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.

The complete reference genome for grapevine (Vitis vinifera L.) genetics and breeding

Grapevine is one of the most economically important crops worldwide.However,the previous versions of the grapevine reference genome tipically consist of thousands of fragments with missing centromeres and telomeres,limiting the accessibility of the repetitive sequences,the centromeric and telomeric regions,and the study of inheritance of important agronomic traits in these regions.Here,we assembled a telomere-to-telomere(T2T)gap-free reference genome for the cultivar PN40024 using PacBio HiFi long reads.The T2T reference genome(PN_T2T)is 69 Mb longer with 9018 more genes identified than the 12X.v0 version.We annotated 67%repetitive sequences,19 centromeres and 36 telomeres,and incorporated gene annotations of previous versions into the PN_T2T assembly.We detected a total of 377 gene clusters,which showed associations with complex traits,such as aroma and disease resistance.Even though PN40024 derives from nine generations of selfing,we still found nine genomic hotspots of heterozygous sites associated with biological processes,such as the oxidation–reduction process and protein phosphorylation.The fully annotated complete reference genome therefore constitutes an important resource for grapevine genetic studies and breeding programs.

Elimination of the yellow pigment gene PSY-E2 tightly linked to the Fusarium head blight resistance gene Fhb7 from Thinopyrum ponticum

Fhb7 is a major gene that was transferred from Thinopyrum ponticum to chromosome 7D of wheat(Triticum aestivum)and confers resistance to both Fusarium head blight(FHB)and Fusarium crown rot(FCR).However,Fhb7 is tightly linked to the PSY-E2 gene,which causes yellow flour,limiting its application in breeding.To break this linkage,marker K-PSY was developed for tagging PSY-E2 and used with Fhb7 markers to identify recombination between the two genes.Screening 21,000 BC1F2 backcross progeny(Chinese Spring ph1bph1b*2/SDAU 2028)revealed two Fhb7^(+)wheat-Tp7el_(2)L lines,Shannong 2–16and Shannong 16–1,that carry a desired truncated Fhb7^(+)translocation segment without PSY-E2.The two lines show levels of resistance to FHB and FCR similar to those of the original translocation line SDAU 2028,but have white flour.To facilitate Fhb7 use in wheat breeding,STS markers were developed and used to isolate Fhb7 on a truncated Tp7el_(2) translocation segment.Near-isogenic lines carrying the Fhb7^(+)segment were generated in the backgrounds of three commercial cultivars,and Fhb7^(+)lines showed increased FHB and FCR resistance without yield penalty.The breakage of the tight linkage between Fhb7 and PSY-E2 via homoeologous recombination provides genetic resources for improvement of wheat resistance to FHB and FCR and permit the large-scale deployment of Fhb7 in breeding using marker-assisted selection.

HbNST1 is a positive regulator of the lignin accumulation in strawflower bracts

Strawflower(Helichrysum bracteatum)capitula have papery bracts and thus have the qualities of a naturally dried flower.The involucral bract cells have a secondary cell wall(SCW)of which a crucial component is lignin.Although the constituents of SCWs have been studied extensively in plants,little is known of the mechanism regulating SCW formation,especially lignin biosynthesis in the involucral bracts of strawflower.In this study,a homolog of NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1,designated Hb NST1,was identified as a positive regulator of lignin biosynthesis in strawflower.The transcript level of Hb NST1 was the highest in the involucral bracts.Subcellular localization analysis indicated that Hb NST1 was localized to the nucleus.Overexpression of Hb NST1 in Chrysanthemum indicum promoted the expression of a gene related to lignin biosynthesis,a homolog of cinnamyl alcohol dehydrogenase,designated Ci CAD,suggesting that Hb NST1 was associated with the accumulation of lignin in the SCW of the involucral bracts.Taken together,the results suggested that Hb NST1 positively regulated lignin accumulation in the involucral bracts and mediated the expression of lignin biosynthesis-related genes in strawflower.

Promotion effect of TGF-β-Zfp423-ApoD pathway on lip sensory recovery after nerve sacrifice caused by nerve collateral compensation

Resection of oral and maxillofacial tumors is often accompanied by the inferior alveolar nerve neurectomy,resulting in abnormal sensation in lower lip.It is generally believed that spontaneous sensory recovery in this nerve injury is difficult.However,during our follow-up,patients with inferior alveolar nerve sacrifice showed different degrees of lower lip sensory recovery.In this study,a prospective cohort study was conducted to demonstrate this phenomenon and analyze the factors influencing sensory recovery.

Recent Progress of Surface Passivation Molecules for Perovskite Solar Cell Applications

Due to the solution processable nature,the prepared perovskite films are polycrystalline with considerable number of defects.These defects,especially defects at interface accelerate the carrier recombination and reduce the carrier collection.Besides,the surface defects also affect the long-term stability of the perovskite solar cells(PVSCs).To solve this problem,surface passivation molecules are introduced at selective interface(the interface between perovskite and carrier selective layer).This review summarizes recent progress of small molecules used in PVSCs.Firstly,different types of defect states in perovskite films are introduced and their effects on device performance are discussed.Subsequently,surface passivation molecules are divided into four categories,and the interaction between the functional groups of the surface passivation molecules and selective defect states in perovskite films are highlighted.Finally,we look into the prospects and challenges in design noble small molecules for PVSCs applications.

VvBBX44 and VvMYBA1 form a regulatory feedback loop to balance anthocyanin biosynthesis in grape

Anthocyanins are essential for the quality of perennial horticultural crops,such as grapes.In grapes,ELONGATED HYPOCOTYL 5(HY5)and MYBA1 are two critical transcription factors that regulate anthocyanin biosynthesis.Our previous work has shown that Vitis vinifera B-box protein 44(VvBBX44)inhibits anthocyanin synthesis and represses VvHY5 expression in grape calli.However,the regulatory mechanism underlying this regulation was unclear.In this study,we found that loss of VvBBX44 function resulted in increased anthocyanin accumulation in grapevine callus.VvBBX44 directly represses VvMYBA1,which activates VvBBX44.VvMYBA1,but not VvBBX44,directly modulates the expression of grape UDP flavonoid 3-O-glucosyltransferase(VvUFGT).We demonstrated that VvBBX44 represses the transcriptional activation of VvUFGT and VvBBX44 induced by VvMYBA1.However,VvBBX44 and VvMYBA1 did not physically interact in yeast.The application of exogenous anthocyanin stimulated VvBBX44 expression in grapevine suspension cells and tobacco leaves.These findings suggest that VvBBX44 and VvMYBA1 form a transcriptional feedback loop to prevent overaccumulation of anthocyanin and reduce metabolic costs.Our work sheds light on the complex regulatory network that controls anthocyanin biosynthesis in grapevine.

水驱动弹头辅助注塑管件壁厚的工艺影响

对水驱动弹头辅助注塑(W-PAIM)中工艺方法和工艺参数对管件壁厚的影响进行了实验研究。对水驱动弹头辅助注塑短射法(W-PAIM-S)、溢流法(W-PAIM--O)及水辅助注塑短射法(WAIM-S)、溢流法(WAIM-O)4种工艺方法进行比较,发现W-PAIM管件比WAIM(水辅助注塑技术)管件壁厚要薄得多,而W-PAIM-O管件壁厚又比W-PAIM-S管件壁厚要更均匀。采用正交试验法考察了熔体温度、注水压力、射胶压力、保压时间、注水延迟时间、模具温度对WPAIM-O管件壁厚的影响规律与大小,发现注水延迟时间和熔体温度对壁厚影响很大,因素影响比合计超过70%;而射胶压力、注水压力及保压时间的影响较小,因素影响比合计不足17%;管件残余壁厚随注水延迟时间的延长逐渐增大,随着熔体温度的升高先减小后增大。

溢流法水驱动弹头辅助注塑管件残余壁厚的形成与工艺参数影响

溢流法水驱动弹头辅助注塑(W-PAIM-O)工艺可制得残余壁厚(RWT)更薄更均匀的管件。采用模拟手段分析了W-PAIM-O工艺中RWT的形成机理,采用单因素实验法考察了熔体温度、熔体注射压力、注水延迟时间、注水压力和模具温度5个工艺参数对RWT的影响。模拟发现,RWT不仅主要取决于弹头的截面尺寸,还与弹头穿透时对侧面熔体的拖曳作用有关;在考察的几个工艺参数中,熔体温度越高,RWT越小,但熔体温度过高时RWT反而增大;熔体注射压力越大RWT越小,但熔体注射压力过大反而使RWT增大;随着模具温度的升高,RWT变薄,模具温度超过一定值后,RWT趋于稳定;RWT随注水延迟时间的延长而变厚;注水压力对W-PAIM-O管件RWT影响较小。

MiR156 regulates anthocyanin biosynthesis through SPL targets and other microRNAs in poplar

Anthocyanins biosynthesized from the flavonoid pathway are types of pigments that are involved in the protection of poplar from biotic and abiotic stresses.Previous researchers studying anthocyanin-related transcription factors and structural genes in poplar have made significant discoveries.However,little is known about the regulatory role of microRNAs in anthocyanin biosynthesis in poplar.Here,we overexpressed miR156 in poplar to study the comprehensive effects of the miR156-SPL module on the biosynthesis of anthocyanins.Small RNA sequencing analysis revealed 228 microRNAs differentially expressed in transgenic poplar plants with dramatically increased miR156 levels.Furthermore,integrated microRNAomic and transcriptomic analysis suggested that two microRNAs,miR160h,and miR858,have the potential to affect anthocyanin accumulation in poplar by regulating auxin response factors and MYB transcription factors,respectively.Additionally,the accumulation of miR160h and miR858 displayed a positive correlation with miR156 levels,suggesting a possible interaction between the miR156-SPL module and these microRNAs in poplar.Last,metabolomics analysis revealed that the levels of anthocyanins,flavones,and flavonols were substantially elevated in transgenic poplar plants overexpressing miR156 compared with the wild type,whereas the total lignin content was reduced in the transgenic plants.Taken together,our results indicate that miR156 can fine tune the anthocyanin biosynthetic pathway via multiple factors,including microRNAs,transcription factors,and the levels of structural genes,in poplar.This provides additional clues for understanding the complex regulatory network of anthocyanin biosynthesis in woody plants.

Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation

Vitis amurensis is a wild Vitis plant that can withstand extreme cold temperatures.However,the accumulation of metabolites during cold acclimation(CA)in V.amurensis remains largely unknown.In this study,plantlets of V.amurensis and V.vinifera cv.Muscat of Hamburg were treated at 4℃ for 24 and 72 h,and changes of metabolites in leaves were detected by gas chromatography coupled with time-of-flight mass spectrometry.Most of the identified metabolites,including carbohydrates,amino acids,and organic acids,accumulated in the two types of grape after CA.Galactinol,raffinose,fructose,mannose,glycine,and ascorbate were continuously induced by cold in V.amurensis,but not in Muscat of Hamburg.Twelve metabolites,including isoleucine,valine,proline,2-oxoglutarate,and putrescine,increased in V.amurensis during CA.More galactinol,ascorbate,2-oxoglutarate,and putrescine,accumulated in V.amurensis,but not in Muscat of Hamburg,during CA,which may be responsible for the excellent cold tolerance in V.amurensis.The expression levels of the genes encodingβ-amylase(BAMY),galactinol synthase(GolS),and raffinose synthase(RafS)were evaluated by quantitative reverse transcription-PCR.The expression BAMY(VIT_02s0012 g00170)and RafS(VIT_05s0077 g00840)were primarily responsible for the accumulation of maltose and raffinose,respectively.The accumulation of galactinol was attributed to different members of GolS in the two grapes.In conclusion,these results show the inherent differences in metabolites between V.amurensis and V.vinifera under CA.

Overexpression of PvWOX3a in switchgrass promotes stem development and increases plant height

Switchgrass(Panicum virgatum L.)is an important perennial,noninvasive,tall ornamental grass that adds color and texture to gardens and landscapes.Moreover,switchgrass has been considered a forage and bioenergy crop because of its vigorous growth,low-input requirements,and broad geography.Here,we identified PvWOX3a from switchgrass,which encodes a WUSCHEL-related homeobox transcription factor.Transgenic overexpression of PvWOX3a in switchgrass increased stem length,internode diameter,and leaf blade length and width,all of which contributed to a 95%average increase in dry weight biomass compared with control plants.Yeast one-hybrid and transient dual-luciferase assays showed that PvWOX3a can repress the expression of gibberellin 2-oxidase and cytokinin oxidase/dehydrogenase through apparently direct interaction with their promoter sequences.These results suggested that overexpression of PvWOX3a could increase gibberellin and cytokinin levels in transgenic switchgrass plants,which promotes cell division,elongation,and vascular bundle development.We also overexpressed PvWOX3a in a transgenic miR156-overexpressing switchgrass line that characteristically exhibited more tillers,thinner internodes,and narrower leaf blades.Double transgenic switchgrass plants displayed significant increases in internode length and diameter,leaf blade width,and plant height but retained a tiller number comparable to that of plants expressing miR156 alone.Ultimately,the double transgenic switchgrass plants produced 174%more dry-weight biomass and 162%more solubilized sugars on average than control plants.These findings indicated that PvWOX3a is a viable potential genetic target for engineering improved shoot architecture and biomass yield of horticulture,fodder,and biofuel crops.

Gene body demethylation increases expression and is associated with self-pruning during grape genome duplication

A colchicine-induced autotetraploid grapevine exhibiting potentially valuable agronomic traits for grape production and breeding,including self-pruning,was identified.This study investigated DNA methylation variation and its role in gene expression during self-pruning in the autotetraploid grapevine.We used RNA-Seq to estimate differentially expressed genes between diploid and autotetraploid grapevine shoot tips.The genes showing increases in the autotetraploid were mainly related to stress response pathways,whereas those showing decreases in the autotetraploid were related to biological metabolism and biosynthesis.Whole-genome bisulfite sequencing was performed to produce single-base methylomes for the diploid and autotetraploid grapevines.Comparison between the methylomes revealed that they were conserved in CG and CHG contexts.In the autotetraploid grapevine,hypodifferentially methylated regions(DMRs)and hyper-DMRs in the gene body increased or decreased gene expression,respectively.Our results indicated that a hypo-DMR in the ACO1 gene body increased its expression and might promote self-pruning.This study reports that hypo-DMRs in the gene body increase gene expression in plants and reveals the mechanism underlying the changes in the modifications affecting gene expression during genome duplication.Overall,our results provide valuable information for understanding the relationships between DNA methylation,gene expression,and autotetraploid breeding in grape.

Arbuscular mycorrhizal symbiosis facilitates apricot seedling(Prunus sibirica L.)growth and photosynthesis in northwest China

Arbuscular mycorrhizal(AM)fungi can successfully enhance photosynthesis(P_(n))and plants growth in agricultural or grassland ecosystems.However,how the symbionts affect species restoration in sunlight-intensive areas remains largely unexplored.Therefore,this study’s objective was to assess the effect of AM fungi on apricot seedling physiology,within a specific time period,in northwest China.In 2010,an experimental field was established in Shaanxi Province,northwest China.The experimental treatments included two AM fungi inoculation levels(0 or 100 g of AM fungal inoculum per seedling),three shade levels(1900,1100,and 550µmol m^(−2) s^(−1)),and three ages(1,3,and 5 years)of transplantation.We examined growth,Pn,and morphological indicators of apricot(Prunus sibirica L.)seedling performances in 2011,2013,and 2015.The colonization rate in mycorrhizal seedlings with similar amounts of shade is higher than the corresponding controls.The mycorrhizal seedling biomass is significantly higher than the corresponding non-mycorrhizal seedling biomass.Generally,P_(n),stomatal conductance(G_(s)),transpiration rate(T_(r)),and water use efficiency are also significantly higher in the mycorrhizal seedlings.Moreover,mycorrhizal seedlings with light shade(LS)have the highest Pn.WUE is increased in non-mycorrhizal seedlings because of the reduction in T_(r),while T_(r) is increased in mycorrhizal seedlings with shade.There is a significant increase in the N,P,and K fractions detected in roots compared with shoots.This means that LS had apparent benefits for mycorrhizal seedlings.Our results also indicate that AM fungi,combined with LS,exert a positive effect on apricot behavior.

Inactivation of FOXO1 induces T follicular cell polarization and involves angioimmunoblastic T cell lymphoma

Objective:Angioimmunoblastic T cell lymphoma(AITL)is an aggressive form of non-Hodgkin lymphoma derived from mature T cells.However,the underlying pathogenesis of AITL remains unresolved.We aimed to explore the role of FOXO1-mediated signaling in the tumorigenesis and progression of AITL.Methods:FOXO1 expression was assessed using immunohistochemistry on a total of 46 AITL tissue samples.Retroviruses encoding FOXO1 shRNA were used to knockdown FOXO1 expression in CD4^+T cells.Flow cytometric assays analyzed the proliferation and survival of FOXO1 knockdown CD4^+T cells.Furthermore,we performed adoptive T-cell transfer experiments to identify whether inactivation of FOXO1 induced neoplastic follicular-helper T(Tfh)cell polarization and function.Results:Patients with low FOXO1 protein levels were prone to have an advanced tumor stage(P=0.049),higher ECOG ps(P=0.024),the presence of bone marrow invasion(P=0.000),and higher IPI(P=0.035).Additionally,the survival rates of patients in the FOXO1 high-expression group were significantly better than those in the FOXO1 low-expression group(χ^2=5.346,P=0.021).We also observed that inactivation of FOXO1 increased CD4^+T cell proliferation and altered the survival and cell-cycle progression of CD4^+T cells.Finally,we confirmed that inactivation of FOXO1 induces Tfh cell programing and function.Conclusions:Inactivation of FOXO1 in AITL plays a key role in the tumorigenesis and progression of AITL.We propose that FOXO1 expression could be a useful prognostic marker in AITL patients to predict poor survival,and to design appropriate therapeutic strategies.

Change of substrate geometry and laminin density stimulates axon branching

Axon branching enables neurons to contact with multiple targets and respond to their microenvironment.Owing to its importance in neuronal network formation,axon branching has been studied extensively during the past decades.It is reported that ECM(Extra Cellular Matrix)components such as laminin,collagen,and tenascin regulate the morphology and motility of neuronal growth cones in culture,but the effects of their distribution and the change of density on axon branching are not well understood.We fabricated chemically homogeneous substrate by microcontact printing(μCP)and inhomogeneous substrate with different laminin density

Finite depth Stokes' first problem of thixotropic fluid

This paper presents a study of the finite depth Stokes＇ first problem for a thixotropic layer. The yield behavior of the thixotropic fluid in this problem is investigated for the first time. The main physical features of this problem are discussed, including the flow field, the wall stress, and the depth of the yield region. It is shown that the yield region appears near the wall, and the yield surface moves from the wall into the flow region and moves back to the wall finally. In contrast to the solution of the Newtonian fluid, the velocity of the thixotropic layer generally does not increase with time monotonously during the start-up process. The classical solution of the Newtonian fluid can be recovered from our results in extreme cases.

Erratum to Inactivation of FOXO1 induces T follicular cell polarization and involves angioimmunoblastic T cell lymphoma

In the published article1,an error appeared in Acknowledgments on page 753.We omitted Natural Science Foundation of Guangdong Province from Acknowledgments,and we updated it as below.We apologize for the errors and for any confusion it may have caused.

Increased long-distance and homo-trans interactions related to H3K27me3 in Arabidopsis hybrids

In plants,the genome structure of hybrids changes compared with their parents,but the effects of these changes in hybrids remain elusive.Comparing reciprocal crosses between Col×C24 and C24×Col in Arabidopsis using high-throughput chromosome conformation capture assay(Hi-C)analysis,we found that hybrid three-dimensional(3D)chromatin organization had more long-distance interactions relative to parents,and this was mainly located in promoter regions and enriched in genes with heterosis-related pathways.The interactions between euchromatin and heterochromatin were increased,and the compartment strength decreased in hybrids.In compartment domain(CD)boundaries,the distal interactions were more in hybrids than their parents.In the hybrids of CURLY LEAF(clf)mutants clfCol×clfC24and clfC24×clfCol,the heterosis phenotype was damaged,and the long-distance interactions in hybrids were fewer than in their parents with lower H3K27me3.ChIP-seq data revealed higher levels of H3K27me3 in the region adjacent to the CD boundary and the same interactional homo-trans sites in the wild-type(WT)hybrids,which may have led to more long-distance interactions.In addition,the differentially expressed genes(DEGs)located in the boundaries of CDs and loop regions changed obviously in WT,and the functional enrichment for DEGs was different between WT and clf in the longdistance interactions and loop regions.Our findings may therefore propose a new epigenetic explanation of heterosis in the Arabidopsis hybrids and provide new insights into crop breeding and yield increase.

Effect of sulfamethazine on the horizontal transfer of plasmid-mediated antibiotic resistance genes and its mechanism of action

As a new type of environmental pollutant,antibiotic resistance genes(ARGs)pose a huge challenge to global health.Horizontal gene transfer(HGT)represents an important route for the spread of ARGs.The widespread use of sulfamethazine(SM2)as a broad-spectrum bacteriostatic agent leads to high residual levels in the environment,thereby increasing the spread of ARGs.Therefore,we chose to study the effect of SM2 on the HGT of ARGs mediated by plasmid RP4 from Escherichia coli(E.coli)HB101 to E.coli NK5449 as well as its mechanism of action.The results showed that compared with the control group,SM2 at concentrations of 10 mg/L and 200 mg/L promoted the HGT of ARGs,but transfer frequency decreased at concentrations of 100 mg/L and 500 mg/L.The transfer frequency at 200 mg/L was 3.04×10^(−5),which was 1.34-fold of the control group.The mechanism of SM2 improving conjugation transfer is via enhancement of the mRNA expression of conjugation genes(trbBP,trfAP)and oxidative stress genes,inhibition of the mRNA expression of vertical transfer genes,up regulation of the outer membrane protein genes(ompC,ompA),promotion of the formation of cell pores,and improvement of the permeability of cell membrane to promote the conjugation transfer of plasmid RP4.The results of this study provide theoretical support for studying the spread of ARGs in the environment.