Lewis acidic ionic liquids were used to catalyze the reaction of epoxypropane with POCl3. Considering the lower cost and catalytic activities, we concluded that [Et3NH]Cl/AlCl3 was the most attractive ionic liquid fro...Lewis acidic ionic liquids were used to catalyze the reaction of epoxypropane with POCl3. Considering the lower cost and catalytic activities, we concluded that [Et3NH]Cl/AlCl3 was the most attractive ionic liquid from an economical point of view. But it would be easily inactivated because of sensitive to water and air. Moreover, it could not be reused easily because of difficulty recovery in the reaction. However, supporting [Et3NH]Cl/AlCl3 catalyst could resolve above problems. Supporting [Et3NH]Cl/ AlCl3 catalyst could be separated by filter easily and reused 5 times in 98% yield. Furthermore, the catalyst was applicable to other epoxy ether cleaving reactions.展开更多
BACKGROUND： Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein （APP） at the mRNA level. In addition, the piperlonguminine （A） and dihydropi...BACKGROUND： Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein （APP） at the mRNA level. In addition, the piperlonguminine （A） and dihydropiperlonguminine （B） components （1 ： 0.8）, which can be separated from Futokadsura stem, selectively inhibit expression of the APP at mRNA and protein levels. OBJECTIVE： Based on previous findings, the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme （BACE1） and APP genes on the production of β-amyloid peptide 42 （Aβ42） in human neuroblastoma cells （SK-N-SH cells） using small interfering RNAs （siRNAs） and A/B components separated from Futokadsura stem, respectively. DESIGN, TIME AND SETTING： A gene interference-based randomized, controlled, in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research, Ministries of Education and Public Health, and Institute of Pharmacologic Research, School of Pharmaceutical Science ＆ Department of Biochemistry, School of Medicine, Shandong University between July 2006 and December 2007. MATERIALS： SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; mouse anti-human BACE1 monoclonal antibody was purchased from R＆D Systems, USA; mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology, USA; and horseradish peroxidase （HRP）-conjugated goat anti-mouse IgG was provided by Sigma, USA. METHODS： The human BACE1 cDNA sequence was obtained from NCBI website （www.ncbi.nlm.nih.gov/sites/entrez）. Three pairs of siRNAs, specific to human BACE1 gene, were synthesized through the use of Silencer pre-designed siRNA specification, and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs （10-50 nmol/L） on SK-N-SH cells. Futokadsura stem was separated and purified with chemical methods, and the crystal was composed of A/B components, with an A to B ratio of 1：0.8. The A/B （1 ： 0.8） components were added to the SK-N-SH cells at different concentrations （13.13, 6.56, and 3.28 mg/mL）. MAIN OUTCOME MEASURES： Using RT-PCR and Western blot methods, BACE1 and APP expression at mRNA and protein levels was detected in SK-N-SH cells following treatment with different siRNAs and concentrations of Futokadsura stem-separated A/B components, respectively. Altered Aβ42 secretion by SK-N-SH cells was determined by ELISA. RESULTS： BACE1 mRNA and protein levels were significantly suppressed by 40 and 50 nmol/L siRNAs at 48 hours post-transfection. A/B components （1 ： 0.8）, which were separated from Futokadsura stem, selectively inhibited mRNA and protein expression of APP in SK-N-SH cells. Aβ42 secretion by SK-N-SH cells was significantly decreased following treatment with siRNAs or A/B components. CONCLUSION： Inhibition of BACE1 and APP genes by various materials and methods efficiently decreased production of Aβ42.展开更多
Graphite was chemically cleaved to graphene by Billups Reaction,and the morphologies and microstructures of graphene were characterized by SEM,Raman and AFM.The results show that the graphite was first functionalized ...Graphite was chemically cleaved to graphene by Billups Reaction,and the morphologies and microstructures of graphene were characterized by SEM,Raman and AFM.The results show that the graphite was first functionalized by 1-iodododecane,which led to the cleavage of the graphene layer in the graphite.The second decoration cleaved the graphite further and graphene was obtained.The heights of the graphene layer were larger than 1 nm due to the organic decoration.展开更多
Background Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy. In this study we investigated the von Willebrand factor-cleaving protease (vWF-cp) activity deficiency in patients with TTP.Me...Background Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy. In this study we investigated the von Willebrand factor-cleaving protease (vWF-cp) activity deficiency in patients with TTP.Methods The plasma or serum vWF-cp activity was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) by detecting the residual collagen binding activity (R-CBA) of von Willebrand factor (vWF) before and after digestion by vWF-cp. Multimers of vWF in plasma of patients with TTP were also analyzed by SDS-agarose electrophoresis. Moreover,the serum vWF-cp activities were compared between the patients with TTP and those with tumors.Results The coefficient of variation for intra-batch and inter-batch of the assay were 3.60% and 8.35%. The plasma and serum vWF-cp activity in healthy individuals were (78.79±9.17)% (n=30) and (79.47±10.78)% (n=53),respectively,while the plasma vWF-cp activity in 5 patients with TTP was markedly decreased [(21.83±19.98)%,P <0.001]. The unusually large vWF multimers were observed in two plasma samples of the patients with TTP. Although the vWF-cp activities in patients with benign and malignant tumors were also decreased ( P <0.03 and P <0.001,respectively),they were relatively high in comparison with that of TTP patients ( P <0.001).Conclusion Measurement of the vWF-cp activity using R-CBA is a simple and rapid method for diagnosing TTP. The vWF-cp activity in patients with TTP was markedly lower than those of patients with tumors.展开更多
The C-glycosidic bond that connects the sugar moiety with aglycone is difficult to be broken or made due to its inert nature.The knowledge of C-glycoside breakdown and synthesis is very limited.Recently,the enzyme Dgp...The C-glycosidic bond that connects the sugar moiety with aglycone is difficult to be broken or made due to its inert nature.The knowledge of C-glycoside breakdown and synthesis is very limited.Recently,the enzyme Dgp A/B/C cascade from a human intestinal bacterium PUE was identified to specifically cleave the C-glycosidic bond of puerarin(daidzein-8-C-glucoside).Here we investigated how puerarin is recognized and oxidized by Dgp A based on crystal structures of Dgp A with or without substrate and biochemical characterization.More strikingly,we found that apart from being a C-glycoside cleaving enzyme,Dgp A/B/C is capable of efficiently converting O-to C-glycoside showing the activity as a structure isomerase.A possible mechanistic model was proposed dependently of the simulated complex structure of Dgp B/C with 3’’-oxo-daidzin and structure-based mutagenesis.Our findings not only shed light on understanding the enzyme-mediated C-glycosidic bond breakage and formation,but also may help to facilitate stereospecific C-glycoside synthesis in pharmaceutical industry.展开更多
A 4′-chlorobenzenesulfonyl chloride-pyrrolecarboxamide hybrid compound was synthesized and its DNA cleaving activity was investigated using a pBluescript SK DNA under UV irradiation. This compound showed potent DNA c...A 4′-chlorobenzenesulfonyl chloride-pyrrolecarboxamide hybrid compound was synthesized and its DNA cleaving activity was investigated using a pBluescript SK DNA under UV irradiation. This compound showed potent DNA cleaving activity.展开更多
In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of A...In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of Alzheimer’s disease remains unclear.This study aimed to explore the effect of GGT5 on cognitive function and brain pathology in an APP/PS1 mouse model of Alzheimer’s disease,as well as the underlying mechanism.We observed a significant reduction in GGT5 expression in two in vitro models of Alzheimer’s disease(Aβ_(1-42)-treated hCMEC/D3 and bEnd.3 cells),as well as in the APP/PS1 mouse model.Additionally,injection of APP/PS1 mice with an adeno-associated virus encoding GGT5 enhanced hippocampal synaptic plasticity and mitigated cognitive deficits.Interestingly,increasing GGT5 expression in cerebrovascular endothelial cells reduced levels of both soluble and insoluble amyloid-βin the brains of APP/PS1 mice.This effect may be attributable to inhibition of the expression ofβ-site APP cleaving enzyme 1,which is mediated by nuclear factor-kappa B.Our findings demonstrate that GGT5 expression in cerebrovascular endothelial cells is inversely associated with Alzheimer’s disease pathogenesis,and that GGT5 upregulation mitigates cognitive deficits in APP/PS1 mice.These findings suggest that GGT5 expression in cerebrovascular endothelial cells is a potential therapeutic target and biomarker for Alzheimer’s disease.展开更多
Noise-induced hearing loss is the primary non-genetic factor contributing to auditory dysfunction.However,there are currently no effective pharmacological interventions for patients with noise-induced hearing loss.Her...Noise-induced hearing loss is the primary non-genetic factor contributing to auditory dysfunction.However,there are currently no effective pharmacological interventions for patients with noise-induced hearing loss.Here,we present evidence suggesting that the lysine-specific demethylase 1 inhibitor–tranylcypromine is an otoprotective agent that could be used to treat noise-induced hearing loss,and elucidate its underlying regulatory mechanisms.We established a mouse model of permanent threshold shift hearing loss by exposing the mice to white broadband noise at a sound pressure level of 120 d B for 4 hours.We found that tranylcypromine treatment led to the upregulation of Sestrin2(SESN2)and activation of the autophagy markers light chain 3B and lysosome-associated membrane glycoprotein 1 in the cochleae of mice treated with tranylcypromine.The noise exposure group treated with tranylcypromine showed significantly lower average auditory brainstem response hearing thresholds at click,4,8,and 16 k Hz frequencies compared with the noise exposure group treated with saline.These findings indicate that tranylcypromine treatment resulted in increased SESN2,light chain 3B,and lysosome-associated membrane glycoprotein 1 expression after noise exposure,leading to a reduction in levels of 4-hydroxynonenal and cleaved caspase-3,thereby reducing noise-induced hair cell loss.Additionally,immunoblot analysis demonstrated that treatment with tranylcypromine upregulated SESN2 expression via the autophagy pathway.Tranylcypromine treatment also reduced the production of NOD-like receptor family pyrin domaincontaining 3(NLRP3)production.In conclusion,our results showed that tranylcypromine treatment ameliorated cochlear inflammation by promoting the expression of SESN2,which induced autophagy,thereby restricting NLRP3-related inflammasome signaling,alleviating cochlear hair cell loss,and protecting hearing function.These findings suggest that inhibiting lysine-specific demethylase 1 is a potential therapeutic strategy for preventing hair cell loss and noise-induced hearing loss.展开更多
The postharvest senescence phase of table grapes comprises a series of biological processes.MicroRNAs(miRNAs)regulate downstream genes at the post-transcriptional level;however,whether miRNAs are involved in postharve...The postharvest senescence phase of table grapes comprises a series of biological processes.MicroRNAs(miRNAs)regulate downstream genes at the post-transcriptional level;however,whether miRNAs are involved in postharvest grape senescence remains unclear.We used small RNA sequencing to identify postharvest-related miRNAs in‘Red Globe'(Vitis vinifera)grapes harvested after 0,30,and 60 d of storage at 4℃(RG0,RG30,RG60).In total,42 known and 219 novel miRNA candidates were obtained.During fruit senescence,the expression of PC-3p-3343_1921,mi R2950,miR395k,miR2111,miR159c,miR169q,PC-5p-1112_4500,and miR167b changed signifcantly(P<0.05).Degradation sequencing identifed 218 targets associated with cell wall organization,tricarboxylic acid(TCA)cycling,pathogen defense,carbon metabolism,hormone signaling,the anthocyanin metabolism pathway,and energy regulation,of which ARF6,GRF3,TCP2,CP1,MYBA2,and WRKY72 were closely related to fruit senescence.We also verified that VIT_00s2146g00010,VIT_02s0012g01750,and VIT_03s0038g00160 with unknown functions are cleaved by senescence-related PC-5p-1112_4500 via the dual luciferase assay,and the transient transformation of grape berries showed that they regulate berry senescence.These results deepen our understanding of the role of mi RNAs in regulating grape berry senescence and prolonging the shelf life of horticultural products.Based on these results,we propose a new theoretical strategy for delaying the postharvest senescence of horticultural products by regulating the expression of key miRNAs(e.g.,PC-5p-1112_4500),thereby extending their shelf life.展开更多
基金A Project Funded by Jiangsu Natural Science Foundation of China(No.BK2011369)
文摘Lewis acidic ionic liquids were used to catalyze the reaction of epoxypropane with POCl3. Considering the lower cost and catalytic activities, we concluded that [Et3NH]Cl/AlCl3 was the most attractive ionic liquid from an economical point of view. But it would be easily inactivated because of sensitive to water and air. Moreover, it could not be reused easily because of difficulty recovery in the reaction. However, supporting [Et3NH]Cl/AlCl3 catalyst could resolve above problems. Supporting [Et3NH]Cl/ AlCl3 catalyst could be separated by filter easily and reused 5 times in 98% yield. Furthermore, the catalyst was applicable to other epoxy ether cleaving reactions.
基金the National Natural Science Foundation of China,No. NSFC-3027164
文摘BACKGROUND： Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein （APP） at the mRNA level. In addition, the piperlonguminine （A） and dihydropiperlonguminine （B） components （1 ： 0.8）, which can be separated from Futokadsura stem, selectively inhibit expression of the APP at mRNA and protein levels. OBJECTIVE： Based on previous findings, the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme （BACE1） and APP genes on the production of β-amyloid peptide 42 （Aβ42） in human neuroblastoma cells （SK-N-SH cells） using small interfering RNAs （siRNAs） and A/B components separated from Futokadsura stem, respectively. DESIGN, TIME AND SETTING： A gene interference-based randomized, controlled, in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research, Ministries of Education and Public Health, and Institute of Pharmacologic Research, School of Pharmaceutical Science ＆ Department of Biochemistry, School of Medicine, Shandong University between July 2006 and December 2007. MATERIALS： SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; mouse anti-human BACE1 monoclonal antibody was purchased from R＆D Systems, USA; mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology, USA; and horseradish peroxidase （HRP）-conjugated goat anti-mouse IgG was provided by Sigma, USA. METHODS： The human BACE1 cDNA sequence was obtained from NCBI website （www.ncbi.nlm.nih.gov/sites/entrez）. Three pairs of siRNAs, specific to human BACE1 gene, were synthesized through the use of Silencer pre-designed siRNA specification, and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs （10-50 nmol/L） on SK-N-SH cells. Futokadsura stem was separated and purified with chemical methods, and the crystal was composed of A/B components, with an A to B ratio of 1：0.8. The A/B （1 ： 0.8） components were added to the SK-N-SH cells at different concentrations （13.13, 6.56, and 3.28 mg/mL）. MAIN OUTCOME MEASURES： Using RT-PCR and Western blot methods, BACE1 and APP expression at mRNA and protein levels was detected in SK-N-SH cells following treatment with different siRNAs and concentrations of Futokadsura stem-separated A/B components, respectively. Altered Aβ42 secretion by SK-N-SH cells was determined by ELISA. RESULTS： BACE1 mRNA and protein levels were significantly suppressed by 40 and 50 nmol/L siRNAs at 48 hours post-transfection. A/B components （1 ： 0.8）, which were separated from Futokadsura stem, selectively inhibited mRNA and protein expression of APP in SK-N-SH cells. Aβ42 secretion by SK-N-SH cells was significantly decreased following treatment with siRNAs or A/B components. CONCLUSION： Inhibition of BACE1 and APP genes by various materials and methods efficiently decreased production of Aβ42.
基金Supported by the National Natural Youth Program Foundation of China(No.10804036,10904050)the Program for New Century Excellent Talents in University,China(No.NCET-09-0156)
文摘Graphite was chemically cleaved to graphene by Billups Reaction,and the morphologies and microstructures of graphene were characterized by SEM,Raman and AFM.The results show that the graphite was first functionalized by 1-iodododecane,which led to the cleavage of the graphene layer in the graphite.The second decoration cleaved the graphite further and graphene was obtained.The heights of the graphene layer were larger than 1 nm due to the organic decoration.
文摘Background Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy. In this study we investigated the von Willebrand factor-cleaving protease (vWF-cp) activity deficiency in patients with TTP.Methods The plasma or serum vWF-cp activity was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) by detecting the residual collagen binding activity (R-CBA) of von Willebrand factor (vWF) before and after digestion by vWF-cp. Multimers of vWF in plasma of patients with TTP were also analyzed by SDS-agarose electrophoresis. Moreover,the serum vWF-cp activities were compared between the patients with TTP and those with tumors.Results The coefficient of variation for intra-batch and inter-batch of the assay were 3.60% and 8.35%. The plasma and serum vWF-cp activity in healthy individuals were (78.79±9.17)% (n=30) and (79.47±10.78)% (n=53),respectively,while the plasma vWF-cp activity in 5 patients with TTP was markedly decreased [(21.83±19.98)%,P <0.001]. The unusually large vWF multimers were observed in two plasma samples of the patients with TTP. Although the vWF-cp activities in patients with benign and malignant tumors were also decreased ( P <0.03 and P <0.001,respectively),they were relatively high in comparison with that of TTP patients ( P <0.001).Conclusion Measurement of the vWF-cp activity using R-CBA is a simple and rapid method for diagnosing TTP. The vWF-cp activity in patients with TTP was markedly lower than those of patients with tumors.
基金supported by grants from National Natural Science Foundation of China(No.81073018 and 81274044)to Rufeng WangStartup fund program at Beijing University of Chinese Medicine(90011451310011)key research fund for drug discovery in Chinese medicine at Beijing University of Chinese Medicine(1000061223476)to Wenfu Ma。
文摘The C-glycosidic bond that connects the sugar moiety with aglycone is difficult to be broken or made due to its inert nature.The knowledge of C-glycoside breakdown and synthesis is very limited.Recently,the enzyme Dgp A/B/C cascade from a human intestinal bacterium PUE was identified to specifically cleave the C-glycosidic bond of puerarin(daidzein-8-C-glucoside).Here we investigated how puerarin is recognized and oxidized by Dgp A based on crystal structures of Dgp A with or without substrate and biochemical characterization.More strikingly,we found that apart from being a C-glycoside cleaving enzyme,Dgp A/B/C is capable of efficiently converting O-to C-glycoside showing the activity as a structure isomerase.A possible mechanistic model was proposed dependently of the simulated complex structure of Dgp B/C with 3’’-oxo-daidzin and structure-based mutagenesis.Our findings not only shed light on understanding the enzyme-mediated C-glycosidic bond breakage and formation,but also may help to facilitate stereospecific C-glycoside synthesis in pharmaceutical industry.
文摘A 4′-chlorobenzenesulfonyl chloride-pyrrolecarboxamide hybrid compound was synthesized and its DNA cleaving activity was investigated using a pBluescript SK DNA under UV irradiation. This compound showed potent DNA cleaving activity.
基金supported by STI2030-Major Projects,No.2021ZD 0201801(to JG)Shanxi Province Basic Research Program,No.20210302123429(to QS).
文摘In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of Alzheimer’s disease remains unclear.This study aimed to explore the effect of GGT5 on cognitive function and brain pathology in an APP/PS1 mouse model of Alzheimer’s disease,as well as the underlying mechanism.We observed a significant reduction in GGT5 expression in two in vitro models of Alzheimer’s disease(Aβ_(1-42)-treated hCMEC/D3 and bEnd.3 cells),as well as in the APP/PS1 mouse model.Additionally,injection of APP/PS1 mice with an adeno-associated virus encoding GGT5 enhanced hippocampal synaptic plasticity and mitigated cognitive deficits.Interestingly,increasing GGT5 expression in cerebrovascular endothelial cells reduced levels of both soluble and insoluble amyloid-βin the brains of APP/PS1 mice.This effect may be attributable to inhibition of the expression ofβ-site APP cleaving enzyme 1,which is mediated by nuclear factor-kappa B.Our findings demonstrate that GGT5 expression in cerebrovascular endothelial cells is inversely associated with Alzheimer’s disease pathogenesis,and that GGT5 upregulation mitigates cognitive deficits in APP/PS1 mice.These findings suggest that GGT5 expression in cerebrovascular endothelial cells is a potential therapeutic target and biomarker for Alzheimer’s disease.
基金supported by the National Key Research and Development Program of China,No.2022YFC2402701(to WC)Key International(Regional)Joint Research Program of the National Natural Science Foundation of China,No.81820108009(to SY)+5 种基金the National Natural Science Foundation of China,Nos.81970890(to WC)and 82371148(to WG)Fujian Provincial Healthcare Young and Middle-aged Backbone Talent Training Project,No.2023GGA035(to XC)Spring City Planthe High-level Talent Promotion and Training Project of Kunming,No.2022SCP001(to SY)the Natural Science Foundation of Hainan Province of China,No.824MS052(to XS)the Sixth Medical Center of Chinese PLA General Hospital Innovation Cultivation,No.CXPY202116(to LX)。
文摘Noise-induced hearing loss is the primary non-genetic factor contributing to auditory dysfunction.However,there are currently no effective pharmacological interventions for patients with noise-induced hearing loss.Here,we present evidence suggesting that the lysine-specific demethylase 1 inhibitor–tranylcypromine is an otoprotective agent that could be used to treat noise-induced hearing loss,and elucidate its underlying regulatory mechanisms.We established a mouse model of permanent threshold shift hearing loss by exposing the mice to white broadband noise at a sound pressure level of 120 d B for 4 hours.We found that tranylcypromine treatment led to the upregulation of Sestrin2(SESN2)and activation of the autophagy markers light chain 3B and lysosome-associated membrane glycoprotein 1 in the cochleae of mice treated with tranylcypromine.The noise exposure group treated with tranylcypromine showed significantly lower average auditory brainstem response hearing thresholds at click,4,8,and 16 k Hz frequencies compared with the noise exposure group treated with saline.These findings indicate that tranylcypromine treatment resulted in increased SESN2,light chain 3B,and lysosome-associated membrane glycoprotein 1 expression after noise exposure,leading to a reduction in levels of 4-hydroxynonenal and cleaved caspase-3,thereby reducing noise-induced hair cell loss.Additionally,immunoblot analysis demonstrated that treatment with tranylcypromine upregulated SESN2 expression via the autophagy pathway.Tranylcypromine treatment also reduced the production of NOD-like receptor family pyrin domaincontaining 3(NLRP3)production.In conclusion,our results showed that tranylcypromine treatment ameliorated cochlear inflammation by promoting the expression of SESN2,which induced autophagy,thereby restricting NLRP3-related inflammasome signaling,alleviating cochlear hair cell loss,and protecting hearing function.These findings suggest that inhibiting lysine-specific demethylase 1 is a potential therapeutic strategy for preventing hair cell loss and noise-induced hearing loss.
基金supported by the Natural Science Foundation of Ningxia,China(2024AAC02039)the Scientific and Technological Innovation Leadership Talent Program of Ningxia,China(2022GKLRLX07)+1 种基金the National Natural Science Foundation of China(32260727 and 32371924)China Agriculture Research System(CARS-29-zp-6)。
文摘The postharvest senescence phase of table grapes comprises a series of biological processes.MicroRNAs(miRNAs)regulate downstream genes at the post-transcriptional level;however,whether miRNAs are involved in postharvest grape senescence remains unclear.We used small RNA sequencing to identify postharvest-related miRNAs in‘Red Globe'(Vitis vinifera)grapes harvested after 0,30,and 60 d of storage at 4℃(RG0,RG30,RG60).In total,42 known and 219 novel miRNA candidates were obtained.During fruit senescence,the expression of PC-3p-3343_1921,mi R2950,miR395k,miR2111,miR159c,miR169q,PC-5p-1112_4500,and miR167b changed signifcantly(P<0.05).Degradation sequencing identifed 218 targets associated with cell wall organization,tricarboxylic acid(TCA)cycling,pathogen defense,carbon metabolism,hormone signaling,the anthocyanin metabolism pathway,and energy regulation,of which ARF6,GRF3,TCP2,CP1,MYBA2,and WRKY72 were closely related to fruit senescence.We also verified that VIT_00s2146g00010,VIT_02s0012g01750,and VIT_03s0038g00160 with unknown functions are cleaved by senescence-related PC-5p-1112_4500 via the dual luciferase assay,and the transient transformation of grape berries showed that they regulate berry senescence.These results deepen our understanding of the role of mi RNAs in regulating grape berry senescence and prolonging the shelf life of horticultural products.Based on these results,we propose a new theoretical strategy for delaying the postharvest senescence of horticultural products by regulating the expression of key miRNAs(e.g.,PC-5p-1112_4500),thereby extending their shelf life.
