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Numerous studies have reported that abnormal cadherin 3 (CDH3) and microRNA (miRNA/miR)-665 expression can induce the progression of gastric cancer (GC). However, the mechanism of interaction between miR-665 and CDH3 in GC requires further investigation. The present study aimed to investigate the influence of miR-665 and CDH3 in GC development. The effect of miR-665 and CDH3 on GC tissues and cell lines was examined using reverse transcription-quantitative PCR. Subsequently, CDH3 protein expression in GC cell lines was detected using western blotting. To confirm the association between miR-665 and CDH3, a dual-luciferase reporter assay system was employed. Cell proliferation and adhesion were analyzed using BrdU ELISA, MTT and cell adhesion assays. Finally, caspase-3 activity assay kit and FITC apoptosis detection kit were used for the determination of apoptosis of GC cells. The current findings confirmed the upregulation of CDH3 expression in GC cell lines and tissues. Experimental results indicated that CDH3 overexpression increased cell proliferation and adhesion, but decreased the apoptosis level of the cells. Similarly, the miR-665 inhibitor enhanced cell proliferation and adhesion, but inhibited apoptosis of GC cells. Additionally, it was observed that CDH3 was a direct target of miR-665 in GC cells and that miR-665 inhibited CDH3 expression, thereby repressing the progression of GC. In conclusion, the present study suggested that by targeting CDH3, miR-665 suppressed the progression of GC. These findings may provide a significant theoretical basis for future GC clinical therapy.

A Gram-positive, aerobic, non-motile, non-spore-forming, short rod-shaped strain, NEAU-LLCT, was isolated from cow dung in Shangzhi city, Heilongjiang Province, northeast China and identified by a polyphasic taxonomic study. Colonies was light yellow, round, with entire margin. Strain NEAU-LLCT was grown at 15–45℃ and pH 6.0–10.0. NaCl concentration ranged from 0 to 5% (W/V). The 16S rRNA gene sequence of NEAU-LLCT showed the high similarities with Microbacterium kyungheense JCM 18735T (98.5%), Microbacterium trichothecenolyticum JCM 1358T (98.3%) and Microbacterium jejuense JCM 18734T (98.2%). The whole-cell sugars were glucose, rhamnose, and ribose. The menaquinones contained MK-12 and MK-13. Ornithine, glutamic acid, lysine, and a small amount of alanine and glycine were the amino acids in the hydrolyzed products of the cell wall. The major fatty acids were iso-C16:0, iso-C18:0, anteiso-C15:0 and anteiso-C17:0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The genome of NEAU-LLCT was 4,369,375 bp and G + C content is 70.28 mol%. A combination of DNA-DNA hybridization result and some phenotypic characteristics demonstrated that strain NEAU-LLCT could be distinguished from its closely related strains. Therefore, the strain NEAU-LLCT was considered to represent a novel specie and was named Microbacterium helvum sp. (Type strain NEAU-LLCT =CCTCC AA 2018026T = JCM 32661T).

Background: Gastric cancer (GC) is a malignant tumor of the digestive tract. Hypoxia plays an important role in the development of cancer, including GC. The present study aimed to investigate the role of circular RNA SLAMF6 (circSLAMF6) in the progression of GC under hypoxia. Methods: The expression of circSLAMF6, microRNA-204-5p (miR-204-5p) and myosin heavy chain 9 (MYH9) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). GC cells were maintained under hypoxia (1% O2) for experiments in vitro. Glucose consumption and lactate production were determined by a Glucose Assay Kit and a Lactate Assay Kit, respectively. Levels of all protein were detected by Western blot. Cell migration and invasion were examined by Transwell assay. The interaction between miR-204-5p and circSLAMF6 or MYH9 was analyzed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Murine xenograft model was established to explore the role of circSLAMF6 in vivo. Results: CircSLAMF6 expression was increased in GC cells under hypoxia. Hypoxia promoted glycolysis, migration, and invasion in GC cells, which were reversed by circSLAMF6 knockdown. CircSLAMF6 was validated as a miR-204-5p sponge, and MYH9 was a target of miR-204-5p. Functionally, miR-204-5p inhibitor weakened the inhibition of circSLAMF6 knockdown on GC cell progression under hypoxia. Besides, MYH9 depletion suppressed glycolysis, migration, and invasion in GC cells under hypoxia. Importantly, circSLAMF6 deficiency inhibited tumor growth in vivo by regulating the miR-204-5p/MYH9 axis. Conclusion: CircSLAMF6 was involved in glycolysis, migration, and invasion by regulating the miR-204-5p/MYH9 axis in GC cells under hypoxia.

Background Antibiotic resistance genes (ARGs) can spread among pathogens via horizontal gene transfer, resulting in imparities in their distribution even within the same species. Therefore, a pan-genome approach to analyzing resistomes is necessary for thoroughly characterizing patterns of ARGs distribution within particular pathogen populations. Software tools are readily available for either ARGs identification or pan-genome analysis, but few exist to combine the two functions. Results We developed Pan Resistome Analysis Pipeline (PRAP) for the rapid identification of antibiotic resistance genes from various formats of whole genome sequences based on the CARD or ResFinder databases. Detailed annotations were used to analyze pan-resistome features and characterize distributions of ARGs. The contribution of different alleles to antibiotic resistance was predicted by a random forest classifier. Results of analysis were presented in browsable files along with a variety of visualization options. We demonstrated the performance of PRAP by analyzing the genomes of 26 Salmonella enterica isolates from Shanghai, China. Conclusions PRAP was effective for identifying ARGs and visualizing pan-resistome features, therefore facilitating pan-genomic investigation of ARGs. This tool has the ability to further excavate potential relationships between antibiotic resistance genes and their phenotypic traits.

Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin.

Isochorismate synthase (ICS) is a crucial enzyme in the salicylic acid (SA) synthesis pathway. The full-length complementary DNA (cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site (MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction (qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography (HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.

In the present study, the effects and molecular mechanisms of thymoquinone (TQ) on colon cancer cells were investigated. Cell viability was determined using a Cell Counting Kit-8 assay, and the results revealed that treatment with TQ significantly decreased cell viability in COLO205 and HCT116 cells in a dose-dependent manner. TQ treatment additionally sensitized COLO205 and HCT116 cells to cisplatin therapy in a concentration-dependent manner. To investigate the molecular mechanisms of TQ action, western blot analysis was used to determine the levels of phosphorylated p65 and nuclear factor-κB (NF-κB)-regulated gene products vascular endothelial growth factor (VEGF), c-Myc and B-cell lymphoma 2 (Bcl-2). The results indicated that TQ treatment significantly decreased the level of phosphorylated p65 in the nucleus, which indicated the inhibition of NF-κB activation by TQ treatment. Treatment with TQ also decreased the expression levels of VEGF, c-Myc and Bcl-2. In addition, the inhibition of NF-κB activation with a specific inhibitor, pyrrolidine dithiocarbamate, potentiated the induction of cell death and caused a chemosensitization effect of TQ in colon cancer cells. Overall, the results of the present study suggested that TQ induced cell death and chemosensitized colon cancer cells by inhibiting NF-κB signaling.

The aim of the present study was to investigate the effect and molecular mechanism of tanshinone IIA (TSA) on colon cancer cells. Cell viability was determined using Cell Counting kit-8 assay and the results demonstrated that TSA treatment significantly decreased the cell viability of HCT1116 and COLO205 cells in a dose-dependent manner. TSA treatment also sensitized HCT1116 and COLO205 cells to fluorouracil therapy in a concentration-dependent manner. Western blotting was performed in order to investigate the molecular mechanisms of TSA action and determine the level of phosporylated p65 and nuclear factor-κB (NF-κB)-regulated genes, including vascular endothelial growth factor (VEGF), c-Myc, cyclooxygenase-2 (COX-2) and B-cell lymphoma-2 (Bcl-2). The results revealed that TSA treatment greatly decreased the level of phosphorylated p65 in the nucleus, which indicated the inhibition of NF-κB activation by TSA treatment. TSA also decreased the expression levels of VEGF, c-Myc, COX-2 and Bcl-2. Furthermore, the inhibition of NF-κB activation with the specific inhibitor, pyrrolidine dithiocarbamate, increased the induction of cell death and chemosensitization effect of TSA in colon cancer cells. In conclusion, these results suggest that TSA induces cell death and chemosensitizes colon cancer cells through the suppression of NF-κB signaling.

The yft1 tomato mutant has a yellow-fruited phenotype controlled by a recessive gene of YFT1 allele, which has been shown by map-based cloning to be a homolog of ETHYLENE INSENSITIVE 2 (EIN2). Genetic lesion of YFT1 allele in yft1 is attributed to a 573 bp DNA fragment (IF573) insertion at 1,200 bp downstream of the transcription start site. Transcriptomic analysis revealed that YFT1 lesion resulted in 5,053 differentially expressed genes (DEGs) in yft1 pericarp compared with the M82 wild type cultivar. These were annotated as being involved in ethylene synthesis, chromoplast development, and carotenoid synthesis. The YFT1 lesion caused a reduction in its own transcript levels in yft1 and impaired ethylene emission and signal transduction, delayed chromoplast development and decreased carotenoid accumulation. The molecular mechanism underlying the downregulated YFT1 allele in yft1 was examined at both RNA and DNA levels. The IF573 event appeared to introduce two negative regulatory sequences located at -272 to -173 bp and -172 to -73 bp in the YFT1 allele promoter, causing alterative splicing due to introduction of aberrant splicing sites, and breaking upstream open reading frames (uORF) structure in the 5'-UTR. Those results a new provided insight into molecular regulation of color formation in tomato fruit.

Drought is one of the most severe abiotic factors restricting plant growth and yield. Numerous genes functioning in drought response are regulated by abscisic acid (ABA) dependent and independent pathways, but knowledge of interplay between the two pathways is still limited. Here, we integrated transcriptome sequencing and network analyses to explore interplays between ABA-dependent and ABA-independent pathways responding to drought stress in Arabidopsis thaliana. We identified 211 ABA-dependent differentially expressed genes (DEGs) and 1,118 ABA-independent DEGs under drought stress. Functional analysis showed that ABA-dependent DEGs were significantly enriched in expected biological processes in response to water deprivation and ABA stimulus, while ABA-independent DEGs were preferentially enriched in response to jasmonic acid (JA), salicylic acid (SA) and gibberellin (GA) stimuli. We found significantly enriched interactions between ABA-dependent and ABA-independent pathways with 94 genes acting as core interacting components by combining network analyses. A link between ABA and JA signaling mediated through a direct interaction of the ABA responsive elements-binding factor ABF3 with the basic helix-loop-helix transcription factor MYC2 was validated by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Our study provides a systematic view of the interplay between ABA-dependent and ABA-independent pathways in response to drought stress.

Phenylalanine ammonia-lyase (PAL) is the key enzyme in the biosynthetic pathway of salicylic acid (SA). In this study, a full-length cDNA of PAL gene (named as AaPAL1) was cloned from Artemisia annua. The gene contains an open reading frame of 2,151 bps encoding 716 amino acids. Comparative and bioinformatics analysis revealed that the polypeptide protein of AaPAL1 was highly homologous to PALs from other plant species. Southern blot analysis revealed that it belonged to a gene family with three members. Quantitative RT-PCR analysis of various tissues of A. annua showed that AaPAL1 transcript levels were highest in the young leaves. A 1160-bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including W-box, TGACG-motif, and TC-rich repeats. Quantitative RT-PCR indicated that AaPAL1 was upregulated by salinity, drought, wounding, and SA stresses, which were corroborated positively with the identified cis-elements within the promoter region. AaPAL1 was successfully expressed in Escherichia. coli and the enzyme activity of the purified AaPAL1 was approximately 287.2 U/mg. These results substantiated the involvement of AaPAL1 in the phenylalanine pathway.

Silique shattering resistance is one of the most important agricultural traits in oil crop breeding. Seed shedding from siliques prior to and during harvest causes devastating losses in oilseed yield. Lignin biosynthesis in the silique walls is thought to affect silique-shattering resistance in oil crops. Here, we identified and characterized B. napus LATE FLOWERING (BnLATE), which encodes a Cys2/His2-type zinc-finger protein. Heterologous expression of BnLATE under the double enhanced CaMV 35S promoter (D35S) in wild-type Arabidopsis plants resulted in a marked decrease in lignification in the replum, valve layer (carpel) and dehiscence zone. pBnLATE::GUS activity was strong in the yellowing silique walls of transgenic lines. Furthermore, the expression pattern of BnLATE and the lignin content gradient in the silique walls at 48 days after pollination (DAP) of 73290, a B. napus silique shattering-resistant line, are similar to those in transgenic Arabidopsis lines expressing BnLATE. Transcriptome sequencing of the silique walls revealed that genes encoding peroxidases, which polymerize monolignols and lignin in the phenylpropanoid pathway, were down-regulated at least two-fold change in the D35S::BnLATE transgenic lines. pBnLATE::BnLATE transgenic lines were further used to identify the function of BnLATE, and the results showed that lignification in the carpel and dehiscence zone of yellowing silique also remarkably decreased compared with the wild-type control, the silique shattering-resistance and expression pattern of peroxidase genes are very similar to results with D35S::BnLATE. These results suggest that BnLATE is a negative regulator of lignin biosynthesis in the yellowing silique walls, and promotes silique-shattering resistance in B. napus through restraining the polymerization of monolignols and lignin.

Cryptochromes (CRY) are blue-light photoreceptors that mediate various light responses in plants and animals. It has long been demonstrated that Arabidopsis CRY (CRY1 and CRY2) C termini (CCT1 and CCT2) mediate light signaling through direct interaction with COP1. Most recently, CRY1 N terminus (CNT1) has been found to be involved in CRY1 signaling independent of CCT1, and implicated in the inhibition of gibberellin acids (GA)/brassinosteroids (BR)/auxin-responsive gene expression. Here, we performed RNA-Seq assay using transgenic plants expressing CCT1 fused to β-glucuronidase (GUS-CCT1, abbreviated as CCT1), which exhibit a constitutively photomorphogenic phenotype, and compared the results with those obtained previously from cry1cry2 mutant and the transgenic plants expressing CNT1 fused to nuclear localization signal sequence (NLS)-tagged YFP (CNT1-NLS-YFP, abbreviated as CNT1), which display enhanced responsiveness to blue light. We found that 2,903 (67.85％) of the CRY-regulated genes are regulated by CCT1 and that 1,095 of these CCT1-regulated genes are also regulated by CNT1. After annotating the gene functions, we found that CCT1 is involved in mediating CRY1 regulation of phytohormone-responsive genes, like CNT1, and that about half of the up-regulated genes by GA/BR/auxin are down-regulated by CCT1 and CNT1, consistent with the antagonistic role for CRY1 and these phytohormones in regulating hypocotyl elongation. Physiological studies showed that both CCT1 and CNT1 are likely involved in mediating CRY1 reduction of seedlings sensitivity to GA under blue light. Furthermore, protein expression studies demonstrate that the inhibition of GA promotion of HY5 degradation by CRY1 is likely mediated by CCT1, but not by CNT1. These results give genome-wide transcriptome information concerning the signaling mechanism of CRY1, unraveling possible involvement of its C and N termini in its regulation of response of GA and likely other phytohormones.

Using a comparative genomic method, 38 and 8 fragments were verified as specific identification targets for Salmonella Typhimurium and Enteritidis, respectively. Primer sets were designed based on these sequences and evaluated by PCR assays. Two primer sets targeting the STM4495 and SEN1392 genes with high specificity were selected for S. Typhimurium and Enteritidis, respectively, and a multiplex PCR method was developed for the identification of these bacteria based on these two primer sets and another primer set targeting the srfC gene specific for Salmonella enterica. The multiplex PCR also included an internal amplification control (IAC), which was constructed by psy gene from Chlorella protothecoides for process control to monitor potential PCR inhibitors. The sensitivity of this multiplex PCR was 89 fg and 138 fg of DNA per PCR for S. Typhimurium and S. Enteritidis, respectively. The detection limits were as low as 22–23 CFU per PCR for pure cultures of both serovars. Moreover, positive results were observed from milk samples that were initially contaminated with 2–3 CFU Salmonella after 12 h of enrichment at 37 °C. These results demonstrate that the comparative genomic method is a valuable tool for identifying new specific targets, a necessary step for developing rapid and accurate detection methods for foodborne pathogens.

Artemisinin has attracted interest due to its medicinal value in treating malaria and its potential for use against certain cancers and viral diseases. Trichome density and capacity determine artemisinin content in Artemisia annua plants. Thus, the ATP-binding cassette transporter G (ABCG) subfamily involved in trichome cuticle development may also influence artemisinin accumulation. In this study, putative A. annua ABC transporter unigenes were identified and classified from the unigene sequences up to date in the National Center for Biotechnology Information database, and nine putative A. annua ABCG transporter unigenes that may be involved in cuticle development were selected for expression analyses. Two of them, AaABCG6 and AaABCG7, showed parallel expression pattern as two artemisinin biosynthesis-specific genes (amorpha-4, 11-diene synthase and a cytochrome P450-dependent hydroxylase, CYP71AV1) in different tissues and different leaf development stages and also showed similar induction in the plants after methyl jasmonate or abscisic acid treatments. Identification of these putative A. annua ABCG transporter unigenes could provide the basis for cloning of the full-length genes and further functional investigation to find the artemisinin relevant transporters, which could be used for improving artemisinin yield in both A. annua plants and heterologous systems using transgenic technology.

The AP2/ERF-domain transcription factor ORCA2 from Catharanthus roseus was demonstrated earlier to regulate the expressions of Str gene, an important gene involved in the terpenoid indole alkaloids biosynthetic pathway in C. roseus cells. Therefore, the factor was postulated to play an important role in the production of secondary metabolites in plants. To investigate the effect of over expression of ORCA2 on the TIAs biosynthesis in C. roseus hair roots, transformation of ORCA2 gene was conducted with the disarmed Agrobacterium rhizogenes C58C1 harboring pCAMBIA1304+, a plasmid that contains the Orca2 gene, a Gus gene and an Hpt gene all under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Transgenic hairy root cultures expressing Orca2 gene were obtained and demonstrated by genomic- polymerase chain reaction (PCR) analysis for the integration of the Orca2 gene in the C. roseus genome, by real-time quantitative PCR (RT-QPCR) and β-glucuronidase (GUS) staining for the expression of the foreign genes. Metabolite analysis using high performance liquid chromatography(HPLC) analysis established that the average content of catharanthine and vindoline in the transgenic hairy root extracts was increased up to 2.03 and 3.67-fold in comparison to the control lines, respectively. However, vinblastine could not been detected in the transgenic and control hairy root cultures by HPLC. Key words: Catharanthus roseus, ORCA2, hairy root, overexpression, terpenoid indole alkaloids (TIAs), AP2/ERF-domain transcription factor.

A 5'-nuclease real-time PCR assay using a minor groove binding probe was developed for the detection of Salmonella enterica from artificially contaminated foods. S. enterica-specific sequences were identified by a comparative genomic approach. Several species-specific target sequences were evaluated for specificity. A real-time PCR assay was developed targeting a nucleotide sequence within the putative type III secretion ATP synthase gene (ssaN). An internal amplification control (IAC) probe was designed by randomly shuffling the target probe sequence and a single-stranded oligonucleotide was synthesized to serve as an IAC. The assay demonstrated 100% inclusivity for the 40 Salmonella strains tested and 100% exclusivity for 24 non-Salmonella strains. The detection limit of the real-time PCR assay was 41.2 fg/PCR with Salmonella Typhimurium genomic DNA and 18.6 fg/PCR using Salmonella Enteritidis genomic DNA; 8 and 4 genome equivalents, respectively. In the presence of a natural background flora derived from chicken meat enrichment cultures, the sample preparation and PCR method were capable of detecting as few as 130 Salmonella cfu/mL. Using the developed real-time PCR method to detect Salmonella in artificially contaminated chicken, liquid egg and peanut butter samples, as few as 1 cfu/10 g of sample was detectable after a brief (6h) non-selective culture enrichment.

ERF transcription factors can bind GCC boxes or non-GCC cis elements to regulate biotic and abiotic stress responses. Here, we report that an ERF transcription factor gene (GbERF2) was cloned by suppression subtraction hybridization from sea-island cotton after Verticillium dahliae attack. The GbERF2 cDNA has a total length of 1143 bp with an open reading frame of 597 bp. The genomic sequence of GbERF2 contains an intron of 515 bp. The gene encodes a predicated polypeptide of 198 amino acids with a molecular weight of 22.5 kDa and a calculated pI of 9.82. The GbERF2 protein has a highly conserved ERF domain while the nucleotide and amino acid sequences have low homology with other ERF plant proteins. An RNA blot revealed that GbERF2 is constitutively expressed in different tissues, but is higher in the leaves. High levels of GbERF2 transcripts rapidly accumulated when the plants were exposed to exogenous ethylene treatment and V. dahliae infection, while there was only a slight accumulation in response to salt, cold, drought and water stresses. In contrast, GbERF2 transcripts declined in response to exogenous abscisic acid (ABA) treatment. GbERF2 transgenic tobacco plants constitutively accumulated higher levels of pathogenesis-related gene transcripts, such as PR-1b, PR2 and PR4. The resistance of transgenic tobacco to fungal infection by Alternaria longipes was enhanced. However, the resistance to bacterial infection by Pseudomonas syringae pv. tabaci was not improved. These results show that GbERF2 plays an important role in response to ethylene stress and fungal attack in cotton.

A novel cDNA clone encoding a COR413-like gene was isolated by suppression subtraction hybridization and cDNA library screening from sea-island cotton (Gossypium barbadense). This gene (designated as GbCOR413, Accession number: AY761065) has a total length of 893 bp with an open reading frame of 600 bp, encoding a predicated polypeptide of 200 amino acids with a molecular weight of 22.74 kDa and a predicated pI of 9.2. Bioinformatics analyses revealed that this gene belonged to a novel stress-regulated multi-spanning transmembrane protein family without signal peptide. By means of semi-quantities RT-PCR analysis, the expression of GbCOR413 under short-term cold treatment at 4°C, water submergence and abscic acid treatment was investigated. Our studies suggested that the cloned gene was a new member of COR gene family which was slowly responsive to cold stress in cotton.

A computer-based analysis was conducted to assess the characteristics of amino acid repeats in Arabidopsis and rice. Our analysis showed a negative gradient in amino acid repeat distribution along the direction of translation in plants. Repeat occurrences are obviously associated with position in plant proteins but are not consistent with the corresponding amino acid contents. These repeats are encoded by the mixed synonymous codons rather than the uninterrupted reiterations of a single codon, and both Arabidopsis and rice have gradients in their distribution. Functional investigation showed that these repeat-containing proteins are preferentially involved in transcription regulation and protein ubiquitination but significantly underrepresented in the processes of DNA recombination and DNA replication. These data reveal that the direction-related mutation bias and functional selection have influenced the distribution of amino acid repeats in plants.Key words: amino acid repeats, amino acid usage, distributional gradient, regulation of transcription, protein ubiquitination.

A full-length cDNA encoding taxadienol acetyl transferase (designated as TmTAT), that catalyzes the first acylation step in Taxol biosynthesis, was cloned from young leaves of Taxus x media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTAT was 1459 bp and contained a 1317-bp open reading frame (ORF) encoding a protein of 439 amino acids. The deduced protein had a calculated molecular weight of about 49 kDa and an isoelectric point (pI) of 5.3, similar to acetyl transferases from Taxus cuspidata and Taxus chinensis. Sequence comparison analysis revealed that the TmTAT had high similarity with other members of plant transferase family. Phylogenetic tree analysis showed that TmTAT had close relationship with acetyl transferases from T. chinenisis and T. cuspidata. Tissue expression pattern analysis revealed that TmTAT expressed strongly in leaves, weak in stems and no expression could be detected in fruits, indicating that TmTAT was a tissue-specific gene.

A full-length cDNA clone with high homology with Brassica napus peptide methionine sulfoxide reductase (PMSR) gene (BnPMSR), which could reverse oxidation of methionine residues into MetSO in protein, was cloned by suppression subtraction hybridization and cDNA library screening from Gossypium barbadense. This gene (named as cMsrA; Accession number: AY224208) had a total length of 1059 bp with an open reading frame of 765 bp, and encoded a predicted polypeptide of 255 amino acids with a molecular weight of 28.4 kDa. The cMsrA protein shared 68.6, 66.3 and 65.8% identity with those PMSR proteins isolated from B. napus, A. thaliana and L. sativa respectively. Expression patterns revealed that cMsrA was enriched later and weaker in resistant variety 7124 (Gossypium barbadense) than sensitive variety Ejing-1 (G. hirsutum) under salt treatment and pathogens attack. These results indicated that cMsrA played an important role in protecting the cells against oxidative damage during the pathogens and salt stress.

An AGAMOUS (AG)-like gene, GbAGL2, was isolated from Gossypium barbadense and characterized. Alignment and phylogenetic analysis indicated that GbAGL2 shared high homology with AG-subfamily genes and belonged to a C-class gene family. DNA gel blot analysis showed that GbAGL2 belonged to a low-copy gene family. Reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) revealed that GbAGL2 was highly expressed in reproductive tissues including ovules and carpels, but barely expressed in vegetative tissues. In addition, GbAGL2 expression in a cotton cultivar XuZhou142 (wt) (XZ142, G. hirsutum L.) and its fibreless mutant XZ142 (fl) was examined. RNA in situ hybridization analysis indicated that GbAGL2 transcripts were preferentially restricted to outer ovule integuments, carpels and fibres. These expression patterns implied that GbAGL2 might participate in the development of the carpel and ovule. Furthermore, Arabidopsis transformation was performed and modifications occurred in flowers, and the silique length of transgenic plants also increased slightly, suggesting that the GbAGL2 gene may have a positive effect on the development of the ovary or ovule. Our findings suggest that GbAGL2 might not only specify the identity of floral organs but also play a potential key role in ovary or fibre development in cotton.

The E8 gene is related to ethylene biosynthesis in plants. To explore the effect of the expression pattern of the E8 gene on different E8 promoters, the molecular evolution of E8 promoters was investigated. A total of 16 E8 promoters were cloned from 16 accessions of seven tomato species, and were further analysed. The results from 19 E8 promoters including three previously cloned E8 promoters (X13437, DQ317599 and AF515784) showed that the size of the E8 promoters varied from 2101 bp (LA2150) to 2256 bp (LA2192); their sequences shared 69.9% homology and the average A/T content was 74.9%. Slide-window analysis divided E8 promoters into three regions — A, B and C — and the sequence identity in these regions was 72.5%, 41.2% and 70.8%, respectively. By searching the cis-elements of E8 promoters in the PLACE database, mutant nucleotides were found in some functional elements, and deletions or insertions were also found in regions responsible for ethylene biosysnthesis (−1702 to −1274) and the negative effect region (−1253 to −936). Our results indicate that the size of the functional region for ethylene biosynthesis in the E8 promoter could be shortened from 429 bp to 113 bp (−1612 to −1500). The results of molecular evolution analysis showed that the 19 E8 promoters could be classified into four clade groups, which is basically consistent with evolution of the tomato genome. Southern blot analysis results showed that the copy number of E8 promoters in tomato and some other wild species changed from 1 to 4. Taken together, our study provides important information for further elucidating the E8 gene expression pattern in tomato, analysing functional elements in the E8 promoter and reconstructing the potent E8 promoter.

A new lipase gene designated as SlLipA was isolated from Serratia liquefaciens S33 DB-1 by the genomic-walking method. The cloned gene contained an open reading frame (ORF) of 1,845 bp encoding 615 amino acids with a conserved GXSXG motif. Genome sequence analysis showed that an aldo/keto reductase gene closed to the SlLipA gene. The lipase gene was cloned into the expression vector pPICZalphaA and successfully integrated into the heterologous host, methylotrophic yeast Pichia pastoris GS115. Five transformants could be expressed as secreted recombinant proteins with the high activity on Triglyceride-Agarose plate and as candidates to produce the recombinant enzyme. A C-terminal His tag was used for its purification. The lipase activity of different transformants against substrate para-nitrophenyl laurate (p-NPL) varied from 14 to 16 U ml(-1). For the substrates para-nitrophenyl caprate (p-NPC), p-NPL, para-nitrophenyl myristate (p-NPM), para-nitrophenyl palmitate (p-NPP), and para-nitrophenyl stearate (p-NPS), the specific activity was shown to be preferred to long acyl chain length of p-NPS.

Background A new method for the prediction of protein structural classes is constructed based on Rough Sets algorithm, which is a rule-based data mining method. Amino acid compositions and 8 physicochemical properties data are used as conditional attributes for the construction of decision system. After reducing the decision system, decision rules are generated, which can be used to classify new objects. Results In this study, self-consistency and jackknife tests on the datasets constructed by G.P. Zhou (Journal of Protein Chemistry, 1998, 17: 729–738) are used to verify the performance of this method, and are compared with some of prior works. The results showed that the rough sets approach is very promising and may play a complementary role to the existing powerful approaches, such as the component-coupled, neural network, SVM, and LogitBoost approaches. Conclusion The results with high success rates indicate that the rough sets approach as proposed in this paper might hold a high potential to become a useful tool in bioinformatics.

Motivation: Simple sequence repeats or microsatellites have been found abundantly in many genomes. However, the significance of distribution preference has not been completely understood. Completion of the Arabidopsis genome sequencing allows us to better understand and characterize microsatellites. Results: Microsatellite distribution was more abundant in 5′-flanking regions of genes compared with that expected in the whole genome, with an over-representation of AG and AAG repeats; there were clear differences from distributions in 3′-flanks and coding fractions, where triplet frequencies evidently corresponded to codon usage. We identified 1140 full-length genes that contained at least one locus of AG or AAG repeats in their upstream sequences, and whose functional characteristics were significantly associated with the repeats. This observation indicates that selective pressure markedly differed in the three transcribed regions, with positive selection of AG and AAG repeats in 5′-flanks close to those genes whose products are preferentially involved in transcription.

Gpr97 is an orphan adhesion GPCR and is highly conserved among species. Up to now, its physiological function remains largely unknown. Here, we show that Gpr97 deficiency results in an extensive reduction in B220+ lymphocytes in mice. More intensive analyses reveal an expanded marginal zone but a decreased follicular B-cell population in Gpr97−/−spleen, which displays disorganized architecture characterized by diffuse, irregular B-cell areas and the absence of discrete perifollicular marginal and mantle zones. In vivo functional studies reveal that the mutant mice could generate antibody responses to T cell-dependent and independent antigens, albeit enhanced response to the former and weakened response to the latter. By screening for the molecular events involved in the observed phenotypes, we found that lambda 5 expression is downregulated and its upstream inhibitor Aiolos is increased in the spleen of mutant mice, accompanied by significantly enhanced phosphorylation and nuclear translocation of cAMP response element-binding protein. Interestingly, increased constitutive Nf-κb p50/p65 expression and activity were observed in Gpr97−/− spleen, implicating a crucial role of Gpr97 in regulating Nf-κb activity. These findings uncover a novel biological function of Gpr97 in regulating B-cell development, implying Gpr97 as a potential therapeutic target for treatment of immunological disorders.