AIM： To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3＇-portion of hepatitis E virus （HEV） open reading frame 2 （ORF2） in plastids of to...AIM： To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3＇-portion of hepatitis E virus （HEV） open reading frame 2 （ORF2） in plastids of tobacco （Nicotiana tabacum cv. SR1）, to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS： Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS： Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 μg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION： HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.展开更多
In plants,submergence from flooding causes hypoxia,which impairs energy production and affects plant growth,productivity,and survival.In Arabidopsis,hypoxia induces nuclear localization of the group VII ethylene-respo...In plants,submergence from flooding causes hypoxia,which impairs energy production and affects plant growth,productivity,and survival.In Arabidopsis,hypoxia induces nuclear localization of the group VII ethylene-responsive transcription factor RELATED TO AP2.12(RAP2.12),following its dissociation from the plasma membrane-anchored ACYL-COA BINDING PRO-TEIN1(ACBP1)and ACBP2.Here,we show that polyunsaturated linolenoyl-Co A(18:3-Co A)regulates RAP2.12release from the plasma membrane.Submergence caused a significant increase in 18:3-Co A,but a significant decrease in 18:0-,18:1-,and 18:2-Co A.Application of 18:3-Co A promoted nuclear accumulation of the green fluorescent protein(GFP)fusions RAP2.12-GFP,HYPOXIA-RESPONSIVE ERF1-GFP,and RAP2.3-GFP,and enhanced transcript levels of hypoxia-responsive genes.Plants with decreased ACBP1 and ACBP2(acbp1 ACBP2-RNAi,produced by ACBP2 RNA interference in the acbp1 mutant)had reduced tolerance to hypoxia and impaired 18:3-Co A-induced expression of hypoxia-related genes.In knockout mutants and overexpression lines of LONG-CHAIN ACYL-COA SYNTHASE2(LACS2)and FATTY ACID DE-SATURASE 3(FAD3),the acyl-Co A pool size and 18:3-Co A levels were closely related to ERF-VII-mediated signaling and hypoxia tolerance.These findings demonstrate that polyunsaturation of long-chain acyl-Co As functions as important mechanism in the regulation of plant hypoxia signaling,by modulating ACBP–ERF-VII dynamics.展开更多
基金Supported by a grant from the Hong Kong Research Grant Council, No. 7342/03M to YX Zhou and E Lam
文摘AIM： To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3＇-portion of hepatitis E virus （HEV） open reading frame 2 （ORF2） in plastids of tobacco （Nicotiana tabacum cv. SR1）, to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS： Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS： Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 μg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION： HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.
基金supported by the National Natural Science Foundation of China(Projects 31725004,31670276,and 31461143001 to S.X.Project 31700220 to L.J.X.)the Natural Science Foundation of Guangdong Province,China(Project 2015A030313122 to Q.F.C.)+1 种基金Sun Yat-Sen University(Projects 17lgpy110 and Plant KF02 to L.J.X.)supported by the Wilson and Amelia Wong Endowment Fund。
文摘In plants,submergence from flooding causes hypoxia,which impairs energy production and affects plant growth,productivity,and survival.In Arabidopsis,hypoxia induces nuclear localization of the group VII ethylene-responsive transcription factor RELATED TO AP2.12(RAP2.12),following its dissociation from the plasma membrane-anchored ACYL-COA BINDING PRO-TEIN1(ACBP1)and ACBP2.Here,we show that polyunsaturated linolenoyl-Co A(18:3-Co A)regulates RAP2.12release from the plasma membrane.Submergence caused a significant increase in 18:3-Co A,but a significant decrease in 18:0-,18:1-,and 18:2-Co A.Application of 18:3-Co A promoted nuclear accumulation of the green fluorescent protein(GFP)fusions RAP2.12-GFP,HYPOXIA-RESPONSIVE ERF1-GFP,and RAP2.3-GFP,and enhanced transcript levels of hypoxia-responsive genes.Plants with decreased ACBP1 and ACBP2(acbp1 ACBP2-RNAi,produced by ACBP2 RNA interference in the acbp1 mutant)had reduced tolerance to hypoxia and impaired 18:3-Co A-induced expression of hypoxia-related genes.In knockout mutants and overexpression lines of LONG-CHAIN ACYL-COA SYNTHASE2(LACS2)and FATTY ACID DE-SATURASE 3(FAD3),the acyl-Co A pool size and 18:3-Co A levels were closely related to ERF-VII-mediated signaling and hypoxia tolerance.These findings demonstrate that polyunsaturation of long-chain acyl-Co As functions as important mechanism in the regulation of plant hypoxia signaling,by modulating ACBP–ERF-VII dynamics.
