UBL-UBA protein functions as a shuttle factor in the 26S ubiquitin degradation pathway,playing a critical role in plant growth and development,and responding to various biotic and abiotic stresses.Although RAD23,a typ...UBL-UBA protein functions as a shuttle factor in the 26S ubiquitin degradation pathway,playing a critical role in plant growth and development,and responding to various biotic and abiotic stresses.Although RAD23,a type of UBL-UBA protein,has been extensively studied in several plants,there is currently no comprehensive analysis available for kiwifruit(Actinidia chinensis).In this study,we identified six AcRAD23 genes in kiwifruit and further analyzed their phylogenetic relationships,gene structure,conserved motif composition and cis-acting element in the promoter.Subcellular localization experiments revealed that all AcRAD23 were localized in the nucleus and the cell membranes.Quantitative real-time PCR(qRT-PCR)analysis demonstrated differential expression patterns of these Ac RAD23 genes across different tissues and under various stress conditions(drought,waterlogging,salt stress,etc.),with AcRAD23D1 showing the highest responsiveness to abiotic stress.Additionally,we investigated the biological function of Ac RAD23D1 using VIGS-mediated gene silencing methods under drought stress conditions.Suppression of AcRAD23D1 expression resulted in reduced relative water content(RWC)but increased malondialdehyde(MDA)content and relative electrolyte leakage(REL)levels in D1-VIGS lines compared to control lines.Furthermore,D1-VIGS lines exhibited a higher accumulation of reactive oxygen species(ROS)along with decreased superoxide dismutase(SOD)and peroxidase(POD)enzyme activities.These findings suggest that AcRAD23D1 may play a positive role in regulating kiwifruit's response to drought stress.Our results provide new insights into the potential involvement of AcRAD23 under abiotic stress conditions while offering a theoretical foundation for understanding the molecular mechanisms underlying kiwifruit's adaptation to stresses.展开更多
Radiation sensitivity proteins-23 （RAD23） are DNA repair factors participate in the ubiquitin/proteasome system （UPS）. Although the genome-wide analysis of RAD23 family members has been conducted in some species, ...Radiation sensitivity proteins-23 （RAD23） are DNA repair factors participate in the ubiquitin/proteasome system （UPS）. Although the genome-wide analysis of RAD23 family members has been conducted in some species, little is known about RAD23 genes in apple （Malusxdomestica Borkh.）. We analyzed this gene family in M. domestica in terms of genomic locations, protein and promoter structures, and expressions in response to stresses. Various members showed a ubiqui- tous pattern of expression in all selected apple parts. Their expressions were altered under chilling, heat, and hydrogen peroxide treatments, as well as abscisic acid （ABA） treatment and water deficiency, suggesting their possible roles in plant stress responses. These results provide essential information about RAD23 genes in apple and will contribute to further functional studies.展开更多
目的构建白念珠菌RAD23高表达菌株,探索其高表达对细胞耐受遗传毒性试剂胁迫的影响。方法采用瞬时CRISPR/Cas9系统,分别于体外扩增Cas9基因、sgRNA以及含MET3启动子的修复DNA,通过醋酸锂法转化白念珠菌野生型菌株,于SD-Ura-Met-Cys培养...目的构建白念珠菌RAD23高表达菌株,探索其高表达对细胞耐受遗传毒性试剂胁迫的影响。方法采用瞬时CRISPR/Cas9系统,分别于体外扩增Cas9基因、sgRNA以及含MET3启动子的修复DNA,通过醋酸锂法转化白念珠菌野生型菌株,于SD-Ura-Met-Cys培养基筛选转化子,通过菌落PCR验证基因型,通过Western blot检测Rad23相对表达量,利用平板表型试验检测RAD23高表达对细胞耐受遗传毒性试剂的影响。结果成功构建了RAD23高表达菌株;与野生型菌株相比,高表达菌株中RAD23蛋白相对表达量约为野生型菌株的7倍(10.20 VS 1.46);在UV胁迫条件下,RAD23高表达菌株存活率显著下降(与野生型菌株相比,下降约1 000倍)。结论利用CRISPR/Cas9系统快速构建了白念珠菌RAD23高表达菌株,且RAD23高表达会抑制细胞对遗传毒性试剂UV的耐受能力。展开更多
Protein Rad23, a nucleotide excision repair factor, mainly involves in repairing the DNA damage from environment, such as UV light. The function of Rad23 protein involved in DNA damage repair from many environmental f...Protein Rad23, a nucleotide excision repair factor, mainly involves in repairing the DNA damage from environment, such as UV light. The function of Rad23 protein involved in DNA damage repair from many environmental factors has been studied extensively, but it is not clear from ultraviolet irradiation. To further investigate the photo-protective function of Rad23 protein on HeLa cells damaged from UV light irradiation, firstly, HeLa cells were irradiated by UV light and incubated with the fusion protein of pCold-Rad23, then the cell viability and apoptosis rate were detected by MTT and Hoechst33342/Pl fluorescent staining, respectively. The results show that the recombinant Rad23 protein can protect the HeLa cells from UV irradiation, and inhibit the apoptosis of HeLa cell by UV irradiation.展开更多
基金financially supported by the National Natural Science Foundation of China(32472679)the Sichuan Science and Technology Department Projects(2023ZHCG0098,2024JDRC0011)+2 种基金the Chengdu Science and Technology Department Project,China(2024-YF0500408-SN)the Postdoctoral Fellowship Program of China Postdoctoral Science Foundation(GZC20231871)the Special Project for the Double Support Plan of Discipline Construction at Sichuan Agricultural University,China(2024ZYTS021)。
文摘UBL-UBA protein functions as a shuttle factor in the 26S ubiquitin degradation pathway,playing a critical role in plant growth and development,and responding to various biotic and abiotic stresses.Although RAD23,a type of UBL-UBA protein,has been extensively studied in several plants,there is currently no comprehensive analysis available for kiwifruit(Actinidia chinensis).In this study,we identified six AcRAD23 genes in kiwifruit and further analyzed their phylogenetic relationships,gene structure,conserved motif composition and cis-acting element in the promoter.Subcellular localization experiments revealed that all AcRAD23 were localized in the nucleus and the cell membranes.Quantitative real-time PCR(qRT-PCR)analysis demonstrated differential expression patterns of these Ac RAD23 genes across different tissues and under various stress conditions(drought,waterlogging,salt stress,etc.),with AcRAD23D1 showing the highest responsiveness to abiotic stress.Additionally,we investigated the biological function of Ac RAD23D1 using VIGS-mediated gene silencing methods under drought stress conditions.Suppression of AcRAD23D1 expression resulted in reduced relative water content(RWC)but increased malondialdehyde(MDA)content and relative electrolyte leakage(REL)levels in D1-VIGS lines compared to control lines.Furthermore,D1-VIGS lines exhibited a higher accumulation of reactive oxygen species(ROS)along with decreased superoxide dismutase(SOD)and peroxidase(POD)enzyme activities.These findings suggest that AcRAD23D1 may play a positive role in regulating kiwifruit's response to drought stress.Our results provide new insights into the potential involvement of AcRAD23 under abiotic stress conditions while offering a theoretical foundation for understanding the molecular mechanisms underlying kiwifruit's adaptation to stresses.
基金supported by the National High Technology Research and Development Program of China(863 Program,2011AA100204)the earmarked fund for the China Agriculture Research System(CARS-28)
文摘Radiation sensitivity proteins-23 （RAD23） are DNA repair factors participate in the ubiquitin/proteasome system （UPS）. Although the genome-wide analysis of RAD23 family members has been conducted in some species, little is known about RAD23 genes in apple （Malusxdomestica Borkh.）. We analyzed this gene family in M. domestica in terms of genomic locations, protein and promoter structures, and expressions in response to stresses. Various members showed a ubiqui- tous pattern of expression in all selected apple parts. Their expressions were altered under chilling, heat, and hydrogen peroxide treatments, as well as abscisic acid （ABA） treatment and water deficiency, suggesting their possible roles in plant stress responses. These results provide essential information about RAD23 genes in apple and will contribute to further functional studies.
文摘目的构建白念珠菌RAD23高表达菌株,探索其高表达对细胞耐受遗传毒性试剂胁迫的影响。方法采用瞬时CRISPR/Cas9系统,分别于体外扩增Cas9基因、sgRNA以及含MET3启动子的修复DNA,通过醋酸锂法转化白念珠菌野生型菌株,于SD-Ura-Met-Cys培养基筛选转化子,通过菌落PCR验证基因型,通过Western blot检测Rad23相对表达量,利用平板表型试验检测RAD23高表达对细胞耐受遗传毒性试剂的影响。结果成功构建了RAD23高表达菌株;与野生型菌株相比,高表达菌株中RAD23蛋白相对表达量约为野生型菌株的7倍(10.20 VS 1.46);在UV胁迫条件下,RAD23高表达菌株存活率显著下降(与野生型菌株相比,下降约1 000倍)。结论利用CRISPR/Cas9系统快速构建了白念珠菌RAD23高表达菌株,且RAD23高表达会抑制细胞对遗传毒性试剂UV的耐受能力。
基金Project(31171176)supported by the National Natural Science Foundation of China
文摘Protein Rad23, a nucleotide excision repair factor, mainly involves in repairing the DNA damage from environment, such as UV light. The function of Rad23 protein involved in DNA damage repair from many environmental factors has been studied extensively, but it is not clear from ultraviolet irradiation. To further investigate the photo-protective function of Rad23 protein on HeLa cells damaged from UV light irradiation, firstly, HeLa cells were irradiated by UV light and incubated with the fusion protein of pCold-Rad23, then the cell viability and apoptosis rate were detected by MTT and Hoechst33342/Pl fluorescent staining, respectively. The results show that the recombinant Rad23 protein can protect the HeLa cells from UV irradiation, and inhibit the apoptosis of HeLa cell by UV irradiation.
