In vitro maturation(IvM)of human oocytes offers cost efficiency and minimal invasiveness,serving as a valuable supplementary tool in assisted reproduction for fertility preservation,ovarian hyperstimulation syndrome p...In vitro maturation(IvM)of human oocytes offers cost efficiency and minimal invasiveness,serving as a valuable supplementary tool in assisted reproduction for fertility preservation,ovarian hyperstimulation syndrome prevention,and other reproductive strategies.Despite its availability for three decades,the clinical use of IVM remains limited due to efficacy and safety concerns.This study examines the DNA methylation profile of IVM oocytes collected during laparoscopic/hysteroscopic surgeries compared to in vivo matured oocytes via reduced representation bisulfite sequencing.Results indicate IVM oocytes exhibit a higher global methylation level.Differentially methylated regions(DMRs)analysis reveals that the in vitro group displays more hypermethylated and fewer hypomethylated DMRs compared to the in vivo group.Additionally,the in vitro group exhibits a higher level of non-CpG methylation than the in vivo group.However,no significant correlation between methylation levels and transcriptional activity in these oocytes is found,especially for those specific imprinted genes or genes related to embryonic development.These findings shed light on the epigenetic landscape of IvM oocytes,contributing to the ongoing assessment of their clinical feasibility and safety in assisted reproduction.展开更多
Fragment velocity distribution is an important parameter affecting the terminal effects of warheads.The rarefaction wave,end cap,and its confinement state can significantly affect the fragmentation of the cylindrical ...Fragment velocity distribution is an important parameter affecting the terminal effects of warheads.The rarefaction wave,end cap,and its confinement state can significantly affect the fragmentation of the cylindrical charge casing.Most of the existing studies have performed experiments and simulations considering the rarefaction wave and unfixed end caps;research on fixed end caps and sufficient theoretical explanations are limited.In this work,the effects of rarefaction waves,end caps,and their fixed states,on the fragment velocity distribution,were studied via experimentation and simulation,and reasonable theoretical explanations were provided.The results show that the rarefaction wave and end caps affect the fragment velocity by changing the pressure states of the detonation products.At the initiation end,the fragment velocities of casings with unfixed initiation ends are 33.3%(300 m/s)greater than that of casings without end caps,because of the weakening of the attenuation effect of the rarefaction wave.The fragment velocities of the casings with fixed initiation ends are 8.3%(100 m/s)greater than that of casings with unfixed initiation ends.At the non-initiation end,the fragment velocities are 24.8%(297 m/s)greater than that of a casing without end caps,and the reflecting shock wave generated by the fixed non-initiation end increases the fragment velocity by 7.3%(113 m/s),compared to the theoretical velocity.This work provides a basis for the structural design and analysis of the terminal effects of warheads.展开更多
A disintegrin and metalloprotease 17(ADAM17)is a membrane-bound enzyme that cleaves cell-surface proteins.Here,we discovered that neuronal ADAM17-mediated signaling supports the reduction of inhibitory presynaptic inp...A disintegrin and metalloprotease 17(ADAM17)is a membrane-bound enzyme that cleaves cell-surface proteins.Here,we discovered that neuronal ADAM17-mediated signaling supports the reduction of inhibitory presynaptic inputs to the pre-sympathetic glutamatergic neural hub,located in the paraventricular nucleus of the hypothalamus(PVN),upon stimulation by angiotensin II(Ang-II).For Ang-II-induced disinhibition,targeting microglial migration had an effect similar to ADAM17 knockout in glutamatergic neurons.Ang-II promoted neuron-mediated chemotaxis of microglia via neuronal CX3CL1 and ADAM17.Inhibiting microglial chemotaxis by targeting CX3CR1 abolished the Ang-II-induced microglial displacement of GABAergic presynaptic terminals and significantly blunted Ang-II’s pressor response.Using conditional and targeted knockout models of ADAM17,an increase in the contact between pre-sympathetic neurons and reactive microglia in the PVN was demonstrated to be neuronal ADAM17-dependent during the developmental stage of salt-sensitive hypertension.Collectively,this study provides evidence that neuronal ADAM17-mediated microglial chemotaxis facilitates the disinhibition of pre-sympathetic glutamatergic tone upon hormonal stimulation.展开更多
The strong vertical discontinuities pose a fundamental challenge to optimizing stimulated reservoir volume(SRV)in multilayered reservoirs.This research proposes a radial borehole-assisted horizontal well fracturing te...The strong vertical discontinuities pose a fundamental challenge to optimizing stimulated reservoir volume(SRV)in multilayered reservoirs.This research proposes a radial borehole-assisted horizontal well fracturing technology,which is expected to achieve effective vertical stimulation and commingled production across multiple pay zones.Under different geological and engineering conditions,the vertical propagation behavior of hydraulic fractures guided by radial boreholes can be determined by adjusting the interlayered lithologies and radial borehole configurations in experimental samples.Experimental results reveal four fracture network patterns:passivated,cross-layer,skip-layer,and hybrid fractures in the radial borehole fracturing.The radial boreholes perform better fracture guiding performances in the high-brittleness interlayers,which form cross-layer and hybrid fracture networks to improve the growth height.Hydraulic fractures tend to propagate from high-strength to low-strength layers under radial borehole guidance.When radial boreholes interconnect multiple lithology layers,hydraulic fractures initiate preferentially in lower-strength zones rather than remaining confined to borehole root ends.Increased radial borehole length and diameter facilitate fracture skip-layer initiation and cross-layer propagation,while multiple borehole branches enhance fracture penetration across high-strength interlayers.Radial boreholes with inclination angles below 30°enhance fracture height by generating cross-layer and hybrid fracture networks.Furthermore,an inter-borehole phase angle of less than 180°facilitates single-wing fracture cross-layer propagation.Fracture height is primarily governed by radial borehole length,followed by quantity,inclination angle,and diameter.Based on the geometric similarity criteria,the recommended parameters for radial borehole-assisted fracturing in a 5 1/2-inch horizontal well include a length>15 m,an inclination angle<30°,and a diameter>52 mm to ensure effective stimulation across three or more pay zones.Finally,the field-scale numerical model was developed to simulate the optimized radial borehole fracturing and demonstrate the technical superiority.These findings are expected to provide an in-depth understanding of the effective stimulation in multilayered reservoirs.展开更多
As the frontier of multidimensional transportation systems,urban air mobility(UAM)is receiving increasing attention from international organizations,governments,and stakeholders in industry and academia owing to its h...As the frontier of multidimensional transportation systems,urban air mobility(UAM)is receiving increasing attention from international organizations,governments,and stakeholders in industry and academia owing to its high efficiency,low carbon footprint,and operational flexibility.Vertical take-off and landing(VTOL)infrastructure is the core facility that enables UAM and is therefore essential for its safe,efficient,and large-scale commercial implementation.However,the key technologies for establishing low-altitude VTOL infrastructure are still nascent,and government,industry,and academia have yet to harmonize the corresponding construction,management,and operation standards.To address this gap,we herein systematically review the related progress and trends,comprehensively surveying the key technologies of establishing VTOL infrastructure serving unmanned aerial vehicles(UAVs)and electric VTOL aircraft from three complementary perspectives of ground-side,airspace-side,and communication,navigation,surveillance,and information services.In the light of future UAM operations characterized by diverse vehicle types and dense air traffic,we propose a conceptual design for a public multioperator VTOL site to provide constructive insights into the sustainable growth of the low-altitude economy.展开更多
BACKGROUND:Serum osmolality is a prognostic indicator in critically ill patients.This study aimed to evaluate the association between high osmolality and 28-day mortality in patients with cardiac arrest(CA)admitted to...BACKGROUND:Serum osmolality is a prognostic indicator in critically ill patients.This study aimed to evaluate the association between high osmolality and 28-day mortality in patients with cardiac arrest(CA)admitted to the intensive care unit(ICU).METHODS:Baseline data of adult patients with CA who were admitted to the ICU from 2008 to 2019 were collected from the Medical Information Mart for Intensive Care(MIMIC)-IV.Patients were divided into survivor and non-survivor groups according to the 28-day prognosis.Serum concentrations of sodium,potassium,glucose,and urea nitrogen on the fi rst day of ICU admission were used to determine serum osmolarity.The primary endpoint of this study was 28-day all-cause mortality.Propensity score matching(PSM)analysis was performed to reduce bias between the survivor and nonsurvivor groups.RESULTS:Among the 798 included CA patients,the high osmolarity on the first day of ICU admission remained significantly associated with increased 28-day mortality(62.0%vs.35.5%,P<0.001)and reduced cumulative survival(log-rank P<0.05)after PSM.Cox regression identifi ed the high osmolarity on the fi rst day of ICU admission as an independent predictor.High serum osmolarity on the fi rst day of ICU admission eff ectively predicted 1-,3-,7-,and 28-day all-cause mortality,with the strongest predictive performance for 1-day mortality both before and after PSM(all P<0.05).CONCLUSION:In this study,elevated serum osmolarity on the first day of ICU admission was independently associated with increased 28-day mortality in CA patients and could serve as a prognostic marker.展开更多
With the global push for energy conservation and the rapid development of low-power,flexible and wearable optical displays,the demand for electrochromic technology has surged.Gel polymer electrolytes(GPEs),a crucial c...With the global push for energy conservation and the rapid development of low-power,flexible and wearable optical displays,the demand for electrochromic technology has surged.Gel polymer electrolytes(GPEs),a crucial component of electrochromic devices(ECDs),show great promise in applications.This is attributed to their efficient ion-transport capabilities,excellent mechanical properties and strong adhesion.All of these characteristics are conducive to enhancing the safety of the devices,streamlining the packaging process,significantly improving the electrochromic performance of ECDs and boosting their commercial application potential.This review provides a comprehensive overview of GPEs for ECDs,focusing on their basic designs,functional modifications and practical applications.Firstly,this review outlines the fundamental design of GPEs for ECDs,encompassing key performance index,classification,gelation mechanism and preparation methods.Building on this foundation,it provides an in-depth discussion of functionalized GPEs developed to enhance device performance or expand functionality,including electrochromic,temperature-responsive,photo-responsive and stretchable self-healing GPE.Furthermore,the integration of GPEs into various ECD applications,including smart windows,displays,energy storage devices and wearable electronic,are summarized to highlight the advantages that the design of GPEs brings to the practical application of ECDs.Finally,based on the summary of GPEs employed for ECDs,the challenges and development expectations in this direction were indicated.展开更多
Agricultural science and technology parks(ASTPs) represent an important growth pole in China’s agricultural modernization.Clarifying their diffusion laws can optimize the technological diffusion process and improve i...Agricultural science and technology parks(ASTPs) represent an important growth pole in China’s agricultural modernization.Clarifying their diffusion laws can optimize the technological diffusion process and improve its efficiency. Our study uses disaggregated spatial information in its model to analyze ASTP technology diffusion in a heterogeneous space. We constructed a comprehensive index system to evaluate the diffusion environmental quality and introduced the heterogeneous diffusion equation to calculate the technological diffusion probability. We applied this framework to a real-world scenario: the apple planting technology diffusion of the Yangling ASTP in the Loess Plateau, China. The results indicated: (1)the technological diffusion environment of the Loess Plateau advantageous apple producing area showed strong spatial heterogeneity caused by climate, topography, and external transportation links.(2) Under the combined effects of distance and spatial heterogeneity, the spatial diffusion pattern of the Yangling ASTP apple technology was expansion diffusion supplemented by hierarchical diffusion and banded diffusion, and (3) ASTP technology diffusion showed a strong distance attenuation effect, and the frictional effect of distance can be decreased by improving the diffusion environmental quality. These laws can promote regional balanced ASTP-driven development.展开更多
Softening is one of the key fruit quality traits,which results from the selective expression of cell wall metabolism genes during ripening.The identification of transcription factors(TFs)that regulate fruit softening ...Softening is one of the key fruit quality traits,which results from the selective expression of cell wall metabolism genes during ripening.The identification of transcription factors(TFs)that regulate fruit softening is an important field in order to understand and control fruit softening.In tomato,NAC(NAM,ATAF,and CUC)TFs members have been demonstrated to be involved in fruit ripening regulation,including NAC-NOR(nonripening),NOR-like1,SlNAC4,SlNAC1.Here,we generated slnac4 mutant knockout(CR-SlNAC4)tomato plant by a clustered regularly interspaced short palindromic repeats genomic targeting system(CRISPR/Cas9)and SlNAC4 overexpressing(OE-SlNAC4)plant.In addition to confirming the previously reported results that SlNAC4 positively regulates fruit ripening,we found that SlNAC4 has a strong effect on tomato fruit softening.Compared with the control fruit,fruit softening was inhibited in slnac4 fruit and conversely was accelerated in OE-SlNAC4 tomato fruit.Through RNA-sequencing(RNA-seq)analysis,we found that expression levels of SlEXP1(expansin)and SlCEL2(endo-β-1,4 glucanase)genes involved in cell wall metabolism were significantly different in WT(wild type)/slnac4 and WT/OE-SlNAC4 fruit.Further study showed that these genes contained a NAC TF binding domain in their promoter regions.In vitro electrophoretic mobility shift assays(EMSA)and dual-luciferase reporter assays(DLR)demonstrated that these two genes were the direct targets of SlNAC4 binding and transactivation.The results enriched the function of SlNAC4 and provided a new dimension in understanding the regulation of tomato fruit softening.展开更多
基金supported by funding from the National Natural Science Foundation of China(81971349 and 81300456).
文摘In vitro maturation(IvM)of human oocytes offers cost efficiency and minimal invasiveness,serving as a valuable supplementary tool in assisted reproduction for fertility preservation,ovarian hyperstimulation syndrome prevention,and other reproductive strategies.Despite its availability for three decades,the clinical use of IVM remains limited due to efficacy and safety concerns.This study examines the DNA methylation profile of IVM oocytes collected during laparoscopic/hysteroscopic surgeries compared to in vivo matured oocytes via reduced representation bisulfite sequencing.Results indicate IVM oocytes exhibit a higher global methylation level.Differentially methylated regions(DMRs)analysis reveals that the in vitro group displays more hypermethylated and fewer hypomethylated DMRs compared to the in vivo group.Additionally,the in vitro group exhibits a higher level of non-CpG methylation than the in vivo group.However,no significant correlation between methylation levels and transcriptional activity in these oocytes is found,especially for those specific imprinted genes or genes related to embryonic development.These findings shed light on the epigenetic landscape of IvM oocytes,contributing to the ongoing assessment of their clinical feasibility and safety in assisted reproduction.
基金the support of the Youth Scientific Research Projects of the Basic Research Program of Shanxi Province(Grant Nos.202303021222111,202303021222113)the China Postdoctoral Science Foundation(Grant No.2025M770001).
文摘Fragment velocity distribution is an important parameter affecting the terminal effects of warheads.The rarefaction wave,end cap,and its confinement state can significantly affect the fragmentation of the cylindrical charge casing.Most of the existing studies have performed experiments and simulations considering the rarefaction wave and unfixed end caps;research on fixed end caps and sufficient theoretical explanations are limited.In this work,the effects of rarefaction waves,end caps,and their fixed states,on the fragment velocity distribution,were studied via experimentation and simulation,and reasonable theoretical explanations were provided.The results show that the rarefaction wave and end caps affect the fragment velocity by changing the pressure states of the detonation products.At the initiation end,the fragment velocities of casings with unfixed initiation ends are 33.3%(300 m/s)greater than that of casings without end caps,because of the weakening of the attenuation effect of the rarefaction wave.The fragment velocities of the casings with fixed initiation ends are 8.3%(100 m/s)greater than that of casings with unfixed initiation ends.At the non-initiation end,the fragment velocities are 24.8%(297 m/s)greater than that of a casing without end caps,and the reflecting shock wave generated by the fixed non-initiation end increases the fragment velocity by 7.3%(113 m/s),compared to the theoretical velocity.This work provides a basis for the structural design and analysis of the terminal effects of warheads.
基金supported by the National Natural Science Foundation of China(82100454,32271016,82101586,and 81872563)the National Heart,Lung,Blood,and Sleep Institute(HL163588).
文摘A disintegrin and metalloprotease 17(ADAM17)is a membrane-bound enzyme that cleaves cell-surface proteins.Here,we discovered that neuronal ADAM17-mediated signaling supports the reduction of inhibitory presynaptic inputs to the pre-sympathetic glutamatergic neural hub,located in the paraventricular nucleus of the hypothalamus(PVN),upon stimulation by angiotensin II(Ang-II).For Ang-II-induced disinhibition,targeting microglial migration had an effect similar to ADAM17 knockout in glutamatergic neurons.Ang-II promoted neuron-mediated chemotaxis of microglia via neuronal CX3CL1 and ADAM17.Inhibiting microglial chemotaxis by targeting CX3CR1 abolished the Ang-II-induced microglial displacement of GABAergic presynaptic terminals and significantly blunted Ang-II’s pressor response.Using conditional and targeted knockout models of ADAM17,an increase in the contact between pre-sympathetic neurons and reactive microglia in the PVN was demonstrated to be neuronal ADAM17-dependent during the developmental stage of salt-sensitive hypertension.Collectively,this study provides evidence that neuronal ADAM17-mediated microglial chemotaxis facilitates the disinhibition of pre-sympathetic glutamatergic tone upon hormonal stimulation.
基金supported by the National Natural Science Foundation of China(Nos.U24B6001,52421002,52474016,and 52020105001)Research on Key Technologies for Exploration and Development of Dry Geothermal Resources(No.2022DJ5503)Deep-land National Science and Technology Major Project of China(No.2024ZD1003504).
文摘The strong vertical discontinuities pose a fundamental challenge to optimizing stimulated reservoir volume(SRV)in multilayered reservoirs.This research proposes a radial borehole-assisted horizontal well fracturing technology,which is expected to achieve effective vertical stimulation and commingled production across multiple pay zones.Under different geological and engineering conditions,the vertical propagation behavior of hydraulic fractures guided by radial boreholes can be determined by adjusting the interlayered lithologies and radial borehole configurations in experimental samples.Experimental results reveal four fracture network patterns:passivated,cross-layer,skip-layer,and hybrid fractures in the radial borehole fracturing.The radial boreholes perform better fracture guiding performances in the high-brittleness interlayers,which form cross-layer and hybrid fracture networks to improve the growth height.Hydraulic fractures tend to propagate from high-strength to low-strength layers under radial borehole guidance.When radial boreholes interconnect multiple lithology layers,hydraulic fractures initiate preferentially in lower-strength zones rather than remaining confined to borehole root ends.Increased radial borehole length and diameter facilitate fracture skip-layer initiation and cross-layer propagation,while multiple borehole branches enhance fracture penetration across high-strength interlayers.Radial boreholes with inclination angles below 30°enhance fracture height by generating cross-layer and hybrid fracture networks.Furthermore,an inter-borehole phase angle of less than 180°facilitates single-wing fracture cross-layer propagation.Fracture height is primarily governed by radial borehole length,followed by quantity,inclination angle,and diameter.Based on the geometric similarity criteria,the recommended parameters for radial borehole-assisted fracturing in a 5 1/2-inch horizontal well include a length>15 m,an inclination angle<30°,and a diameter>52 mm to ensure effective stimulation across three or more pay zones.Finally,the field-scale numerical model was developed to simulate the optimized radial borehole fracturing and demonstrate the technical superiority.These findings are expected to provide an in-depth understanding of the effective stimulation in multilayered reservoirs.
基金supported by the National Natural Science Foundation of China(No.U2333214).
文摘As the frontier of multidimensional transportation systems,urban air mobility(UAM)is receiving increasing attention from international organizations,governments,and stakeholders in industry and academia owing to its high efficiency,low carbon footprint,and operational flexibility.Vertical take-off and landing(VTOL)infrastructure is the core facility that enables UAM and is therefore essential for its safe,efficient,and large-scale commercial implementation.However,the key technologies for establishing low-altitude VTOL infrastructure are still nascent,and government,industry,and academia have yet to harmonize the corresponding construction,management,and operation standards.To address this gap,we herein systematically review the related progress and trends,comprehensively surveying the key technologies of establishing VTOL infrastructure serving unmanned aerial vehicles(UAVs)and electric VTOL aircraft from three complementary perspectives of ground-side,airspace-side,and communication,navigation,surveillance,and information services.In the light of future UAM operations characterized by diverse vehicle types and dense air traffic,we propose a conceptual design for a public multioperator VTOL site to provide constructive insights into the sustainable growth of the low-altitude economy.
基金funded by the Shenzhen Science and Technology Program(JCYJ20230807112007014 to PG)the Shenzhen Key Medical Discipline Construction Fund(SZXK046 to PG).
文摘BACKGROUND:Serum osmolality is a prognostic indicator in critically ill patients.This study aimed to evaluate the association between high osmolality and 28-day mortality in patients with cardiac arrest(CA)admitted to the intensive care unit(ICU).METHODS:Baseline data of adult patients with CA who were admitted to the ICU from 2008 to 2019 were collected from the Medical Information Mart for Intensive Care(MIMIC)-IV.Patients were divided into survivor and non-survivor groups according to the 28-day prognosis.Serum concentrations of sodium,potassium,glucose,and urea nitrogen on the fi rst day of ICU admission were used to determine serum osmolarity.The primary endpoint of this study was 28-day all-cause mortality.Propensity score matching(PSM)analysis was performed to reduce bias between the survivor and nonsurvivor groups.RESULTS:Among the 798 included CA patients,the high osmolarity on the first day of ICU admission remained significantly associated with increased 28-day mortality(62.0%vs.35.5%,P<0.001)and reduced cumulative survival(log-rank P<0.05)after PSM.Cox regression identifi ed the high osmolarity on the fi rst day of ICU admission as an independent predictor.High serum osmolarity on the fi rst day of ICU admission eff ectively predicted 1-,3-,7-,and 28-day all-cause mortality,with the strongest predictive performance for 1-day mortality both before and after PSM(all P<0.05).CONCLUSION:In this study,elevated serum osmolarity on the first day of ICU admission was independently associated with increased 28-day mortality in CA patients and could serve as a prognostic marker.
基金supported by the National Natural Science Foundation of China(52103299)。
文摘With the global push for energy conservation and the rapid development of low-power,flexible and wearable optical displays,the demand for electrochromic technology has surged.Gel polymer electrolytes(GPEs),a crucial component of electrochromic devices(ECDs),show great promise in applications.This is attributed to their efficient ion-transport capabilities,excellent mechanical properties and strong adhesion.All of these characteristics are conducive to enhancing the safety of the devices,streamlining the packaging process,significantly improving the electrochromic performance of ECDs and boosting their commercial application potential.This review provides a comprehensive overview of GPEs for ECDs,focusing on their basic designs,functional modifications and practical applications.Firstly,this review outlines the fundamental design of GPEs for ECDs,encompassing key performance index,classification,gelation mechanism and preparation methods.Building on this foundation,it provides an in-depth discussion of functionalized GPEs developed to enhance device performance or expand functionality,including electrochromic,temperature-responsive,photo-responsive and stretchable self-healing GPE.Furthermore,the integration of GPEs into various ECD applications,including smart windows,displays,energy storage devices and wearable electronic,are summarized to highlight the advantages that the design of GPEs brings to the practical application of ECDs.Finally,based on the summary of GPEs employed for ECDs,the challenges and development expectations in this direction were indicated.
基金Under the auspices of the National Natural Science Foundation of China(No.41771129)Social Science Foundation of Shaanxi(No.2015D055)Social Science Research Project on Major Theoretical and Practical Issues of Shaanxi(No.2020Z026)。
文摘Agricultural science and technology parks(ASTPs) represent an important growth pole in China’s agricultural modernization.Clarifying their diffusion laws can optimize the technological diffusion process and improve its efficiency. Our study uses disaggregated spatial information in its model to analyze ASTP technology diffusion in a heterogeneous space. We constructed a comprehensive index system to evaluate the diffusion environmental quality and introduced the heterogeneous diffusion equation to calculate the technological diffusion probability. We applied this framework to a real-world scenario: the apple planting technology diffusion of the Yangling ASTP in the Loess Plateau, China. The results indicated: (1)the technological diffusion environment of the Loess Plateau advantageous apple producing area showed strong spatial heterogeneity caused by climate, topography, and external transportation links.(2) Under the combined effects of distance and spatial heterogeneity, the spatial diffusion pattern of the Yangling ASTP apple technology was expansion diffusion supplemented by hierarchical diffusion and banded diffusion, and (3) ASTP technology diffusion showed a strong distance attenuation effect, and the frictional effect of distance can be decreased by improving the diffusion environmental quality. These laws can promote regional balanced ASTP-driven development.
文摘Softening is one of the key fruit quality traits,which results from the selective expression of cell wall metabolism genes during ripening.The identification of transcription factors(TFs)that regulate fruit softening is an important field in order to understand and control fruit softening.In tomato,NAC(NAM,ATAF,and CUC)TFs members have been demonstrated to be involved in fruit ripening regulation,including NAC-NOR(nonripening),NOR-like1,SlNAC4,SlNAC1.Here,we generated slnac4 mutant knockout(CR-SlNAC4)tomato plant by a clustered regularly interspaced short palindromic repeats genomic targeting system(CRISPR/Cas9)and SlNAC4 overexpressing(OE-SlNAC4)plant.In addition to confirming the previously reported results that SlNAC4 positively regulates fruit ripening,we found that SlNAC4 has a strong effect on tomato fruit softening.Compared with the control fruit,fruit softening was inhibited in slnac4 fruit and conversely was accelerated in OE-SlNAC4 tomato fruit.Through RNA-sequencing(RNA-seq)analysis,we found that expression levels of SlEXP1(expansin)and SlCEL2(endo-β-1,4 glucanase)genes involved in cell wall metabolism were significantly different in WT(wild type)/slnac4 and WT/OE-SlNAC4 fruit.Further study showed that these genes contained a NAC TF binding domain in their promoter regions.In vitro electrophoretic mobility shift assays(EMSA)and dual-luciferase reporter assays(DLR)demonstrated that these two genes were the direct targets of SlNAC4 binding and transactivation.The results enriched the function of SlNAC4 and provided a new dimension in understanding the regulation of tomato fruit softening.
