Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed...Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene （zein） from maize endosperm protein with lysine-rich gene （Cflr） from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.展开更多
Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bac...Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bacteria display libraries against the protective antigen VP2 were constructed and screened.VP2 specific scFvs were selected following three rounds of screening procedures.Selected scFvs were characterized by FCM and ELISA.Seven scFvs showed high affinity and specific binding to CPV.Moreover,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scFv-2 showed the neutralizing ability with a titer of 32768.This study generated the first canine-origin neutralizing scFv against CPV.The neutralizing scFv would be constructed into full-length antibody in the future.This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.展开更多
Trehalose synthase is an important functional enzyme in the synthesis of trehalose in organisms and also participates in plant stress-resistant physiological processes.The transcriptomic study showed that a trehalose-...Trehalose synthase is an important functional enzyme in the synthesis of trehalose in organisms and also participates in plant stress-resistant physiological processes.The transcriptomic study showed that a trehalose-6-phosphate synthase gene was responsive to salt and alkaline stresses in Glycine soja.To dissect the molecular mechanisms of this enzyme in plant responses to stresses,the PCR technique was used to clone a trehalose-6-phosphate synthase gene from Glycine soja and it was designated as the GsTPS9.The full-length cDNA of this gene was 2583bp which encoded 861 amino acids.The sequence and structure analyses indicated that the GsTPS9 had high homology with Glycine max GmTPS9.The qRT-PCR analysis revealed that the GsTPS9 gene was expressed in Glycine soja roots,stems and leaves,and the highest expression level was in roots;the GsTPS9 gene had different responses under the stresses of NaCl,NaHCO_(3),PEG6000,ABA,MeJA and SA.This study laid the foundation for revealing the mechanism of the TPS in plant signal transduction pathways.展开更多
The enzyme myo-inositol-1-phosphate synthase（MIPS EC 5.5.1.4） catalyzes the first step of myo-inositol biosynthesis, a product that plays crucial roles in plants as an osmoprotectant, transduction molecule, cell wal...The enzyme myo-inositol-1-phosphate synthase（MIPS EC 5.5.1.4） catalyzes the first step of myo-inositol biosynthesis, a product that plays crucial roles in plants as an osmoprotectant, transduction molecule, cell wall constituent and production of stress related molecule. Previous reports highlighted an important role of MIPS family genes in abiotic stresses particularly under salt stress tolerance in several plant species; however, little is known about the cellular and physiological functions of MIPS2 genes under abiotic conditions. In this study, a novel salt stress responsive gene designated Gs MIPS2 from wild soybean Glycine soja 07256 was functionally characterized contained an open reading frame（ORF） of 1 533 bp coding a peptide sequence of 510 amino acids along with mass of 56 445 ku. Multiple sequence alignment analysis revealed its 92%-99% similarity with other MIPS family members in legume proteins. Quantitative real-time PCR results demonstrated that Gs MIPS2 was induced by salt stress and expressed in roots of soybean. The positive function of Gs MIPS2 under salt response at different growth stages of transgenic Arabidopsis was also elucidated. The results showed that Gs MIPS2 transgenic lines displayed increased tolerance as compared to WT and atmips2 mutant lines under salt stress. Furthermore, the expression levels of some salt stress responsive marker genes, including KIN1, RD29 A, RD29 B, P5 Cs and COR47 were significantly up-regulated in Gs MIPS2 overexpression lines than wild type and atmips2 mutant. Collectively, these results suggested that Gs MIPS2 gene was a positive regulator of plant tolerance to salt stress. This was the first report to demonstrate that overexpression of Gs MIPS2 gene from wild soybean improved salt tolerance in transgenic Arabidopsis.展开更多
Bacillus subtilis is a representative probiotic widely used in food, medicine, livestock and other industries. In this experiment a strain of Bacillus subtilis was isolated and identified, and its ability to degrade c...Bacillus subtilis is a representative probiotic widely used in food, medicine, livestock and other industries. In this experiment a strain of Bacillus subtilis was isolated and identified, and its ability to degrade cellulose was also measured. The results showed that Bacillus subtilis had strong capacity to degrade cellulose(62.3% was degraded) and weak capacity to degrade hemicellulose(17.2% was degraded), while it could hardly degrade lignin. The total protein amount in the fermentation medium with cellulose-rich substrate reached by 9.4% after Bacillus subtilis fermentation, compared with that without cellulose-rich substrate. Furthermore, the amounts of Met, Lys and Leu reached by 31.4%, 42.2% and 4.9%, respectively. At 36 h of fermentation, the activity of cellulase reached the highest, and at this time the activity of the enzyme was obtained at 7.19 U · mL-1. The mRNA expression level of the cellulase gene was detected by qPCR, and the experimental group with cellulose substrate was about 2.5 times more than that of the non-cellulose substrate control group. These above results demonstrated that this strain of Bacillus subtilis had a strong ability to degrade cellulose, and synthesize more proteins and restrictive essential amino acids. This study revealed that Bacillus subtilis was a new alternative to ferment cellulose substrates to produce commercial feed or feed additives.展开更多
Bacillus lincheniformis is a representative probiotic, widely used in food, medicine and livestock industries and other industries. In this experiment, a strain of Bacillus lincheniformis was isolated and identified, ...Bacillus lincheniformis is a representative probiotic, widely used in food, medicine and livestock industries and other industries. In this experiment, a strain of Bacillus lincheniformis was isolated and identified, which was used to ferment celluloserich substrate. The effects were analyzed. The experimental results showed that Bacillus lincheniformis had the capacity to degrade cellulose(32.8% was degraded) and less capacity to degrade hemicellulose(7.9% was degraded), which could not degrade ligin. The total protein contents in the products fermented by Bacillus lincheniformis with cellulose-rich substrate riched by 11.6%, compared with the fermented products with no cellulose-rich substrate, furthermore, the contents of Met, Lys and Leu riched by 14%, 17.5% and 4.1%, respectively. These results showed that Bacillus lincheniformis had the ability to degrade cellulose and it provided experimental data for further utilization.展开更多
Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV S...Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV SYG-61 were isolated.The genes of scFv antibodies were derived from goslings immunized with GPV SYG-61,and scFvs were subcloned into a pBSD vector for the construction of pBSD-scFv libraries.The pBSD-scFv libraries were screened following three rounds using VP2(protective antigen of GPV)as the bait by flow cytometry(FCM).After screening,the 15 clones with high mean fluorescence intensity(MFI)were isolated and sequenced.These 15 scFvs were expressed by pET-28a(+)in E.coli.The specificity and affinity of the 15 purified scFvs were successfully confirmed by ELISA.In the preliminary neutralization experiment on primary goose embryo fibroblast(GEF)in vitro,three of the 15 purified scFvs(named scFv-10,scFv-11 and scFv-50)showed significant neutralizing capacities.The study generated the first goose-origin neutralizing scFv against GPV and laid the foundation for the appearance of full-length goose-origin neutralizing monoclonal antibody against GPV.展开更多
基金Supported by the Funding of High Technology Project of Ministry of Science and Technology of China(863 Project,2013AA102504-03)
文摘Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene （zein） from maize endosperm protein with lysine-rich gene （Cflr） from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.
基金Supported by the National Key R&D Program of China(2017YFD0501102,2016YFD0501003)Science and Technology Department of Heilongjiang Province(GX18B018)Education Department of Heilongjiang Province(TSTAU-R2018017)。
文摘Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bacteria display libraries against the protective antigen VP2 were constructed and screened.VP2 specific scFvs were selected following three rounds of screening procedures.Selected scFvs were characterized by FCM and ELISA.Seven scFvs showed high affinity and specific binding to CPV.Moreover,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scFv-2 showed the neutralizing ability with a titer of 32768.This study generated the first canine-origin neutralizing scFv against CPV.The neutralizing scFv would be constructed into full-length antibody in the future.This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.
基金Supported by the National Natural Science Foundation of China(31670272)Heilongjiang Provincial Natural Science Foundation(C2017014)。
文摘Trehalose synthase is an important functional enzyme in the synthesis of trehalose in organisms and also participates in plant stress-resistant physiological processes.The transcriptomic study showed that a trehalose-6-phosphate synthase gene was responsive to salt and alkaline stresses in Glycine soja.To dissect the molecular mechanisms of this enzyme in plant responses to stresses,the PCR technique was used to clone a trehalose-6-phosphate synthase gene from Glycine soja and it was designated as the GsTPS9.The full-length cDNA of this gene was 2583bp which encoded 861 amino acids.The sequence and structure analyses indicated that the GsTPS9 had high homology with Glycine max GmTPS9.The qRT-PCR analysis revealed that the GsTPS9 gene was expressed in Glycine soja roots,stems and leaves,and the highest expression level was in roots;the GsTPS9 gene had different responses under the stresses of NaCl,NaHCO_(3),PEG6000,ABA,MeJA and SA.This study laid the foundation for revealing the mechanism of the TPS in plant signal transduction pathways.
基金Supported by "863" Project(2008AA10Z153)the National Natural Science Foundation of China(31171578)+1 种基金Heilongjiang Provincial Higher School Science and Technology Innovation Team Building Program(2011TD005)the National Basic Scientific Talent Training Fund Projects(J1210069)
文摘The enzyme myo-inositol-1-phosphate synthase（MIPS EC 5.5.1.4） catalyzes the first step of myo-inositol biosynthesis, a product that plays crucial roles in plants as an osmoprotectant, transduction molecule, cell wall constituent and production of stress related molecule. Previous reports highlighted an important role of MIPS family genes in abiotic stresses particularly under salt stress tolerance in several plant species; however, little is known about the cellular and physiological functions of MIPS2 genes under abiotic conditions. In this study, a novel salt stress responsive gene designated Gs MIPS2 from wild soybean Glycine soja 07256 was functionally characterized contained an open reading frame（ORF） of 1 533 bp coding a peptide sequence of 510 amino acids along with mass of 56 445 ku. Multiple sequence alignment analysis revealed its 92%-99% similarity with other MIPS family members in legume proteins. Quantitative real-time PCR results demonstrated that Gs MIPS2 was induced by salt stress and expressed in roots of soybean. The positive function of Gs MIPS2 under salt response at different growth stages of transgenic Arabidopsis was also elucidated. The results showed that Gs MIPS2 transgenic lines displayed increased tolerance as compared to WT and atmips2 mutant lines under salt stress. Furthermore, the expression levels of some salt stress responsive marker genes, including KIN1, RD29 A, RD29 B, P5 Cs and COR47 were significantly up-regulated in Gs MIPS2 overexpression lines than wild type and atmips2 mutant. Collectively, these results suggested that Gs MIPS2 gene was a positive regulator of plant tolerance to salt stress. This was the first report to demonstrate that overexpression of Gs MIPS2 gene from wild soybean improved salt tolerance in transgenic Arabidopsis.
基金Supported by the Key Applied Technology Research and Development Program of Heilongjiang Province(GA15B203)Student Innovation and Pioneer Training Program(SIPT)of Northeast Agricultural University(SIPT,201610224153)
文摘Bacillus subtilis is a representative probiotic widely used in food, medicine, livestock and other industries. In this experiment a strain of Bacillus subtilis was isolated and identified, and its ability to degrade cellulose was also measured. The results showed that Bacillus subtilis had strong capacity to degrade cellulose(62.3% was degraded) and weak capacity to degrade hemicellulose(17.2% was degraded), while it could hardly degrade lignin. The total protein amount in the fermentation medium with cellulose-rich substrate reached by 9.4% after Bacillus subtilis fermentation, compared with that without cellulose-rich substrate. Furthermore, the amounts of Met, Lys and Leu reached by 31.4%, 42.2% and 4.9%, respectively. At 36 h of fermentation, the activity of cellulase reached the highest, and at this time the activity of the enzyme was obtained at 7.19 U · mL-1. The mRNA expression level of the cellulase gene was detected by qPCR, and the experimental group with cellulose substrate was about 2.5 times more than that of the non-cellulose substrate control group. These above results demonstrated that this strain of Bacillus subtilis had a strong ability to degrade cellulose, and synthesize more proteins and restrictive essential amino acids. This study revealed that Bacillus subtilis was a new alternative to ferment cellulose substrates to produce commercial feed or feed additives.
基金Supported by the Key Applied Technology Research and Development Program of Heilongjiang Province(GA15B203)Student Innovation and Pioneer Training Program(SIPT)of Northeast Agricultural University(SIPT,201610224153)
文摘Bacillus lincheniformis is a representative probiotic, widely used in food, medicine and livestock industries and other industries. In this experiment, a strain of Bacillus lincheniformis was isolated and identified, which was used to ferment celluloserich substrate. The effects were analyzed. The experimental results showed that Bacillus lincheniformis had the capacity to degrade cellulose(32.8% was degraded) and less capacity to degrade hemicellulose(7.9% was degraded), which could not degrade ligin. The total protein contents in the products fermented by Bacillus lincheniformis with cellulose-rich substrate riched by 11.6%, compared with the fermented products with no cellulose-rich substrate, furthermore, the contents of Met, Lys and Leu riched by 14%, 17.5% and 4.1%, respectively. These results showed that Bacillus lincheniformis had the ability to degrade cellulose and it provided experimental data for further utilization.
基金Supported by the National Key R&D Program of China(2017YFD0501102,20I7YFD050I103-03 and 2017YFD0501004)Science and Technology Department of Heilongjiang Province(GX18B018)Education Department ofHeilongjiang Province(TSTAU-R2018017)。
文摘Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV SYG-61 were isolated.The genes of scFv antibodies were derived from goslings immunized with GPV SYG-61,and scFvs were subcloned into a pBSD vector for the construction of pBSD-scFv libraries.The pBSD-scFv libraries were screened following three rounds using VP2(protective antigen of GPV)as the bait by flow cytometry(FCM).After screening,the 15 clones with high mean fluorescence intensity(MFI)were isolated and sequenced.These 15 scFvs were expressed by pET-28a(+)in E.coli.The specificity and affinity of the 15 purified scFvs were successfully confirmed by ELISA.In the preliminary neutralization experiment on primary goose embryo fibroblast(GEF)in vitro,three of the 15 purified scFvs(named scFv-10,scFv-11 and scFv-50)showed significant neutralizing capacities.The study generated the first goose-origin neutralizing scFv against GPV and laid the foundation for the appearance of full-length goose-origin neutralizing monoclonal antibody against GPV.
