Detection performance of PCR for Legionella pneumophila in environmental samples: a systematic review and meta-analysis.

BACKGROUND: Legionellosis remains a public health problem. The most common diagnostic method to detect Legionella pneumophila (L. pneumophila) is culture. Polymerase chain reaction (PCR) is a fast and accurate method for this detection in environmental samples. METHODS: Four databases were searched for studies that evaluated the detection efficiency of PCR in L. pneumophila. The quality evaluation was conducted using Review Manager 5.3. We used Meta-DiSc 1.4 software and the Stata 15.0 software to create forest plots, a meta-regression, a bivariate boxplot and a Deeks' funnel plot. RESULTS: A total of 18 four-fold tables from 16 studies were analysed. The overall pooled sensitivity and specificity of PCR was 94% and 72%, respectively. The positive likelihood ratio (RLR) and negative likelihood ratio (NLR) was 2.73 and 0.12, respectively. The result of the diagnostic odds ratio (DOR) was 22.85 and the area under the curve (AUC) was 0.7884. CONCLUSION: Establishing a laboratory diagnostic tool for L. pneumophila detection is important for epidemiological studies. In this work, PCR demonstrated a promising diagnostic accuracy for L. pneumophila.

Genome-Wide Characterization of High-Affinity Nitrate Transporter 2 (NRT2) Gene Family in Brassica napus.

Nitrate transporter 2 (NRT2) plays an essential role in Nitrogen (N) uptake, transport, utilization, and stress resistance. In this study, the NRT2 gene family in two sequenced Brassica napus ecotypes were identified, including 31 genes in 'Zhongshuang11' (BnaZSNRT2s) and 19 in 'Darmor-bzh' (BnaDarNRT2s). The candidate genes were divided into three groups (Group I-III) based on phylogenetic analyses, supported by a conserved intron-exon structure in each group. Collinearity analysis revealed that the large expansion of BnaZSNRT2s attributed to allopolyploidization of ancestors Brassica rapa and Brassica oleracea, and small-scale duplication events in B. napus. Transcription factor (TF) binding site prediction, cis-element analysis, and microRNA prediction suggested that the expressions of BnaZSNRT2s are regulated by multiple factors, and the regulatory pattern is relatively conserved in each group and is tightly connected between groups. Expression assay showed the diverse and differentiated spatial-temporal expression profiles of BnaZSNRT2s in Group I, but conserved patterns were observed in Group II/III; and the low nitrogen (LN) stress up-regulated expression profiles were presented in Group I-III, based on RNA-seq data. RT-qPCR analyses confirmed that BnaZSNRT2.5A-1 and BnaZSNRT2.5C-1 in Group II were highly up-regulated under LN stress in B. napus roots. Our results offer valid information and candidates for further functional BnaZSNRT2s studies.

Genome-wide characterization, expression analyses, and functional prediction of the NPF family in Brassica napus.

BACKGROUND: NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family (NPF) members are essential transporters for many substrates in plants, including nitrate, hormones, peptides, and secondary metabolites. Here, we report the global characterization of NPF in the important oil crop Brassica napus, including that for phylogeny, gene/protein structures, duplications, and expression patterns. RESULTS: A total of 199 B. napus (BnaNPFs) NPF-coding genes were identified. Phylogenetic analyses categorized these genes into 11 subfamilies, including three new ones. Sequence feature analysis revealed that members of each subfamily contain conserved gene and protein structures. Many hormone-/abiotic stress-responsive cis-acting elements and transcription factor binding sites were identified in BnaNPF promoter regions. Chromosome distribution analysis indicated that BnaNPFs within a subfamily tend to cluster on one chromosome. Syntenic relationship analysis showed that allotetraploid creation by its ancestors (Brassica rapa and Brassica oleracea) (57.89%) and small-scale duplication events (39.85%) contributed to rapid BnaNPF expansion in B. napus. A genome-wide spatiotemporal expression survey showed that NPF genes of each Arabidopsis and B. napus subfamily have preferential expression patterns across developmental stages, most of them are expressed in a few organs. RNA-seq analysis showed that many BnaNPFs (32.66%) have wide exogenous hormone-inductive profiles, suggesting important hormone-mediated patterns in diverse bioprocesses. Homologs in a clade or branch within a given subfamily have conserved organ/spatiotemporal and hormone-inductive profiles, indicating functional conservation during evolution. qRT-PCR-based comparative expression analysis of the 12 BnaNPFs in the NPF2-1 subfamily between high- and low-glucosinolate (GLS) content B. napus varieties revealed that homologs of AtNPF2.9 (BnaNPF2.12, BnaNPF2.13, and BnaNPF2.14), AtNPF2.10 (BnaNPF2.19 and BnaNPF2.20), and AtNPF2.11 (BnaNPF2.26 and BnaNPF2.28) might be involved in GLS transport. qRT-PCR further confirmed the hormone-responsive expression profiles of these putative GLS transporter genes. CONCLUSION: We identified 199 B. napus BnaNPFs; these were divided into 11 subfamilies. Allopolyploidy and small-scale duplication events contributed to the immense expansion of BnaNPFs in B. napus. The BnaNPFs had preferential expression patterns in different tissues/organs and wide hormone-induced expression profiles. Four BnaNPFs in the NPF2-1 subfamily may be involved in GLS transport. Our results provide an abundant gene resource for further functional analysis of BnaNPFs.

Genome-wide survey of the bHLH super gene family in Brassica napus.

BACKGROUND: The basic helix-loop-helix (bHLH) gene family is one of the largest transcription factor families in plants and is functionally characterized in diverse species. However, less is known about its functions in the economically important allopolyploid oil crop, Brassica napus. RESULTS: We identified 602 potential bHLHs in the B. napus genome (BnabHLHs) and categorized them into 35 subfamilies, including seven newly separated subfamilies, based on phylogeny, protein structure, and exon-intron organization analysis. The intron insertion patterns of this gene family were analyzed and a total of eight types were identified in the bHLH regions of BnabHLHs. Chromosome distribution and synteny analyses revealed that hybridization between Brassica rapa and Brassica oleracea was the main expansion mechanism for BnabHLHs. Expression analyses showed that BnabHLHs were widely in different plant tissues and formed seven main patterns, suggesting they may participate in various aspects of B. napus development. Furthermore, when roots were treated with five different hormones (IAA, auxin; GA3, gibberellin; 6-BA, cytokinin; ABA, abscisic acid and ACC, ethylene), the expression profiles of BnabHLHs changed significantly, with many showing increased expression. The induction of five candidate BnabHLHs was confirmed following the five hormone treatments via qRT-PCR. Up to 246 BnabHLHs from nine subfamilies were predicted to have potential roles relating to root development through the joint analysis of their expression profiles and homolog function. CONCLUSION: The 602 BnabHLHs identified from B. napus were classified into 35 subfamilies, and those members from the same subfamily generally had similar sequence motifs. Overall, we found that BnabHLHs may be widely involved in root development in B. napus. Moreover, this study provides important insights into the potential functions of the BnabHLHs super gene family and thus will be useful in future gene function research.

Genome-Wide Survey and Expression Analysis of the KT/HAK/KUP Family in Brassica napus and Its Potential Roles in the Response to K+ Deficiency.

The KT/HAK/KUP (HAK) family is the largest potassium (K+) transporter family in plants, which plays key roles in K+ uptake and homeostasis, stress resistance, and root and embryo development. However, the HAK family has not yet been characterized in Brassica napus. In this study, 40 putative B. napus HAK genes (BnaHAKs) are identified and divided into four groups (Groups I-III and V) on the basis of phylogenetic analysis. Gene structure analysis revealed 10 conserved intron insertion sites across different groups. Collinearity analysis demonstrated that both allopolyploidization and small-scale duplication events contributed to the large expansion of BnaHAKs. Transcription factor (TF)-binding network construction, cis-element analysis, and microRNA prediction revealed that the expression of BnaHAKs is regulated by multiple factors. Analysis of RNA-sequencing data further revealed extensive expression profiles of the BnaHAKs in groups II, III, and V, with limited expression in group I. Compared with group I, most of the BnaHAKs in groups II, III, and V were more upregulated by hormone induction based on RNA-sequencing data. Reverse transcription-quantitative polymerase reaction analysis revealed that the expression of eight BnaHAKs of groups I and V was markedly upregulated under K+-deficiency treatment. Collectively, our results provide valuable information and key candidate genes for further functional studies of BnaHAKs.

Global Survey and Expressions of the Phosphate Transporter Gene Families in Brassica napus and Their Roles in Phosphorus Response.

Phosphate (Pi) transporters play critical roles in Pi acquisition and homeostasis. However, currently little is known about these genes in oil crops. In this study, we aimed to characterize the five Pi transporter gene families (PHT1-5) in allotetraploid Brassica napus. We identified and characterized 81 putative PHT genes in B. napus (BnaPHTs), including 45 genes in PHT1 family (BnaPHT1s), four BnaPHT2s, 10 BnaPHT3s, 13 BnaPHT4s and nine BnaPHT5s. Phylogenetic analyses showed that the largest PHT1 family could be divided into two groups (Group I and II), while PHT4 may be classified into five, Groups I-V. Gene structure analysis revealed that the exon-intron pattern was conservative within the same family or group. The sequence characteristics of these five families were quite different, which may contribute to their functional divergence. Transcription factor (TF) binding network analyses identified many potential TF binding sites in the promoter regions of candidates, implying their possible regulating patterns. Collinearity analysis demonstrated that most BnaPHTs were derived from an allopolyploidization event (~40.7%) between Brassica rapa and Brassica oleracea ancestors, and small-scale segmental duplication events (~39.5%) in the descendant. RNA-Seq analyses proved that many BnaPHTs were preferentially expressed in leaf and flower tissues. The expression profiles of most colinearity-pairs in B. napus are highly correlated, implying functional redundancy, while a few pairs may have undergone neo-functionalization or sub-functionalization during evolution. The expression levels of many BnaPHTs tend to be up-regulated by different hormones inductions, especially for IAA, ABA and 6-BA treatments. qRT-PCR assay demonstrated that six BnaPHT1s (BnaPHT1.11, BnaPHT1.14, BnaPHT1.20, BnaPHT1.35, BnaPHT1.41, BnaPHT1.44) were significantly up-regulated under low- and/or rich- Pi conditions in B. napus roots. This work analyzes the evolution and expression of the PHT family in Brassica napus, which will help further research on their role in Pi transport.

Evolutionary and Comparative Expression Analyses of TCP Transcription Factor Gene Family in Land Plants.

The plant-specific Teosinte-branched 1/Cycloidea/Proliferating (TCP) transcription factor genes are involved in plants' development, hormonal pathways, and stress response but their evolutionary history is uncertain. The genome-wide analysis performed here for 47 plant species revealed 535 TCP candidates in terrestrial plants and none in aquatic plants, and that TCP family genes originated early in the history of land plants. Phylogenetic analysis divided the candidate genes into Classes I and II, and Class II was further divided into CYCLOIDEA (CYC) and CINCINNATA (CIN) clades; CYC is more recent and originated from CIN in angiosperms. Protein architecture, intron pattern, and sequence characteristics were conserved in each class or clade supporting this classification. The two classes significantly expanded through whole-genome duplication during evolution. Expression analysis revealed the conserved expression of TCP genes from lower to higher plants. The expression patterns of Class I and CIN genes in different stages of the same tissue revealed their function in plant development and their opposite effects in the same biological process. Interaction network analysis showed that TCP proteins tend to form protein complexes, and their interaction networks were conserved during evolution. These results contribute to further functional studies on TCP family genes.

Global Analysis of WOX Transcription Factor Gene Family in Brassica napus Reveals Their Stress- and Hormone-Responsive Patterns.

The plant-specific WUSCHEL-related homeobox (WOX) transcription factor gene family is important for plant growth and development but little studied in oil crops. We identified and characterized 58 putative WOX genes in Brassica napus (BnWOXs), which were divided into three major clades and nine subclades based on the gene structure and conserved motifs. Collinearity analysis revealed that most BnWOXs were the products of allopolyploidization and segmental duplication events. Gene structure analysis indicated that introns/exons and protein motifs were conserved in each subclade and RNA sequencing revealed that BnWOXs had narrow expression profiles in major tissues and/or organs across different developmental stages. The expression pattern of each clade was highly conserved and similar to that of the sister and orthologous pairs from Brassica rapa and Brassica oleracea. Quantitative real-time polymerase chain reaction showed that members of the WOX4 subclade were induced in seedling roots by abiotic and hormone stresses, indicating their contribution to root development and abiotic stress responses. 463 proteins were predicted to interact with BnWOXs, including peptides regulating stem cell homeostasis in meristems. This study provides insights into the evolution and expression of the WOX gene family in B. napus and will be useful in future gene function research.

[GC-MS analysis of volatile aromatic substances in Chaenomeles speciosa fruit].

OBJECTIVE: To analyze the volatile aromatic substances in Chaenomeles speciosa fruit produced in Chongqing in order to provide the characteristic data for it's resources development and flavors chemistry research. METHODS: The volatile aromatic substances were extracted by steam distillation from Chaenomeles speciosa fresh fruit and seperated and identified by GC-MS. RESULTS: 106 volatile aromatic substances were seperated and identified, mainly including esters, alcohols, carboxylic acids, alkanes and alkenes, ketones, which made a great contribution to flavor of Chaenomeles speciosa fresh fruit. CONCLUSION: This study elucidated the composition of volatile aromatic substances in Chaenomeles speciosa fruit produced in Chongqing, which could provide basic information for exploitation and utilization of it's flavor substances.

Monoclonal antibodies against recombinant Der f 3 reveal localization of Der f 3 in the gut and faecal pellets of Dermatophagoides farinae.

Home dust mite derived materials are known to be a major source of problematic inhalant allergens. The aim of this study was to determine the localization of the group 3 allergen, Der f 3, within Dermatophagoides farinae, in order to assess the relative importance of excreted materials and nonexcreted body components as allergen sources. Recombinant Der f 3 (rDer f 3) was expressed in bacteria and purified as an immunogen for production of monoclonal antibodies (mAb) against it. Dermatophagoides farinae mites and their faecal pellets were embedded in paraffin, and serial sections were immunoprobed with mAb clone 3D3 against Der f 3. D. farinae midgut mucosa, gut contents and faecal pellets were strongly immunopositive for Der f 3. Der f 3 immunoreactive products were not detected in any other internal organs of the mite. These results suggest that Der f 3 allergen may be synthesized in and secreted from the digestive tract and excreted from the mite's body in the faecal pellets.

Genome-wide identification and expression profiling of the carotenoid cleavage dioxygenase (CCD) gene family in Brassica napus L.

Carotenoid cleavage dioxygenase (CCD), a key enzyme in carotenoid metabolism, cleaves carotenoids to form apo-carotenoids, which play a major role in plant growth and stress responses. CCD genes had not previously been systematically characterized in Brassica napus (rapeseed), an important oil crop worldwide. In this study, we identified 30 BnCCD genes and classified them into nine subgroups based on a phylogenetic analysis. We identified the chromosomal locations, gene structures, and cis-promoter elements of each of these genes and performed a selection pressure analysis to identify residues under selection. Furthermore, we determined the subcellular localization, physicochemical properties, and conserved protein motifs of the encoded proteins. All the CCD proteins contained a retinal pigment epithelial membrane protein (RPE65) domain. qRT-PCR analysis of expression of 20 representative BnCCD genes in 16 tissues of the B. napus cultivar Zhong Shuang 11 ('ZS11') revealed that members of the BnCCD gene family possess a broad range of expression patterns. This work lays the foundation for functional studies of the BnCCD gene family.

Differential Alternative Splicing Genes and Isoform Regulation Networks of Rapeseed (Brassica napus L.) Infected with Sclerotinia sclerotiorum.

Alternative splicing (AS) is a post-transcriptional level of gene expression regulation that increases transcriptome and proteome diversity. How the AS landscape of rapeseed (Brassica napus L.) changes in response to the fungal pathogen Sclerotinia sclerotiorum is unknown. Here, we analyzed 18 RNA-seq libraries of mock-inoculated and S. sclerotiorum-inoculated susceptible and tolerant B. napus plants. We found that infection increased AS, with intron retention being the main AS event. To determine the key genes functioning in the AS response, we performed a differential AS (DAS) analysis. We identified 79 DAS genes, including those encoding splicing factors, defense response proteins, crucial transcription factors and enzymes. We generated coexpression networks based on the splicing isoforms, rather than the genes, to explore the genes' diverse functions. Using this weighted gene coexpression network analysis alongside a gene ontology enrichment analysis, we identified 11 modules putatively involved in the pathogen defense response. Within these regulatory modules, six DAS genes (ascorbate peroxidase 1, ser/arg-rich protein 34a, unknown function 1138, nitrilase 2, v-atpase f, and amino acid transporter 1) were considered to encode key isoforms involved in the defense response. This study provides insight into the post-transcriptional response of B. napus to S. sclerotiorum infection.

Identification and characterization of a novel delta6-fatty acid desaturase gene from Rhizopus nigricans.

Fatty acid composition of fungi is analysed through the gas chromatography technique. With specific activity a novel enzyme Delta6-fatty acid desaturase was screened and isolated from Rhizopus nigricans. In this study R. nigricans was identified as a fungal species that produced plentiful gamma-linolenic acid. A 1,475 bp full-length cDNA, designated as RnD6D here, with high homology to fungal Delta6-fatty acid desaturase genes was isolated from R. nigricans using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1,380 bp encoding a deduced polypeptide of 459 amino acids. Bioinformatics analysis characterized the putative RnD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, hydropathy profile and a cytochrome b5-like domain in the N-terminus. The corresponding genomic sequence of RnD6D was 1,689 bp with 4 introns, which was at least one intron more than other fungal Delta6-fatty acid desaturase genes. To elucidate the function of this novel putative desaturase, the coding sequence was expressed in Saccharomyces cerevisiae strain INVScl. A novel peak corresponding to gamma-linolenic acid methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The result demonstrated that the coding produced Delta6-fatty acid desaturase activity of RnD6D which led to the accumulation of gamma-linolenic acid. The functionally active RnD6D gene cloned here defined a novel Delta6-fatty acid desaturase from R. nigricans.

[QTL for seed germination rate and related physiological traits of Brassica napus L].

One hundred and eighty-three recombinant inbred lines from the cross between GH06 and P174 were used for genetic analysis of seed germination rate and physiological trait analysis of Brassica napus L. Composite interval mapping (CIM) was applied to identify QTL associated with seed germination rate (GR) of the seeds that stored for two years (STY), one year (SOY), and fresh seeds (FS), respectively. The activity of lipases, seed conductivity, reducing sugar content, total sugar content, and root vitality of STY and FS were investigated. The QTL for seed GR of various stored seeds were different. Three QTLs for STY were detected on the linkage group (LG) 9, 14, and 17. Two QTL for SOY were mined on LG 5 and 9. Two QTLs for FS were detected on LG 4 and 18. The germination rate of seeds from three years was significantly different, and the QTL of GR was not identical, which indicated that the seed germination was controlled by many loci. Furthermore, the seed germination rate was negatively correlated with seed conductivity, which means that measurement of seed conductivity can be used to estimate GR, and the study of conductivity is important for GR research.

Brassica napus Infected with Leptosphaeria maculans.

Alternative splicing (AS) is a post-transcriptional regulatory process that enhances transcriptome diversity, thereby affecting plant growth, development, and stress responses. To identify the new transcripts and changes in the isoform-level AS landscape of rapeseed (Brassica napus) infected with the fungal pathogen Leptosphaeria maculans, we compared eight RNA-seq libraries prepared from mock-inoculated and inoculated B. napus cotyledons and stems. The AS events that occurred in stems were almost the same as those in cotyledons, with intron retention representing the most common AS pattern. We identified 1892 differentially spliced genes between inoculated and uninoculated plants. We performed a weighted gene co-expression network analysis (WGCNA) to identify eight co-expression modules and their Hub genes, which are the genes most connected with other genes within each module. There are nine Hub genes, encoding nine transcription factors, which represent key regulators of each module, including members of the NAC, WRKY, TRAF,AP2/ERF-ERF, C2H2,C2C2-GATA, HMG, bHLH, and C2C2-CO-like families. Finally, 52 and 117 alternatively spliced genes in cotyledons and stems were also differentially expressed between mock-infected and infected materials, such as HMG and C2C2-Dof; which have dual regulatory mechanisms in response to L. maculans. The splicing of the candidate genes identified in this study could be exploited to improve resistance to L. maculans.

Identification and Characterization of Major Constituents in Different-Colored Rapeseed Petals by UPLC-HESI-MS/MS.

Oilseed rape (Brassica napus L.) is the second highest yielding oil crop worldwide. In addition to being used as an edible oil and a feed for livestock, rapeseed has high ornamental value. In this study, we identified and characterized the main floral major constituents, including phenolic acids and flavonoids components, in rapeseed accessions with different-colored petals. A total of 144 constituents were identified using ultrahigh-performance liquid chromatography-HESI-mass spectrometry (UPLC-HESI-MS/MS), 57 of which were confirmed and quantified using known standards and mainly contained phenolic acids, flavonoids, and glucosinolates compounds. Most of the epicatechin, quercetin, and isorhamnetin derivates were found in red and pink petals of B. napus, while kaempferol derivates were in yellow and pale white petals. Moreover, petal-specific compounds, including a putative hydroxycinnamic acid derivative, sinapoyl malate, 1-O-sinapoyl-ß-d-glucose, feruloyl glucose, naringenin-7-O-glucoside, cyanidin-3-glucoside, cyanidin-3,5-di-O-glucoside, petunidin-3-O-ß-glucopyranoside, isorhamnetin-3-O-glucoside, kaempferol-3-O-glucoside-7-O-glucoside, quercetin-3,4'-O-di-ß-glucopyranoside, quercetin-3-O-glucoside, and delphinidin-3-O-glucoside, might contribute to a variety of petal colors in B. napus. In addition, bound phenolics were tentatively identified and contained three abundant compounds (p-coumaric acid, ferulic acid, and 8-O-4'-diferulic acid). These results provide insight into the molecular mechanisms underlying petal color and suggest strategies for breeding rapeseed with a specific petal color in the future.

Genome-Wide Identification and Expression Profiling of Cytokinin Oxidase/Dehydrogenase (CKX) Genes Reveal Likely Roles in Pod Development and Stress Responses in Oilseed Rape (Brassica napus L.).

Cytokinin oxidase/dehydrogenases (CKXs) play a critical role in the irreversible degradation of cytokinins, thereby regulating plant growth and development. Brassica napus is one of the most widely cultivated oilseed crops worldwide. With the completion of whole-genome sequencing of B. napus, genome-wide identification and expression analysis of the BnCKX gene family has become technically feasible. In this study, we identified 23 BnCKX genes and analyzed their phylogenetic relationships, gene structures, conserved motifs, protein subcellular localizations, and other properties. We also analyzed the expression of the 23 BnCKX genes in the B. napus cultivar Zhong Shuang 11 ('ZS11') by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), revealing their diverse expression patterns. We selected four BnCKX genes based on the results of RNA-sequencing and qRT-PCR and compared their expression in cultivated varieties with extremely long versus short siliques. The expression levels of BnCKX5-1, 5-2, 6-1, and 7-1 significantly differed between the two lines and changed during pod development, suggesting they might play roles in determining silique length and in pod development. Finally, we investigated the effects of treatment with the synthetic cytokinin 6-benzylaminopurine (6-BA) and the auxin indole-3-acetic acid (IAA) on the expression of the four selected BnCKX genes. Our results suggest that regulating BnCKX expression is a promising way to enhance the harvest index and stress resistance in plants.

Molecular cloning of two genes encoding cinnamate 4-hydroxylase (C4H) from oilseed rape (Brassica napus).

Cinnamate 4-hydroxylase (C4H) is a key enzyme of phenylpropanoid pathway, which synthesizes numerous secondary metabolites to participate in development and adaption. Two C4H isoforms, the 2192-bp BnC4H-1 and 2108-bp BnC4H-2, were cloned from oilseed rape (Brassica napus). They both have two introns and a 1518-bp open reading frame encoding a 505-amino-acid polypeptide. BnC4H-1 is 57.73 kDa with an isoelectric point of 9.11, while 57.75 kDa and 9.13 for BnC4H-2. They share only 80.6% identities on nucleotide level but 96.6% identities and 98.4% positives on protein level. Showing highest homologies to Arabidopsis thaliana C4H, they possess a conserved p450 domain and all P450-featured motifs, and are identical to typical C4Hs at substrate-recognition sites and active site residues. They are most probably associated with endoplasmic reticulum by one or both of the N- and C-terminal transmembrane helices. Phosphorylation may be a necessary post-translational modification. Their secondary structures are dominated by alpha helices and random coils. Most helices locate in the central region, while extended strands mainly distribute before and after this region. Southern blot indicated about 9 or more C4H paralogs in B. napus. In hypocotyl, cotyledon, stem, flower, bud, young- and middle-stage seed, they are co-dominantly expressed. In root and old seed, BnC4H-2 is dominant over BnC4H-1, with a reverse trend in leaf and pericarp. Paralogous C4H numbers in Brassicaceae genomes and possible roles of conserved motifs in 5' UTR and the 2nd intron are discussed.

Cloning and molecular characterization of a functional flavonoid 3'-hydroxylase gene from Brassica napus.

A flavonoid 3'-hydroxylase (F3'H) gene, denoted BnF3'H-1, was cloned from oilseed rape (Brassica napus). The gene of 3038 base pairs (bp) contains 3 introns. The complementary DNA (cDNA) consists of 1820bp and has an open reading frame of 1536bp encoding a polypeptide of 511 amino acids with a molecular weight of 56.62kDa and an isoelectric point of 7.08. BnF3'H-1 shows high homology to known F3'H genes, especially F3'H from Arabidopsis thaliana. Untranslated regions (UTRs) may play important roles in regulating the expression of BnF3'H-1. Besides containing a Kozak sequence, the first 77-bp region is C-rich but G-poor, and the 26-bp 5'-UTR contains 3 sites of ACCACT-like sequences. Alternative polyadenylation in the 3'-UTR is adopted by this gene to generate heterogeneous transcripts. Conserved domain search and motif characterization identified BnF3'H-1 as a cytochrome P450. All F3'H-featured motifs, VVVAAS, GGEK and VDVKG, are unchanged in BnF3'H-1. The N-terminal signal peptide/anchor and 3 transmembrane helices were predicted in BnF3'H-1, and its subcellular localization is most probably at the endoplasmic reticulum. Since 16 phosphorylation sites could be predicted, phosphorylation may be a necessary post-translational modification of BnF3'H-1. The secondary structure is dominated by alpha-helices and random coils. Most helices are located in the middle region, while extended strands mainly intersperse in terminal regions. DNA gel blot analysis indicated that 2 different F3'H genes might exist in B. napus. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and RNA gel blot analysis showed that flowers have the highest F3'H expression, followed by pericarp and seed, and lower levels in some other organs. This species-featured expression pattern is in obedience to multiple functional roles that F3'H gene(s) play(s) in various organs of B. napus. The BnF3'H-1 coding region was expressed in Escherichia coli, and enzyme activity of the His-tagged protein was demonstrated by monitoring the conversion of the substrate naringenin using high-performance liquid chromatography (HPLC), suggesting that BnF3'H-1 is catalytically functional. RT-PCR analysis suggests that transcription level of the F3'H gene(s) is not the reason for the different seed colorations found in near-isogenic lines (black-seeded L1 and yellow-seeded L2) of B. napus.

[Cloning and expression of a delta6-fatty acid desaturase gene from Rhizopus stolonifer in Saccharomyces cervisiae].

Fatty acid composition of fungi is analysed through the gas chromatography( GC) technique. With specific activity a novel enzyme delta6-fatty acid desaturase was screened and isolated from Rhizopus stolonifer. In this study R. stolonifer was identified as a fungal species that produced plentiful gamma-linolenic acid. A 1475bp full-length cDNA, designated as RnD6D here, with high homology to fungal delta6-fatty acid desaturase genes was isolated from R. stolonifer using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1380bp encoding a deduced polypeptide of 459 amino acids. Bioinformatics analysis characterized the putative RnD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, hydropathy profile and a cytochrome b5-like domain in the N-terminus. To elucidate the function of this novel putative desaturase, the coding sequence was expressed in Saccharomyces cerevisiae strain INVScl. A novel peak corresponding to gamma-linolenic acid(GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The percentage of this new GLA was 12.25% of total fatty acids. The result demonstrated that the coding produced delta6-fatty acid desaturase activity of RnD6D which led to the accumulation of gamma-linolenic acid.