Effects of dissolved oxygen on intestinal bacterial community and immunity of Atlantic salmon Salmo salar

Dissolved oxygen(DO)is one of most important factors which affect wide range physiologic features of including immune responses and intestinal bacterial community.However,the underlying mechanisms remain enigmatic.To address this question,the intestinal bacterial community compositions and the immune features of Atlantic salmon(Salmo salar)grown in recirculating aquaculture systems(RAS)were characterized.Fish were reared under different DO saturation levels,e.g.,200% saturation named high group(H),100%saturation named control group(CK),and 60%saturation named lower group(L).Large variations in the operational taxonomic units(OTUs)frequency distribution for the intestinal bacterial community of Atlantic salmon were observed.The intestinal bacterial community of all groups was dominated mainly by three phyla,e.g.,Proteobacteria.Firmicutes,and Bacteroidetes.Interestingly,Acinetobacter baumannii,an opportunistic pathogen of salmon was increased significantly in L group.We further monitored the immunity features of fish under different DO levels.The results show that leucocyte number,cortisol level,the expressions of interleukin-1β(IL-1β),Toll-like receptor 4(TLR4),and nucleotide-binding oligomerization domain like protein 2(NOD2)were higher at significant levels in the L group than those in the other two groups.TLR4 and NOD2 are usually related with the bacterial infections;therefore,it is reasonable to believe that the stronger immune responses observed in the L group might be related with the higher abundance of A.baumannii in the inte stine of Atlantic salmon.Overall,these findings demonstrated that low DO level may induce stronger immunity responses in Atlantic salmon.

Sequence stratigraphy, paleogeography, and coal accumulation regularity of major coal-accumulating periods in China

There are 9 major coal-accumulating periods during geological history in China,including the Early Carboniferous,Late Carboniferous-Early Permian,Middle Permian,Late Permian,Late Triassic,Early-Middle Jurassic,Early Cretaceous,Paleogene and Neogene.The coal formed in these periods were developed in different coal-accumulating areas(CAA)including the North China,South China,Northwest China,Northeast China,the Qinghai–Tibet area,and China offshore area.In this paper,we investigated depositional environments,sequence stratigraphy,lithofacies paleogeography and coal accumulation pattern of five major coal-accumulating periods including the Late Carboniferous to Middle Permian of the North China CAA,the Late Permian of the South China CAA,the Late Triassic of the South China CAA,the Early-Middle Jurassic of the North and Northwest China CAA,and the Early Cretaceous in the Northeast China CAA.According to distribution of the coal-bearing strata and the regional tectonic outlines,we have identified distribution range of the coal-forming basins,sedimentary facies types and coal-accumulating models.The sequence stratigraphic frameworks of the major coal-accumulating periods were established based on recognition of a variety of sequence boundaries.The distribution of thick coals and migration patterns of the coal-accumulating centers in the sequence stratigraphic framework were analyzed.The lithofacies paleogeography maps based on third-order sequences were reconstructed and the distribution of coal accumulation centers and coal-rich belts were predicted.

Razumikhin-Type Theorems on General Decay Stability of Impulsive Stochastic Functional Differential Systems with Markovian Switching

In this paper, the Razumikhin approach is applied to the study of both p-th moment and almost sure stability on a general decay for a class of impulsive stochastic functional differential systems with Markovian switching. Based on the Lyapunov-Razumikhin methods, some sufficient conditions are derived to check the stability of impulsive stochastic functional differential systems with Markovian switching. One numerical example is provided to demonstrate the effectiveness of the results.

Razumikhin-Type Theorems on p-th Moment Stability for Stochastic Switching Nonlinear Systems with Delay

This paper mainly tends to utilize Razumikhin-type theorems to investigate p-th moment stability for a class of stochastic switching nonlinear systems with delay. Based on the Lyapunov-Razumik- hin methods, some sufficient conditions are derived to check the stability of stochastic switching nonlinear systems with delay. One numerical example is provided to demonstrate the effectiveness of the results.

解锁作物多样性:通过基因组编辑增加遗传变异

The germplasm resource repository harbors an extensive collection of genetic variations,providing a crucial foundation for the survival and sustainable development of humankind.Throughout history,major agricultural breakthroughs have relied on safeguarding,exploring,and harnessing germplasm resources.However,the pursuit of high yields in modern agriculture has led to a continuous reduction in biodiversity,resulting in monocultures and an undesirable homogeneity of breeding materials.As a consequence,germplasm resources are facing the alarming prospect of accelerated loss leading to a decline in crop diversity.Furthermore,modern agricultural varieties encounter formidable challenges in terms of adapting to unfavorable growing conditions,such as environmental heterogeneity and the prevalence of pests and pathogens(Fig.1a).Enhancing the genetic variability of modern crops becomes paramount for fostering innovation within germplasm resources and ensuring food security.

Creating one-line hybrid crops by synthetic apomixis

Hybrid breeding plays an important role in increasing crop yields by taking advantage of hybrid vigor,which helps crops grow with superior yields across various climates and contributes to ensuring secure food production.Hybrid seed production is relatively straightforward and simple in cross-pollinating plants such as maize but is difficult in self-pollinating plants such as rice,wheat,and soybean.Therefore,breeders have developed a three-line breeding strategy,utilizing a sterile line,a maintainer line,and a restorer line,for breeding hybrid rice(Figure 1A;Yuan,1966).

Enabling technology and core theory of synthetic biology

Synthetic biology provides a new paradigm for life science research(“build to learn”)and opens the future journey of biotechnology(“build to use”).Here,we discuss advances of various principles and technologies in the mainstream of the enabling technology of synthetic biology,including synthesis and assembly of a genome,DNA storage,gene editing,molecular evolution and de novo design of function proteins,cell and gene circuit engineering,cell-free synthetic biology,artificial intelligence(AI)-aided synthetic biology,as well as biofoundries.We also introduce the concept of quantitative synthetic biology,which is guiding synthetic biology towards increased accuracy and predictability or the real rational design.We conclude that synthetic biology will establish its disciplinary system with the iterative development of enabling technologies and the maturity of the core theory.

Identification of two migratory colon ILC2 populations differentially expressing IL-17A and IL-5/IL-13

Group 2 innate lymphoid cells(ILC2s)play important tissue resident roles in anti-parasite immunity,allergic immune response,tissue homeostasis,and tumor immunity.ILC2s are considered tissue resident cells with little proliferation at steady state.Recent studies have shown that a subset of small intestinal ILC2s could leave their residing tissues,circulate and migrate to different organs,including lung,liver,mesenteric LN and spleen,upon activation.However,it remains unknown whether other ILC populations with migratory behavior exist.In this study,we find two major colon ILC2 populations with potential to migrate to the lung in response to IL-25 stimulation.One subset expresses IL-17A and resembles inflammatory ILC2s(iILC2s)but lacks CD27 expression,whereas the other expresses CD27 but not IL-17A.In addition,the IL-17A^(+)ILC2s express lower levels of CD127,CD25,and ST2 than CD27^(+)ILC2s,which express higher levels of IL-5 and IL-13.Surprisingly,we found that both colon ILC2 populations still maintained their colonic features of preferential expression of IL-17A and CD27,IL-5/IL-13,respectively.Together,our study identifies two migratory colon ILC2 subsets with unique surface markers and cytokine profiles which are critical in regulating lung and colon immunity and homeostasis.

Anthocyanin-assisted Agrobacterium infiltration for the rapid evaluation of genome editing efficiencies across multiple plant species

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.

Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System

Transcription activator-like effector nucleases （TALENs） and clustered regularly interspaced short palindromic repeats （CRISPR）/ CRISPR-associated （Cas） systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmlPKIA, ZmlPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmlPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies （13.1%） similar to those obtained with TALENs （9.1%）. Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.

Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems

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.

Manipulating mRNA splicing by base editing in plants

Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.

Current and future editing reagent delivery systems for plant genome editing

Many genome editing tools have been developed and new ones are anticipated; some have been extensively applied in plant genetics, biotechnology and breeding, especially the CRISPR/Cas9 system. These technologies have opened up a new era for crop improvement due to their precise editing of user-specified sequences related to agronomic traits. In this review, we will focus on an update of recent developments in the methodologies of editing reagent delivery, and consider the pros and cons of current delivery systems. Finally, we will reflect on possible future directions.

An Uncanonical CCCH-Tandem Zinc-Finger Protein Represses Secondary Wall Synthesis and Controls Mechanical Strength in Rice

Secondary walls, which represent the bulk of biomass, have a large impact on plant growth and adaptation to environments. Secondary wall synthesis is switched and regulated by a sophisticated signaling transduction network. However, there is limited understanding of these regulatory pathways. Here, we report that ILAl-interacting protein 4 （lIP4） can repress secondary wall synthesis, lIP4 is a phosphorylation sub- strate of an Raf-like MAPKKK, but its function is unknown. By generating lip4 mutants and relevant transgenic plants, we found that lesions in lIP4 enhance secondary wall formation. Gene expression and transactivation activity assays revealed that lIP4 negatively regulates the expression of MYB61 and CESAs but does not bind their promoters, lIP4 interacts with NAC29/NAC31, the upstream regulators of secondary wall synthesis, and suppresses the downstream regulatory pathways in plants. Mutagenesis analyses showed that phosphomimic UP4 proteins translocate from the nucleus to the cytoplasm, which releases interacting NACs and attenuates its repression function. Moreover, we revealed that liPs are evolutionarily conserved and share unreported CCCH motifs, referred to as uncanonical CCCH-tandem zinc-finger proteins. Collectively, our study provides mechanistic insights into the control of secondary wall synthesis and presents an opportunity for improving relevant agronomic traits in crops.

Robust genome editing of CRISPR-Cas9 at NAG PAMs in rice

Dear Editor,The CRISPR-Cas9(clustered regularly interspaced short palindromic repeats/Cas9)system has been widely used for a variety of applications,including targeted gene knockout,gene insertion,gene replacement and base editing.Despite its wide use,the genome editing using CRISPR-Cas9 is performed almost exclusively at sites containing canonical NGG protospacer adjacent motifs(PAMs).To overcome the PAM constraint of the CRISPR-Cas9 system,many attempts have been made to develop various Cas9 orthologs

Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture.

Genome editing provides novel strategies for improving plant traits,but relies on current genetic transformation and plant regeneration procedures,which can be inefficient.We have engineered a barley stripe mosaic virus(BSMV)-based sgRNA delivery vector(BSMV-sg)that is effective in performing heritable genome editing in Cas9-transgenic wheat plants.Mutated progenies were present in the next generation at frequencies ranging from 12.9%to 100%in three different wheat varieties,and 53.8%to 100%of mutants were virus-free.We also achieved multiplex mutagenesis in progeny using a pool of BSMV-sg vectors harboring different sgRNAs.Furthermore,we devised a virus-induced transgene-free editing procedure(VITF-Edit)to generate Cas9-free wheat mutants by crossing BSMV-infected Cas9-transgenic wheat pollen with wild-type wheat.Our study provides a robust,convenient and tissue culture-free approach for genome editing in wheat through virus infection.

Generation of thermosensitive male-sterile maize by targeted knockout of the ZmTMS5 gene

Maize（Zea mays L.）is one of the most important cereal crops,with a global production of 1.02 billion tons in 2013（Baldaufa et al.,2016）.Heterosis is widely used to increase the productivity of maize,and the first commercial hybrid maize was introduced in the 1930s（Duvick,2001）.

The vernalization-induced long non-coding RNA VAS functions with the transcription factor TaRF2b to promote TaVRN1 expression for flowering in hexaploid wheat

Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.

CRISPR editing-mediated antiviral immunity: a versatile source of resistance to combat plant virus infections

Virus infection poses a constant threat on global crop productivity.It is estimated that virus diseases account for at least 10% of crop losses worldwide.Development of resistant crops using naturally evolved resistance genes has been a main measure for controlling plant virus epidemics.However,isolation of plant resistance genes is costly and time-consuming.Furthermore,most plant resistance genes are pathogen race-specific,with very few of them being broadly specific (Langner et al.,2018).Consequently,artificial immunity with engineered resistance to virus infections has received substantial research,which includes pathogenderived resistance,introducing single or stacked transgene into crops,and RNA interference (Shepherd et al.,2009).However,these strategies have so far achieved limited success due to narrow availability and/or fitness costs on host plants.Therefore,it is urgent to develop novel and more efficient strategies to combat plant virus infections.Recent breakthrough of CRISPR/Cas-based DNA and RNA editing tools provides a promising direction for engineering artificial immunity to plant viruses.There are now increasing reports demonstrating that CRISPR/Cas systems can be harnessed to develop antiviral immunity in plants with high efficiency and broad specificity (Table S1 in Supporting Information).

Genome editing in plants with MAD7 nuclease

MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.