Cotton bolls exhibit the lowest insecticidal efficacy among all organs of Bt cotton,which would ultimately affect the yield formation.The objective of this study was to investigate the effects of different urea concen...Cotton bolls exhibit the lowest insecticidal efficacy among all organs of Bt cotton,which would ultimately affect the yield formation.The objective of this study was to investigate the effects of different urea concentrations on the seed Bt protein contents,seed cotton yield and the corresponding protein metabolism mechanism.The experiments were conducted during 2017–2018 cotton growing seasons.Two cultivars,Sikang 3(hybrid,SK3)and Sikang 1(conventional,SK1),were treated with six urea concentrations and their seed Bt protein contents were compared during boll formation period.The urea spray concentration had a significant effect on the seed Bt toxin content and seed cotton yield.Spraying of either 5 or 6%urea led to higher insecticidal protein contents and higher seed cotton yield for both cultivars.Moreover,the highest amino acid and soluble protein contents,as well as GPT and GOT activities,and lower protease and peptidase activities were observed at the 5 to 6%urea levels.Significant positive correlations between the seed Bt toxin and amino acid contents,and between the seed Bt toxin content and GPT activities were detected.The lower boll worm number and hazard boll rate were also observed with the 5 to 6%urea treatments,which may be the reason why nitrogen spraying increased the seed cotton yield.Therefore,our results suggested that the seed Bt toxin content and insect resistance were impacted markedly by external nitrogen application,and 5 to 6%urea had the greatest effect on insect resistance.展开更多
Background: In order to uncover the mechanism of significantly reduced insect resistance at the late developmental stage in cotton(Gossypium hirsutum L.),the relationship between boll setting rate under different plan...Background: In order to uncover the mechanism of significantly reduced insect resistance at the late developmental stage in cotton(Gossypium hirsutum L.),the relationship between boll setting rate under different planting densities and Bacillus thuringiensis(Bt)insecticidal concentrations in the boll wall were investigated in the present study.Two studies were arranged at Yangzhou,China during the 2017–2018 cotton growth seasons.Five planting densities(15000,25000,45000,60000 and 75000 plants per hectare)and the flower-removal treatment were imposed separately on Bt cotton cultivar Sikang3 to arrange different boll setting rates,and the boll setting rates and Bt toxin content were compared.Results: Higher boll setting rate together with lower Bt toxin contents in boll wall was observed under low planting density,whereas lower boll setting rate and higher Bt toxin contents were found under high planting density.Also,higher Bt protein concentration was associated with higher soluble protein content,glutamic-pyruvic transaminase(GPT),and glutamic oxaloacetate transaminase(GOT)activities,but lower amino acid content,and protease and peptidase activities.It was further confirmed that a higher boll setting rate with lower Bt protein content under flower-removal.Conclusions: This study demonstrated that the insecticidal efficacy of boll walls was significantly impacted by boll formation.Reduced protein synthesis and enhanced protein degradation were related to the reduced Bt toxin concentration.展开更多
A method of extracting and purifying Cry1Ab protein(Bt toxin) from Cry1Ab transgenic rice was established. Most of the Bt toxin present in the tissue of Cry1Ab transgenic rice was extracted effectively with a solution...A method of extracting and purifying Cry1Ab protein(Bt toxin) from Cry1Ab transgenic rice was established. Most of the Bt toxin present in the tissue of Cry1Ab transgenic rice was extracted effectively with a solution of 50 mmol/LNa_2CO_3 and NaHCO_3. The crude protein containing Bt toxin was obtained after the pretreatment of Cry1Ab transgenic rice with ultra-filtration, ammonium sulfate precipitation and centrifugation. The dialysed crude protein was futher separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The protein bound on DEAE Sephadex A-50 gel was eluted with buffer solution B(10 mmol/L tris-HCl buffer+1.0 mmol/L EDTA, pH=8.0) mixed with 0.1, 0.3, 0.5 and 0.8 mol/L NaCl in a discontinuous gradient elution mode. The peak of the Bt toxin eluted from the columns was identified by ELISA and bioassayed with larvae of tobacco hornworms and silkworms. The purity and the bioactivity of the Bt toxin were determined by means of SDS-PAGE and larvicidal assay, respectively. The purity of the Bt toxin obtained by this method is high, and its insecticidal activity is retained after the toxin is purified.展开更多
Investigations were done on the usefulness of Envirologix CrylAb/CrylAc Plate kits for quantitative analysis of Bt toxin content in transgenic rice grains. Two transgenic rice lines: Kemingdao 1 (KMD1) and Kemingdao 2...Investigations were done on the usefulness of Envirologix CrylAb/CrylAc Plate kits for quantitative analysis of Bt toxin content in transgenic rice grains. Two transgenic rice lines: Kemingdao 1 (KMD1) and Kemingdao 2 (KMD2), transformed with a crylAb gene, and their parental variety, cv. Xiushui 11, were used as positive and negative samples. Results showed that the correlation coefficients as high as 0. 985 - 0. 998, significant at probability level of 0. 05 or 0.01, were obtained for linear regression equations by using the appended calibrators of the kits. No significant differences were detected for values of same rice sample obtained from different trials or by using different lots of the product (kit). The detectable Bt toxin content by this method could be as low as 0.5 ng/g. The Envirologix Kit could be useful for rapid quantitative detection of Bt toxin in rice grains because of its preciseness, simplicity and time saving.展开更多
A promoter of the PNZIP(Pharbitis nil leucine zipper)gene(1.459 kb)was cloned from Pharbitis nil and fused to the GUS(b-glucuronidase)and Bacillus thuringiensis endotoxin(Cry9C)genes.Several transgenic PNZIP::GUS and ...A promoter of the PNZIP(Pharbitis nil leucine zipper)gene(1.459 kb)was cloned from Pharbitis nil and fused to the GUS(b-glucuronidase)and Bacillus thuringiensis endotoxin(Cry9C)genes.Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation.Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants.In contrast,GUS staining in the reproductive structures such as petals,anther,and immature seeds of PNZIP::GUS cotton was very faint.Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus(Ca MV)35S::Cry9C line were selected for enzyme-linked immunosorbent assay(ELISA)and insect bioassays.Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from24.6 to 45.5μg g^(-1) fresh weight.In green tissues such as the leaves,boll rinds,and bracts of the PNZIP::Cry9C line,the Cry9C protein accumulated up to 50.2,39.7,and 48.3μg g^(-1) fresh weight respectively.In contrast,seeds of the PNZIP::Cry9C line(PZ1.3)accumulated only 0.26μg g^(-1) fresh weight of the Cry9C protein,which was 100 times lower than that recorded for the seeds of the Ca MV 35S::Cry9C line.The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm.The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds.These features should allay public concerns about the safety of transgenic foods.We propose the future utility of PNZIP as an economical,environmentally friendly promoter in cotton biotechnology.展开更多
基金the National Natural Science Foundation of China(31671613 and 31901462)the National Key Research and Development Program of China(2018YFD0100406 and 2017YFD0201306)+3 种基金the Natural Science Foundation of Jiangsu Province,China(BK20191439)the Natural Science Foundation of Jiangsu Higher Education Institutions,China(17KJA210003)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Cotton bolls exhibit the lowest insecticidal efficacy among all organs of Bt cotton,which would ultimately affect the yield formation.The objective of this study was to investigate the effects of different urea concentrations on the seed Bt protein contents,seed cotton yield and the corresponding protein metabolism mechanism.The experiments were conducted during 2017–2018 cotton growing seasons.Two cultivars,Sikang 3(hybrid,SK3)and Sikang 1(conventional,SK1),were treated with six urea concentrations and their seed Bt protein contents were compared during boll formation period.The urea spray concentration had a significant effect on the seed Bt toxin content and seed cotton yield.Spraying of either 5 or 6%urea led to higher insecticidal protein contents and higher seed cotton yield for both cultivars.Moreover,the highest amino acid and soluble protein contents,as well as GPT and GOT activities,and lower protease and peptidase activities were observed at the 5 to 6%urea levels.Significant positive correlations between the seed Bt toxin and amino acid contents,and between the seed Bt toxin content and GPT activities were detected.The lower boll worm number and hazard boll rate were also observed with the 5 to 6%urea treatments,which may be the reason why nitrogen spraying increased the seed cotton yield.Therefore,our results suggested that the seed Bt toxin content and insect resistance were impacted markedly by external nitrogen application,and 5 to 6%urea had the greatest effect on insect resistance.
基金Natural Science Research of Jiangsu Higher Education Institutions of China(17KJA210003)The Project#31671613 and#31901462 supported by National Natural Science Foundation of China,Project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD)+1 种基金Natural Science Foundation of Jiangsu Province(BK20191439)Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX19_2106).
文摘Background: In order to uncover the mechanism of significantly reduced insect resistance at the late developmental stage in cotton(Gossypium hirsutum L.),the relationship between boll setting rate under different planting densities and Bacillus thuringiensis(Bt)insecticidal concentrations in the boll wall were investigated in the present study.Two studies were arranged at Yangzhou,China during the 2017–2018 cotton growth seasons.Five planting densities(15000,25000,45000,60000 and 75000 plants per hectare)and the flower-removal treatment were imposed separately on Bt cotton cultivar Sikang3 to arrange different boll setting rates,and the boll setting rates and Bt toxin content were compared.Results: Higher boll setting rate together with lower Bt toxin contents in boll wall was observed under low planting density,whereas lower boll setting rate and higher Bt toxin contents were found under high planting density.Also,higher Bt protein concentration was associated with higher soluble protein content,glutamic-pyruvic transaminase(GPT),and glutamic oxaloacetate transaminase(GOT)activities,but lower amino acid content,and protease and peptidase activities.It was further confirmed that a higher boll setting rate with lower Bt protein content under flower-removal.Conclusions: This study demonstrated that the insecticidal efficacy of boll walls was significantly impacted by boll formation.Reduced protein synthesis and enhanced protein degradation were related to the reduced Bt toxin concentration.
基金Supported by the National Science Foundation of China(No.2 0 1770 2 1and 2 0 2 770 31)
文摘A method of extracting and purifying Cry1Ab protein(Bt toxin) from Cry1Ab transgenic rice was established. Most of the Bt toxin present in the tissue of Cry1Ab transgenic rice was extracted effectively with a solution of 50 mmol/LNa_2CO_3 and NaHCO_3. The crude protein containing Bt toxin was obtained after the pretreatment of Cry1Ab transgenic rice with ultra-filtration, ammonium sulfate precipitation and centrifugation. The dialysed crude protein was futher separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The protein bound on DEAE Sephadex A-50 gel was eluted with buffer solution B(10 mmol/L tris-HCl buffer+1.0 mmol/L EDTA, pH=8.0) mixed with 0.1, 0.3, 0.5 and 0.8 mol/L NaCl in a discontinuous gradient elution mode. The peak of the Bt toxin eluted from the columns was identified by ELISA and bioassayed with larvae of tobacco hornworms and silkworms. The purity and the bioactivity of the Bt toxin were determined by means of SDS-PAGE and larvicidal assay, respectively. The purity of the Bt toxin obtained by this method is high, and its insecticidal activity is retained after the toxin is purified.
文摘Investigations were done on the usefulness of Envirologix CrylAb/CrylAc Plate kits for quantitative analysis of Bt toxin content in transgenic rice grains. Two transgenic rice lines: Kemingdao 1 (KMD1) and Kemingdao 2 (KMD2), transformed with a crylAb gene, and their parental variety, cv. Xiushui 11, were used as positive and negative samples. Results showed that the correlation coefficients as high as 0. 985 - 0. 998, significant at probability level of 0. 05 or 0.01, were obtained for linear regression equations by using the appended calibrators of the kits. No significant differences were detected for values of same rice sample obtained from different trials or by using different lots of the product (kit). The detectable Bt toxin content by this method could be as low as 0.5 ng/g. The Envirologix Kit could be useful for rapid quantitative detection of Bt toxin in rice grains because of its preciseness, simplicity and time saving.
基金the National Natural Science Foundation of China (31171592, 31371673)Fundamental Research Funds for the Central Universities (2013PY064)
文摘A promoter of the PNZIP(Pharbitis nil leucine zipper)gene(1.459 kb)was cloned from Pharbitis nil and fused to the GUS(b-glucuronidase)and Bacillus thuringiensis endotoxin(Cry9C)genes.Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation.Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants.In contrast,GUS staining in the reproductive structures such as petals,anther,and immature seeds of PNZIP::GUS cotton was very faint.Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus(Ca MV)35S::Cry9C line were selected for enzyme-linked immunosorbent assay(ELISA)and insect bioassays.Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from24.6 to 45.5μg g^(-1) fresh weight.In green tissues such as the leaves,boll rinds,and bracts of the PNZIP::Cry9C line,the Cry9C protein accumulated up to 50.2,39.7,and 48.3μg g^(-1) fresh weight respectively.In contrast,seeds of the PNZIP::Cry9C line(PZ1.3)accumulated only 0.26μg g^(-1) fresh weight of the Cry9C protein,which was 100 times lower than that recorded for the seeds of the Ca MV 35S::Cry9C line.The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm.The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds.These features should allay public concerns about the safety of transgenic foods.We propose the future utility of PNZIP as an economical,environmentally friendly promoter in cotton biotechnology.
