Volume 68, Issue 3

Reactive oxygen species (ROS) are continuously produced by the metabolically active cells of seeds, and apparently play important roles in biological processes such as germination and dormancy. Germination and ROS accumulation appear to be linked, and seed germination success may be closely associated with internal ROS contents and the activities of ROS-scavenging systems. Although ROS were long considered hazardous molecules, their functions as cell signaling compounds are now well established and widely studied in plants. In seeds, ROS have important roles in endosperm weakening, the mobilization of seed reserves, protection against pathogens, and programmed cell death. ROS may also function as messengers or transmitters of environmental cues during seed germination. Little is currently known, however, about ROS biochemistry or their functions or the signaling pathways during these processes, which are to be considered in the present review.

It is not easy to manipulate biosynthetic genes of Bacillus thuringiensis since there is a powerful methyl-specific restriction system in this microorganism. In this study, a PCR-based system was used to delete polyphosphate kinase gene (ppk) of Bacillus thuringiensis israelensis (Bti) by replacing the wild-type gene with a cassette containing the apramycin resistance gene as selectable marker. λ-Red was used to promote recombination in Escherichia coli between a PCR-amplified apramycin resistance cassette (linear deletion cassette selectable in E. coli and Bti) and Bti DNA on a plasmid. The isolated mutant plasmid was transferred to Bti by conjugation. Double cross-over transformants were screened for their antibiotic resistance and the mutation was proven by PCR, southern blot hybridization and RT-PCR. The described method, which uses the advantage of quick plasmid construction in E. coli and simple transformation of linear deletion cassette, is very useful to delete entire gene/genes of Bti without any polar effects on genes transcriptionally downstream.

The paper presents the results of comprehensive analyses concerning the phenotypic variability of the primary thallus in five chosen Cladonia species in relation to habitat conditions. Morphology and anatomy, as well as the cortex ultrasculpture, of primary squamules were examined in individual specimens from sunny and shady populations of each species. The Mann-Whitney U test and Principal Component Analysis (PCA) revealed several clear correlations between the type of habitat and the organisation of squamules. Significant differences refer mainly to anatomical features, especially to epinecral layer frequency and cortex thickness. On the contrary, examination of the surface ultrasculpture of squamules under SEM did not show any important differences between populations. Regardless of the habitat factors, a full range of cortical surface rugosity in samples from both habitats was noticed, and a high variability often refers to a singular squamule. The results indicate that some features are largely modified by environmental factors and/or depend on the development stage of individual specimens, and examination of these relationships should be the first step towards the evaluation of the taxonomic usefulness of the considered features.

The first specimens of Allium elmaliense were collected in Elmal? (Antalya/Turkey) in 2001 and were described as new species in 2004. However, Allium elmaliense was claimed as synonym of Allium cyrilli in 2006. This study was undertaken to reveal the differences between these taxa utilizing morphological, palynological, and chromosome characters and genomic differences based on the DNA analyses along with the ecological preference studies conducted during 2006 and 2011. The results clearly indicated differences between these two taxa in terms of morphological characters, pollen, seed surfaces and niche preferences. Chromosome morphology and Td-DAMD-PCR fingerprinting studies revealed that Allium elmaliense Deniz & Sümbül is a distinct species and not a synonym of A. cyrilli Ten.

The present study considers genetic diversity of 38 populations in 4 Cirsium species of the genus Cirsium Mill. (Asteraceae), occurring in different ecological regions and tries to compare degree of genetic variability among the species with wide geographical distribution versus endemic C. pyramidale showing confined geographical distribution. The results showed that the endemic species has similar value of genetic diversity parameters as the species with wider distribution. We also studied the possible admixture nature of these populations and tried to understand the relation between genetic changes, geographical distribution and polyploidy level and chromosome pairing in these species. ISSR analysis showed population difference in allele composition and frequency. Clustering and PcoA ordination produced different groupings in each species, while STRUCTURE and reticulation analyses revealed high degree of genetic admixture and gene exchange among populations as well as allelic rearrangement. No significant correlation was observed between geographical distance and genetic distance of the populations and AMOVA test revealed no significant difference among populations in each species studied. However, high amount of within population variation occurred in all 4 species indicating their cross-pollination nature and high genetic admixture. The populations also varied in chiasma frequency and chromosome pairing as well as the occurrence of heterozygote translocations all creating more variability to be used by plants for local adaptation.

For the protection or re-establishment of species-rich grasslands, the limiting factors controlling species richness have increasingly become of scientific interest. This study aims to analyze the role of disturbance for the occurrence of a low-competitive herb, Centaurium erythraea Rafn, in mesotrophic, lowland grasslands in NW Germany. We sampled a total of 38 plots with presence of C. erythraea and 24 control (random) plots in semi-natural grasslands. As a proxy for disturbance, we estimated the cover of bare ground and Ellenberg flooding indicator species and measured the distance between the plot and the nearest path. Moreover, we counted the number of C. erythraea individuals within each plot. In the GLM analyses the distance from path was the only predictor; both the presence of the species and the number of individuals decreased with the distance from path. The grasslands at the path edges had the highest disturbance intensity. Here horse riding, military-vehicle traffic and regular sod-cutting directly create bare ground. In general, disturbance creating bare ground seems to be the key factor enabling germination and growth of C. erythraea in mesotrophic grasslands and other low-competitive short-lived species. Disturbance enhances the expression of the seed bank, favours the development of the shade-avoiding and low-growing rosettes and oppresses tall-growing competitors. Therefore we suggest grazing as the best management method.

Photosynthesis, chlorophyll (Chl) fluorescence, and antioxidant enzymes were measured in the mulberry (Morus spp.) cultivars Da 10, Hongguo 2, Anza 1, and Taiwan 72C002, which were subjected to salinity and high-temperature stress (STS; 0.1%, 0.3%, and 0.5% NaCl concentrations, 34.5°C–40.5°C/27.8°C–29.2°C day/night temperatures). Control plants were watered with 1 L of full-strength Hoagland’s nutrient solution with no added NaCl. Net photosynthetic rate (P N), stomatal conductance (g s), and effective quantum yield of photosystem II photochemistry (ΦPSII) increased in Anza 1 and Taiwan 72C002 under 0.1% STS but decreased in Da 10 and Hongguo 2 compared with the control. However, all the above parameters, including Chl content, maximum quantum yield of photosystem II photochemistry (Fv/Fm), nonphotochemical quenching (NPQ), and maximum carboxylation velocity of Rubisco (V cmax, decreased in Taiwan 72C002, Honggua 2, and Da 10 under 0.3% and 0.5% STS, suggesting that photoinhibition occurred under severe STS. Under STS, there were no significant changes in P N, Fv/Fm, ΦPSII, ascorbate peroxidase (APX) activity, superoxide dismutase (SOD) activity, catalase activity, superoxide anion radical (O2−) content, malondialdehyde (MDA) content, soluble sugar content (SSC), and leaf biomass in Anza 1 even at 0.5% STS, showing that Anza 1 displays high resistance to STS. In addition, peroxidase activity was significantly higher in Anza 1 than in the other mulberry cultivars. Significant adverse effects of severe salinity on photosynthesis and Chl fluorescence parameters were observed in Da 10. Additionally, SOD, peroxidase, and APX activities were lower in Da 10, whereas O2− and MDA contents were higher in comparison with the other mulberry cultivars under 0.3% and 0.5% STS, suggesting that Da 10 had low resistance to STS. These results show that 0.1% STS had a positive effect on photosynthesis and Chl fluorescence parameters in Anza 1 and Taiwan 72C002, and higher peroxidase activity can to a certain extent explain the higher STS tolerance in Anza 1. Damages to DSM photosystems might be related to lower SOD, POD, and APX activities, which resulted in the accumulation of reactive oxygen species.

This investigation was carried on to find out the changes occurred in Stevia rebaudiana in response to paclobutrazol (PBZ; 0–4 mg L−1) treatment and drought stress. Polyethylene glycol (PEG; 0–6 % w/v) was used to stimulate drought stress. Drought stress reduced fresh and dry weight, water content, chlorophylls, carotenoids, anthocyanins, water soluble carbohydrates, reducing sugar and proline amounts. Electrolyte leakage, MDA, α-tocopherol and glycine betaine contents increased in drought-stressed plants. The activity of P5CS and PDH enzymes and protein content showed no significant changes under drought stress. PBZ (with or without PEG) treatments decreased fresh and dry weight and water content. In PBZ-treated plants, less pigments was damaged by drought stress. PBZ treatment reduced the negative effect of drought stress on lipid peroxidation which resulted in lower electrolyte leakage and MDA content, compared to the same PEG level without PBZ. PBZ (with or without PEG) treatments increased glycine betaine, α-tocopherol, proline and protein contents. The amount of water soluble carbohydrates, reducing sugar and activity of P5CS and PDH were not affected by PBZ treatments. SDS-PAGE analysis revealed that drought stress increased a 25 kD protein with a critical function in plant development under stresses. According to the results, PEG provoked a severe drought stress in S. rebaudiana that could partly be restored by PBZ treatment.

The assays commonly used to determine ATP content in biological samples generally measure total cellular ATP content, but not the different subcellular pools. In this study a new simple method for measuring ATP content in a cytosol-enriched fraction (CEF) was developed, based on a rapid cytosolic ATP extraction (by an isotonic grinding medium that preserves organelle integrity) and its detection monitoring the NADPH fluorescence generated via hexokinase/glucose-6-phosphate dehydrogenase coupled reactions. Four protocols, differing for timing of NADPH generation and for either the presence or absence of some inhibitors of ATP and NADPH metabolism, were compared by determining CEF-ATP, as well as total ATP, in durum wheat (Triticum durum Desf.) etiolated seedlings. The best protocol was the one adopting both simultaneous NADPH generation and use of inhibitors during tissue homogenization. This protocol also showed higher performance than the classical trichloroacetic acid extraction. Using the new method, CEF-ATP content was assessed in control, salt- and osmotic-stressed seedlings, resulting 2.68 ± 0.04, 1.69 ± 0.12 (−40%) and 1.35 ± 0.16 (−50%) μmol/g dry weight, respectively. Finally, the effects of this stress-dependent decrease of cytosolic ATP were evaluated with respect to a possible modulation of two mitochondrial energy-dissipating systems, the uncoupling protein (PUCP) and the K+ channel (PmitoKATP), both inhibited by cytosolic ATP. Experiments carried out at different physiological ATP concentrations suggest that the decreased cytosolic ATP content occurring under hyperosmotic stress may contribute to attenuate inhibition of PmitoKATP, thus promoting its activity (up to about 90%), but not of PUCP, that appears to lose ATP sensitivity under stress condition.

Infusions of the leaves and seeds of Annona crassiflora Mart. are commonly employed in the treatment of diarrhoea, snakebites, tumours and disorders of the hair and scalp. The aim of the present study was to investigate the cytotoxic and genotoxic properties of ethanolic extracts of A. crassiflora by evaluating their effects on germination, root elongation, chromosome structure and the cell division of Lactuca sativa (lettuce). The experiment followed a randomized design involving the treatment of L. sativa seeds with ethanolic extracts from leaves and seeds of A. crassiflora applied at ten concentrations (0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg/L) and with five repetitions per treatment. Seeds of L. sativa exposed for 48 h to A. crassiflora leaf extract at concentrations ≥ 0.1 mg/L, or to seed extracts at concentrations ≥ 0.2 mg/L, showed germination percentages that were significantly (p < 0.05) lower than those of seeds exposed to aqueous ethanol control. Exposure of L. sativa seedlings to leaf (but not seed) extracts of A. crassiflora produced significant (p < 0.05) reductions in the mitotic indices of root meristem cells of lettuce and induced chromosome and nuclear abnormalities in the root cells. The presence of chromosome stickiness, bridges, fragments, laggard chromosomes and nuclear condensation were also observed. The cytogenetic effects observed suggest that folkloric medicines prepared with extracts of the leaves or seeds of A. crassiflora should be employed with caution.

Despite the growing awareness of the ecological importance of epibiont-host associations, detailed inventories for planktonic hosts are rare. Here, we provide an updated checklist of the peritrich and suctorian epibiont ciliates (Ciliophora) on the cladocerans (Crustacea: Cladocera). Thirty-nine species of peritrich ciliates (of which 34 are assigned to species) and three species of suctorian ciliates are found to be epibionts on the Cladocera. Fifty-eight cladoceran taxa are known to be hosts of the ciliate epibionts, 33 of these hosts (57%) are planktonic. Seven taxa were determined to the level of genus. Complete species designations were geographically biased (38 of 51 species) towards European sites, suggesting poor taxonomic knowledge beyond Europe. Also, the recently discovered continental endemism of cladoceran hosts could indicate that associated ciliates are more diverse than previously appreciated.

Headwater streams are important resources in production of organic matter, but zooplankton of headwater streams has rarely been studied. In the present study spatial changes in zooplankton communities between upper headwater forest section and downstream meadow section of four small streams were examined (NW Poland). The environmental conditions of stream riparian zone, e.g., the presence of leaf litter, pools, slackwaters and wetlands had a great influence on the spatial changes in zooplankton. Low gradient of stream bed, longer water residence time and larger surfaces of wetlands and slackwaters also positively affected zooplankton communities, especially in the meadow section. Along the streams, from the stream headwater to the downstream-meadow section, significant spatial changes in zooplankton communities were observed. Significantly higher number of taxa and density of zooplankton were observed in meadow sections than in headwater forest sections. In the forest sections, there was a definite domination of benthic and littoral rotifers in the zooplankton composition, while in the meadow sections, planktonic rotifers dominated. Cladocerans were observed only in downstream meadow section; two littoral species Coronatella rectangula and Peracantha truncata and one planktonic Daphnia magna which probably were washed from close pools of small stagnant water bodies. Copepods were noted along the entire length of streams. The occasional presence of planktonic rotifers in the forest section was probably caused by their washout from a few small stagnant water bodies.

The present study investigates the zooplankton community dynamics and the abiotic environment in the eutrophic Lake Lysimachia (western Greece). The lake is considered to be recovering from eutrophication after the termination of an urban sewage inflow in 2000, and its waters are replenished constantly from the nearby oligotrophic Lake Trichonis. The results show that, although a decrease in nutrient concentrations was observed compared to the past, the lake still has eutrophic characteristics. This was reflected in the zooplankton community which is typical of those found in eutrophic lakes where rotifers prevail. Similarities among this lake and other nearby lakes were found considering the zooplankton community composition and seasonal variation. However, Lake Lysimachia is inhabited also by a number of different and even unique species (e.g., Moina micrura), suggesting that this ecosystem may be an important biodiversity refuge. Most of the zooplankton species were correlated with water temperature and, to a lesser extent, eutrophication key-water quality variables. Although there are few available data on the zooplankton of the lake, the abundance and composition of the community presenting characteristics indicative of intermediate trophic conditions and suggesting that the lake is probably under a kind of “biological” recovery.

In spring 2005 we investigated the collembolan communities in the 50-m-deep collapse doline of the perennial ice cave Silická ľadnica in the Slovak Karst (Slovakia). Samples were taken at seven sites along a 117.5-m-long transect on the slope from the ice-bearing cave mouth at the bottom of the doline up to the terrain surface at 500 m above sea level (a.s.l.). The temperature above the soil surface (+0.6 to +13.6°C) positively correlated with altitude. Species numbers (ranging from 20–32) and diversity indices were highest at sites in the middle of the slope with rendzina and well developed organic profiles. A Kruskal-Wallis ANOVA revealed significant differences in abundance between the sites. Mean abundance near the permafrost zone at the bottom of the doline was significantly higher than at the sites further upslope. The abundances of some eurytopic and forest species were significantly correlated with soil temperature. Cluster analysis and the IndVal method indicated differences in the structure of Collembolan communities along the transect. The community at the coldest site had the lowest species richness and the highest mean abundance of individuals. A total of ten montane species were recorded, with a lower number near the permafrost zone compared to the micro-climatically more favourable middle section of the gradient.

Structure and density of soil microarthropod communities (Oribatida and Collembola) were studied in one natural beech forest and one spruce monoculture planted on a former beech stand in South Bohemia (Czech Republic). The spruce monoculture establishment increased microarthropod densities (93,000 ind. m−2 in the natural beech forest vs. 400,540 ind. m−2 in the spruce monoculture for Oribatida; 66,360 ind. m−2 in the natural beech forest vs. 136,360 ind. m−2 in the spruce monoculture for Collembola); additionally, it changed greatly the community structure in terms of species composition and functional traits. In the spruce monoculture, groups susceptible to disturbance were suppressed. The oribatid trophic structure changed as well with opportunistic herbifungivorous species increasing in the monoculture at the expense of fungivorous species. Similarly, hemiedaphic collembolans increased in the monoculture at the expense of euedaphic species. We conclude that the “functional approach” seems to be fruitful in revealing soil fauna response to environmental change.

In this study we describe how the river morphology of seven High Tatras streams has been influenced in different ways by a severe windstorm that occurred in November 2004. A control site situated in an undisturbed area is compared with six sites in windstorm damaged areas. In 2009–2010 River Habitat Survey (RHS) was used to assess the character of 500 m lengths of stream and to derive associated morphological indices (HQA, HMS sensu Raven 1998, RHS indices sensu Vaughan 2010). Large amounts of large woody debris in the channel influenced the streams despite most of it having been removed after the windstorm. Streams situated in deforested valley slopes were probably more affected by erosion; they had higher amounts of transported inorganic matter and also depositional bar features. Some changes in land-use had occurred with streams situated on the south-east side of the High Tatras having a more uniform banktop vegetation structure than the other streams. Distinct secondary succession of bank top vegetation was observed along windstorm influenced streams. The highest percentage of shrubs occurred where impacted streams remained untouched. Morphologically, the streams most affected by windstorm are those situated in deforested steep valley slopes that are affected by erosion and siltation.

Hydrobiological research of high mountain streams in the High Tatras was carried out in 2009–2010. We evaluated the influence of windstorm on caddisfly assemblages. To assess the influence of windstorm we focused on river morphology using the RHS method and evaluation of TAM, TOM and BOM (CPOM, FPOM, UFPOM) amount in seven streams. Site 1 was a control and the other six were disturbed by the windstorm in different ways. The most remarkable differences compared to the control site was in feeding structure at sites most affected by erosion. In these streams there was a noticeable dominance of predators from the family Rhyacophilidae (mainly Rhyacophila tristis; sites 3, 4, 5, 6) and a remarkable decrease of passive filter feeders (site 4, 5). Using Spearman coefficient we confirmed a positive correlation between the proportion of predators and amount of TAM caused by erosion of steep deforested slopes of windstorm affected sites. In contrast, we did not observe such an increase in the proportion of predators at site 7, which was affected by both windstorm and subsequently by fire. This might be explained by the shallow vee valley with no observed erosion, where this site is situated. We also found a negative correlation between predators and evenness, indicating unstable community structure clearing succesion in the streams disturbed by erosion, and a negative correlation between passive filter feeders and UFPOM. We found out that overhanging tree boughs and LWD had an influence on species composition. RHS was a useful tool in characterising the influence of hydromorphology on caddisfly assemblages.

Competition is a basic type of interaction between species. Because complete competitors cannot exist in one habitat, closely related species must show at least slight differences in their biology or ecology. In the present study, we analyzed the habitat use of the two closely related bush-cricket species Tettigonia caudata and T. viridissima which often occur syntopically. The meso- and microhabitat use of males was compared in an agricultural landscape in NE Germany. Males of both species were found to use similar mesohabitats and to prefer dense and high vegetation. Microhabitats differed significantly in two aspects: Microhabitats of T. caudata had a higher proportion of herbs, while T. viridissima used higher song posts. These differences in microhabitats might explain the co-occurrence of the bush-crickets in open habitats. However, variation between the two species might also take place during other life-cycle stages such as during egg development.

The West-Carpathian endemic Cyclamen fatrense is a forests species. Its distribution area lies at the northern margin of distribution area of the Cyclamen genus. Several studies have focused on the pollinators and consumers of representatives of this genus, but they mention only some more important animal species. The aim of our study was to determine the species spectrum of animals having a relationship to C. fatrense in its natural environment and to define mutual relationships between this plant and animals based on comparison of phenologic manifestations of cyclamen and known ecologic data about its visitors. In course of different phenophases of C. fatrense we recorded 127 invertebrate taxa on this plant. Among them 7 taxa were identified as consumers, 29 taxa as potential consumers, 5 taxa as potential pollinators and 91 had an indifferent relationship to the cyclamen. Damaging or direct consummation of cyclamens by vertebrates has not been observed.

The toxicity and deterrent effects of phytoheamagglutinin (Phaseolus vulgaris agglutinin, PHA) to the grain aphid (Sitobion avenae F.) was investigated in the laboratory by use of liquid diets or gels containing various PHA concentrations. The addition of PHA to the liquid diet increased the aphid pre-reproductive period, mortality, and generation time and decreased aphid fecundity and intrinsic rate of natural increase. Aphids fed on sucrose-agarose gels with higher concentrations of PHA exhibited no phloem-feeding behavior. The number of gel penetrations was reduced and duration of the pathway phase was increased as PHA concentration increased. These results indicate that PHA is detrimental to S. avenae and that the effect of PHA is at least partially explained by suppression of feeding behaviour. PHA could be useful for managing S. avenae.

During many insemination interventions semen coagulates already within the insemination needle, which considerably lengthens the duration of inseminating a single queen bee. Considering this, the authors decided to determine the type and activity of proteases and their inhibitors in normal and coagulated sperm. The samples were collected from mature and old drones. The sperm proteins were isolated in 1% Triton X-100. The samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor activity detection by means of the modified Felicioli method. The mixing of non-coagulated semen from different drones increased protein concentration. The activities of proteases were decreased in normal sperm samples as compared with a corresponding rise in the sperm mixture from many drones. The non-coagulated sperm samples were found to contain aspartic and serine proteases. Additionally, thiolic and metallic proteases were also found in the coagulated sperm samples. There was a rise in protease inhibitor activity at pH 3.0 and 12.0, and a fall at pH 7.0 after mixing the sperm samples collected from numerous drones. Oscillation in these activities stemmed from sperm coagulation.

In order to determine an unknown fish population from the Vrana Lake, mitochondrial cytochrome b gene and non-coding nuclear region Cyfun P were investigated. Stabile population of Bulldog rudd, Scardinius dergle Heckel & Kner, the endemic Croatian freshwater fish in the Krka River, was genetically characterized with the same markers in order to compare it with the material from the Vrana Lake. Genetic markers were sequenced and aligned with the similar ones obtained from the GenBank in order to determine taxonomic and phylogenetic position of these two species. A significant discrepancy between nuclear genetic markers of our specimens and the sequence from the GenBank was found. Phylogenetic analysis suggested that the specimens from the Vrana Lake belong to the species S. hesperidicus. Morphometric characteristics, the maximum length and body mass showed new maximum values for both S. dergle and S. hesperidicus.

The present study was aimed to compare and contrast the histochemical, histological and ultrastructural variations (microanatomical differences) in the nephrons of selected snake species, Eryx jaculus (Boidae), Psammophis sibilans (Colubridae), Naja haje (Elapidae) and Echis pyramidum (Viperidae) from Egypt. The structural studies were carried out by conventional light and electron microscopy. The nephron, the renal unit of snakes, consists of renal corpuscle, proximal tubule, intermediate segment, distal tubule and collecting tubule. The renal corpuscles have large capillaries with clear and dark fenestrated endothelial cells. The proximal tubule showed long microvilli, cytoplasmic vacuoles, developed endoplasmic reticulum and abundant mitochondria. The intermediate segment was lined by ciliated cells. The lining cells of the distal tubules showed few microvilli, abundant dense mitochondria and clear vesicles of mucous appeared in the terminal portion. The collecting tubules consisted of mucous cells. In summary, the ultra-structure studies of nephrons revealed several interspecies similarities and also some intra-species differences in species of snakes.

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full-length cDNA of GRP78 from silkworm Bombyx mori (BmGRP78). It is 2645 bp, including an open reading frame (ORF) of 1977 bp encoding a polypeptide of 658 amino acids. We sequenced the ORF sequence from genomic DNA, and found only one intron (104 bp) existed in Bmgrp78 gene structure. The deduced amino acid sequence of Bmgrp78 carries the ER retention signal KDEL at its C-terminus and is highly homologous to GRP78 of Fenneropenaeus chinensis (83.59%) and Homo sapiens (80.27%). RT-PCR analysis shows that Bmgrp78 is ubiquitously expressed in tissues of silkworm. Heat shock over 35°C notably enhanced the expression of Bmgrp78 in head tissues. In response to starvation, the transcript level of Bmgrp78 in head tissues was 2.8-fold (at WS stage) and 3.2-fold (at SD1 stage) higher than the control level, but it remained stable in the midgut tissues. After silkworms were challenged by bacteria, the relative expression level of Bmgrp78 in midgut was up-regulated and reached maximal level at 24 hour after injection, and remained higher level than that of controls at about 48 hour post challenge. We infer that BmGRP78 may play important roles in chaperoning, protein folding and immune function of silkworm.

T-vectors play an important role in cloning of polymerase chain reaction products. In the present study, a novel pUEG-T vector was developed using enhanced green fluorescent protein (EGFP) as an indicator. To improve EGFP-based green-white screening, the lipoprotein mutant promoter, a strong constitutive promoter, was utilized to control the expression of egfp gene. Two other efficient expression elements, the ColE1 replication origin of pUC18 and the expression cassette of pET-28a, were also integrated into pUEG-T vector. Expression analysis demonstrated the efficient accumulation of active EGFPs in Escherichia coli DH5α cells carrying the T-vector precursor pUEG. In T-A cloning using pUEG-T vector, white colonies containing foreign DNA and green colonies having no insertion could be handily distinguished under normal white light, without any chemical inducer or chromogenic substrate. Furthermore, no false positive was observed in any of the tested white colonies. This proves that pUEG-T is an inexpensive, convenient and efficient T-vector.