本文对蒺藜苜蓿叶绿体基因组全序列密码子进行分析，筛选出50条CDS（coding DNA sequence）利用Codo-nW软件进行分析其密码子使用模式。结果显示，蒺藜苜蓿叶绿体基因组密码子第3位碱基GC含量为26．9％，即第3位密码子富含A和U，ENC值...本文对蒺藜苜蓿叶绿体基因组全序列密码子进行分析，筛选出50条CDS（coding DNA sequence）利用Codo-nW软件进行分析其密码子使用模式。结果显示，蒺藜苜蓿叶绿体基因组密码子第3位碱基GC含量为26．9％，即第3位密码子富含A和U，ENC值在37．11～51．91之间密码子偏好性较弱。相对同义密码子使用度分析显示RSCU值大于1的密码子有23个，其中以A和U为结尾20个。中性绘图分析显示GC12与GC3的相关系数为0．341，相关性不显著，回归系数为0．4843；单基因ENC比值多分布在-0．05～0．05，即大部分基因ENC值离ENC期望值较近；对应性分析，第一轴显示了12．50％的差异为主要影响因素，第一轴与ENC和GC。的相关系数分别为0．091和-0．092，均相关不显著。综合这几项分析发现蒺藜苜蓿叶绿体基因组密码子偏好性主要受到突变的影响，但是并不是唯一的影响因素，其他因素对密码子偏好性也可能有一定的影响。最终通过高表达优越密码子方法确定得出UUA、UUG、CCU等23个密码子为最优密码子，为之后对外源基因进行改造，提高其在叶绿体中的表达效率奠定了基础。展开更多
In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmac...In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 （CYP72A67） involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethyl- methanesulfonate （EMS） mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s （CYP72A68） responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities （leaves/roots） of sapogenin synthesis.展开更多
The R2R3-MYB genes make up one of the largest transcription factor families in plants, and play regulatory roles in various biological processes such as development, metabolism and defense response. Although genome-wi...The R2R3-MYB genes make up one of the largest transcription factor families in plants, and play regulatory roles in various biological processes such as development, metabolism and defense response. Although genome-wide analyses of this gene family have been conducted in several species, R2R3-MYB genes have not been systematically analyzed in Medicago truncatula, a sequenced model legume plant. Here, we performed a comprehensive, genome-wide computational analysis of the structural characteristics, phylogeny, functions and expression patterns of M. truncatula R2R3-MYB genes. DNA binding domains are highly conserved among the 155 putative MtR2R3-MYB proteins that we identified. Chromosomal location analysis revealed that these genes were distributed across all eight chromosomes. Results showed that the expansion of the MtR2R3-MYB family was mainly attributable to segmental duplication and tandem duplication. A comprehensive classification was performed based on phylogenetic analysis of the R2R3-MYB gene families in M. truncatula, Arabidopsis thaliana and other plant species. Evolutionary relationships within clades were supported by clade-specific conserved motifs outside the MYB domain. Species-specific clades have been gained or lost during evolution, resulting in functional divergence. Also, tissue-specific expression patterns were investigated. The functions of stress response-related clades were further verified by the changes in transcript levels of representative R2R3-MYB genes upon treatment with abiotic and biotic stresses. This study is the first report on identification and characterization of R2R3-MYB gene family based on the genome of M. truncatula, and will facilitate functional analysis of this gene family in the future.展开更多
以蒺藜苜蓿(Medica go truncatula)种子为试验材料,通过测定种子生活力、发芽率、吸水率及粗提物的生物活性;并用不同浓度的赤霉素(GA_3)浸种后4℃低温层积或同浓度硝普钠(SNP)浸种,揭示蒺藜苜蓿种子休眠原因及破除方法。结果表明:蒺藜...以蒺藜苜蓿(Medica go truncatula)种子为试验材料,通过测定种子生活力、发芽率、吸水率及粗提物的生物活性;并用不同浓度的赤霉素(GA_3)浸种后4℃低温层积或同浓度硝普钠(SNP)浸种,揭示蒺藜苜蓿种子休眠原因及破除方法。结果表明:蒺藜苜蓿种子具有生活力;30 d内裸粒种子发芽率为100%;破皮种子的吸水率显著高于完整种子;随着蒺藜苜蓿各部分粗提物浓度的升高,白三叶(Tri folium repens)草种子萌发和幼苗生长显著降低;0.1~0.7 mmo·L^(-1)的GA_3浸种后低温层积或同浓度SNP浸种均可以显著提高种子的发芽率,其中以0.1 mmol·L^(-1)GA_3浸种后低温层积或同浓度的SNP处理效果最好。表明蒺藜苜蓿种子的休眠属于综合性休眠,可用GA_3浸种后低温层积或SNP浸种来解除其休眠。展开更多
Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid （ABA） but how this hormone controls seed germination is...Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid （ABA） but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.展开更多
We identified de novo sucrose transporter （SUT） genes involved in long-distance transport of sucrose from photosynthetic source leaves towards sink organs in the model leguminous species Medicago truncatula. The ide...We identified de novo sucrose transporter （SUT） genes involved in long-distance transport of sucrose from photosynthetic source leaves towards sink organs in the model leguminous species Medicago truncatula. The iden- tification and functional analysis of sugar transporters provide key information on mechanisms that underlie carbon partitioning in plant-microorganism interactions. In that way, full-length sequences of the M. truncatula SUT （MtSUT） family were retrieved and biochemical characterization of MtSUT members was performed by heterologous expression in yeast. The MtSUT family now comprises six genes which distribute among Dicotyledonous clades. MtSUTI-1 and MtSUT4-1 are key members in regard to their expression profiles in source leaves and sink roots and were characterized as functional H~/sucrose transporters. Physiological and molecular responses to phosphorus supply and inoculation by the arbuscular mycorrhizal fungus （AMF） Glomus intraradices was studied by gene expression and sugar quantification analyses. Sucrose represents the main sugar transport form in M. truncatula and the expression profiles of MtSUTI-1, MtSUT2, and MtSUT4-1 highlight a fine-tuning regulation for beneficial sugar fluxes towards the fungal symbiont. Taken together, these results suggest distinct functions for proteins from the SUT1, SUT2, and SUT4 clades in plant and in bio- trophic interactions.展开更多
Synonymous codon usage pattern presumably reflects gene expression optimization as a result of molecular evolution. Though much attention has been paid to various model organisms ranging from prokaryotes to eukaryotes...Synonymous codon usage pattern presumably reflects gene expression optimization as a result of molecular evolution. Though much attention has been paid to various model organisms ranging from prokaryotes to eukaryotes, codon usage has yet been extensively investigated for model legume Medicago truncatula. In present study, 39 531 available coding sequences （CDSs） from M. truncatula were examined for codon usage bias （CUB）. Based on analyses including neutrality plots, effective number of codons plots, and correlations between optimal codons frequency and codon adaptation index, we conclude that natural selection is a major driving force in M. truncatula CUB. We have identified 30 optimal codons encoding 18 amino acids based on relative synonymous codon usage. These optimal codons characteristically end with A or T, except for AGG and TTG encoding arginine and leucine respectively. Optimal codon usage is positively correlated with the GC content at three nucleotide positions of codons and the GC content of CDSs. The abundance of expressed sequence tag is a proxy for gene expression intensity in the legume, but has no relatedness with either CDS length or GC content. Collectively, we unravel the synonymous codon usage pattern in M. truncatula, which may serve as the valuable information on genetic engineering of the model legume and forage crop.展开更多
文摘本文对蒺藜苜蓿叶绿体基因组全序列密码子进行分析，筛选出50条CDS（coding DNA sequence）利用Codo-nW软件进行分析其密码子使用模式。结果显示，蒺藜苜蓿叶绿体基因组密码子第3位碱基GC含量为26．9％，即第3位密码子富含A和U，ENC值在37．11～51．91之间密码子偏好性较弱。相对同义密码子使用度分析显示RSCU值大于1的密码子有23个，其中以A和U为结尾20个。中性绘图分析显示GC12与GC3的相关系数为0．341，相关性不显著，回归系数为0．4843；单基因ENC比值多分布在-0．05～0．05，即大部分基因ENC值离ENC期望值较近；对应性分析，第一轴显示了12．50％的差异为主要影响因素，第一轴与ENC和GC。的相关系数分别为0．091和-0．092，均相关不显著。综合这几项分析发现蒺藜苜蓿叶绿体基因组密码子偏好性主要受到突变的影响，但是并不是唯一的影响因素，其他因素对密码子偏好性也可能有一定的影响。最终通过高表达优越密码子方法确定得出UUA、UUG、CCU等23个密码子为最优密码子，为之后对外源基因进行改造，提高其在叶绿体中的表达效率奠定了基础。
文摘In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 （CYP72A67） involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethyl- methanesulfonate （EMS） mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s （CYP72A68） responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities （leaves/roots） of sapogenin synthesis.
基金supported by the National Natural Science Foundation of China(31372362)
文摘The R2R3-MYB genes make up one of the largest transcription factor families in plants, and play regulatory roles in various biological processes such as development, metabolism and defense response. Although genome-wide analyses of this gene family have been conducted in several species, R2R3-MYB genes have not been systematically analyzed in Medicago truncatula, a sequenced model legume plant. Here, we performed a comprehensive, genome-wide computational analysis of the structural characteristics, phylogeny, functions and expression patterns of M. truncatula R2R3-MYB genes. DNA binding domains are highly conserved among the 155 putative MtR2R3-MYB proteins that we identified. Chromosomal location analysis revealed that these genes were distributed across all eight chromosomes. Results showed that the expansion of the MtR2R3-MYB family was mainly attributable to segmental duplication and tandem duplication. A comprehensive classification was performed based on phylogenetic analysis of the R2R3-MYB gene families in M. truncatula, Arabidopsis thaliana and other plant species. Evolutionary relationships within clades were supported by clade-specific conserved motifs outside the MYB domain. Species-specific clades have been gained or lost during evolution, resulting in functional divergence. Also, tissue-specific expression patterns were investigated. The functions of stress response-related clades were further verified by the changes in transcript levels of representative R2R3-MYB genes upon treatment with abiotic and biotic stresses. This study is the first report on identification and characterization of R2R3-MYB gene family based on the genome of M. truncatula, and will facilitate functional analysis of this gene family in the future.
文摘Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid （ABA） but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.
文摘We identified de novo sucrose transporter （SUT） genes involved in long-distance transport of sucrose from photosynthetic source leaves towards sink organs in the model leguminous species Medicago truncatula. The iden- tification and functional analysis of sugar transporters provide key information on mechanisms that underlie carbon partitioning in plant-microorganism interactions. In that way, full-length sequences of the M. truncatula SUT （MtSUT） family were retrieved and biochemical characterization of MtSUT members was performed by heterologous expression in yeast. The MtSUT family now comprises six genes which distribute among Dicotyledonous clades. MtSUTI-1 and MtSUT4-1 are key members in regard to their expression profiles in source leaves and sink roots and were characterized as functional H~/sucrose transporters. Physiological and molecular responses to phosphorus supply and inoculation by the arbuscular mycorrhizal fungus （AMF） Glomus intraradices was studied by gene expression and sugar quantification analyses. Sucrose represents the main sugar transport form in M. truncatula and the expression profiles of MtSUTI-1, MtSUT2, and MtSUT4-1 highlight a fine-tuning regulation for beneficial sugar fluxes towards the fungal symbiont. Taken together, these results suggest distinct functions for proteins from the SUT1, SUT2, and SUT4 clades in plant and in bio- trophic interactions.
基金supported by the National Basic Research Program of China (2014CB138702)the National Natural Science Foundation of China (31502001)
文摘Synonymous codon usage pattern presumably reflects gene expression optimization as a result of molecular evolution. Though much attention has been paid to various model organisms ranging from prokaryotes to eukaryotes, codon usage has yet been extensively investigated for model legume Medicago truncatula. In present study, 39 531 available coding sequences （CDSs） from M. truncatula were examined for codon usage bias （CUB）. Based on analyses including neutrality plots, effective number of codons plots, and correlations between optimal codons frequency and codon adaptation index, we conclude that natural selection is a major driving force in M. truncatula CUB. We have identified 30 optimal codons encoding 18 amino acids based on relative synonymous codon usage. These optimal codons characteristically end with A or T, except for AGG and TTG encoding arginine and leucine respectively. Optimal codon usage is positively correlated with the GC content at three nucleotide positions of codons and the GC content of CDSs. The abundance of expressed sequence tag is a proxy for gene expression intensity in the legume, but has no relatedness with either CDS length or GC content. Collectively, we unravel the synonymous codon usage pattern in M. truncatula, which may serve as the valuable information on genetic engineering of the model legume and forage crop.
