Development of Molecular Marker Linked with Cercospora Leaf Spot (CLS) Disease Resistance in Vigna radiata, Cloning, and Expression for Evaluating Antifungal Activity against Cercospora canescens

We developed a molecular marker for MAS of mungbean resistant varieties against CLS from the consensus sequence(MB-CLsRG)of identified RGAs(MB-ClsRCaG1 and MB-ClsRCaG2).The MB-CLsRG sequence-specific primer pair was used to screen Cercospora leaf spot(CLS)resistant varieties of mungbean in genomic analysis that showed congruency with phenotypic screening.Validation of molecular marker linkage with CLS resistance was performed using rtPCR in transcriptomic analysis.The sequenced PCR products showed 100%homology with MB-CLsRG sequence and putative disease resistance proteins that confirmed the linkage of molecular marker with CLS resistance in mungbean.The antifungal potential of MB-CLsRG gene encoding protein was assessed.The MB-CLsRG gene sequence was cloned in the E.coli expression vector for recombinant protein production.The recombinant protein was then investigated for its in vitro antifungal potential against Cercospora canescens.The in vitro investigation showed strong antifungal activity of recombinant protein as it restricted the growth of fungal mycelial mass.The results validated the linkage of developed marker with CLS-resistant mungbean varieties;therefore,it can be used to screen resistant varieties from a large population in MAS.Moreover,the recombinant protein of the MB-CLsRG gene sequence revealed antifungal potential,which proved the gene sequence could be suitable to use in transgenic plants technology to develop fungal-resistant transgenic crops.

Cloning, E. coli Expression and Molecular Analysis of a Novel Sesquiterpene Synthase Gene from Artemisia annua

A 1 886 bp full-length sesquiterpene synthase (AaSES) cDNA was cloned front a high-yield Artemisia annua L. strain 001 by a rapid amplification of cDNA end (RACE) strategy. AaSES is 59% identical to Artemisia cyclase cDNA clone cASC125, 50% identical to epi-cedrol synthase from A. annua , 48% identical to amorpha-4, 11-diene synthase from A. annua, 39% identical to the 5-epi-aristolechene synthase from tabacco, 38 % identical to vetispiradiene synthase front H. muticus, 41 % identical to the, delta-cadinene synthase from cotton. The coding region of the cDNA was inserted into a procaryotic expression vector pET-30a and overexpressed in E. coli BL21 ( DE3). The cyclase proteins extracted front bacterial culture were found largely in an insoluble protein fraction. AaSES expresses in leaves, stems a-rid flowers, not in roots as indicated by Northern blotting analysis.

Molecular Cloning and Bioinformatics Analysis of msrA Gene from Vibrio alginolyticus Strain HY9901

[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of msrA,and its bioinformatics analysis was carried out.[Results]The full length of msrA gene was 639 bp,encoding 212 amino acids,and its theoretical molecular weight was about 23729.60 Da.The protein had a stable structure,and it was hydrophobic overall.The structure of signal peptides at the N terminal of the amino acid sequence was predicted,and it was found that there was no signal peptide cleavage site and no transmembrane region.The amino acid sequence of MsrA contained multiple signal binding sites.Protein subcellular localization showed that MsrA protein was most likely located in the cytoplasm.Homology analysis showed that MsrA of V.alginolyticus had high homology with other Vibrio species,and the highest homology with V.alginolyticus.In the prediction of functional domains,MsrA had the function of methionine sulfoxide reduction.In secondary structure prediction,MsrA contained random coils at a proportion of 46.70%,which was the highest.The similarity between the tertiary structure model of MsrA and template Q87SW6.1.A was 89.15%.PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites.[Conclusions]This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress.

Molecular Cloning of sodB Gene from Vibrio alginolyticus HY9901 and Its Bioinformatics Analysis

Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.

Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.

Na^+/K^+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress

Na^＋/K^＋-ATPases are membrane-associated enzymes responsible for the active transport of Na^＋ and K^＋ ions across cell membranes, generating chemical and electrical gradients. These enzymes＇ α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na^＋/K^＋-ATPase α-subunit eDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction （RT-PCR） and rapid amplification of eDNA end methods. Analysis of the nucleotide sequence revealed that the eDNA had a full-length of 3 833 base pairs （bp）, with an open reading frame of 3 120 bp, 5＇ untranslated region （UTR） of 317 bp, and 3＇ UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na^＋/K^＋-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of arnino acid sequences showed that the P. tritubereulatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit＇s transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab＇s Na^＋/K^＋-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

Distribution of Chitinolytic Enzyme in the Organs and Molecular Cloning of a Novel Chitinase Gene from the Kidney of Marbled Rockfish <i>Sebastiscus marmoratus</i>

Actinopterygii express two types of chitinase (acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2)) that are active at acidic pHs and involved in digestion in the stomach. We proposed the existence of a new fish chitinase that has a non-digestive function. In this study, we used Sebastiscus marmoratus, for which characteristics and cDNA cloning of chitinase isozymes (SmChi-1, SmChi-2) in the stomach have been reported. Initially, we examined the distribution of chitinase and β-N-acetylhexosaminidase (Hex) in the body and then we tried to clone novel chitinase cDNA from the kidney. Chitinase and Hex activities were measured using pNP-(GlcNAc)n, (n = 2, 3) and pNP-GlcNAc as substrates, respectively. Total RNA was extracted from the kidney. RT-PCR was performed to obtain chitinase cDNA fragments using reverse transcriptase with an oligo dT primer. The RACE method was used to obtain sequences of the upstream and downstream regions of cDNA. The full-length chitinase cDNA was determined using PrimeSTAR&reg;?Max DNA polymerase with proofreading activity. High chitinase activity was observed in the stomach, as previously reported. In addition, relatively high activity was observed in the liver, spleen, kidney, and heart. In contrast, Hex activity was detected in all organs. This result is consistent with the report that Hex is related to body-wide metabolism. Full-length cDNA (SmChi-3) of the novel chitinase was obtained from the kidney, which contained 1440 bp open reading frames. The domain structure of SmChi-3 was assumed to be similar to those of SmChi-1 and SmChi-2. SmChi-1 and SmChi-2 have a serine and glycine-rich linker region, which is characteristic of AMCase. In contrast, SmChi-3 contained no apparent sequence in the linker region. Phylogenetic analysis revealed the existence of a new chitinase group, which was named fish chitinase-3 (FCase-3) and differed from AFCase-1 and AFCase-2.

Porcine LEM domain-containing 3: Molecular cloning, functional characterization, and polymorphism associated with ear size

Ear size exhibits remarkable diversity in pig breeds. LEM domain-containing 3 （LEMD3） on chromosome 5 is considered as an important candidate for porcine ear size. This is the first study on cloning and characterization of LEMD3 cDNA. The complete cDNA contains 4 843 bp, including a 2 736-bp open reading frame （ORF）, a 37-bp 5＂-untranslated region （UTR） and a 2070-bp 3＂-UTR. The complete LEMD3 gene is 126241-bp and contains 13 exons and 12 introns. The ORF encodes a deduced LEMD3 protein of 911 amino acids, which shares 82-94% nucleic acid and 51-96% amino acid identity with other species. A phylogenetic tree constructed based on the amino acid sequences revealed that the porcine LEMD3 protein was closely related with cattle LEMD3. Resequencing of the ORF and promoter of LEMD3 from Minzhu pig and Large White revealed three single nucleotide polymorphisms （SNPs）： L964C〉A in the complete coding region, L4625A〉G in the 3＂ UTR, and L-394T〉C in the promoter region. Genome-wide association study （GWAS） revealed that all of SNPs were shown significant association with ear size in Large WhitexMinzhu pig intercross population. With conditional GWAS, -Iogl0（P-value） decreased by more than 80% when each of three SNPs was included as a fixed effect. These results suggested direct involvement of LEMD3 or close linkage to the causative mutation for ear size. The findings of this study might form the basis for understanding the genetic mechanism of ear size variation in pigs and provide potential molecular markers for screening ear size diversity in pig breeds.

The molecular cloning and restriction enzyme mapping of retinitis pigmentosa 3 (RP3) locus

Objective: To clone retinitis pigmentosa region by yeast artificial chromosome (YAC) and establish therestriction enzyme physical map. Methods: The ornithine transcarbamoylase (OTC ) cDNA probe. which is closelylinked to the RP3 locus, was chosen to screen the X chromosome YAC library by colony in situ hybridization. Size determination, sequence tagged site (STS) analysis and long range physical mapping were performed with positive YACs. The results obtained were used to map these YACs. Results: We built a 1. 6 Mb YAC contig cont aming infor mation on RP3 range. restriction enzyme sites, CpG islands location and YAC position. Conclusion: Thework provides a good basis for identification and cloning of the RP3 gene.

Chondrichthyes Chitinase: Molecular Cloning, Distribution, and Phylogenetic Analysis

We have previously reported the presence of three types of chitinase (acidic fish chitinase-1: AFCase-1, acidic fish chitinase-2: AFCase-2, fish chitinase-3: FCase-3) in Actinopterygii. In the present research, we report the identification of the novel chitinase genes HjChi (ORF: 1380 bp) and DkChi (ORF: 1440 bp) from the stomach of Chondrichthyes, Japanese bullhead shark (Heterodontus japonicas) and Kwangtung skate (Dipturus kwangtungensis), respectively. Organ-specific expression analysis identified the stomach-specific expression of HjChi, whereas DkChi was expressed widely in all organs. Chitinase activity was measured using pNP-(GlcNAc)n (n = 2, 3) as a substrate and β-N-acetylhexosaminidase (Hex) activity was measured using pNPGlcNAc. Relatively high values of chitinase activity were observed in the stomach, spleen, and gonads of the Japanese bullhead shark, H. japonicas , compared with that observed in the stomach of the Kwangtung skate D. kwangtungensis . However, Hex activity was detected throughout the body of both species. The optimal pH of chitinase in both the Japanese bullhead shark, H. japonicas, and the Kwangtung skate, D. kwangtungensis, were 3.5 - 5.5 and 3.5 - 4.0, respectively, and 4.0 for Hex in both species. Phylogenetic analysis revealed that Chondrichthyes chitinase forms a unique group (Chondrichthyes chitinase). These results suggested that the possibility of the formation of chitinase groups for each class in the phylogenetic analysis based on the observation of class-specific chitinase.

Association Mapping, QTL Mapping and Positional Cloning in Maize: Applications in Molecular Breeding

Natural variation is at the core of plant breeding. Genetic or linkage mapping is the traditional method for identifying loci/genes responsible for variati on in complex traits. More recently, association mapping or

Cost-Effective Method of Gene Synthesis by Sequencing from Microchip-Derived Oligos for Droplet Cloning

Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy.

The chemical environment and structural ordering in liquid Mg-Y-Zn system:An ab-initio molecular dynamics investigation of melt for the formation mechanism of LPSO structure

In an effort to clarify the formation mechanism of LPSO structure in Mg-Y-Zn alloy,the chemical environment and structural ordering in liquid Mg-rich Mg-Y-Zn system are investigated with the aid of ab-initio molecular dynamics simulation.In liquid Mg-rich Mg-Y alloys,the strong Mg-Y interaction is determined,which promotes the formation of fivefold symmetric local structure.For Mg-Zn alloys,the weak Mg-Zn interaction results in the fivefold symmetry weakening in the liquid structure.Due to the coexistence of Y and Zn,the strong attractive interaction is introduced in liquid Mg-Y-Zn ternary alloy,and contributes to the clustering of Mg,Y,Zn launched from Zn.What is more,the distribution of local structures becomes closer to that in pure Mg compared with that in binary Mg-Y and Mg-Zn alloys.These results should relate to the origins of the Y/Zn segregation zone and close-packed stacking mode in LPSO structure,which provides a new insight into the formation mechanism of LPSO structure at atomic level.

Gene Cloning and Bioinformatics Analysis of phoR Gene from Vibrio alginolyticus HY9901

PhoR is a histidine kinase in a two-component regulatory system that regulates phosphorus metabolic pathways and undertakes the key mission of information transmission in pathogenic bacteria.The full-length phoR gene was successfully cloned from the Vibrio alginolyticus HY9901 strain.A comprehensive analysis of the cloned gene was conducted using bioinformatics.Sequence analysis revealed that the total length of the phoR gene(GenBank accession No.:KJ958404.1)is 1299 bp,with the coding region containing a total of 432 amino acid residues.The phylogenetic tree of PhoR revealed that it belongs to the same subclade as V.diabolicus.The SMART program was employed for the purpose of functional domain prediction,which revealed that PhoR possesses three major functional domains:PAS(amino acids 98-166),HisKA(amino acids 205-272),and HATPase_c(amino acids 317-429).

Gates joint locally connected network for accurate and robust reconstruction in optical molecular tomography

Optical molecular tomography(OMT)is a potential pre-clinical molecular imaging technique with applications in a variety of biomedical areas,which can provide non-invasive quantitative three-dimensional(3D)information regarding tumor distribution in living animals.The construction of optical transmission models and the application of reconstruction algorithms in traditional model-based reconstruction processes have affected the reconstruction results,resulting in problems such as low accuracy,poor robustness,and long-time consumption.Here,a gates joint locally connected network(GLCN)method is proposed by establishing the mapping relationship between the inside source distribution and the photon density on surface directly,thus avoiding the extra time consumption caused by iteration and the reconstruction errors caused by model inaccuracy.Moreover,gates module was composed of the concatenation and multiplication operators of three different gates.It was embedded into the network aiming at remembering input surface photon density over a period and allowing the network to capture neurons connected to the true source selectively by controlling three different gates.To evaluate the performance of the proposed method,numerical simulations were conducted,whose results demonstrated good performance in terms of reconstruction positioning accuracy and robustness.

Microwave irradiation-induced alterations in physicochemical properties and methane adsorption capability of coals:An experimental study using carbon molecular sieve

In order to comprehend the applicability of microwave irradiation for recovering coalbed methane,it is necessary to evaluate the microwave irradiation-induced alterations in coals with varying levels of metamorphism.In this work,the carbon molecular sieve combined with KMnO_(4)oxidation was selected to fabricate carbon molecular sieve with diverse oxidation degrees,which can serve as model substances toward coals.Afterwards,the microwave irradiation dependences of pores,functional groups,and highpressure methane adsorption characteristics of model substances were studied.The results indicated that microwave irradiation causes rearrangement of oxygen-containing functional groups,which could block the micropores with a size of 0.40-0.60 nm in carbon molecular sieve;meanwhile,naphthalene and phenanthrene generated by macro-molecular structure pyrolysis due to microwave irradiation could block the micropores with a size of 0.70-0.90 nm.These alterations in micropore structure weaken the saturated methane adsorption capacity of oxidized carbon molecular sieve by 2.91%-23.28%,suggesting that microwave irradiation could promote methane desorption.Moreover,the increased mesopores found for oxidized carbon molecular sieve after microwave irradiation could benefit CH4 diffusion.In summary,the oxidized carbon molecular sieve can act as model substances toward coals with different ranks.Additionally,microwave irradiation is a promising technology to enhance coalbed methane recovery.

Integrating Levels of Hierarchical Organization in Porous Organic Molecular Materials

Porous organic molecular materials(POMMs)are an emergent class of molecular-based materials characterized by the formation of extended porous frameworks,mainly held by non-covalent interactions.POMMs represent a variety of chemical families,such as hydrogen-bonded organic frameworks,porous organic salts,porous organic cages,C-H···πmicroporous crystals,supramolecular organic frameworks,π-organic frameworks,halogen-bonded organic framework,and intrinsically porous molecular materials.In some porous materials such as zeolites and metal organic frameworks,the integration of multiscale has been adopted to build materials with multifunctionality and optimized properties.Therefore,considering the significant role of hierarchy in porous materials and the growing importance of POMMs in the realm of synthetic porous materials,we consider it appropriate to dedicate for the first time a critical review covering both topics.Herein,we will provide a summary of literature examples showcasing hierarchical POMMs,with a focus on their main synthetic approaches,applications,and the advantages brought forth by introducing hierarchy.

Ab initio nonadiabatic molecular dynamics study on spin–orbit coupling induced spin dynamics in ferromagnetic metals

Understanding the photoexcitation induced spin dynamics in ferromagnetic metals is important for the design of photo-controlled ultrafast spintronic device.In this work,by the ab initio nonadiabatic molecular dynamics simulation,we have studied the spin dynamics induced by spin–orbit coupling(SOC)in Co and Fe using both spin-diabatic and spin-adiabatic representations.In Co system,it is found that the Fermi surface(E_(F))is predominantly contributed by the spin-minority states.The SOC induced spin flip will occur for the photo-excited spin-majority electrons as they relax to the E_(F),and the spin-minority electrons tend to relax to the EFwith the same spin through the electron–phonon coupling(EPC).The reduction of spin-majority electrons and the increase of spin-minority electrons lead to demagnetization of Co within100 fs.By contrast,in Fe system,the E_(F) is dominated by the spin-majority states.In this case,the SOC induced spin flip occurs for the photo-excited spin-minority electrons,which leads to a magnetization enhancement.If we move the E_(F) of Fe to higher energy by 0.6eV,the E_(F) will be contributed by the spin-minority states and the demagnetization will be observed again.This work provides a new perspective for understanding the SOC induced spin dynamics mechanism in magnetic metal systems.

Effects of low molecular weight polysaccharide from Sargassum thunbergii against palmitic acid-induced intracellular lipid accumulation in 3T3-L1 adipocyte and HepG2 cells

Low molecular weight polysaccharides can be isolated from Sargassum thunbergii(LMPST)and in vitro experiments were conducted to evaluate the inhibitory effects on lipids.Two natures of LMPST were attained from S.thunbergii and appraised their LMPST on palmitic acid(PA)induced lipid accretion in Hep G2,and 3T3-L1 cells.LMPST treatment lessened lipid deposition and intracellular free fatty acid and triglyceride intensities in PA-treated above mentioned cells.The mechanistic study publicized that LMPST2 significantly suppressed adipogenesis and stimulated the PA-treated 3T3-L1 cells occupied in the lipolysis pathway.Furthermore,in PA-treated Hep G2 cells,the free fatty acid oxidation was significantly increased by LMPST2.Given these constructive properties of LMPST2 from S.thunbergii,is a potential candidate for diminishing the intracellular lipids,and for a therapeutic agent in those conditions.

Biomimetic MXene membranes with negatively thermo-responsive switchable 2D nanochannels for graded molecular sieving

Negatively thermo-responsive 2D membranes,which mimic the stomatal opening/closing of plants,have drawn substantial interest for tunable molecular separation processes.However,these membranes are still restricted significantly on account of low water permeability and poor dynamic tunability of 2D nanochannels under temperature stimulation.Here,we present a biomimetic negatively thermo-responsive MXene membrane by covalently grafting poly(N-isopropylacrylamide)(PNIPAm)onto MXene nanosheets.The uniformly grafted PNIPAm polymer chains can enlarge the interlayer spacings for increasing water permeability while also allowing more tunability of 2D nanochannels for enhancing the capability of gradually separating multiple molecules of different sizes.As expected,the constructed membrane exhibits ultrahigh water permeance of 95.6 L m^(-2) h^(-1) bar^(-1) at 25℃,which is eight-fold higher than the state-of-the-art negatively thermoresponsive 2D membranes.Moreover,the highly temperature-tunable 2D nanochannels enable the constructed membrane to perform excellent graded molecular sieving for dye-and antibiotic-based ternary mixtures.This strategy provides new perspectives in engineering smart 2D membrane and expands the scope of temperature-responsive membranes,showing promising applications in micro/nanofluidics and molecular separation.