CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean

CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean

Effect of Exogenous Phosphate on the Lability and Phytoavailability of Arsenic in Soils

The effect of exogenous phosphate (P, 200 mg·kg-1 soil) on the lability and phyto-availability of arsenic (As) was studied using the diffusive gradients in thin films (DGT) technique. Lettuce were grown on the As-amended soils following the stabilization of soil labile As after 90 day’s incubation. Phosphate (P) application generally facilitated plant growth except one grown on P-sufficient soil. Soil labile As concentration increased in all the soils after P application due to a competition effect. Plant As concentration increased in red soils collected from Hunan Province, while decreases were observed in the other soils. Even though, an overall trend of decrease was obtained in As phytoavailability along with the increase of DGT-measured soil labile P/As molar ratio. The functional equation between P/As and As phytoavailability provided a critical value of 1.7, which could be used as a guidance for rational P fertilization, thus avoiding overfertilization

Soil ridging combined with biochar or calcium-magnesium-phosphorus fertilizer application: Enhanced interaction with Ca, Fe and Mn in new soil habitat reduces uptake of As and Cd in rice

peer reviewedReducing the bioavailability of both cadmium (Cd) and arsenic (As) in paddy fields is a worldwide challenge. The authors investigated whether ridge cultivation combined with biochar or calcium-magnesium-phosphorus (CMP) fertilizer effectively reduces the accumulation of Cd and As in rice grains. Field trial showed that applying biochar or CMP on the ridges was similar to the continuous flooding, which maintained grain Cd at a low level, but grain As was reduced by 55.6%, 46.8% (IIyou28) and 61.9%, 59.3% (Ruiyou 399). Compared with ridging alone, the application of biochar or CMP decreased grain Cd by 38.7%, 37.8% (IIyou28) and 67.58%, 60.98% (Ruiyou399), and reduced grain As by 38.9%, 26.9% (IIyou28) and 39.7%, 35.5% (Ruiyou 399). Microcosm experiment showed that applying biochar and CMP on the ridges decreased As in soil solution by 75.6% and 82.5%, respectively, and kept Cd at a comparably low level at 0.13–0.15 μg L−1. Aggregated boosted tree (ABT) analysis revealed that ridge cultivation combined with soil amendments altered soil pH, redox state (Eh) and enhanced the interaction of Ca, Fe, Mn with As and Cd, which promoted the concerted reduction of As and Cd bioavailability. Application of biochar on the ridges enhanced the effects of Ca and Mn to maintain a low level of Cd, and enhanced the effects of pH to reduce As in soil solution. Similar to ridging alone, applying CMP on the ridges enhanced the effects of Mn to reduce As in soil solution, and enhanced the effects of pH and Mn to maintain Cd at a low level. Ridging also promoted the association of As with poorly/well-crystalline Fe/Al and the association of Cd on Mn-oxides. This study provides an effective and environmentally friendly method to decrease Cd and As bioavailability in paddy fields and mitigate Cd and As accumulation in rice grain

Helium-induced damage in MAB phase MoAlB and Fe2AlB2: first-principles simulation

Similar to M _n+1 AX _n (MAX, M: transition metal, A: A group element, X: C or N, n = 1 ∼ 3) phase materials, MAB (M: transition metal, A: A group element, B: B) phases also exhibit excellent comprehensive mechanical and thermal properties that are applicable to future nuclear reactors. The origination and growth conditions of He bubbles under irradiation in MAB phase MoAlB and Fe _2 AlB _2 have been calculated through first-principles theory in this work. In general, Fe _2 AlB _2 may present lower single/di-vacancy formation energies and a consequent higher He bubble number density. The final He bubble shape and comparative average size of MoAlB and Fe _2 AlB _2 have been predicted as well. In MoAlB there will form large platelet-like He bubbles and small spherical ones. In Fe _2 AlB _2 there will form spherical He bubbles with different sizes. These He bubbles can all further link via interlayer vacancies into string-like shape. Fe _2 AlB _2 also possesses higher He-induced embrittlement tendency than MoAlB

Evolutionary game study on multi-agent value co-creation of service-oriented digital transformation in the construction industry.

The service-oriented digital transformation of the construction industry is a development trend of cross-border industrial integration and transformation and upgrading in the digital economy environment, and collaborative value creation among stakeholders is seen as a strategic imperative to promote this process. This study aims to achieve efficient collaborative value co-creation and accelerate the digital transformation process of the construction industry by exploring the collaborative strategies and evolution laws of value co-creators in the digital service ecosystem of the construction industry. Based on evolutionary game theory and methods, this paper analyzes the evolutionary stability strategies and conditions of each participant in the service-oriented value chain at different stages of the digital transformation of the construction industry. It is found that with the improvement of the level of digitalization, the degree of cooperation among game players continues to increase until a stable state of full cooperation is achieved. The initial willingness of the game players to cooperate accelerates the speed of the system's evolution to the stable state of full cooperation in the middle stage of digital transformation. Additionally, the improvement of the construction process digitalization level can subvert the evolution result of full non-coordination caused by a low initial willingness to cooperate. The research conclusions and corresponding countermeasures and suggestions can provide a strategic reference for the service-oriented digital transformation of the construction industry

Splicing Method of Micro-Nano-Scale Pore Radius Distribution in Tight Sandstone Reservoir

Accurate characterization of the micro- and nano-pore radius values in a tight sandstone reservoir is the key work to reasonably evaluate reservoir properties. The previous exploration of pore-stitching methods is mainly based on the morphological extension of similar segments. However, few scholars compare and verify the image and non-image stitching methods, so they cannot clarify the application scope of different pore-stitching methods. In this study, the pore structures of eight selected tight sandstone samples were evaluated using high-pressure mercury injection, nuclear magnetic resonance, scanning electron microscope, and the helium porosity test. Then, the C-value fitting, interpolation fitting, and morphological fitting were used to establish high-pressure mercury injection and Nuclear Magnetic Resonance (NMR) pore distribution curves to evaluate the differences among the micro-nano-scale pore radius values determined by the three fitting methods. Finally, the pore radius distribution is extracted from the binary image of Scanning Electron Microscope (SEM). After correcting the helium porosity data, the application scope of different fitting methods is evaluated by using the mean standard deviation verification method, and the optimal solution of the stitching method of pore radius distribution in each application scope is found. Compared to other studies, this research demonstrated three relatively simple methods for the determination of the full range of pore size distributions, providing a reliable method to evaluate the prerequisites of the range of application. This study provides a new idea for the micro-nano-scale pore radius splicing method of a tight sandstone reservoir, and the research results can provide a reference for the actual reservoir evaluation of oil and gas fields