoto Encyclopedia of Gene and Genomes(KEGG)analyses showed that the differentially expressed genes were mainly involved in biological processes,such as carbon and amino acid metabolism.For example,genes encoding 6-phosphofructokinase,pyruvate kinase,glutamic pyruvate transaminase,pyruvate dehydrogenase,citrate synthase,isocitrate dehydrogenase,2-oxoglutarate dehydrogenase,glutamate synthase,1-pyrroline-5-carboxylate dehydrogenase,glutamic oxaloacetic transaminase,amino-acid N-acetyltransferase,and acetylornithine deacetylase were all significantly upregulated.The DPC treatment increased pyruvate,α-ketoglutarate,and oxaloacetate by increasing the operational rate of the glycolytic pathway of the citric acid cycle.It also significantly upregulated the genes encoding glutamate synthase,pyrrolidine-5-carboxylic acid dehydrogenase,glutamate oxaloacetate transaminase,and N-acetylglutamate synthetase,while it downregulated the genes encoding glutamine synthetase.Therefore,the synthesis of aspartic acid,glutamic acid,pyruvate,and arginine increased after treatment with DPC,and the Cry1Ac protein content was increased by regulating carbon and amino acid metabolism.
文摘A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API-B genes was constructed. These two insect-resistant genes were transferred into two cotton ( Gossypium hirsutum L.) varieties ( or lines) via Agrobacterium-mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0% - 99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Ac and API-B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect-resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API-B with that expressing Cry1Ac only revealed that the insect-resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect-resistant genes reported here is reasonable.
