Soil organic carbon(SOC)is the most important indicators of soil quality and health.Identifying the spatial distribution of SOC and its influencing factors in cropland is crucial to understand the terrestrial carbon c...Soil organic carbon(SOC)is the most important indicators of soil quality and health.Identifying the spatial distribution of SOC and its influencing factors in cropland is crucial to understand the terrestrial carbon cycle and optimize agronomic management.Yunnan Province,characterized by mountainous topography and varied elevation,is one of the highest SOC regions in China.Yet its SOC stock of cropland and influencing factors has not been fully studied due to the lack of adequate soil investigation.In this study,the digital mapping of SOC at 1 km resolution and the estimation of total SOC stock in cropland of Yunnan Province was undertaken using 8637 topsoil(0-20 cm)samples and a series of spatial data through Random Forest(RF)model.It was showed that across the cropland of Yunnan Province,the mean SOC density and total stock were 4.84 kg m^(-2) and 337.5 Mt,respectively.The spatial distribution indicated that relatively high SOC density regions resided in the northwest and northeast parts of Yunnan Province.Elevation(19.5%),temperature(17.3%),rainfall(14.5%),and Topographic wetness index(9.9%)were the most important factors which controlled spatial variability of SOC density.Agronomic practices(e.g.,crop straw treatments,fertilizer management)should be optimized for the sustainable development of crop production with high SOC sequestration capacity in Yunnan Province.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
[ Objective ] To figure out the optimal setting height of gyplure trappers for controlling such pests as Spodoptera litura, Helicoverpa assuha, Helicoverpa armigera Hubner and Agrotis ypsilon that endanger tobacco lea...[ Objective ] To figure out the optimal setting height of gyplure trappers for controlling such pests as Spodoptera litura, Helicoverpa assuha, Helicoverpa armigera Hubner and Agrotis ypsilon that endanger tobacco leaves, and screen trappers suitable for the tobacco production. [ Method] In the growth season of to- bacco plants, moth trapper, new moth trapper lure/insect inlet are set up at four different heights （70, 110, 150 and 190 cm vertically from soil surface in the field）, to investigate the quantity of trapped moths. [ Result ] For both trappers, the height of trapping the most moths is 70 - 150 cm, so the initial height of lure/ insect inlet should be 120 cm, considering the 1-m mature tobacco plant height and the 20-cm distance between lure/insect inlet and crop canopy. [ Conclusion] Mother trapper is cheaper, and the new moth trapper is convenient and requires less management cost. The former is favorable for trapping S. litura, and the latter for H. assulta, H. armigera Hubner, and both of them are suitable for trapping A. ypsilon. In tobacco producing regions of Yunnan, population bases of the four noctuids endangering tobacco leaves show the sequence of S. litura 〉 A. ypsilon 〉 H. armigera Hubner 〉 H. assuha. But in tobacco production, H. armigera Hubner, H. assuha, and A. ypsilon cause the most serious damages, so the four noctuids must be controlled. Key words Tobacco noctuid; Sex lure; Trapper; Setting height展开更多
In order to validate the control effects of integrated technology of medicine and fertilizer on tobacco bacterial wilt,field experiments were conducted by using youmeili and tetracycline as the test agents.The results...In order to validate the control effects of integrated technology of medicine and fertilizer on tobacco bacterial wilt,field experiments were conducted by using youmeili and tetracycline as the test agents.The results obtained were listed as follows:(1)The incidence rate of bacterial wilt ascended more quickly after July 10 th,the incidence of CK reached 78.30%on July 25 th.(2)The control effect of“youmeili 3 g+tetramycin 200 times liquid”reached 69.47%,significantly higher than other treatments.(3)The output value of“Youmeil 3 g+tetramycin 200 times liquid”served as 1635.66 yuan/667m^2,with the highest economic character in all treatments.In a word,integrated technology of medicine and fertilizer was a feasible approach to control tobacco bacterial wilt.展开更多
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
Pleiotropic drug resistance(PDR) transporters are widely distributed membrane proteins catalyzing the export or import of a diverse array of molecules, and are involved in many plant responses to biotic and abiotic st...Pleiotropic drug resistance(PDR) transporters are widely distributed membrane proteins catalyzing the export or import of a diverse array of molecules, and are involved in many plant responses to biotic and abiotic stresses. However, it is unclear whether PDRs are involved in Nicotiana attenuata resistance to the necrotic fungal pathogen Alternaria alternata. In this study, transcriptional levels of both NaPDR1 and NaPDR1-like were highly induced in N. attenuata leaves after A. alternata inoculation. Interestingly,silencing NaPDR1 or NaPDR1-like individually had little effect on N. attenuata resistance to A. alternata;however, when both genes were co-silenced plants became highly susceptible to the fungus, which was associated with elevated JA and ethylene responses. Neither NaPDR1 nor NaPDR1-like was significantly elicited by exogenous treatment with methyl jasmonate(MeJA), whereas both were highly induced by ethylene. The elicitation levels of both genes by A. alternata were significantly reduced in plants with impaired JA or ethylene signaling pathways. Thus, we conclude that both NaPDR1 and NaPDR1-like function redundantly to confer resistance against A. alternata in N. attenuata, and the elicitation of the transcripts of both genes by the fungus is partially dependent on ethylene and jasmonate signaling.展开更多
Tobacco(Nicotiana tabacum) is one of the most economically important nonfood crops,and flue-cured tobacco accounts for approximately 80% of world tobacco production.An extremely narrow genetic diversity in the tobacco...Tobacco(Nicotiana tabacum) is one of the most economically important nonfood crops,and flue-cured tobacco accounts for approximately 80% of world tobacco production.An extremely narrow genetic diversity in the tobacco pool has led to a low efficiency of PCR-based molecular markers(such as AFLP and SSR).Diversity Arrays Technology(DArT) is a high-throughput hybridisation-based marker system that has been developed in many plants including wheat,which,like tobacco,has a complex genome.In this study,we developed a tobacco DArT chip that included 7680 representative sequence tags based on typical tobacco accessions.The 1076 DArT markers of flue-cured tobacco were identified and most(82.1%) of their polymorphism information contents(PICs) were greater than 0.4.An integrated linkage map that included 851 markers(238 DArT and 613 SSR),which is the highest density map of flue-cured tobacco to date,was constructed.This chip-based DArT system provides an alternative in high-throughput marker genotyping for tobacco.展开更多
Diversity arrays technology （DART） is a microarray-based marker system that achieves high throughput by reducing the complexity of the genome. A DArT chip has recently been developed for tobacco. In this study, we g...Diversity arrays technology （DART） is a microarray-based marker system that achieves high throughput by reducing the complexity of the genome. A DArT chip has recently been developed for tobacco. In this study, we genotyped 267 flue-cured cultivars/landraces, including 121 Chinese accessions over five decades from widespread geographic regions in China, 103 from the Americas, and 43 other foreign cultivars, using the newly developed chip. Three hundred and thirty polymorphic DArT makers were selected and used for a phylogenetic analysis, which sug- gested that the 267 accessions could be classified into two subgroups, which could each be further divided into 2-4 sections. Eight elite cultivars, which account for 83% of the area of Chinese tobacco production, were all found in one subgroup. Two high-quality cultivars, HHDJY and Cuibil, were grouped together in one section, while six other high-yield cultivars were grouped into another section. The 330 DArT marker clones were sequenced and close to 95% of them are within non-repetitive regions. Finally, the implications of this study for Chinese flue-cured tobacco breeding and production programs were discussed.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
Molecularly imprinted polymers(MIPs)for solid-phase extraction and pre-concentration of esculetin have been successfully prepared by the bulk polymerization method using esculetin as a template molecule.Polymers of va...Molecularly imprinted polymers(MIPs)for solid-phase extraction and pre-concentration of esculetin have been successfully prepared by the bulk polymerization method using esculetin as a template molecule.Polymers of varying composition were prepared using different monomers(4-vinylpyridine,methacrylic acid,and acrylamide),ethylene glycol dimethacrylate as the cross-linker,2,2-azobis(2-methylpropinitrile)as the initiator,and different porogen solvents(ethanol,acetone/methanol,and acetonitrile).The best polymer was obtained when 4-vinylpyridine was used as the monomer and acetone/methanol(3:2)as the porogen solvent,whereas the template:-monomer:-cross-linker ratio was 1:4:20.The imprinting factor of the selected MIPs for esculetin was 3.77.The polymers were evaluated according to their selective recognition properties for esculetin and structurally-related compounds(esculin,scopoletin,coumarin,and 7-methoxycoumarin).Chemical and morphological characterizations of the polymers were investigated by FTIR and scanning electron microscope,which confirmed a high degree of polymerization.Surface area,pore volume,and pore size of the polymer were investigated by Brunauer-Emmett-Teller analysis.MIPs were also successfully used as solid-phase adsorbent materials for the extraction of esculetin from tobacco leaves.Esculetin contents in dried tobacco leaves were found to be(9.27±0.17)μg g-1.展开更多
The phylogenetic diversity of phosphate solubilizing bacteria （PSB） distributed in P-rich soils in the Dianchi Lake drainage area of China was characterized, and the tricalcium phosphate （TCP） solubilizing activit...The phylogenetic diversity of phosphate solubilizing bacteria （PSB） distributed in P-rich soils in the Dianchi Lake drainage area of China was characterized, and the tricalcium phosphate （TCP） solubilizing activities of isolated PSB were determined. Among 1 328 bacteria isolated from 100 P-rich soil samples, 377 isolates （28.39% of the total） that exhibited TCP solubilization activity were taken as PSB. These PSB showed different abilities to solubilize TCP, with the concentrations of solubilized P in bacterial cultures varying from 33.48 to 69.63 mg L^（-1）. A total of 123 PSB isolates, with relatively high TCP solubilization activity （〉 54.00 mg L^（-1））, were submitted for restriction fragment length polymorphism （RFLP） analysis, which revealed 32 unique RFLP patterns. Based on these patterns, 62 representative isolates, one to three from each RFLP pattern, were selected for 16S rRNA sequencing. Phylogenetic analysis placed the 123 PSB into three bacterial phyla, namely Proteobacteria, Aetinobacteria and Firmicutes. Members of Proteobacteria were the dominant PSB, where 107 isolates represented by 26 RFLP patterns were associated with the genera of Burkholderia, Pseudomonas, Acinetobacter, Enterobacter, Pantoea, Serratia, Klebsiella, Leclercia, Raoultella and Cedeeea. Firmicutes were the subdominant group, in which 13 isolates were affiliated with the genera of Bacillus and Brevibacterium. The remaining 3 isolates were identified as three species of the genus Arthrobacter. This research extends the knowledge on PSB in P-rich soils and broadens the spectrum of PSB for the development of environmentally friendly biophosphate fertilizers.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
Being sessile,plants have evolved sophisticated mechanisms to balance between growth and defense to survive in the harsh environment.The transition from growth to defense is commonly achieved by factors,such as protei...Being sessile,plants have evolved sophisticated mechanisms to balance between growth and defense to survive in the harsh environment.The transition from growth to defense is commonly achieved by factors,such as protein kinases(PKs)and transcription factors,that initiate signal transduction and regulate specialized metabolism.Plants produce an array of lineage-specific specialized metabolites for chemical defense and stress tolerance.Some of these molecules are also used by humans as drugs.However,many of these defense-responsive metabolites are toxic to plant cells and inhibitory to growth and development.Plants have,thus,evolved complex regulatory networks to balance the accumulation of the toxic metabolites.Perception of external stimuli is a vital part of the regulatory network.Protein kinase-mediated signaling activates a series of defense responses by phosphorylating the target pro-teins and translating the stimulus into downstream cellular signaling.As biosynthesis of specialized metabolites is triggered when plants perceive stimuli,a possible connection between PKs and spe-cial ized meta bolism is well recognized.However,the roles of PKs in plant specialized metabolism have not received much attention until recently.Here,we summarize the recent advances in understanding PKs in plant specialized metabolism.We aim to highlight how the stimulatory signals are transduced,leading to the biosynthesis of corresponding metabolites.We discuss the post-translational regulation of specialized metabolism and provide insights into the mechanisms by which plants respond to the external signals.In addition,we propose possible strategies to increase the production of plant spe-cial ized metabolites in biotechnological applications using PKs.展开更多
基金funded by the Science and Technology Projects of Yunnan Province,China(2017YN06 and 2018BB019)the Key Research&Development project of Yunnan Province(2018530000241017)the National Natural Science Foundation of China(31671642)。
文摘Soil organic carbon(SOC)is the most important indicators of soil quality and health.Identifying the spatial distribution of SOC and its influencing factors in cropland is crucial to understand the terrestrial carbon cycle and optimize agronomic management.Yunnan Province,characterized by mountainous topography and varied elevation,is one of the highest SOC regions in China.Yet its SOC stock of cropland and influencing factors has not been fully studied due to the lack of adequate soil investigation.In this study,the digital mapping of SOC at 1 km resolution and the estimation of total SOC stock in cropland of Yunnan Province was undertaken using 8637 topsoil(0-20 cm)samples and a series of spatial data through Random Forest(RF)model.It was showed that across the cropland of Yunnan Province,the mean SOC density and total stock were 4.84 kg m^(-2) and 337.5 Mt,respectively.The spatial distribution indicated that relatively high SOC density regions resided in the northwest and northeast parts of Yunnan Province.Elevation(19.5%),temperature(17.3%),rainfall(14.5%),and Topographic wetness index(9.9%)were the most important factors which controlled spatial variability of SOC density.Agronomic practices(e.g.,crop straw treatments,fertilizer management)should be optimized for the sustainable development of crop production with high SOC sequestration capacity in Yunnan Province.
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.
基金Sponsored by Yunnan Provincial Tobacco Monopoly Administration(Company)Scientific and Technological Program(2012YN04,2010YN09)State Tobacco Monopoly Bureau(110200902065)
文摘[ Objective ] To figure out the optimal setting height of gyplure trappers for controlling such pests as Spodoptera litura, Helicoverpa assuha, Helicoverpa armigera Hubner and Agrotis ypsilon that endanger tobacco leaves, and screen trappers suitable for the tobacco production. [ Method] In the growth season of to- bacco plants, moth trapper, new moth trapper lure/insect inlet are set up at four different heights （70, 110, 150 and 190 cm vertically from soil surface in the field）, to investigate the quantity of trapped moths. [ Result ] For both trappers, the height of trapping the most moths is 70 - 150 cm, so the initial height of lure/ insect inlet should be 120 cm, considering the 1-m mature tobacco plant height and the 20-cm distance between lure/insect inlet and crop canopy. [ Conclusion] Mother trapper is cheaper, and the new moth trapper is convenient and requires less management cost. The former is favorable for trapping S. litura, and the latter for H. assulta, H. armigera Hubner, and both of them are suitable for trapping A. ypsilon. In tobacco producing regions of Yunnan, population bases of the four noctuids endangering tobacco leaves show the sequence of S. litura 〉 A. ypsilon 〉 H. armigera Hubner 〉 H. assuha. But in tobacco production, H. armigera Hubner, H. assuha, and A. ypsilon cause the most serious damages, so the four noctuids must be controlled. Key words Tobacco noctuid; Sex lure; Trapper; Setting height
基金Supported by Science and Technology Project of Yunnan Tobacco Company(2015YN07,2017YN26)~~
文摘In order to validate the control effects of integrated technology of medicine and fertilizer on tobacco bacterial wilt,field experiments were conducted by using youmeili and tetracycline as the test agents.The results obtained were listed as follows:(1)The incidence rate of bacterial wilt ascended more quickly after July 10 th,the incidence of CK reached 78.30%on July 25 th.(2)The control effect of“youmeili 3 g+tetramycin 200 times liquid”reached 69.47%,significantly higher than other treatments.(3)The output value of“Youmeil 3 g+tetramycin 200 times liquid”served as 1635.66 yuan/667m^2,with the highest economic character in all treatments.In a word,integrated technology of medicine and fertilizer was a feasible approach to control tobacco bacterial wilt.
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 NSFC (Grant No. 31670262)Key Project of Applied Basic Research Program of Yunnan (Grant No. 2014FA040)+2 种基金100-Oversea-Top-Talents Recruitment plan of Yunnan to jinsong WuProject of Applied Basic Research Program of Yunnan(Grant No. 2017FB048) to Lan Mafounding (Grant No. 110201603008) to Dunhuang Fang
文摘Pleiotropic drug resistance(PDR) transporters are widely distributed membrane proteins catalyzing the export or import of a diverse array of molecules, and are involved in many plant responses to biotic and abiotic stresses. However, it is unclear whether PDRs are involved in Nicotiana attenuata resistance to the necrotic fungal pathogen Alternaria alternata. In this study, transcriptional levels of both NaPDR1 and NaPDR1-like were highly induced in N. attenuata leaves after A. alternata inoculation. Interestingly,silencing NaPDR1 or NaPDR1-like individually had little effect on N. attenuata resistance to A. alternata;however, when both genes were co-silenced plants became highly susceptible to the fungus, which was associated with elevated JA and ethylene responses. Neither NaPDR1 nor NaPDR1-like was significantly elicited by exogenous treatment with methyl jasmonate(MeJA), whereas both were highly induced by ethylene. The elicitation levels of both genes by A. alternata were significantly reduced in plants with impaired JA or ethylene signaling pathways. Thus, we conclude that both NaPDR1 and NaPDR1-like function redundantly to confer resistance against A. alternata in N. attenuata, and the elicitation of the transcripts of both genes by the fungus is partially dependent on ethylene and jasmonate signaling.
基金supported by Yunnan Provincial Tobacco Company(08A05//2011YN04)China National Tobacco Company (110200701023/110201201003(JY-03))
文摘Tobacco(Nicotiana tabacum) is one of the most economically important nonfood crops,and flue-cured tobacco accounts for approximately 80% of world tobacco production.An extremely narrow genetic diversity in the tobacco pool has led to a low efficiency of PCR-based molecular markers(such as AFLP and SSR).Diversity Arrays Technology(DArT) is a high-throughput hybridisation-based marker system that has been developed in many plants including wheat,which,like tobacco,has a complex genome.In this study,we developed a tobacco DArT chip that included 7680 representative sequence tags based on typical tobacco accessions.The 1076 DArT markers of flue-cured tobacco were identified and most(82.1%) of their polymorphism information contents(PICs) were greater than 0.4.An integrated linkage map that included 851 markers(238 DArT and 613 SSR),which is the highest density map of flue-cured tobacco to date,was constructed.This chip-based DArT system provides an alternative in high-throughput marker genotyping for tobacco.
基金supported by the China National Tobacco Corporation (Nos. 110200701023 and 110201101010-JY-04)the Yunnan Provincial Tobacco Company (Nos. 08A05 and 2010YN02), China
文摘Diversity arrays technology （DART） is a microarray-based marker system that achieves high throughput by reducing the complexity of the genome. A DArT chip has recently been developed for tobacco. In this study, we genotyped 267 flue-cured cultivars/landraces, including 121 Chinese accessions over five decades from widespread geographic regions in China, 103 from the Americas, and 43 other foreign cultivars, using the newly developed chip. Three hundred and thirty polymorphic DArT makers were selected and used for a phylogenetic analysis, which sug- gested that the 267 accessions could be classified into two subgroups, which could each be further divided into 2-4 sections. Eight elite cultivars, which account for 83% of the area of Chinese tobacco production, were all found in one subgroup. Two high-quality cultivars, HHDJY and Cuibil, were grouped together in one section, while six other high-yield cultivars were grouped into another section. The 330 DArT marker clones were sequenced and close to 95% of them are within non-repetitive regions. Finally, the implications of this study for Chinese flue-cured tobacco breeding and production programs were discussed.
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supported by the Yunnan Green Tobacco Production Research(2011YN01)the China Equipment and Education Resources System(CERS-1-75)
文摘Molecularly imprinted polymers(MIPs)for solid-phase extraction and pre-concentration of esculetin have been successfully prepared by the bulk polymerization method using esculetin as a template molecule.Polymers of varying composition were prepared using different monomers(4-vinylpyridine,methacrylic acid,and acrylamide),ethylene glycol dimethacrylate as the cross-linker,2,2-azobis(2-methylpropinitrile)as the initiator,and different porogen solvents(ethanol,acetone/methanol,and acetonitrile).The best polymer was obtained when 4-vinylpyridine was used as the monomer and acetone/methanol(3:2)as the porogen solvent,whereas the template:-monomer:-cross-linker ratio was 1:4:20.The imprinting factor of the selected MIPs for esculetin was 3.77.The polymers were evaluated according to their selective recognition properties for esculetin and structurally-related compounds(esculin,scopoletin,coumarin,and 7-methoxycoumarin).Chemical and morphological characterizations of the polymers were investigated by FTIR and scanning electron microscope,which confirmed a high degree of polymerization.Surface area,pore volume,and pore size of the polymer were investigated by Brunauer-Emmett-Teller analysis.MIPs were also successfully used as solid-phase adsorbent materials for the extraction of esculetin from tobacco leaves.Esculetin contents in dried tobacco leaves were found to be(9.27±0.17)μg g-1.
基金the National Key Sciences and Technology Program for Water Solutions, China (Nos.2009ZX07102-004 and2012ZX07102-003)the National Natural Science Foundation of China (Nos.30970100 and 31160376)+1 种基金the Zhengzhou Tobacco Research Institute of China (No.122009CZ0420)the Department of Science and Technology of Yunnan Province, China (Nos.2011FA002and 11N010905)
文摘The phylogenetic diversity of phosphate solubilizing bacteria （PSB） distributed in P-rich soils in the Dianchi Lake drainage area of China was characterized, and the tricalcium phosphate （TCP） solubilizing activities of isolated PSB were determined. Among 1 328 bacteria isolated from 100 P-rich soil samples, 377 isolates （28.39% of the total） that exhibited TCP solubilization activity were taken as PSB. These PSB showed different abilities to solubilize TCP, with the concentrations of solubilized P in bacterial cultures varying from 33.48 to 69.63 mg L^（-1）. A total of 123 PSB isolates, with relatively high TCP solubilization activity （〉 54.00 mg L^（-1））, were submitted for restriction fragment length polymorphism （RFLP） analysis, which revealed 32 unique RFLP patterns. Based on these patterns, 62 representative isolates, one to three from each RFLP pattern, were selected for 16S rRNA sequencing. Phylogenetic analysis placed the 123 PSB into three bacterial phyla, namely Proteobacteria, Aetinobacteria and Firmicutes. Members of Proteobacteria were the dominant PSB, where 107 isolates represented by 26 RFLP patterns were associated with the genera of Burkholderia, Pseudomonas, Acinetobacter, Enterobacter, Pantoea, Serratia, Klebsiella, Leclercia, Raoultella and Cedeeea. Firmicutes were the subdominant group, in which 13 isolates were affiliated with the genera of Bacillus and Brevibacterium. The remaining 3 isolates were identified as three species of the genus Arthrobacter. This research extends the knowledge on PSB in P-rich soils and broadens the spectrum of PSB for the development of environmentally friendly biophosphate fertilizers.
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Being sessile,plants have evolved sophisticated mechanisms to balance between growth and defense to survive in the harsh environment.The transition from growth to defense is commonly achieved by factors,such as protein kinases(PKs)and transcription factors,that initiate signal transduction and regulate specialized metabolism.Plants produce an array of lineage-specific specialized metabolites for chemical defense and stress tolerance.Some of these molecules are also used by humans as drugs.However,many of these defense-responsive metabolites are toxic to plant cells and inhibitory to growth and development.Plants have,thus,evolved complex regulatory networks to balance the accumulation of the toxic metabolites.Perception of external stimuli is a vital part of the regulatory network.Protein kinase-mediated signaling activates a series of defense responses by phosphorylating the target pro-teins and translating the stimulus into downstream cellular signaling.As biosynthesis of specialized metabolites is triggered when plants perceive stimuli,a possible connection between PKs and spe-cial ized meta bolism is well recognized.However,the roles of PKs in plant specialized metabolism have not received much attention until recently.Here,we summarize the recent advances in understanding PKs in plant specialized metabolism.We aim to highlight how the stimulatory signals are transduced,leading to the biosynthesis of corresponding metabolites.We discuss the post-translational regulation of specialized metabolism and provide insights into the mechanisms by which plants respond to the external signals.In addition,we propose possible strategies to increase the production of plant spe-cial ized metabolites in biotechnological applications using PKs.
