[目的]研究‘南通小方柿’(Diospyros kaki Linn.‘Nantongxiaofangshi’)乙醇脱氢酶基因DkADH1的功能,阐明其在柿果脱涩过程中的作用。[方法]构建了DkADH1基因植物双元表达载体,通过农杆菌介导法将该基因转入番茄中。以转基因和非转基...[目的]研究‘南通小方柿’(Diospyros kaki Linn.‘Nantongxiaofangshi’)乙醇脱氢酶基因DkADH1的功能,阐明其在柿果脱涩过程中的作用。[方法]构建了DkADH1基因植物双元表达载体,通过农杆菌介导法将该基因转入番茄中。以转基因和非转基因番茄株系的不同组织为材料,钨酸钠-钼酸钠比色法测定可溶性单宁含量,并用实时荧光定量PCR(qRT-PCR)检测原花青素(PA)合成途径相关基因的表达情况。[结果]经PCR和RT-PCR检测,获得了3个转DkADH1基因番茄株系。过量表达DkADH1基因的转基因番茄植株的叶片、花、果实中可溶性单宁含量均显著低于非转基因番茄植株。qRTPCR显示:转基因植株不同组织中PA合成途径相关基因F3'5'H、LAR、MYB4和PAL的表达量与非转基因植株相比均显著下调。[结论]DkADH1能降低植物可溶性单宁的含量并抑制其生物合成途径相关基因的表达,与柿果脱涩密切相关。展开更多
目的构建ADH1B慢病毒表达载体并建立ADH1B稳定过表达的人肝癌细胞株HepG2.2.15。方法根据ADH1B基因序列设计引物,PCR扩增并连接于GV492载体质粒交换转化DH5α感受态,选取阳性克隆转化子进行测序鉴定。采用三质粒系统包装ADH1B慢病毒载...目的构建ADH1B慢病毒表达载体并建立ADH1B稳定过表达的人肝癌细胞株HepG2.2.15。方法根据ADH1B基因序列设计引物,PCR扩增并连接于GV492载体质粒交换转化DH5α感受态,选取阳性克隆转化子进行测序鉴定。采用三质粒系统包装ADH1B慢病毒载体转染293T细胞收集上清,测定滴度。感染人肝癌细胞HepG2.2.15,嘌呤霉素筛选得到过表达ADH1B的稳定细胞株,设立空白组、阴性对照组和过表达组,通过荧光显微镜观察各组GFP表达情况,并用Western blot及RT-qPCR从蛋白、mRNA水平对ADH1B基因表达进行检测。结果通过PCR、酶切鉴定及基因测序成功构建ADH1B过表达慢病毒载体,检测病毒滴度为2×109TU·mL-1。利用此病毒悬液感染HepG2.2.15细胞,成功筛选到ADH1B稳定表达的HepG2.2.15细胞株。Western blot及RT-qPCR结果显示:重组慢病毒载体LV-ADH1B能够有效感染HepG2.2.15细胞,与空白组和阴性对照组细胞相比,在过表达组细胞ADH1B m RNA和蛋白中表达量均升高。结论成功构建ADH1B基因的重组慢病毒载体LV-ADH1B,并筛选出稳定过表达ADH1B基因的人肝癌细胞株HepG2.2.15。展开更多
Our previous studies demonstrated that oral vitamin A supplementation during late-stage pregnancy and the neonatal stage enhances birth weight,growth performance,and mRNA expression related to muscle and preadipocyte ...Our previous studies demonstrated that oral vitamin A supplementation during late-stage pregnancy and the neonatal stage enhances birth weight,growth performance,and mRNA expression related to muscle and preadipocyte development in beef cattle.The alcohol dehydrogenase 1C(ADH1C)c.-64T>C genotype also correlated with vitamin A concentration in beef production.This study aimed to investigate the effects of vitamin A supplementation on the muscle development and vitamin A metabolism in weaned beef calves with different ADH1C genotypes.Twenty male calves(90 d of age;initial BW:89.03 kg[SD 8.60])were stratified according to ADH1C genotype and vitamin A treatment(duration:3 months)and randomly assigned to 4 groups with a 22 factorial arrangement.Vitamin A treatments included the following:control(10,000 IU/kg of as-fed,a.TT type;b.TC type);treatment(40,000 IU/kg of as-fed,c.TT type;and d.TC type).Parameters including BW,FI,blood,longissimus dorsi muscle,and liver status during the experimental period were analyzed using the generalized linear model(GLM)procedure and Tukey's test by SAS 9.4 program.Serum vitamin A was significantly increased(P<0.05)in the vitamin A treatment group at 4 and 6 months of age.TT type calves showed higher serum vitamin A concentration(P<0.05)than the TC type calves.Serum triglyceride and non-esterified fatty acid(NEFA)levels increased(P<0.05)in the treatment group compared with the control at 6 months of age.However,BW,ADG and FI showed no differences between the groups.In addition,mRNA expression in longissimus dorsi muscle revealed upregulation of paired box 7(PAX7)(P<0.05)after the vitamin A treatment period based on biopsy results.Both ADH1C and aldehyde dehydrogenase(ALDH)1A1 mRNA expression was downregulated(P<0.01)by vitamin A supplementation.The TC type of ADH1C showed higher mRNA expression than the TT type.However,no effect was observed on adipogenic mRNA expression(preadipocyte factor-1[PREF-1],peroxisome proliferator-activated receptor gamma[PPARg],fatty acid binding protein 4[FABP4])in all groups.Our findings suggest that weaned calves treated with vitamin A may promote the storage of satellite cells by elevating PAX7 gene expression in the muscle.The TC type calves may show increased capacity for vitamin A metabolism,which can be used in genetically customizing feed management to maximize beef production in the calves.展开更多
Louis Pasteur first reported that living cells switch from aerobic to anaerobic metabolism under low-oxygen conditions,but the underlying regulatory mechanism remains to be fully elucidated.ALCOHOL DEHYDROGENASE 1(ADH...Louis Pasteur first reported that living cells switch from aerobic to anaerobic metabolism under low-oxygen conditions,but the underlying regulatory mechanism remains to be fully elucidated.ALCOHOL DEHYDROGENASE 1(ADH1)encodes a key enzyme in ethanolic fermentation and is upregulated under hypoxia.In this study,we searched for Arabidopsis thaliana mutants with defects in hypoxia-induced ADH1 expression and identified the IQ DOMAIN containing protein 22(IQD22)as a crucial regulator of ADH1-mediated hypoxia tolerance.The iqd22 mutant plants were hypersensitive to submergence and hypoxic stress as compared with the wild-type plants,whereas IQD22 overexpressors were more tolerant.We showed that under hypoxia,IQD22 enhances the interaction between the calcium-dependent protein kinase CPK12 and the ETHYLENE RESPONSE FACTOR(ERF)-VII-type transcription factor RELATED TO AP2.12(RAP2.12)to upregulate hypoxia-responsive genes,including ADH1.Moreover,we found that IQD22 interacts with calmodulins(CaMs)in vivo and facilitates their association with ADH1,stimulating its abundance in response to hypoxia.Metabolic profiling revealed that hypoxia causes significant increase in glycolytic metabolites but greatly lower ethanol accumulation in the iqd22-2 mutant.Genetic analysis showed that disruption of ADH1 suppresses the improved hypoxia-tolerance phenotype of IQD22 overexpressors.Taken together,these results indicate that IQD22 functions in the CaM-ADH1 and CPK12-RAP2.12 regulatory modules,which coordinately mediate calcium-dependent activation of anaerobic respiration to control metabolic flux during hypoxia.展开更多
文摘[目的]研究‘南通小方柿’(Diospyros kaki Linn.‘Nantongxiaofangshi’)乙醇脱氢酶基因DkADH1的功能,阐明其在柿果脱涩过程中的作用。[方法]构建了DkADH1基因植物双元表达载体,通过农杆菌介导法将该基因转入番茄中。以转基因和非转基因番茄株系的不同组织为材料,钨酸钠-钼酸钠比色法测定可溶性单宁含量,并用实时荧光定量PCR(qRT-PCR)检测原花青素(PA)合成途径相关基因的表达情况。[结果]经PCR和RT-PCR检测,获得了3个转DkADH1基因番茄株系。过量表达DkADH1基因的转基因番茄植株的叶片、花、果实中可溶性单宁含量均显著低于非转基因番茄植株。qRTPCR显示:转基因植株不同组织中PA合成途径相关基因F3'5'H、LAR、MYB4和PAL的表达量与非转基因植株相比均显著下调。[结论]DkADH1能降低植物可溶性单宁的含量并抑制其生物合成途径相关基因的表达,与柿果脱涩密切相关。
文摘目的构建ADH1B慢病毒表达载体并建立ADH1B稳定过表达的人肝癌细胞株HepG2.2.15。方法根据ADH1B基因序列设计引物,PCR扩增并连接于GV492载体质粒交换转化DH5α感受态,选取阳性克隆转化子进行测序鉴定。采用三质粒系统包装ADH1B慢病毒载体转染293T细胞收集上清,测定滴度。感染人肝癌细胞HepG2.2.15,嘌呤霉素筛选得到过表达ADH1B的稳定细胞株,设立空白组、阴性对照组和过表达组,通过荧光显微镜观察各组GFP表达情况,并用Western blot及RT-qPCR从蛋白、mRNA水平对ADH1B基因表达进行检测。结果通过PCR、酶切鉴定及基因测序成功构建ADH1B过表达慢病毒载体,检测病毒滴度为2×109TU·mL-1。利用此病毒悬液感染HepG2.2.15细胞,成功筛选到ADH1B稳定表达的HepG2.2.15细胞株。Western blot及RT-qPCR结果显示:重组慢病毒载体LV-ADH1B能够有效感染HepG2.2.15细胞,与空白组和阴性对照组细胞相比,在过表达组细胞ADH1B m RNA和蛋白中表达量均升高。结论成功构建ADH1B基因的重组慢病毒载体LV-ADH1B,并筛选出稳定过表达ADH1B基因的人肝癌细胞株HepG2.2.15。
基金the National Research Foundation of Korea(NRF)grant funded by the Korea government(MSIT)(2020R1A2B5B02001843).
文摘Our previous studies demonstrated that oral vitamin A supplementation during late-stage pregnancy and the neonatal stage enhances birth weight,growth performance,and mRNA expression related to muscle and preadipocyte development in beef cattle.The alcohol dehydrogenase 1C(ADH1C)c.-64T>C genotype also correlated with vitamin A concentration in beef production.This study aimed to investigate the effects of vitamin A supplementation on the muscle development and vitamin A metabolism in weaned beef calves with different ADH1C genotypes.Twenty male calves(90 d of age;initial BW:89.03 kg[SD 8.60])were stratified according to ADH1C genotype and vitamin A treatment(duration:3 months)and randomly assigned to 4 groups with a 22 factorial arrangement.Vitamin A treatments included the following:control(10,000 IU/kg of as-fed,a.TT type;b.TC type);treatment(40,000 IU/kg of as-fed,c.TT type;and d.TC type).Parameters including BW,FI,blood,longissimus dorsi muscle,and liver status during the experimental period were analyzed using the generalized linear model(GLM)procedure and Tukey's test by SAS 9.4 program.Serum vitamin A was significantly increased(P<0.05)in the vitamin A treatment group at 4 and 6 months of age.TT type calves showed higher serum vitamin A concentration(P<0.05)than the TC type calves.Serum triglyceride and non-esterified fatty acid(NEFA)levels increased(P<0.05)in the treatment group compared with the control at 6 months of age.However,BW,ADG and FI showed no differences between the groups.In addition,mRNA expression in longissimus dorsi muscle revealed upregulation of paired box 7(PAX7)(P<0.05)after the vitamin A treatment period based on biopsy results.Both ADH1C and aldehyde dehydrogenase(ALDH)1A1 mRNA expression was downregulated(P<0.01)by vitamin A supplementation.The TC type of ADH1C showed higher mRNA expression than the TT type.However,no effect was observed on adipogenic mRNA expression(preadipocyte factor-1[PREF-1],peroxisome proliferator-activated receptor gamma[PPARg],fatty acid binding protein 4[FABP4])in all groups.Our findings suggest that weaned calves treated with vitamin A may promote the storage of satellite cells by elevating PAX7 gene expression in the muscle.The TC type calves may show increased capacity for vitamin A metabolism,which can be used in genetically customizing feed management to maximize beef production in the calves.
基金supported by the National Key R&D Program of China(Project 2024YFD1200800)the National Natural Science Foundation of China(pProjects 32321163646,U22A20458,and 32202468)the Natural Science Foundation of Guangdong Province(pProjects 2023A1515012038).
文摘Louis Pasteur first reported that living cells switch from aerobic to anaerobic metabolism under low-oxygen conditions,but the underlying regulatory mechanism remains to be fully elucidated.ALCOHOL DEHYDROGENASE 1(ADH1)encodes a key enzyme in ethanolic fermentation and is upregulated under hypoxia.In this study,we searched for Arabidopsis thaliana mutants with defects in hypoxia-induced ADH1 expression and identified the IQ DOMAIN containing protein 22(IQD22)as a crucial regulator of ADH1-mediated hypoxia tolerance.The iqd22 mutant plants were hypersensitive to submergence and hypoxic stress as compared with the wild-type plants,whereas IQD22 overexpressors were more tolerant.We showed that under hypoxia,IQD22 enhances the interaction between the calcium-dependent protein kinase CPK12 and the ETHYLENE RESPONSE FACTOR(ERF)-VII-type transcription factor RELATED TO AP2.12(RAP2.12)to upregulate hypoxia-responsive genes,including ADH1.Moreover,we found that IQD22 interacts with calmodulins(CaMs)in vivo and facilitates their association with ADH1,stimulating its abundance in response to hypoxia.Metabolic profiling revealed that hypoxia causes significant increase in glycolytic metabolites but greatly lower ethanol accumulation in the iqd22-2 mutant.Genetic analysis showed that disruption of ADH1 suppresses the improved hypoxia-tolerance phenotype of IQD22 overexpressors.Taken together,these results indicate that IQD22 functions in the CaM-ADH1 and CPK12-RAP2.12 regulatory modules,which coordinately mediate calcium-dependent activation of anaerobic respiration to control metabolic flux during hypoxia.
