The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of t...The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.展开更多
Jasmonic acid(JA)plays important roles in plant resistance to insect herbivores.One important derivative of JA is 12-OH-JA,which is produced by two independent pathways:direct hydroxylation of JA by jasmonate-induced ...Jasmonic acid(JA)plays important roles in plant resistance to insect herbivores.One important derivative of JA is 12-OH-JA,which is produced by two independent pathways:direct hydroxylation of JA by jasmonate-induced oxygenases(JOXs)or hydrolyzation of 12-OH-JA-Ile.Yet the function of 12-OH-JA in plant-herbivore interactions remains largely unknown.In this study,we silenced four JOX homologs independently in the wild tobacco Nicotiana attenuata by virus-induced gene silencing(VIGS),and found that all four JOX homologs are involved in JA hydroxylation.Simultaneously silencing the four JA hydroxylases in VIGS-NaJOXs plants decreased herbivory-induced 12-OH-JA by 33%,but JA and JA-Ile levels increased by 45%and 30%,respectively,compared to those in control plants.Compared to direct hydroxylation from JA,hydrolyzation from 12-OH-JA-Ile is equally important for herbivory-induced 12-OHJA accumulation:in the 12-OH-JA-Ile deficient irJAR4/6 plants,12-OH-JA decreased 34%.Moreover,VIGSNaJOXs plants exhibited enhanced resistance to the generalist herbivore Spodoptera litura.The poor larval performance was strongly correlated with high levels of several JA-Ile-dependent direct defense metabolites in the VIGS-NaJOXs plants.When we simultaneously silenced all four JA hydroxylases in the JAIle-deficient irJAR4/6 background,the enhanced herbivore resistance diminished,demonstrating that enhanced herbivore resistance resulted from elevated JA-Ile levels.Given that silencing these NaJOX-like genes did not detectably alter plant growth but highly increased plant defense levels,we propose that JOX genes are potential targets for genetic improvement of herbivore-resistant crops.展开更多
The bHLH transcription factors play pivotal roles in plant growth and development,production of secondary metabolites and responses to various environmental stresses.Although the bHLH genes have been well studied in m...The bHLH transcription factors play pivotal roles in plant growth and development,production of secondary metabolites and responses to various environmental stresses.Although the bHLH genes have been well studied in model plant species,a comprehensive investigation of the bHLH genes is required for tobacco with newly obtained high-quality genome.In the present study,a total of 309 NtbHLH genes were identified and can be divided into 23 subfamilies.The conserved amino acids which are essential for their function were predicted for the NtbHLH proteins.Moreover,the NtbHLH genes were conserved during evolution through analyzing the gene structures and conserved motifs.A total of 265 NtbHLH genes were localized in the 24 tobacco chromosomes while the remained 44 NtbHLH genes were mapped to the scaffolds due to the complexity of tobacco genome.Moreover,transcripts of NtbHLH genes were obviously tissue-specific expressed from the gene-chip data from 23 tobacco tissues,and expressions of 20 random selected NtbHLH genes were further confirmed by quantitative real-time PCR,indicating their potential functions in the plant growth and development.Importantly,overexpressed NtbHLH86 gene confers improve drought tolerance in tobacco indicating that it might be involved in the regulation of drought stress.Therefore,our findings here provide a valuable information on the characterization of NtbHLH genes and further investigation of their functions in tobacco.展开更多
In order to select the strain that can degrade nitrite, the screening plate with nitrite was used as the sole nitrogen source to screen the strain with ability to degrade nitrite. A strain with nitrite degrading capac...In order to select the strain that can degrade nitrite, the screening plate with nitrite was used as the sole nitrogen source to screen the strain with ability to degrade nitrite. A strain with nitrite degrading capacity was isolated from the sludge of a shrimp-farming pond in Hepu City, Guangxi Zhuang Autonomous Region. The isolated strain was identified based on colonial morphology, physiological and biochemical characteristics, and 16S rRNA sequence. Results showed that the strain could grow well on the culture medium containing 2.3 g/L nitrite. According to the morphological characteristics, nitrogen source requirements and evolutionary tree of the 16S rRNA sequence, the isolated strain was identified as Acinetobacter radioresistens, which was named HPAR132 strain. This study laid the foundation for further investigation of nitrite-oxidizing bacterium HPAR132.展开更多
CRISPR/Cas is a simple,robust,versatile tool for plant biology studies and precision plant breeding.However,establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa(M...CRISPR/Cas is a simple,robust,versatile tool for plant biology studies and precision plant breeding.However,establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa(Medicago sativa L.),the most important forage legume worldwide,remains a formidable challenge.Here,we systematically identified endogenous U6 promoters in alfalfa through transient expression via Agrobacterium-mediated infiltration of alfalfa leaves.We further demonstrated the efficacy of the three most active promoters for genome editing using an optimized alfalfa hairy root system.Subsequently,we established an improved CRISPR/Cas9 multiplex system containing three or four tandemly arrayed MsU6-promoter-driven polycistronic tRNA-sgRNA(PTG)expression cassettes,each consisting of three tRNA-sgRNA units,to simultaneously edit three or four alfalfa genes,coupled with the visual reporter RH1 or RUBY.This toolkit showed efficient multiplex editing in the hairy root system with visual selection.We successfully obtained regenerated,red-colored shoots resulting from the stable transformation of alfalfa.These results highlight the potential application of the visual reporter system for the stable transformation of alfalfa.Our improved CRISPR/Cas9 multiplex system enables convenient,high-efficiency multiplex genome editing in alfalfa,providing a versatile toolset to facilitate functional studies of multiple genes and gene families for basic research and the genetic improvement of alfalfa.展开更多
Recessive resistance mediated by mutations in the eukaryotic translation initiation factor 4E(eIF4E),has proven effective against diverse potyviruses and is extensively utilized in breeding programs.However,the rise o...Recessive resistance mediated by mutations in the eukaryotic translation initiation factor 4E(eIF4E),has proven effective against diverse potyviruses and is extensively utilized in breeding programs.However,the rise of resistance-breaking(RB)strains and emerging potyviral species necessitates the development of more durable and broad-spectrum resistance strategies.In this study,our field survey in Yunnan,China,identified potato virus Y(PVY)RB isolates,as well as the prevalence of tobacco vein banding mosaic virus(TVBMV)and chilli veinal mottle virus(ChiVMV),in tobacco carrying the recessive va locus,which lacks the eIF4E1-S susceptibility gene,due to a chromosomal deletion.Protein interaction and viral infection assays demonstrated that both eIF4E1-S and eIFiso4E-T are used by PVY RB as susceptibility factors for infection,with the combined inactivation of these genes confering durable resistance.Similarly,the knockout of eIFiso4E-S,in the va genetic background,provided effective resistance to TVBMV and reduced susceptibility to ChiVMV.Notably,pyramiding mutations in eIFiso4E-S and eIFiso4E-T,in va tobacco,generated plants exhibiting robust,broad-spectrum resistance,to all three viruses,without compromising plant development.These findings underscore the potential of stacking eIF4E mutations to engineer durable,broad-spectrum resistance to potyviruses in tobacco,offering a promising strategy for crop improvement.展开更多
Targeting-induced local lesions in genomes(TILLING)is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants.Although TILLING has been developed for many diploid plants...Targeting-induced local lesions in genomes(TILLING)is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants.Although TILLING has been developed for many diploid plants,the technology has been used in very few polyploid species due to their genomic complexity.Here,we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco(Nicotiana tabacum L.)using an ethyl methanesulfonate(EMS)-mutagenized population of 1,536 individuals.We optimized the procedures for endonuclease preparation,leaf tissue sampling,DNA extraction,normalization,pooling,PCR amplification,heteroduplex formation,and capillary electrophoresis.In a test screen using seven target genes with eight PCR fragments,we obtained 118 mutants.The mutation density was estimated to be approximately one mutation per 106kb on average.Phenotypic analyses showed that mutations in two heavy metal transporter genes,HMA2S and HMA4T,led to reduced accumulation of cadmium and zinc,which was confirmed independently using CRISPR/Cas9 to generate knockout mutants.Our results demonstrate that this powerful TILLING platform(available at http://www.croptilling.org)can be used in tobacco to facilitate functional genomics applications.展开更多
An investigation on the optimization of parental RNA interference （RNAi） conditions for hunchback （hb） gene in Locusta migratoria manilensis （Meyen） was conducted. Double stranded RNA （dsRNA） corresponding to ...An investigation on the optimization of parental RNA interference （RNAi） conditions for hunchback （hb） gene in Locusta migratoria manilensis （Meyen） was conducted. Double stranded RNA （dsRNA） corresponding to hb gene was injected into haemocoel of female adults ofL. migratoria manilensis. Embryos developed from the eggs laid by the injected adults on the 7th day after eclosion showed observable effects of RNAi for hb. The silencing effect after delivery treatment of dsRNA for hb gene was maintained for more than 21 days. A significant decrease of hb transcripts was further confirmed by Northern blot analysis. The dose of dsRNA/insect at 2μg could trigger 96.9% RNAi effects, while silencing appeared to have no dependence on the size of dsRNA. Results suggest that parental RNAi could be employed to efficiently identify the developmental gene functions in L. migratoria manilensis.展开更多
In the attempt to discover new genes involved in the floral development in monocotyledonousin species,we have cloned and characterized the homologous PISTALLATA-like(PI-like)gene from Phalaenopsis hybrid cultivar name...In the attempt to discover new genes involved in the floral development in monocotyledonousin species,we have cloned and characterized the homologous PISTALLATA-like(PI-like)gene from Phalaenopsis hybrid cultivar named PhPI9(Phalaenopsis PI STILLATA#9).The cDNA of PhPI9 has a fragment of 834 bp and has 60%identity with the PISTILATA from Arabidopsis.The deduced amino acid sequence of PhPI9 had the typical PI-motif.It also formed a subclade with other monocot PI-type genes in phylogenetic analysis.Southern analysis showed that PhPI9 was present in the Phalaenopsis orchid genome as a single copy.Furthermore,it was expressed only in the lip of the Phalaenopsis flower and no expression was detected in vegetative organs.Thus,as a B-function MADS-box gene,PhPI9 specifies floral organ identity in orchids.展开更多
基金supported by the National Natural Science Foundation of China(32001532 and 31860411)the National Key Research and Development Program of China,(2022YFF1000020)+1 种基金Hunan Seed Industry Innovation Project(2021NK1012)the Yunnan Tobacco Company Project(2020530000241009)。
文摘The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.
基金supported by the Key Project of Applied Basic Research Program of Yunnan(2017FA015)the Young Academic and Technical Leader Raising Foundation of Yunnan Province(no. 2017HB063)+1 种基金the Yunnan Academy of Tobacco Agricultural Sciences(2018530000241002 and 2019530000241003)the Biotechnology Experimental Center at the Kunming Institute of Botany,CAS,for supporting plant cultivation.
文摘Jasmonic acid(JA)plays important roles in plant resistance to insect herbivores.One important derivative of JA is 12-OH-JA,which is produced by two independent pathways:direct hydroxylation of JA by jasmonate-induced oxygenases(JOXs)or hydrolyzation of 12-OH-JA-Ile.Yet the function of 12-OH-JA in plant-herbivore interactions remains largely unknown.In this study,we silenced four JOX homologs independently in the wild tobacco Nicotiana attenuata by virus-induced gene silencing(VIGS),and found that all four JOX homologs are involved in JA hydroxylation.Simultaneously silencing the four JA hydroxylases in VIGS-NaJOXs plants decreased herbivory-induced 12-OH-JA by 33%,but JA and JA-Ile levels increased by 45%and 30%,respectively,compared to those in control plants.Compared to direct hydroxylation from JA,hydrolyzation from 12-OH-JA-Ile is equally important for herbivory-induced 12-OHJA accumulation:in the 12-OH-JA-Ile deficient irJAR4/6 plants,12-OH-JA decreased 34%.Moreover,VIGSNaJOXs plants exhibited enhanced resistance to the generalist herbivore Spodoptera litura.The poor larval performance was strongly correlated with high levels of several JA-Ile-dependent direct defense metabolites in the VIGS-NaJOXs plants.When we simultaneously silenced all four JA hydroxylases in the JAIle-deficient irJAR4/6 background,the enhanced herbivore resistance diminished,demonstrating that enhanced herbivore resistance resulted from elevated JA-Ile levels.Given that silencing these NaJOX-like genes did not detectably alter plant growth but highly increased plant defense levels,we propose that JOX genes are potential targets for genetic improvement of herbivore-resistant crops.
基金funded by the National Natural Science Foundation of China(grant number 31760072 to G.Bai,and grant number 31860413 to H.Xie)Yunnan Applied Basic Research Project(grant number 202001AT070010 to G.Bai)the Yunnan Academy of Tobacco Agricultural Sciences(grant numbers YNTC-2016YN22 and CNTC-110202001025(JY08)to H.Xie,YNTC-2016YN24,YNTC-2015YN02,YNTC-2018530000241002,and YNTC-2019530000241003 to D.-H.Yang).
文摘The bHLH transcription factors play pivotal roles in plant growth and development,production of secondary metabolites and responses to various environmental stresses.Although the bHLH genes have been well studied in model plant species,a comprehensive investigation of the bHLH genes is required for tobacco with newly obtained high-quality genome.In the present study,a total of 309 NtbHLH genes were identified and can be divided into 23 subfamilies.The conserved amino acids which are essential for their function were predicted for the NtbHLH proteins.Moreover,the NtbHLH genes were conserved during evolution through analyzing the gene structures and conserved motifs.A total of 265 NtbHLH genes were localized in the 24 tobacco chromosomes while the remained 44 NtbHLH genes were mapped to the scaffolds due to the complexity of tobacco genome.Moreover,transcripts of NtbHLH genes were obviously tissue-specific expressed from the gene-chip data from 23 tobacco tissues,and expressions of 20 random selected NtbHLH genes were further confirmed by quantitative real-time PCR,indicating their potential functions in the plant growth and development.Importantly,overexpressed NtbHLH86 gene confers improve drought tolerance in tobacco indicating that it might be involved in the regulation of drought stress.Therefore,our findings here provide a valuable information on the characterization of NtbHLH genes and further investigation of their functions in tobacco.
文摘In order to select the strain that can degrade nitrite, the screening plate with nitrite was used as the sole nitrogen source to screen the strain with ability to degrade nitrite. A strain with nitrite degrading capacity was isolated from the sludge of a shrimp-farming pond in Hepu City, Guangxi Zhuang Autonomous Region. The isolated strain was identified based on colonial morphology, physiological and biochemical characteristics, and 16S rRNA sequence. Results showed that the strain could grow well on the culture medium containing 2.3 g/L nitrite. According to the morphological characteristics, nitrogen source requirements and evolutionary tree of the 16S rRNA sequence, the isolated strain was identified as Acinetobacter radioresistens, which was named HPAR132 strain. This study laid the foundation for further investigation of nitrite-oxidizing bacterium HPAR132.
基金supported by grants from the Biological Breeding-National Science and Technology Major Project(2022ZD04011,2023ZD04073)National Natural Science Foundation of China(32325035,32071864)+4 种基金Fundamental Research Funds for Central Non-profit Scientific Institution(1610392024008)Central Laboratory of Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Hainan Seed Industry Laboratory(B23CJ0208)Yunnan Academy of Tobacco Agricultural Sciences(CNTC-YC2022530000241013,CNTC110201501015(JY-02))Hohhot Key R&D Project(2023-JBGS-S-1)Inner Mongolia Center of Pratacultural Technology Innovation(CCPTZX2023B01).
文摘CRISPR/Cas is a simple,robust,versatile tool for plant biology studies and precision plant breeding.However,establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa(Medicago sativa L.),the most important forage legume worldwide,remains a formidable challenge.Here,we systematically identified endogenous U6 promoters in alfalfa through transient expression via Agrobacterium-mediated infiltration of alfalfa leaves.We further demonstrated the efficacy of the three most active promoters for genome editing using an optimized alfalfa hairy root system.Subsequently,we established an improved CRISPR/Cas9 multiplex system containing three or four tandemly arrayed MsU6-promoter-driven polycistronic tRNA-sgRNA(PTG)expression cassettes,each consisting of three tRNA-sgRNA units,to simultaneously edit three or four alfalfa genes,coupled with the visual reporter RH1 or RUBY.This toolkit showed efficient multiplex editing in the hairy root system with visual selection.We successfully obtained regenerated,red-colored shoots resulting from the stable transformation of alfalfa.These results highlight the potential application of the visual reporter system for the stable transformation of alfalfa.Our improved CRISPR/Cas9 multiplex system enables convenient,high-efficiency multiplex genome editing in alfalfa,providing a versatile toolset to facilitate functional studies of multiple genes and gene families for basic research and the genetic improvement of alfalfa.
基金supported by National Natural Science Foundation of China(31860490)YNTC funds(2023530000241007 and 2017YN02)+1 种基金Yunnan Daguan Lab(YNDG202302ZY01)CLZ is supported by the Postdoctoral Fellowship Program of CPSF under grant number GZC20241527。
文摘Recessive resistance mediated by mutations in the eukaryotic translation initiation factor 4E(eIF4E),has proven effective against diverse potyviruses and is extensively utilized in breeding programs.However,the rise of resistance-breaking(RB)strains and emerging potyviral species necessitates the development of more durable and broad-spectrum resistance strategies.In this study,our field survey in Yunnan,China,identified potato virus Y(PVY)RB isolates,as well as the prevalence of tobacco vein banding mosaic virus(TVBMV)and chilli veinal mottle virus(ChiVMV),in tobacco carrying the recessive va locus,which lacks the eIF4E1-S susceptibility gene,due to a chromosomal deletion.Protein interaction and viral infection assays demonstrated that both eIF4E1-S and eIFiso4E-T are used by PVY RB as susceptibility factors for infection,with the combined inactivation of these genes confering durable resistance.Similarly,the knockout of eIFiso4E-S,in the va genetic background,provided effective resistance to TVBMV and reduced susceptibility to ChiVMV.Notably,pyramiding mutations in eIFiso4E-S and eIFiso4E-T,in va tobacco,generated plants exhibiting robust,broad-spectrum resistance,to all three viruses,without compromising plant development.These findings underscore the potential of stacking eIF4E mutations to engineer durable,broad-spectrum resistance to potyviruses in tobacco,offering a promising strategy for crop improvement.
基金supported by Yunnan Province Tobacco funding(2014YN05,2014YN06,2016YN25,2017YN03)Bureau of National Tobacco funding(110201401006[JY-06],110201601031[JY-05],110201401005[JY-05]).
文摘Targeting-induced local lesions in genomes(TILLING)is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants.Although TILLING has been developed for many diploid plants,the technology has been used in very few polyploid species due to their genomic complexity.Here,we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco(Nicotiana tabacum L.)using an ethyl methanesulfonate(EMS)-mutagenized population of 1,536 individuals.We optimized the procedures for endonuclease preparation,leaf tissue sampling,DNA extraction,normalization,pooling,PCR amplification,heteroduplex formation,and capillary electrophoresis.In a test screen using seven target genes with eight PCR fragments,we obtained 118 mutants.The mutation density was estimated to be approximately one mutation per 106kb on average.Phenotypic analyses showed that mutations in two heavy metal transporter genes,HMA2S and HMA4T,led to reduced accumulation of cadmium and zinc,which was confirmed independently using CRISPR/Cas9 to generate knockout mutants.Our results demonstrate that this powerful TILLING platform(available at http://www.croptilling.org)can be used in tobacco to facilitate functional genomics applications.
文摘An investigation on the optimization of parental RNA interference （RNAi） conditions for hunchback （hb） gene in Locusta migratoria manilensis （Meyen） was conducted. Double stranded RNA （dsRNA） corresponding to hb gene was injected into haemocoel of female adults ofL. migratoria manilensis. Embryos developed from the eggs laid by the injected adults on the 7th day after eclosion showed observable effects of RNAi for hb. The silencing effect after delivery treatment of dsRNA for hb gene was maintained for more than 21 days. A significant decrease of hb transcripts was further confirmed by Northern blot analysis. The dose of dsRNA/insect at 2μg could trigger 96.9% RNAi effects, while silencing appeared to have no dependence on the size of dsRNA. Results suggest that parental RNAi could be employed to efficiently identify the developmental gene functions in L. migratoria manilensis.
文摘In the attempt to discover new genes involved in the floral development in monocotyledonousin species,we have cloned and characterized the homologous PISTALLATA-like(PI-like)gene from Phalaenopsis hybrid cultivar named PhPI9(Phalaenopsis PI STILLATA#9).The cDNA of PhPI9 has a fragment of 834 bp and has 60%identity with the PISTILATA from Arabidopsis.The deduced amino acid sequence of PhPI9 had the typical PI-motif.It also formed a subclade with other monocot PI-type genes in phylogenetic analysis.Southern analysis showed that PhPI9 was present in the Phalaenopsis orchid genome as a single copy.Furthermore,it was expressed only in the lip of the Phalaenopsis flower and no expression was detected in vegetative organs.Thus,as a B-function MADS-box gene,PhPI9 specifies floral organ identity in orchids.
