Genetic transformation is one of the key steps in the molecular breeding of chrysanthemum,which relies on an optimal regeneration and transformation system.However,the regeneration system of different chrysanthemum cu...Genetic transformation is one of the key steps in the molecular breeding of chrysanthemum,which relies on an optimal regeneration and transformation system.However,the regeneration system of different chrysanthemum cultivars varies,and the regeneration time of most cultivars is long.To screen cultivars with highly efficient regeneration,leaves and shoot tip thin cell layers(tTCL)from eight chrysanthemum cultivars with different flower colors and flower types were cultured on Murashige and Skoog media(MS)supplemented with 1.0-5.0 mg L^(−1)6-benzylaminopurine(6-BA)and 0.1-1.0 mg L^(−1)α-naphthaleneacetic(NAA).The results showed that the most efficient regeneration media were MS+6-BA 1.0 mg L^(−1)+NAA 0.5 mg L^(−1)for leaf explants and MS+6-BA 5.0 mg L^(−1)+NAA 0.1 mg L^(−1)for tTCL explants.Subsequently,another 13 chrysanthemum cultivars were screened by using the media,and finally,three cultivars with high regeneration efficiency were obtained from 21 cultivars.Among these,C1 had the highest regeneration efficiency:the regeneration rate of leaf explants reached 80.0%after 42 days of culture,and the regeneration rate of tTCL explants reached 100%after 31 days of culture.Furthermore,we also established the transformation system for C1 as follows:preculturing for one day,infecting with Agrobacterium suspension(OD_(600)=0.6)for 10 min,and cultivating in the regeneration medium with 350 mg L^(−1)carbenicillin and 10 mg L^(−1)kanamycin,thus ultimately achieving a transformation rate of 4.0%.In this study,a new chrysanthemum cultivar with an efficient regeneration and transformation system was screened,which is beneficial to enrich the flower color of chrysanthemum transgenic plant recipients and to the functional research of flower color or type-related genes.展开更多
Chrysanthemum×morifolium is a horticultural crop which plays a vital role in theflower industry with signifi-cant economic value and has a cultivation history of over three thousand years in China.The accumulation ...Chrysanthemum×morifolium is a horticultural crop which plays a vital role in theflower industry with signifi-cant economic value and has a cultivation history of over three thousand years in China.The accumulation of anthocyanins is always affected by light.Here,we revealed that anthocyanin accumulation is highly dependent on light in‘2021135’genotype chrysanthemum,while it is light-independent in‘2001402’genotype chrysanthe-mum.However,no literature has been reported regarding the non-photosensitive chrysanthemum in anthocya-nins light-independent synthesis pathways.Through the phenotype analysis of 44 F1 generations,we found that light-independence is a dominant trait which can be stable inherited by progeny.The transcriptome of the rayflorets of‘2021135’and‘2001402’under light and bagging treatment were sequenced and analyzed.Based on weighted gene co-expression network analysis(WGCNA),K-means analysis,and Real-Time Quantitative Poly-merase Chain Reaction(RT-qPCR)analysis,16 genes were highly correlated with the anthocyanin content.The anthocyanin content of rayflorets treated with different light-quality conditions indicated that blue light signifi-cantly affected anthocyanin accumulations.Through Yeast one-hybrid analysis,CmBIC1.1 and CmBIC1.2 can directly regulate the anthocyanin structural gene CmCHS2.In our study,we revealed the important characteristics of light-independent anthocyanin synthesis in chrysanthemums and screened regulatory factors in light-depen-dent and light-independent anthocyanin synthesis pathways.The results laid the groundwork for subsequent ana-lysis of the molecular mechanism involved in the light-independent synthesis of anthocyanins in chrysanthemums.展开更多
The caseinolytic protease complex ClpP1P2 is crucial for protein homeostasis in mycobacteria and stress response and virulence of the pathogens.Its role as a potential drug target for combating tuberculosis(TB)has jus...The caseinolytic protease complex ClpP1P2 is crucial for protein homeostasis in mycobacteria and stress response and virulence of the pathogens.Its role as a potential drug target for combating tuberculosis(TB)has just begun to be substantiated in drug discovery research.We conducted a biochemical screening targeting the ClpP1P2 using a library of compounds phenotypically active against Mycobacterium tuberculosis(Mtb).The screening identified a phenyl ester compound GDI-5755,inhibiting the growth of Mtb and M.bovis BCG,the model organism of mycobacteria.GDI-5755 covalently modified the activesite serine residue of ClpP1,rendering the peptidase inactive,which was delineated through protein mass spectrometry and kinetic analyses.GDI-5755 exerted antibacterial activity by inhibiting ClpP1P2 in the bacteria,which could be demonstrated through a minimum inhibitory concentration(MIC)shift assay with a clpP1 CRISPRi knockdown(clpP1-KD)mutant GH189.The knockdown also remarkably heightened the mutant's sensitivity to ethionamide and meropenem,but not to many other TB drugs.On the other hand,a comparative proteomic analysis of wild-type cells exposed to GDI-5755 revealed the dysregulated proteome,specifically showing changes in the expression levels of multiple TB drug targets,including EthA,LdtMt2,and PanD.Subsequent evaluation confirmed the synergistic activity of GDI-5755 when combined with the TB drugs to inhibit mycobacterial growth.Our findings indicate that small-molecule inhibitors targeting ClpP1P2,when used alongside existing TB medications,could represent novel therapeutic strategies.展开更多
基金This work is supported by Fundamental Research Funds for the Central Universities(Grant No.2021ZY34)the National Natural Science Foundation of China(Grant No.31870693).
文摘Genetic transformation is one of the key steps in the molecular breeding of chrysanthemum,which relies on an optimal regeneration and transformation system.However,the regeneration system of different chrysanthemum cultivars varies,and the regeneration time of most cultivars is long.To screen cultivars with highly efficient regeneration,leaves and shoot tip thin cell layers(tTCL)from eight chrysanthemum cultivars with different flower colors and flower types were cultured on Murashige and Skoog media(MS)supplemented with 1.0-5.0 mg L^(−1)6-benzylaminopurine(6-BA)and 0.1-1.0 mg L^(−1)α-naphthaleneacetic(NAA).The results showed that the most efficient regeneration media were MS+6-BA 1.0 mg L^(−1)+NAA 0.5 mg L^(−1)for leaf explants and MS+6-BA 5.0 mg L^(−1)+NAA 0.1 mg L^(−1)for tTCL explants.Subsequently,another 13 chrysanthemum cultivars were screened by using the media,and finally,three cultivars with high regeneration efficiency were obtained from 21 cultivars.Among these,C1 had the highest regeneration efficiency:the regeneration rate of leaf explants reached 80.0%after 42 days of culture,and the regeneration rate of tTCL explants reached 100%after 31 days of culture.Furthermore,we also established the transformation system for C1 as follows:preculturing for one day,infecting with Agrobacterium suspension(OD_(600)=0.6)for 10 min,and cultivating in the regeneration medium with 350 mg L^(−1)carbenicillin and 10 mg L^(−1)kanamycin,thus ultimately achieving a transformation rate of 4.0%.In this study,a new chrysanthemum cultivar with an efficient regeneration and transformation system was screened,which is beneficial to enrich the flower color of chrysanthemum transgenic plant recipients and to the functional research of flower color or type-related genes.
基金supported by General Project of 2021 Science and Technology Plan of Beijing Municipal Education Comission(KM202111418001)Beijing Natural Science Foundation,China(Grant No.6212022).
文摘Chrysanthemum×morifolium is a horticultural crop which plays a vital role in theflower industry with signifi-cant economic value and has a cultivation history of over three thousand years in China.The accumulation of anthocyanins is always affected by light.Here,we revealed that anthocyanin accumulation is highly dependent on light in‘2021135’genotype chrysanthemum,while it is light-independent in‘2001402’genotype chrysanthe-mum.However,no literature has been reported regarding the non-photosensitive chrysanthemum in anthocya-nins light-independent synthesis pathways.Through the phenotype analysis of 44 F1 generations,we found that light-independence is a dominant trait which can be stable inherited by progeny.The transcriptome of the rayflorets of‘2021135’and‘2001402’under light and bagging treatment were sequenced and analyzed.Based on weighted gene co-expression network analysis(WGCNA),K-means analysis,and Real-Time Quantitative Poly-merase Chain Reaction(RT-qPCR)analysis,16 genes were highly correlated with the anthocyanin content.The anthocyanin content of rayflorets treated with different light-quality conditions indicated that blue light signifi-cantly affected anthocyanin accumulations.Through Yeast one-hybrid analysis,CmBIC1.1 and CmBIC1.2 can directly regulate the anthocyanin structural gene CmCHS2.In our study,we revealed the important characteristics of light-independent anthocyanin synthesis in chrysanthemums and screened regulatory factors in light-depen-dent and light-independent anthocyanin synthesis pathways.The results laid the groundwork for subsequent ana-lysis of the molecular mechanism involved in the light-independent synthesis of anthocyanins in chrysanthemums.
基金supported by the Bill&Melinda Gates Foundation(grant number:INV-008249)GHDDI sponsors in Beijing,China.
文摘The caseinolytic protease complex ClpP1P2 is crucial for protein homeostasis in mycobacteria and stress response and virulence of the pathogens.Its role as a potential drug target for combating tuberculosis(TB)has just begun to be substantiated in drug discovery research.We conducted a biochemical screening targeting the ClpP1P2 using a library of compounds phenotypically active against Mycobacterium tuberculosis(Mtb).The screening identified a phenyl ester compound GDI-5755,inhibiting the growth of Mtb and M.bovis BCG,the model organism of mycobacteria.GDI-5755 covalently modified the activesite serine residue of ClpP1,rendering the peptidase inactive,which was delineated through protein mass spectrometry and kinetic analyses.GDI-5755 exerted antibacterial activity by inhibiting ClpP1P2 in the bacteria,which could be demonstrated through a minimum inhibitory concentration(MIC)shift assay with a clpP1 CRISPRi knockdown(clpP1-KD)mutant GH189.The knockdown also remarkably heightened the mutant's sensitivity to ethionamide and meropenem,but not to many other TB drugs.On the other hand,a comparative proteomic analysis of wild-type cells exposed to GDI-5755 revealed the dysregulated proteome,specifically showing changes in the expression levels of multiple TB drug targets,including EthA,LdtMt2,and PanD.Subsequent evaluation confirmed the synergistic activity of GDI-5755 when combined with the TB drugs to inhibit mycobacterial growth.Our findings indicate that small-molecule inhibitors targeting ClpP1P2,when used alongside existing TB medications,could represent novel therapeutic strategies.
