Caihua Dong

Expressing a gene encoding wheat oxalate oxidase enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus).

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO2 and H2O2. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat (Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H2O2 levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum.

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana

BackgroundPlant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana.ResultsHere we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species.ConclusionThis study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Overexpression of Brassica napus MPK4 Enhances Resistance to Sclerotinia sclerotiorum in Oilseed Rape

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus) resulting in significant economic losses. Studies on the Arabidopsis thaliana MPK4 loss-of-function mutant have implicated that AtMPK4 is involved in plant defense regulation, and its effect on disease resistance varies in different plant-pathogen interactions. In this study, we isolated a B. napus mitogen-activated protein kinase, BnMPK4, and found that BnMPK4 along with PDF1.2 are inducible in resistant line Zhongshuang9 but both are consistently suppressed in susceptible line 84039 after inoculation with S. sclerotiorum. Transgenic oilseed rape overexpressing BnMPK4 markedly enhances resistance to S. sclerotiorum and Botrytis cinerea. Further experiments showed that transgenic plants inhibited growth of S. sclerotiorum and constitutively activated PDF1.2 but decreased H2O2 production and constitutively suppressed PR-1 expression. Treatment of roots of the transgenic plants with H2O2 solution resulted in enhanced susceptibility to the two pathogens. Our results support the idea that MPK4 positively regulates jasmonic acid-mediated defense response, which might play an important role in resistance to S. sclerotiorum in oilseed rape.

Brassica GLABRA2 genes: analysis of function related to seed oil content and development of functional markers.

Regulation of seed oil accumulation in oilseed rape (Brassica napus) has important economic significance. However, few genes have been characterized that affect final seed oil content. Through a mutant identification, the class IV homeodomain-ZIP transcription factor GLABRA2 (GL2) has been found to regulate seed oil accumulation in Arabidopsis, in addition to its role in trichome development. In this study, we isolated four distinct orthologues of GL2 from B. napus (AC-genome), B. rapa (A) and B. oleracea (C), using an overlapping-PCR strategy. The four GL2 orthologues were very similar, with 96.10–99.69% identity in exon regions, 75.45–93.84% in intron regions, 97.34–99.87% in amino acid sequences. Alignments of the four genes revealed that the A-genome sequences of BnaA.GL2.a from B. napus and BraA.GL2.a from B. rapa are more similar than the others, and likewise the C-genome sequences of BnaC.GL2.b from B. napus and BolC.GL2.a from B. oleracea are more similar. BnaA.GL2.a and BraA.GL2.a from the A-genome are highly expressed in roots, whilst BnaC.GL2.b and BolC.GL2.a from the C-genome are preferentially expressed in seeds. Transgenic ectopic overexpression and suppression of BnaC.GL2.b in Arabidopsis allowed further investigation of the effect on seed oil content. Overexpression generated two phenotypes: the wild-type-like and the gl2-mutant-like (an Arabidopsis glabrous mutant of gl2-2), with increases in seed oil content of 3.5–5.0% in the gl2-mutant-like transgenic plants. Suppression resulted in increases of 2.5–6.1% in seed oil content, and reduced trichome number at the leaf margins. These results suggest that BnaC.GL2.b can negatively regulate oil accumulation in Arabidopsis seeds. As a result of comparing the four GL2 genes, three A/C-genome-specific primer sets were developed and a C-genome-specific EcoRV cleavage site was identified, which can be used as functional markers to distinguish these orthologues within Brassica species. The genes identified and their molecular markers developed in this study will be valuable both for oilseed rape breeding focusing on improvement of seed oil content, and for detecting gene flow between populations.

A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes

BackgroundTo facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.ResultsTwo or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.ConclusionsThe method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.

Analysis of expression sequence tags from a full-length-enriched cDNA library of developing sesame seeds (Sesamum indicum)

BackgroundSesame (Sesamum indicum) is one of the most important oilseed crops with high oil contents and rich nutrient value. However, genetic improvement efforts in sesame could not get benefit from molecular biology technology due to poor DNA and RNA sequence resources. In this study, we carried out a large scale of expressed sequence tags (ESTs) sequencing from developing sesame seeds and further conducted analysis on seed storage products-related genes.ResultsA normalized and full-length enriched cDNA library from 5 ~ 30 days old immature seeds was constructed and randomly sequenced, leading to generation of 41,248 expressed sequence tags (ESTs) which then formed 4,713 contigs and 27,708 singletons with 44.9% uniESTs being putative full-length open reading frames. Approximately 26,091 of all these uniESTs have significant matches to the counterparts in Nr database of GenBank, and 21,628 of them were assigned to one or more Gene ontology (GO) terms. Homologous genes involved in oil biosynthesis were identified including some conservative transcription factors regulating oil biosynthesis such as LEAFY COTYLEDON1 (LEC1), PICKLE (PKL), WRINKLED1 (WRI1) and majority of them were found for the first time in sesame seeds. One hundred and 17 ESTs were identified possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin. In total, 9,347 putative functional genes from developing seeds were identified, which accounts for one third of total genes in the sesame genome. Further analysis of the uniESTs identified 1,949 non-redundant simple sequence repeats (SSRs).ConclusionsThis study has provided an overview of genes expressed during sesame seed development. This collection of sesame full-length cDNAs covered a wide variety of genes in seeds, in particular, candidate genes involved in biosynthesis of sesame oils and lignans. These EST sequences enriched with full length will contribute to comparative genomic studies on sesame and other oilseed plants and serve as an abundant information platform for functional marker development and functional gene study.

Cysteine Protease 51 (CP51), an anther-specific cysteine protease gene, is essential for pollen exine formation in Arabidopsis

Cysteine proteases play important roles in intracellular protein degradation, programmed cell death and responses to environmental stimuli in plant. Although subclassification and biochemical analysis of major plant papain-like cysteine proteases (PLCPs) have been studied, the biological function of many PLCPs remained unknown. In this study, we identified a PLCP gene Cysteine Protease 51 (CP51) which participates in exine formation and anther development in Arabidopsis thaliana. Promoter-GUS fusion detection showed its specific expression in anthers at stages 9–12. RNA interference (RNAi) transgenic plants with reduced CP51 transcriptional levels exhibited a male sterile phenotype with aborted microspores, shortened siliques and fewer or no seeds. Cytological analysis indicated that pollen abortion occurred due to defective pollen exine and the tapetum degraded earlier during the transition from the uninucleated stage to the binucleated stage. Scanning electron microscopy demonstrated that aborted microspores lacked complete or normal reticulate exine, and the intine membrane was extruded in the pollens of CP51-RNAi plants. Transmission electron microscopy further revealed that the tapetum degeneration was initiated early and that normal tectum connections to the bacula were missing in anthers of CP51-RNAi plants. Taken together, these results suggested that CP51 critically mediates tapetum stability and pollen exine formation.

ocsESTdb: a database of oil crop seed EST sequences for comparative analysis and investigation of a global metabolic network and oil accumulation metabolism

BackgroundOil crop seeds are important sources of fatty acids (FAs) for human and animal nutrition. Despite their importance, there is a lack of an essential bioinformatics resource on gene transcription of oil crops from a comparative perspective. In this study, we developed ocsESTdb, the first database of expressed sequence tag (EST) information on seeds of four large-scale oil crops with an emphasis on global metabolic networks and oil accumulation metabolism that target the involved unigenes.DescriptionA total of 248,522 ESTs and 106,835 unigenes were collected from the cDNA libraries of rapeseed (Brassica napus), soybean (Glycine max), sesame (Sesamum indicum) and peanut (Arachis hypogaea). These unigenes were annotated by a sequence similarity search against databases including TAIR, NR protein database, Gene Ontology, COG, Swiss-Prot, TrEMBL and Kyoto Encyclopedia of Genes and Genomes (KEGG). Five genome-scale metabolic networks that contain different numbers of metabolites and gene–enzyme reaction–association entries were analysed and constructed using Cytoscape and yEd programs. Details of unigene entries, deduced amino acid sequences and putative annotation are available from our database to browse, search and download. Intuitive and graphical representations of EST/unigene sequences, functional annotations, metabolic pathways and metabolic networks are also available. ocsESTdb will be updated regularly and can be freely accessed at http://ocri-genomics.org/ocsESTdb/.ConclusionocsESTdb may serve as a valuable and unique resource for comparative analysis of acyl lipid synthesis and metabolism in oilseed plants. It also may provide vital insights into improving oil content in seeds of oil crop species by transcriptional reconstruction of the metabolic network.