AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line.METHODS: p27 gene was obtained by RT-PCR, and its sequence was approv...AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line.METHODS: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line.p27-pcDNA3.1-K562 cell clone was screened by G418 after transfection, p27 protein was identified by Western blot.MTT was used to detect the survival rate of the cell. Flow cytometry was used to detect cell cycle and apoptosis index.RESULTS: The expression of p27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. A strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells as compared with that of the control (pcDNA3.1-K562 cells). The cells at G0/G1 phase were significantly increased, and cells at S phase were greatly declined.The apoptosis index was increased greatly after p27-pcDNA3.1-K562 cells were treated with STI571, and survival rate of the cell was markedly declined (0.35-0.58,P<0.05-0.048 vs STI571-K562 cell, 0.35-0.72, P<0.01-0.001 vs p27-K562 cell).CONCLUSION: p27 and STI571 have a synergistic action on inhibition of proliferation and induction of apoptosis on K562 cells.展开更多
Leaf senescence is the final stage of leaf development, where the nutrients and energy of senescent leaves are redistributed to developing tissues or organs for plant growth, reproduction, and defense. Outer leaves ar...Leaf senescence is the final stage of leaf development, where the nutrients and energy of senescent leaves are redistributed to developing tissues or organs for plant growth, reproduction, and defense. Outer leaves are photosynthetic organs that usually senesce at the late heading stage in Chinese cabbage, and premature leaf senescence often reduces leafy head yield and quality. In this study, 11 premature leaf senescence mutants were screened from an ethyl methanesulfonate-mutagenized population of the double haploid line ‘FT' in Chinese cabbage. At the early heading stage, the mutants exhibited edge yellowing within its outer leaves, and at the mature stage, its leafy head weight decreased significantly. Genetic analysis revealed that the mutated trait of all 11 mutants corresponds to single gene recessive inheritance. Semi-diallel cross tests showed that 5 of the 11 were allelic mutants. MutMap and Kompetitive Allele Specific PCR genotyping revealed that BraA01g001400.3C was the candidate gene, which is orthologous of Arabidopsis SUPPRESSOR OF rps4-RLD 1, encoding an immune regulator, so we named it as BrSRFR1. All the BrSRFR1 in the five allelic mutants exhibited single nucleotide polymorphisms at different positions on their exons and led to premature translation termination, which confirmed that defect in BrSRFR1 led to premature leaf senescence. These results verify the role of Br SRFR1 on leaf senescence and provide a new insight into the mechanisms of leaf senescence in Chinese cabbage, which reveals a novel function of SRFR1 in plant development.展开更多
[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of m...[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of msrA,and its bioinformatics analysis was carried out.[Results]The full length of msrA gene was 639 bp,encoding 212 amino acids,and its theoretical molecular weight was about 23729.60 Da.The protein had a stable structure,and it was hydrophobic overall.The structure of signal peptides at the N terminal of the amino acid sequence was predicted,and it was found that there was no signal peptide cleavage site and no transmembrane region.The amino acid sequence of MsrA contained multiple signal binding sites.Protein subcellular localization showed that MsrA protein was most likely located in the cytoplasm.Homology analysis showed that MsrA of V.alginolyticus had high homology with other Vibrio species,and the highest homology with V.alginolyticus.In the prediction of functional domains,MsrA had the function of methionine sulfoxide reduction.In secondary structure prediction,MsrA contained random coils at a proportion of 46.70%,which was the highest.The similarity between the tertiary structure model of MsrA and template Q87SW6.1.A was 89.15%.PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites.[Conclusions]This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress.展开更多
Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indic...Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.展开更多
Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthes...Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy.展开更多
Although the gut microflora is thought to be an essential factor in the development of ulcerative colitis (UC), the entire gut microflora occurring in UC remains unknown. Most studies use feces to represent the microf...Although the gut microflora is thought to be an essential factor in the development of ulcerative colitis (UC), the entire gut microflora occurring in UC remains unknown. Most studies use feces to represent the microflora distribution;however, here we analyzed the bacterial diversity in colonic mucus from UC patients receiving colectomy surgery and control patients. The diversity of microflora was investigated using a combination of terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of the 16S rRNA gene sequences. In the T-RFLP analysis, the number of terminal restriction fragments (T-RFs) decreased significantly in UC patients when compared to control samples. Also in the clone library analysis, the number of operational taxonomic units (OTU) and the Shannon diversity index were reduced significantly in UC patients. These molecular analyses reveal an overall dysbiosis in UC patients. No specific pathogen was found, and a strong negative correlation in relative abundance of bacterial populations was observed between the phyla Bacteroidetes and Firmicutes in the UC patients. This is the first report showing a significant correlation between these two phyla, which may be important characteristics in the pathogenesis of UC.展开更多
In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic spe...In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.展开更多
An EMS(ethy methanesulfonate)-induced lethal etiolated(le)mutant obtained from the rice variety Zhongjian 100 was characterized by lethal etiolated phenotypes,with significantly reduced levels of chlorophyll a,chlorop...An EMS(ethy methanesulfonate)-induced lethal etiolated(le)mutant obtained from the rice variety Zhongjian 100 was characterized by lethal etiolated phenotypes,with significantly reduced levels of chlorophyll a,chlorophyll b,total chlorophyll,and carotenoids.Additionally,the mutant displayed a significantly decreased number of chloroplast grana,along with irregular and less-stacked grana lamellae.The le mutant showed markedly diminished root length,root surface area,and root volume compared with the wild type.It also exhibited significantly lower catalase activity and total protein content,while peroxidase activity was significantly higher.Using the map-based cloning method,we successfully mapped the LE gene to a 48-kb interval between markers RM16107 and RM16110 on rice chromosome 3.A mutation(from T to C)was identified at nucleotide position 692 bp of LOC_Os03g59640(ChlD),resulting in a change from leucine to proline.By crossing HM133(a pale green mutant with a single-base substitution of A for G in exon 10 of ChlD subunit)with a heterozygous line of le(LEle),we obtained two plant lines heterozygous at both the LE and HM133 loci.Among 15 transgenic plants,3 complementation lines displayed normal leaf color with significantly higher total chlorophyll,chlorophyll a,and chlorophyll b contents.The mutation in le led to a lethal etiolated phenotype,which has not been observed in other ChlD mutants.The mutation in the AAA+domain of ChlD disrupted the interaction of ChlDle with ChlI as demonstrated by a yeast two-hybrid assay,leading to the loss of ChlD function and hindering chlorophyll synthesis and chloroplast development.Consequently,this disruption is responsible for the lethal etiolated phenotype in the mutant.展开更多
For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids w...For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids were encoded.Bioinformatics analysis showed that the VrPR protein belonged to the NADB Rossmann superfamily,which was one of the non-transmembrane hydrophilic proteins.VrPR was assumed to have 44 amino acid phosphorylation sites and be contained in chloroplasts.The VrPR secondary structure comprised of random coil,αhelix,βangle,and extended chain,all of which were quite compatible with the anticipated tertiary structure.Moreover,analysis of the phylogenetic tree indicated that the soybean PR(Glyma.12G222200)and VrPR were closely related.Furthermore,chlorophyll content in leaves is markedly increased in Arabidopsis when VrPR is overexpressed.Ourfindings will serve as a reference for more functional studies on the PR genes in mung bean.展开更多
[Objectives]The paper was to clone and analyze bioinformatics of ndk gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the ndk gene sequence of V.alginolyticus HY9901.The full le...[Objectives]The paper was to clone and analyze bioinformatics of ndk gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the ndk gene sequence of V.alginolyticus HY9901.The full length of ndk gene was amplified by PCR and bioinformatics analysis was performed.MEGA 5.0 software was used to construct NDK phylogenetic tree by neighbor-joining method.SWISS-MODEL program was used to obtain the three-dimensional structural model of single subunit from NDK protein.[Results]The ndk gene,molecular structural formula C702H1094N192O214S7,was 426 bp in total,encoding 141 amino acids,with the theoretical molecular weight of 15.87199 kD and the theoretical pI value of 5.13.The prediction results of protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite showed that NDK mainly existed in the cytoplasm,and the protein was unstable and hydrophobic.There was neither signal peptide cleavage site,nor transmembrane region and KEGG metabolic pathway.The amino acid sequence had two protein kinase C phosphorylation sites,a casein kinaseⅡphosphorylation site,a N-myristoylation site,three microbody C-terminal target signal sites,and a nucleoside diphosphate kinase active site.Homology analysis showed that the NDK of V.alginolyticus had high homology with that of V.diabolicus,with a similarity of 98.58%.Analysis of the structural functional domain revealed that the protein had one NDK structural functional domain.The prediction results of secondary structure showed that theα-helix,random coil,β-sheet and extended strand accounted for 53.19%,28.37%,7.09%and 11.35%,respectively.Analysis of NDK protein via STRING database demonstrated that the proteins interacting with NDK protein were NrdA,NrdB,GmK,CmK,TmK,PyrG,PyrH,RelA,FolE and SpoT.[Conclusions]The study plays a positive role in the prevention and control of vibriosis and the improvement of the current aquaculture environment.展开更多
[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The ...[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.展开更多
[Objectives]To amplify the h-ns gene of Vibrio alginolyticus and analyze it by bioinformatics.[Methods]According to the h-ns gene sequence of V.alginolyticus HY9901,a pair of specific primers were designed and amplifi...[Objectives]To amplify the h-ns gene of Vibrio alginolyticus and analyze it by bioinformatics.[Methods]According to the h-ns gene sequence of V.alginolyticus HY9901,a pair of specific primers were designed and amplified by PCR.[Results]The h-ns gene was 408 bp in length and 135 amino acids were encoded.The predicted theoretical protein molecular weight was about 14.98 kD,and the isoelectric point was 4.99.Protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite predictions showed that H-NS was located outside the cell membrane,and the protein was unstable and hydrophobic.There was no signal peptide cleavage site,no transmembrane region and no KEGG metabolic pathway.The amino acid sequence contained three phosphorylation sites,one N-terminal myristoylation site and three microsomal C-terminal target signal sites.Using MEGA 5.0,H-NS phylogenetic tree was constructed by ortho-connection method.The results showed that H-NS of V.alginolyticus was closer to H-NS of Vibrio diabolicus.Using SWISS-MODEL,the three-dimensional structure model of H-NS subunit was simulated,which was similar to the crystal structure of Salmonella typhimurium H-NS1-83.[Conclusions]This study lays a foundation for exploring the regulation mechanism of V.alginolyticus H-NS protein on bacterial virulence in the future.展开更多
Scientists from Shanghai Institutes of Biological Science under theChinese Academy of Sciences (CAS) have cloned dentinogenesisgene, which is believed responsible for a genetic tooth disease.The disease, Dentinogenesi...Scientists from Shanghai Institutes of Biological Science under theChinese Academy of Sciences (CAS) have cloned dentinogenesisgene, which is believed responsible for a genetic tooth disease.The disease, Dentinogenesis imperfecta 1, one of the most commongenetic tooth problems, causes brittle teeth for one out of every six toeight thousand humans in the world. There is no effective treatment展开更多
[Objective] Using molecular biotechnology to clone the sus scrofa GPX2 gene. [Method] Using total RNA of sus scrofa duodenum as template, degenerated primer pairs were designed according to the homology alignment anal...[Objective] Using molecular biotechnology to clone the sus scrofa GPX2 gene. [Method] Using total RNA of sus scrofa duodenum as template, degenerated primer pairs were designed according to the homology alignment analysis of GPX2 gene of human, rat, mouse, dog and cattle. A sus scrofa GPX2 gene sequence of 330 bp was obtained by RT-PCR application method. Primes were designed respectively according to the known sequence, sus scrofa GPX2 gene was isolated and cloned by 3-RACE and 5-RACE method and analyzed the gene sequence. [Result] A mRNA sequence of 924 bp was successfully cloned and isolated in this research. This sequence contained complete 3＇end and had higher sequence homology with human,mouse,cattle and dog GPX2 gene, and there was codon called TGA which encoding Sec on the position of No. 114-116 gene. [Conclusion] Sequence alignment analysis showed that the cloned gene was sus scrofa GPX2 gene （ NCBI GenBank database, the sequence number was D098982）.展开更多
[Objective] The aim was to clone the cDNA and DNA sequences of the CCR （Cinnamoyl-CoA reductase） gene which involves in lignin biosynthesis, from Pennisetum purpureum, and to make comprehensive analysis on these seq...[Objective] The aim was to clone the cDNA and DNA sequences of the CCR （Cinnamoyl-CoA reductase） gene which involves in lignin biosynthesis, from Pennisetum purpureum, and to make comprehensive analysis on these sequences. [Method] CCR sequences were cloned from P. purpureum by using conventional RT-PCR and RACE （Rapid Amplification of cDNA Ends） methods; and the bioinformatic analyses of the CCR were conducted by means of NCBI, ProtParam ProtScale, TMHMM, TargetP, SignalP, Pfam20.0, Prosite, Swiss-Model, ClustalW2, DNAman, DNAstar and MEGA5. [Result] The cloned PpCCR （P. purpureum CCR） cDNA sequence was 1 316 bp, including a 1 110 bp ORF and 206 bp 3’-UTR. The cloned DNA sequence from PpCCR was 6 133 bp in full-length, containing five exons and four introns. Bioinformatic analysis indicated that PpCCR encoded a polypeptide of 369 amino acids, the secondary structure of which was primarily composed of random coil and α-helix, belonging to NAD-dependent epimerase/dehydratase family, and its co-factor binding sites and substrate binding sites were highly conserved. [Conclusion] DNA and cDNA sequences of CCR gene were obtained from P. purpureum, which had the typical characteristics of other homologous genes. The obtained bioinformatic data provided theoretical references for the further analysis of CCR and better application of P. purpureum in the future.展开更多
基金Supported by the National Natural Science Foundation of China,No. 39770336
文摘AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line.METHODS: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line.p27-pcDNA3.1-K562 cell clone was screened by G418 after transfection, p27 protein was identified by Western blot.MTT was used to detect the survival rate of the cell. Flow cytometry was used to detect cell cycle and apoptosis index.RESULTS: The expression of p27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. A strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells as compared with that of the control (pcDNA3.1-K562 cells). The cells at G0/G1 phase were significantly increased, and cells at S phase were greatly declined.The apoptosis index was increased greatly after p27-pcDNA3.1-K562 cells were treated with STI571, and survival rate of the cell was markedly declined (0.35-0.58,P<0.05-0.048 vs STI571-K562 cell, 0.35-0.72, P<0.01-0.001 vs p27-K562 cell).CONCLUSION: p27 and STI571 have a synergistic action on inhibition of proliferation and induction of apoptosis on K562 cells.
基金supported by the National Natural Science Foundation of China (Grant No.31972405)Graduate Student Innovation Cultivation Project of Shenyang Agricultural University (Grant No.2021YCXB16)。
文摘Leaf senescence is the final stage of leaf development, where the nutrients and energy of senescent leaves are redistributed to developing tissues or organs for plant growth, reproduction, and defense. Outer leaves are photosynthetic organs that usually senesce at the late heading stage in Chinese cabbage, and premature leaf senescence often reduces leafy head yield and quality. In this study, 11 premature leaf senescence mutants were screened from an ethyl methanesulfonate-mutagenized population of the double haploid line ‘FT' in Chinese cabbage. At the early heading stage, the mutants exhibited edge yellowing within its outer leaves, and at the mature stage, its leafy head weight decreased significantly. Genetic analysis revealed that the mutated trait of all 11 mutants corresponds to single gene recessive inheritance. Semi-diallel cross tests showed that 5 of the 11 were allelic mutants. MutMap and Kompetitive Allele Specific PCR genotyping revealed that BraA01g001400.3C was the candidate gene, which is orthologous of Arabidopsis SUPPRESSOR OF rps4-RLD 1, encoding an immune regulator, so we named it as BrSRFR1. All the BrSRFR1 in the five allelic mutants exhibited single nucleotide polymorphisms at different positions on their exons and led to premature translation termination, which confirmed that defect in BrSRFR1 led to premature leaf senescence. These results verify the role of Br SRFR1 on leaf senescence and provide a new insight into the mechanisms of leaf senescence in Chinese cabbage, which reveals a novel function of SRFR1 in plant development.
基金Supported National Natural Science Foundation of China(32073015)Undergraduate Training Program for Innovation and Entrepreneurship of Guangdong Ocean University(CXXL2024007)+2 种基金Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802)Postgraduate Education Innovation Program of Guangdong Ocean University(No.202446)Postgraduate Education Innovation Program of Guangdong Province(YJYH[2022]1).
文摘[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of msrA,and its bioinformatics analysis was carried out.[Results]The full length of msrA gene was 639 bp,encoding 212 amino acids,and its theoretical molecular weight was about 23729.60 Da.The protein had a stable structure,and it was hydrophobic overall.The structure of signal peptides at the N terminal of the amino acid sequence was predicted,and it was found that there was no signal peptide cleavage site and no transmembrane region.The amino acid sequence of MsrA contained multiple signal binding sites.Protein subcellular localization showed that MsrA protein was most likely located in the cytoplasm.Homology analysis showed that MsrA of V.alginolyticus had high homology with other Vibrio species,and the highest homology with V.alginolyticus.In the prediction of functional domains,MsrA had the function of methionine sulfoxide reduction.In secondary structure prediction,MsrA contained random coils at a proportion of 46.70%,which was the highest.The similarity between the tertiary structure model of MsrA and template Q87SW6.1.A was 89.15%.PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites.[Conclusions]This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityNational Natural Science Foundation of China(32073015)+1 种基金Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2024007)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.
文摘Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy.
文摘Although the gut microflora is thought to be an essential factor in the development of ulcerative colitis (UC), the entire gut microflora occurring in UC remains unknown. Most studies use feces to represent the microflora distribution;however, here we analyzed the bacterial diversity in colonic mucus from UC patients receiving colectomy surgery and control patients. The diversity of microflora was investigated using a combination of terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of the 16S rRNA gene sequences. In the T-RFLP analysis, the number of terminal restriction fragments (T-RFs) decreased significantly in UC patients when compared to control samples. Also in the clone library analysis, the number of operational taxonomic units (OTU) and the Shannon diversity index were reduced significantly in UC patients. These molecular analyses reveal an overall dysbiosis in UC patients. No specific pathogen was found, and a strong negative correlation in relative abundance of bacterial populations was observed between the phyla Bacteroidetes and Firmicutes in the UC patients. This is the first report showing a significant correlation between these two phyla, which may be important characteristics in the pathogenesis of UC.
基金supported by grants from the Significant Special Found of "13115" S&T Innovation Project of Shaanxi Province,China(2007 ZDKG-01)"13115" Technology Innovation Engineering and Engineering Technology Research Center of Shaanxi Province,China(2008 ZDGC-02)the Special Capital for the Construction of Modern Agriculture Technical System of Shaanxi Province,China (NYCYTX-001)
文摘In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.
基金supported by the National Natural Science Foundation of China(Grant No.32072049)Open Foundation of State Key Laboratory of Rice Biology,China(Grant No.20210208).
文摘An EMS(ethy methanesulfonate)-induced lethal etiolated(le)mutant obtained from the rice variety Zhongjian 100 was characterized by lethal etiolated phenotypes,with significantly reduced levels of chlorophyll a,chlorophyll b,total chlorophyll,and carotenoids.Additionally,the mutant displayed a significantly decreased number of chloroplast grana,along with irregular and less-stacked grana lamellae.The le mutant showed markedly diminished root length,root surface area,and root volume compared with the wild type.It also exhibited significantly lower catalase activity and total protein content,while peroxidase activity was significantly higher.Using the map-based cloning method,we successfully mapped the LE gene to a 48-kb interval between markers RM16107 and RM16110 on rice chromosome 3.A mutation(from T to C)was identified at nucleotide position 692 bp of LOC_Os03g59640(ChlD),resulting in a change from leucine to proline.By crossing HM133(a pale green mutant with a single-base substitution of A for G in exon 10 of ChlD subunit)with a heterozygous line of le(LEle),we obtained two plant lines heterozygous at both the LE and HM133 loci.Among 15 transgenic plants,3 complementation lines displayed normal leaf color with significantly higher total chlorophyll,chlorophyll a,and chlorophyll b contents.The mutation in le led to a lethal etiolated phenotype,which has not been observed in other ChlD mutants.The mutation in the AAA+domain of ChlD disrupted the interaction of ChlDle with ChlI as demonstrated by a yeast two-hybrid assay,leading to the loss of ChlD function and hindering chlorophyll synthesis and chloroplast development.Consequently,this disruption is responsible for the lethal etiolated phenotype in the mutant.
基金This research was funded by National Natural Science Foundation of China,Grant Number(31871576)National Keypoint Research and Invention Program of the Thirteenth,Grant Number(2019YFD1002205)The APC was funded by National Keypoint Research and Invention Program of the Thirteenth.
文摘For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids were encoded.Bioinformatics analysis showed that the VrPR protein belonged to the NADB Rossmann superfamily,which was one of the non-transmembrane hydrophilic proteins.VrPR was assumed to have 44 amino acid phosphorylation sites and be contained in chloroplasts.The VrPR secondary structure comprised of random coil,αhelix,βangle,and extended chain,all of which were quite compatible with the anticipated tertiary structure.Moreover,analysis of the phylogenetic tree indicated that the soybean PR(Glyma.12G222200)and VrPR were closely related.Furthermore,chlorophyll content in leaves is markedly increased in Arabidopsis when VrPR is overexpressed.Ourfindings will serve as a reference for more functional studies on the PR genes in mung bean.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityNational Natural Science Foundationof China(32073015)+2 种基金Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Train-ing Program of Guangdong Ocean University(CXXL2022005)UndergraduateInnovation Team of Guangdong Ocean University(CCTD201802)。
文摘[Objectives]The paper was to clone and analyze bioinformatics of ndk gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the ndk gene sequence of V.alginolyticus HY9901.The full length of ndk gene was amplified by PCR and bioinformatics analysis was performed.MEGA 5.0 software was used to construct NDK phylogenetic tree by neighbor-joining method.SWISS-MODEL program was used to obtain the three-dimensional structural model of single subunit from NDK protein.[Results]The ndk gene,molecular structural formula C702H1094N192O214S7,was 426 bp in total,encoding 141 amino acids,with the theoretical molecular weight of 15.87199 kD and the theoretical pI value of 5.13.The prediction results of protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite showed that NDK mainly existed in the cytoplasm,and the protein was unstable and hydrophobic.There was neither signal peptide cleavage site,nor transmembrane region and KEGG metabolic pathway.The amino acid sequence had two protein kinase C phosphorylation sites,a casein kinaseⅡphosphorylation site,a N-myristoylation site,three microbody C-terminal target signal sites,and a nucleoside diphosphate kinase active site.Homology analysis showed that the NDK of V.alginolyticus had high homology with that of V.diabolicus,with a similarity of 98.58%.Analysis of the structural functional domain revealed that the protein had one NDK structural functional domain.The prediction results of secondary structure showed that theα-helix,random coil,β-sheet and extended strand accounted for 53.19%,28.37%,7.09%and 11.35%,respectively.Analysis of NDK protein via STRING database demonstrated that the proteins interacting with NDK protein were NrdA,NrdB,GmK,CmK,TmK,PyrG,PyrH,RelA,FolE and SpoT.[Conclusions]The study plays a positive role in the prevention and control of vibriosis and the improvement of the current aquaculture environment.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityGrants from Shenzhen Science and Technology Project(JCYJ20190813104207152)+3 种基金National Natural Science Foundation of China(32073015)Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2022005)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityNational Natural Science Foundation of China(32073015)+2 种基金Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2022005)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To amplify the h-ns gene of Vibrio alginolyticus and analyze it by bioinformatics.[Methods]According to the h-ns gene sequence of V.alginolyticus HY9901,a pair of specific primers were designed and amplified by PCR.[Results]The h-ns gene was 408 bp in length and 135 amino acids were encoded.The predicted theoretical protein molecular weight was about 14.98 kD,and the isoelectric point was 4.99.Protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite predictions showed that H-NS was located outside the cell membrane,and the protein was unstable and hydrophobic.There was no signal peptide cleavage site,no transmembrane region and no KEGG metabolic pathway.The amino acid sequence contained three phosphorylation sites,one N-terminal myristoylation site and three microsomal C-terminal target signal sites.Using MEGA 5.0,H-NS phylogenetic tree was constructed by ortho-connection method.The results showed that H-NS of V.alginolyticus was closer to H-NS of Vibrio diabolicus.Using SWISS-MODEL,the three-dimensional structure model of H-NS subunit was simulated,which was similar to the crystal structure of Salmonella typhimurium H-NS1-83.[Conclusions]This study lays a foundation for exploring the regulation mechanism of V.alginolyticus H-NS protein on bacterial virulence in the future.
文摘Scientists from Shanghai Institutes of Biological Science under theChinese Academy of Sciences (CAS) have cloned dentinogenesisgene, which is believed responsible for a genetic tooth disease.The disease, Dentinogenesis imperfecta 1, one of the most commongenetic tooth problems, causes brittle teeth for one out of every six toeight thousand humans in the world. There is no effective treatment
文摘[Objective] Using molecular biotechnology to clone the sus scrofa GPX2 gene. [Method] Using total RNA of sus scrofa duodenum as template, degenerated primer pairs were designed according to the homology alignment analysis of GPX2 gene of human, rat, mouse, dog and cattle. A sus scrofa GPX2 gene sequence of 330 bp was obtained by RT-PCR application method. Primes were designed respectively according to the known sequence, sus scrofa GPX2 gene was isolated and cloned by 3-RACE and 5-RACE method and analyzed the gene sequence. [Result] A mRNA sequence of 924 bp was successfully cloned and isolated in this research. This sequence contained complete 3＇end and had higher sequence homology with human,mouse,cattle and dog GPX2 gene, and there was codon called TGA which encoding Sec on the position of No. 114-116 gene. [Conclusion] Sequence alignment analysis showed that the cloned gene was sus scrofa GPX2 gene （ NCBI GenBank database, the sequence number was D098982）.
基金Supported by the National Natural Science Foundation of China(30972138)the Guangdong Natural Science Foundation(9451064201003804)~~
文摘[Objective] The aim was to clone the cDNA and DNA sequences of the CCR （Cinnamoyl-CoA reductase） gene which involves in lignin biosynthesis, from Pennisetum purpureum, and to make comprehensive analysis on these sequences. [Method] CCR sequences were cloned from P. purpureum by using conventional RT-PCR and RACE （Rapid Amplification of cDNA Ends） methods; and the bioinformatic analyses of the CCR were conducted by means of NCBI, ProtParam ProtScale, TMHMM, TargetP, SignalP, Pfam20.0, Prosite, Swiss-Model, ClustalW2, DNAman, DNAstar and MEGA5. [Result] The cloned PpCCR （P. purpureum CCR） cDNA sequence was 1 316 bp, including a 1 110 bp ORF and 206 bp 3’-UTR. The cloned DNA sequence from PpCCR was 6 133 bp in full-length, containing five exons and four introns. Bioinformatic analysis indicated that PpCCR encoded a polypeptide of 369 amino acids, the secondary structure of which was primarily composed of random coil and α-helix, belonging to NAD-dependent epimerase/dehydratase family, and its co-factor binding sites and substrate binding sites were highly conserved. [Conclusion] DNA and cDNA sequences of CCR gene were obtained from P. purpureum, which had the typical characteristics of other homologous genes. The obtained bioinformatic data provided theoretical references for the further analysis of CCR and better application of P. purpureum in the future.
