Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

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.

SSA-over-array(SSoA):A stacked DRAM architecture for nearmemory computing

Aiming to enhance the bandwidth in near-memory computing,this paper proposes a SSA-over-array(SSoA)architecture.By relocating the secondary sense amplifier(SSA)from dynamic random access memory(DRAM)to the logic die and repositioning the DRAM-to-logic stacking interface closer to the DRAM core,the SSoA overcomes the layout and area limitations of SSA and master DQ(MDQ),leading to improvements in DRAM data-width density and frequency,significantly enhancing bandwidth density.The quantitative evaluation results show a 70.18 times improvement in bandwidth per unit area over the baseline,with a maximum bandwidth of 168.296 Tbps/Gb.We believe the SSoA is poised to redefine near-memory computing development strategies.

根癌农杆菌介导的蒙古冰草稳定遗传转化体系建立

蒙古冰草(Agropyron mongolicum)亦称沙芦草,为禾本科(Poaceae)小麦族(Triticeae)冰草属(Agropyron)多年生疏丛型牧草,具有饲用价值高、抗寒耐旱以及耐盐、耐瘠薄、耐风沙等特性,是改良天然草场的适宜草种与挖掘优良耐逆基因资源的重要材料。然而,目前尚未建立蒙古冰草高效遗传转化体系,制约了该物种的基因资源鉴定与遗传改良应用。以蒙农1号蒙古冰草种子为来源的高再生效率的胚性愈伤系#89为外植体,建立了根癌农杆菌(Agrobacterium tumefaciens)EHA105介导的蒙古冰草稳定遗传转化体系,转化效率达30%。此外,针对多次继代后蒙古冰草愈伤系再生能力退化的难题,通过在再生培养基中添加1 mg·L^(-1) ABA或提高蔗糖浓度至45 g·L^(-1),成功将再生能力衰退的蒙古冰草愈伤系再生效率由5%分别提高至35%与42%。研究结果为后续蒙古冰草基因编辑体系建立、基因功能鉴定和新品种培育奠定了重要技术基础。

野大麦高效组培快繁及农杆菌介导的愈伤侵染体系建立

野大麦(Hordeum brevisubulatum)为禾本科大麦属多年生草本植物,具有较强的抗寒和耐盐碱能力,是挖掘抗逆基因的优良种质资源。但目前尚未见野大麦遗传转化体系的报道。该研究以蒙农1号杂交野大麦成熟胚为外植体诱导愈伤组织,建立了野大麦高效组培快繁体系,分化率达70%,快繁系数为35。在此基础上,利用根癌农杆菌(Agrobacterium tumefaciens)EHA105菌株侵染筛选获得的高质量愈伤系YZ101,通过优化侵染条件使侵染率接近30%。该体系的建立为野大麦功能基因组研究与分子设计育种奠定了基础。

3D printing of silk fibroin-based hybrid scaffold treated with platelet rich plasma for bone tissue engineering

3D printing/bioprinting are promising techniques to fabricate scaffolds with well controlled and patient-specific structures and architectures for bone tissue engineering.In this study,we developed a composite bioink consisting of silk fibroin(SF),gelatin(GEL),hyaluronic acid(HA),and tricalcium phosphate(TCP)and 3D bioprinted the silk fibroin-based hybrid scaffolds.The 3D bioprinted scaffolds with dual crosslinking were further treated with human platelet-rich plasma(PRP)to generate PRP coated scaffolds.Live/Dead and MTT assays demonstrated that PRP treatment could obviously promote the cell growth and proliferation of human adipose derived mesenchymal stem cells(HADMSC).In addition,the treatment of PRP did not significantly affect alkaline phosphatase(ALP)activity and expression,but significantly upregulated the gene expression levels of late osteogenic markers.This study demonstrated that the 3D printing of silk fibroin-based hybrid scaffolds,in combination with PRP post-treatment,might be a more efficient strategy to promote osteogenic differentiation of adult stem cells and has significant potential to be used for bone tissue engineering.

3D printing of multilayered scaffolds for rotator cuff tendon regeneration

Repairing massive rotator cuff tendon defects remains a challenge due to the high retear rate after surgical intervention.3D printing has emerged as a promising technique that enables the fabrication of engineered tissues with heterogeneous structures and mechanical properties,as well as controllable microenvironments for tendon regeneration.In this study,we developed a new strategy for rotator cuff tendon repair by combining a 3D printed scaffold of polylactic-co-glycolic acid(PLGA)with cell-laden collagen-fibrin hydrogels.We designed and fabricated two types of scaffolds:one featuring a separate layer-by-layer structure and another with a tri-layered structure as a whole.Uniaxial tensile tests showed that both types of scaffolds had improved mechanical properties compared to single-layered PLGA scaffolds.The printed scaffold with collagen-fibrin hydrogels effectively supported the growth,proliferation,and tenogenic differentiation of human adipose-derived mesenchymal stem cells.Subcutaneous implantation of the multilayered scaffolds demonstrated their excellent in vivo biocompatibility.This study demonstrates the feasibility of 3D printing multilayered scaffolds for application in rotator cuff tendon regeneration.

Mechanical defect identification for gas‐insulated switchgear equipment based on time‐frequency vibration signal analysis

Mechanical defect is an important reason for the failure of gas‐insulated switchgear(GIS)equipment.Based on the time‐frequency characteristic vibration signal analysis on five kinds of mechanical defects,a novel intelligent algorithm model combining complementary ensemble empirical mode decomposition(CEEMD)and genetic al-gorithm improved kernel fuzzy mean clustering(GAKFCM)was proposed to identify the mechanical defect type.First,the mechanical defect platform and detection sys-tem were built.Then CEEMD and IMF sensitivity factors were used to analyse the time‐frequency signal of five kinds of vibration defects,and the feature extraction was performed on the processed vibration signals.Finally,the mechanical vibration defect recognition model was established based on the GAKFCM algorithm and its validity was verified.Results show that the developed detection system can detect mechanical vibration signals sensitively.Singular values,frequency band lines and entropy can reflect the energy attenuation and distribution differences for different type of me-chanical defect vibration signals.The proposed GAKFCM clustering model combining the above vibration feature parameters can effectively find and diagnose the mechanical defect of GIS equipment.Its recognition accuracy reaches 96.74%,especially for the loose contact seat bolts and poor contact failures of the disconnector.

Insight into the mechanism of fracture properties modulated by microstructure in the myofibrillar protein and polysaccharide gel systems

The objective of this study was to investigate the mechanism of fracture properties modulated by microstructure in the myofibrillar protein(MP)and polysaccharides gel systems.Compare to the modified starch,the dietary fiber significantly improved the fracture stress and reduced the fracture strain at same concentration.The treatment with 2%dietary fiber had the highest value of fracture stress and the lowest value of fracture strain,which were 259 g and 1.12 respectively.From the skeleton structure,the Raman spectroscopy result showed that dietary fiber addition significantly reduced the intensity at 2945 cm−1,which suggested that the aggregation of hydrophobic groups was improved.The SEM showed that the treatment with 2%dietary fiber had the highest fractal dimension value of 1.7772 and the lowest lacunary value of 0.258.From the filling structure,the paraffin section showed that the polysac-charides were just simply trapped in MP gel networks and formed numerous large volumes and no-elastic of cavities.The principal component analysis suggested that the compactness of three-dimensional gel networks determined fracture stress of composite gel.The no-no-elastic of cavities formed by modified starch and dietary fiber resulted in the reduction of fracture strain.These results would promote the development of innovative nutritional meat product formulation with satisfied textural property.