Duplex-specific nuclease(DSN)is a necessary component to digest double-stranded DNA(dsDNA)and DNA in DNA-RNA hybrid duplexes specifically,and widely used in food analysis and biological sample treatment.However,few ca...Duplex-specific nuclease(DSN)is a necessary component to digest double-stranded DNA(dsDNA)and DNA in DNA-RNA hybrid duplexes specifically,and widely used in food analysis and biological sample treatment.However,few cases were reported to produce DSN harnessing genetic engineering,which limited the application and pushed up the cost of DSN.In this study,Pandalus borealis DSN gene was expressed by the famous microbial host,Komagataella phaffii,through three steps including promoter and signal peptide selection,DSN gene expression cassette increasing,cultivation optimization to reach 2.6×10^(5)U/μL in fermentation broth.Then,a simple purification process was carried out to prepare the freeze drying DSN for characterization.This DSN was claimed as Km value of 399.04 ng/μL,Vmax of 1.7×10^(6)μmol/(μL⋅min),optimal temperature of 37℃,and activated by Mg^(2+)and Mn^(2+).Therefore,this DSN was profiled for its application which was supported by re-combinant K.phaffii sufficiently.展开更多
Highly sensitive and reliable detection of multiple myeloma remains a major challenge in liquid biopsy. Herein, for the first time, quantum dot-molecular beacon (QD-MB) functionalized MoS_(2) (QD-MB @MoS_(2)) fluoresc...Highly sensitive and reliable detection of multiple myeloma remains a major challenge in liquid biopsy. Herein, for the first time, quantum dot-molecular beacon (QD-MB) functionalized MoS_(2) (QD-MB @MoS_(2)) fluorescent probes were designed for the dual detection of multiple myeloma (MM)-related miRNA-155 and miRNA-150. The results indicate that the two probes can effectively detect miRNA-155 and miRNA-150 simultaneously with satisfactory recovery rates, and the limit of detections (LODs) of miRNA-155 and miRNA-150 in human serum are low to 7.19 fM and 5.84 fM, respectively. These results indicate that our method is the most sensitive detection so far reported and that the designed fluorescent probes with signal amplification strategies can achieve highly sensitive detection of MM-related miRNAs for MM diagnosis.展开更多
Transcription profiling assays have revealed substantial changes in gene expression during plant-microbe interactions,but it is often time-and labor-consuming to define the causative roles of the differentially expres...Transcription profiling assays have revealed substantial changes in gene expression during plant-microbe interactions,but it is often time-and labor-consuming to define the causative roles of the differentially expressed genes that finetune the plant responses to diverse pathogens.We improved the duplex-specific nuclease-mediated transcriptome subtraction method and integrated it with SMART cDNA library construction technology to generate normalized libraries consisting of full-length cDNAs derived from transcripts upregulated in the rice-Magnaporthe oryzae interaction.By adding BP recombination sites to the sequence of primers used for the synthesis of fulllength cDNAs,we were able to transfer the full-length cDNAs of the library to an expression binary vector with CaMV 35S promoter,which allowed a subsequent screening for candidate genes potentially involved in the disease process by Agrobacterium-mediated transient assay.Results showed that the newly established approach preferentially suppressed the cDNA abundance of most constitutive genes and enriched or normalized that of the upregulated ones.Subsequent screening with transient expression in planta isolated 61 clones,which carry fulllength cDNAs of 32 distinct genes,were capable of causing cell death on Nicotiana benthamiana.When transiently expressed in barley leaf,five of the identified genes were able to induce chlorosis,and additional 11 genes were found to promote disease symptoms caused by infection with the blast pathogen while the others did not enhance the symptoms of pathogen infection.These observations demonstrate that the subtractive hybridizationassisted functional screening is an efficient approach that provides initial leads to the role of candidate genes in the complex process of plant-microbe interactions.展开更多
基金supported by the National Key Research and Devel-opment Program,China(2025YFA0922200),National Natural Science Foundation of China(No.32572515,W2512043).
文摘Duplex-specific nuclease(DSN)is a necessary component to digest double-stranded DNA(dsDNA)and DNA in DNA-RNA hybrid duplexes specifically,and widely used in food analysis and biological sample treatment.However,few cases were reported to produce DSN harnessing genetic engineering,which limited the application and pushed up the cost of DSN.In this study,Pandalus borealis DSN gene was expressed by the famous microbial host,Komagataella phaffii,through three steps including promoter and signal peptide selection,DSN gene expression cassette increasing,cultivation optimization to reach 2.6×10^(5)U/μL in fermentation broth.Then,a simple purification process was carried out to prepare the freeze drying DSN for characterization.This DSN was claimed as Km value of 399.04 ng/μL,Vmax of 1.7×10^(6)μmol/(μL⋅min),optimal temperature of 37℃,and activated by Mg^(2+)and Mn^(2+).Therefore,this DSN was profiled for its application which was supported by re-combinant K.phaffii sufficiently.
基金The authors acknowledge the generous financial support from the National Natural Science Foundation of China(61971207,61975070,51902143,61775088)Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD),Key Research and Development Project of Jiangsu Province(BE2018062,BE2019033)+6 种基金Natural Science Foundation of Jiangsu Province(BK20191467)Post-graduate Research&Practice Innovation Program of Jiangsu Province(KYCX20_2229)International S&T Cooperation Program of Jiangsu Province(BZ2019063,BZ2020045,BZ2020030)Natural Science Foundation of the Jiangsu Higher Education Institutes of China(19KJB430018,20KJA430003)Special Project for Technology Innovation of Xuzhou City(KC19250,KC20201,KC20244)Open Project of State Key Laboratory of Advanced Materials and Electronic Components(FHR-JS-202011017)We also thank Dr.Zhiling Yan for his help and support on the collection of blood samples.WC would like to thank the support from Solgro Inc.,Guangxi Jialouyuan Medical Inc.and UT Arlington distinguished award.
文摘Highly sensitive and reliable detection of multiple myeloma remains a major challenge in liquid biopsy. Herein, for the first time, quantum dot-molecular beacon (QD-MB) functionalized MoS_(2) (QD-MB @MoS_(2)) fluorescent probes were designed for the dual detection of multiple myeloma (MM)-related miRNA-155 and miRNA-150. The results indicate that the two probes can effectively detect miRNA-155 and miRNA-150 simultaneously with satisfactory recovery rates, and the limit of detections (LODs) of miRNA-155 and miRNA-150 in human serum are low to 7.19 fM and 5.84 fM, respectively. These results indicate that our method is the most sensitive detection so far reported and that the designed fluorescent probes with signal amplification strategies can achieve highly sensitive detection of MM-related miRNAs for MM diagnosis.
基金partially supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China(Grant No.20130008110005).
文摘Transcription profiling assays have revealed substantial changes in gene expression during plant-microbe interactions,but it is often time-and labor-consuming to define the causative roles of the differentially expressed genes that finetune the plant responses to diverse pathogens.We improved the duplex-specific nuclease-mediated transcriptome subtraction method and integrated it with SMART cDNA library construction technology to generate normalized libraries consisting of full-length cDNAs derived from transcripts upregulated in the rice-Magnaporthe oryzae interaction.By adding BP recombination sites to the sequence of primers used for the synthesis of fulllength cDNAs,we were able to transfer the full-length cDNAs of the library to an expression binary vector with CaMV 35S promoter,which allowed a subsequent screening for candidate genes potentially involved in the disease process by Agrobacterium-mediated transient assay.Results showed that the newly established approach preferentially suppressed the cDNA abundance of most constitutive genes and enriched or normalized that of the upregulated ones.Subsequent screening with transient expression in planta isolated 61 clones,which carry fulllength cDNAs of 32 distinct genes,were capable of causing cell death on Nicotiana benthamiana.When transiently expressed in barley leaf,five of the identified genes were able to induce chlorosis,and additional 11 genes were found to promote disease symptoms caused by infection with the blast pathogen while the others did not enhance the symptoms of pathogen infection.These observations demonstrate that the subtractive hybridizationassisted functional screening is an efficient approach that provides initial leads to the role of candidate genes in the complex process of plant-microbe interactions.
