[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta （DE3）. [Method] Specific primers were designed according to NCBI database information and the conserved sequences of c...[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta （DE3）. [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta （DE3）. Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences （304 and 853 bp respectively） of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops.展开更多
Root nodule symbiosis(RNS)is a mutualistic association formed between nitrogen-fixing rhizobia or Frankia and host plants limited to four orders within Rosid I―Fabales,Fagales,Cucurbitales,and Rosales―which comprise...Root nodule symbiosis(RNS)is a mutualistic association formed between nitrogen-fixing rhizobia or Frankia and host plants limited to four orders within Rosid I―Fabales,Fagales,Cucurbitales,and Rosales―which comprise the so-called‘Nitrogen Fixing Nodulation Clade’(NFNC).The majority of nodulation studies have focused on Leguminosae,given their agricultural and environmental importance,as well as the widespread occurrence of nodulation among members of this family.Endowing cereal crops with nitrogen fixation,like Leguminosae,presents a strategy to reduce the detrimental effects of synthetic fertilizer overuse.Different hypotheses on the origin of RNS have been proposed;however,key genetic innovations underlying the evolution of RNS,even in Leguminosae,have been rarely reported.In this review,we begin by examining current knowledge of genetic innovations―including gene gain,gene loss,and the acquisition or loss of conserved noncoding sequences(CNS)in preexisting genes.We explore the available evidence supporting these genetic innovations underlying the evolution of RNS in Leguminosae and offer the phylogenomics approach that could be applied to uncover these genetic innovations.Finally,we conclude by proposing a model of genetic innovations underlying the evolution of RNS in Leguminosae and consider the potential implications for the development of nitrogen-fixing crops.展开更多
Conserved non-coding sequences(CNSs)are integral elements of transcriptional regulation.Transcriptional tuning of PLETHORA(PLT)genes that encode master regulators of plant development is vital for embryogenesis and me...Conserved non-coding sequences(CNSs)are integral elements of transcriptional regulation.Transcriptional tuning of PLETHORA(PLT)genes that encode master regulators of plant development is vital for embryogenesis and meristematic function.However,how the expression of PLT genes is modulated through CNSs remains unclear.Through motif-based mining of upstream sequences in 120 angiosperm ge-nomes,we identified 21 conserved and lineage-specific CNSs,two of which are unusually long,similar,and colinear within eudicots.Using Arabidopsis thaliana,we demonstrate that these two deeply conserved elements,which we named BOX1 and BOX2,control PLT1 and PLT2 expression.CRISPR mutants within these elements specifically reduced PLT expression levels,and reporter lines revealed that deletion of either or both BOXes altered and/or abrogated the PLT2 expression pattern in the root tip,affecting the abil-ity to rescue the plt1 plt2 double mutant.We further show that the influence of these elements on expres-sion patterns is already exerted during embryogenesis and functional in the context of the early embryo.Finally,we reveal the existence of a BOX-mediated autoregulatory feedback loop that,in large part,explains CNS influence on expression patterns.Wethus uncover a transcriptional mechanism by which genes encoding master regulators of embryo and root meristem development are regulated.展开更多
One method for identifying noncoding regulatory regions of a genome is to quantify rates of divergence between related species, as functional sequence will generally diverge more slowly. Most approaches to identifying...One method for identifying noncoding regulatory regions of a genome is to quantify rates of divergence between related species, as functional sequence will generally diverge more slowly. Most approaches to identifying these conserved noncoding sequences （CNSs） based on alignment have had relatively large minimum sequence lengths （≥15 bp） compared with the average length of known transcription factor binding sites. To circumvent this constraint, STAG-CNS that can simultaneously integrate the data from the promoters of conserved orthologous genes in three or more species was developed. Using the data from up to six grass species made it possible to identify conserved sequences as short as 9 bp with false discovery rate ≤0.05. These CNSs exhibit greater overlap with open chromatin regions identified using DNase I hypersensitivity assays, and are enriched in the promoters of genes involved in transcriptional regulation. STAG-CNS was further employed to characterize loss of conserved noncoding sequences associated with retained duplicate genes from the ancient maize polyploidy. Genes with fewer retained CNSs show lower overall expression, although this bias is more apparent in samples of complex organ systems containing many cell types, suggesting that CNS loss may correspond to a reduced number of expression contexts rather than lower expression levels across the entire ancestral expression domain.展开更多
Plant genomes contain a large fraction of noncoding sequences.The discovery and annotation of conserved noncoding sequences(CNSs)in plants is an ongoing challenge.Here we report the application of comparative genomics...Plant genomes contain a large fraction of noncoding sequences.The discovery and annotation of conserved noncoding sequences(CNSs)in plants is an ongoing challenge.Here we report the application of comparative genomics to systematically identify CNSs in 50 well-annotated Gramineae genomes using rice(Oryza sativa)as the reference.We conduct multiple-way whole-genome alignments to the rice genome.The rice genome is annotated as 20 conservation states(CSs)at single-nucleotide resolution using a multivariate hidden Markov model(Cons HMM)based on the multiple-genome alignments.Different states show distinct enrichments for various genomic features,and the conservation scores of CSs are highly correlated with the level of associated chromatin accessibility.We find that at least 33.5%of the rice genome is highly under selection,with more than 70%of the sequence lying outside of coding regions.A catalog of 855,366 regulatory CNSs is generated,and they significantly overlapped with putative active regulatory elements such as promoters,enhancers,and transcription factor binding sites.Collectively,our study provides a resource for elucidating functional noncoding regions of the rice genome and an evolutionary aspect of regulatory sequences in higher plants.展开更多
In this paper, an infinite sequence of conservation laws for a generalized variable-coefficient fifth-order Korteweg-de Vries equation in fluids are constructed based on the Backlund transformation. Hirota bilinear fo...In this paper, an infinite sequence of conservation laws for a generalized variable-coefficient fifth-order Korteweg-de Vries equation in fluids are constructed based on the Backlund transformation. Hirota bilinear form and symbolic computation are applied to obtain three kinds of solutions. Variable coefficients can affect the conserved density, associated flux, and appearance of the characteristic lines. Effects of the wave number on the soliton structures are also discussed and types of soliton structures, e.g., the double-periodic soliton, parallel soliton and soliton complexes, are presented.展开更多
The 5’-end of the mitochondrial control region sequences of three flatfishes (Pleuronectiformes: Pleuronectidae) were amplified and sequenced. These sequences were compared with those of other three Pleuronectids spe...The 5’-end of the mitochondrial control region sequences of three flatfishes (Pleuronectiformes: Pleuronectidae) were amplified and sequenced. These sequences were compared with those of other three Pleuronectids species retrieved from GenBank. A phylogenetic tree was constructed based on the partial control region sequences. The results of phyloge- netic analysis are consistent with those of conventional systematics. Compared to previous studies, the structure of the 5’-end of mitochondrial control region was analyzed. The terminal associated sequence motif and its complementary motif were i- dentified at the 5’-end of the sequences. A conserved sequence block, named as CM5’d, was identified in the 5’-end of con- trol region sequences in all Pleuronectids. Another central conserved sequence block, named as CSB-F, was detected in the central conserved blocks.展开更多
Physical contact between genes distant on chromosomes is a potentially important way for genes to coordinate their expressions.To investigate the potential importance of distant contacts,we performed high-throughput c...Physical contact between genes distant on chromosomes is a potentially important way for genes to coordinate their expressions.To investigate the potential importance of distant contacts,we performed high-throughput chromatin conformation capture(Hi-C)experiments on leaf nuclei isolated from Brassica rapa and Brassica oleracea.We then combined our results with published Hi-C data from Arabidopsis thaliana.We found that distant genes come into physical contact and do so preferentially between the proximal promoter of one gene and the downstream region of another gene.Genes with higher numbers of conserved noncoding sequences(CNSs)nearby were more likely to have contact with distant genes.With more CNSs came higher numbers of transcription factor binding sites and more histone modifications associated with the activity.In addition,for the genes we studied,distant contacting genes with CNSs were more likely to be transcriptionally coordinated.These observations suggest that CNSs may enrich active histone modifications and recruit transcription factors,correlating with distant contacts to ensure coordinated expression.This study advances our knowledge of gene contacts and provides insights into the relationship between CNSs and distant gene contacts in plants.展开更多
The family GH126,best represented by the amylolytic enzyme CPF_2247 from Clostridium perfringens,exclusively includes proteins of bacterial origin,covering predominantly the phylum Bacillota.Although all the members s...The family GH126,best represented by the amylolytic enzyme CPF_2247 from Clostridium perfringens,exclusively includes proteins of bacterial origin,covering predominantly the phylum Bacillota.Although all the members should adopt the catalytic(α/α)6-barrel domain,neither the catalytic machinery nor the reaction mechanism has been determined as yet.The limited biochemical characterization,especially some uncertainty concerning the endo-vs exo-mode of action and retaining vs inverting mechanism,combined with the sequence-structural resemblance of GH126 members to inverting β-glucanases from families GH8 and GH48(the clan GH-M),may lead to misclassification of putative proteins.The present study was therefore designed in an effort to identify unique sequence-structural features that would definitively differentiate family GH126 from both GH8 and GH48.To achieve this,a sequence logo,representing the seven GH126 conserved sequence regions established previously,was created using 1665 GH126 sequences.The logo was compared with GH8 and GH48 logos based on,respectively,86 and 63 selected enzymes.An invariant tyrosine residue in CSR-6 was identified as a reliable marker for the family GH126.In addition,protein BLAST searches identified 87 putative proteins that taxonomically extend the family GH126 not only outside Bacillota,but also outside Bacteria to include representatives of archaeons and eukaryotes(fungi).Evolutionary analysis of the 434 sequences representing all the three families GH126 with GH8 and GH48,including the BLAST hits,revealed an intermediate group.In the future,it may either define a new GH family closely related to GH126,or at least constitute a future GH126 subfamily.展开更多
Plant cystatins or phytocystatins are cysteine proteinase inhibitors exist widely in different plant species. Because they can kill insects by inhibiting the digestive function of the cysteine proteinase in gut, they ...Plant cystatins or phytocystatins are cysteine proteinase inhibitors exist widely in different plant species. Because they can kill insects by inhibiting the digestive function of the cysteine proteinase in gut, they are believed to play an important role in plant’s defense against pests. Phytocystatins contain the conserved QXVXG motif and show some features on their sequence different to animal cystatins. After sequencing the protein directly and the cDNA clone, a large number of plant cystatins have been characterized. A multialignment with BLAST software and a detail analysis of 38 phytocystatins show that phytocystatins possess a specific conserved amino acid sequence [LRVI]-[AGT]-[RQKE]-[FY]- [AS]-[VI]-X-[EGHDQV]-[HYFQ]-N different to the conserved sequence demonstrated by Margis in 1998. This conserved sequence can be enough to detect with exclusivity phytocystatin sequences on protein data banks. A classification of these phytocystatins is performed and they can be divided into 3 groups according to their features on amino acid sequence, and the group-I can be still divided into 3 subgroups based on the feature of their amino acid and genomic sequence. By the CLUSTALX software, the most conserved nucleotide sequences of phytocystatins were found, which could be used to design the degenerate premiers to search new phytocystatins with PCR reaction.展开更多
Major outbreaks of severe acute respiratory syndrome(SARS)and coronavirus disease 2019(COVID-19),together with the continuous risk of zoonotic spillover of animal sarbecoviruses,underscore the urgent need for vaccines...Major outbreaks of severe acute respiratory syndrome(SARS)and coronavirus disease 2019(COVID-19),together with the continuous risk of zoonotic spillover of animal sarbecoviruses,underscore the urgent need for vaccines that confer broad protection across the sarbecovirus subgenus.Current immunogen selection strategies for pansarbecovirus vaccine development predominantly rely on phylogenetic or spike sequence conservation analyses,which often fail to accurately predict the breadth of cross-neutralization.To overcome this limitation,we systematically evaluated cross-neutralization profiles among 25 representative sarbecoviruses from clades 1 and 3 via guinea pig antisera individually raised against full-length spike proteins in pseudovirus neutralization assays while excluding clade 2 viruses lacking known receptor usage.Neutralization profiling revealed four distinct immunogenic clusters that diverged from traditional phylogenetic relationships.Antisera induced by the palm civet-derived SARS-CoV-1 strain SZ1 broadly neutralized all clade 1a viruses,whereas full coverage of clade 1b viruses required at least two distinct immunogens.Remarkably,sera elicited by multiple clade 1 immunogens also neutralized clade 3 viruses despite no prior exposure to clade 3 antigens.Guided by these findings,we proposed a minimal trivalent immunogen combination—SZ1,SARS-CoV-2,and PCoV-GX—that elicited broad neutralization against both clade 1 and clade 3.This rational approach eliminates the need for additional clade 3-specific antigens and provides a preclinical framework for developing next-generation pansarbecovirus vaccines.展开更多
基金Supported by Scientific Research Fund for Doctoral Program of Wuhan Polytechnic University (2006696)~~
文摘[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta （DE3）. [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta （DE3）. Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences （304 and 853 bp respectively） of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops.
基金supported by the National Natural Science Foundation of China(32300512)and the Xplorer Prize.
文摘Root nodule symbiosis(RNS)is a mutualistic association formed between nitrogen-fixing rhizobia or Frankia and host plants limited to four orders within Rosid I―Fabales,Fagales,Cucurbitales,and Rosales―which comprise the so-called‘Nitrogen Fixing Nodulation Clade’(NFNC).The majority of nodulation studies have focused on Leguminosae,given their agricultural and environmental importance,as well as the widespread occurrence of nodulation among members of this family.Endowing cereal crops with nitrogen fixation,like Leguminosae,presents a strategy to reduce the detrimental effects of synthetic fertilizer overuse.Different hypotheses on the origin of RNS have been proposed;however,key genetic innovations underlying the evolution of RNS,even in Leguminosae,have been rarely reported.In this review,we begin by examining current knowledge of genetic innovations―including gene gain,gene loss,and the acquisition or loss of conserved noncoding sequences(CNS)in preexisting genes.We explore the available evidence supporting these genetic innovations underlying the evolution of RNS in Leguminosae and offer the phylogenomics approach that could be applied to uncover these genetic innovations.Finally,we conclude by proposing a model of genetic innovations underlying the evolution of RNS in Leguminosae and consider the potential implications for the development of nitrogen-fixing crops.
基金funded by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek(GSGT.2019.019 to M.K.)supported by the Office of Science of the US Department of Energy(Contract No.DE-AC02-05CH11231).
文摘Conserved non-coding sequences(CNSs)are integral elements of transcriptional regulation.Transcriptional tuning of PLETHORA(PLT)genes that encode master regulators of plant development is vital for embryogenesis and meristematic function.However,how the expression of PLT genes is modulated through CNSs remains unclear.Through motif-based mining of upstream sequences in 120 angiosperm ge-nomes,we identified 21 conserved and lineage-specific CNSs,two of which are unusually long,similar,and colinear within eudicots.Using Arabidopsis thaliana,we demonstrate that these two deeply conserved elements,which we named BOX1 and BOX2,control PLT1 and PLT2 expression.CRISPR mutants within these elements specifically reduced PLT expression levels,and reporter lines revealed that deletion of either or both BOXes altered and/or abrogated the PLT2 expression pattern in the root tip,affecting the abil-ity to rescue the plt1 plt2 double mutant.We further show that the influence of these elements on expres-sion patterns is already exerted during embryogenesis and functional in the context of the early embryo.Finally,we reveal the existence of a BOX-mediated autoregulatory feedback loop that,in large part,explains CNS influence on expression patterns.Wethus uncover a transcriptional mechanism by which genes encoding master regulators of embryo and root meristem development are regulated.
文摘One method for identifying noncoding regulatory regions of a genome is to quantify rates of divergence between related species, as functional sequence will generally diverge more slowly. Most approaches to identifying these conserved noncoding sequences （CNSs） based on alignment have had relatively large minimum sequence lengths （≥15 bp） compared with the average length of known transcription factor binding sites. To circumvent this constraint, STAG-CNS that can simultaneously integrate the data from the promoters of conserved orthologous genes in three or more species was developed. Using the data from up to six grass species made it possible to identify conserved sequences as short as 9 bp with false discovery rate ≤0.05. These CNSs exhibit greater overlap with open chromatin regions identified using DNase I hypersensitivity assays, and are enriched in the promoters of genes involved in transcriptional regulation. STAG-CNS was further employed to characterize loss of conserved noncoding sequences associated with retained duplicate genes from the ancient maize polyploidy. Genes with fewer retained CNSs show lower overall expression, although this bias is more apparent in samples of complex organ systems containing many cell types, suggesting that CNS loss may correspond to a reduced number of expression contexts rather than lower expression levels across the entire ancestral expression domain.
基金supported by the Nanjing University Deng Feng Scholars Programthe Priority Academic Program Development(PAPD)of Jiangsu Higher Education Institutionsthe National Natural Science Foundation of China(32070656)。
文摘Plant genomes contain a large fraction of noncoding sequences.The discovery and annotation of conserved noncoding sequences(CNSs)in plants is an ongoing challenge.Here we report the application of comparative genomics to systematically identify CNSs in 50 well-annotated Gramineae genomes using rice(Oryza sativa)as the reference.We conduct multiple-way whole-genome alignments to the rice genome.The rice genome is annotated as 20 conservation states(CSs)at single-nucleotide resolution using a multivariate hidden Markov model(Cons HMM)based on the multiple-genome alignments.Different states show distinct enrichments for various genomic features,and the conservation scores of CSs are highly correlated with the level of associated chromatin accessibility.We find that at least 33.5%of the rice genome is highly under selection,with more than 70%of the sequence lying outside of coding regions.A catalog of 855,366 regulatory CNSs is generated,and they significantly overlapped with putative active regulatory elements such as promoters,enhancers,and transcription factor binding sites.Collectively,our study provides a resource for elucidating functional noncoding regions of the rice genome and an evolutionary aspect of regulatory sequences in higher plants.
基金Supported by the National Natural Science Foundation of China under Grant No.60772023by the Slpported Project under Grant No.SKLSDE-2010ZX-07 of the State Key Laboratory of Software Development Environment,Beijing University of Aeronautics and As tronautics+2 种基金by the Specialized Research Fund for the Doctoral Program of Higher Educatioi under Grant No.200800130006Chinese Ministry of Education,and by the Innovation Foundation for Ph.D.Graduates under Grant Nos.30-0350 and 30-0366Beijing University of Aeronautics and Astronautics
文摘In this paper, an infinite sequence of conservation laws for a generalized variable-coefficient fifth-order Korteweg-de Vries equation in fluids are constructed based on the Backlund transformation. Hirota bilinear form and symbolic computation are applied to obtain three kinds of solutions. Variable coefficients can affect the conserved density, associated flux, and appearance of the characteristic lines. Effects of the wave number on the soliton structures are also discussed and types of soliton structures, e.g., the double-periodic soliton, parallel soliton and soliton complexes, are presented.
基金the Shandong Foundation of Sciences(No.Y2000D04) the National Key Basic Research Program from the Ministry of Science and Technology of China(No.G19990437).
文摘The 5’-end of the mitochondrial control region sequences of three flatfishes (Pleuronectiformes: Pleuronectidae) were amplified and sequenced. These sequences were compared with those of other three Pleuronectids species retrieved from GenBank. A phylogenetic tree was constructed based on the partial control region sequences. The results of phyloge- netic analysis are consistent with those of conventional systematics. Compared to previous studies, the structure of the 5’-end of mitochondrial control region was analyzed. The terminal associated sequence motif and its complementary motif were i- dentified at the 5’-end of the sequences. A conserved sequence block, named as CM5’d, was identified in the 5’-end of con- trol region sequences in all Pleuronectids. Another central conserved sequence block, named as CSB-F, was detected in the central conserved blocks.
基金supported by the National Key Research and Development Program of China(2022YFF1003003)the Agricultural Science and Technology Innovation Program(ASTIP)+1 种基金the Central Public-interest Scientific Institution Basal Research Fund(Y2022PT23)the China Postdoctoral Science Foundation(2019M650918)。
文摘Physical contact between genes distant on chromosomes is a potentially important way for genes to coordinate their expressions.To investigate the potential importance of distant contacts,we performed high-throughput chromatin conformation capture(Hi-C)experiments on leaf nuclei isolated from Brassica rapa and Brassica oleracea.We then combined our results with published Hi-C data from Arabidopsis thaliana.We found that distant genes come into physical contact and do so preferentially between the proximal promoter of one gene and the downstream region of another gene.Genes with higher numbers of conserved noncoding sequences(CNSs)nearby were more likely to have contact with distant genes.With more CNSs came higher numbers of transcription factor binding sites and more histone modifications associated with the activity.In addition,for the genes we studied,distant contacting genes with CNSs were more likely to be transcriptionally coordinated.These observations suggest that CNSs may enrich active histone modifications and recruit transcription factors,correlating with distant contacts to ensure coordinated expression.This study advances our knowledge of gene contacts and provides insights into the relationship between CNSs and distant gene contacts in plants.
基金financially supported by the Grant No.2/0146/21 from the Slovak Grant Agency VEGAthe Grant No.FPPV-35-2024 from the University of SS.Cyril and Methodius in Trnava.
文摘The family GH126,best represented by the amylolytic enzyme CPF_2247 from Clostridium perfringens,exclusively includes proteins of bacterial origin,covering predominantly the phylum Bacillota.Although all the members should adopt the catalytic(α/α)6-barrel domain,neither the catalytic machinery nor the reaction mechanism has been determined as yet.The limited biochemical characterization,especially some uncertainty concerning the endo-vs exo-mode of action and retaining vs inverting mechanism,combined with the sequence-structural resemblance of GH126 members to inverting β-glucanases from families GH8 and GH48(the clan GH-M),may lead to misclassification of putative proteins.The present study was therefore designed in an effort to identify unique sequence-structural features that would definitively differentiate family GH126 from both GH8 and GH48.To achieve this,a sequence logo,representing the seven GH126 conserved sequence regions established previously,was created using 1665 GH126 sequences.The logo was compared with GH8 and GH48 logos based on,respectively,86 and 63 selected enzymes.An invariant tyrosine residue in CSR-6 was identified as a reliable marker for the family GH126.In addition,protein BLAST searches identified 87 putative proteins that taxonomically extend the family GH126 not only outside Bacillota,but also outside Bacteria to include representatives of archaeons and eukaryotes(fungi).Evolutionary analysis of the 434 sequences representing all the three families GH126 with GH8 and GH48,including the BLAST hits,revealed an intermediate group.In the future,it may either define a new GH family closely related to GH126,or at least constitute a future GH126 subfamily.
基金Supported by The National Basic Research Program (973 Program) ( G1999016003), Graduate Students’ Research Foundation of BeijingForestry University and the Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University
文摘Plant cystatins or phytocystatins are cysteine proteinase inhibitors exist widely in different plant species. Because they can kill insects by inhibiting the digestive function of the cysteine proteinase in gut, they are believed to play an important role in plant’s defense against pests. Phytocystatins contain the conserved QXVXG motif and show some features on their sequence different to animal cystatins. After sequencing the protein directly and the cDNA clone, a large number of plant cystatins have been characterized. A multialignment with BLAST software and a detail analysis of 38 phytocystatins show that phytocystatins possess a specific conserved amino acid sequence [LRVI]-[AGT]-[RQKE]-[FY]- [AS]-[VI]-X-[EGHDQV]-[HYFQ]-N different to the conserved sequence demonstrated by Margis in 1998. This conserved sequence can be enough to detect with exclusivity phytocystatin sequences on protein data banks. A classification of these phytocystatins is performed and they can be divided into 3 groups according to their features on amino acid sequence, and the group-I can be still divided into 3 subgroups based on the feature of their amino acid and genomic sequence. By the CLUSTALX software, the most conserved nucleotide sequences of phytocystatins were found, which could be used to design the degenerate premiers to search new phytocystatins with PCR reaction.
基金supported by the Natural Science Foundation of China(grant no.82172244)the Major Project of Guangzhou National Laboratory(GZNL2024A01019).
文摘Major outbreaks of severe acute respiratory syndrome(SARS)and coronavirus disease 2019(COVID-19),together with the continuous risk of zoonotic spillover of animal sarbecoviruses,underscore the urgent need for vaccines that confer broad protection across the sarbecovirus subgenus.Current immunogen selection strategies for pansarbecovirus vaccine development predominantly rely on phylogenetic or spike sequence conservation analyses,which often fail to accurately predict the breadth of cross-neutralization.To overcome this limitation,we systematically evaluated cross-neutralization profiles among 25 representative sarbecoviruses from clades 1 and 3 via guinea pig antisera individually raised against full-length spike proteins in pseudovirus neutralization assays while excluding clade 2 viruses lacking known receptor usage.Neutralization profiling revealed four distinct immunogenic clusters that diverged from traditional phylogenetic relationships.Antisera induced by the palm civet-derived SARS-CoV-1 strain SZ1 broadly neutralized all clade 1a viruses,whereas full coverage of clade 1b viruses required at least two distinct immunogens.Remarkably,sera elicited by multiple clade 1 immunogens also neutralized clade 3 viruses despite no prior exposure to clade 3 antigens.Guided by these findings,we proposed a minimal trivalent immunogen combination—SZ1,SARS-CoV-2,and PCoV-GX—that elicited broad neutralization against both clade 1 and clade 3.This rational approach eliminates the need for additional clade 3-specific antigens and provides a preclinical framework for developing next-generation pansarbecovirus vaccines.
