Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus

The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2–70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus.     

Molecular cloning and characterization of a cDNA encoding nitrate reductase from Chlorella ellipsoidea (Chlorophyta)

Nitrate reductase (NR) genes have beencloned from higher plants, fungi and algae.Based on seven of the amino acid residuesmost strongly conserved between Chlorella vulgaries and Chlamydomonasreinhardtii NR gene, a degenerate primerwas designed. This degenerate primer wasused to amplify the corresponding homologyin Chlorella ellipsoidea. A 3304 bpfull-length cDNA was cloned by rapidamplification of cDNA ends (RACE). Thededuced amino acid sequence of this cDNAhas a high degree of similarity withpreviously identified members of the NRgene. This suggests that the amplified cDNAencodes a functional NR. Northern blotexpression analysis suggests that this geneis strongly induced by nitrate, but isrepressed by ammonium. The nucleotidesequence data reported in this paper willappear in the DDBJ/EMBL/GeneBank databasesunder accession number AY275834.     

Molecular cloning, mRNA expression and enzymatic characterization of cathepsin F from olive flounder (Paralichthys olivaceus)

Cathepsin F is a recently described papain-like cysteine protease of unknown function, and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. In the present study, the cDNA of olive flounder (Paralichthys olivaceus) cathepsin F (PoCtF) was cloned by the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) approaches. The PoCtF gene was determined to consist of the 1844 bp nucleotide sequence which encodes for a 475-amino acid polypeptide. The results of RT-PCR analysis revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however the PoCtF expressions increased significantly in gill at 3 h post-injection with lipopolysaccharide (LPS). Also, immunostaining using anti-PoCtF antibody was strongest on the epidermal mucus in the fin.The cDNA encoding mature enzyme of PoCtF was expressed in Escherichia coli using the pGEX-4 T-1 expression vector system. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC, a substrate commonly used for functional characterization of cysteine proteinases, and the optimal pH for the protease activity was 7.5. The findings of the present study suggest that PoCtF has a higher optimum pH than mammalian cathepsin F, and PoCtF is an interesting target for future investigations of the role of cathepsin F in the epidermal mucus and fish innate immune system.     

Molecular cloning and analysis of a cDNA coding for nucleoside diphosphate kinase from ryegrass

A full-length cDNA, LpNDPK, encoding ryegrass nucleoside diphosphate kinase (EC 2.7.4.6) has been cloned and sequenced. The nucleotide sequence of the clone contains an open reading frame of 450 nucleotides encoding a protein of 150 amino acid residues with a calculated molecular mass of 16.5 kDa and a P_i of 6.62. The LpNDPK encoded protein possesses substantial homology with nucleoside diphosphate kinases (NDPKs) isolated and cloned form other sources; the highest identity (86 percnt;) was observed with NDPK from sugarcane (Saccharum officinarum). Amino acid comparisons with other NDPKs show that the presented ryegrass NDPK sequence also contains several motifs and specific residues crucial for catalytic activity which are highly conserved among other NDPKs. RT-PCR expression analysis using primers covering the coding region of LpNDPK revealed that the ryegrass NDPK gene is equally expressed in stem, leaf, and flower tissue.     

Molecular cloning and characterization of a mannose-binding lectin gene from Crinum asiaticum

Full-length cDNA of a mannose-binding lectin or agglutinin gene was cloned from a traditional Chinese medicinal herb Crinum asiaticum var. sinicum through RACE-PCR cloning. The full-length cDNA of C. asiaticum agglutinin (caa) was 820 bp and contained a 528 bp open reading frame encoding a lectin precursor (preproprotein) of 175 amino acid residues with a 22 aa signal peptide. The coding region of the caa gene was high in G/C content. The first 20 bp of the 5' UTR had a dC content of 50%, which was a typical feature of the leader sequence. By cutting away the signal peptide, the CAA proprotein was 15.79 kDa with a pl of 9.27 and contained 3 mannose-binding sites (QDNY). Random coil and extended strand constituted interlaced domination of the main part of the secondary structure. B-lectin conserved domain existed within N24 to G130. Predicted three-dimensional structure of CAA proprotein was very similar to that of GNA (Galanthus nivalis agglutinin). It is significant that besides certain homologies to known monocot mannose-binding lectins from Amaryllidaceae, Orchidaceae, Alliaceae and Liliaceae, caa also showed high similarity to gastrodianin type antifungal proteins. No intron was detected within the region of genomic sequence corresponding to the caa full-length cDNA. Southern blot analysis indicated that the caa gene belonged to a low-copy gene family. Northern blot analysis demonstrated that caa mRNA was constitutively expressed in all the tested tissue types including the root, bulb, leaf, rachise, flower and fruit tissues.     

Molecular cloning, expression analysis and enzymatic characterization of cathepsin K from olive flounder (Paralichthys olivaceus)

We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow. However, PoCtK expression was significantly increased in the muscle and gill at 3–24 h post-injection with bacterial lipopolysaccharide (LPS). The cDNA encoding for the mature enzyme of PoCtK was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified via the cleavage of the synthetic peptide Z-Gly-Pro-Arg-MCA, zymography, and the collagen degradation assay. The optimum pH for the protease activity was 8, and the recombinant PoCtK enzyme degraded collagen types I, II, III, IV, and VI and acid-soluble collagen from olive flounder muscle in the presence of chondroitin 4-sulphate (C-4S). Therefore, our data indicate that cathepsin K may play a role in the immune system of fish skin and muscle, in addition to its principal bone-specific function as a collagenolytic enzyme.     

The effects of 2-bromopalmitate on the fatty acid composition in differentiating adipocytes of red sea bream (Pagrus major)

To determine whether external factors affect the adipogenic function of fish adipocytes, the effects of 2-bromopalmitate (a PPAR agonist) on the fatty acid composition in differentiating adipocytes of red sea bream were investigated in vitro. In the presence of 2-bromopalmitate, the red sea bream adipocytes were differentiated and the effects on the fatty acid composition and the adipogenic gene expression were analyzed. With the level of 2-bromopalmitate, the content of 16:1n-7, a delta-9 desaturation product, increased in association with the increase in a stearoyl CoA desaturase (SCD) gene expression level while the triglyceride accumulation was not affected. Subsequently, the effects on the bioconversion of the n-3 and n-6 fatty acids, which are main series of dietary essential fatty acids, were examined. In the presence of 300 μM of 18:3n-3 or 18:2n-6, red sea bream stromal-vascular cells accumulated the lipid in the cytoplasm within 3 days by the fatty acid uptake with the increase of corresponding fatty acid contents. Furthermore, in both the 18:3n-3 and 18:2n-6 stored cells, the products of delta-6 desaturation (18:4n-3 and 18:3n-6, respectively) and C_18–20 elongation (20:3n-3 and 20:2n-6, respectively) were detected. However, neither the delta-6 desatutration nor C_18–20 elongation of 18:3n-3 and 18:2n-6 were enhanced by 2-bromopalmitate treatment. In conclusion, the results indicate that the adipocyte function in fish, e.g. adipogenic gene expression and fatty acid composition, can be modified by external factors and a main effect of 2-bromopalmitate is the increase in the content of delta-9 desaturation product by stimulating the SCD gene expression.     

华北平原地区景观格局对麦田害螨种群数量的影响

农业生产的集约化经营导致农田景观格局日趋单一,而农田景观格局的变化势必对害虫种群产生深刻的影响,阐明景观因子对害虫种群的作用是通过生境管理进行害虫控制的基础。以华北平原地区的山东省为研究区域,24个县级单元为样点,通过对卫星遥感影像和土地覆盖分类数据的分析,获取了样点单元的景观格局指数,同时定点调查了样点单元的麦田害螨种群数量。利用相关性分析明确了影响麦田中两种害螨—麦岩螨(Petrobia latens(Müller))和麦圆叶爪螨(Penthaleus major(Duges))种群发生的主要景观因子。研究结果表明景观因子对麦田中两种害螨种群均有显著影响,而两种害螨对景观因子的响应并不一致。麦岩螨的发生量与森林的最大斑块面积指数和平均斑块面积均呈显著正相关,而与森林类的形状和水体的景观形状指数均存在显著负相关;麦圆叶爪螨的发生量同水体的总面积、斑块面积比例、最大斑块面积指数以及县域范围的平均斑块面积均呈显著负相关,而与水体类的形状呈显著正相关。因此在麦田害螨发生较重的地区,在区域性景观规划时,可以通过优化农田周围的森林和水体管理,不利于其种群发生,从而达到对麦田害螨种群生态调控的目的。     

Molecular cloning and expression of retinoic-acid synthesizing enzyme raldh2 from Takifugu rubripes

Retinaldehyde dehydrogenases (raldhs) synthesize retinoic acid (RA), which is required for pattern formation and organogenesis during embryogenesis. To elucidate the common role of RA on vertebrate embryos, we first sought to clone a homologous gene to human raldh2 from fugu, Takifugu rubripes. We cloned a 1837 bp cDNA that encodes fugu raldh. The deduced amino acid sequence of the fugu raldh comprises 502 amino acids. The fugu Raldh showed highest sequence identity to zebrafish, Danio rerio, Raldh2 (79.9%). The fugu Raldh also showed high sequence identity to other vertebrate Raldh2: Xenopus laevis (77.2%), human (77.4%), mouse (74.3%) and chick (73.9%). Comparative genomic analysis showed that the gene arrangement around fugu raldh agreed with that of human raldh2. Fugu raldh mRNA was expressed through embryogenesis similarly to raldh2 in other vertebrates. These results and phylogenetic analyses suggest that pufferfish raldh is a fugu orthologue of other species' raldh2.     

Extra- and intra-cellular ice formation of red seabream (Pagrus major) embryos at different cooling rates

The ice crystal formation is assumed as the most lethal factor for the failure of fish embryo cryopreservation and intracellular ice formation (IIF) plays a central role in cell injury during cooling. The objectives were to observe the morphological changes of red seabream (Pagrus major) embryo during the cooling–thawing process, and to examine the effect of cryoprotectant and cooling rate on the temperatures of oil globule ice formation (T_OIF), extra-cellular ice formation (T_EIF) and intracellular ice formation (T_IIF) using cryomicroscope. After thawing, the morphological changes of embryos were observed and recorded by the video attachment and monitor under the microscope. During the cooling process, three representative phenomena were observed: oil globule gradually turned bright firstly, then the whole field of view flashed and the embryo blackened. Cooling rate affect the temperature of both extra- and intra-cellular ice formations. T_EIF and T_IIF at high cooling rate were much lower than that at low cooling rate. And the value of T_EIF − T_IIF increased from 0.45 to 11.11 °C with the increase of cooling rate from 3 to130 °C/min. Taken together, our results suggested that high cooling rate with proper cryoprotectant would be a good option for red seabream embryo cryopreservation.     

Molecular cloning,characterization, and expression of a cDNA encoding an endochitinase gene from the mycoparasite Stachybotrys elegans

Stachybotrys elegans is a mycoparasite of the soilborne plant pathogenic fungus Rhizoctonia solani. The mycoparasitic activity of S. elegans is correlated with the production of cell wall degrading enzymes such as chitinases. This report details the cloning by RACE-PCR and characterization of a full-length cDNA clone, sechi44, that appears to encode an extracellular endochitinase. An analysis of the sechi44 sequence indicates that this gene contains a 1269-bp ORF and encodes a 423-aa polypeptide. The SECHI44 protein has a calculated molecular weight of 44.1kDa and pI of 5.53. Since the SECHI44 protein also appears to encode a signal peptide, an extracellular location for the corresponding protein is predicted. Comparison of SECHI44 sequence with known sequences of fungal endochitinases revealed that SECHI44 is grouped with endochitinases from other mycoparasites. Real-time quantitative RT-PCR analysis showed an elevated level of expression of sechi44 (21-fold) in chitin-rich (induced) as compared to no-carbon (non-induced) culture conditions. In dual culture, the temporal expression of sechi44 increased after 2 days of contact with R. solani, reaching a 10-fold increase after 9 days, followed by a decrease to basic expression level at 12 days. Interestingly, inhibition of sechi44 expression was observed when S. elegans hyphae were in close proximity with R. solani hyphae.     

Leishmania major: Secreted antigens of Leishmania major promastigotes shift the immune response of the C57BL/6 mice toward Th2 in vitro

BALB/c mice are sensitive to Leishmaniamajor infection, while C57BL/6 mice are resistant and able to mount an effective immune response against the parasite. Since the secreted antigens of L. major suppress the proliferation of BALB/c mice lymphocytes in vitro, we analyzed their effects on the immune system of resistant C57BL/6 mice. Secreted antigens were semi-purified and two fractions with immunosuppressive activity were isolated. 15 μg/ml of fraction could suppress 60% of lymphocyte proliferation and prevent the stimulated lymphocytes entering from G1 phase into the S phase of the cell cycle. These fractions decreased the production of IFN-γ, increased IL-4 level in the lymphocyte culture and down-regulated the nitric oxide production by activated macrophages. These results may suggest that L. major parasite by secreting immunosuppressive factors could down-regulate the immune system of both sensitive and resistant mice for own survival advantage.     

Molecular cloning and nucleotide sequence of Thermus thermophilus HB8 trpE and trpG

The trpE gene of Thermus thermophilus HB8 was cloned by complementation of an Escherichia coli tryptophan auxotroph. The E. coli harboring the cloned gene produced the anthranilate synthase I, which was heat-stable and enzymatically active at higher temperature. The nucleotide sequence of the trpE gene and its flanking regions was determined. The trpE gene was preceded by an attenuator-like structure and followed by the trpG gene, with a short gap between them. No other gene essential for tryptophan biosynthesis was observed after the trpG gene. The amino-acid sequences of the T. themophilus anthranilate synthase I and II deduced from the nucleotide sequence were compared with those of other organisms.     

Isolation of infective promastigotes of Leishmania major from long-term culture by cocultivation with macrophage cell line

Research on Leishmania–macrophage interaction is mainly focused on the impact of the parasite on macrophages and several known virulent factors have been described. Furthermore, studies on macrophage revealed several defense mechanisms including various cytokines which are released by macrophages to defend against parasite. In the present study, a new aspect of this interaction was evaluated: parasite characteristics, which emerge when they were cocultivated with macrophage. Two promastigote characteristics, survival at high temperature (32 °C) and infectivity rate were the focus of this study. In this study, an in vitro coculture model for promastigotes with macrophage cell line, J774 A1, was introduced using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane. After 5–7 days of coculturing at 32 °C, a few promastigotes survived longer than control group. Once this population of parasite was cultured at optimal temperature (26 °C), the emerged new clone was much more infective for J774 A1 cell line in comparison with the original one. Having this system and using the new clone of promastigotes, parasite infectivity rate was raised from 1–2% of original clone to 35–45%. Using this new introduced technique, infective promastigotes were isolated from 9 month old frequently sub-cultured clone of Leishmania major. This coculturing system allows investigators to prepare infective promastigotes from the frequently cultured parasites. Molecular and biochemical mechanisms of this phenomenon need to be investigated.     

cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

High toxicity of the novel bloom-forming species Chattonella ovata (Raphidophyceae) to cultured fish

A toxicological study of an axenic cell line of novel species Chattonella ovata Y. Hara et Chihara (Raphidophyceae) revealed that cultured species of sea bream (Pagrus major), horse mackerel (Trachurus japonicus), and yellowtail (Seriola quinqueradiata) were killed by 4.1–6.8 × 10³, 5.4 × 10³, and 2.8 × 10³ cells/mL, respectively. The sensitivity of the gill lamellae to C. ovata differed among the fish species tested. This finding revealed that C. ovata was highly toxic to the cultured fish. Histological examination showed that edema and hyperplasia of the secondary gill lamellae of red sea bream and horse mackerel occurred when exposed to, or killed by C. ovata, whereas severe damage in the gill lamellae was not observed in yellowtail. Chattonella produced high amounts of superoxide anion radicals and hydrogen peroxide, possibly responsible for the fish death observed. Based on the results of this study and occurrence of a red tide by this organism in China in 2001, we consider this organism to be one of the harmful algae in coastal waters. This is the first report demonstrating that C. ovata is highly toxic to fish, and that it produces superoxide and hydrogen peroxide.