The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of ...The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.展开更多
In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong...In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong” with RT-PCR and RACE methods and named as RrGlu. The full-length cDNA is 1380 bp with an open reading frame of 1041 bp, encoding 346 amino acids. The derived protein has a molecular weight of 37.85 kD, a calculated pI of 9.12, a pfam00332 conserved domain at position 36 - 345, and belongs to glycosyl hydrolase family 17. The derived protein is a hydrophilic protein secreted into the vacuole. There is a signal peptide cleavage site at position 34 - 35, a transmembrane domain at position 13 - 32, six Ser phosphorylation sites, three Thr phosphorylation sites, three Tyr phosphorylation sites, one N-glycosylation site, and five O-glycosylation sites. There are 31.50% α-helixes, 30.92% random coil, 25.14% extended peptide chain, and 12.43% β-corner structure. This protein and the Glu protein from eight other species, including Prunus persica, share a sequence homology of greater than 72%;all of the proteins contain a pfam00332 conserved domain and a β-1,3-glucanase active center sequence (LIVM)-X-(LIVMFYW)3-(STAG)-E-(ST)-G-W-P-(ST)-X-G. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of R. rugosa pollination incompatibility and improve the theory and techniques of breeding ornamental R. rugosa.展开更多
Trichomes are specialized structures that originate from epidermal cells of organs in higher plants.The cotton fiber is a unique single-celled trichome that elongates from the seed coat epidermis.Cotton(Gossypium hirs...Trichomes are specialized structures that originate from epidermal cells of organs in higher plants.The cotton fiber is a unique single-celled trichome that elongates from the seed coat epidermis.Cotton(Gossypium hirsutum)fibers and trichomes are models for cell differentiation.In an attempt to elucidate the intercellular factors that regulate fiber and trichome cell development,we identified a plasmodesmal β-1,3-glucanase gene(designated GhPdBG)controlling the opening and closing of plasmodesmata in cotton fibers.Structural and evolutionary analysis showed haplotypic variation in the promoter region of the GhPdBG gene among 352 cotton accessions,but high conservation in the coding region.GhPdBG was expressed predominantly in cotton fibers and localized to plasmodesmata(PD).Expression patterns of PdBG that corresponded to PD permeability were apparent during fiber development in G.hirsutum and G.barbadense.The PdBG-mediated opening-closure of PD appears to be involved in fiber development and may account for the contrasting fiber traits of these two species.Ectopic expression of GhPdBG revealed that it functions in regulating fiber and trichome length and/or density by modulating plasmodesmatal permeability.This finding suggests that plasmodesmal targeting of GhPdBG,as a switch of intercellular channels,regulates single-celled fiber and trichome development in cotton.展开更多
In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was ...In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.展开更多
Pod width influences pod size,shape,yield,and consumer preference in snap beans(Phaseolus vulgaris L.).In this study,we map Pv PW1,a quantitative trait locus associated with pod width in snap beans,through genotyping ...Pod width influences pod size,shape,yield,and consumer preference in snap beans(Phaseolus vulgaris L.).In this study,we map Pv PW1,a quantitative trait locus associated with pod width in snap beans,through genotyping and phenotyping of recombinant plants.We identify Phvul.006G072800,encoding theβ-1,3-glucanase 9 protein,as the causal gene for Pv PW1.The Pv PW1^(G3555)allele is found to positively regulate pod width,as revealed by an association analysis between pod width phenotype and the Pv PW1^(G3555C)genotype across 17 bi-parental F_(2)populations.In total,97.7%of the 133 wide pod accessions carry Pv PW1^(G3555),while 82.1%of the 78 narrow pod accessions carry Pv PW1C3555,indicating strong selection pressure on Pv PW1 during common bean breeding.Re-sequencing data from 59 common bean cultivars identify an 8-bp deletion in the intron linked to Pv PW1C3555,leading to the development of the In Del marker of Pv M436.Genotyping 317 common bean accessions with Pv M436 demonstrated that accessions with Pv M436^(247)and Pv M436^(227)alleles have wider pods compared to those with Pv M436^(219)allele,establishing Pv M436 as a reliable marker for molecular breeding in snap beans.These findings highlight Pv PW1 as a critical gene regulating pod width and underscore the utility of Pv M436 in marker-assisted selection for snap bean breeding.展开更多
Pichia membranefaciens Hansen and Candida guilliermondii (Cast) Langeronet Guerra are two antagonists of R. stolonifer on harvested nectarine and peach fruits. In this study, β-1,3-glucanase and chitinase activities ...Pichia membranefaciens Hansen and Candida guilliermondii (Cast) Langeronet Guerra are two antagonists of R. stolonifer on harvested nectarine and peach fruits. In this study, β-1,3-glucanase and chitinase activities of the antagonists were induced in vitro and in vivo. The highest β-1, 3-glucanase activity was detected in Lilly-Barnett minimal salt medium supplemented with glucose in combination with CWP of R. stolonifer as a carbon source. The β-1,3-glucanase activity of P. membranefaciens reached the maximum level, being 114.0 SU (specific activity unit), and that of C. guilliermondii reached 103.2 SU. The lowest β-1,3-glucanase activity was observed in the medium containing glucose as sole car-bon source. P. membranefaciens was able to produce signifi-cantly higher levels of chitinase (exochitinase and endochiti-nase) in vitro than C. guilliermondii grown in Czapeck mini-mal medium. An increase in β-1,3-glucanase and chitinase activity was also triggered by wounding, adding of carbon sources展开更多
Plant b-1,3-glucanases are members of the pathogenesis-related protein 2（PR-2） family,which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses.One of the differenti...Plant b-1,3-glucanases are members of the pathogenesis-related protein 2（PR-2） family,which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses.One of the differentially expressed proteins（spot 842） identified in a recent proteomic comparison between five pairs of closely related maize（Zea mays L.） lines differing in aflatoxin resistance was further investigated in the present study.Here,the corresponding cDNA was cloned from maize and designated as ZmGns.ZmGns encodes a protein of338 amino acids containing a potential signal peptide.The expression of Zm Gns was detectible in all tissues studied with the highest level in silks.ZmGns was significantly induced by biotic stresses including three bacteria and the fungus Aspergillus flavus.ZmGns was also induced by most abiotic stresses tested and growth hormones including salicylic acid.In vivo,ZmGns showed a significant inhibitory activity against thebacterial pathogen Pseudomonas syringae pv.tomato DC3000 and fungal pathogen Botrytis cinerea when it overexpressed in Arabidopsis.Its high level of expression in the silk tissue and its induced expression by phytohormone treatment,as well as by bacterial and fungal infections,suggest it plays a complex role in maize growth,development,and defense.展开更多
The optimization of cultural conditions for β glucanase production by Bacillus subtilis ZJF 1A5 was investigated in flask trials. Temperature had great effect on β glucanase production which maximized...The optimization of cultural conditions for β glucanase production by Bacillus subtilis ZJF 1A5 was investigated in flask trials. Temperature had great effect on β glucanase production which maximized at optimal temperature of 37℃ and decreased significantly when temperature was over 37℃.Charge quantity affected β glucanase production significantly. Adding oxygen vector N dodecane or acetic ether benefited β glucanase production, but it depended on the concentration and charge quantity. The results of fractional factorial design showed that age and size of inoculum and shaking speed were the key factors affecting β glucanase production and the cultivation time span to reach the highest β glucanase activity. The optimal cultural conditions for β glucanase production obtained with CCD were as follows: inoculum age and size (16 h, 3.82%(v/v)), shaking speed 210 r/min, charge quantity of 30 mL in 250 mL flask and initial pH 7.0, cultured at 37℃ for 50 h. Repeated experimental results accorded with those predicted by a second order polynomial model. The amount of β glucanase, α amylase and neutral protease produced by B subtilis ZJF 1A5 was associated partially with cell growth. Those three enzymes' activities increased following the cell growth and increased significantly when cells entered the stationary phase.展开更多
The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system （polyethylene...The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system （polyethylene glycol （PEG）/MgSO4） was examined with regard to the effects of PEG molecular weight （MW） and concentration, MgSO4 concentration, pH and NaC1 concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of β-glucanase with the PEG/MgSO4 system. MgSO4 concentration influenced the partition and extraction of β-glucanase significantly, pH had little effect on β-glucanase or proteases partition but affected a-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of β-glucanase but had very significant effects on the partitioning of α-amylase and on the neutral proteases. The partition behaviors of β-glucanase, α-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying β-glucanase was developed, which achieved β-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.展开更多
In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by ...In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.展开更多
文摘The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.
文摘In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong” with RT-PCR and RACE methods and named as RrGlu. The full-length cDNA is 1380 bp with an open reading frame of 1041 bp, encoding 346 amino acids. The derived protein has a molecular weight of 37.85 kD, a calculated pI of 9.12, a pfam00332 conserved domain at position 36 - 345, and belongs to glycosyl hydrolase family 17. The derived protein is a hydrophilic protein secreted into the vacuole. There is a signal peptide cleavage site at position 34 - 35, a transmembrane domain at position 13 - 32, six Ser phosphorylation sites, three Thr phosphorylation sites, three Tyr phosphorylation sites, one N-glycosylation site, and five O-glycosylation sites. There are 31.50% α-helixes, 30.92% random coil, 25.14% extended peptide chain, and 12.43% β-corner structure. This protein and the Glu protein from eight other species, including Prunus persica, share a sequence homology of greater than 72%;all of the proteins contain a pfam00332 conserved domain and a β-1,3-glucanase active center sequence (LIVM)-X-(LIVMFYW)3-(STAG)-E-(ST)-G-W-P-(ST)-X-G. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of R. rugosa pollination incompatibility and improve the theory and techniques of breeding ornamental R. rugosa.
基金the State Key Laboratory of Cotton Biology Open Fund(CB2021A04)the Agricultural Seed Project of Shandong Province(2020LZGC002)the Science Foundation of Shandong Province(ZR2020MC107)。
文摘Trichomes are specialized structures that originate from epidermal cells of organs in higher plants.The cotton fiber is a unique single-celled trichome that elongates from the seed coat epidermis.Cotton(Gossypium hirsutum)fibers and trichomes are models for cell differentiation.In an attempt to elucidate the intercellular factors that regulate fiber and trichome cell development,we identified a plasmodesmal β-1,3-glucanase gene(designated GhPdBG)controlling the opening and closing of plasmodesmata in cotton fibers.Structural and evolutionary analysis showed haplotypic variation in the promoter region of the GhPdBG gene among 352 cotton accessions,but high conservation in the coding region.GhPdBG was expressed predominantly in cotton fibers and localized to plasmodesmata(PD).Expression patterns of PdBG that corresponded to PD permeability were apparent during fiber development in G.hirsutum and G.barbadense.The PdBG-mediated opening-closure of PD appears to be involved in fiber development and may account for the contrasting fiber traits of these two species.Ectopic expression of GhPdBG revealed that it functions in regulating fiber and trichome length and/or density by modulating plasmodesmatal permeability.This finding suggests that plasmodesmal targeting of GhPdBG,as a switch of intercellular channels,regulates single-celled fiber and trichome development in cotton.
文摘In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA28070000)the Natural Science Foundation of Heilongjiang Province(TD 2023C005)the National Natural Science Foundation of China(32372029 and 32001506)。
文摘Pod width influences pod size,shape,yield,and consumer preference in snap beans(Phaseolus vulgaris L.).In this study,we map Pv PW1,a quantitative trait locus associated with pod width in snap beans,through genotyping and phenotyping of recombinant plants.We identify Phvul.006G072800,encoding theβ-1,3-glucanase 9 protein,as the causal gene for Pv PW1.The Pv PW1^(G3555)allele is found to positively regulate pod width,as revealed by an association analysis between pod width phenotype and the Pv PW1^(G3555C)genotype across 17 bi-parental F_(2)populations.In total,97.7%of the 133 wide pod accessions carry Pv PW1^(G3555),while 82.1%of the 78 narrow pod accessions carry Pv PW1C3555,indicating strong selection pressure on Pv PW1 during common bean breeding.Re-sequencing data from 59 common bean cultivars identify an 8-bp deletion in the intron linked to Pv PW1C3555,leading to the development of the In Del marker of Pv M436.Genotyping 317 common bean accessions with Pv M436 demonstrated that accessions with Pv M436^(247)and Pv M436^(227)alleles have wider pods compared to those with Pv M436^(219)allele,establishing Pv M436 as a reliable marker for molecular breeding in snap beans.These findings highlight Pv PW1 as a critical gene regulating pod width and underscore the utility of Pv M436 in marker-assisted selection for snap bean breeding.
基金This work was supported by the National Natural Science Foundation of China (Grant No. 30170663) Young Scientists Creative Group for Life Science and Biological Technology of the Chinese Academy of Sciences.
文摘Pichia membranefaciens Hansen and Candida guilliermondii (Cast) Langeronet Guerra are two antagonists of R. stolonifer on harvested nectarine and peach fruits. In this study, β-1,3-glucanase and chitinase activities of the antagonists were induced in vitro and in vivo. The highest β-1, 3-glucanase activity was detected in Lilly-Barnett minimal salt medium supplemented with glucose in combination with CWP of R. stolonifer as a carbon source. The β-1,3-glucanase activity of P. membranefaciens reached the maximum level, being 114.0 SU (specific activity unit), and that of C. guilliermondii reached 103.2 SU. The lowest β-1,3-glucanase activity was observed in the medium containing glucose as sole car-bon source. P. membranefaciens was able to produce signifi-cantly higher levels of chitinase (exochitinase and endochiti-nase) in vitro than C. guilliermondii grown in Czapeck mini-mal medium. An increase in β-1,3-glucanase and chitinase activity was also triggered by wounding, adding of carbon sources
基金supported by USDA cooperative agreement 58-6435-6-055
文摘Plant b-1,3-glucanases are members of the pathogenesis-related protein 2（PR-2） family,which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses.One of the differentially expressed proteins（spot 842） identified in a recent proteomic comparison between five pairs of closely related maize（Zea mays L.） lines differing in aflatoxin resistance was further investigated in the present study.Here,the corresponding cDNA was cloned from maize and designated as ZmGns.ZmGns encodes a protein of338 amino acids containing a potential signal peptide.The expression of Zm Gns was detectible in all tissues studied with the highest level in silks.ZmGns was significantly induced by biotic stresses including three bacteria and the fungus Aspergillus flavus.ZmGns was also induced by most abiotic stresses tested and growth hormones including salicylic acid.In vivo,ZmGns showed a significant inhibitory activity against thebacterial pathogen Pseudomonas syringae pv.tomato DC3000 and fungal pathogen Botrytis cinerea when it overexpressed in Arabidopsis.Its high level of expression in the silk tissue and its induced expression by phytohormone treatment,as well as by bacterial and fungal infections,suggest it plays a complex role in maize growth,development,and defense.
文摘The optimization of cultural conditions for β glucanase production by Bacillus subtilis ZJF 1A5 was investigated in flask trials. Temperature had great effect on β glucanase production which maximized at optimal temperature of 37℃ and decreased significantly when temperature was over 37℃.Charge quantity affected β glucanase production significantly. Adding oxygen vector N dodecane or acetic ether benefited β glucanase production, but it depended on the concentration and charge quantity. The results of fractional factorial design showed that age and size of inoculum and shaking speed were the key factors affecting β glucanase production and the cultivation time span to reach the highest β glucanase activity. The optimal cultural conditions for β glucanase production obtained with CCD were as follows: inoculum age and size (16 h, 3.82%(v/v)), shaking speed 210 r/min, charge quantity of 30 mL in 250 mL flask and initial pH 7.0, cultured at 37℃ for 50 h. Repeated experimental results accorded with those predicted by a second order polynomial model. The amount of β glucanase, α amylase and neutral protease produced by B subtilis ZJF 1A5 was associated partially with cell growth. Those three enzymes' activities increased following the cell growth and increased significantly when cells entered the stationary phase.
基金Project (No. 20276064) supported by the National Nature ScienceFoundation of China
文摘The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system （polyethylene glycol （PEG）/MgSO4） was examined with regard to the effects of PEG molecular weight （MW） and concentration, MgSO4 concentration, pH and NaC1 concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of β-glucanase with the PEG/MgSO4 system. MgSO4 concentration influenced the partition and extraction of β-glucanase significantly, pH had little effect on β-glucanase or proteases partition but affected a-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of β-glucanase but had very significant effects on the partitioning of α-amylase and on the neutral proteases. The partition behaviors of β-glucanase, α-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying β-glucanase was developed, which achieved β-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.
基金Project supported by the National Natural Science Foundation of China (No. 20276064) and Natural Science Foundation of ZhejiangProvince (No. Z304076), China
文摘In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.
