[Objective] The aim of this study is to understand the genetic characteristics of a grain shape mutant and its possible role in genetic improvement of grain yield in rice. [Method] On the basis of the collection of T-...[Objective] The aim of this study is to understand the genetic characteristics of a grain shape mutant and its possible role in genetic improvement of grain yield in rice. [Method] On the basis of the collection of T-DNA tag lines, the progeny of homozygous plants carrying T-DNA insertion were screened for mutants with mutated phenotypes. The genetic analysis of the mutant and test for the linkage between the mutated phenotype and the T-DNA insertion were carried out to determine its genetic characteristics. [Result] In the present study, a grain shape mutant induced by T-DNA insertion in rice was identified, which showed small grain. Genetic analysis of the mutant showed that the two types of phenotype, normal and small grain in the segregating populations derived from the T-DNA heterozygotes, fit the ratio of 3∶1. Test for Basta resistance showed that all the mutants were resistant while the normal plants segregated for resistant and susceptible by the ratio of 2∶1. The results indicated that the mutant phenotype cosegregated with Bar gene. The small grain mutant caused by T-DNA insertion was confirmed by PCR amplification aiming at T-DNA. [Conclusion] The grain shape mutant is useful for isolation of the tagged gene and genetic improvement in rice.展开更多
A rice （Oryza sativa） T-DNA insertion population, which included more than 63 000 independent transgenic lines and 8 840 identified flanking sequence tags （FSTs） that were mapped onto the rice genome, was develope...A rice （Oryza sativa） T-DNA insertion population, which included more than 63 000 independent transgenic lines and 8 840 identified flanking sequence tags （FSTs） that were mapped onto the rice genome, was developed to systemi- cally study the rice seed quality control. Genome-wide analysis of the FST distribution showed that T-DNA insertions were positively correlated with expressed genes, but negatively with transposable elements and small RNAs. In addition, the recovered T-DNAs were preferentially located at the untranslated region of the expressed genes. More than 11 000 putative homozygous lines were obtained through multi-generations of planting and resistance screening, and measurement of seed quality of around half of them, including the contents of starch, amylose, protein and fat, with a nondestructive near-infrared spectroscopy method, identified 551 mutants with unique or multiple altered param- eters of seed quality. Analysis of the corresponding FSTs showed that genes participating in diverse functions, including metabolic processes and transcriptional regulation, were involved, indicating that seed quality is regulated by a complex network.展开更多
Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot(FCR)in wheat and poses a significant threat to wheat production in terms of grain yield and quality.However,the mechanism by which F....Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot(FCR)in wheat and poses a significant threat to wheat production in terms of grain yield and quality.However,the mechanism by which F.pseudograminearum infects wheat remains unclear.In this study,we aimed to elucidate these mechanisms by constructing a T-DNA insertion mutant library for the highly virulent strain WZ-8A of F.pseudograminearum.By screening this mutant library,we identified nine independent mutants that displayed impaired pathogenesis in barley leaves.Among these mutants,one possessed a disruption in the gene FpRCO1 that is an ortholog of Saccharomyces cerevisiae RCO1,encoding essential component of the Rpd3S histone deacetylase complex in F.pseudograminearum.To further investigate the role of FpRCO1 in F.pseudograminearum,we employed a split-marker approach to knock out FpRCO1 in F.pseudograminearum WZ-8A.FpRCO1 deletion mutants exhibit reduced vegetative growth,conidium production,and virulence in wheat coleoptiles and barley leaves,whereas the complementary strain restores these phenotypes.Moreover,under stress conditions,the FpRCO1 deletion mutants exhibited increased sensitivity to NaCl,sorbitol,and SDS,but possessed reduced sensitivity to H_(2)O_(2)compared to these characteristics in the wild-type strain.RNA-seq analysis revealed that deletion of FpRCO1 affected gene expression(particularly the downregulation of TRI gene expression),thus resulting in significantly reduced deoxynivalenol(DON)production.In summary,our findings highlight the pivotal role of FpRCO1 in regulating vegetative growth and development,asexual reproduction,DON production,and pathogenicity of F.pseudograminearum.This study provides valuable insights into the molecular mechanisms underlying F.pseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.展开更多
bZIP transcription factor family is one of the largest groups of the plant transcription factor families and plays an important role in plant growth and adaption to the abiotic stresses. In this study, two AtbZIP1 mut...bZIP transcription factor family is one of the largest groups of the plant transcription factor families and plays an important role in plant growth and adaption to the abiotic stresses. In this study, two AtbZIP1 mutant Arabidopsis （bzipl） were used with T-DNA inserted into two different sites, designated as SALK-556773 and SALK-660942, in order to identify different effects on AtbZIP1 gene expression by different T-DNA insertion sites. PCR and RT-PCR results revealed that T-DNA insertion in CDS region could effectively inhibit AtbZIP1 gene expression, while T-DNA insertion in 3＇-UTR couldn＇t. The phenotype analysis further confirmed the differences and showed that T-DNA insertion in CDS region decreased plants＇ drought resistance, while in 3＇-UTR couldn＇t. The phenotype assays also suggested that AtbZIP1 held pivotal roles in plant response to drought stress.展开更多
The use of transfer DNA(T-DNA)as amutagen has been developed for tagging genes inmany crops,and results showed that T-DNAinsertion is a random event,and that theinserted genes are stable through multiplegenerations.Th...The use of transfer DNA(T-DNA)as amutagen has been developed for tagging genes inmany crops,and results showed that T-DNAinsertion is a random event,and that theinserted genes are stable through multiplegenerations.Through sequencing PCR-amplifiedfragments adjacent to the inserted elements,wecan construct the T-DNA flanking database,which would be useful for cloning the genestagged by T-DNA.展开更多
Plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter-Binding Protein-Like (SPL) were identified to play essential roles in the es...Plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter-Binding Protein-Like (SPL) were identified to play essential roles in the establishment of plant architecture. However, the roles and regulation of miR156 is not well understood yet. Here, we identified a T-DNA insertion mutant Osmtd1 (Oryza sativa multi-tillering and dwarf mutant). Osmtd1 produced more tillers and displayed short stature phenotype. We determined that the dramatic morphological changes were caused by a single T-DNA insertion in Osmtd1. Further analysis revealed that the T-DNA insertion was located in the gene Os08g34258 encoding a putative inhibitor I family protein. Os08g34258 was knocked out and OsmiR156f was significantly upregulated in Osmtd1. Overexpression of Os08g34258 in Osmtd1 complemented the defects of the mutant architecture, while overexpression of OsmiR156f in wild-type rice phenocopied Osmtd1. We showed that the expression of OsSPL3, OsSPL12, and OsSPL14 were significantly downregulated in Osmtd1 or OsmiR156f overexpressed lines, indicating that OsSPL3, OsSPL12, and OsSPL14 were possibly direct target genes of OsmiR156f. Our results suggested that OsmiR156f controlled plant architecture by mediating plant stature and tiller outgrowth and may be regulated by an unknown protease inhibitor I family protein.展开更多
Plant respiration is characterized by two pathways for electron transfer to O2, namely the cytochrome pathway （CP） that is linked to ATP production, and the alternative pathway （AP）, where electrons from ubiquinol...Plant respiration is characterized by two pathways for electron transfer to O2, namely the cytochrome pathway （CP） that is linked to ATP production, and the alternative pathway （AP）, where electrons from ubiquinol are directly transferred to O2 via an alternative oxidase （AOX） without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T- DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOXIA. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOXIA expression in the wild-type, led to an increased expression of AOXID in leaves of the aoxla-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosyn- thesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild- type, which was only marginally affected by the inhibitor. It thus appears that AOX1 D was unable to fully compensate for the loss of AOXIA when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex （GDC）, suggests that the aoxla mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOXIA to optimize pho- tosynthesis when treated with antimycin A. We suggest that the aoxla mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.展开更多
With the completion of the rice （Oryza sativa L.） genome-sequencing project, the rice research community proposed to characterize the func- tion of every predicted gene in rice by 2020. One of the most effective and...With the completion of the rice （Oryza sativa L.） genome-sequencing project, the rice research community proposed to characterize the func- tion of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.展开更多
Three T-DNA insertional embryonic lethal mutants from NASC (The Nottingham Arabidopsis Stock Center)were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion. ...Three T-DNA insertional embryonic lethal mutants from NASC (The Nottingham Arabidopsis Stock Center)were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion. The N4081 mutant has a segregation ratio of 1:3.04in average and one T-DNA insertion site according to our assay It was therefore chosen for further analysis. To isolate the joint fragment of T-DNA and plan DNA, the plasmid rescue technique waJs used. pEL-7, one of plasmids from left border of T-DNA, which contained pBR322 was selected from ampicillin plate. The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot. Restriction analysis confirmed the presence of known sites of EcoRI, PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid, pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA. The Southern Blot indicated the hybridization band in both of them. Furthermore, the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A Sequencer. The results showed the 822 bp fragment contained a 274 bp sequence, which is 99.6%homolog (273bp/274 bp) to Ti plasmid pTi 15955 DNA.Ten bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA.Taken together, pEL-7 should contain a joint fragment of T-DNA and flanking plant DNA. This plasmid DNA could be used for the isolation of plant gene, which will be helpful to elucidate the relationship between gene function and plant embryo development.展开更多
Transformation by Agrobacterium tumefaciens, an important tool in modern plant research, involves the integration of T-DNA initially present on a plasmid in agrobacteria into the genome of plant cells. The process of ...Transformation by Agrobacterium tumefaciens, an important tool in modern plant research, involves the integration of T-DNA initially present on a plasmid in agrobacteria into the genome of plant cells. The process of attachment of the agrobacteria to plant cells and the transport of T-DNA into the cell and further to the nucleus has been well described. However, the exact mechanism of integration into the host＇s DNA is still unclear, although several models have been proposed. During confirmation of T-DNA insertion alleles from the GABI-Kat collection of Arabidopsis thaliana mutants, we have generated about 34 000 sequences from the junctions between inserted T-DNA and adjacent genome regions. Here, we describe the evaluation of this dataset with regard to existing models for T-DNA integration. The results suggest that integration into the plant genome is mainly mediated by the endogenous plant DNA repair machinery. The observed integration events showed characteristics highly similar to those of repair sites of double- strand breaks with respect to microhomology and deletion sizes. In addition, we describe unexpected integration events, such as large deletions and inversions at the integration site that are relevant for correct interpretation of results from T-DNA insertion mutants in reverse genetics experiments.展开更多
Agrobacterium tumefaciens-mediated DNA transiormation method was applied to transform Noclulisporium sylviforme fusant HDF-68, a taxol-produeing fungus. We constructed a binary vector pBI121-43 canting a hygromycin-re...Agrobacterium tumefaciens-mediated DNA transiormation method was applied to transform Noclulisporium sylviforme fusant HDF-68, a taxol-produeing fungus. We constructed a binary vector pBI121-43 canting a hygromycin-resistant gene cassette between the right and left borders of T-DNA, Optimal co-cultivation of N.sylviforrne with A. tumefaciens containing pBI121-43 led to 110- 130 hygromycin-resistant transformants per＂ million eonidia. Putative transformants were found to be mitotically stable. The molecular analysis of transformants demonstrated the random integration of single copy of the T-DNA into the host genome. This transformation system serves as a basic tool for insertional mutagenesis in N. sylviforme fusant HDF-68, and the development of such svstem lays a solid foundation for constructing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.展开更多
In our previous studies,a nonpathogenic mutant of Magnaporthe grisea,A1-412,which was defective in appressorium formation,penetration and infectious growth,was obtained by T-DNA insertional mutagenesis. Here we report...In our previous studies,a nonpathogenic mutant of Magnaporthe grisea,A1-412,which was defective in appressorium formation,penetration and infectious growth,was obtained by T-DNA insertional mutagenesis. Here we reported the identification and characterization of the corresponding gene. Se- quence analysis of the genomic DNA franking T-DNA isolated by TAIL-PCR technique showed that T-DNA was inserted into the promoter region of the predicted G protein γ-subunit gene MGG1 (for Magnaporthe grisea G protein Gamma subunit). MGG1 is predicted to encode a 93-aa protein with a typical G-protein gamma like domain (GGL) and C-terminal CAAX box. The amino-acid sequence of MGG1 is highly identical to the Gγ subunits of other filamentous fungi. Further phenotypic investigation of A1-412 showed that ex- ogenous cAMP could induce appressorium formation,although the formed appressoria were abnormal in shape and unable to penetrate onion epidermis or rice leaves. Moreover,few perithecia were observed when A1-412 was crossed with the appropriate mat- ing-type strain. The above phenotypes in A1-412 were partially complemented by reintroduction of the gene MGG1. Our results indicate that the G-protein gamma subunit MGG1 may be involved in regulating morphogenesis,mating and pathogenicity in M. grisea.展开更多
Dear Editor, Forward genetic screens are commonly used as unbiased tools to isolate genes responsible for a phenotype of interest. In Arabidopsis thaliana, especially T-DNA activation tagging pop- ulations are freque...Dear Editor, Forward genetic screens are commonly used as unbiased tools to isolate genes responsible for a phenotype of interest. In Arabidopsis thaliana, especially T-DNA activation tagging pop- ulations are frequently employed. These populations are gener- ated using vectors containing multiple copies of the constitutive 35S promoters derived from cau and often result in isolation i flower mosaic virus （35S CaMV） of dominant gain-of-function alleles （Weigel et al., 2000; Nakazawa et al., 2003）. This allows the study of members of large gene families that are often func- tionally redundant and, therefore, hard to identify in loss-of- function screens. Moreover, due to the dominant nature,展开更多
An efficient TfOH-catalyzed O—H insertion reaction of α-aryl diazoesters with carboxylic acids is reported.This metal-free protocol provides an operationally simple method for a one-pot assembly of diverse α-acylox...An efficient TfOH-catalyzed O—H insertion reaction of α-aryl diazoesters with carboxylic acids is reported.This metal-free protocol provides an operationally simple method for a one-pot assembly of diverse α-acyloxy esters in moderate to high yields with a broad substrate scope.All starting materials are readily available,and the reactions can be conducted in the open air at room temperature.展开更多
A blue light-induced formal insertion reaction ofα-siloxy carbene into the C—H bond of 1,3-diketones has been reported.Under the irradiation of blue light,acylsilane converts toα-siloxy carbene,which then undergoes...A blue light-induced formal insertion reaction ofα-siloxy carbene into the C—H bond of 1,3-diketones has been reported.Under the irradiation of blue light,acylsilane converts toα-siloxy carbene,which then undergoes formal C—H bond insertion reaction with the enol form of 1,3-diketone.This method uses readily available and relative stable acylsilane as car-bene precursor,which features a simple and metal-free approach under mild conditions.Moreover,the synthetic potential of this protocol has been demonstrated by performing the reaction on a gram scale with comparable high yield.展开更多
The switchable cross-coupling of indoles and pyridotriazoles through carbene insertion at C_(2)-or C_(3)-positon has been developed in this paper.This highly site-selective C-H carbenoid functionalization is determine...The switchable cross-coupling of indoles and pyridotriazoles through carbene insertion at C_(2)-or C_(3)-positon has been developed in this paper.This highly site-selective C-H carbenoid functionalization is determined by both the Rh-catalyst species and auxiliary groups.[Cp∗RhCl_(2)]_(2) and coordinating pyrimidyl group direct the C-H carbenoid functionalization to occur at the C_(2)-position,while Rh2OAc4 and noncoordinating benzyl group lead the reaction to occur at the C_(3)-position of the indoles.This regioselective C−H functionalization strategy is of significant importance for the discovery of indole drugs.展开更多
BACKGROUND Adenoma detection rate(ADR),a key colonoscopy quality metric,varies with patient demographics and procedural factors.AIM To identify independent predictors of≥25%ADR,develop a risk model,and propose withdr...BACKGROUND Adenoma detection rate(ADR),a key colonoscopy quality metric,varies with patient demographics and procedural factors.AIM To identify independent predictors of≥25%ADR,develop a risk model,and propose withdrawal durations based on different insertion times.METHODS We retrospectively analyzed 830 cases using logistic regression and identified four key factors,validated in a prospective cohort of 5699 patients.Their importance was confirmed using random forest(RF),extreme gradient boosting(XGBoost)and light gradient boosting machine(LightGBM).Attempts to determine targetachieving withdrawal time by grouping cases based on insertion time and Cox regression were inconclusive.Using the 5699-case dataset,we developed a predictive model combining support vector machine(SVM)with XGBoost.We built a Shiny app using this model for clinical application.RESULTS Multivariate logistic regression identified age[odds ratio(OR)=1.05;95%confidence interval(CI):1.03-1.08;P<0.001],male(OR=1.79;95%CI:1.32-2.41;P=0.005),higher endoscopist experience(OR=1.79;95%CI:1.20-2.68;P=0.005),and longer withdrawal time(P<0.001)as independent risk factors for colorectal adenoma.A nomogram demonstrated strong discrimination[area under the curve(AUC)=0.720],with robust calibration and decision-curve performance.Feature importance via RF,XGBoost,and LightGBM confirmed key predictors.A hybrid model combining SVM regression for withdrawal-time estimation and XGBoost classification achieved stable results,with XGBoost reporting AUCs of 0.640 in training and 0.610 in testing,and similar validation outcomes.Deployed via a Shiny app for clinical use.However,model discrimination was modest(AUC:0.61-0.64),suggesting that clinical utility requires further refinement.CONCLUSION A hybrid SVM-XGBoost model using four key endoscopic factors was independently validated and is available as a Shiny app,delivering real-time decision support to streamline endoscopy and enhance clinical outcomes.展开更多
Uronic acids are prevalent components of crucial glycoconjugates,pivotal in various biological processes.In nature,NDP-uronic acids,the nucleosides-activated uronic acids,serve as glycosylation donors catalyzed by uro...Uronic acids are prevalent components of crucial glycoconjugates,pivotal in various biological processes.In nature,NDP-uronic acids,the nucleosides-activated uronic acids,serve as glycosylation donors catalyzed by uronosyltransferases(UATs)to construct glycans containing uronic acids.Despite their biological importance,the synthesis of naturally occurring NDP-uronic acids on a large scale remains challenging.Here,we developed an oxidation reaction insertion strategy for the efficient synthesis of NDP-uronic acids,and 11 NDP-uronic acids were successfully prepared in good yield and on a large scale.The prepared NDP-uronic acids can be used to explore new uronosyltransferases and synthesize uronic acids containing carbohydrates for fundamental research.展开更多
Objective:To explore the evidence-based nursing optimization strategy for catheter tip positioning during peripherally inserted central catheter(PICC)insertion in patients with persistent left superior vena cava(PLSVC...Objective:To explore the evidence-based nursing optimization strategy for catheter tip positioning during peripherally inserted central catheter(PICC)insertion in patients with persistent left superior vena cava(PLSVC).Methods:For one ovarian cancer patient with PICC malposition in the coronary sinus(CS)due to PLSVC,multimodal imaging techniques were integrated to accurately locate the catheter tip.The catheter position was adjusted based on evidence(withdrawing 5 cm),and a standardized nursing process was established,including personalized health education,catheter fixation and displacement monitoring,complication monitoring,establishment of a specialized disease information archive system,and formulation of a follow-up plan.Results:The catheter tip was successfully withdrawn from the coronary sinus(at the T8 level)to the middle and lower part of the PLSVC(at the T6 vertebral level),and the catheter functioned normally after adjustment.No complications such as arrhythmia or thrombosis occurred during the 332-day chemotherapy period.Conclusion:The PICC tip in PLSVC patients should be positioned in the middle and lower part of the PLSVC(at the T5–T7 vertebral level).This new standard can effectively avoid CS-related complications.The integration of multi-modal imaging techniques and evidence-based nursing management are key to ensuring safe infusion.展开更多
T-DNA insertion mutants have been widely used to define gene functions in Arabidopsis and in other plants. Here, we report an unexpected phenomenon of epigenetic suppression of T-DNA insertion mutants in Arabidopsis. ...T-DNA insertion mutants have been widely used to define gene functions in Arabidopsis and in other plants. Here, we report an unexpected phenomenon of epigenetic suppression of T-DNA insertion mutants in Arabidopsis. When the two T-DNA insertion mutants, yucl-1 and ag-TD, were crossed together, the defects in all of the ag-TD plants in the F2 popumation were partially suppressed regardless of the presence of yucl-1. Conversion of ag-TD to the suppressed ag- TD （named as ag-TD＊） did not follow the laws of Mendelian genetics. The ag-TD＊ could be stably transmitted for many generations without reverting to ag-TD, and ag-TD^＊ had the capacity to convert ag-TD to ag-TD^＊. We show that epige-netic suppression of T-DNA mutants is not a rare event, but certain structural features in the T-DNA mutants are needed in order for the suppression to take place. The suppressed T-DNA mutants we observed were all intronic T-DNA mutants and the T-DNA fragments in both the trigger T-DNA as well as in the suppressed T-DNA shared stretches of identical sequences. We demonstrate that the suppression of intronic T-DNA mutants is mediated by trans-interactions between two T-DNA insertions. This work shows that caution is needed when intronic T-DNA mutants are used.展开更多
文摘[Objective] The aim of this study is to understand the genetic characteristics of a grain shape mutant and its possible role in genetic improvement of grain yield in rice. [Method] On the basis of the collection of T-DNA tag lines, the progeny of homozygous plants carrying T-DNA insertion were screened for mutants with mutated phenotypes. The genetic analysis of the mutant and test for the linkage between the mutated phenotype and the T-DNA insertion were carried out to determine its genetic characteristics. [Result] In the present study, a grain shape mutant induced by T-DNA insertion in rice was identified, which showed small grain. Genetic analysis of the mutant showed that the two types of phenotype, normal and small grain in the segregating populations derived from the T-DNA heterozygotes, fit the ratio of 3∶1. Test for Basta resistance showed that all the mutants were resistant while the normal plants segregated for resistant and susceptible by the ratio of 2∶1. The results indicated that the mutant phenotype cosegregated with Bar gene. The small grain mutant caused by T-DNA insertion was confirmed by PCR amplification aiming at T-DNA. [Conclusion] The grain shape mutant is useful for isolation of the tagged gene and genetic improvement in rice.
文摘A rice （Oryza sativa） T-DNA insertion population, which included more than 63 000 independent transgenic lines and 8 840 identified flanking sequence tags （FSTs） that were mapped onto the rice genome, was developed to systemi- cally study the rice seed quality control. Genome-wide analysis of the FST distribution showed that T-DNA insertions were positively correlated with expressed genes, but negatively with transposable elements and small RNAs. In addition, the recovered T-DNAs were preferentially located at the untranslated region of the expressed genes. More than 11 000 putative homozygous lines were obtained through multi-generations of planting and resistance screening, and measurement of seed quality of around half of them, including the contents of starch, amylose, protein and fat, with a nondestructive near-infrared spectroscopy method, identified 551 mutants with unique or multiple altered param- eters of seed quality. Analysis of the corresponding FSTs showed that genes participating in diverse functions, including metabolic processes and transcriptional regulation, were involved, indicating that seed quality is regulated by a complex network.
基金supported by grants from the National Natural Science Foundation of China(31901835)the Science and Technology Planning Project of Henan Province of China(212102110145)the International(Regional)Cooperation and Exchange Program of the National Natural Science Foundation of China(31961143018).
文摘Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot(FCR)in wheat and poses a significant threat to wheat production in terms of grain yield and quality.However,the mechanism by which F.pseudograminearum infects wheat remains unclear.In this study,we aimed to elucidate these mechanisms by constructing a T-DNA insertion mutant library for the highly virulent strain WZ-8A of F.pseudograminearum.By screening this mutant library,we identified nine independent mutants that displayed impaired pathogenesis in barley leaves.Among these mutants,one possessed a disruption in the gene FpRCO1 that is an ortholog of Saccharomyces cerevisiae RCO1,encoding essential component of the Rpd3S histone deacetylase complex in F.pseudograminearum.To further investigate the role of FpRCO1 in F.pseudograminearum,we employed a split-marker approach to knock out FpRCO1 in F.pseudograminearum WZ-8A.FpRCO1 deletion mutants exhibit reduced vegetative growth,conidium production,and virulence in wheat coleoptiles and barley leaves,whereas the complementary strain restores these phenotypes.Moreover,under stress conditions,the FpRCO1 deletion mutants exhibited increased sensitivity to NaCl,sorbitol,and SDS,but possessed reduced sensitivity to H_(2)O_(2)compared to these characteristics in the wild-type strain.RNA-seq analysis revealed that deletion of FpRCO1 affected gene expression(particularly the downregulation of TRI gene expression),thus resulting in significantly reduced deoxynivalenol(DON)production.In summary,our findings highlight the pivotal role of FpRCO1 in regulating vegetative growth and development,asexual reproduction,DON production,and pathogenicity of F.pseudograminearum.This study provides valuable insights into the molecular mechanisms underlying F.pseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.
基金Supported by National Natural Science Foundation of China (30570990)National Major Project for Cultivation of Transgenic Crops (20082x08004)+1 种基金Key Research Plan of Heilongjiang Province (GA06B103)Innovation Research Group of NEAU (CXT004)
文摘bZIP transcription factor family is one of the largest groups of the plant transcription factor families and plays an important role in plant growth and adaption to the abiotic stresses. In this study, two AtbZIP1 mutant Arabidopsis （bzipl） were used with T-DNA inserted into two different sites, designated as SALK-556773 and SALK-660942, in order to identify different effects on AtbZIP1 gene expression by different T-DNA insertion sites. PCR and RT-PCR results revealed that T-DNA insertion in CDS region could effectively inhibit AtbZIP1 gene expression, while T-DNA insertion in 3＇-UTR couldn＇t. The phenotype analysis further confirmed the differences and showed that T-DNA insertion in CDS region decreased plants＇ drought resistance, while in 3＇-UTR couldn＇t. The phenotype assays also suggested that AtbZIP1 held pivotal roles in plant response to drought stress.
文摘The use of transfer DNA(T-DNA)as amutagen has been developed for tagging genes inmany crops,and results showed that T-DNAinsertion is a random event,and that theinserted genes are stable through multiplegenerations.Through sequencing PCR-amplifiedfragments adjacent to the inserted elements,wecan construct the T-DNA flanking database,which would be useful for cloning the genestagged by T-DNA.
基金supported by the National Natural Science Foundation of China (no. 91317312 and 91117006)Open Foundation Project for Hunan Provincial Higher Institutional Innovation Platform (no. 09K052)Hunan Provincial Key Laboratory for Crop Germplasm Innovation and Utilization (no. 12KFXM05)
文摘Plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter-Binding Protein-Like (SPL) were identified to play essential roles in the establishment of plant architecture. However, the roles and regulation of miR156 is not well understood yet. Here, we identified a T-DNA insertion mutant Osmtd1 (Oryza sativa multi-tillering and dwarf mutant). Osmtd1 produced more tillers and displayed short stature phenotype. We determined that the dramatic morphological changes were caused by a single T-DNA insertion in Osmtd1. Further analysis revealed that the T-DNA insertion was located in the gene Os08g34258 encoding a putative inhibitor I family protein. Os08g34258 was knocked out and OsmiR156f was significantly upregulated in Osmtd1. Overexpression of Os08g34258 in Osmtd1 complemented the defects of the mutant architecture, while overexpression of OsmiR156f in wild-type rice phenocopied Osmtd1. We showed that the expression of OsSPL3, OsSPL12, and OsSPL14 were significantly downregulated in Osmtd1 or OsmiR156f overexpressed lines, indicating that OsSPL3, OsSPL12, and OsSPL14 were possibly direct target genes of OsmiR156f. Our results suggested that OsmiR156f controlled plant architecture by mediating plant stature and tiller outgrowth and may be regulated by an unknown protease inhibitor I family protein.
文摘Plant respiration is characterized by two pathways for electron transfer to O2, namely the cytochrome pathway （CP） that is linked to ATP production, and the alternative pathway （AP）, where electrons from ubiquinol are directly transferred to O2 via an alternative oxidase （AOX） without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T- DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOXIA. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOXIA expression in the wild-type, led to an increased expression of AOXID in leaves of the aoxla-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosyn- thesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild- type, which was only marginally affected by the inhibitor. It thus appears that AOX1 D was unable to fully compensate for the loss of AOXIA when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex （GDC）, suggests that the aoxla mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOXIA to optimize pho- tosynthesis when treated with antimycin A. We suggest that the aoxla mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.
基金supported by the National Natural Science Foundation of China(30970172)the 863 Project Grant2012AA10A304the Program for New Century Excellent Talents in University
文摘With the completion of the rice （Oryza sativa L.） genome-sequencing project, the rice research community proposed to characterize the func- tion of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.
文摘Three T-DNA insertional embryonic lethal mutants from NASC (The Nottingham Arabidopsis Stock Center)were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion. The N4081 mutant has a segregation ratio of 1:3.04in average and one T-DNA insertion site according to our assay It was therefore chosen for further analysis. To isolate the joint fragment of T-DNA and plan DNA, the plasmid rescue technique waJs used. pEL-7, one of plasmids from left border of T-DNA, which contained pBR322 was selected from ampicillin plate. The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot. Restriction analysis confirmed the presence of known sites of EcoRI, PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid, pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA. The Southern Blot indicated the hybridization band in both of them. Furthermore, the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A Sequencer. The results showed the 822 bp fragment contained a 274 bp sequence, which is 99.6%homolog (273bp/274 bp) to Ti plasmid pTi 15955 DNA.Ten bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA.Taken together, pEL-7 should contain a joint fragment of T-DNA and flanking plant DNA. This plasmid DNA could be used for the isolation of plant gene, which will be helpful to elucidate the relationship between gene function and plant embryo development.
文摘Transformation by Agrobacterium tumefaciens, an important tool in modern plant research, involves the integration of T-DNA initially present on a plasmid in agrobacteria into the genome of plant cells. The process of attachment of the agrobacteria to plant cells and the transport of T-DNA into the cell and further to the nucleus has been well described. However, the exact mechanism of integration into the host＇s DNA is still unclear, although several models have been proposed. During confirmation of T-DNA insertion alleles from the GABI-Kat collection of Arabidopsis thaliana mutants, we have generated about 34 000 sequences from the junctions between inserted T-DNA and adjacent genome regions. Here, we describe the evaluation of this dataset with regard to existing models for T-DNA integration. The results suggest that integration into the plant genome is mainly mediated by the endogenous plant DNA repair machinery. The observed integration events showed characteristics highly similar to those of repair sites of double- strand breaks with respect to microhomology and deletion sizes. In addition, we describe unexpected integration events, such as large deletions and inversions at the integration site that are relevant for correct interpretation of results from T-DNA insertion mutants in reverse genetics experiments.
基金the National Natural Science Foundation of China(No.30570025)the Education Department of Heilongjiang Province(No.10551238)
文摘Agrobacterium tumefaciens-mediated DNA transiormation method was applied to transform Noclulisporium sylviforme fusant HDF-68, a taxol-produeing fungus. We constructed a binary vector pBI121-43 canting a hygromycin-resistant gene cassette between the right and left borders of T-DNA, Optimal co-cultivation of N.sylviforrne with A. tumefaciens containing pBI121-43 led to 110- 130 hygromycin-resistant transformants per＂ million eonidia. Putative transformants were found to be mitotically stable. The molecular analysis of transformants demonstrated the random integration of single copy of the T-DNA into the host genome. This transformation system serves as a basic tool for insertional mutagenesis in N. sylviforme fusant HDF-68, and the development of such svstem lays a solid foundation for constructing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.
基金the National Key Basic Research and Development Program （Grant No. 2006CB101901） the National Natural Science Foundation of China （Grant Nos. 30570054 ＆ 30270861）
文摘In our previous studies,a nonpathogenic mutant of Magnaporthe grisea,A1-412,which was defective in appressorium formation,penetration and infectious growth,was obtained by T-DNA insertional mutagenesis. Here we reported the identification and characterization of the corresponding gene. Se- quence analysis of the genomic DNA franking T-DNA isolated by TAIL-PCR technique showed that T-DNA was inserted into the promoter region of the predicted G protein γ-subunit gene MGG1 (for Magnaporthe grisea G protein Gamma subunit). MGG1 is predicted to encode a 93-aa protein with a typical G-protein gamma like domain (GGL) and C-terminal CAAX box. The amino-acid sequence of MGG1 is highly identical to the Gγ subunits of other filamentous fungi. Further phenotypic investigation of A1-412 showed that ex- ogenous cAMP could induce appressorium formation,although the formed appressoria were abnormal in shape and unable to penetrate onion epidermis or rice leaves. Moreover,few perithecia were observed when A1-412 was crossed with the appropriate mat- ing-type strain. The above phenotypes in A1-412 were partially complemented by reintroduction of the gene MGG1. Our results indicate that the G-protein gamma subunit MGG1 may be involved in regulating morphogenesis,mating and pathogenicity in M. grisea.
文摘Dear Editor, Forward genetic screens are commonly used as unbiased tools to isolate genes responsible for a phenotype of interest. In Arabidopsis thaliana, especially T-DNA activation tagging pop- ulations are frequently employed. These populations are gener- ated using vectors containing multiple copies of the constitutive 35S promoters derived from cau and often result in isolation i flower mosaic virus （35S CaMV） of dominant gain-of-function alleles （Weigel et al., 2000; Nakazawa et al., 2003）. This allows the study of members of large gene families that are often func- tionally redundant and, therefore, hard to identify in loss-of- function screens. Moreover, due to the dominant nature,
文摘An efficient TfOH-catalyzed O—H insertion reaction of α-aryl diazoesters with carboxylic acids is reported.This metal-free protocol provides an operationally simple method for a one-pot assembly of diverse α-acyloxy esters in moderate to high yields with a broad substrate scope.All starting materials are readily available,and the reactions can be conducted in the open air at room temperature.
文摘A blue light-induced formal insertion reaction ofα-siloxy carbene into the C—H bond of 1,3-diketones has been reported.Under the irradiation of blue light,acylsilane converts toα-siloxy carbene,which then undergoes formal C—H bond insertion reaction with the enol form of 1,3-diketone.This method uses readily available and relative stable acylsilane as car-bene precursor,which features a simple and metal-free approach under mild conditions.Moreover,the synthetic potential of this protocol has been demonstrated by performing the reaction on a gram scale with comparable high yield.
基金CAMS Innovation Fund for Medical Sciences(CIFMS)(Nos.2022-I2M-1-013,2022-I2M-1-014,2022-I2M-2-002).
文摘The switchable cross-coupling of indoles and pyridotriazoles through carbene insertion at C_(2)-or C_(3)-positon has been developed in this paper.This highly site-selective C-H carbenoid functionalization is determined by both the Rh-catalyst species and auxiliary groups.[Cp∗RhCl_(2)]_(2) and coordinating pyrimidyl group direct the C-H carbenoid functionalization to occur at the C_(2)-position,while Rh2OAc4 and noncoordinating benzyl group lead the reaction to occur at the C_(3)-position of the indoles.This regioselective C−H functionalization strategy is of significant importance for the discovery of indole drugs.
基金Supported by the Young and Middle-Aged Talents Program of Wuxi Health Commission,No.BJ2020011Cohort Research Program of Wuxi Medical Center,Nanjing Medical University,No.WMCC202314Wuxi People’s Hospital 2024“Wild Goose Array Talent”Reserve Discipline Leader,No.2024-YZ-HBDTR-YC-2024.
文摘BACKGROUND Adenoma detection rate(ADR),a key colonoscopy quality metric,varies with patient demographics and procedural factors.AIM To identify independent predictors of≥25%ADR,develop a risk model,and propose withdrawal durations based on different insertion times.METHODS We retrospectively analyzed 830 cases using logistic regression and identified four key factors,validated in a prospective cohort of 5699 patients.Their importance was confirmed using random forest(RF),extreme gradient boosting(XGBoost)and light gradient boosting machine(LightGBM).Attempts to determine targetachieving withdrawal time by grouping cases based on insertion time and Cox regression were inconclusive.Using the 5699-case dataset,we developed a predictive model combining support vector machine(SVM)with XGBoost.We built a Shiny app using this model for clinical application.RESULTS Multivariate logistic regression identified age[odds ratio(OR)=1.05;95%confidence interval(CI):1.03-1.08;P<0.001],male(OR=1.79;95%CI:1.32-2.41;P=0.005),higher endoscopist experience(OR=1.79;95%CI:1.20-2.68;P=0.005),and longer withdrawal time(P<0.001)as independent risk factors for colorectal adenoma.A nomogram demonstrated strong discrimination[area under the curve(AUC)=0.720],with robust calibration and decision-curve performance.Feature importance via RF,XGBoost,and LightGBM confirmed key predictors.A hybrid model combining SVM regression for withdrawal-time estimation and XGBoost classification achieved stable results,with XGBoost reporting AUCs of 0.640 in training and 0.610 in testing,and similar validation outcomes.Deployed via a Shiny app for clinical use.However,model discrimination was modest(AUC:0.61-0.64),suggesting that clinical utility requires further refinement.CONCLUSION A hybrid SVM-XGBoost model using four key endoscopic factors was independently validated and is available as a Shiny app,delivering real-time decision support to streamline endoscopy and enhance clinical outcomes.
基金financially supported by National Natural Science Foundation of China(No.22207113 to J.Zhang)Guangdong Basic and Applied Basic Research Foundation(No.2021A1515110588to J.Zhang)Natural Science Foundation of Shanghai Municipality(No.22ZR1474000 to L.Wen)。
文摘Uronic acids are prevalent components of crucial glycoconjugates,pivotal in various biological processes.In nature,NDP-uronic acids,the nucleosides-activated uronic acids,serve as glycosylation donors catalyzed by uronosyltransferases(UATs)to construct glycans containing uronic acids.Despite their biological importance,the synthesis of naturally occurring NDP-uronic acids on a large scale remains challenging.Here,we developed an oxidation reaction insertion strategy for the efficient synthesis of NDP-uronic acids,and 11 NDP-uronic acids were successfully prepared in good yield and on a large scale.The prepared NDP-uronic acids can be used to explore new uronosyltransferases and synthesize uronic acids containing carbohydrates for fundamental research.
文摘Objective:To explore the evidence-based nursing optimization strategy for catheter tip positioning during peripherally inserted central catheter(PICC)insertion in patients with persistent left superior vena cava(PLSVC).Methods:For one ovarian cancer patient with PICC malposition in the coronary sinus(CS)due to PLSVC,multimodal imaging techniques were integrated to accurately locate the catheter tip.The catheter position was adjusted based on evidence(withdrawing 5 cm),and a standardized nursing process was established,including personalized health education,catheter fixation and displacement monitoring,complication monitoring,establishment of a specialized disease information archive system,and formulation of a follow-up plan.Results:The catheter tip was successfully withdrawn from the coronary sinus(at the T8 level)to the middle and lower part of the PLSVC(at the T6 vertebral level),and the catheter functioned normally after adjustment.No complications such as arrhythmia or thrombosis occurred during the 332-day chemotherapy period.Conclusion:The PICC tip in PLSVC patients should be positioned in the middle and lower part of the PLSVC(at the T5–T7 vertebral level).This new standard can effectively avoid CS-related complications.The integration of multi-modal imaging techniques and evidence-based nursing management are key to ensuring safe infusion.
文摘T-DNA insertion mutants have been widely used to define gene functions in Arabidopsis and in other plants. Here, we report an unexpected phenomenon of epigenetic suppression of T-DNA insertion mutants in Arabidopsis. When the two T-DNA insertion mutants, yucl-1 and ag-TD, were crossed together, the defects in all of the ag-TD plants in the F2 popumation were partially suppressed regardless of the presence of yucl-1. Conversion of ag-TD to the suppressed ag- TD （named as ag-TD＊） did not follow the laws of Mendelian genetics. The ag-TD＊ could be stably transmitted for many generations without reverting to ag-TD, and ag-TD^＊ had the capacity to convert ag-TD to ag-TD^＊. We show that epige-netic suppression of T-DNA mutants is not a rare event, but certain structural features in the T-DNA mutants are needed in order for the suppression to take place. The suppressed T-DNA mutants we observed were all intronic T-DNA mutants and the T-DNA fragments in both the trigger T-DNA as well as in the suppressed T-DNA shared stretches of identical sequences. We demonstrate that the suppression of intronic T-DNA mutants is mediated by trans-interactions between two T-DNA insertions. This work shows that caution is needed when intronic T-DNA mutants are used.
