Rationale:If one of the partners is having balanced autosomal translocation,it is usually observed that the offspring inherit either normal chromosomes,balanced translocation identical to one of the parent or unbalanc...Rationale:If one of the partners is having balanced autosomal translocation,it is usually observed that the offspring inherit either normal chromosomes,balanced translocation identical to one of the parent or unbalanced chromosomal rearrangements of the same parental chromosome having translocation.Concern:A unique case presented with history of 8 miscarriages for genetic counseling.The last abortus material evaluation showed monosomy of chromosome X(Turner syndrome)in all the analyzed cells.There was a history of infertility and also repeated second trimester abortions on the paternal side.On the maternal side,there was a history of intellectual disability.Diagnose:History of repeated abnormal pregnancy outcomes.Wife’s karyotype is normal;however,husband shows translocation between chromosome 4 and 22.Intervention:Peripheral blood sample around 3 mL was collected for karyotype.Embryo biopsy was done and DNA was extracted and processed for whole exome sequencing.Outcomes:Wife’s karyotype is normal and husband has translocation between chromosome 4 and 22.Surprisingly,the entire pregnancy outcome including embryo screening has different,complete or partial aneuploidies of chromosomes other than chromosome 4 and 22.Main lesson:Though the translocation in one of the parent is balanced,we have to think beyond traditional ways for evaluating a couple with repeated pregnancy loss as we cannot predict the errors at cell division.Option of in vitro fertilization and preimplantation genetic diagnosis in couples having balanced translocations should be discussed so that early intervention can prevent the agony of pregnancy loss.展开更多
The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of t...The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.展开更多
Metagenomics has opened new avenues for exploring the genetic potential of uncultured microorganisms,which may serve as promising sources of enzymes and natural products for industrial applications.Identifying enzymes...Metagenomics has opened new avenues for exploring the genetic potential of uncultured microorganisms,which may serve as promising sources of enzymes and natural products for industrial applications.Identifying enzymes with improved catalytic properties from the vast amount of available metagenomic data poses a significant challenge that demands the development of novel computational and functional screening tools.The catalytic properties of all enzymes are primarily dictated by their structures,which are predominantly determined by their amino acid sequences.However,this aspect has not been fully considered in the enzyme bioprospecting processes.With the accumulating number of available enzyme sequences and the increasing demand for discovering novel biocatalysts,structural and functional modeling can be employed to identify potential enzymes with novel catalytic properties.Recent efforts to discover new polysaccharide-degrading enzymes from rumen metagenome data using homology-based searches and machine learning-based models have shown significant promise.Here,we will explore various computational approaches that can be employed to screen and shortlist metagenome-derived enzymes as potential biocatalyst candidates,in conjunction with the wet lab analytical methods traditionally used for enzyme characterization.展开更多
Chemotherapy is one of the main therapeutic modalities for cancer patients.Cisplatin(CDDP),as one of thefirst-line drugs,is of great importance in the chemotherapy of various tumors.However,a significant percentage of c...Chemotherapy is one of the main therapeutic modalities for cancer patients.Cisplatin(CDDP),as one of thefirst-line drugs,is of great importance in the chemotherapy of various tumors.However,a significant percentage of cancer patients are resistant to CDDP treatment.Due to the CDDP side effects on normal tissues,the diagnosis of CDDP resistance is required to suggest the most efficient therapeutic strategies for cancer patients.Several molecular mechanisms and signaling pathways are associated with CDDP response.The PI3K/AKT signaling pathway has a pivotal role in the transmission of extracellular signals into the cells to regulate various pathophysiological processes such as cell proliferation,migration,and drug resistance.In the present review,we summarized all of the studies which have been reported on the role of PI3K/AKT pathway in regulation of CDDP response.It was shown that the PI3K/AKT pathway is mainly involved in CDDP response in lung,ovarian,and gastrointestinal cancers.It was also observed that the non-coding RNAs have a key role in CDDP response by regulation of PI3K/AKT pathway.This review paves the way for suggesting a PI3K/AKT-related panel marker for the prediction of CDDP response in different cancer patients.展开更多
5-flurouracil(5-FU)-based chemotherapy is the main pharmacological therapy for advanced colorectal cancer(CRC).Despite significant progress in the treatment of CRC during the last decades,5-FU drug resistance remains ...5-flurouracil(5-FU)-based chemotherapy is the main pharmacological therapy for advanced colorectal cancer(CRC).Despite significant progress in the treatment of CRC during the last decades,5-FU drug resistance remains the most important cause of failure in CRC therapy.Resistance to 5-FU is a complex and multistep process.Different mechanisms including microsatellite instability,increased expression level of key enzyme thymidylate synthase and its polymorphism,increased level of 5-FU-activating enzymes and mutation of TP53 are proposed as the main determinants of resistance to 5-FU in CRC cells.Recently,microribonucleic acids(miRNA)and their alterations were found to have a crucial role in 5-FU resistance.In this regard,the miRNA-mediated mechanisms of 5-FU drug resistance reside among the new fields of pharmacogenetics of CRC drug response that has not been completely discovered.Identification of the biological markers that are related to response to 5-FU-based chemotherapy is an emerging field of precision medicine.This approach will have an important role in defining those patients who are most likely to benefit from 5-FU-based chemotherapy in the future.Thereby,the identification of 5-FU drug resistance mechanisms is an essential step to predict and eventually overcome resistance.In the present comprehensive review,we will summarize the latest knowledge regarding the molecular determinants of response to 5-FU-based chemotherapy in CRC by emphasizing the role of miRNAs.展开更多
Diabetes mellitus during pregnancy is associated with an increased risk of multiple congenital anomalies in progeny.There are sufficient evidence suggesting that the children of diabetic women exhibit intellectual and...Diabetes mellitus during pregnancy is associated with an increased risk of multiple congenital anomalies in progeny.There are sufficient evidence suggesting that the children of diabetic women exhibit intellectual and behavioral abnormalities accompanied by modification of hippocampus structure and function.Although,the exact mechanism by which maternal diabetes affects the developing hippocampus remains to be defined.Multiple biological alterations,including hyperglycemia,hyperinsulinemia,oxidative stress,hypoxia,and iron deficiency occur in pregnancies with diabetes and affect the development of central nervous system(CNS) of the fetus.The conclusion from several studies is that disturbance in glucose and insulin homeostasis in mothers and infants are major teratogenic factor in the development of CNS.Insulin and Insulin-like growth factor-1(IGF-1) are two key regulators of CNS function and development.Insulin and IGF-1 receptors(IR and IGF1 R,respectively) are distributed in a highly specific pattern with the high density in some brain regions such as hippocampus.Recent researches have clearly established that maternal diabetes disrupts the regulation of both IR and IGF1 R in the hippocampus of rat newborn.Dissecting out the mechanisms responsible for maternal diabetes-related changes in the development of hippocampus is helping to prevent from impaired cognitive and memory functions in offspring.展开更多
INTRODUCTIONHepatocellular transplantation (HCT) could providea therapeutic alternative to orthotopic livertransplantation(OLT) in the treatment of hepaticmetabolic defects and experimental hepaticfailure.Under approp...INTRODUCTIONHepatocellular transplantation (HCT) could providea therapeutic alternative to orthotopic livertransplantation(OLT) in the treatment of hepaticmetabolic defects and experimental hepaticfailure.Under appropriate conditions,theengrafted liver cells can continue to express liver-specific functions for an indefinite period of time.展开更多
Jasmonic acid(JA)plays important roles in plant resistance to insect herbivores.One important derivative of JA is 12-OH-JA,which is produced by two independent pathways:direct hydroxylation of JA by jasmonate-induced ...Jasmonic acid(JA)plays important roles in plant resistance to insect herbivores.One important derivative of JA is 12-OH-JA,which is produced by two independent pathways:direct hydroxylation of JA by jasmonate-induced oxygenases(JOXs)or hydrolyzation of 12-OH-JA-Ile.Yet the function of 12-OH-JA in plant-herbivore interactions remains largely unknown.In this study,we silenced four JOX homologs independently in the wild tobacco Nicotiana attenuata by virus-induced gene silencing(VIGS),and found that all four JOX homologs are involved in JA hydroxylation.Simultaneously silencing the four JA hydroxylases in VIGS-NaJOXs plants decreased herbivory-induced 12-OH-JA by 33%,but JA and JA-Ile levels increased by 45%and 30%,respectively,compared to those in control plants.Compared to direct hydroxylation from JA,hydrolyzation from 12-OH-JA-Ile is equally important for herbivory-induced 12-OHJA accumulation:in the 12-OH-JA-Ile deficient irJAR4/6 plants,12-OH-JA decreased 34%.Moreover,VIGSNaJOXs plants exhibited enhanced resistance to the generalist herbivore Spodoptera litura.The poor larval performance was strongly correlated with high levels of several JA-Ile-dependent direct defense metabolites in the VIGS-NaJOXs plants.When we simultaneously silenced all four JA hydroxylases in the JAIle-deficient irJAR4/6 background,the enhanced herbivore resistance diminished,demonstrating that enhanced herbivore resistance resulted from elevated JA-Ile levels.Given that silencing these NaJOX-like genes did not detectably alter plant growth but highly increased plant defense levels,we propose that JOX genes are potential targets for genetic improvement of herbivore-resistant crops.展开更多
In order to select the strain that can degrade nitrite, the screening plate with nitrite was used as the sole nitrogen source to screen the strain with ability to degrade nitrite. A strain with nitrite degrading capac...In order to select the strain that can degrade nitrite, the screening plate with nitrite was used as the sole nitrogen source to screen the strain with ability to degrade nitrite. A strain with nitrite degrading capacity was isolated from the sludge of a shrimp-farming pond in Hepu City, Guangxi Zhuang Autonomous Region. The isolated strain was identified based on colonial morphology, physiological and biochemical characteristics, and 16S rRNA sequence. Results showed that the strain could grow well on the culture medium containing 2.3 g/L nitrite. According to the morphological characteristics, nitrogen source requirements and evolutionary tree of the 16S rRNA sequence, the isolated strain was identified as Acinetobacter radioresistens, which was named HPAR132 strain. This study laid the foundation for further investigation of nitrite-oxidizing bacterium HPAR132.展开更多
The bHLH transcription factors play pivotal roles in plant growth and development,production of secondary metabolites and responses to various environmental stresses.Although the bHLH genes have been well studied in m...The bHLH transcription factors play pivotal roles in plant growth and development,production of secondary metabolites and responses to various environmental stresses.Although the bHLH genes have been well studied in model plant species,a comprehensive investigation of the bHLH genes is required for tobacco with newly obtained high-quality genome.In the present study,a total of 309 NtbHLH genes were identified and can be divided into 23 subfamilies.The conserved amino acids which are essential for their function were predicted for the NtbHLH proteins.Moreover,the NtbHLH genes were conserved during evolution through analyzing the gene structures and conserved motifs.A total of 265 NtbHLH genes were localized in the 24 tobacco chromosomes while the remained 44 NtbHLH genes were mapped to the scaffolds due to the complexity of tobacco genome.Moreover,transcripts of NtbHLH genes were obviously tissue-specific expressed from the gene-chip data from 23 tobacco tissues,and expressions of 20 random selected NtbHLH genes were further confirmed by quantitative real-time PCR,indicating their potential functions in the plant growth and development.Importantly,overexpressed NtbHLH86 gene confers improve drought tolerance in tobacco indicating that it might be involved in the regulation of drought stress.Therefore,our findings here provide a valuable information on the characterization of NtbHLH genes and further investigation of their functions in tobacco.展开更多
AIMTo identify the disease-causing gene mutation in a Chinese pedigree with autosomal dominant cone-rod dystrophy (adCORD).METHODSA southern Chinese adCORD pedigree including 9 affected individuals was studied. Whole-...AIMTo identify the disease-causing gene mutation in a Chinese pedigree with autosomal dominant cone-rod dystrophy (adCORD).METHODSA southern Chinese adCORD pedigree including 9 affected individuals was studied. Whole-exome sequencing (WES), coupling the Agilent whole-exome capture system to the Illumina HiSeq 2000 DNA sequencing platform was used to search the specific gene mutation in 3 affected family members and 1 unaffected member. After a suggested variant was found through the data analysis, the putative mutation was validated by Sanger DNA sequencing of samples from all available family members.RESULTSThe results of both WES and Sanger sequencing revealed a novel nonsense mutation c.C766T (p.Q256X) within exon 5 of CRX gene which was pathogenic for adCORD in this family. The mutation could affect photoreceptor-specific gene expression with a dominant-negative effect and resulted in loss of the OTX tail, thus the mutant protein occupies the CRX-binding site in target promoters without establishing an interaction and, consequently, may block transactivation.CONCLUSIONAll modes of Mendelian inheritance in CORD have been observed, and genetic heterogeneity is a hallmark of CORD. Therefore, conventional genetic diagnosis of CORD would be time-consuming and labor-intensive. Our study indicated the robustness and cost-effectiveness of WES in the genetic diagnosis of CORD.展开更多
Introduction: Beta-thalassemia is characterized by absence or reduced synthesis of the β-globin. Carriers of β-thalas- semia, typically have microcytic hypochromic anemia and elevated hemoglobin HbA2 and normal HbF ...Introduction: Beta-thalassemia is characterized by absence or reduced synthesis of the β-globin. Carriers of β-thalas- semia, typically have microcytic hypochromic anemia and elevated hemoglobin HbA2 and normal HbF level. On the other hand carriers of severe alpha-thalassemia also have similar CBC parameters to that of β-thalassemia with normal HbA2 level. Co-presence of mutations in the β-globin and delta-globin genes (point mutations or deletions) usually give normal HbA2 and elevated HbF level. We report a β-thal carrier with normal level of HbA2 and increased level of HbF who had a point mutation in CD39 on the beta-globin gene and a point mutation in CD27 on the δ-globin gene named Hb-Yialousa. Materials & Methods: An individual with low hematological indices, normal HbA2 and elevated HbF was referred to our center as routine premarital screening program. Mutations in the β-globin and δ-globin genes were screened using ARMS and sequencing methods. Results: The mutation in β- and δ-globin genes were identified as CD39 and CD27 (HbYialousa) respectively. No point mutation or deletion in α-globin gene was identified. Discussion: We showed that normal HBA2 with elevated HbF level is due to co-inheritance of delta-globin gene mutation with mutation in the β-globin gene. When screening for β-thalassemia, one has to either rule out presence of α-globin gene mutation of mutation in the delta-globin gene.展开更多
AIM: To generate DNA-aptamers binding to Methicillinresistant Staphylococcus aureus(MRSA).METHODS: The Cell-Systematic Evolution of Ligands by Exponential Enrichment(SELEX) technology was used to run the selection aga...AIM: To generate DNA-aptamers binding to Methicillinresistant Staphylococcus aureus(MRSA).METHODS: The Cell-Systematic Evolution of Ligands by Exponential Enrichment(SELEX) technology was used to run the selection against MRSA bacteria and develop target-specific aptamers. MRSA bacteria were targeted while Enterococcus faecalis bacteria were used for counter selection during that process. Binding assays to determine the right aptamer candidates as well as binding assays on clinical samples were performed through flow cytometry and analyzed using the FlowJ o software. The characterization of the aptamers was done by determination of their Kd values and determined by analysis of flow data at different aptamer concentration using Sigma Plot. Finally, the recognitionof the complex Gold-nanoparticle-aptamer to the bacteria cells was observed using transmission electron microscopy(TEM).RESULTS: During the cell-SELEX selection process, 17 rounds were necessary to generate enrichment of the pool. While the selection was run using fixed cells, it was shown that the binding of the pools with live cells was giving similar results. After sequencing and analysis of the two last pools, four sequences were identified to be aptamer candidates. The characterization of those aptamers showed that based on their Kd values, DTMRSA4 presented the best binding with a Kd value of 94.61 ± 18.82 nmol/L. A total of ten clinical samples of MRSA, S. aureus and Enterococcus faecalis were obtained to test those aptamers and determine their binding on a panel of samples. DTMRSA1 and DTMRSA3 showed the best results regarding their specificity to MRSA, DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding to MRSA cells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding property.CONCLUSION: A total of four aptamers that bind to MRSA were obtained with Kd values ranking from 94 to 200 nmol/L.展开更多
Exosomes are small vesicles that carry molecules from one cell to another.They have many features that make them interesting for research,such as their stability,low immunogenicity,size of the nanoscale,toxicity,and s...Exosomes are small vesicles that carry molecules from one cell to another.They have many features that make them interesting for research,such as their stability,low immunogenicity,size of the nanoscale,toxicity,and selective delivery.Exosomes can also interact with viruses in diverse ways.Emerging research highlights the significant role of exosomes in viral infections,particularly in the context of diseases like COVID-19,HIV,HBV and HCV.Understanding the intricate interplay between exosomes and the human immune system holds great promise for the development of effective antiviral therapies.An important aspect is gaining clarity on how exosomes influence the immune system and enhance viral infectivity through their inherent characteristics.By leveraging the innate properties of exosomes,viruses exploit the machinery involved in exosome biogenesis to set replication,facilitate the spread of infection,and eliminate immune responses.They can either help or hinder viral infection by modulating the immune system.This review summarizes the recent findings on how exosomes mediate viral infection and how they can be used for diagnosis or therapy.This could lead to new clinical applications of exosomes in disease management.展开更多
Analysis of the frequency of antigen-specific cytotoxic T lymphocytes (CTLs) ex vivo is largely dependent on the use of MHC/peptide tetramers. However, the latter reagents have not been widely available, most likely b...Analysis of the frequency of antigen-specific cytotoxic T lymphocytes (CTLs) ex vivo is largely dependent on the use of MHC/peptide tetramers. However, the latter reagents have not been widely available, most likely because of their costly and time-consuming production. In this report we utilized an economic strategy to construct HLA/peptide tetramers with recombinant peptide-linked β2 microglobulin (β2m). The HLA-A2-restricted, melanoma antigen MARTI-derived pep- tide MART127-35( AAGIGILTV) was fused to the N terminus of human β2m through a 15-amino acid (aa)-long linker before being refolded with the recombinant biotinylated HLA-A2 heavy chain ectodomain. The resulted 2-component (2C) monomer was then tetramerized with phycoerythin-labeled streptavidin. The experimental result showed that the 2C HLA-A2/ MART127-35 monomer was shown to bind to the HLA class complex-specific monoclonal antibody W6/32 and the HLA-A2/ MART127-35 complex-specific single chain antibody fragment (scFv) 8.3, suggesting the correctness of its specificity. Fur- thermore, the 2C HLA-A2/MART127-35 tetramer detected a specific CD8+ T cell population in HLA-A2-restricted melanoma infiltrating lymphocytes as the conventional 3C HLA-A2/MART127-35 tetramer. The yield of 2C HLA-A2/MART127-35 monomer was 2. 5 times more than that of the conventional 3C monomer. Taken together, these data indicate that the HLA-A2/ MART127-35 tetramer can be generated conveniently through the use of MART127-35 peptide-β2 m fusion proteins, which can fa- cilitate the monitoring of HLA-A2-restricted, MART1-specific CTL responses in patients with melanoma.展开更多
CRISPR/Cas is a simple,robust,versatile tool for plant biology studies and precision plant breeding.However,establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa(M...CRISPR/Cas is a simple,robust,versatile tool for plant biology studies and precision plant breeding.However,establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa(Medicago sativa L.),the most important forage legume worldwide,remains a formidable challenge.Here,we systematically identified endogenous U6 promoters in alfalfa through transient expression via Agrobacterium-mediated infiltration of alfalfa leaves.We further demonstrated the efficacy of the three most active promoters for genome editing using an optimized alfalfa hairy root system.Subsequently,we established an improved CRISPR/Cas9 multiplex system containing three or four tandemly arrayed MsU6-promoter-driven polycistronic tRNA-sgRNA(PTG)expression cassettes,each consisting of three tRNA-sgRNA units,to simultaneously edit three or four alfalfa genes,coupled with the visual reporter RH1 or RUBY.This toolkit showed efficient multiplex editing in the hairy root system with visual selection.We successfully obtained regenerated,red-colored shoots resulting from the stable transformation of alfalfa.These results highlight the potential application of the visual reporter system for the stable transformation of alfalfa.Our improved CRISPR/Cas9 multiplex system enables convenient,high-efficiency multiplex genome editing in alfalfa,providing a versatile toolset to facilitate functional studies of multiple genes and gene families for basic research and the genetic improvement of alfalfa.展开更多
Recessive resistance mediated by mutations in the eukaryotic translation initiation factor 4E(eIF4E),has proven effective against diverse potyviruses and is extensively utilized in breeding programs.However,the rise o...Recessive resistance mediated by mutations in the eukaryotic translation initiation factor 4E(eIF4E),has proven effective against diverse potyviruses and is extensively utilized in breeding programs.However,the rise of resistance-breaking(RB)strains and emerging potyviral species necessitates the development of more durable and broad-spectrum resistance strategies.In this study,our field survey in Yunnan,China,identified potato virus Y(PVY)RB isolates,as well as the prevalence of tobacco vein banding mosaic virus(TVBMV)and chilli veinal mottle virus(ChiVMV),in tobacco carrying the recessive va locus,which lacks the eIF4E1-S susceptibility gene,due to a chromosomal deletion.Protein interaction and viral infection assays demonstrated that both eIF4E1-S and eIFiso4E-T are used by PVY RB as susceptibility factors for infection,with the combined inactivation of these genes confering durable resistance.Similarly,the knockout of eIFiso4E-S,in the va genetic background,provided effective resistance to TVBMV and reduced susceptibility to ChiVMV.Notably,pyramiding mutations in eIFiso4E-S and eIFiso4E-T,in va tobacco,generated plants exhibiting robust,broad-spectrum resistance,to all three viruses,without compromising plant development.These findings underscore the potential of stacking eIF4E mutations to engineer durable,broad-spectrum resistance to potyviruses in tobacco,offering a promising strategy for crop improvement.展开更多
Hepatitis E virus （HEV） genotype 4 was originally identified in China. Its neutralization antigenic epitopes have not been characterized. Recently, we identified a neutralizing monoclonal antibody （mAb） IG10, whic...Hepatitis E virus （HEV） genotype 4 was originally identified in China. Its neutralization antigenic epitopes have not been characterized. Recently, we identified a neutralizing monoclonal antibody （mAb） IG10, which was generated following immunization of mice with p166Chn, a recombinant protein comprising 464-629 amino acids （aa） of the HEV genotype 4 capsid protein. In this study, a panel of 22 N- and/or C-terminal truncated and 6 site-directed mutated p166Chn proteins were prepared. Only those N- or C-terminal truncated proteins containing the region 477-613 aa could react with the mAb 1G10, suggesting the neutralization epitope of HEV genotype 4 is located between aa477 and aa613. However, a both N- and C-terminal truncated protein, pN477-C613, neither reacted to 1G10 nor elicited neutralizing antibodies in mice, while another both terminal truncated protein, pN472-C617, did, suggesting the flanking regions of the pN477-C613 could help to stabilize and allow presentation of the neutralization epitope to the immune system. Substituting Leu477 and/or Leu613 with the polar, uncharged threonine （Thr） caused 〉 50% reduction of the mutants＇ immunoreactivity to IG10, whereas replacement by hydrophobic phenylalanine （Phe） made little impact on the immunoreactivity, revealing functional associations between hydrophobicity of aa at positions 477 and 613 and the antigenicity of p166Chn. These data suggested Leu477 and Leu613 are critical in forming the neutralization epitope of HEV genotype 4. Cellular ＆ Molecular Immunology. 2008;5（6）：447-456.展开更多
An investigation on the optimization of parental RNA interference （RNAi） conditions for hunchback （hb） gene in Locusta migratoria manilensis （Meyen） was conducted. Double stranded RNA （dsRNA） corresponding to ...An investigation on the optimization of parental RNA interference （RNAi） conditions for hunchback （hb） gene in Locusta migratoria manilensis （Meyen） was conducted. Double stranded RNA （dsRNA） corresponding to hb gene was injected into haemocoel of female adults ofL. migratoria manilensis. Embryos developed from the eggs laid by the injected adults on the 7th day after eclosion showed observable effects of RNAi for hb. The silencing effect after delivery treatment of dsRNA for hb gene was maintained for more than 21 days. A significant decrease of hb transcripts was further confirmed by Northern blot analysis. The dose of dsRNA/insect at 2μg could trigger 96.9% RNAi effects, while silencing appeared to have no dependence on the size of dsRNA. Results suggest that parental RNAi could be employed to efficiently identify the developmental gene functions in L. migratoria manilensis.展开更多
Targeting-induced local lesions in genomes(TILLING)is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants.Although TILLING has been developed for many diploid plants...Targeting-induced local lesions in genomes(TILLING)is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants.Although TILLING has been developed for many diploid plants,the technology has been used in very few polyploid species due to their genomic complexity.Here,we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco(Nicotiana tabacum L.)using an ethyl methanesulfonate(EMS)-mutagenized population of 1,536 individuals.We optimized the procedures for endonuclease preparation,leaf tissue sampling,DNA extraction,normalization,pooling,PCR amplification,heteroduplex formation,and capillary electrophoresis.In a test screen using seven target genes with eight PCR fragments,we obtained 118 mutants.The mutation density was estimated to be approximately one mutation per 106kb on average.Phenotypic analyses showed that mutations in two heavy metal transporter genes,HMA2S and HMA4T,led to reduced accumulation of cadmium and zinc,which was confirmed independently using CRISPR/Cas9 to generate knockout mutants.Our results demonstrate that this powerful TILLING platform(available at http://www.croptilling.org)can be used in tobacco to facilitate functional genomics applications.展开更多
基金support from ICMR-National Institute for Research in Reproductive Health(ICMR-NIRRH).
文摘Rationale:If one of the partners is having balanced autosomal translocation,it is usually observed that the offspring inherit either normal chromosomes,balanced translocation identical to one of the parent or unbalanced chromosomal rearrangements of the same parental chromosome having translocation.Concern:A unique case presented with history of 8 miscarriages for genetic counseling.The last abortus material evaluation showed monosomy of chromosome X(Turner syndrome)in all the analyzed cells.There was a history of infertility and also repeated second trimester abortions on the paternal side.On the maternal side,there was a history of intellectual disability.Diagnose:History of repeated abnormal pregnancy outcomes.Wife’s karyotype is normal;however,husband shows translocation between chromosome 4 and 22.Intervention:Peripheral blood sample around 3 mL was collected for karyotype.Embryo biopsy was done and DNA was extracted and processed for whole exome sequencing.Outcomes:Wife’s karyotype is normal and husband has translocation between chromosome 4 and 22.Surprisingly,the entire pregnancy outcome including embryo screening has different,complete or partial aneuploidies of chromosomes other than chromosome 4 and 22.Main lesson:Though the translocation in one of the parent is balanced,we have to think beyond traditional ways for evaluating a couple with repeated pregnancy loss as we cannot predict the errors at cell division.Option of in vitro fertilization and preimplantation genetic diagnosis in couples having balanced translocations should be discussed so that early intervention can prevent the agony of pregnancy loss.
基金supported by the National Natural Science Foundation of China(32001532 and 31860411)the National Key Research and Development Program of China,(2022YFF1000020)+1 种基金Hunan Seed Industry Innovation Project(2021NK1012)the Yunnan Tobacco Company Project(2020530000241009)。
文摘The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.
基金Funding was provided by the Agricultural Biotechnology Research Institute of Iran(ABRII),Swedish Research Council(Vetenskapsrådet grant no.:2017-05019)the BBSRC Institute Strategic Programme Gut Microbes and Health(BB/r012490/1,its constituent project BBS/e/F/000Pr10355).
文摘Metagenomics has opened new avenues for exploring the genetic potential of uncultured microorganisms,which may serve as promising sources of enzymes and natural products for industrial applications.Identifying enzymes with improved catalytic properties from the vast amount of available metagenomic data poses a significant challenge that demands the development of novel computational and functional screening tools.The catalytic properties of all enzymes are primarily dictated by their structures,which are predominantly determined by their amino acid sequences.However,this aspect has not been fully considered in the enzyme bioprospecting processes.With the accumulating number of available enzyme sequences and the increasing demand for discovering novel biocatalysts,structural and functional modeling can be employed to identify potential enzymes with novel catalytic properties.Recent efforts to discover new polysaccharide-degrading enzymes from rumen metagenome data using homology-based searches and machine learning-based models have shown significant promise.Here,we will explore various computational approaches that can be employed to screen and shortlist metagenome-derived enzymes as potential biocatalyst candidates,in conjunction with the wet lab analytical methods traditionally used for enzyme characterization.
文摘Chemotherapy is one of the main therapeutic modalities for cancer patients.Cisplatin(CDDP),as one of thefirst-line drugs,is of great importance in the chemotherapy of various tumors.However,a significant percentage of cancer patients are resistant to CDDP treatment.Due to the CDDP side effects on normal tissues,the diagnosis of CDDP resistance is required to suggest the most efficient therapeutic strategies for cancer patients.Several molecular mechanisms and signaling pathways are associated with CDDP response.The PI3K/AKT signaling pathway has a pivotal role in the transmission of extracellular signals into the cells to regulate various pathophysiological processes such as cell proliferation,migration,and drug resistance.In the present review,we summarized all of the studies which have been reported on the role of PI3K/AKT pathway in regulation of CDDP response.It was shown that the PI3K/AKT pathway is mainly involved in CDDP response in lung,ovarian,and gastrointestinal cancers.It was also observed that the non-coding RNAs have a key role in CDDP response by regulation of PI3K/AKT pathway.This review paves the way for suggesting a PI3K/AKT-related panel marker for the prediction of CDDP response in different cancer patients.
文摘5-flurouracil(5-FU)-based chemotherapy is the main pharmacological therapy for advanced colorectal cancer(CRC).Despite significant progress in the treatment of CRC during the last decades,5-FU drug resistance remains the most important cause of failure in CRC therapy.Resistance to 5-FU is a complex and multistep process.Different mechanisms including microsatellite instability,increased expression level of key enzyme thymidylate synthase and its polymorphism,increased level of 5-FU-activating enzymes and mutation of TP53 are proposed as the main determinants of resistance to 5-FU in CRC cells.Recently,microribonucleic acids(miRNA)and their alterations were found to have a crucial role in 5-FU resistance.In this regard,the miRNA-mediated mechanisms of 5-FU drug resistance reside among the new fields of pharmacogenetics of CRC drug response that has not been completely discovered.Identification of the biological markers that are related to response to 5-FU-based chemotherapy is an emerging field of precision medicine.This approach will have an important role in defining those patients who are most likely to benefit from 5-FU-based chemotherapy in the future.Thereby,the identification of 5-FU drug resistance mechanisms is an essential step to predict and eventually overcome resistance.In the present comprehensive review,we will summarize the latest knowledge regarding the molecular determinants of response to 5-FU-based chemotherapy in CRC by emphasizing the role of miRNAs.
文摘Diabetes mellitus during pregnancy is associated with an increased risk of multiple congenital anomalies in progeny.There are sufficient evidence suggesting that the children of diabetic women exhibit intellectual and behavioral abnormalities accompanied by modification of hippocampus structure and function.Although,the exact mechanism by which maternal diabetes affects the developing hippocampus remains to be defined.Multiple biological alterations,including hyperglycemia,hyperinsulinemia,oxidative stress,hypoxia,and iron deficiency occur in pregnancies with diabetes and affect the development of central nervous system(CNS) of the fetus.The conclusion from several studies is that disturbance in glucose and insulin homeostasis in mothers and infants are major teratogenic factor in the development of CNS.Insulin and Insulin-like growth factor-1(IGF-1) are two key regulators of CNS function and development.Insulin and IGF-1 receptors(IR and IGF1 R,respectively) are distributed in a highly specific pattern with the high density in some brain regions such as hippocampus.Recent researches have clearly established that maternal diabetes disrupts the regulation of both IR and IGF1 R in the hippocampus of rat newborn.Dissecting out the mechanisms responsible for maternal diabetes-related changes in the development of hippocampus is helping to prevent from impaired cognitive and memory functions in offspring.
基金the grant of National Natural Science Foundation of China,No.39600129
文摘INTRODUCTIONHepatocellular transplantation (HCT) could providea therapeutic alternative to orthotopic livertransplantation(OLT) in the treatment of hepaticmetabolic defects and experimental hepaticfailure.Under appropriate conditions,theengrafted liver cells can continue to express liver-specific functions for an indefinite period of time.
基金supported by the Key Project of Applied Basic Research Program of Yunnan(2017FA015)the Young Academic and Technical Leader Raising Foundation of Yunnan Province(no. 2017HB063)+1 种基金the Yunnan Academy of Tobacco Agricultural Sciences(2018530000241002 and 2019530000241003)the Biotechnology Experimental Center at the Kunming Institute of Botany,CAS,for supporting plant cultivation.
文摘Jasmonic acid(JA)plays important roles in plant resistance to insect herbivores.One important derivative of JA is 12-OH-JA,which is produced by two independent pathways:direct hydroxylation of JA by jasmonate-induced oxygenases(JOXs)or hydrolyzation of 12-OH-JA-Ile.Yet the function of 12-OH-JA in plant-herbivore interactions remains largely unknown.In this study,we silenced four JOX homologs independently in the wild tobacco Nicotiana attenuata by virus-induced gene silencing(VIGS),and found that all four JOX homologs are involved in JA hydroxylation.Simultaneously silencing the four JA hydroxylases in VIGS-NaJOXs plants decreased herbivory-induced 12-OH-JA by 33%,but JA and JA-Ile levels increased by 45%and 30%,respectively,compared to those in control plants.Compared to direct hydroxylation from JA,hydrolyzation from 12-OH-JA-Ile is equally important for herbivory-induced 12-OHJA accumulation:in the 12-OH-JA-Ile deficient irJAR4/6 plants,12-OH-JA decreased 34%.Moreover,VIGSNaJOXs plants exhibited enhanced resistance to the generalist herbivore Spodoptera litura.The poor larval performance was strongly correlated with high levels of several JA-Ile-dependent direct defense metabolites in the VIGS-NaJOXs plants.When we simultaneously silenced all four JA hydroxylases in the JAIle-deficient irJAR4/6 background,the enhanced herbivore resistance diminished,demonstrating that enhanced herbivore resistance resulted from elevated JA-Ile levels.Given that silencing these NaJOX-like genes did not detectably alter plant growth but highly increased plant defense levels,we propose that JOX genes are potential targets for genetic improvement of herbivore-resistant crops.
文摘In order to select the strain that can degrade nitrite, the screening plate with nitrite was used as the sole nitrogen source to screen the strain with ability to degrade nitrite. A strain with nitrite degrading capacity was isolated from the sludge of a shrimp-farming pond in Hepu City, Guangxi Zhuang Autonomous Region. The isolated strain was identified based on colonial morphology, physiological and biochemical characteristics, and 16S rRNA sequence. Results showed that the strain could grow well on the culture medium containing 2.3 g/L nitrite. According to the morphological characteristics, nitrogen source requirements and evolutionary tree of the 16S rRNA sequence, the isolated strain was identified as Acinetobacter radioresistens, which was named HPAR132 strain. This study laid the foundation for further investigation of nitrite-oxidizing bacterium HPAR132.
基金funded by the National Natural Science Foundation of China(grant number 31760072 to G.Bai,and grant number 31860413 to H.Xie)Yunnan Applied Basic Research Project(grant number 202001AT070010 to G.Bai)the Yunnan Academy of Tobacco Agricultural Sciences(grant numbers YNTC-2016YN22 and CNTC-110202001025(JY08)to H.Xie,YNTC-2016YN24,YNTC-2015YN02,YNTC-2018530000241002,and YNTC-2019530000241003 to D.-H.Yang).
文摘The bHLH transcription factors play pivotal roles in plant growth and development,production of secondary metabolites and responses to various environmental stresses.Although the bHLH genes have been well studied in model plant species,a comprehensive investigation of the bHLH genes is required for tobacco with newly obtained high-quality genome.In the present study,a total of 309 NtbHLH genes were identified and can be divided into 23 subfamilies.The conserved amino acids which are essential for their function were predicted for the NtbHLH proteins.Moreover,the NtbHLH genes were conserved during evolution through analyzing the gene structures and conserved motifs.A total of 265 NtbHLH genes were localized in the 24 tobacco chromosomes while the remained 44 NtbHLH genes were mapped to the scaffolds due to the complexity of tobacco genome.Moreover,transcripts of NtbHLH genes were obviously tissue-specific expressed from the gene-chip data from 23 tobacco tissues,and expressions of 20 random selected NtbHLH genes were further confirmed by quantitative real-time PCR,indicating their potential functions in the plant growth and development.Importantly,overexpressed NtbHLH86 gene confers improve drought tolerance in tobacco indicating that it might be involved in the regulation of drought stress.Therefore,our findings here provide a valuable information on the characterization of NtbHLH genes and further investigation of their functions in tobacco.
基金Supported by the Zhejiang Provincial Natural Science Foundation of China (No.LY12H12001)the Ningbo Key Foundation of Society Development (No.2014C50091)+2 种基金the Ningbo Natural Science Foundation (No.2012A610192)the Ningbo Yinzhou District S&T Foundation (No.YK2013-90)the Shenzhen Municipal Government of China (No.GJHZ20130417140916986)
文摘AIMTo identify the disease-causing gene mutation in a Chinese pedigree with autosomal dominant cone-rod dystrophy (adCORD).METHODSA southern Chinese adCORD pedigree including 9 affected individuals was studied. Whole-exome sequencing (WES), coupling the Agilent whole-exome capture system to the Illumina HiSeq 2000 DNA sequencing platform was used to search the specific gene mutation in 3 affected family members and 1 unaffected member. After a suggested variant was found through the data analysis, the putative mutation was validated by Sanger DNA sequencing of samples from all available family members.RESULTSThe results of both WES and Sanger sequencing revealed a novel nonsense mutation c.C766T (p.Q256X) within exon 5 of CRX gene which was pathogenic for adCORD in this family. The mutation could affect photoreceptor-specific gene expression with a dominant-negative effect and resulted in loss of the OTX tail, thus the mutant protein occupies the CRX-binding site in target promoters without establishing an interaction and, consequently, may block transactivation.CONCLUSIONAll modes of Mendelian inheritance in CORD have been observed, and genetic heterogeneity is a hallmark of CORD. Therefore, conventional genetic diagnosis of CORD would be time-consuming and labor-intensive. Our study indicated the robustness and cost-effectiveness of WES in the genetic diagnosis of CORD.
文摘Introduction: Beta-thalassemia is characterized by absence or reduced synthesis of the β-globin. Carriers of β-thalas- semia, typically have microcytic hypochromic anemia and elevated hemoglobin HbA2 and normal HbF level. On the other hand carriers of severe alpha-thalassemia also have similar CBC parameters to that of β-thalassemia with normal HbA2 level. Co-presence of mutations in the β-globin and delta-globin genes (point mutations or deletions) usually give normal HbA2 and elevated HbF level. We report a β-thal carrier with normal level of HbA2 and increased level of HbF who had a point mutation in CD39 on the beta-globin gene and a point mutation in CD27 on the δ-globin gene named Hb-Yialousa. Materials & Methods: An individual with low hematological indices, normal HbA2 and elevated HbF was referred to our center as routine premarital screening program. Mutations in the β-globin and δ-globin genes were screened using ARMS and sequencing methods. Results: The mutation in β- and δ-globin genes were identified as CD39 and CD27 (HbYialousa) respectively. No point mutation or deletion in α-globin gene was identified. Discussion: We showed that normal HBA2 with elevated HbF level is due to co-inheritance of delta-globin gene mutation with mutation in the β-globin gene. When screening for β-thalassemia, one has to either rule out presence of α-globin gene mutation of mutation in the delta-globin gene.
文摘AIM: To generate DNA-aptamers binding to Methicillinresistant Staphylococcus aureus(MRSA).METHODS: The Cell-Systematic Evolution of Ligands by Exponential Enrichment(SELEX) technology was used to run the selection against MRSA bacteria and develop target-specific aptamers. MRSA bacteria were targeted while Enterococcus faecalis bacteria were used for counter selection during that process. Binding assays to determine the right aptamer candidates as well as binding assays on clinical samples were performed through flow cytometry and analyzed using the FlowJ o software. The characterization of the aptamers was done by determination of their Kd values and determined by analysis of flow data at different aptamer concentration using Sigma Plot. Finally, the recognitionof the complex Gold-nanoparticle-aptamer to the bacteria cells was observed using transmission electron microscopy(TEM).RESULTS: During the cell-SELEX selection process, 17 rounds were necessary to generate enrichment of the pool. While the selection was run using fixed cells, it was shown that the binding of the pools with live cells was giving similar results. After sequencing and analysis of the two last pools, four sequences were identified to be aptamer candidates. The characterization of those aptamers showed that based on their Kd values, DTMRSA4 presented the best binding with a Kd value of 94.61 ± 18.82 nmol/L. A total of ten clinical samples of MRSA, S. aureus and Enterococcus faecalis were obtained to test those aptamers and determine their binding on a panel of samples. DTMRSA1 and DTMRSA3 showed the best results regarding their specificity to MRSA, DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding to MRSA cells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding property.CONCLUSION: A total of four aptamers that bind to MRSA were obtained with Kd values ranking from 94 to 200 nmol/L.
文摘Exosomes are small vesicles that carry molecules from one cell to another.They have many features that make them interesting for research,such as their stability,low immunogenicity,size of the nanoscale,toxicity,and selective delivery.Exosomes can also interact with viruses in diverse ways.Emerging research highlights the significant role of exosomes in viral infections,particularly in the context of diseases like COVID-19,HIV,HBV and HCV.Understanding the intricate interplay between exosomes and the human immune system holds great promise for the development of effective antiviral therapies.An important aspect is gaining clarity on how exosomes influence the immune system and enhance viral infectivity through their inherent characteristics.By leveraging the innate properties of exosomes,viruses exploit the machinery involved in exosome biogenesis to set replication,facilitate the spread of infection,and eliminate immune responses.They can either help or hinder viral infection by modulating the immune system.This review summarizes the recent findings on how exosomes mediate viral infection and how they can be used for diagnosis or therapy.This could lead to new clinical applications of exosomes in disease management.
文摘Analysis of the frequency of antigen-specific cytotoxic T lymphocytes (CTLs) ex vivo is largely dependent on the use of MHC/peptide tetramers. However, the latter reagents have not been widely available, most likely because of their costly and time-consuming production. In this report we utilized an economic strategy to construct HLA/peptide tetramers with recombinant peptide-linked β2 microglobulin (β2m). The HLA-A2-restricted, melanoma antigen MARTI-derived pep- tide MART127-35( AAGIGILTV) was fused to the N terminus of human β2m through a 15-amino acid (aa)-long linker before being refolded with the recombinant biotinylated HLA-A2 heavy chain ectodomain. The resulted 2-component (2C) monomer was then tetramerized with phycoerythin-labeled streptavidin. The experimental result showed that the 2C HLA-A2/ MART127-35 monomer was shown to bind to the HLA class complex-specific monoclonal antibody W6/32 and the HLA-A2/ MART127-35 complex-specific single chain antibody fragment (scFv) 8.3, suggesting the correctness of its specificity. Fur- thermore, the 2C HLA-A2/MART127-35 tetramer detected a specific CD8+ T cell population in HLA-A2-restricted melanoma infiltrating lymphocytes as the conventional 3C HLA-A2/MART127-35 tetramer. The yield of 2C HLA-A2/MART127-35 monomer was 2. 5 times more than that of the conventional 3C monomer. Taken together, these data indicate that the HLA-A2/ MART127-35 tetramer can be generated conveniently through the use of MART127-35 peptide-β2 m fusion proteins, which can fa- cilitate the monitoring of HLA-A2-restricted, MART1-specific CTL responses in patients with melanoma.
基金supported by grants from the Biological Breeding-National Science and Technology Major Project(2022ZD04011,2023ZD04073)National Natural Science Foundation of China(32325035,32071864)+4 种基金Fundamental Research Funds for Central Non-profit Scientific Institution(1610392024008)Central Laboratory of Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Hainan Seed Industry Laboratory(B23CJ0208)Yunnan Academy of Tobacco Agricultural Sciences(CNTC-YC2022530000241013,CNTC110201501015(JY-02))Hohhot Key R&D Project(2023-JBGS-S-1)Inner Mongolia Center of Pratacultural Technology Innovation(CCPTZX2023B01).
文摘CRISPR/Cas is a simple,robust,versatile tool for plant biology studies and precision plant breeding.However,establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa(Medicago sativa L.),the most important forage legume worldwide,remains a formidable challenge.Here,we systematically identified endogenous U6 promoters in alfalfa through transient expression via Agrobacterium-mediated infiltration of alfalfa leaves.We further demonstrated the efficacy of the three most active promoters for genome editing using an optimized alfalfa hairy root system.Subsequently,we established an improved CRISPR/Cas9 multiplex system containing three or four tandemly arrayed MsU6-promoter-driven polycistronic tRNA-sgRNA(PTG)expression cassettes,each consisting of three tRNA-sgRNA units,to simultaneously edit three or four alfalfa genes,coupled with the visual reporter RH1 or RUBY.This toolkit showed efficient multiplex editing in the hairy root system with visual selection.We successfully obtained regenerated,red-colored shoots resulting from the stable transformation of alfalfa.These results highlight the potential application of the visual reporter system for the stable transformation of alfalfa.Our improved CRISPR/Cas9 multiplex system enables convenient,high-efficiency multiplex genome editing in alfalfa,providing a versatile toolset to facilitate functional studies of multiple genes and gene families for basic research and the genetic improvement of alfalfa.
基金supported by National Natural Science Foundation of China(31860490)YNTC funds(2023530000241007 and 2017YN02)+1 种基金Yunnan Daguan Lab(YNDG202302ZY01)CLZ is supported by the Postdoctoral Fellowship Program of CPSF under grant number GZC20241527。
文摘Recessive resistance mediated by mutations in the eukaryotic translation initiation factor 4E(eIF4E),has proven effective against diverse potyviruses and is extensively utilized in breeding programs.However,the rise of resistance-breaking(RB)strains and emerging potyviral species necessitates the development of more durable and broad-spectrum resistance strategies.In this study,our field survey in Yunnan,China,identified potato virus Y(PVY)RB isolates,as well as the prevalence of tobacco vein banding mosaic virus(TVBMV)and chilli veinal mottle virus(ChiVMV),in tobacco carrying the recessive va locus,which lacks the eIF4E1-S susceptibility gene,due to a chromosomal deletion.Protein interaction and viral infection assays demonstrated that both eIF4E1-S and eIFiso4E-T are used by PVY RB as susceptibility factors for infection,with the combined inactivation of these genes confering durable resistance.Similarly,the knockout of eIFiso4E-S,in the va genetic background,provided effective resistance to TVBMV and reduced susceptibility to ChiVMV.Notably,pyramiding mutations in eIFiso4E-S and eIFiso4E-T,in va tobacco,generated plants exhibiting robust,broad-spectrum resistance,to all three viruses,without compromising plant development.These findings underscore the potential of stacking eIF4E mutations to engineer durable,broad-spectrum resistance to potyviruses in tobacco,offering a promising strategy for crop improvement.
基金supported by grants from the National Natural Science Foundation of China (No. 30271231, No. 30671858)the High Technology Research Program of Jiangsu Province, China (BG2006607)partially supported by the National High Technology Research and Development Program of China (863 Program, 2006AA02A235)
文摘Hepatitis E virus （HEV） genotype 4 was originally identified in China. Its neutralization antigenic epitopes have not been characterized. Recently, we identified a neutralizing monoclonal antibody （mAb） IG10, which was generated following immunization of mice with p166Chn, a recombinant protein comprising 464-629 amino acids （aa） of the HEV genotype 4 capsid protein. In this study, a panel of 22 N- and/or C-terminal truncated and 6 site-directed mutated p166Chn proteins were prepared. Only those N- or C-terminal truncated proteins containing the region 477-613 aa could react with the mAb 1G10, suggesting the neutralization epitope of HEV genotype 4 is located between aa477 and aa613. However, a both N- and C-terminal truncated protein, pN477-C613, neither reacted to 1G10 nor elicited neutralizing antibodies in mice, while another both terminal truncated protein, pN472-C617, did, suggesting the flanking regions of the pN477-C613 could help to stabilize and allow presentation of the neutralization epitope to the immune system. Substituting Leu477 and/or Leu613 with the polar, uncharged threonine （Thr） caused 〉 50% reduction of the mutants＇ immunoreactivity to IG10, whereas replacement by hydrophobic phenylalanine （Phe） made little impact on the immunoreactivity, revealing functional associations between hydrophobicity of aa at positions 477 and 613 and the antigenicity of p166Chn. These data suggested Leu477 and Leu613 are critical in forming the neutralization epitope of HEV genotype 4. Cellular ＆ Molecular Immunology. 2008;5（6）：447-456.
文摘An investigation on the optimization of parental RNA interference （RNAi） conditions for hunchback （hb） gene in Locusta migratoria manilensis （Meyen） was conducted. Double stranded RNA （dsRNA） corresponding to hb gene was injected into haemocoel of female adults ofL. migratoria manilensis. Embryos developed from the eggs laid by the injected adults on the 7th day after eclosion showed observable effects of RNAi for hb. The silencing effect after delivery treatment of dsRNA for hb gene was maintained for more than 21 days. A significant decrease of hb transcripts was further confirmed by Northern blot analysis. The dose of dsRNA/insect at 2μg could trigger 96.9% RNAi effects, while silencing appeared to have no dependence on the size of dsRNA. Results suggest that parental RNAi could be employed to efficiently identify the developmental gene functions in L. migratoria manilensis.
基金supported by Yunnan Province Tobacco funding(2014YN05,2014YN06,2016YN25,2017YN03)Bureau of National Tobacco funding(110201401006[JY-06],110201601031[JY-05],110201401005[JY-05]).
文摘Targeting-induced local lesions in genomes(TILLING)is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants.Although TILLING has been developed for many diploid plants,the technology has been used in very few polyploid species due to their genomic complexity.Here,we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco(Nicotiana tabacum L.)using an ethyl methanesulfonate(EMS)-mutagenized population of 1,536 individuals.We optimized the procedures for endonuclease preparation,leaf tissue sampling,DNA extraction,normalization,pooling,PCR amplification,heteroduplex formation,and capillary electrophoresis.In a test screen using seven target genes with eight PCR fragments,we obtained 118 mutants.The mutation density was estimated to be approximately one mutation per 106kb on average.Phenotypic analyses showed that mutations in two heavy metal transporter genes,HMA2S and HMA4T,led to reduced accumulation of cadmium and zinc,which was confirmed independently using CRISPR/Cas9 to generate knockout mutants.Our results demonstrate that this powerful TILLING platform(available at http://www.croptilling.org)can be used in tobacco to facilitate functional genomics applications.
