Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.I...Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.In this study,we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa,a variety widely cultivated in Northeast China.Subsequently,we created a single transcript CRISPR/Cas9(CRISPR_2.0)toolkit,incorporating multiplex gRNAs,designed for gene editing in Gongnong 1.Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter,a widely employed polymeraseⅡconstitutive promoter known for strong transgene expression in dicots.To assess the toolkit’s efficiency,we targeted PALM1,a gene associated with a recognizable multifoliate phenotype.Utilizing the CRISPR_2.0 toolkit,we directed PALM1 editing at two sites in the wild-type Gongnong 1.Results indicated a 35.1%occurrence of editing events all in target 2 alleles,while no mutations were detected at target 1 in the transgenic-positive lines.To explore more efficient sgRNAs,we developed a rapid,reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation,incorporating the visible reporter MtLAP1.This screening system demonstrated that most purple visible hairy roots underwent gene editing.Notably,sgRNA3,with an 83.0%editing efficiency,was selected using the visible hairy root system.As anticipated,tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype.These palm1 lines demonstrated an average crude protein yield increase of 21.5%compared to trifoliolate alfalfa.Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.展开更多
通过实验探究鱼洗现象的物理原理,采用Cool Edit Pro 2.0软件作为工具探究其影响因素。实验结果表明:洗壁上的驻波是二维驻波;鱼洗产生四柱水花时振动频率取决于鱼洗中的水量,与水面半径无关;当鱼洗振动产生水花时,整个鱼洗对水花的喷...通过实验探究鱼洗现象的物理原理,采用Cool Edit Pro 2.0软件作为工具探究其影响因素。实验结果表明:洗壁上的驻波是二维驻波;鱼洗产生四柱水花时振动频率取决于鱼洗中的水量,与水面半径无关;当鱼洗振动产生水花时,整个鱼洗对水花的喷溅都是有贡献的,而不仅仅是洗壁起作用。展开更多
作为一种研究复杂系统的有效手段,基于Agent的建模仿真方法得到了广泛应用。但目前还没有一种得到广泛认可的建模仿真规范来指导和约束Agent仿真模型的设计、开发、集成和运行,不利于提高Agent仿真模型的可重用性和开发效率。提出将采...作为一种研究复杂系统的有效手段,基于Agent的建模仿真方法得到了广泛应用。但目前还没有一种得到广泛认可的建模仿真规范来指导和约束Agent仿真模型的设计、开发、集成和运行,不利于提高Agent仿真模型的可重用性和开发效率。提出将采用模型驱动架构和基于组件设计思想的仿真模型可移植性规范(simu-lation model portability standard 2.0,SMP2)引入到基于Agent的建模仿真中,通过把Agent仿真中的元素映射到SMP2的模型设计和开发模式架构下,并根据Agent仿真特性对SMP2进行相应扩展,来支持模型驱动的组件化的Agent仿真模型开发。利用一个应用实例,验证了提出方法的有效性。展开更多
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA26030301)Hohhot Key R&D Project(2023-JBGSS-1),the National Natural Science Foundation of China(U23A200206,32071864,32325035)+1 种基金the Taishan Scholar Program of Shandong(to Chunxiang Fu)the Shandong Provincial Natural Science Foundation(ZR202210270038)。
文摘Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.In this study,we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa,a variety widely cultivated in Northeast China.Subsequently,we created a single transcript CRISPR/Cas9(CRISPR_2.0)toolkit,incorporating multiplex gRNAs,designed for gene editing in Gongnong 1.Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter,a widely employed polymeraseⅡconstitutive promoter known for strong transgene expression in dicots.To assess the toolkit’s efficiency,we targeted PALM1,a gene associated with a recognizable multifoliate phenotype.Utilizing the CRISPR_2.0 toolkit,we directed PALM1 editing at two sites in the wild-type Gongnong 1.Results indicated a 35.1%occurrence of editing events all in target 2 alleles,while no mutations were detected at target 1 in the transgenic-positive lines.To explore more efficient sgRNAs,we developed a rapid,reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation,incorporating the visible reporter MtLAP1.This screening system demonstrated that most purple visible hairy roots underwent gene editing.Notably,sgRNA3,with an 83.0%editing efficiency,was selected using the visible hairy root system.As anticipated,tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype.These palm1 lines demonstrated an average crude protein yield increase of 21.5%compared to trifoliolate alfalfa.Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.
文摘作为一种研究复杂系统的有效手段,基于Agent的建模仿真方法得到了广泛应用。但目前还没有一种得到广泛认可的建模仿真规范来指导和约束Agent仿真模型的设计、开发、集成和运行,不利于提高Agent仿真模型的可重用性和开发效率。提出将采用模型驱动架构和基于组件设计思想的仿真模型可移植性规范(simu-lation model portability standard 2.0,SMP2)引入到基于Agent的建模仿真中,通过把Agent仿真中的元素映射到SMP2的模型设计和开发模式架构下,并根据Agent仿真特性对SMP2进行相应扩展,来支持模型驱动的组件化的Agent仿真模型开发。利用一个应用实例,验证了提出方法的有效性。
