High temperature stress is one of the major environmental factors that affect the growth and development of plants. Although WRKY transcription factors play a critical role in stress responses, there are few studies o...High temperature stress is one of the major environmental factors that affect the growth and development of plants. Although WRKY transcription factors play a critical role in stress responses, there are few studies on the regulation of heat stress by WRKY transcription factors,especially in tomato. Here, we identified a group I WRKY transcription factor, SlWRKY3, involved in thermotolerance in tomato. First, SlWRKY3 was induced and upregulated under heat stress. Accordingly, overexpression of SlWRKY3 led to an increase, whereas knock-out of SlWRKY3 resulted in decreased tolerance to heat stress. Overexpression of SlWRKY3 accumulated less reactive oxygen species(ROS), whereas knock-out of SlWRKY3 accumulated more ROS under heat stress. This indicated that SlWRKY3 positively regulates heat stress in tomato. In addition,SlWRKY3 activated the expression of a range of abiotic stress-responsive genes involved in ROS scavenging, such as a SlGRXS1 gene cluster.Further analysis showed that SlWRKY3 can bind to the promoters of the SlGRXS1 gene cluster and activate their expression. Collectively, these results imply that SlWRKY3 is a positive regulator of thermotolerance through direct binding to the promoters of the SlGRXS1 gene cluster and activating their expression and ROS scavenging.展开更多
A recombinant strain, Escherichia coli JM109-AN1, was obtained by constructing of a genomic library of the total DNA of Delftia sp. AN3 in E. coli JM109 and screening for catechol 2,3-dioxygenase activity. This recomb...A recombinant strain, Escherichia coli JM109-AN1, was obtained by constructing of a genomic library of the total DNA of Delftia sp. AN3 in E. coli JM109 and screening for catechol 2,3-dioxygenase activity. This recombinant strain could grow on aniline as sole carbon, nitrogen and energy source. Enzymatic assays revealed that the exogenous genes including aniline dioxygenase （AD） and catechol 2,3-dioxygenase （C230） genes could well express in the recombinant strain with the activities of AD and C230 up to 0.31 U/mg wet cell and 1.92 U/mg crude proteins, respectively. The AD or C23O of strain AN3 could only catalyze aniline or catechol but not any other substituted substrates. This recombinant strain contained a recombinant plasmid, pKC505-AN1, in which a 29.7-kb DNA fragment from Delftia sp. AN3 was inserted. Sequencing and open reading frame （orfs） analysis of this 29.7 kb fragment revealed that it contained at least 27 orfs, among them a gene cluster （consisting of at least 16 genes, named danQTA1A2BRDCEFG1HIJKG2） was responsible for the complete metabolism of aniline to TCA-cycle intermediates. This gene cluster could be divided into two main parts, the upper sequences consisted of 7 genes （danQTA1A2BRD） were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, and the central genes （danCEFG1HIJKG2） were expected to encode meta-cleavage pathway enzymes for catechol degradation to TCA-cycle intermediates. Unlike clusters tad from Delftia tsuruhatensis AD9 and tdn from Pseudomonas putida UCC22, in this gene cluster, all the genes were in the same transcriptional direction. There was only one set of C230 gene （danC） and ferredoxin-like protein gene （danD）. The presence of only one set of these two genes and specificity of AD and C230 might be the reason for strain AN3 could only degrade aniline. The products of danQTA1A2BRDC showed 99%-100% identity to those from Delftia acidovorans 7N, and 50%-85% identity to those of tad cluster from D. tsuruhatensis AD9 in amino acid residues. Besides this dan cluster, the 29.7 kb fragment also contained genes encoding the trans-membrane transporter and transposases which might be needed for transposition of the gene cluster. Pulsed-field gel electrophoresis （PFGE） and plasmid curing experiments suggested that the dan cluster might be encoded on the chromosome of strain AN3. The GenBank accession number for the dan cluster of Delftia sp. AN3 is DQ661649.展开更多
The use of pan-ethnic-group products form knowledge primarily depends on a designer＇s subjective experience without user participation. The majority of studies primarily focus on the detection of the perceptual deman...The use of pan-ethnic-group products form knowledge primarily depends on a designer＇s subjective experience without user participation. The majority of studies primarily focus on the detection of the perceptual demands of consumers from the target product category. A pan-ethnic-group products form gene clustering method based on emotional semantic is constructed. Consumers＇ perceptual images of the pan-ethnic-group products are obtained by means of product form gene extraction and coding and computer aided product form clustering technology. A case of form gene clustering about the typical pan-ethnic-group products is investigated which indicates that the method is feasible. This paper opens up a new direction for the future development of product form design which improves the agility of product design process in the era of Industry 4.0.展开更多
AIM： To identify the gene （s） related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism. METHODS： Transposon-mediated mutagenesis and tagging method and cassette P...AIM： To identify the gene （s） related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism. METHODS： Transposon-mediated mutagenesis and tagging method and cassette PCR-based chromosomal walking method were adopted to isolate the mutant strain （s） of B8 that lost the antagonistic activity and to clone DNA fragments around Tn5 insertion site. Sequence compiling and open reading frame （ORF） finding were done with DNAStar program and homologous sequence and conserved domain searches were performed with BlastN or BlastP programs at www.ncbi.nlm.nih.gov. To verify the gene involved in the antagonistic activity, complementation of a full-length clone of the anrFgene to the mutant B8F strain was used. RESULTS： A 3 321 bp contig around the Tn5 insertion site was obtained and an ORF of 2 634 bp in length designated as anrFgene encoding for a 877 aa polyketide synthase-like protein was identified. It had a homology of 83% at the nucleotide level and 79% ID/87% SIM at the protein level, to the admM gene of Pantoea agglomerans andrimid biosynthetic gene cluster （AY192157）. The Tn5 was inserted at 2 420 bp of the gene corresponding to the COG3319 （the thioesterase domain of type I polyketide synthase） coding region on BSF. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrFgene to the mutant B8F. CONCLUSION： The anrFgene obtained is related to the antagonistic activity of BS, and the antagonistic substances produced by B8 are andrimid and/or its analogs.展开更多
Actinobacillus pleuropneumoniae(APP)is the major pathogen of porcine contagious pleuropneumoniae(PCP).The QseB/QseC two-component system(TCS)consists of the regulator QseB and the kinase QseC,which relates to quorum s...Actinobacillus pleuropneumoniae(APP)is the major pathogen of porcine contagious pleuropneumoniae(PCP).The QseB/QseC two-component system(TCS)consists of the regulator QseB and the kinase QseC,which relates to quorum sensing(QS)and virulence in some bacteria.Here,we investigated the role of QseB/QseC in apf gene cluster(apfABCD)expression of APP.Our results have showed that QseB/QseC TCS can potentially regulate the expression of apf gene cluster.The△qseBC,△apfA,△apfB,△apfC and△apfD strains are more sensitive to acidic and osmotic stressful conditions,and exhibite lower biofilm formation ability than wild-type(WT)strain,whereas the complemented strains show similar phenotype to the wr strain.In additon,the mutants have defective antiphagocytosis,adhesion and invasion when they come into contact with the host cells.In experimental animal models of infection,mice infected with△qseBC,△apfA,△apfB,△apfC and △apfD strains showed lower mortality and bacterial loads in the lung and the blood than those infected with wr strain.In conclusion,our results suggest that QseB/QseC TCS contributes to stress resistance,biofilm formation,phagocytosis,adhesion,invasion and virulence by downregulating expression of apf gene cluster in A.pleuropneumoniae.展开更多
Streptomyces has enormous potential to produce novel natural products(NPs)as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters(BGCs).However,the lack of efficient gen...Streptomyces has enormous potential to produce novel natural products(NPs)as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters(BGCs).However,the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery.Here,we developed an easy-to-use,highly flexible DNA assembly toolkit for gene cluster engineering.The DNA assembly toolkit is compatible with various DNA assembling approaches including Biobrick,Golden Gate,CATCH,yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly.This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters.To demonstrate the utility of this toolkit,we quantified a library of modular regulatory parts,and engineered a gene cluster(act)using characterized promoters that led to increased production.Overall,this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.展开更多
Among plant metabolites,phenolamides,which are conjugates of hydroxycinnamic acid derivatives and polyamines,play important roles in plant adaptation to abiotic and biotic stresses.However,the molecular mechanisms und...Among plant metabolites,phenolamides,which are conjugates of hydroxycinnamic acid derivatives and polyamines,play important roles in plant adaptation to abiotic and biotic stresses.However,the molecular mechanisms underlying phenolamide metabolism and regulation as well as the effects of domestication and breeding on phenolamide diversity in tomato remain largely unclear.In this study,we performed a metabolite-based genome-wide association study and identified two biosynthetic gene clusters(BGC7 and BGC11)containing 12 genes involved in phenolamide metabolism,including four biosynthesis genes(two 4CL genes,one C3H gene,and one CPA gene),seven decoration genes(five AT genes and two UGT genes),and one transport protein gene(DTX29).Using gene co-expression network analysis we further discovered that SlMYB13 positively regulates the expression of two gene clusters,thereby promoting phenolamide accumulation.Genetic and physiological analyses showed that BGC7,BGC11 and SlMYB13 enhance drought tolerance by enhancing scavenging of reactive oxygen species and increasing abscisic acid content in tomato.Natural variation analysis suggested that BGC7,BGC11 and SlMYB13 were negatively selected during tomato domestication and improvement,leading to reduced phenolamide content and drought tolerance of cultivated tomato.Collectively,our study discovers a key mechanism of phenolamide biosynthesis and regulation in tomato and reveals that crop domestication and improvement shapes metabolic diversity to affect plant environmental adaptation.展开更多
Biosynthetic gene clusters(BGCs)are regions of a genome where genes involved in a biosynthetic pathway are in proximity.The origin and evolution of plant BGCs as well as their role in specialized metabolism remain lar...Biosynthetic gene clusters(BGCs)are regions of a genome where genes involved in a biosynthetic pathway are in proximity.The origin and evolution of plant BGCs as well as their role in specialized metabolism remain largely unclear.In this study,we have assembled a chromosome-scale genome of Japanese catnip(Schizonepeta tenuifolia)and discovered a BGC that contains multiple copies of genes involved in four adjacent steps in the biosynthesis of p-menthane monoterpenoids.This BGC has an unprecedented bipartite structure,with mirrored biosynthetic regions separated by 260 kilobases.This bipartite BGC includes identical copies of a gene encoding an old yellow enzyme,a type of flavin-dependent reductase.In vitro assays and virus-induced gene silencing revealed that this gene encodes the missing isopiperitenone reductase.This enzyme evolved from a completely different enzyme family to isopiperitenone reductase from closely related Mentha spp.,indicating convergent evolution of this pathway step.Phylogenomic analysis revealed that this bipartite BGC has emerged uniquely in the S.tenuifolia lineage and through insertion of pathway genes into a region rich in monoterpene synthases.The cluster gained its bipartite structure via an inverted duplication.The discovered bipartite BGC for p-menthane biosynthesis in S.tenuifolia has similarities to the recently described duplicated p-menthane biosynthesis gene pairs in the Mentha longifolia genome,providing an example of the convergent evolution of gene order.This work expands our understanding of plant BGCs with respect to both form and evolution,and highlights the power of BGCs for gene discovery in plant biosynthetic pathways.展开更多
Higher-order chromatin organization is essential for transcriptional regulation,genome stability maintenance,and other genome functions.Increasing evidence has revealed significant differences in 3D chromatin organiza...Higher-order chromatin organization is essential for transcriptional regulation,genome stability maintenance,and other genome functions.Increasing evidence has revealed significant differences in 3D chromatin organization between plants and animals.However,the extent,pattern,and rules of chromatin organization in plants are still unclear.In this study,we systematically identified and characterized long-range chromatin loops in the Arabidopsis 3D genome.We identified hundreds of long-range cis chromatin loops and found their anchor regions are closely associated with H3K27me3 epigenetic modifications.Furthermore,we demonstrated that these chromatin loops are dependent on Polycomb group(PcG)proteins,suggesting that the Polycomb repressive complex2(PRC2)complex is essential for establishing and maintaining these novel loops.Although most of these PcG-medicated chromatin loops are stable,many of these loops are tissue-specific or dynamically regulated by different treatments.Interestingly,tandemly arrayed gene clusters and metabolic gene clusters are enriched in anchor regions.Long-range H3K27me3-marked chromatin interactions are associated with the coregulation of specific gene clusters.Finally,we also identified H3K27me3-associated chromatin loops associated with gene clusters in Oryza sativa and Glycine max,indicating that these long-range chromatin loops are conserved in plants.Our results provide novel insights into genome evolution and transcriptional coregulation in plants.展开更多
Methylobacterium species,the representative bacteria distributed in phyllosphere region of plants,often synthesize carotenoids to resist harmful UV radiations.Methylobacterium extorquens is known to produce a caroteno...Methylobacterium species,the representative bacteria distributed in phyllosphere region of plants,often synthesize carotenoids to resist harmful UV radiations.Methylobacterium extorquens is known to produce a carotenoid pigment and recent research revealed that this carotenoid has a C_(30) backbone.However,its exact structure remains unknown.In the present study,the carotenoid produced by M.extorquens AM1 was isolated and its structure was determined as 4-[2-O-11Z-octadecenoyl-β-glucopyranosyl]-4,4′-diapolycopenedioc acid(1),a glycosylated C_(30) carotenoid.Furthermore,the genes related to the C_(30)carotenoid synthesis were investigated.Squalene,the precursor of the C_(30) carotenoid,is synthesized by the co-occurrence of META1p1815,META1p1816 and META1p1817.Further overexpression of the genes related to squalene synthesis improved the titer of carotenoid 1.By using gene deletion and gene complementation experiments,the glycosyltransferase META1p3663 and acyltransferase META1p3664 were firstly confirmed to catalyze the tailoring steps from 4,4′-diapolycopene-4,4′-dioic acid to carotenoid 1.In conclusion,the structure and biosynthetic genes of carotenoid 1 produced by M.extorquens AM1 were firstly characterized in this work,which shed lights on engineering M.extorquens AM1 for producing carotenoid 1 in high yield.展开更多
Jujube witches’broom(JWB)caused by phytoplasma has a severely negative effect on multiple metabolisms in jujube.The GST gene family in plants participates in the regulation of a variety of biotic and abiotic stresses...Jujube witches’broom(JWB)caused by phytoplasma has a severely negative effect on multiple metabolisms in jujube.The GST gene family in plants participates in the regulation of a variety of biotic and abiotic stresses.This study aims to identify and reveal the changes in the jujube GST gene family in response to phytoplasma infection.Here,70 ZjGSTs were identified in the jujube genome and divided into 8 classes.Among them,the Tau-class,including 44 genes,was the largest.Phylogenetic analysis indicated that Tau-class genes were highly conserved among species,such as Arabidopsis,cotton,chickpea,and rice.Through chromosome location analysis,37.1%of genes were clustered,and 8 of 9 gene clusters were composed of Tau class members.Through RT-PCR,qRT-PCR and enzyme activity detection,the results showed that the expression of half(20/40)of the tested ZjGSTs was inhibited by phytoplasma infection in field and tissue culture conditions,and GST activity was also significantly reduced.In the resistant and susceptible varieties under phytoplasma infection,ZjGSTU49-ZjGSTU54 in the cluster IV showed opposite expression patterns,which may be due to functional divergence during evolution.Some upregulated genes(ZjGSTU45,ZjGSTU49,ZjGSTU59,and ZjGSTU70)might be involved in the process of jujube against JWB.The yeast two-hybrid results showed that all 6 Tauclass proteins tested could form homodimers or heterodimers.Overall,the comprehensive analysis of the jujube GST gene family revealed that ZjGSTs responded actively to phytoplasma infection.Furthermore,some screened genes(ZjGSTU24,ZjGSTU49-52,ZjGSTU70,and ZjDHAR10)will contribute to further functional studies of jujube-phytoplasma interactions.展开更多
Background The oxidative modification of low-density lipoprotein in theartery wall is currently believed to be central to the pathogenesis of atherosclerosis. Paraoxonase(PON1), an enzyme located on high-density lipop...Background The oxidative modification of low-density lipoprotein in theartery wall is currently believed to be central to the pathogenesis of atherosclerosis. Paraoxonase(PON1), an enzyme located on high-density lipoprotein (HDL) , can prevent low-density lipoprotein(LDL) from oxidation at a certain extent. Recent studies show two other members of paraoxonase genefamily, PON2 and PON3, possess antioxidant properties similar to PON1. The aim of the present studywas to explore the role of PON gene cluster on coronary heart disease (CHD) in Chinese Han women.Methods Seven polymorphisms including PON1 -107C > T, -162G > A, -831G > A, R160G, Q192R, PON2S311C, and PON3 -133C > A were genotyped in 184 female patients with CHD and 239 female controls.The plasma PON1 activity toward phenylacetate was determined in 50 cases and 50 controls randomlyselected. Results The plasma PON1 activities were significantly lower in cases than in controls.Individual SNP analysis showed that cases had significantly higher frequencies of PON1 -107T, -831Gand PON2 311S alleles than controls. The genotype distributions of -107C >T were also significantlydifferent between two groups. The odds ratios for the development of CHD were 1. 66 for -107TCcarriers and 2. 0 for -107TT carriers, compared with -107CC carriers. Haplotype analyses showed thatthe distributions of haplotypes comprised of PON1 -107C > T and PON2 S311C were significantlydifferent between cases and controls, with cases having higher frequency of T-S haplotype (44.8% vs.36.3%, P =0.013). The T-S haplotype remained significantly associated with CHD after adjustingenvironmental risk factors (P = 0.0069). Conclusions This association study suggested that lowerplasma PON1 activity increased the risk of CHD in Chinese woman, which may be mediated by the higherfrequency of -107T allele in cases. Haplotype analyses indicated that there might be somesynergistic effects between the PON1 -107C > T and PON2 S311C polymorphisms.展开更多
Genomic clustering of non-homologous genes for the biosynthesis of plant defensive compounds is an emerging theme, but insights into their formation and physiological function remain limited. Here we report the identi...Genomic clustering of non-homologous genes for the biosynthesis of plant defensive compounds is an emerging theme, but insights into their formation and physiological function remain limited. Here we report the identification of a newly discovered hydroxycinnamoyl tyramine(HT) gene cluster in rice.This cluster contains a pyridoxamine 50-phosphate oxidase(Os PDX3) producing the cofactor pyridoxal50-phosphate(PLP), a PLP-dependent tyrosine decarboxylase(Os Ty DC1), and two duplicated hydroxycinnamoyl transferases(Os THT1 and Os THT2). These members were combined to represent an enzymological innovation gene cluster. Natural variation analysis showed that the abundance of the toxic tyramine intermediate of the gene cluster among different rice accessions is mainly determined by the coordinated transcription of Os Ty DC1 and Os THT1. Further pathogen incubation assays demonstrated that the end products of the HT gene cluster displayed enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. Oryzae(Xoo) and fungal pathogen Magnaporthe oryzae(M. oryzae), and the enhanced resistance is associated with the boost of phytoalexins and the activation of defense response. The unique presence of the HT gene cluster in Oryza AA genome, together with the enrichment of transposon elements within this gene cluster region, provides an evolutionary background to accelerate cluster member combinations. Our study not only discovered a gene cluster involved in the phenylpropanoid metabolism but also addressed the key aspects of gene cluster formation. In addition, our results provide a new metabolic pool for plant defense against pathogens.展开更多
Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase ...Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.展开更多
The human genome contains millions of DNA regulatory elements and a large number of gene clusters,most of which have not been tested experimentally.The clustered regularly interspaced short palindromic repeats(CRISPR)...The human genome contains millions of DNA regulatory elements and a large number of gene clusters,most of which have not been tested experimentally.The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)programed with a synthetic single-guide RNA(sgRNA)emerges as a method for genome editing in virtually any organisms.Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs.Specifically,we found that,in cultured human cells and mice,efficient precise inversions of DNA fragments ranging in size froma few tens of bp to hundreds of kb could be generated.In addition,DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks(DSBs)on two homologous chromosomes(chromatids).Moreover,junctions of combinatorial inversions and duplications of the protocadherin(Pcdh)gene clusters induced by Cas9 with four sgRNAs could be detected.In mice,we obtained founders with alleles of precise inversions,duplications,and deletions of DNA fragments of variable sizes by CRISPR.Interestingly,we found that very efficient inversions were mediated by microhomology-mediated end joining(MMEJ)through short inverted repeats.We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice.Finally,we applied this CRISPR method to a regulatory element of the Pcdha cluster and found a new role in the regulation of members of the Pcdhg cluster.This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.展开更多
Phenolamides(PAs), a diverse group of specialized metabolites, including hydroxycinnamoylputrescine(HP), hydroxycinnamoylagmatine, and hydroxycinnamoyltryptamine, are important in plant resistance to biotic stress. Ho...Phenolamides(PAs), a diverse group of specialized metabolites, including hydroxycinnamoylputrescine(HP), hydroxycinnamoylagmatine, and hydroxycinnamoyltryptamine, are important in plant resistance to biotic stress. However, the genes involved in the biosynthesis and modulation of PAs have not been fully elucidated. This study identified an HP biosynthetic gene cluster in rice(Oryza sativa) comprising one gene(Os ODC) encoding a decarboxylase and two tandem-duplicated genes(Os PHT3 and Os PHT4)encoding putrescine hydroxycinnamoyl acyltransferases coexpressed in different tissues. Os ODC catalyzes the conversion of ornithine to putrescine, which is used in HP biosynthesis involving Os PHT3 and Os PHT4. Os PHT3 or Os PHT4 overexpression causes HP accumulation and cell death and putrescine hydroxycinnamoyl acyltransferases(PHT) activity-dependent resistance against the fungal pathogen Magnaporthe oryzae. Os ODC overexpression plants also confer enhanced resistance to M. oryzae.Notably, the basic leucine zipper transcription factor APIP5, a negative regulator of cell death, directly binds to the Os PHT4 promoter, repressing its transcription. Moreover, APIP5 suppression induces Os PHT4 expression and HP accumulation. Comparative genomic analysis revealed that the HP biosynthetic gene cluster is conserved in monocots. These results characterized a previously unidentified monocot-specific gene cluster that is involved in HP biosynthesis and contributes to defense and cell death in rice.展开更多
By using 304 recombinant inbred lines derived from indica rice cross Zhong 156/Gumei 2, a linkage map consisting of 177 marker loci and covering 12 rice chromosomes was constructed and employed for mapping genes confe...By using 304 recombinant inbred lines derived from indica rice cross Zhong 156/Gumei 2, a linkage map consisting of 177 marker loci and covering 12 rice chromosomes was constructed and employed for mapping genes conferring blast resistance in rice. Genomic location of gene Pi25(t) conferring neck blast resistance to the Chinese isolate 92-183 (race ZC15) was verified to be located between markers A7 and RG456 on chromosome 6, with genetic distances of 1.7 cM and 1.5 cM to A7 and RG456, respectively. Leaf blast resistance of Gumei 2 to the Philippine isolate Ca89 (lineage 4) was found to be controlled by a single gene. The gene tentatively designated as Pi26(\) was located between makers B10 and R674 on chromosome 6, with genetic distances of 5.7 cM and 25.8 cM to B10 and R674 respectively. Resistant alleles at both gene loci were derived from Gumei 2, indicating an existence of resistance gene cluster in Gumei 2.展开更多
Benzoxazinoids are a class of protective and allelopathic plant secondary metabolites that have been identified in multiple grass species and are encoded by the Bx biosynthetic gene cluster(BGC)in maize.Data mining of...Benzoxazinoids are a class of protective and allelopathic plant secondary metabolites that have been identified in multiple grass species and are encoded by the Bx biosynthetic gene cluster(BGC)in maize.Data mining of 41 high-quality grass genomes identified complete Bx clusters(containing genes Bx1–Bx5 and Bx8)in three genera(Zea,Echinochloa,and Dichanthelium)of Panicoideae and partial clusters in Triticeae.The Bx cluster probably originated from gene duplication and chromosomal translocation of native homologs of Bx genes.An ancient Bx cluster that included additional Bx genes(e.g.,Bx6)is presumed to have been present in ancestral Panicoideae.The ancient Bx cluster was putatively gained by the Triticeae ancestor via horizontal transfer(HT)from the ancestral Panicoideae and later separated into multiple segments on different chromosomes.Bx6 appears to have been under less constrained selection compared with the Bx cluster during the evolution of Panicoideae,as evidenced by the fact that it was translocated away from the Bx cluster in Zea mays,moved to other chromosomes in Echinochloa,and even lost in Dichanthelium.Further investigations indicate that purifying selection and polyploidization have shaped the evolutionary trajectory of Bx clusters in the grass family.This study provides the first candidate case of HT of a BGC between plants and sheds new light on the evolution of BGCs.展开更多
Beauvericin, a cyclohexadepsipeptide-possessing natural product with synergistic antifungal, insecticidal, and cytotoxic activities. We isolated and characterized the fpBeas gene cluster, devoted to beauvericin biosyn...Beauvericin, a cyclohexadepsipeptide-possessing natural product with synergistic antifungal, insecticidal, and cytotoxic activities. We isolated and characterized the fpBeas gene cluster, devoted to beauvericin biosynthesis, from the filamentous fungus Fusarium proliferatum LF061. Targeted inactivation of the F. proliferatum genomic copy of fpBeas abolished the production of beauvericin. Comparative sequence analysis of the FpBEAS showed 74% similarity with the BbBEAS that synthesizes the cyclic trimeric ester beauvericin in Beauveria bassiana, which assembles N-methyl-dipeptidol monomer intermediates by the programmed iterative use of the nonribosomal peptide synthetase modules. Differences between the organization of the beauvericin loci in F. proliferaturm and B. bassiana revealed the mechanism for high production of beauvericin in F. proliferatum. Our work provides new insights into beauvericin biosynthesis, and may lead to beauvericin overproduction and creation of new analogs via synthetic biology approaches.展开更多
Cardiac hypertrophy is the strongest predictor of the development of heart failure, and anti-hypertrophic treatment holds the key to improving the clinical syndrome and increasing the survival rates for heart failure....Cardiac hypertrophy is the strongest predictor of the development of heart failure, and anti-hypertrophic treatment holds the key to improving the clinical syndrome and increasing the survival rates for heart failure. The paraoxonase(PON) gene cluster(PC) protects against atherosclerosis and coronary artery diseases. However, the role of PC in the heart is largely unknown. To evaluate the roles of PC in cardiac hypertrophy, transgenic mice carrying the intact human PON1, PON2, and PON3 genes and their flanking sequences were studied. We demonstrated that the PC transgene(PC-Tg) protected mice from cardiac hypertrophy induced by Ang II; these mice had reduced heart weight/body weight ratios, decreased left ventricular wall thicknesses and increased fractional shortening compared with wild-type(WT) control. The same protective tendency was also observed with an Apoe^(-/-)background. Mechanically, PC-Tg normalized the disequilibrium of matrix metalloproteinases(MMPs)/tissue inhibitors of MMPs(TIMPs) in hypertrophic hearts, which might contribute to the protective role of PC-Tg in cardiac fibrosis and, thus, protect against cardiac remodeling. Taken together, our results identify a novel anti-hypertrophic role for the PON gene cluster, suggesting a possible strategy for the treatment of cardiac hypertrophy through elevating the levels of the PON gene family.展开更多
基金supported by grants from the National Key Research&Development Plan,China (Grant Nos.2021YFD1200201,2022YFD1200502)National Natural Science Foundation of China(31972426,31991182)+3 种基金Key Project of Hubei Hongshan Laboratory(Grant No.2021hszd007)Wuhan Major Project of Key Technologies in Biological Breeding (Grant No.2022021302024852)Fundamental Research Funds for the Central Universities,China (Grant No.2662022YLPY001)International Cooperation Promotion Plan of Shihezi University (Grant No.GJHZ202104)。
文摘High temperature stress is one of the major environmental factors that affect the growth and development of plants. Although WRKY transcription factors play a critical role in stress responses, there are few studies on the regulation of heat stress by WRKY transcription factors,especially in tomato. Here, we identified a group I WRKY transcription factor, SlWRKY3, involved in thermotolerance in tomato. First, SlWRKY3 was induced and upregulated under heat stress. Accordingly, overexpression of SlWRKY3 led to an increase, whereas knock-out of SlWRKY3 resulted in decreased tolerance to heat stress. Overexpression of SlWRKY3 accumulated less reactive oxygen species(ROS), whereas knock-out of SlWRKY3 accumulated more ROS under heat stress. This indicated that SlWRKY3 positively regulates heat stress in tomato. In addition,SlWRKY3 activated the expression of a range of abiotic stress-responsive genes involved in ROS scavenging, such as a SlGRXS1 gene cluster.Further analysis showed that SlWRKY3 can bind to the promoters of the SlGRXS1 gene cluster and activate their expression. Collectively, these results imply that SlWRKY3 is a positive regulator of thermotolerance through direct binding to the promoters of the SlGRXS1 gene cluster and activating their expression and ROS scavenging.
文摘A recombinant strain, Escherichia coli JM109-AN1, was obtained by constructing of a genomic library of the total DNA of Delftia sp. AN3 in E. coli JM109 and screening for catechol 2,3-dioxygenase activity. This recombinant strain could grow on aniline as sole carbon, nitrogen and energy source. Enzymatic assays revealed that the exogenous genes including aniline dioxygenase （AD） and catechol 2,3-dioxygenase （C230） genes could well express in the recombinant strain with the activities of AD and C230 up to 0.31 U/mg wet cell and 1.92 U/mg crude proteins, respectively. The AD or C23O of strain AN3 could only catalyze aniline or catechol but not any other substituted substrates. This recombinant strain contained a recombinant plasmid, pKC505-AN1, in which a 29.7-kb DNA fragment from Delftia sp. AN3 was inserted. Sequencing and open reading frame （orfs） analysis of this 29.7 kb fragment revealed that it contained at least 27 orfs, among them a gene cluster （consisting of at least 16 genes, named danQTA1A2BRDCEFG1HIJKG2） was responsible for the complete metabolism of aniline to TCA-cycle intermediates. This gene cluster could be divided into two main parts, the upper sequences consisted of 7 genes （danQTA1A2BRD） were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, and the central genes （danCEFG1HIJKG2） were expected to encode meta-cleavage pathway enzymes for catechol degradation to TCA-cycle intermediates. Unlike clusters tad from Delftia tsuruhatensis AD9 and tdn from Pseudomonas putida UCC22, in this gene cluster, all the genes were in the same transcriptional direction. There was only one set of C230 gene （danC） and ferredoxin-like protein gene （danD）. The presence of only one set of these two genes and specificity of AD and C230 might be the reason for strain AN3 could only degrade aniline. The products of danQTA1A2BRDC showed 99%-100% identity to those from Delftia acidovorans 7N, and 50%-85% identity to those of tad cluster from D. tsuruhatensis AD9 in amino acid residues. Besides this dan cluster, the 29.7 kb fragment also contained genes encoding the trans-membrane transporter and transposases which might be needed for transposition of the gene cluster. Pulsed-field gel electrophoresis （PFGE） and plasmid curing experiments suggested that the dan cluster might be encoded on the chromosome of strain AN3. The GenBank accession number for the dan cluster of Delftia sp. AN3 is DQ661649.
基金Supported by National Key Technology R&D Program,China(Grant No.2015BAH21F01)National 111 Project,China(Grant No.B13044)
文摘The use of pan-ethnic-group products form knowledge primarily depends on a designer＇s subjective experience without user participation. The majority of studies primarily focus on the detection of the perceptual demands of consumers from the target product category. A pan-ethnic-group products form gene clustering method based on emotional semantic is constructed. Consumers＇ perceptual images of the pan-ethnic-group products are obtained by means of product form gene extraction and coding and computer aided product form clustering technology. A case of form gene clustering about the typical pan-ethnic-group products is investigated which indicates that the method is feasible. This paper opens up a new direction for the future development of product form design which improves the agility of product design process in the era of Industry 4.0.
基金Supported by the National Natural Science Foundation of China,No. 39870034the National High Technology Research and Development Program of China, the 863 Program, No. 104-04-01-01the Major Project of Science and Technology Development of Zhejiang Province, No. 021102529
文摘AIM： To identify the gene （s） related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism. METHODS： Transposon-mediated mutagenesis and tagging method and cassette PCR-based chromosomal walking method were adopted to isolate the mutant strain （s） of B8 that lost the antagonistic activity and to clone DNA fragments around Tn5 insertion site. Sequence compiling and open reading frame （ORF） finding were done with DNAStar program and homologous sequence and conserved domain searches were performed with BlastN or BlastP programs at www.ncbi.nlm.nih.gov. To verify the gene involved in the antagonistic activity, complementation of a full-length clone of the anrFgene to the mutant B8F strain was used. RESULTS： A 3 321 bp contig around the Tn5 insertion site was obtained and an ORF of 2 634 bp in length designated as anrFgene encoding for a 877 aa polyketide synthase-like protein was identified. It had a homology of 83% at the nucleotide level and 79% ID/87% SIM at the protein level, to the admM gene of Pantoea agglomerans andrimid biosynthetic gene cluster （AY192157）. The Tn5 was inserted at 2 420 bp of the gene corresponding to the COG3319 （the thioesterase domain of type I polyketide synthase） coding region on BSF. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrFgene to the mutant B8F. CONCLUSION： The anrFgene obtained is related to the antagonistic activity of BS, and the antagonistic substances produced by B8 are andrimid and/or its analogs.
基金supported by grants from the Technique Innovation Program of Hubei Province(No.2018ABA108)the National Pig Industry Technology System(No.CARS-35).
文摘Actinobacillus pleuropneumoniae(APP)is the major pathogen of porcine contagious pleuropneumoniae(PCP).The QseB/QseC two-component system(TCS)consists of the regulator QseB and the kinase QseC,which relates to quorum sensing(QS)and virulence in some bacteria.Here,we investigated the role of QseB/QseC in apf gene cluster(apfABCD)expression of APP.Our results have showed that QseB/QseC TCS can potentially regulate the expression of apf gene cluster.The△qseBC,△apfA,△apfB,△apfC and△apfD strains are more sensitive to acidic and osmotic stressful conditions,and exhibite lower biofilm formation ability than wild-type(WT)strain,whereas the complemented strains show similar phenotype to the wr strain.In additon,the mutants have defective antiphagocytosis,adhesion and invasion when they come into contact with the host cells.In experimental animal models of infection,mice infected with△qseBC,△apfA,△apfB,△apfC and △apfD strains showed lower mortality and bacterial loads in the lung and the blood than those infected with wr strain.In conclusion,our results suggest that QseB/QseC TCS contributes to stress resistance,biofilm formation,phagocytosis,adhesion,invasion and virulence by downregulating expression of apf gene cluster in A.pleuropneumoniae.
基金supported by the National Key Research and Development Program of China[2020YFA0906900,2018YFA0900700]Natural Science Foundation of China[31500069]+1 种基金the Chinese Academy of Sciences[No.QYZDB-SSW-SMC050,No.XDB0480000 of the Strategic Priority Research Program]CAS Youth Interdisciplinary Team and the Shenzhen Science and Technology Innovation Committee[No.JCYJ20180507182241844,JCHZ20200005,DWKF20190009].
文摘Streptomyces has enormous potential to produce novel natural products(NPs)as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters(BGCs).However,the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery.Here,we developed an easy-to-use,highly flexible DNA assembly toolkit for gene cluster engineering.The DNA assembly toolkit is compatible with various DNA assembling approaches including Biobrick,Golden Gate,CATCH,yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly.This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters.To demonstrate the utility of this toolkit,we quantified a library of modular regulatory parts,and engineered a gene cluster(act)using characterized promoters that led to increased production.Overall,this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.
基金supported by grants from the National Key Research and Development Program of China(2022YFF1001900)the Hainan Province Science and Technology Special Fund(no.ZDYF2022XDNY144)+4 种基金the Hainan Provincial Academician Innovation Platform Project(no.HD-YSZX-202004)the Young Elite Scientists Sponsorship Program by CAST(no.2019QNRC001)the Hainan University Startup Fund(no.KYQD(ZR)21025)the Collaborative Innovation Center of Nanfan and High-Efficiency Tropical Agriculture,Hainan University(no.XTCX2022NYB06)the Innovation Project of Postgraduates of Hainan Province(no.Qhyb2022-56).
文摘Among plant metabolites,phenolamides,which are conjugates of hydroxycinnamic acid derivatives and polyamines,play important roles in plant adaptation to abiotic and biotic stresses.However,the molecular mechanisms underlying phenolamide metabolism and regulation as well as the effects of domestication and breeding on phenolamide diversity in tomato remain largely unclear.In this study,we performed a metabolite-based genome-wide association study and identified two biosynthetic gene clusters(BGC7 and BGC11)containing 12 genes involved in phenolamide metabolism,including four biosynthesis genes(two 4CL genes,one C3H gene,and one CPA gene),seven decoration genes(five AT genes and two UGT genes),and one transport protein gene(DTX29).Using gene co-expression network analysis we further discovered that SlMYB13 positively regulates the expression of two gene clusters,thereby promoting phenolamide accumulation.Genetic and physiological analyses showed that BGC7,BGC11 and SlMYB13 enhance drought tolerance by enhancing scavenging of reactive oxygen species and increasing abscisic acid content in tomato.Natural variation analysis suggested that BGC7,BGC11 and SlMYB13 were negatively selected during tomato domestication and improvement,leading to reduced phenolamide content and drought tolerance of cultivated tomato.Collectively,our study discovers a key mechanism of phenolamide biosynthesis and regulation in tomato and reveals that crop domestication and improvement shapes metabolic diversity to affect plant environmental adaptation.
基金supported by the National Natural Science Foundation of China(grant nos.81973435 and 81473313)the National Natural Science Foundation for Young Scientists of China(grant no.81903756)+2 种基金the Open Project of the Natural Science Foundation of Nanjing University of Chinese Medicine(no.NZY81903756)research on ecological planting and quality assurance of Jiangsu Dao-di herbs(2021)and a Jiangsu Government Scholarship for Overseas Studies(JS-2020-044).We also acknowledge support from the BBSRC(BBN006452/1)and UKRI(MR/S01862X/1).
文摘Biosynthetic gene clusters(BGCs)are regions of a genome where genes involved in a biosynthetic pathway are in proximity.The origin and evolution of plant BGCs as well as their role in specialized metabolism remain largely unclear.In this study,we have assembled a chromosome-scale genome of Japanese catnip(Schizonepeta tenuifolia)and discovered a BGC that contains multiple copies of genes involved in four adjacent steps in the biosynthesis of p-menthane monoterpenoids.This BGC has an unprecedented bipartite structure,with mirrored biosynthetic regions separated by 260 kilobases.This bipartite BGC includes identical copies of a gene encoding an old yellow enzyme,a type of flavin-dependent reductase.In vitro assays and virus-induced gene silencing revealed that this gene encodes the missing isopiperitenone reductase.This enzyme evolved from a completely different enzyme family to isopiperitenone reductase from closely related Mentha spp.,indicating convergent evolution of this pathway step.Phylogenomic analysis revealed that this bipartite BGC has emerged uniquely in the S.tenuifolia lineage and through insertion of pathway genes into a region rich in monoterpene synthases.The cluster gained its bipartite structure via an inverted duplication.The discovered bipartite BGC for p-menthane biosynthesis in S.tenuifolia has similarities to the recently described duplicated p-menthane biosynthesis gene pairs in the Mentha longifolia genome,providing an example of the convergent evolution of gene order.This work expands our understanding of plant BGCs with respect to both form and evolution,and highlights the power of BGCs for gene discovery in plant biosynthetic pathways.
基金supported by the National Natural Science Foundation of China(31970614 and 32270288 to W.Q.)Director’s Award of Peking University Institute of Advanced Agricultural Sciences,Shandong Development Fund of Science&TechnologyAward of Natural Science Foundation of Shandong Province(ZR2021ZD30)。
文摘Higher-order chromatin organization is essential for transcriptional regulation,genome stability maintenance,and other genome functions.Increasing evidence has revealed significant differences in 3D chromatin organization between plants and animals.However,the extent,pattern,and rules of chromatin organization in plants are still unclear.In this study,we systematically identified and characterized long-range chromatin loops in the Arabidopsis 3D genome.We identified hundreds of long-range cis chromatin loops and found their anchor regions are closely associated with H3K27me3 epigenetic modifications.Furthermore,we demonstrated that these chromatin loops are dependent on Polycomb group(PcG)proteins,suggesting that the Polycomb repressive complex2(PRC2)complex is essential for establishing and maintaining these novel loops.Although most of these PcG-medicated chromatin loops are stable,many of these loops are tissue-specific or dynamically regulated by different treatments.Interestingly,tandemly arrayed gene clusters and metabolic gene clusters are enriched in anchor regions.Long-range H3K27me3-marked chromatin interactions are associated with the coregulation of specific gene clusters.Finally,we also identified H3K27me3-associated chromatin loops associated with gene clusters in Oryza sativa and Glycine max,indicating that these long-range chromatin loops are conserved in plants.Our results provide novel insights into genome evolution and transcriptional coregulation in plants.
基金the National Key R&D Program of China(grant No.2021YFC2103500)National Natural Science Foundation of China(grant No.22078169)+1 种基金Natural Science Foundation of Shandong Province,China(ZR2021MC074,ZR2020MC008)Shandong Provincial Key Research and Development Plan(2021ZDSYS28).
文摘Methylobacterium species,the representative bacteria distributed in phyllosphere region of plants,often synthesize carotenoids to resist harmful UV radiations.Methylobacterium extorquens is known to produce a carotenoid pigment and recent research revealed that this carotenoid has a C_(30) backbone.However,its exact structure remains unknown.In the present study,the carotenoid produced by M.extorquens AM1 was isolated and its structure was determined as 4-[2-O-11Z-octadecenoyl-β-glucopyranosyl]-4,4′-diapolycopenedioc acid(1),a glycosylated C_(30) carotenoid.Furthermore,the genes related to the C_(30)carotenoid synthesis were investigated.Squalene,the precursor of the C_(30) carotenoid,is synthesized by the co-occurrence of META1p1815,META1p1816 and META1p1817.Further overexpression of the genes related to squalene synthesis improved the titer of carotenoid 1.By using gene deletion and gene complementation experiments,the glycosyltransferase META1p3663 and acyltransferase META1p3664 were firstly confirmed to catalyze the tailoring steps from 4,4′-diapolycopene-4,4′-dioic acid to carotenoid 1.In conclusion,the structure and biosynthetic genes of carotenoid 1 produced by M.extorquens AM1 were firstly characterized in this work,which shed lights on engineering M.extorquens AM1 for producing carotenoid 1 in high yield.
基金supported by grants from the National Key R&D Program Project Funding(Grant No.2018YFD1000607)the Foundation for 100 Innovative Talents of Hebei Province(Grant No.SLRC2019031)+1 种基金the National Natural Science Foundation of China(Grant No.31772285)the Hebei Province Innovation Foundation for Postgraduates(Grant No.CXZZBS2020097)。
文摘Jujube witches’broom(JWB)caused by phytoplasma has a severely negative effect on multiple metabolisms in jujube.The GST gene family in plants participates in the regulation of a variety of biotic and abiotic stresses.This study aims to identify and reveal the changes in the jujube GST gene family in response to phytoplasma infection.Here,70 ZjGSTs were identified in the jujube genome and divided into 8 classes.Among them,the Tau-class,including 44 genes,was the largest.Phylogenetic analysis indicated that Tau-class genes were highly conserved among species,such as Arabidopsis,cotton,chickpea,and rice.Through chromosome location analysis,37.1%of genes were clustered,and 8 of 9 gene clusters were composed of Tau class members.Through RT-PCR,qRT-PCR and enzyme activity detection,the results showed that the expression of half(20/40)of the tested ZjGSTs was inhibited by phytoplasma infection in field and tissue culture conditions,and GST activity was also significantly reduced.In the resistant and susceptible varieties under phytoplasma infection,ZjGSTU49-ZjGSTU54 in the cluster IV showed opposite expression patterns,which may be due to functional divergence during evolution.Some upregulated genes(ZjGSTU45,ZjGSTU49,ZjGSTU59,and ZjGSTU70)might be involved in the process of jujube against JWB.The yeast two-hybrid results showed that all 6 Tauclass proteins tested could form homodimers or heterodimers.Overall,the comprehensive analysis of the jujube GST gene family revealed that ZjGSTs responded actively to phytoplasma infection.Furthermore,some screened genes(ZjGSTU24,ZjGSTU49-52,ZjGSTU70,and ZjDHAR10)will contribute to further functional studies of jujube-phytoplasma interactions.
文摘Background The oxidative modification of low-density lipoprotein in theartery wall is currently believed to be central to the pathogenesis of atherosclerosis. Paraoxonase(PON1), an enzyme located on high-density lipoprotein (HDL) , can prevent low-density lipoprotein(LDL) from oxidation at a certain extent. Recent studies show two other members of paraoxonase genefamily, PON2 and PON3, possess antioxidant properties similar to PON1. The aim of the present studywas to explore the role of PON gene cluster on coronary heart disease (CHD) in Chinese Han women.Methods Seven polymorphisms including PON1 -107C > T, -162G > A, -831G > A, R160G, Q192R, PON2S311C, and PON3 -133C > A were genotyped in 184 female patients with CHD and 239 female controls.The plasma PON1 activity toward phenylacetate was determined in 50 cases and 50 controls randomlyselected. Results The plasma PON1 activities were significantly lower in cases than in controls.Individual SNP analysis showed that cases had significantly higher frequencies of PON1 -107T, -831Gand PON2 311S alleles than controls. The genotype distributions of -107C >T were also significantlydifferent between two groups. The odds ratios for the development of CHD were 1. 66 for -107TCcarriers and 2. 0 for -107TT carriers, compared with -107CC carriers. Haplotype analyses showed thatthe distributions of haplotypes comprised of PON1 -107C > T and PON2 S311C were significantlydifferent between cases and controls, with cases having higher frequency of T-S haplotype (44.8% vs.36.3%, P =0.013). The T-S haplotype remained significantly associated with CHD after adjustingenvironmental risk factors (P = 0.0069). Conclusions This association study suggested that lowerplasma PON1 activity increased the risk of CHD in Chinese woman, which may be mediated by the higherfrequency of -107T allele in cases. Haplotype analyses indicated that there might be somesynergistic effects between the PON1 -107C > T and PON2 S311C polymorphisms.
基金supported by the National Science Fund for Distinguished Young Scholars of China (31625021)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (31821005)+1 种基金the State Key Program of National Natural Science Foundation of China (31530052)the Hainan University Startup Fund (KYQD(ZR)1866)。
文摘Genomic clustering of non-homologous genes for the biosynthesis of plant defensive compounds is an emerging theme, but insights into their formation and physiological function remain limited. Here we report the identification of a newly discovered hydroxycinnamoyl tyramine(HT) gene cluster in rice.This cluster contains a pyridoxamine 50-phosphate oxidase(Os PDX3) producing the cofactor pyridoxal50-phosphate(PLP), a PLP-dependent tyrosine decarboxylase(Os Ty DC1), and two duplicated hydroxycinnamoyl transferases(Os THT1 and Os THT2). These members were combined to represent an enzymological innovation gene cluster. Natural variation analysis showed that the abundance of the toxic tyramine intermediate of the gene cluster among different rice accessions is mainly determined by the coordinated transcription of Os Ty DC1 and Os THT1. Further pathogen incubation assays demonstrated that the end products of the HT gene cluster displayed enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. Oryzae(Xoo) and fungal pathogen Magnaporthe oryzae(M. oryzae), and the enhanced resistance is associated with the boost of phytoalexins and the activation of defense response. The unique presence of the HT gene cluster in Oryza AA genome, together with the enrichment of transposon elements within this gene cluster region, provides an evolutionary background to accelerate cluster member combinations. Our study not only discovered a gene cluster involved in the phenylpropanoid metabolism but also addressed the key aspects of gene cluster formation. In addition, our results provide a new metabolic pool for plant defense against pathogens.
基金supported by grants from the Ministry of Science and Technology of China(2013CB734001 and 2015CB150600)the National Natural Science Foundation of China(31370097 and 31571281)
文摘Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.
基金supported by grants to Q.W.from the National Natural Science Foundation of China(31171015 and 31470820)the Science and Technology Commission of Shanghai Municipality(13XD1402000 and 14JC1403600).
文摘The human genome contains millions of DNA regulatory elements and a large number of gene clusters,most of which have not been tested experimentally.The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)programed with a synthetic single-guide RNA(sgRNA)emerges as a method for genome editing in virtually any organisms.Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs.Specifically,we found that,in cultured human cells and mice,efficient precise inversions of DNA fragments ranging in size froma few tens of bp to hundreds of kb could be generated.In addition,DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks(DSBs)on two homologous chromosomes(chromatids).Moreover,junctions of combinatorial inversions and duplications of the protocadherin(Pcdh)gene clusters induced by Cas9 with four sgRNAs could be detected.In mice,we obtained founders with alleles of precise inversions,duplications,and deletions of DNA fragments of variable sizes by CRISPR.Interestingly,we found that very efficient inversions were mediated by microhomology-mediated end joining(MMEJ)through short inverted repeats.We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice.Finally,we applied this CRISPR method to a regulatory element of the Pcdha cluster and found a new role in the regulation of members of the Pcdhg cluster.This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.
基金supported by grants from the National Natural Science Foundation of China (31822041 and 31972225)the National Key Research and Development Program of China (2016YFD0100600) to Yuese Ning+5 种基金the National Natural Science Foundation of China (U20A2021) to Ruyi Wangthe National Natural Science Foundation of China (31801692) to Fan Zhangthe State Key Program of National Natural Science Foundation of China (31530052)the National Science Fund for Distinguished Young Scholars (31625021)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (31821005)the Hainan University Start up Fund (KYQD(ZR)1866 to Jie Luo。
文摘Phenolamides(PAs), a diverse group of specialized metabolites, including hydroxycinnamoylputrescine(HP), hydroxycinnamoylagmatine, and hydroxycinnamoyltryptamine, are important in plant resistance to biotic stress. However, the genes involved in the biosynthesis and modulation of PAs have not been fully elucidated. This study identified an HP biosynthetic gene cluster in rice(Oryza sativa) comprising one gene(Os ODC) encoding a decarboxylase and two tandem-duplicated genes(Os PHT3 and Os PHT4)encoding putrescine hydroxycinnamoyl acyltransferases coexpressed in different tissues. Os ODC catalyzes the conversion of ornithine to putrescine, which is used in HP biosynthesis involving Os PHT3 and Os PHT4. Os PHT3 or Os PHT4 overexpression causes HP accumulation and cell death and putrescine hydroxycinnamoyl acyltransferases(PHT) activity-dependent resistance against the fungal pathogen Magnaporthe oryzae. Os ODC overexpression plants also confer enhanced resistance to M. oryzae.Notably, the basic leucine zipper transcription factor APIP5, a negative regulator of cell death, directly binds to the Os PHT4 promoter, repressing its transcription. Moreover, APIP5 suppression induces Os PHT4 expression and HP accumulation. Comparative genomic analysis revealed that the HP biosynthetic gene cluster is conserved in monocots. These results characterized a previously unidentified monocot-specific gene cluster that is involved in HP biosynthesis and contributes to defense and cell death in rice.
文摘By using 304 recombinant inbred lines derived from indica rice cross Zhong 156/Gumei 2, a linkage map consisting of 177 marker loci and covering 12 rice chromosomes was constructed and employed for mapping genes conferring blast resistance in rice. Genomic location of gene Pi25(t) conferring neck blast resistance to the Chinese isolate 92-183 (race ZC15) was verified to be located between markers A7 and RG456 on chromosome 6, with genetic distances of 1.7 cM and 1.5 cM to A7 and RG456, respectively. Leaf blast resistance of Gumei 2 to the Philippine isolate Ca89 (lineage 4) was found to be controlled by a single gene. The gene tentatively designated as Pi26(\) was located between makers B10 and R674 on chromosome 6, with genetic distances of 5.7 cM and 25.8 cM to B10 and R674 respectively. Resistant alleles at both gene loci were derived from Gumei 2, indicating an existence of resistance gene cluster in Gumei 2.
基金This work was supported by grants from the Zhejiang Natural Science Foundation(LZ17C130001)the Jiangsu Collaborative Innovation Center for Modern Crop Production,111 Project(B17039).
文摘Benzoxazinoids are a class of protective and allelopathic plant secondary metabolites that have been identified in multiple grass species and are encoded by the Bx biosynthetic gene cluster(BGC)in maize.Data mining of 41 high-quality grass genomes identified complete Bx clusters(containing genes Bx1–Bx5 and Bx8)in three genera(Zea,Echinochloa,and Dichanthelium)of Panicoideae and partial clusters in Triticeae.The Bx cluster probably originated from gene duplication and chromosomal translocation of native homologs of Bx genes.An ancient Bx cluster that included additional Bx genes(e.g.,Bx6)is presumed to have been present in ancestral Panicoideae.The ancient Bx cluster was putatively gained by the Triticeae ancestor via horizontal transfer(HT)from the ancestral Panicoideae and later separated into multiple segments on different chromosomes.Bx6 appears to have been under less constrained selection compared with the Bx cluster during the evolution of Panicoideae,as evidenced by the fact that it was translocated away from the Bx cluster in Zea mays,moved to other chromosomes in Echinochloa,and even lost in Dichanthelium.Further investigations indicate that purifying selection and polyploidization have shaped the evolutionary trajectory of Bx clusters in the grass family.This study provides the first candidate case of HT of a BGC between plants and sheds new light on the evolution of BGCs.
基金supported by the National Basic Research Program of China (2013CB734000, 2012CB725200)the CAS Pillar Program (XDA04074000)+1 种基金Ministry of Science and Technology of China (2011ZX11102-011-11)ZHANG LiXin is an Awardee for the National Distinguished Young Scholar Program in China
文摘Beauvericin, a cyclohexadepsipeptide-possessing natural product with synergistic antifungal, insecticidal, and cytotoxic activities. We isolated and characterized the fpBeas gene cluster, devoted to beauvericin biosynthesis, from the filamentous fungus Fusarium proliferatum LF061. Targeted inactivation of the F. proliferatum genomic copy of fpBeas abolished the production of beauvericin. Comparative sequence analysis of the FpBEAS showed 74% similarity with the BbBEAS that synthesizes the cyclic trimeric ester beauvericin in Beauveria bassiana, which assembles N-methyl-dipeptidol monomer intermediates by the programmed iterative use of the nonribosomal peptide synthetase modules. Differences between the organization of the beauvericin loci in F. proliferaturm and B. bassiana revealed the mechanism for high production of beauvericin in F. proliferatum. Our work provides new insights into beauvericin biosynthesis, and may lead to beauvericin overproduction and creation of new analogs via synthetic biology approaches.
基金supported by the National Natural Science Foundation of China(81422002,91339201,31271227,31571193)the National Science and Technology Support Project(2013YQ0309230502,2014BAI02B01)the Beijing Nova Program(XX2013064)
文摘Cardiac hypertrophy is the strongest predictor of the development of heart failure, and anti-hypertrophic treatment holds the key to improving the clinical syndrome and increasing the survival rates for heart failure. The paraoxonase(PON) gene cluster(PC) protects against atherosclerosis and coronary artery diseases. However, the role of PC in the heart is largely unknown. To evaluate the roles of PC in cardiac hypertrophy, transgenic mice carrying the intact human PON1, PON2, and PON3 genes and their flanking sequences were studied. We demonstrated that the PC transgene(PC-Tg) protected mice from cardiac hypertrophy induced by Ang II; these mice had reduced heart weight/body weight ratios, decreased left ventricular wall thicknesses and increased fractional shortening compared with wild-type(WT) control. The same protective tendency was also observed with an Apoe^(-/-)background. Mechanically, PC-Tg normalized the disequilibrium of matrix metalloproteinases(MMPs)/tissue inhibitors of MMPs(TIMPs) in hypertrophic hearts, which might contribute to the protective role of PC-Tg in cardiac fibrosis and, thus, protect against cardiac remodeling. Taken together, our results identify a novel anti-hypertrophic role for the PON gene cluster, suggesting a possible strategy for the treatment of cardiac hypertrophy through elevating the levels of the PON gene family.
