Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。...Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。对突变株和野生型分别进行不同浓度过氧化氢(H2O2)处理,结果表明:与野生型菌株R1相比,dps突变株在低浓度H2O2(≤10mmol/L)条件下存活率急剧下降,而高浓度(≥30mmol/L)下则完全致死。Native-PAGE活性染色结果显示,稳定生长期dps突变株体内两种过氧化氢酶(KatA和KatB)的活性较野生型R1分别上调2.3倍和2.6倍。通过质粒构建和大肠杆菌诱导表达,获得可溶性Dps蛋白。体外结合和DNA保护实验结果显示:Dps具有明显的DNA结合功能,并能保护质粒DNA免受羟自由基攻击。本研究证明,Dps蛋白在耐辐射奇球菌抗氧化体系中发挥重要作用,可能对该菌极端抗性机制有重要贡献。展开更多
Loss-of-function mutants are fundamental resources for gene function studies.However,it is difficult to generate viable and heritable knockout mutants for essential genes.Here,we show that targeted editing of the C-te...Loss-of-function mutants are fundamental resources for gene function studies.However,it is difficult to generate viable and heritable knockout mutants for essential genes.Here,we show that targeted editing of the C-terminal sequence of the embryo lethal gene MITOGEN-ACTIVATED PROTEIN KINASES 1(OsMPK1)results in weak mutants.This C-terminal-edited osmpk1 mutants displayed severe developmental defects and altered disease resistance but generated tens of viable seeds that inherited the mutations.Using the same C-terminal editing approach,we also obtained viable mutants for a wallassociated protein kinase(Os07g0493200)and a leucine-rich repeat receptor-like protein kinase(Os01g0239700),while the null mutations of these genes were lethal.These data suggest that protein kinase activity could be reduced by introducing frameshift mutations adjacent to the C-terminus,which could generate valuable resources for gene function studies and tune protein kinase activity for signaling pathway engineering.展开更多
COVID-19 is caused by coronavirus SARS-CoV-2.Current systemic vaccines generally pro-vide limited protection against viral replication and shedding within the airway.Recombinant VSV(rVSV)is an effective vector which i...COVID-19 is caused by coronavirus SARS-CoV-2.Current systemic vaccines generally pro-vide limited protection against viral replication and shedding within the airway.Recombinant VSV(rVSV)is an effective vector which inducing potent and comprehensive immunities.Currently,there are two clinical trials investigating COVID-19vaccines based on VSV vectors.These vaccines were developed with spike protein of WA1 which administrated intramuscularly.Although intranasal route is ideal for activating mucosal immunity with VSV vector,safety is of concern.Thus,a highly attenuated rVSV with three amino acids mutations in matrix protein(VSV_(MT))was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern.It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently.VSV_(MT) indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein.With a single-dosed intranasal inoculation of rVSV_(ΔGMT)-S_(Δ21),potent SARS-CoV-2specific neutraliza-tion antibodies could be stimulated in animals,particularly in term of mucosal and cellular immunity.Strikingly,the chimeric VSV encoding S_(Δ21) of Delta-variant can induce more potent immune responses compared with those encoding S_(Δ21) of Omicron-or WA1-strain.VSV_(MT) is a promising platform to develop a mucosal vaccine for countering COVID-19.展开更多
OsMADS32 is a monocot specific MIKCc type MADS‐box gene that plays an important role in regulating rice floral meristem and organs identity, a crucial process for reproductive success and rice yield. However, its und...OsMADS32 is a monocot specific MIKCc type MADS‐box gene that plays an important role in regulating rice floral meristem and organs identity, a crucial process for reproductive success and rice yield. However, its underlying mechanism of action remains to be clarified. Here, we characterized a hypomorphic mutant allele of OsMADS32/CFO1, cfo1‐3 and identified its function in controlling rice flower development by bioinformatics and protein‐protein interaction analysis. The cfo1‐3 mutant produces defective flowers, including loss of lodicule identity, formation of ectopic lodicule or hull‐like organs and decreased stamen number, mimicking phenotypes related to the mutation of B class genes. Molecular characterization indicated that mis‐splicing of OsMADS32 transcripts in the cfo1‐3 mutant resulted in an extra eight amino acids in the K‐domain of OsMADS32 protein. By yeast two hybrid and bimolecular fluorescence comple-mentation assays, we revealed that the insertion of eight amino acids or deletion of the internal region in the K1 subdomain of OsMADS32 affects the interaction between OsMADS32 with PISTILLATA （PI）‐like proteins OsMADS2 and OsMADS4. This work provides new insight into the mecha-nism by which OsMADS32 regulates rice lodicule and stamen identity, by interaction with two PI‐like proteins via its K domain.展开更多
文摘Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。对突变株和野生型分别进行不同浓度过氧化氢(H2O2)处理,结果表明:与野生型菌株R1相比,dps突变株在低浓度H2O2(≤10mmol/L)条件下存活率急剧下降,而高浓度(≥30mmol/L)下则完全致死。Native-PAGE活性染色结果显示,稳定生长期dps突变株体内两种过氧化氢酶(KatA和KatB)的活性较野生型R1分别上调2.3倍和2.6倍。通过质粒构建和大肠杆菌诱导表达,获得可溶性Dps蛋白。体外结合和DNA保护实验结果显示:Dps具有明显的DNA结合功能,并能保护质粒DNA免受羟自由基攻击。本研究证明,Dps蛋白在耐辐射奇球菌抗氧化体系中发挥重要作用,可能对该菌极端抗性机制有重要贡献。
基金supported by the National Natural Science Foundation of China(32293243)Fundamental Research Funds for the Central Universities(2021ZKPY002,2662023PY006)supported by Hainan Yazhou Bay Seed Laboratory and the China National Seed Group(project B23YQ1516).
文摘Loss-of-function mutants are fundamental resources for gene function studies.However,it is difficult to generate viable and heritable knockout mutants for essential genes.Here,we show that targeted editing of the C-terminal sequence of the embryo lethal gene MITOGEN-ACTIVATED PROTEIN KINASES 1(OsMPK1)results in weak mutants.This C-terminal-edited osmpk1 mutants displayed severe developmental defects and altered disease resistance but generated tens of viable seeds that inherited the mutations.Using the same C-terminal editing approach,we also obtained viable mutants for a wallassociated protein kinase(Os07g0493200)and a leucine-rich repeat receptor-like protein kinase(Os01g0239700),while the null mutations of these genes were lethal.These data suggest that protein kinase activity could be reduced by introducing frameshift mutations adjacent to the C-terminus,which could generate valuable resources for gene function studies and tune protein kinase activity for signaling pathway engineering.
基金supported by Biomedicine and Technology Supporting Project of Shanghai Science and Technology Innovation Plan(Grant Nos.22S11902200 and 20S11904900,China)Open grant of Engineering Research Center of Cell&Therapeutic Antibody,Ministry of Education,Shanghai Jiao Tong University(Grant No.19X110020009-003,China).
文摘COVID-19 is caused by coronavirus SARS-CoV-2.Current systemic vaccines generally pro-vide limited protection against viral replication and shedding within the airway.Recombinant VSV(rVSV)is an effective vector which inducing potent and comprehensive immunities.Currently,there are two clinical trials investigating COVID-19vaccines based on VSV vectors.These vaccines were developed with spike protein of WA1 which administrated intramuscularly.Although intranasal route is ideal for activating mucosal immunity with VSV vector,safety is of concern.Thus,a highly attenuated rVSV with three amino acids mutations in matrix protein(VSV_(MT))was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern.It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently.VSV_(MT) indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein.With a single-dosed intranasal inoculation of rVSV_(ΔGMT)-S_(Δ21),potent SARS-CoV-2specific neutraliza-tion antibodies could be stimulated in animals,particularly in term of mucosal and cellular immunity.Strikingly,the chimeric VSV encoding S_(Δ21) of Delta-variant can induce more potent immune responses compared with those encoding S_(Δ21) of Omicron-or WA1-strain.VSV_(MT) is a promising platform to develop a mucosal vaccine for countering COVID-19.
基金supported by the Funds from National Natural Science Foundation of China (30971739,31270222,31230051,and J1210047)Key Project on Basic Research from Science and Technology Commission of Shanghai (14JC1403900)the Innovation Program of Shanghai Municipal Education Commission (13ZZ018)
文摘OsMADS32 is a monocot specific MIKCc type MADS‐box gene that plays an important role in regulating rice floral meristem and organs identity, a crucial process for reproductive success and rice yield. However, its underlying mechanism of action remains to be clarified. Here, we characterized a hypomorphic mutant allele of OsMADS32/CFO1, cfo1‐3 and identified its function in controlling rice flower development by bioinformatics and protein‐protein interaction analysis. The cfo1‐3 mutant produces defective flowers, including loss of lodicule identity, formation of ectopic lodicule or hull‐like organs and decreased stamen number, mimicking phenotypes related to the mutation of B class genes. Molecular characterization indicated that mis‐splicing of OsMADS32 transcripts in the cfo1‐3 mutant resulted in an extra eight amino acids in the K‐domain of OsMADS32 protein. By yeast two hybrid and bimolecular fluorescence comple-mentation assays, we revealed that the insertion of eight amino acids or deletion of the internal region in the K1 subdomain of OsMADS32 affects the interaction between OsMADS32 with PISTILLATA （PI）‐like proteins OsMADS2 and OsMADS4. This work provides new insight into the mecha-nism by which OsMADS32 regulates rice lodicule and stamen identity, by interaction with two PI‐like proteins via its K domain.
