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.
