Purpose:Under-foot impact loadings can cause serious lower limb injuries in many activities,such asautomobile collisions and underbody explosions to military vehicles.The present study aims to comparethe biomechanical...Purpose:Under-foot impact loadings can cause serious lower limb injuries in many activities,such asautomobile collisions and underbody explosions to military vehicles.The present study aims to comparethe biomechanical responses of the mainstream vehicle occupant dummies with the human body lowerlimb model and analyze their robustness and applicability for assessing lower limb injury risk in underfoot impact loading environments.Methods:The Hybrid III model,the test device for human occupant restraint(THOR)model,and a hybridhuman body model with the human active lower limb model were adopted for under-foot impactanalysis regarding different impact velocities and initial lower limb postures.Results:The results show that the 2 dummy models have larger peak tibial axial force and highersensitivity to the impact velocities and initial postures than the human lower limb model.In particular,the Hybrid III dummy model presented extremely larger peak tibial axial forces than the human lowerlimb model.In the case of minimal difference in tibial axial force,Hybrid III's tibial axial force(7.5 KN)isstill 312.5%that of human active lower limb's(2.4 KN).Even with closer peak tibial axial force values,thebiomechanical response curve shapes of the THOR model show significant differences from the humanlower limb model.Conclusion:Based on the present results,the Hybrid III dummy cannot be used to evaluate the lowerlimb injury risk in under-foot loading environments.In contrast,potential improvement in ankle biofidelity and related soft tissues of the THOR dummy can be implemented in the future for betterapplicability.展开更多
CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deleti...CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease(T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.展开更多
MicroRNAs （miRNAs） are endogenous non-coding small RNAs that silence genes through mRNA degradation or translational inhibition. The phytohormone abscisic acid （ABA） is essential for plant development and adaptati...MicroRNAs （miRNAs） are endogenous non-coding small RNAs that silence genes through mRNA degradation or translational inhibition. The phytohormone abscisic acid （ABA） is essential for plant development and adaptation to abiotic and biotic stresses. In Arabidopsis, miRNAs are implicated in ABA functions. However, ABA-responsive miRNAs have not been systematically studied in rice. Here high throughput sequencing of small RNAs revealed that 107 miRNAs were differentially expressed in the rice ABA deficient mutant, Osabal. Of these, 13 were confirmed by stem-loop RT-PCR. Among them, miR1425-5P, miR169a, miR169n, miR390-5P, miR397a and miR397b were up-regulated, but miR162b reduced in expression in Osabal. The targets of these 13 miRNAs were predicted and validated by gene expression profiling. Interestingly, the expression levels of these miRNAs and their targets were regulated by ABA. Cleavage sites were detected on 7 of the miRNA targets by 5＇-Rapid Amplification of cDNA Ends （5＇-RACE）. Finally, miR162b and its target OsTRE1 were shown to affect rice resistance to drought stress, suggesting that miR162b increases resistance to drought by targeting OsTRE1. Our work provides important information for further characterization and functional analysis of ABA-responsive miRNAs in rice.展开更多
MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both acade...MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.展开更多
Pytohormone abscisic acid （ABA） plays important roles in defense responses. Nonetheless, how ABA regulates plant resistance to biotrophic fungi remains largely unknown. Arabidopsis ABA-deficient mutants, aba2-1 and ...Pytohormone abscisic acid （ABA） plays important roles in defense responses. Nonetheless, how ABA regulates plant resistance to biotrophic fungi remains largely unknown. Arabidopsis ABA-deficient mutants, aba2-1 and aba3-1, displayed enhanced resistance to the biotrophic powdery mildew fungus Golovinomyces cichoracearum. Moreover, exogenously administered ABA increased the susceptibility of Arabidopsis to G. cichoracearum. Arabidopsis ABA perception components mutants, abil-1 and abi2-1, also displayed similar phenotypes to ABA-deficient mutants in resistance to G. cichoracearum. However, the resistance to G. cichoracearum is not changed in downstream ABA signaling transduction mutants, abi3-1, abi4-1, and abi5-1. Microscopic examination revealed that hyphal growth and conidiophore production of G. cichoracearum were compromised in the ABA deficient mutants, even though pre-penetration and penetration growth of the fimgus were not affected. In addition, salicylic acid （SA） and MPK3 are found to be involved in ABA-regulated resistance to G. cichoracearurn. Our work demonstrates that ABA negatively regulates post-penetration resistance of Arabidopsis to powdery mildew fungus G. cichoracearum, probably through antagonizing the function of SA.展开更多
The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9(SpCas9)and its variants.The detailed mechanism remains unknown.Here,based on in vitro...The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9(SpCas9)and its variants.The detailed mechanism remains unknown.Here,based on in vitro cleavage assays and base editing analysis,we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1).We also show that these nicks are made on the target DNA strand.These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets.This system provides a novel tool for achieving trait stacking in plants.展开更多
Vascular plants are characterized by the existence of phloem and xylem vascular tissues that are specialized for water and nutrient transporting and mechanical support.The xylem is composed of vessels and tracheids fo...Vascular plants are characterized by the existence of phloem and xylem vascular tissues that are specialized for water and nutrient transporting and mechanical support.The xylem is composed of vessels and tracheids formed by non-living cells whose cellulose cell walls are stiffened with lignin.The xylem tissues across different parts of the plant are interconnected to form a continuous water-conducting system.Vascular plants possess true stems,leaves,and roots,enabling them to expand to diverse habitats and to become the dominant type of land plants.However,to adapt to new habitats,vascular plants,being sessile,have to face some extreme environmental conditions.Pathogens are a common and constant threat confronting plants as they migrate to new environments.To survive,plants have developed complex immune systems during an evolutionary arms race against pathogens.展开更多
Dear Editor, The general way to probe functions of a protein in vivo is to perturb its level and then observe subsequent phe- notypic changes. In plants, modulation of protein level is mainly carried out at DNA or RN...Dear Editor, The general way to probe functions of a protein in vivo is to perturb its level and then observe subsequent phe- notypic changes. In plants, modulation of protein level is mainly carried out at DNA or RNA level, which is indirect and thus affected by stability of the target protein. Thus, experimental approaches to perturb protein level directly are needed, but still limited in plants. In mammalian cells, a technique to modulate protein level directly has been developed. Engineered destabilizing domain （DD） of the human FKBP12 protein can confer instability to other pro- teins when fused to it. A small synthetic molecule ligand Shield 1 （Shldl） can bind DD and shield it from degrada- tion. The level of DD fused protein can be controlled by adjusting Shldl concentration （Banaszynski et al., 2006）.展开更多
基金This work is supported by the National Natural Science Foundation of China(Grant No.51875187,32171305)Hunan YouthTalent Program(Grant No.2020RC3016)+1 种基金Natural Science Foundation of Chongqing(Grant No.cstc2021jcyj-msxmX0109)Chongqing Technology Innovation and Application DevelopmentProject(CSTB2023YSZX-JSX0003).
文摘Purpose:Under-foot impact loadings can cause serious lower limb injuries in many activities,such asautomobile collisions and underbody explosions to military vehicles.The present study aims to comparethe biomechanical responses of the mainstream vehicle occupant dummies with the human body lowerlimb model and analyze their robustness and applicability for assessing lower limb injury risk in underfoot impact loading environments.Methods:The Hybrid III model,the test device for human occupant restraint(THOR)model,and a hybridhuman body model with the human active lower limb model were adopted for under-foot impactanalysis regarding different impact velocities and initial lower limb postures.Results:The results show that the 2 dummy models have larger peak tibial axial force and highersensitivity to the impact velocities and initial postures than the human lower limb model.In particular,the Hybrid III dummy model presented extremely larger peak tibial axial forces than the human lowerlimb model.In the case of minimal difference in tibial axial force,Hybrid III's tibial axial force(7.5 KN)isstill 312.5%that of human active lower limb's(2.4 KN).Even with closer peak tibial axial force values,thebiomechanical response curve shapes of the THOR model show significant differences from the humanlower limb model.Conclusion:Based on the present results,the Hybrid III dummy cannot be used to evaluate the lowerlimb injury risk in under-foot loading environments.In contrast,potential improvement in ankle biofidelity and related soft tissues of the THOR dummy can be implemented in the future for betterapplicability.
基金supported by grants from the National Transgenic Science and Technology Program of China(2019ZX08010-003,2019ZX08010-001,2018ZX0801002B)the National Key Research and Development Program of China(2016YFD0100602)+1 种基金the Scientific Program of Beijing Municipal Commission of Science and Technology(Z171100001517001)the National Natural Science Foundation of China(31672015)to J.L.Q。
文摘CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease(T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.
基金supported by the National Natural Science Foundation of China(Nos.31071673, 31371929)the Chinese Academy of Sciences(No. KSCX2-EW-N-06)
文摘MicroRNAs （miRNAs） are endogenous non-coding small RNAs that silence genes through mRNA degradation or translational inhibition. The phytohormone abscisic acid （ABA） is essential for plant development and adaptation to abiotic and biotic stresses. In Arabidopsis, miRNAs are implicated in ABA functions. However, ABA-responsive miRNAs have not been systematically studied in rice. Here high throughput sequencing of small RNAs revealed that 107 miRNAs were differentially expressed in the rice ABA deficient mutant, Osabal. Of these, 13 were confirmed by stem-loop RT-PCR. Among them, miR1425-5P, miR169a, miR169n, miR390-5P, miR397a and miR397b were up-regulated, but miR162b reduced in expression in Osabal. The targets of these 13 miRNAs were predicted and validated by gene expression profiling. Interestingly, the expression levels of these miRNAs and their targets were regulated by ABA. Cleavage sites were detected on 7 of the miRNA targets by 5＇-Rapid Amplification of cDNA Ends （5＇-RACE）. Finally, miR162b and its target OsTRE1 were shown to affect rice resistance to drought stress, suggesting that miR162b increases resistance to drought by targeting OsTRE1. Our work provides important information for further characterization and functional analysis of ABA-responsive miRNAs in rice.
基金supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020101 and XDA24020310)the National Natural Science Foundation of China(31672015,31788103)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(2020000003)。
文摘MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB11030500 to Jin-Long Qiu)the National Natural Science Foundation of China(31071673 to Jin-Long Qiu)
文摘Pytohormone abscisic acid （ABA） plays important roles in defense responses. Nonetheless, how ABA regulates plant resistance to biotrophic fungi remains largely unknown. Arabidopsis ABA-deficient mutants, aba2-1 and aba3-1, displayed enhanced resistance to the biotrophic powdery mildew fungus Golovinomyces cichoracearum. Moreover, exogenously administered ABA increased the susceptibility of Arabidopsis to G. cichoracearum. Arabidopsis ABA perception components mutants, abil-1 and abi2-1, also displayed similar phenotypes to ABA-deficient mutants in resistance to G. cichoracearum. However, the resistance to G. cichoracearum is not changed in downstream ABA signaling transduction mutants, abi3-1, abi4-1, and abi5-1. Microscopic examination revealed that hyphal growth and conidiophore production of G. cichoracearum were compromised in the ABA deficient mutants, even though pre-penetration and penetration growth of the fimgus were not affected. In addition, salicylic acid （SA） and MPK3 are found to be involved in ABA-regulated resistance to G. cichoracearurn. Our work demonstrates that ABA negatively regulates post-penetration resistance of Arabidopsis to powdery mildew fungus G. cichoracearum, probably through antagonizing the function of SA.
基金supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020102)the National Transgenic Science and Technology Program(2018ZX0801002B)+2 种基金the National Natural Science Foundation of China(31788103 and 31971370)the Chinese Academy of Sciences(QYZDY-SSW-SMC030)the National Key R&D Program of China(2018YFA0900600,2016YFD0100102-11,and 2016YFD0100605)。
文摘The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9(SpCas9)and its variants.The detailed mechanism remains unknown.Here,based on in vitro cleavage assays and base editing analysis,we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1).We also show that these nicks are made on the target DNA strand.These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets.This system provides a novel tool for achieving trait stacking in plants.
基金supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDPB16 and XDA24020101 to J.-L.Q.)。
文摘Vascular plants are characterized by the existence of phloem and xylem vascular tissues that are specialized for water and nutrient transporting and mechanical support.The xylem is composed of vessels and tracheids formed by non-living cells whose cellulose cell walls are stiffened with lignin.The xylem tissues across different parts of the plant are interconnected to form a continuous water-conducting system.Vascular plants possess true stems,leaves,and roots,enabling them to expand to diverse habitats and to become the dominant type of land plants.However,to adapt to new habitats,vascular plants,being sessile,have to face some extreme environmental conditions.Pathogens are a common and constant threat confronting plants as they migrate to new environments.To survive,plants have developed complex immune systems during an evolutionary arms race against pathogens.
文摘Dear Editor, The general way to probe functions of a protein in vivo is to perturb its level and then observe subsequent phe- notypic changes. In plants, modulation of protein level is mainly carried out at DNA or RNA level, which is indirect and thus affected by stability of the target protein. Thus, experimental approaches to perturb protein level directly are needed, but still limited in plants. In mammalian cells, a technique to modulate protein level directly has been developed. Engineered destabilizing domain （DD） of the human FKBP12 protein can confer instability to other pro- teins when fused to it. A small synthetic molecule ligand Shield 1 （Shldl） can bind DD and shield it from degrada- tion. The level of DD fused protein can be controlled by adjusting Shldl concentration （Banaszynski et al., 2006）.
