Baculovirus <Emphasis Type="Italic">per os</Emphasis> infectivity factors are involved in HearNPV ODVs infection of HzAM1 cells <Emphasis Type="Italic">in vitro</Emphasis>

Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry. Foundation items: National Nature Science Foundations of China (30325002, 30470075); National Basic Research Priorities Program of China (2003CB1140).     

Molecular analysis of the genus <Emphasis Type="Italic">Anoxybacillus</Emphasis> based on sequence similarity of the genes <Emphasis Type="Italic">recN</Emphasis>, <Emphasis Type="Italic">flaA</Emphasis>, and <Emphasis Type="Italic">ftsY</Emphasis>

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550–600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3–42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.     

Expression of the <Emphasis Type="Italic">Escherichia coli</Emphasis> heat-labile enterotoxin B subunit in transgenic watercress (<Emphasis Type="Italic">Nasturtium officinale</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A gene encoding the B subunit of the enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) was adapted to the optimized plant coding sequence, and fused to the endoplasmic reticulum retention signal SEKDEL in order to enhance its expression level and protein assembly in plants. The synthetic LTB (sLTB) gene was placed into a plant expression vector under the control of the CaMV 35S promoter, and subsequently introduced into the watercress (Nasturtium officinale L.) plant by the Agrobacterium-mediated transformation method. The integration of the sLTB gene into the genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification. The assembly of plant-produced LTB protein was detected by western blot analysis. The highest amount of LTB protein produced in transgenic watercress leaf tissue was approximately 1.3% of the total soluble plant protein. G_M1-ganglioside enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to G_M1-ganglioside, which is the receptor for biologically active LTB on the cell surface, suggesting that the plant-synthesized LTB subunits formed biologically active pentamers.     

High-resolution Mapping and Analysis of the Resistance Locus <Emphasis Type="Italic">Rpi-abpt</Emphasis> Against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Potato

Introduction of more durable resistance against Phytophthora infestans causing late blight into the cultivated potato is of importance for sustainable agriculture. We identified a new monogenically inherited resistance locus that is localized on chromosome 4. The resistance is derived from an ABPT clone, which is originally a complex quadruple hybrid in which Solanum acaule, S. bulbocastanum, S. phureja and S. tuberosum were involved. Resistance data of the original resistant accessions of the wild species and analysis of mobility of AFLP markers linked to the resistance locus suggest that the resistance locus is originating from S. bulbocastanum. A population of 1383 genotypes was screened with two AFLP markers flanking the Rpi-abpt locus and 98 recombinants were identified. An accurate high-resolution map was constructed and the Rpi-abpt locus was localized in a 0.5 cM interval. One AFLP marker was found to co-segregate with the Rpi-abpt locus. Its DNA sequence was highly similar with sequences found on a tomato BAC containing several resistance gene analogues on chromosome 4 and its translated protein sequence appeared to be homologous to several disease resistance related proteins. The results indicated that the Rpi-abpt gene is a member of an R gene cluster.     

Production of transgenic <Emphasis Type="Italic">Pinus armandii</Emphasis> plants harbouring <Emphasis Type="Italic">btCry</Emphasis>III(<Emphasis Type="Italic">A</Emphasis>) gene

A synthetic chimeric gene SbtCryIII(A) encoding the insecticidal protein btCryIII(A), was transformed into Pinus armandii embryos and embryogenic calli using Agrobacterium tumefaciens. Polymerase chain reaction and genomic DNA Southern blot analysis showed that the SbtCryIII(A) gene was integrated into the genome of transgenic Pinus armandii plants, and Northern blot analysis indicated that the SbtCryIII(A) gene was transcribed.     

Organization of phenylalanine ammonia lyase (<Emphasis Type="Italic">PAL</Emphasis>), acidic <Emphasis Type="Italic">PR-5</Emphasis> and osmotin-like (<Emphasis Type="Italic">OSM</Emphasis>) defence-response gene families in the potato genome

Defence-response (DR) genes are candidates for the genetic functions underlying quantitative resistance to plant pathogens. The organization of three DR gene families encoding phenylalanine ammonia lyase (PAL), acidic PR-(pathogenesis-related) protein 5, and basic PR-5, or osmotin-like (OSM), proteins was studied in the potato genome. A bacterial artificial chromosome (BAC) library containing ~50,000 clones was constructed from high-molecular weight genomic DNA of the diploid potato clone PD59, a hybrid between Solanum tuberosum and S. phureja. BAC clones carrying one or more copies of the DR genes were identified and characterized by Southern hybridization, sequence analysis and genetic mapping. PAL, acidic PR-5 and OSM (basic PR-5) genes were all organized into gene families of varying complexity. The PAL gene family consisted of at least 16 members, several of which were physically linked. Four acidic PR-5 homologous were localized to a 45-kb segment on potato chromosome XII. One of these, PR-5/319, codes for the acidic thaumatin-like protein C found in intercellular fluids of potato. Nine OSM genes were organized at two loci: eight form a 90-kb cluster on chromosome VIII, and a single gene was found on chromosome XI. The topology of a phylogenetic tree based on PR-5 and OSM protein sequences from Solanaceae suggests a mode of evolution for these gene families. The results will form the basis for further studies on the potential role of these defence-related loci in quantitative resistance to pathogens.     

Gene <Emphasis Type="Italic">RAD31</Emphasis> is identical to gene <Emphasis Type="Italic">MEC1</Emphasis> of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The earlier identified gene RAD31 was mapped on the right arm of chromosome II in the region of gene MEC1 localization. Epistatic analysis demonstrated that the rad31 mutation is an allele of the MEC1 gene, which allows further designation of the rad31 mutation as mec1-212. Mutation mec1-212, similar to deletion alleles of this gene, causes sensitivity to hydroxyurea, disturbs the check-point function, and suppresses UV-induced mutagenesis. However, this mutation significantly increases the frequency of spontaneous canavanine-resistance mutations induced by disturbances in correcting errors of DNA replication and repair, which distinguishes it from all identified alleles of gene MEC1.     

Molecular cloning,expression in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> of Attacin A gene from <Emphasis Type="Italic">Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.     

Factors affecting <Emphasis Type="Italic">in vitro</Emphasis> propagation and field establishment of <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

The effect of plant growth regulators (PGRs), gelling agents, sucrose and heat shock on shoot multiplication, shoot growth, rooting and subsequent survival of Chlorophytum borivilianum Sant. et Fernand was evaluated. Benzyladenine (BA) was found to be better cytokinin over kinetin (KIN) for shoot multiplication. Sucrose concentrations from 116–290 mM in the basal medium (BM) promoted shoot multiplication. Heat shock (50 °C, 1 h) also promoted shoot multiplication at these sucrose concentrations on both BM medium and BM supplemented with 5.0 μM BA. Beneficial effect of sucrose was also observed on rooting of shoots on BM as well as BM supplemented with 5.0 μM indole-3-butyric acid (IBA). Phytagel as a gelling agent was found to be more effective for shoot proliferation and growth compared to agar. Amongst various soil mixtures tested, higher survival of plants was observed in soil containing Vermicompost. It was interesting to note that a maximum plant survival (> 95 %) was observed when plants were directly transferred to net-house (irradiance reduced to 50 % with green net, without humidity and temperature control) than poly-house (with humidity and temperature control). Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis of regenerated plants showed genetic similarity to mother plant.     

Characterization of <Emphasis Type="Italic">chsA,</Emphasis> a new gene controlling the chemotactic response in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7

We report, here, the characterization of a mutant strain of Azospirillum brasilense Sp7 impaired in surface motility and chemotactic response. Presence of flagella in the mutant strain was confirmed by western blot analysis, using antisera raised against the polar and lateral flagellins, and by electron microscopy. Genetic complementation and nucleotide sequencing led to the identification of a new gene, named chsA. The deduced translation product, ChsA protein, contained a PAS sensory domain and an EAL domain. As ChsA displayed characteristic signaling protein architecture, it is thought that this protein is a component of the signaling pathway controlling chemotaxis in Azospirillum.     

The expression of analgesic-antitumor peptide (<Emphasis Type="Italic">AGAP</Emphasis>) from Chinese <Emphasis Type="Italic">Buthus</Emphasis><Emphasis Type="Italic">martensii</Emphasis> Karsch in transgenic tobacco and tomato

The present study aimed to obtain analgesic-antitumor peptide (AGAP) gene expression in plants. The analgesic-antitumor peptide (AGAP) gene was from the venom of Buthus martensii Karsch. Previous studies showed that AGAP has both analgesic and antitumor activities, suggesting that AGAP would be useful in clinical situations as an antitumor drug. Given that using a plant as an expression vector has more advantages than prokaryotic expression, we tried to obtain transgenic plants containing AGAP. In the present study, the AGAP gene was cloned into the plasmid pBI121 to obtain the plant expression vector pBI-AGAP. By tri-parental mating and freeze–thaw transformation, pBI-AGAP was transformed into Agrobacterium tumefaciens LBA4404. Tobacco (Nicotiana tabacum) and tomato (Lycopersicom esculentum) were transformed by the method of Agrobacterium-mediated leaf disc transformation. The transformants were then screened to grow and root on media containing kanamycin. Finally, transformations were confirmed by analysis of PCR, RT-PCR and western blotting. The results showed that the AGAP gene was integrated into the genomic DNA of tobacco and tomato and was successfully expressed. Therefore, the present study suggests a potential industrial application of AGAP expressed in plants.     

Effect of seed-specific expression of the <Emphasis Type="Italic">ipt</Emphasis> Gene on <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. seed composition

A transgenic approach to manipulation of endosperm development has been investigated. Nicotiana tabacum cv. Xanthi, an endosperm-containing dicotyledon, has been used as a model plant and the 2.6 kb wheat high molecular weight (HMW) glutenin subunit 12 promoter has been used fused either to the gus reporter gene (HMWgus construct)—to study promoter characteristics—or to the Agrobacterium ipt gene—to study the effect of cytokinin (CK) over-expression on assimilate accumulation in the seed. In transgenic tobacco the promoter:gus fusion showed that HMW is an endosperm-specific promoter with maximum expression 20 days after anthesis (DAA), corresponding to the mid to late stages of seed development. Transgenic plants containing the HMWipt construct showed no morphological abnormalities but they had an average increase in seed weight and total ethanol-insoluble carbohydrates and protein content of 8.1%, 7.0% and 8.3%, respectively. SDS PAGE analysis demonstrated that the effect on protein accumulation was non-specific. The highest values of the parameters analysed correlated with moderate increases in the levels of biologically active CKs. These results suggest that ectopic expression of small amounts of CKs can be used to increase storage assimilate accumulation without a detrimental effect on development.