Dear Editor,Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)is a powerful and versatile gene editing system that has been extensively utilized in various animals and plants,which holds enormous potent...Dear Editor,Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)is a powerful and versatile gene editing system that has been extensively utilized in various animals and plants,which holds enormous potential and value for scientific research and breeding.However,single-targeted CRISPR can only induce a few base deletions,insertions,or substitution.Ideally,thesemutations result in premature termination of the protein encoded by the target gene,leading to a loss of function[1].展开更多
With the ever-increasing human population and deteriorating environmental conditions,there is an urgent need to breed environmentally friendly and resource-conserving rice cultivars to achieve sustainable agricultural...With the ever-increasing human population and deteriorating environmental conditions,there is an urgent need to breed environmentally friendly and resource-conserving rice cultivars to achieve sustainable agricultural development and food security.However,conventional rice improvement strategies,such as hybrid breeding,are time-consuming and laborious processes and may not be able to keep pace with increasing food demand in the future.Targeted genome-editing technologies,especially clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein(CRISPR/Cas),permit efficient targeted genome modification and offer great promise for the creation of desired plants with higher yield,improved grain quality,and resistance to herbicides,diseases,and insect pests.There is also great potential for tapping heterosis using the CRISPR/Cas technology.In this review,we focus on the most essential applications of CRISPR/Cas genome editing to rice genetic improvement,considering traits such as yield,quality,and herbicide,disease and insect-pest resistance.We discuss applications of CRISPR/Cas to the exploitation of heterosis.Finally,we outline perspectives for future rice breeding using genome-editing technologies.展开更多
BACKGROUND:Studies have demonstrated that the mechanisms underlying cellular apoptosis signal transduction focus on two pathways:intracellular mitochondria and extracellular death receptor.The current evidence support...BACKGROUND:Studies have demonstrated that the mechanisms underlying cellular apoptosis signal transduction focus on two pathways:intracellular mitochondria and extracellular death receptor.The current evidence supports that signal transduction of cellular apoptosis also includes endoplasmic reticulum stress signal transduction. OBJECTIVE:To observe Caspase-12 expression and cellular apoptosis following ischemia in rats with progressive spinal cord compression,and to verify the influence of endoplasmic reticulum stress on the apoptosis induced by spinal cord injury. DESIGN,TIME AND SETTING:A randomized,controlled,animal trial was performed at the Institute of Neuroscience in Chongqing Medical University between January and October in 2006. MATERIALS:Immunohistochemical kit,diaminobenzidine,and TUNEL kit were purchased from Beijing Zhongshan Biotechnology,China;rabbit anti-rat Caspase-12 monoclonal antibody was provided by Santa Cruz,USA. METHODS:Sixty Wistar rats,aged 3-4 months,were randomly assigned to a model group(n=50), which underwent spinal cord compression in the L_1 segment following L_1 laminectomy and articular process excision to establish a model of progressive spinal cord compression,and a sham-surgery group(n=10),which underwent only laminectomy.Starting with the first day after surgery,the rats were locally anesthetized,the skin was opened,and the screw was rotated by 1/4 of a cycle,twice weekly. MAIN OUTCOME MEASURES:At 3,7,14,21,and 28 days after surgery,rats from each group were anesthetized,and the spinal cords were resected.Pathological changes following spinal cord compression were determined using hematoxylin-eosin staining,Nissl dye,and transmission electron microscopy.The TUNEL method was used to observe neuronal apoptosis in the compressed spinal cord segments.Immunohistochemistry and Western blot were utilized to detect Caspase-12 expression in the compressed segments. RESULTS:Cellular swelling,neural degeneration,and altered endoplasmic reticulum structures were observed at 3 days following compression.Symptoms became gradually aggravated with increasing compression time.Compared with the sham-surgery group,the number of apoptotic neurons was remarkably increased in compressed segments of the model group(P<0.05),and Caspase-12 expression was also shown to increase(P<0.05). CONCLUSION:Neuronal apoptosis was a predominant pathological factor resulting in secondary spinal cord injury during progressive spinal cord compression,and Caspase-12 was shown to be possibly involved in neuronal apoptosis induced by progressive spinal cord compression.展开更多
CRISPR-Cas12a offers a convenient tool for multiplex genome editing in rice. However, the CRISPR-Cas12a system displays variable editing efficiency across genomic loci, with marked influence by CRISPR RNAs(crRNAs). To...CRISPR-Cas12a offers a convenient tool for multiplex genome editing in rice. However, the CRISPR-Cas12a system displays variable editing efficiency across genomic loci, with marked influence by CRISPR RNAs(crRNAs). To improve the efficiency of the CRISPR-Cas12a system for multiplex genome editing, we identified various architectures and expression strategies for crRNAs. Transformation of binary vectors loaded with engineered CRISPR-Cas12a systems into rice calli and subsequent sequencing revealed that a modified tRNA-crRNA array not only efficiently achieved rice multiplex genome editing, but also successfully edited target sites that were not edited by the crRNA array. This improvement contributes to the application of the CRISPR-Cas12a system in plant genome editing, especially for genomic loci that have hitherto been difficult to edit.展开更多
BACKGROUND:Tyrosine hydroxylase and phenylethanolamine-n-methyl transferase expression coexist in Purkinje cells of the rat cerebellum.Numerous reports have also been published addressing whether dopamine-beta-hydroxy...BACKGROUND:Tyrosine hydroxylase and phenylethanolamine-n-methyl transferase expression coexist in Purkinje cells of the rat cerebellum.Numerous reports have also been published addressing whether dopamine-beta-hydroxylase(DBH) expression exists in cerebellar Purkinje cells. OBJECTIVE:To investigate the coexistence of DBH and activator protein-2αexpression in rat cerebellar Purkinje cells. DESIGN,TIME AND SETTING:A cell morphological study was performed at the Institute of Neuroscience,Chongqing Medical University,China in May 2007. MATERIALS:Ten healthy Wistar rats,of either gender,aged 14 weeks,served as experimental animals.Rabbit anti-mouse DBH,goat anti-mouse activator protein-2αand rabbit anti-mouseβ-actin (Santa Cruz Biotechnology,Inc.,USA),horseradish peroxidase-labeled goat anti-rabbit IgG, FITC-labeled mouse anti-rabbit IgG,and Cy3-labeled mouse anti-goat IgG(Boster,Wuhan,China), were used in this study. METHODS:Immunohistochemical staining was used to measure the expression of DBH or activator protein-2α,with double-label immunofluorescence being employed to determine coexpression of both,in the cerebellum of 5 randomly selected rats.Western blot assay was utilized to determine the expression of DBH and activator protein-2αin the cerebellum of the remaining 5 rats. MAIN OUTCOME MEASURES:Expression,localization and coexistence of DBH and activator protein-2αin the cerebellum were measured separately. RESULTS:Immunohistochemical staining demonstrated that cerebellar Purkinje cells stained positive for DBH and activator protein-2α.Western blot assay also demonstrated DBH and activator protein-2αexpression in the cerebellum.Double-labeling immunofluorescence showed the coexistence of DBH and activator protein-2αin cerebellar Purkinje cells. CONCLUSION:Norepinephrine and activator protein-2αcoexist in rat cerebellar Purkinje cells.展开更多
Heterosis has long been exploited in the hybrid seed industry,which contributes to high and stable yields of modern agriculture(Huang et al.,2016).However,heterosis phenotypes of hybrid plants are segregated in its of...Heterosis has long been exploited in the hybrid seed industry,which contributes to high and stable yields of modern agriculture(Huang et al.,2016).However,heterosis phenotypes of hybrid plants are segregated in its offspring.Apomixis allows instant fixation and propagation though seeds with heterozygous genotypes,showing great potential in plant breeding and agricultural practice(Ye and Cui,2019).Apomixis naturally occurs in hundreds of plant species,but it is absent in major crop species(Underwood and Mercier,2022).Recently,synthetic apomixis has been engineered in rice by combining Mitosis instead of Meiosis(MiMe)with a mutation of MATRILINEAL or ectopic expression of BABY BOOM1(BBM1),enabling clonal reproduction of F1 hybrids through seeds and stable transmission of heterotic phenotypes over generations(Khanday et al.,2019;Wang et al.,2019;Liu et al.,2022).However,the fertility of both two strategies was significantly reduced compared with that of the wild type,which hinders the application of both strategies in agriculture.In this study,we established synthetic apomixis with a high fertility that is almost comparable to normal hybrid rice.展开更多
Dear Editor,Genetic breeding involves the recombination and selection of various valuable genes.Meiotic crossover(CO)promotes the generation of new allelic combinations on chromosomes,which is essential for breeding e...Dear Editor,Genetic breeding involves the recombination and selection of various valuable genes.Meiotic crossover(CO)promotes the generation of new allelic combinations on chromosomes,which is essential for breeding elite varieties(Wijnker and de Jong,2008).An increase in CO promotes genetic diversity,whereas a decrease can rapidly stabilize excellent traits(Mercier et al.,2015).Furthermore,the complete elimination of CO facilitates heterotic fixation during apomixis(Wang et al.,2019).展开更多
Isolated attosecond pulses(IAPs)are generated via applying amplitude gating on high-order harmonic generation driven by carrier-envelope phase stabilized 5.2 fs pulses with 0.5 mJ pulse energy at 770 nm central wavele...Isolated attosecond pulses(IAPs)are generated via applying amplitude gating on high-order harmonic generation driven by carrier-envelope phase stabilized 5.2 fs pulses with 0.5 mJ pulse energy at 770 nm central wavelength at the Synergetic Extreme Condition User Facility.A continuum ranging from 70 to 100 eV that supports sub-100-attosecond pulse is extracted by Zr foil and Mo/Si multilayer mirror.We demonstrate the characterization of the IAP.The retrieved pulse duration is 86attoseconds.The developed attosecond laser beamline with repetition rate up to 10 kHz is available for users to conduct attosecond photoelectron spectroscopy researches with a capability of coincidence measurement.展开更多
Osteoporosis(OP)is a debilitating skeletal abnormality involving bone remodeling and bone cell homeostasis characterized by decreased bone strength and high fracture risk.A novel therapeutic intervention for OP by man...Osteoporosis(OP)is a debilitating skeletal abnormality involving bone remodeling and bone cell homeostasis characterized by decreased bone strength and high fracture risk.A novel therapeutic intervention for OP by manipulating cellular autophagy-apoptosis processes to promote skeletal homeostasis is presented.Protective effects of the naturally occurring plant extract Liquiritigenin(LG)were demonstrated in an ovariectomy(OvX)-OP mouse model and preosteoblast MC3T3-E1 cells.Micro-CT and histological staining assessments of skeletal phenotype were applied alongside detection of autophagy activity in osteocytes and MC3T3-E1 cells by transmission electron microscopy(TEM).The effects of LG on chloroquine(CQ)-and the apoptosis-inducing TS-treated osteogenic differentiations and status of lysosomes within MC3T3-E1 cells were analyzed by Neutral red,Alizarin red S and alkaline phosphatase(ALP)staining and Western blot assays.Treatment with LG prevented bone loss,increased osteogenic differentiation in vivo and in vitro,and inhibited osteoclast formation to some extent.TEM analyses revealed that LG can improve auto-lysosomal degradation within osteocytes from OVX mice and MC3T3-E1 cells.The abnormal status of lysosomes associated with CQ and TS treatments was notably alleviated by LG which also reduced levels of apoptosis-induced inhibition of osteogenic differentiation and averted abnormal osteogenic differentiation as a consequence of a blockage in autolysosome degradation.Overall,LG stimulates bone growth in Oovx mice through increased osteogenic differentiation and regulation of autophagyapoptosis mechanisms,presenting an auspicious natural therapy for Op.展开更多
In hybrid plants,heterosis often produces large,vigorous plants with high yields;however,hybrid seeds are generated by costly and laborious crosses of inbred parents.Apomixis,in which a plant produces a clone of itsel...In hybrid plants,heterosis often produces large,vigorous plants with high yields;however,hybrid seeds are generated by costly and laborious crosses of inbred parents.Apomixis,in which a plant produces a clone of itself via asexual reproduction through seeds,may produce another revolution in plant biology.Recently,synthetic apomixis enabled clonal reproduction of F_(1) hybrids through seeds in rice(Oryza sativa),but the inheritance of the synthetic apomixis trait and superior heterotic phenotypes across generations remained unclear.Here,we propagated clonal plants to the T_(4) generation and investigated their genetic and molecular stability at each generation.By analyzing agronomic traits,as well as the genome,methylome,transcriptome,and allele-specific transcriptome,we showed that the descendant clonal plants remained stable.Unexpectedly,in addition to normal clonal seeds,the plants also produced a few aneuploids that had eliminated large genomic segments in each generation.Despite the identification of rare aneuploids,the observation that the synthetic apomixis trait is stably transmitted through multiple generations helps confirm the feasibility of using apomixis in the future.展开更多
The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-i...The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM(available at http://www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing(NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers,cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.展开更多
Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions,or for analyzing epistatic relationships in genetic pathways.Using conv...Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions,or for analyzing epistatic relationships in genetic pathways.Using conventional methods,mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring.This process is both time-展开更多
Grain yield is one of the most important and complex trait for genetic improvement in crops; it is known to be controlled by a number of genes known as quantitative trait loci(QTLs). In the past decade, many yield-con...Grain yield is one of the most important and complex trait for genetic improvement in crops; it is known to be controlled by a number of genes known as quantitative trait loci(QTLs). In the past decade, many yield-contributing QTLs have been identified in crops.However, it remains unclear whether those QTLs confer the same yield performance in different genetic backgrounds. Here, we performed CRISPR/Cas_9-mediated QTL editing in five widely-cultivated rice varieties and revealed that the same QTL can have diverse, even opposing, effects on grain yield in different genetic backgrounds.展开更多
The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9) system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain r...The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9) system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction(PCR)/restriction enzyme(RE) assay, T7 endonuclease I(T7EI) assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting(HRM) analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are timeand labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we described a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR(ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.展开更多
The clustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease 9(CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructe...The clustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease 9(CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T_0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T_0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T_0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.展开更多
基金We thank Professor Pengcheng Wei of Anhui Agricultural University for his guidance on the experimental methods.This work was supported by the National Key Research and Development Program of China(Grant No.2022YFD2100101)the Joint NSFC-ISF Research Program(Grant No.32061143022)+1 种基金the 2115 Talent Development Program of China Agricultural University(Grant No.1061-00109017)to HZthe National Natural Science Foundation of China(Grant No.3217180159).
文摘Dear Editor,Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)is a powerful and versatile gene editing system that has been extensively utilized in various animals and plants,which holds enormous potential and value for scientific research and breeding.However,single-targeted CRISPR can only induce a few base deletions,insertions,or substitution.Ideally,thesemutations result in premature termination of the protein encoded by the target gene,leading to a loss of function[1].
基金supported by the National Natural Science Foundation of China(U20A2030)Central Public-interest Scientific Institution Basal Research Fund(Y2020YJ12 and Y2020XK17)+1 种基金Key Research and Development Program of China National Rice Research Institute(CNRRI-2020-01)Foreign Cooperation Project of Ningxia Academy of Agricultural and Forestry Institute(DW-X-2018004)。
文摘With the ever-increasing human population and deteriorating environmental conditions,there is an urgent need to breed environmentally friendly and resource-conserving rice cultivars to achieve sustainable agricultural development and food security.However,conventional rice improvement strategies,such as hybrid breeding,are time-consuming and laborious processes and may not be able to keep pace with increasing food demand in the future.Targeted genome-editing technologies,especially clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein(CRISPR/Cas),permit efficient targeted genome modification and offer great promise for the creation of desired plants with higher yield,improved grain quality,and resistance to herbicides,diseases,and insect pests.There is also great potential for tapping heterosis using the CRISPR/Cas technology.In this review,we focus on the most essential applications of CRISPR/Cas genome editing to rice genetic improvement,considering traits such as yield,quality,and herbicide,disease and insect-pest resistance.We discuss applications of CRISPR/Cas to the exploitation of heterosis.Finally,we outline perspectives for future rice breeding using genome-editing technologies.
基金the National Natural Science Foundation of China,No.30270437Chunhui Program of the Ministry of Education in 2003, No.200407
文摘BACKGROUND:Studies have demonstrated that the mechanisms underlying cellular apoptosis signal transduction focus on two pathways:intracellular mitochondria and extracellular death receptor.The current evidence supports that signal transduction of cellular apoptosis also includes endoplasmic reticulum stress signal transduction. OBJECTIVE:To observe Caspase-12 expression and cellular apoptosis following ischemia in rats with progressive spinal cord compression,and to verify the influence of endoplasmic reticulum stress on the apoptosis induced by spinal cord injury. DESIGN,TIME AND SETTING:A randomized,controlled,animal trial was performed at the Institute of Neuroscience in Chongqing Medical University between January and October in 2006. MATERIALS:Immunohistochemical kit,diaminobenzidine,and TUNEL kit were purchased from Beijing Zhongshan Biotechnology,China;rabbit anti-rat Caspase-12 monoclonal antibody was provided by Santa Cruz,USA. METHODS:Sixty Wistar rats,aged 3-4 months,were randomly assigned to a model group(n=50), which underwent spinal cord compression in the L_1 segment following L_1 laminectomy and articular process excision to establish a model of progressive spinal cord compression,and a sham-surgery group(n=10),which underwent only laminectomy.Starting with the first day after surgery,the rats were locally anesthetized,the skin was opened,and the screw was rotated by 1/4 of a cycle,twice weekly. MAIN OUTCOME MEASURES:At 3,7,14,21,and 28 days after surgery,rats from each group were anesthetized,and the spinal cords were resected.Pathological changes following spinal cord compression were determined using hematoxylin-eosin staining,Nissl dye,and transmission electron microscopy.The TUNEL method was used to observe neuronal apoptosis in the compressed spinal cord segments.Immunohistochemistry and Western blot were utilized to detect Caspase-12 expression in the compressed segments. RESULTS:Cellular swelling,neural degeneration,and altered endoplasmic reticulum structures were observed at 3 days following compression.Symptoms became gradually aggravated with increasing compression time.Compared with the sham-surgery group,the number of apoptotic neurons was remarkably increased in compressed segments of the model group(P<0.05),and Caspase-12 expression was also shown to increase(P<0.05). CONCLUSION:Neuronal apoptosis was a predominant pathological factor resulting in secondary spinal cord injury during progressive spinal cord compression,and Caspase-12 was shown to be possibly involved in neuronal apoptosis induced by progressive spinal cord compression.
基金funded by the National Key Research and Development Program of China(2016YFD0101800)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciencesthe National GMO New Variety Breeding Program of China(2016ZX08011-001)。
文摘CRISPR-Cas12a offers a convenient tool for multiplex genome editing in rice. However, the CRISPR-Cas12a system displays variable editing efficiency across genomic loci, with marked influence by CRISPR RNAs(crRNAs). To improve the efficiency of the CRISPR-Cas12a system for multiplex genome editing, we identified various architectures and expression strategies for crRNAs. Transformation of binary vectors loaded with engineered CRISPR-Cas12a systems into rice calli and subsequent sequencing revealed that a modified tRNA-crRNA array not only efficiently achieved rice multiplex genome editing, but also successfully edited target sites that were not edited by the crRNA array. This improvement contributes to the application of the CRISPR-Cas12a system in plant genome editing, especially for genomic loci that have hitherto been difficult to edit.
基金the National Natural Science Foundation of China.No.30270437
文摘BACKGROUND:Tyrosine hydroxylase and phenylethanolamine-n-methyl transferase expression coexist in Purkinje cells of the rat cerebellum.Numerous reports have also been published addressing whether dopamine-beta-hydroxylase(DBH) expression exists in cerebellar Purkinje cells. OBJECTIVE:To investigate the coexistence of DBH and activator protein-2αexpression in rat cerebellar Purkinje cells. DESIGN,TIME AND SETTING:A cell morphological study was performed at the Institute of Neuroscience,Chongqing Medical University,China in May 2007. MATERIALS:Ten healthy Wistar rats,of either gender,aged 14 weeks,served as experimental animals.Rabbit anti-mouse DBH,goat anti-mouse activator protein-2αand rabbit anti-mouseβ-actin (Santa Cruz Biotechnology,Inc.,USA),horseradish peroxidase-labeled goat anti-rabbit IgG, FITC-labeled mouse anti-rabbit IgG,and Cy3-labeled mouse anti-goat IgG(Boster,Wuhan,China), were used in this study. METHODS:Immunohistochemical staining was used to measure the expression of DBH or activator protein-2α,with double-label immunofluorescence being employed to determine coexpression of both,in the cerebellum of 5 randomly selected rats.Western blot assay was utilized to determine the expression of DBH and activator protein-2αin the cerebellum of the remaining 5 rats. MAIN OUTCOME MEASURES:Expression,localization and coexistence of DBH and activator protein-2αin the cerebellum were measured separately. RESULTS:Immunohistochemical staining demonstrated that cerebellar Purkinje cells stained positive for DBH and activator protein-2α.Western blot assay also demonstrated DBH and activator protein-2αexpression in the cerebellum.Double-labeling immunofluorescence showed the coexistence of DBH and activator protein-2αin cerebellar Purkinje cells. CONCLUSION:Norepinephrine and activator protein-2αcoexist in rat cerebellar Purkinje cells.
基金supported by the National Natural Science Foundation of China(32188102,32025028,and U20A2030)the National Key Research and Development Program of China(2022YFF1003304)+1 种基金the Central Public-interest Scientific Institution Basal Research Fund(Y2022QC20)the Hainan Yazhou Bay Seed Laboratory(B21HJ0215).
文摘Heterosis has long been exploited in the hybrid seed industry,which contributes to high and stable yields of modern agriculture(Huang et al.,2016).However,heterosis phenotypes of hybrid plants are segregated in its offspring.Apomixis allows instant fixation and propagation though seeds with heterozygous genotypes,showing great potential in plant breeding and agricultural practice(Ye and Cui,2019).Apomixis naturally occurs in hundreds of plant species,but it is absent in major crop species(Underwood and Mercier,2022).Recently,synthetic apomixis has been engineered in rice by combining Mitosis instead of Meiosis(MiMe)with a mutation of MATRILINEAL or ectopic expression of BABY BOOM1(BBM1),enabling clonal reproduction of F1 hybrids through seeds and stable transmission of heterotic phenotypes over generations(Khanday et al.,2019;Wang et al.,2019;Liu et al.,2022).However,the fertility of both two strategies was significantly reduced compared with that of the wild type,which hinders the application of both strategies in agriculture.In this study,we established synthetic apomixis with a high fertility that is almost comparable to normal hybrid rice.
文摘Dear Editor,Genetic breeding involves the recombination and selection of various valuable genes.Meiotic crossover(CO)promotes the generation of new allelic combinations on chromosomes,which is essential for breeding elite varieties(Wijnker and de Jong,2008).An increase in CO promotes genetic diversity,whereas a decrease can rapidly stabilize excellent traits(Mercier et al.,2015).Furthermore,the complete elimination of CO facilitates heterotic fixation during apomixis(Wang et al.,2019).
基金supported by the Synergic Extreme Condition User Facility(SECUF),the National Natural Science Foundation of China(Nos.91850209,12034020,92150103,61690221,and 12174435)the National Key R&D Program of China(Nos.2022YFA1604200 and 2017YFB0405202)。
文摘Isolated attosecond pulses(IAPs)are generated via applying amplitude gating on high-order harmonic generation driven by carrier-envelope phase stabilized 5.2 fs pulses with 0.5 mJ pulse energy at 770 nm central wavelength at the Synergetic Extreme Condition User Facility.A continuum ranging from 70 to 100 eV that supports sub-100-attosecond pulse is extracted by Zr foil and Mo/Si multilayer mirror.We demonstrate the characterization of the IAP.The retrieved pulse duration is 86attoseconds.The developed attosecond laser beamline with repetition rate up to 10 kHz is available for users to conduct attosecond photoelectron spectroscopy researches with a capability of coincidence measurement.
基金This study was supported by the National Natural Science Foundation of China(No.81671257,81371221,31600825)81671257,81371221,31600825)Innovation Research Group at Institutions of Higher Education in Chongqing,China(No.CXQTP19019019034)Program for Innovation Team Building at Institutions of Higher Education in Chongqing in 2016.
文摘Osteoporosis(OP)is a debilitating skeletal abnormality involving bone remodeling and bone cell homeostasis characterized by decreased bone strength and high fracture risk.A novel therapeutic intervention for OP by manipulating cellular autophagy-apoptosis processes to promote skeletal homeostasis is presented.Protective effects of the naturally occurring plant extract Liquiritigenin(LG)were demonstrated in an ovariectomy(OvX)-OP mouse model and preosteoblast MC3T3-E1 cells.Micro-CT and histological staining assessments of skeletal phenotype were applied alongside detection of autophagy activity in osteocytes and MC3T3-E1 cells by transmission electron microscopy(TEM).The effects of LG on chloroquine(CQ)-and the apoptosis-inducing TS-treated osteogenic differentiations and status of lysosomes within MC3T3-E1 cells were analyzed by Neutral red,Alizarin red S and alkaline phosphatase(ALP)staining and Western blot assays.Treatment with LG prevented bone loss,increased osteogenic differentiation in vivo and in vitro,and inhibited osteoclast formation to some extent.TEM analyses revealed that LG can improve auto-lysosomal degradation within osteocytes from OVX mice and MC3T3-E1 cells.The abnormal status of lysosomes associated with CQ and TS treatments was notably alleviated by LG which also reduced levels of apoptosis-induced inhibition of osteogenic differentiation and averted abnormal osteogenic differentiation as a consequence of a blockage in autolysosome degradation.Overall,LG stimulates bone growth in Oovx mice through increased osteogenic differentiation and regulation of autophagyapoptosis mechanisms,presenting an auspicious natural therapy for Op.
基金supported by the National Natural Science Foundation of China(U20A2030,32025028,and 32188102)the Central Public-interest Scientific Institution Basal Research Fund(Y2022QC20)+1 种基金the Hainan Yazhou Bay Seed Laboratory(#B21HJ0215)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(CAAS-ZDRW202001).
文摘In hybrid plants,heterosis often produces large,vigorous plants with high yields;however,hybrid seeds are generated by costly and laborious crosses of inbred parents.Apomixis,in which a plant produces a clone of itself via asexual reproduction through seeds,may produce another revolution in plant biology.Recently,synthetic apomixis enabled clonal reproduction of F_(1) hybrids through seeds in rice(Oryza sativa),but the inheritance of the synthetic apomixis trait and superior heterotic phenotypes across generations remained unclear.Here,we propagated clonal plants to the T_(4) generation and investigated their genetic and molecular stability at each generation.By analyzing agronomic traits,as well as the genome,methylome,transcriptome,and allele-specific transcriptome,we showed that the descendant clonal plants remained stable.Unexpectedly,in addition to normal clonal seeds,the plants also produced a few aneuploids that had eliminated large genomic segments in each generation.Despite the identification of rare aneuploids,the observation that the synthetic apomixis trait is stably transmitted through multiple generations helps confirm the feasibility of using apomixis in the future.
基金supported by the National Key Research and Development Program of China (2017YFD0102002)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciencesthe National Natural Science Foundation of China (31401363)
文摘The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM(available at http://www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing(NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers,cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.
基金supported by the Agricultural Science and Technology Innovation Program
文摘Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions,or for analyzing epistatic relationships in genetic pathways.Using conventional methods,mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring.This process is both time-
基金supported by the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences
文摘Grain yield is one of the most important and complex trait for genetic improvement in crops; it is known to be controlled by a number of genes known as quantitative trait loci(QTLs). In the past decade, many yield-contributing QTLs have been identified in crops.However, it remains unclear whether those QTLs confer the same yield performance in different genetic backgrounds. Here, we performed CRISPR/Cas_9-mediated QTL editing in five widely-cultivated rice varieties and revealed that the same QTL can have diverse, even opposing, effects on grain yield in different genetic backgrounds.
基金supported by the National Natural Science Foundation of China (Nos. 31271681 and 3140101312)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences
文摘The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9) system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction(PCR)/restriction enzyme(RE) assay, T7 endonuclease I(T7EI) assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting(HRM) analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are timeand labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we described a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR(ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.
基金supported by the National Natural Science Foundation of China (31271681, 3140101312)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural SciencesJiangsu Agriculture Science and Technology Innovation Fund (CX(13)5075)
文摘The clustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease 9(CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T_0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T_0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T_0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.
