Genome editing in plants is a powerful strategy that can substantially advance functional genomics research,facilitating the discovery,enhancement,and development of novel traits with significant agricultural implicat...Genome editing in plants is a powerful strategy that can substantially advance functional genomics research,facilitating the discovery,enhancement,and development of novel traits with significant agricultural implications.Various methodologies,such as zinc finger nucleases(ZFNs),transcription activator-like effector nucleases(TALENs),and CRISPR/Cas systems,have been developed for this purpose.展开更多
Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional ...Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.展开更多
Pepper(Capsicum annuum)is one of the most important horticultural crops worldwide,which makes the development of an effective protoplast system for transient gene expression highly significant.Typically,plant protopla...Pepper(Capsicum annuum)is one of the most important horticultural crops worldwide,which makes the development of an effective protoplast system for transient gene expression highly significant.Typically,plant protoplasts are initially isolated through enzymatic digestion and then used for transient transformations mediated by polyethylene glycol(PEG).However,PEG-mediated protoplast transformation suffers from low and inconsistent efficiency,is influenced by various factors,and requires greater operator expertise.Here,we present a simple and efficient protoplast system for transient gene expression in C.annuum and Nicotiana benthamiana,without PEG-mediated transfection.This procedure involved using the first and second fully expanded true leaves of pepper and N.benthamiana plants at the six-leaf stage for Agrobacterium infiltration,followed by enzymatic digestion for protoplast isolation.The resulting protoplast transfections achieved remarkably high efficiencies,facilitating functional analyses such as subcellular localization and protein—protein interaction studies(for example,BiFC,Co-IP,and Split-LUC assays).Thus,we have demonstrated a simplified and highly efficient transient expression system for protoplasts and potential wide-ranging applications in C.annuum and N.benthamiana while bypassing PEG-mediated transfection.展开更多
The utility of artificial microRNAs（amiRNAs） to induce loss of gene function has been reported for many plant species,but expression efficiency of the different amiRNA constructs in different transgenic plants was l...The utility of artificial microRNAs（amiRNAs） to induce loss of gene function has been reported for many plant species,but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable,In this study,expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation.A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments.Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs.This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.展开更多
Agrobacterium-mediated plant transformation is widely used in plant genetic engineering.However,its efficiency is limited by plant immunity against Agrobacterium.Chili pepper(Capsicum annuum L.)is an important vegetab...Agrobacterium-mediated plant transformation is widely used in plant genetic engineering.However,its efficiency is limited by plant immunity against Agrobacterium.Chili pepper(Capsicum annuum L.)is an important vegetable that is recalcitrant to Agrobacterium-mediated transformation.In this work,Agrobacterium was found to induce a strong immune response in pepper,which might be the reason for T-DNA being difficult to express in pepper.An Agrobacterium mutant screen was conducted and a point mutation in the hisI gene was identified due to a weak immune response and enhanced transient expression mediated by this Agrobacterium mutant in pepper leaves.Further genetic analysis revealed that histidine biosynthesis deficiency caused by mutations in many genes of this pathway led to reduced pepper cell death,presumably due to reduced bacterial growth.However,mutation analysis of threonine and tryptophan biosynthesis genes showed that the biosynthesis of different amino acids may play different roles in Agrobacterium growth and stimulating the pepper immune response.The possible application of Agrobacterium amino acid biosynthesis mutations in plant biology was discussed.展开更多
Associated with ITU-T G.719,a post-processing method in frequency domain for enhancing the perceptual quality of the decoded transient audio is proposed only to the audio decoder with no side information from the enco...Associated with ITU-T G.719,a post-processing method in frequency domain for enhancing the perceptual quality of the decoded transient audio is proposed only to the audio decoder with no side information from the encoder.The proposed post-filter is used to filter the frequency coefficients of the decoded transient frame and consists of a short-term post-filter and a spectral tilt compensation filter which are derived from linear predictive coding（LPC） predictor based on the discrete cosine transform（DCT） coefficients of the decoded transient frame.As a result,the post-filter in frequency domain shapes the temporal noise in time domain and controls the pre-echo noise effectively while enhancing the transient perception.Listening test results show that the preferring ratio of the post-processed transient signal is higher than that of the original decoded signal at a low bit rate of 32 kbit/s in G.719 and the post-processing module brings a complexity of 12.399 WMOPS to the decoder.展开更多
The rocks surrounding a roadway exhibit some special and complex phenomena with increasing depth of excavation in underground engineering.Quasi-static analysis cannot adequately explain these engineering problems.The ...The rocks surrounding a roadway exhibit some special and complex phenomena with increasing depth of excavation in underground engineering.Quasi-static analysis cannot adequately explain these engineering problems.The computational model of a circular roadway considering the transient effect of excavation unloading is established for these problems.The time factor makes the solution of the problem difficult.Thus,the computational model is divided into a dynamic model and a static model.The Laplace integral transform and inverse transform are performed to solve the dynamic model and elasticity theory is used to analyze the static model.The results from an example show that circumferential stress increases and radial stress decreases with time.The stress difference becomes large gradually in this progress.The displacement increases with unloading time and decreases with the radial depth of surrounding rocks.It can be seen that the development trend of unloading and displacement is similar by comparing their rates.Finally,the results of ANSYS are used to verify the analytical solution.The contrast indicates that the laws of the two methods are basically in agreement.Thus,the analysis can provide a reference for further study.展开更多
In this study, we produced tomato plants overexpressing an invertase inhibitor gene （Sly-INH） from tomato using a simple and efficient transient transformation system. Compared with control plants, the expression of...In this study, we produced tomato plants overexpressing an invertase inhibitor gene （Sly-INH） from tomato using a simple and efficient transient transformation system. Compared with control plants, the expression of Sly-INH was highly upregulated in Sly-INH overexpressing plants, as indicated by real-time polymerase chain reaction （PCR）. Physiological analysis revealed that Sly-INH inhibited the activity of cell wall invertase （CWIN）, which increased sugar accumulation in tomato fruit. Furthermore, Sly-INH mediated sucrose metabolism by regulating CWIN activity. Our results suggest that invertase activity is potentially regulated by the Sly-INH inhibitor at the post-translational level, and they demonstrate that the transient transformation system is an effective method for determining the functions of genes in tomato.展开更多
Populus spp.have long been used as model woody plant species for molecular biology research.However,tissues of poplar are often recalcitrant to experimental procedures for molecular studies.We generated a hormone auto...Populus spp.have long been used as model woody plant species for molecular biology research.However,tissues of poplar are often recalcitrant to experimental procedures for molecular studies.We generated a hormone autotrophic poplar suspension cell line from a hybrid of Populus alba×P.berolinensis‘Yinzhong’,named Qu-2.Qu-2 cells are suitable as a model biological system for studying woody plants.Qu-2 cells have many advantages over suspension cell lines derived so far from any other woody plants.Qu-2 cells are very easy to cultivate and can grow on several common plant culture media without the addition of any plant hormone.They show exceptionally high growth rates,reaching an approximately 150-fold increase in biomass after one week of culturing.Another important unique characteristic of Qu-2 cells is that they can be cryopreserved and readily reactivated.Qu-2 cells are suitable for molecular manipulations such as protoplast production,transient transformation,and RNA-seq analysis.Therefore,Qu-2 cells have the great potential to be an excellent model cell line in tree molecular biological research,ranging from physiology to gene function.The Qu-2 cells will be made available to the plant community for research.展开更多
The analysis of transient linear viscoelastic response of asphalt concrete （AC） is important for engineering applications. The traditional transient response of AC is analyzed in the time domain by performing compli...The analysis of transient linear viscoelastic response of asphalt concrete （AC） is important for engineering applications. The traditional transient response of AC is analyzed in the time domain by performing complicated convolution integral. The frequency domain approach allows one to determine the transient responses by performing simple multi- plication instead of the complicated convolution integral, and it does not require the time derivative of the input excitation, and thus, the approach could greatly reduce the analysis complexity. This study investigated the frequency domain approach in calculating the transient response by utilizing the discrete Fourier transform technique. The accuracy and effectiveness of the frequency domain approach were verified by comparing the analytical and calculated responses for the standard 3-parameter Maxwell model and by comparing the time and frequency domain solutions for AC. The effect of aliasing of the frequency domain approach can effectively reduce by selecting a small sampling interval for the time domain excitation function. A sampling interval is acceptable as long as the amplitude of the Fourier transformed excitation is close to 0 more than half of the sampling rate. The results show that the frequency domain approach provides a simple and accurate way to perform linear viscoelastic analysis of AC.展开更多
CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable...CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.展开更多
基金supported by the Agricultural Gongguan Project of Xinjiang Production and Construction Corps,China(NYHXGG,2023AA102)the Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-CSIAF-202402)+1 种基金the Outstanding Youth Foundation of Henan Scientific Committee,China(222300420097)the China Agriculture Research System of MOF and MARA(CARS-15-02).
文摘Genome editing in plants is a powerful strategy that can substantially advance functional genomics research,facilitating the discovery,enhancement,and development of novel traits with significant agricultural implications.Various methodologies,such as zinc finger nucleases(ZFNs),transcription activator-like effector nucleases(TALENs),and CRISPR/Cas systems,have been developed for this purpose.
基金supported by Biological Breeding of Early Maturing and Disease Resistant Cotton Varieties (NO.2023ZD04041)the Project of China Agriculture Research System (Grant No. CARS-15-06)+2 种基金Natural Science Foundation of Henan Province (Grant No. 232300421041 and 222300420382)National Natural Science Foundation of China (Grant No. U21 A20213)the Central Public-interest Scientific Institution Basal Research Fund (Grant No. 1610162023017 and 1610162023028)。
文摘Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.
基金supported by the National Natural Science Foundation of China(Grant Nos.32472536 and 32302526)the Natural Science Foundation of Anhui Province,China(Grant No.2208085MC64)+2 种基金the Outstanding Innovative Research Team for Molecular Enzymology and Detection in Anhui Provincial Universities(Grant No.2022AH010012)the University Synergy Innovation Program of Anhui Province(Grant No.GXXT-2022-067)the University Natural Science Research Program of Anhui Provincial Education Department(Grant No.KJ2021A0118).
文摘Pepper(Capsicum annuum)is one of the most important horticultural crops worldwide,which makes the development of an effective protoplast system for transient gene expression highly significant.Typically,plant protoplasts are initially isolated through enzymatic digestion and then used for transient transformations mediated by polyethylene glycol(PEG).However,PEG-mediated protoplast transformation suffers from low and inconsistent efficiency,is influenced by various factors,and requires greater operator expertise.Here,we present a simple and efficient protoplast system for transient gene expression in C.annuum and Nicotiana benthamiana,without PEG-mediated transfection.This procedure involved using the first and second fully expanded true leaves of pepper and N.benthamiana plants at the six-leaf stage for Agrobacterium infiltration,followed by enzymatic digestion for protoplast isolation.The resulting protoplast transfections achieved remarkably high efficiencies,facilitating functional analyses such as subcellular localization and protein—protein interaction studies(for example,BiFC,Co-IP,and Split-LUC assays).Thus,we have demonstrated a simplified and highly efficient transient expression system for protoplasts and potential wide-ranging applications in C.annuum and N.benthamiana while bypassing PEG-mediated transfection.
基金supported by the Ministry of Education of China and Agriculture and Agri-Food Canada(MOE-AAFC) PhD student research program
文摘The utility of artificial microRNAs（amiRNAs） to induce loss of gene function has been reported for many plant species,but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable,In this study,expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation.A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments.Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs.This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.
基金supported by the National Key Research and Development Program of China(Grant No.2018YFD1000800)National Natural Science Foundation of China(Grant No.32172600)。
文摘Agrobacterium-mediated plant transformation is widely used in plant genetic engineering.However,its efficiency is limited by plant immunity against Agrobacterium.Chili pepper(Capsicum annuum L.)is an important vegetable that is recalcitrant to Agrobacterium-mediated transformation.In this work,Agrobacterium was found to induce a strong immune response in pepper,which might be the reason for T-DNA being difficult to express in pepper.An Agrobacterium mutant screen was conducted and a point mutation in the hisI gene was identified due to a weak immune response and enhanced transient expression mediated by this Agrobacterium mutant in pepper leaves.Further genetic analysis revealed that histidine biosynthesis deficiency caused by mutations in many genes of this pathway led to reduced pepper cell death,presumably due to reduced bacterial growth.However,mutation analysis of threonine and tryptophan biosynthesis genes showed that the biosynthesis of different amino acids may play different roles in Agrobacterium growth and stimulating the pepper immune response.The possible application of Agrobacterium amino acid biosynthesis mutations in plant biology was discussed.
基金Supported by the Cooperation Between BIT and Ericsson
文摘Associated with ITU-T G.719,a post-processing method in frequency domain for enhancing the perceptual quality of the decoded transient audio is proposed only to the audio decoder with no side information from the encoder.The proposed post-filter is used to filter the frequency coefficients of the decoded transient frame and consists of a short-term post-filter and a spectral tilt compensation filter which are derived from linear predictive coding（LPC） predictor based on the discrete cosine transform（DCT） coefficients of the decoded transient frame.As a result,the post-filter in frequency domain shapes the temporal noise in time domain and controls the pre-echo noise effectively while enhancing the transient perception.Listening test results show that the preferring ratio of the post-processed transient signal is higher than that of the original decoded signal at a low bit rate of 32 kbit/s in G.719 and the post-processing module brings a complexity of 12.399 WMOPS to the decoder.
基金supported by the National Natural Science Foundation of China (Nos.51479108 and 51174196)the National Basic Research Program of China (No.2014CB046300)+1 种基金Shandong University of Science and Technology (No.2012KYTD104)Research Start-up Project of Shandong University of Science and Technology (No.2015RCJJ061)
文摘The rocks surrounding a roadway exhibit some special and complex phenomena with increasing depth of excavation in underground engineering.Quasi-static analysis cannot adequately explain these engineering problems.The computational model of a circular roadway considering the transient effect of excavation unloading is established for these problems.The time factor makes the solution of the problem difficult.Thus,the computational model is divided into a dynamic model and a static model.The Laplace integral transform and inverse transform are performed to solve the dynamic model and elasticity theory is used to analyze the static model.The results from an example show that circumferential stress increases and radial stress decreases with time.The stress difference becomes large gradually in this progress.The displacement increases with unloading time and decreases with the radial depth of surrounding rocks.It can be seen that the development trend of unloading and displacement is similar by comparing their rates.Finally,the results of ANSYS are used to verify the analytical solution.The contrast indicates that the laws of the two methods are basically in agreement.Thus,the analysis can provide a reference for further study.
基金supported by the National Natural Science Foundation of China(Nos.31372054 and 30971999)the State Key Laboratory of Plant Physiology and Biochemistry Open Project of China(No.SKLPPBKF1404)
文摘In this study, we produced tomato plants overexpressing an invertase inhibitor gene （Sly-INH） from tomato using a simple and efficient transient transformation system. Compared with control plants, the expression of Sly-INH was highly upregulated in Sly-INH overexpressing plants, as indicated by real-time polymerase chain reaction （PCR）. Physiological analysis revealed that Sly-INH inhibited the activity of cell wall invertase （CWIN）, which increased sugar accumulation in tomato fruit. Furthermore, Sly-INH mediated sucrose metabolism by regulating CWIN activity. Our results suggest that invertase activity is potentially regulated by the Sly-INH inhibitor at the post-translational level, and they demonstrate that the transient transformation system is an effective method for determining the functions of genes in tomato.
基金supported by the National Key Research and Development Program of China(2016YFD0600106)the National Natural Science Foundation of China(31670675)the Fundamental Research Funds for the Central Universities(2572018CL01 and 2572018CL02).
文摘Populus spp.have long been used as model woody plant species for molecular biology research.However,tissues of poplar are often recalcitrant to experimental procedures for molecular studies.We generated a hormone autotrophic poplar suspension cell line from a hybrid of Populus alba×P.berolinensis‘Yinzhong’,named Qu-2.Qu-2 cells are suitable as a model biological system for studying woody plants.Qu-2 cells have many advantages over suspension cell lines derived so far from any other woody plants.Qu-2 cells are very easy to cultivate and can grow on several common plant culture media without the addition of any plant hormone.They show exceptionally high growth rates,reaching an approximately 150-fold increase in biomass after one week of culturing.Another important unique characteristic of Qu-2 cells is that they can be cryopreserved and readily reactivated.Qu-2 cells are suitable for molecular manipulations such as protoplast production,transient transformation,and RNA-seq analysis.Therefore,Qu-2 cells have the great potential to be an excellent model cell line in tree molecular biological research,ranging from physiology to gene function.The Qu-2 cells will be made available to the plant community for research.
基金sponsored by Inner Mongolia Transportation Research Project(NJ-2014-X)Shanxi Transportation Research Project(2015-1-22)National Natural Science Foundation of China(51208080)
文摘The analysis of transient linear viscoelastic response of asphalt concrete （AC） is important for engineering applications. The traditional transient response of AC is analyzed in the time domain by performing complicated convolution integral. The frequency domain approach allows one to determine the transient responses by performing simple multi- plication instead of the complicated convolution integral, and it does not require the time derivative of the input excitation, and thus, the approach could greatly reduce the analysis complexity. This study investigated the frequency domain approach in calculating the transient response by utilizing the discrete Fourier transform technique. The accuracy and effectiveness of the frequency domain approach were verified by comparing the analytical and calculated responses for the standard 3-parameter Maxwell model and by comparing the time and frequency domain solutions for AC. The effect of aliasing of the frequency domain approach can effectively reduce by selecting a small sampling interval for the time domain excitation function. A sampling interval is acceptable as long as the amplitude of the Fourier transformed excitation is close to 0 more than half of the sampling rate. The results show that the frequency domain approach provides a simple and accurate way to perform linear viscoelastic analysis of AC.
基金supported by the National Natural Science Foundation of China(31788103 and 31971370)the National Key Research and Development Program(2022YFF1002802)the Ministry of Agriculture and Rural Affairs of China,and the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020102).
文摘CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.
