16S rDNA and ERIC （Enterobacteia Repetitive Intergenic Consensus Sequences） based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were d...16S rDNA and ERIC （Enterobacteia Repetitive Intergenic Consensus Sequences） based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus＂ strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn＇t have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.展开更多
Background:Potassium(K)deficiency has become a common field production problem following the widespread adoption of Bacillus thuringiensis(Bt)transgenic cotton(Gossypium hirsutum L.)worldwide.The purpose of this study...Background:Potassium(K)deficiency has become a common field production problem following the widespread adoption of Bacillus thuringiensis(Bt)transgenic cotton(Gossypium hirsutum L.)worldwide.The purpose of this study was to clarify whether the introduction of Bt gene directly reduces the K-use efficiency of cotton to induce K deficiency.Results:The cotton variety,Jihe 321(wild type,WT)and its two Bt(Cry1Ac)-transgenic overexpression lines(OE-29317,OE-29312)were studied in field with low soil-test K+(47.8 mg·kg^(−1)).In the field with low soil-test K+,only OE-29317 had less biomass and K+accumulation than the WT at some growth stages.Both Bt lines produced similar or even greater seed cotton yield than WT in the field.When the Bt gene(~70%)in OE-29317 and OE-29312 plants was silenced by virus-induced gene silencing(VIGS),the VIGS-Bt plants did not produce more biomass than VIGSgreen fluorescent protein(control)plants.Conclusions:The introduction of Bt gene did not necessarily hinder the K use efficiency of the cotton lines under this study.展开更多
Vegetative Insecticidal Proteins(VIPs), a large family of insecticidal proteins, are produced from Bacillus thuringiensis(Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticide...Vegetative Insecticidal Proteins(VIPs), a large family of insecticidal proteins, are produced from Bacillus thuringiensis(Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticides that confer a wider insecticidal spectrum and have stronger activity. This work compared the geographical distribution of Bt strains and their vip3 genes in different climatic zones in China, the tropical(Hainan Province), subtropical(Guangxi Province) and temperate zones(Heilongjiang Province). A total of 156 Bt strains were isolated from 841 soil samples in Hainan Province tropical region, 356 Bt strains from 1 420 soil samples in Guangxi Province and 167 Bt strains from 1 010 soil samples in different geographical regions in Heilongjiang Province. Twenty-two out of 156 strains from tropical Hainan Province and two out of 356 from subtropical Guangxi Province were found to express vip3 genes, while vip3 genes were not expressed from temperate zone in Heilongjiang Province. Restriction Fragment Length Polymorphism(RFLP) was used to identify different types of vip3 genes that were within the same family and three fulllength vip3 genes were isolated. The genes cloned from Bacillus thuringiensis strain SL3 expressed in the transformed E. coli BL21 strain. Through SDS-PAGE, 88.6 ku insecticidal protein was expressed. The bioassays used two-instar larva of Lepidoptera insects(Spodoptera exigua and Agrotis ipsilon) were performed. The results of the bioassays showed that the protein strongly inhibited the body weight increasement on Spodoptera exigua and Agrotis ipsilon in a standard bioassay. Taken together, the results indicated that the distribution of Bt strains and vip3 genes had regional preference. Tropical and subtropical regions were the rich resources of Bt strains and vip3 genes compared with temperate region. These results would undoubtedly facilitate the studies of insecticidal proteins and expand the list of the pest-killing candidates to make fully use of the extremely rich microbial resources. The new vip3 genes isolated in the current study might also help resolve the emerging insecticidal resistance problems.展开更多
Bacillus thuringiensis （Bt） is widely used in insecticides. Bt is a gram positive sporulation bacterium belonging to Bacillaceae family. It produces different insecticidal proteins like Cry toxin, Vip toxin, β-exot...Bacillus thuringiensis （Bt） is widely used in insecticides. Bt is a gram positive sporulation bacterium belonging to Bacillaceae family. It produces different insecticidal proteins like Cry toxin, Vip toxin, β-exotoxin and chitinase, etc. Bt insecticidal crystal proteins （ICPs） are not homologous to other known Vip protein and then act against lepidopteran, dipteran, coleopteran and nematodeslarvae via a unique process. In this experiment, modern high-throughput sequencing technique and sequencing were used and the whole genome sequence of BtLTS290 was obtained. The results compared to the database of GenBank showed that there was a cry57 gene in the genome sequence of BtLTS290. A novel cry57 gene was cloned and named cry57Ab1 （accession number is KF638650） by International Nomenclature Committee of Bt Endotoxin. cry57Ab1 gene could be expressed with the molecular weight of 90 ku. Cry57Ab1 protein had no obvious activity against Spodoptera exigua and Helicoverpa armigera. And Cry57Ab1 protein had a slight insecticidal activity against Ostrinia furnacalis and Plutella xylostella. Furthermore, the domain Ⅱof Cry57Ab1 and Cry1Bb were exchanged by overlapping extension PCR. SDS-PAGE showed that the molecular weight of Cry57Ab/1Bb/57Ab was about 90 ku. The insecticidal activity of Cry57Ab1 protein and Cry57Ab/1Bb/57Ab recombinant protein were determined. The results showed that the insecticidal activity of the recombinant protein to Spodoptera exigua and Helicoverpa armigera was very low, and the corrected mortality was less than 10%. The insecticidal activities against Ostrinia furnacalis and Plutella xylostella were reduced. The corrected mortality of Ostrinia furnacalis was 4.4%, and the corrected mortality rate for Plutella xylostella was 6.7%. Domain Ⅱof cry toxin played a key role on affecting host specifcity.展开更多
Bacillus thuringiensis(Bt)exhibits strong insecticidal activity and is harmless to non-target organisms such as human and animals.Bt becomes the most commonly used environment-friendly insecticidal microorganism.Howev...Bacillus thuringiensis(Bt)exhibits strong insecticidal activity and is harmless to non-target organisms such as human and animals.Bt becomes the most commonly used environment-friendly insecticidal microorganism.However,the insecticidal activities of different Bt strains variy significantly.Therefore,it is particularly important to compare the insecticidal activities of different strains and explore their insecticidal effector mechanisms to expand Bt insecticidal spectrum and enrich transgenic resources.Here,the insecticidal activities of Vip3Aa57 and Vip3Aa62 strains,carrying vegetative insecticidal protein-encoding genes that were inserted into the expression vector pET-21b and transformed into Escherichia coli Rosetta(DE3)strain,expressing 88 ku protein,were compared.Vip3Aa57 protein reportedly displayed body weight inhibition effect on Spodoptera exigua without affecting Heliothis armigera while Vip3Aa62 protein was known to have strong insecticidal activity against S.exigua(LC50=5.124 ng•mg^(-1)).A low H.armigera activity(LC50=870.1 ng•mg^(-1))was observed.The paraffin sectioning results showed that Vip3Aa57 protein affected S.exigua midgut cell morphology.The laser confocal microscopic imaging results showed that Vip3Aa57 bound to receptors in the midgut without damaging the midgut tissue morphology.This study would be conducive for making full use of Bt strains in the soil to compare the insecticidal activities of different Vip insecticidal genes.It could thus provide significant help in revealing the underlying insecticidal mechanisms of Vip3Aa insecticidal genes,developing new insecticidal proteins and delaying pest resistance problems.展开更多
Bacillus thuringiensis is a ubiquitous gram-positive, spore-forming bacterium that forms parasporal crystal during the stationary phase of its growth cycle. These crystal proteins, including Cry and Cyt protein, are t...Bacillus thuringiensis is a ubiquitous gram-positive, spore-forming bacterium that forms parasporal crystal during the stationary phase of its growth cycle. These crystal proteins, including Cry and Cyt protein, are toxic to certain insects. Lately, some problems about Cyt classification, structural characteristic, action mechanism and resistance to Cyt toxin are becoming new hotspots. We review the progress of above problems in several foreign labs.展开更多
Bauxite residue deposit area(BRDA)is a typical abandoned mining wasteland representing extreme hostile environment with increased alkalinity.Microbially-driven neutralization of bauxite residue,based on the microbial ...Bauxite residue deposit area(BRDA)is a typical abandoned mining wasteland representing extreme hostile environment with increased alkalinity.Microbially-driven neutralization of bauxite residue,based on the microbial acid producing metabolisms,is a novel strategy for achieving rapid pH neutralization and thus improving its environmental outcomes.The hypothesis was that these extreme conditions promote microbial communities which are capable of novel ecologically relevant functions.Several alkaliphilic acid producing bacteria were isolated in this study.One strain was selected for its superior growth pattern and acid metabolism(termed EEEL02).Based on the phylogenetic analysis,this strain was identified as Bacillus thuringiensis.The optimized fermentation conditions were as follows:pH 10;NaCl concentration 5%;temperature 25℃;EEEL02 preferred glucose and peptone as carbon and nitrogen sources,respectively.Based on optimal fermentation conditions,EEEL02 induced a significant pH reduction from 10.26 to 5.62 in 5-day incubation test.Acetic acid,propionic acid and CO2(g)were the major acid metabolites of fermentation,suggesting that the pH reduction in bauxite residue may be caused by acid neutralization derived from microbial metabolism.This finding provided the basis of a novel strategy for achieving rapid pH neutralization of bauxite residue.展开更多
Background The pink bollworm,Pectinophora gossypiella(Saunders),is a devastating global pest of cotton that has caused substantial economic damage to Bt Bollgard-Ⅱ~?cotton plants in recent years due to the evolution ...Background The pink bollworm,Pectinophora gossypiella(Saunders),is a devastating global pest of cotton that has caused substantial economic damage to Bt Bollgard-Ⅱ~?cotton plants in recent years due to the evolution of Bt resistance.The associated fitness cost is assumed to be one of the factors delaying the development of resistance against Bt transgenic crops.Hence,the present study was undertaken to assess the biological performance of pink bollworms by comparing the life history and demographic parameters of a resistant(Field-R)and susceptible(Lab-S)population.Results Prolonged larval duration(23.40 days in Field-R vs 18.80 days in Lab-S population),total life cycle(male=50.00 vs 42.80 days;female=53.60 vs 46.20 days),reduced fecundity(100.60 vs 154.20 eggs/female)and fertility(88.00 vs 138.00 fertile eggs/female)was observed.The demographic parameters indicated a significant reduction in the net reproductive rate(184.27 vs 276.72),innate capacity for increase in number(0.11 vs 0.15),finite rate of increase in number(1.12 vs 1.16 female progenies produced/female/day),weekly multiplication rate(2.16 vs 2.86),potential fecundity(545.06 vs 634.11 eggs),number of hypothetical F_2 females(33955.65 vs 76572.41),but longer mean length of generation(47.54 vs 37.74 days)and population doubling time(6.30 vs 4.62 days)in Field-R compared with Lab-S population.A stage-specific life to ble demonstrated the differences in survival rates between susceptible and resistant populations at various life stages,with the resistant population having higher generation mortality(0.22vs 0.19).Conclusions The study confirms the involvement of fitness costs associated with Bt resistance in P.gossypiella.Despite reduced reproductive fitness,the resistant population tried prolonging the larval stage as a compensatory mechanism to repair the damaged host tissues due to Bt intoxication and for accumulation of enough nutrient reserves for normal pupation and adult emergence.Presence of a high proportion of double Bt-resistant larvae in the field coupled with continued noncompliance with refug planting certainly favours the flaring up of this monophagous pest despite the observed fitness costs.The resistance cannot be effectively reversed unless suitable alternative management strategies are deployed.展开更多
基金Supported by Genetically Modified Organisms Breeding Major Projects (2009ZX08009-031B)State Key Laboratory for Biology of Plant Diseases and Insect Pests Open Fund (DKL2010OP13)
文摘16S rDNA and ERIC （Enterobacteia Repetitive Intergenic Consensus Sequences） based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus＂ strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn＇t have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.
文摘Background:Potassium(K)deficiency has become a common field production problem following the widespread adoption of Bacillus thuringiensis(Bt)transgenic cotton(Gossypium hirsutum L.)worldwide.The purpose of this study was to clarify whether the introduction of Bt gene directly reduces the K-use efficiency of cotton to induce K deficiency.Results:The cotton variety,Jihe 321(wild type,WT)and its two Bt(Cry1Ac)-transgenic overexpression lines(OE-29317,OE-29312)were studied in field with low soil-test K+(47.8 mg·kg^(−1)).In the field with low soil-test K+,only OE-29317 had less biomass and K+accumulation than the WT at some growth stages.Both Bt lines produced similar or even greater seed cotton yield than WT in the field.When the Bt gene(~70%)in OE-29317 and OE-29312 plants was silenced by virus-induced gene silencing(VIGS),the VIGS-Bt plants did not produce more biomass than VIGSgreen fluorescent protein(control)plants.Conclusions:The introduction of Bt gene did not necessarily hinder the K use efficiency of the cotton lines under this study.
基金Supported by National Key R&D Projects(2017YFD0201200)Heilongjiang Provincial National Science Foundation(C2016025)
文摘Vegetative Insecticidal Proteins(VIPs), a large family of insecticidal proteins, are produced from Bacillus thuringiensis(Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticides that confer a wider insecticidal spectrum and have stronger activity. This work compared the geographical distribution of Bt strains and their vip3 genes in different climatic zones in China, the tropical(Hainan Province), subtropical(Guangxi Province) and temperate zones(Heilongjiang Province). A total of 156 Bt strains were isolated from 841 soil samples in Hainan Province tropical region, 356 Bt strains from 1 420 soil samples in Guangxi Province and 167 Bt strains from 1 010 soil samples in different geographical regions in Heilongjiang Province. Twenty-two out of 156 strains from tropical Hainan Province and two out of 356 from subtropical Guangxi Province were found to express vip3 genes, while vip3 genes were not expressed from temperate zone in Heilongjiang Province. Restriction Fragment Length Polymorphism(RFLP) was used to identify different types of vip3 genes that were within the same family and three fulllength vip3 genes were isolated. The genes cloned from Bacillus thuringiensis strain SL3 expressed in the transformed E. coli BL21 strain. Through SDS-PAGE, 88.6 ku insecticidal protein was expressed. The bioassays used two-instar larva of Lepidoptera insects(Spodoptera exigua and Agrotis ipsilon) were performed. The results of the bioassays showed that the protein strongly inhibited the body weight increasement on Spodoptera exigua and Agrotis ipsilon in a standard bioassay. Taken together, the results indicated that the distribution of Bt strains and vip3 genes had regional preference. Tropical and subtropical regions were the rich resources of Bt strains and vip3 genes compared with temperate region. These results would undoubtedly facilitate the studies of insecticidal proteins and expand the list of the pest-killing candidates to make fully use of the extremely rich microbial resources. The new vip3 genes isolated in the current study might also help resolve the emerging insecticidal resistance problems.
基金Supported by the National Key R&D Projects(2017YFD0201200)Heilongjiang Provincial National Science Foundation(C2016025)
文摘Bacillus thuringiensis （Bt） is widely used in insecticides. Bt is a gram positive sporulation bacterium belonging to Bacillaceae family. It produces different insecticidal proteins like Cry toxin, Vip toxin, β-exotoxin and chitinase, etc. Bt insecticidal crystal proteins （ICPs） are not homologous to other known Vip protein and then act against lepidopteran, dipteran, coleopteran and nematodeslarvae via a unique process. In this experiment, modern high-throughput sequencing technique and sequencing were used and the whole genome sequence of BtLTS290 was obtained. The results compared to the database of GenBank showed that there was a cry57 gene in the genome sequence of BtLTS290. A novel cry57 gene was cloned and named cry57Ab1 （accession number is KF638650） by International Nomenclature Committee of Bt Endotoxin. cry57Ab1 gene could be expressed with the molecular weight of 90 ku. Cry57Ab1 protein had no obvious activity against Spodoptera exigua and Helicoverpa armigera. And Cry57Ab1 protein had a slight insecticidal activity against Ostrinia furnacalis and Plutella xylostella. Furthermore, the domain Ⅱof Cry57Ab1 and Cry1Bb were exchanged by overlapping extension PCR. SDS-PAGE showed that the molecular weight of Cry57Ab/1Bb/57Ab was about 90 ku. The insecticidal activity of Cry57Ab1 protein and Cry57Ab/1Bb/57Ab recombinant protein were determined. The results showed that the insecticidal activity of the recombinant protein to Spodoptera exigua and Helicoverpa armigera was very low, and the corrected mortality was less than 10%. The insecticidal activities against Ostrinia furnacalis and Plutella xylostella were reduced. The corrected mortality of Ostrinia furnacalis was 4.4%, and the corrected mortality rate for Plutella xylostella was 6.7%. Domain Ⅱof cry toxin played a key role on affecting host specifcity.
基金Supported by the National Key R&D Projects(2017YFD0201200)。
文摘Bacillus thuringiensis(Bt)exhibits strong insecticidal activity and is harmless to non-target organisms such as human and animals.Bt becomes the most commonly used environment-friendly insecticidal microorganism.However,the insecticidal activities of different Bt strains variy significantly.Therefore,it is particularly important to compare the insecticidal activities of different strains and explore their insecticidal effector mechanisms to expand Bt insecticidal spectrum and enrich transgenic resources.Here,the insecticidal activities of Vip3Aa57 and Vip3Aa62 strains,carrying vegetative insecticidal protein-encoding genes that were inserted into the expression vector pET-21b and transformed into Escherichia coli Rosetta(DE3)strain,expressing 88 ku protein,were compared.Vip3Aa57 protein reportedly displayed body weight inhibition effect on Spodoptera exigua without affecting Heliothis armigera while Vip3Aa62 protein was known to have strong insecticidal activity against S.exigua(LC50=5.124 ng•mg^(-1)).A low H.armigera activity(LC50=870.1 ng•mg^(-1))was observed.The paraffin sectioning results showed that Vip3Aa57 protein affected S.exigua midgut cell morphology.The laser confocal microscopic imaging results showed that Vip3Aa57 bound to receptors in the midgut without damaging the midgut tissue morphology.This study would be conducive for making full use of Bt strains in the soil to compare the insecticidal activities of different Vip insecticidal genes.It could thus provide significant help in revealing the underlying insecticidal mechanisms of Vip3Aa insecticidal genes,developing new insecticidal proteins and delaying pest resistance problems.
文摘Bacillus thuringiensis is a ubiquitous gram-positive, spore-forming bacterium that forms parasporal crystal during the stationary phase of its growth cycle. These crystal proteins, including Cry and Cyt protein, are toxic to certain insects. Lately, some problems about Cyt classification, structural characteristic, action mechanism and resistance to Cyt toxin are becoming new hotspots. We review the progress of above problems in several foreign labs.
基金Projects(41877511,41842020)supported by the National Natural Science Foundation of ChinaProject(502221703)supported by the Innovative Project of Independent Exploration of Central South University,China
文摘Bauxite residue deposit area(BRDA)is a typical abandoned mining wasteland representing extreme hostile environment with increased alkalinity.Microbially-driven neutralization of bauxite residue,based on the microbial acid producing metabolisms,is a novel strategy for achieving rapid pH neutralization and thus improving its environmental outcomes.The hypothesis was that these extreme conditions promote microbial communities which are capable of novel ecologically relevant functions.Several alkaliphilic acid producing bacteria were isolated in this study.One strain was selected for its superior growth pattern and acid metabolism(termed EEEL02).Based on the phylogenetic analysis,this strain was identified as Bacillus thuringiensis.The optimized fermentation conditions were as follows:pH 10;NaCl concentration 5%;temperature 25℃;EEEL02 preferred glucose and peptone as carbon and nitrogen sources,respectively.Based on optimal fermentation conditions,EEEL02 induced a significant pH reduction from 10.26 to 5.62 in 5-day incubation test.Acetic acid,propionic acid and CO2(g)were the major acid metabolites of fermentation,suggesting that the pH reduction in bauxite residue may be caused by acid neutralization derived from microbial metabolism.This finding provided the basis of a novel strategy for achieving rapid pH neutralization of bauxite residue.
基金the award of the DST-INSPIRE fellowship(IF190948)by the Department of Science and Technology,Government of India。
文摘Background The pink bollworm,Pectinophora gossypiella(Saunders),is a devastating global pest of cotton that has caused substantial economic damage to Bt Bollgard-Ⅱ~?cotton plants in recent years due to the evolution of Bt resistance.The associated fitness cost is assumed to be one of the factors delaying the development of resistance against Bt transgenic crops.Hence,the present study was undertaken to assess the biological performance of pink bollworms by comparing the life history and demographic parameters of a resistant(Field-R)and susceptible(Lab-S)population.Results Prolonged larval duration(23.40 days in Field-R vs 18.80 days in Lab-S population),total life cycle(male=50.00 vs 42.80 days;female=53.60 vs 46.20 days),reduced fecundity(100.60 vs 154.20 eggs/female)and fertility(88.00 vs 138.00 fertile eggs/female)was observed.The demographic parameters indicated a significant reduction in the net reproductive rate(184.27 vs 276.72),innate capacity for increase in number(0.11 vs 0.15),finite rate of increase in number(1.12 vs 1.16 female progenies produced/female/day),weekly multiplication rate(2.16 vs 2.86),potential fecundity(545.06 vs 634.11 eggs),number of hypothetical F_2 females(33955.65 vs 76572.41),but longer mean length of generation(47.54 vs 37.74 days)and population doubling time(6.30 vs 4.62 days)in Field-R compared with Lab-S population.A stage-specific life to ble demonstrated the differences in survival rates between susceptible and resistant populations at various life stages,with the resistant population having higher generation mortality(0.22vs 0.19).Conclusions The study confirms the involvement of fitness costs associated with Bt resistance in P.gossypiella.Despite reduced reproductive fitness,the resistant population tried prolonging the larval stage as a compensatory mechanism to repair the damaged host tissues due to Bt intoxication and for accumulation of enough nutrient reserves for normal pupation and adult emergence.Presence of a high proportion of double Bt-resistant larvae in the field coupled with continued noncompliance with refug planting certainly favours the flaring up of this monophagous pest despite the observed fitness costs.The resistance cannot be effectively reversed unless suitable alternative management strategies are deployed.
