Carotenoids are the largest group of natural pigments responsible for the yellow,orange,and red colors in plant kernels,fruits,and leaves(Gupta and Hirschberg,2021).In plants,carotenoids are involved in manybiological...Carotenoids are the largest group of natural pigments responsible for the yellow,orange,and red colors in plant kernels,fruits,and leaves(Gupta and Hirschberg,2021).In plants,carotenoids are involved in manybiological processes,such as acting as accessory light-harvesting pigments in photosynthesis,participating in photoprotection,and serving as precursors for the hormones abscisic acid(ABA)and strigolactones(Ruiz-Sola and Rodriguez-Concepcion,2012).展开更多
As an important group of plant cellular organelles, the molecular mechanism of plastid division is poorly understood. Recent studies have revealed that the homologs of ftsZ gene, an essential prokaryotic cell division...As an important group of plant cellular organelles, the molecular mechanism of plastid division is poorly understood. Recent studies have revealed that the homologs of ftsZ gene, an essential prokaryotic cell division gene, are involved in plastid division process of plant cells. Antisense and sense expression constructions were employed to investigate the functions of the two ftsZ genes, NtFtsZ1 and NtFtsZ2, in transgenic Nicotiana tabacum L. plants. Although antisense expression of,NtFtsZs reduced the native protein level obviously, the size and number of chloroplasts in transgenic tobacco plants had no effect. In contrast, overexpression of NtFtsZs in transgenic plants strikingly changed the number and morphology of chloroplasts. Even only 1 - 2 huge chloroplasts could be seen in the mesophyll cells of some overexpression transgenic plants. Analyses of chloroplast ultrastructures and chlorophyll content of different transgenic plants suggested that NtFtsZs gene have no direct influence on the normal development and function of chloroplasts. ne changes in chloroplast morphology must be a compensation for the change in chloroplast number. The different phenotypes of chloroplasts in antisense and sense transgenic plants implied that different members from the same ftsZ gene family may have similar function in controlling plastid division. Meanwhile, the changes of chloroplast morphology in sense transgenic plants represented the possible plastoskeleton function of ftsZ in higher plant.展开更多
The inheritance of plastid DNA in Pharbitis was studied by the method of restriction fragment length polymorphisms (RFLP).Experimental results showed that plastid DNA from Pharbitis was paternally inherited in recipro...The inheritance of plastid DNA in Pharbitis was studied by the method of restriction fragment length polymorphisms (RFLP).Experimental results showed that plastid DNA from Pharbitis was paternally inherited in reciprocal crosses,P. nil ×P. limbata and P. limbata×P. nil hybrids.But,in the cross of P. limbata×P. nil,the possibility of biparental inheritance of plastid DNA could not be roled out in our preliminary experiment.Thus Pharbitis became the third genus among angiosperms characterized with male plastid transmission.The mechanisms of paternal plastids DNA inheritance in Pharbitis is unclear.The authors proposed that dilution,exclusion and/or degeneration of maternal plastid,including their DNA,after fertilization should be considered.展开更多
Complete plastid genomes have been proposed as potential“super-barcodes”for plant identification and delineation,particularly in cases where standard DNA barcodes may be insufficient.However,few studies have systema...Complete plastid genomes have been proposed as potential“super-barcodes”for plant identification and delineation,particularly in cases where standard DNA barcodes may be insufficient.However,few studies have systematically addressed how taxonomic complexity,especially in rapidly radiating lineages with intricate evolutionary histories,might influencethe efficacyof plastome-scale barcodes.Pedicularis is a hyperdiverse genus in the Himalaya-Hengduan Mountains,and previous studies have demonstrated high discriminatory power of the standard barcodes within this genus.Therefore,Pedicularis serves as a model for investigating the key plastome-sequence characteristics and biological phenomena that determine species-discrimination capacity.In this study,we evaluated 292 plastomes representing 96 Pedicularis species to compare the discriminatory power of complete plastid genomes with of standard DNA barcodes.Our results revealed that the traditional standard barcode combination(nrITS+matK+rbcL+trnH-psbA)achieved the highest discrimination rates(81.25%),closely followed by the plastid large single copy(LSC)region(80.21%),then by full plastome,the supermatrix of proteincoding genes,and hypervariable regions(79.17%).Notably,the matK and ycf1 gene alone could discriminate 78.13%of species.Key determinants of species discrimination by integrating alignment length(AL)and the proportion of parsimony-informative sites(PPIS),as well as conserved genes under relaxed selection exhibiting stronger discriminatory capacity.Unlike previous studies that demonstrated superior discrimination rates of plastome-scale barcodes,this study reveals a notable exception of minimal differences between traditional DNA and plastome-scale barcodes that appearing linked to Pedicularis’specificbiological habits and potentially reflectingunique evolutionary patterns in the plastid genome.展开更多
The plastid genome(plastome)represents an indispensable molecular resource for studying plant phylogeny and evolution.Although plastome size is much smaller than that of nuclear genomes,accurately and efficientlyannot...The plastid genome(plastome)represents an indispensable molecular resource for studying plant phylogeny and evolution.Although plastome size is much smaller than that of nuclear genomes,accurately and efficientlyannotating and utilizing plastome sequences remain challenging.Therefore,a streamlined phylogenomic pipeline spanning plastome annotation,phylogenetic reconstruction and comparative genomics would greatly facilitate research utilizing this important organellar genome.Here,we develop PlastidHub,a novel web application employing innovative tools to analyze plastome sequences.In comparison with existing tools,key novel functionalities in PlastidHub include:(1)standardization of quadripartite structure;(2)improvement of annotation flexibility and consistency;(3)quantitative assessment of annotation completeness;(4)diverse extraction modes for canonical and specialized sequences;(5)intelligent screening of molecular markers for biodiversity studies;(6)genelevel visual comparison of structural variations and annotation completeness.PlastidHub features cloud-based web applications that do not require users to install,update,or maintain tools;detailed help documents including user guides,test examples,a static pop-up prompt box,and dynamic pop-up warning prompts when entering unreasonable parameter values;batch processing capabilities for all tools;intermediate results for secondary use;and easy-to-operate task flows between fileupload and download.A key feature of PlastidHub is its interrelated task-based user interface design.Give that PlastidHub is easy to use without specialized computational skills or resources,this new platform should be widely used among botanists and evolutionary biologists,improving and expediting research employing the plastome.PlastidHub is available at https://www.plastidhub.cn.展开更多
The inheritance of chloroplast DNA (cpDNA) in sweet potato (Ipomoea batatas Lain.) was analyzed using DNA restriction fingerprinting. The cpDNA fingerprints of hybrids from reciprocal crosses between Xushu18 and AB78-...The inheritance of chloroplast DNA (cpDNA) in sweet potato (Ipomoea batatas Lain.) was analyzed using DNA restriction fingerprinting. The cpDNA fingerprints of hybrids from reciprocal crosses between Xushu18 and AB78-1 were found to be identical to those of their female parents, which reveals that cpDNA of sweet potato is maternally inherited in this intervarietal crossing. This maternal cpDNA transmission pattern does not accord with the putative one based on former cytological studies. The plastid inheritance in Convolvulaceae has been briefly reviewed in this study, and the utility of DNA restriction fingerprinting analysis in the study of plastid inheritance is also discussed.展开更多
Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the fil...Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the filamentous bacteria indicated that the NtFtsZs could recognize the potential division sites in E. coli and be polymerized with heterogeneous FtsZ from bacteria. The overexpression of NtFtsZs ∶ gfp inhibited the division of host strain cells and resulted in the long filamentous bacterial morphology. These results suggested that eukaryotic ftsZs have similar function to their prokaryotic homologs. Meanwhile, the different deletions of motifs of NtFtsZs are also employed to investigate the functions of these proteins in E. coli . The results showed that the C_terminal domains of NtFtsZs were related to the correct localization of NtFtsZs in E. coli and the N_terminal domains of NtFtsZs were responsible for the polymerization of homogeneous and heterogeneous FtsZ proteins. The significance of these results in understanding the functions of NtFtsZs in plastid division were discussed.展开更多
Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuber and tuberous roots, and fleshy fruit development. Based on previously reported ...Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuber and tuberous roots, and fleshy fruit development. Based on previously reported in vitro assays, β amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was shown often extrachloroplastic in living cells. Recently we have shown for the first time that β_amylase is predominantly immuno_localized to plastids in living cells of developing apple fruit. But it remains to know whether this model of β_amylase compartmentation is more widespread in plant living cells. The present experiment, conducted in tuberous root of sweet potato ( Ipomea batatas Lam. cv. Xushu 18) and via immunogold electron_microscopy technique, showed that β amylase visualized by gold particles was predominantly localized in plastids especially at periphery of starch granules, but the gold particles were scarcely found in other subcellular compartments, indicating that the enzyme is subcellularly compartmented in the same zone as its starch substrates. The density of gold particles (β amylase) in plastids was increasing during growing season, but the predominantly plastid_distributed pattern of β amylase in cells was shown unchanged throughout the tuberous root development. These data prove that the enzyme is compartmented in its functional sites, and so provide evidence to support the possible widespread biological function of the enzyme in catalyzing starch breakdown in plant living cells or at least in living cells of plant storage organs.展开更多
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
The genus Trigonotis comprises nearly 60 species mainly distributed in East and Southeast Asia.China has the largest number of Trigonotis species in the world,with a total of 44 species,of which 38 are endemic.Nutlet ...The genus Trigonotis comprises nearly 60 species mainly distributed in East and Southeast Asia.China has the largest number of Trigonotis species in the world,with a total of 44 species,of which 38 are endemic.Nutlet morphology is useful for the taxonomic delimitation of Trigonotis.However,there are still controversial circumscriptions of nutlet shape in some species.In previous studies,interspecies phylogenetic relationships were inferred using few DNA markers and very few taxa,which possibly led to erroneous or incomplete conclusions.In this study,the nutlet morphology of 39 Trigonotis taxa and the characteristics of 34 complete chloroplast genomes(29 taxa)were investigated and analyzed.Then,the phylogenetic relationships were discussed within this genus based on complete chloroplast genomes.To the best of our knowledge,this study is the first comprehensive analysis of nutlet morphology and complete chloroplast genome of Trigonotis.Based on nutlet morphology,Trigonotis can be divided into two groups:Group 1,hemispherical or oblique tetrahedron with carpopodiums,and Group 2,inverted tetrahedron without carpopodiums.The chloroplast genome of Trigonotis exhibited a typical quadripartite structure,including 84-86 protein-coding,37 transfer RNA,and 8 ribosomal RNA genes,with a total length of 147,247-148,986 bp.Genes in the junctions were well conserved in Trigonotis,similar to those in other Boraginaceae s.str.species.Furthermore,Trigonotis chloroplast genomes showed relatively high diversity,with more conserved genic regions than intergenic regions;in addition,we detected 14 hot spots(Pi>0.005)in non-coding regions.Phylogenetic analyses based on chloroplast genome data identified highly resolved relationships between Trigonotis species.Specifically,Trigonotis was divided into two clades with strong support:one clade included species with hemispherical or oblique tetrahedron nutlets with carpopodiums and bracts,whereas the other clade included species with inverted tetrahedron nutlets without carpopodiums or bracts.Our results may inform future taxonomic,phylogenetic,and evolutionary studies on Boraginaceae.展开更多
Euglena gracilis is a unicellular green eukaryotic microalga that features characteristics of both plants and animals.The photosynthetic function of its chloroplast is easily lost under stress resulting in bleached mu...Euglena gracilis is a unicellular green eukaryotic microalga that features characteristics of both plants and animals.The photosynthetic function of its chloroplast is easily lost under stress resulting in bleached mutants,while the physiological role of their residual plastid DNAs remains unclear.In this study,we obtained five bleached mutants by ofloxacin(Ofl)treatment,identified 12 residual plastid genes in five bleached mutants,and determined the mRNA levels in the wild type E.gracilis(WT)and one bleached mutant(OflB2)under dark and light stimulation conditions by quantitative reverse transcribed PCR(qRTPCR).Results show that the expression of all selected plastid genes in both WT and OflB2 mutant did not change significantly in darkness,while their responses to light stimulation were different.Under the light stimulation conditions,half of the genes did not change significantly,while most of the other genes were down-regulated in OflB2 mutant and up-regulated in WT.Therefore,the bleached mutant retains part of the plastid genome and the plastid relic is responsive to light.Our research will help to understand the functions of residual plastid DNA and evolution of chloroplasts.展开更多
基金supported by the National Key Research and Development Program of China(2022YFD1200704-3)Crop Varietal Improvement and Insect Pests Control by Nuclear Radiation,the Sichuan Province Science and Technology Program(2022NSFSC0018,2021YFYZ0011,2020YJ0249,MZGC20230108)the Biological Breeding Program of State Key of Sichuan Agricultural University(SKL-ZY202234).
文摘Carotenoids are the largest group of natural pigments responsible for the yellow,orange,and red colors in plant kernels,fruits,and leaves(Gupta and Hirschberg,2021).In plants,carotenoids are involved in manybiological processes,such as acting as accessory light-harvesting pigments in photosynthesis,participating in photoprotection,and serving as precursors for the hormones abscisic acid(ABA)and strigolactones(Ruiz-Sola and Rodriguez-Concepcion,2012).
文摘As an important group of plant cellular organelles, the molecular mechanism of plastid division is poorly understood. Recent studies have revealed that the homologs of ftsZ gene, an essential prokaryotic cell division gene, are involved in plastid division process of plant cells. Antisense and sense expression constructions were employed to investigate the functions of the two ftsZ genes, NtFtsZ1 and NtFtsZ2, in transgenic Nicotiana tabacum L. plants. Although antisense expression of,NtFtsZs reduced the native protein level obviously, the size and number of chloroplasts in transgenic tobacco plants had no effect. In contrast, overexpression of NtFtsZs in transgenic plants strikingly changed the number and morphology of chloroplasts. Even only 1 - 2 huge chloroplasts could be seen in the mesophyll cells of some overexpression transgenic plants. Analyses of chloroplast ultrastructures and chlorophyll content of different transgenic plants suggested that NtFtsZs gene have no direct influence on the normal development and function of chloroplasts. ne changes in chloroplast morphology must be a compensation for the change in chloroplast number. The different phenotypes of chloroplasts in antisense and sense transgenic plants implied that different members from the same ftsZ gene family may have similar function in controlling plastid division. Meanwhile, the changes of chloroplast morphology in sense transgenic plants represented the possible plastoskeleton function of ftsZ in higher plant.
文摘The inheritance of plastid DNA in Pharbitis was studied by the method of restriction fragment length polymorphisms (RFLP).Experimental results showed that plastid DNA from Pharbitis was paternally inherited in reciprocal crosses,P. nil ×P. limbata and P. limbata×P. nil hybrids.But,in the cross of P. limbata×P. nil,the possibility of biparental inheritance of plastid DNA could not be roled out in our preliminary experiment.Thus Pharbitis became the third genus among angiosperms characterized with male plastid transmission.The mechanisms of paternal plastids DNA inheritance in Pharbitis is unclear.The authors proposed that dilution,exclusion and/or degeneration of maternal plastid,including their DNA,after fertilization should be considered.
基金supported by the National Natural Science Foundation of China(32371700,32071670 and 31870196)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB31000000)+4 种基金the Science and Technology Basic Resources Investigation Program of China(2021FY100200)Yunnan Revitalization Talent Support Program“Young Talent”and“Innovation Team”Projects(202405AS350019)the 14th Five-Year Plan of Xishuangbanna Tropical Botanical Garden,Chinese Academy of Science(XTBG-1450101)the Key R&D program of Yunnan Province,China(202103AC100003)the Key Basic Research program of Yunnan Province,China(202101BC070003).
文摘Complete plastid genomes have been proposed as potential“super-barcodes”for plant identification and delineation,particularly in cases where standard DNA barcodes may be insufficient.However,few studies have systematically addressed how taxonomic complexity,especially in rapidly radiating lineages with intricate evolutionary histories,might influencethe efficacyof plastome-scale barcodes.Pedicularis is a hyperdiverse genus in the Himalaya-Hengduan Mountains,and previous studies have demonstrated high discriminatory power of the standard barcodes within this genus.Therefore,Pedicularis serves as a model for investigating the key plastome-sequence characteristics and biological phenomena that determine species-discrimination capacity.In this study,we evaluated 292 plastomes representing 96 Pedicularis species to compare the discriminatory power of complete plastid genomes with of standard DNA barcodes.Our results revealed that the traditional standard barcode combination(nrITS+matK+rbcL+trnH-psbA)achieved the highest discrimination rates(81.25%),closely followed by the plastid large single copy(LSC)region(80.21%),then by full plastome,the supermatrix of proteincoding genes,and hypervariable regions(79.17%).Notably,the matK and ycf1 gene alone could discriminate 78.13%of species.Key determinants of species discrimination by integrating alignment length(AL)and the proportion of parsimony-informative sites(PPIS),as well as conserved genes under relaxed selection exhibiting stronger discriminatory capacity.Unlike previous studies that demonstrated superior discrimination rates of plastome-scale barcodes,this study reveals a notable exception of minimal differences between traditional DNA and plastome-scale barcodes that appearing linked to Pedicularis’specificbiological habits and potentially reflectingunique evolutionary patterns in the plastid genome.
基金the Natural Science Foundation of Shandong Province(ZR2020QC022)the Science and Technology Basic Resources Investigation Program of China(No.2019FY100900)+2 种基金the Major Program for Basic Research Project of Yunnan Province(202401BC070001)Yunnan Revitalization Talent Support Program:Yunling Scholar Project to Tingshuang Yithe open research project of“Cross Cooperative Team”of the Germplasm Bank of Wild Species,Kunming Institute of Botany,Chinese Academy of Sciences.
文摘The plastid genome(plastome)represents an indispensable molecular resource for studying plant phylogeny and evolution.Although plastome size is much smaller than that of nuclear genomes,accurately and efficientlyannotating and utilizing plastome sequences remain challenging.Therefore,a streamlined phylogenomic pipeline spanning plastome annotation,phylogenetic reconstruction and comparative genomics would greatly facilitate research utilizing this important organellar genome.Here,we develop PlastidHub,a novel web application employing innovative tools to analyze plastome sequences.In comparison with existing tools,key novel functionalities in PlastidHub include:(1)standardization of quadripartite structure;(2)improvement of annotation flexibility and consistency;(3)quantitative assessment of annotation completeness;(4)diverse extraction modes for canonical and specialized sequences;(5)intelligent screening of molecular markers for biodiversity studies;(6)genelevel visual comparison of structural variations and annotation completeness.PlastidHub features cloud-based web applications that do not require users to install,update,or maintain tools;detailed help documents including user guides,test examples,a static pop-up prompt box,and dynamic pop-up warning prompts when entering unreasonable parameter values;batch processing capabilities for all tools;intermediate results for secondary use;and easy-to-operate task flows between fileupload and download.A key feature of PlastidHub is its interrelated task-based user interface design.Give that PlastidHub is easy to use without specialized computational skills or resources,this new platform should be widely used among botanists and evolutionary biologists,improving and expediting research employing the plastome.PlastidHub is available at https://www.plastidhub.cn.
文摘The inheritance of chloroplast DNA (cpDNA) in sweet potato (Ipomoea batatas Lain.) was analyzed using DNA restriction fingerprinting. The cpDNA fingerprints of hybrids from reciprocal crosses between Xushu18 and AB78-1 were found to be identical to those of their female parents, which reveals that cpDNA of sweet potato is maternally inherited in this intervarietal crossing. This maternal cpDNA transmission pattern does not accord with the putative one based on former cytological studies. The plastid inheritance in Convolvulaceae has been briefly reviewed in this study, and the utility of DNA restriction fingerprinting analysis in the study of plastid inheritance is also discussed.
文摘Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the filamentous bacteria indicated that the NtFtsZs could recognize the potential division sites in E. coli and be polymerized with heterogeneous FtsZ from bacteria. The overexpression of NtFtsZs ∶ gfp inhibited the division of host strain cells and resulted in the long filamentous bacterial morphology. These results suggested that eukaryotic ftsZs have similar function to their prokaryotic homologs. Meanwhile, the different deletions of motifs of NtFtsZs are also employed to investigate the functions of these proteins in E. coli . The results showed that the C_terminal domains of NtFtsZs were related to the correct localization of NtFtsZs in E. coli and the N_terminal domains of NtFtsZs were responsible for the polymerization of homogeneous and heterogeneous FtsZ proteins. The significance of these results in understanding the functions of NtFtsZs in plastid division were discussed.
文摘Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuber and tuberous roots, and fleshy fruit development. Based on previously reported in vitro assays, β amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was shown often extrachloroplastic in living cells. Recently we have shown for the first time that β_amylase is predominantly immuno_localized to plastids in living cells of developing apple fruit. But it remains to know whether this model of β_amylase compartmentation is more widespread in plant living cells. The present experiment, conducted in tuberous root of sweet potato ( Ipomea batatas Lam. cv. Xushu 18) and via immunogold electron_microscopy technique, showed that β amylase visualized by gold particles was predominantly localized in plastids especially at periphery of starch granules, but the gold particles were scarcely found in other subcellular compartments, indicating that the enzyme is subcellularly compartmented in the same zone as its starch substrates. The density of gold particles (β amylase) in plastids was increasing during growing season, but the predominantly plastid_distributed pattern of β amylase in cells was shown unchanged throughout the tuberous root development. These data prove that the enzyme is compartmented in its functional sites, and so provide evidence to support the possible widespread biological function of the enzyme in catalyzing starch breakdown in plant living cells or at least in living cells of plant storage organs.
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This project was funded by the Science and Technology Basic Work,Ministry of Science and Technology,China(2013FY112100 to Q.L.)National Natural Science Foundation of China(31700175 to Z.W.)Fostering Project for Young Teachers of Zhengzhou University,China(JC21343014 to Z.W.).
文摘The genus Trigonotis comprises nearly 60 species mainly distributed in East and Southeast Asia.China has the largest number of Trigonotis species in the world,with a total of 44 species,of which 38 are endemic.Nutlet morphology is useful for the taxonomic delimitation of Trigonotis.However,there are still controversial circumscriptions of nutlet shape in some species.In previous studies,interspecies phylogenetic relationships were inferred using few DNA markers and very few taxa,which possibly led to erroneous or incomplete conclusions.In this study,the nutlet morphology of 39 Trigonotis taxa and the characteristics of 34 complete chloroplast genomes(29 taxa)were investigated and analyzed.Then,the phylogenetic relationships were discussed within this genus based on complete chloroplast genomes.To the best of our knowledge,this study is the first comprehensive analysis of nutlet morphology and complete chloroplast genome of Trigonotis.Based on nutlet morphology,Trigonotis can be divided into two groups:Group 1,hemispherical or oblique tetrahedron with carpopodiums,and Group 2,inverted tetrahedron without carpopodiums.The chloroplast genome of Trigonotis exhibited a typical quadripartite structure,including 84-86 protein-coding,37 transfer RNA,and 8 ribosomal RNA genes,with a total length of 147,247-148,986 bp.Genes in the junctions were well conserved in Trigonotis,similar to those in other Boraginaceae s.str.species.Furthermore,Trigonotis chloroplast genomes showed relatively high diversity,with more conserved genic regions than intergenic regions;in addition,we detected 14 hot spots(Pi>0.005)in non-coding regions.Phylogenetic analyses based on chloroplast genome data identified highly resolved relationships between Trigonotis species.Specifically,Trigonotis was divided into two clades with strong support:one clade included species with hemispherical or oblique tetrahedron nutlets with carpopodiums and bracts,whereas the other clade included species with inverted tetrahedron nutlets without carpopodiums or bracts.Our results may inform future taxonomic,phylogenetic,and evolutionary studies on Boraginaceae.
基金Supported by the National Natural Science Foundation of China(No.31670116)the Guangdong Innovation Research Team Fund(No.2014ZT05S078)the Shenzhen Grant Plan for Science&Technology(Nos.JCYJ20160308095910917,JCYJ20170818100339597,JCYJ 20130329114940668)。
文摘Euglena gracilis is a unicellular green eukaryotic microalga that features characteristics of both plants and animals.The photosynthetic function of its chloroplast is easily lost under stress resulting in bleached mutants,while the physiological role of their residual plastid DNAs remains unclear.In this study,we obtained five bleached mutants by ofloxacin(Ofl)treatment,identified 12 residual plastid genes in five bleached mutants,and determined the mRNA levels in the wild type E.gracilis(WT)and one bleached mutant(OflB2)under dark and light stimulation conditions by quantitative reverse transcribed PCR(qRTPCR).Results show that the expression of all selected plastid genes in both WT and OflB2 mutant did not change significantly in darkness,while their responses to light stimulation were different.Under the light stimulation conditions,half of the genes did not change significantly,while most of the other genes were down-regulated in OflB2 mutant and up-regulated in WT.Therefore,the bleached mutant retains part of the plastid genome and the plastid relic is responsive to light.Our research will help to understand the functions of residual plastid DNA and evolution of chloroplasts.
