cDNA Fragments Research Articles

Cloning and sequencing analysis on the cDNA fragment SI800 separated from rye which related with salt tolerance

SI_(800)(Salt Induced(800 bp),SI_(800)) was a cDNA fragment,which separated from rye leaves by DDRT—PCR.In this study,we purified SI_(800) by low temperature agarose electrophoresis and linked to pGEM—T vector then transformed to Escherichia coli(JM 109).30 positive clones were obtained.2 positive clones were selected randomly after test by digestion,PCR and sequenced.The result indicated that SI_(800) was composed of(735 bp).DNA sequence analysis showed(68%) homologue to uxaB gene for altronate oxidoreductase in GenBank.

Dust mite allergen Der f 4: Expression, characterization, andIgE binding in pediatric asthma.

House dust mite hypersensitivity affects millions of people worldwide, and although many allergens produced by house dust mite species have been identified, some of the less potent allergens remain to be studied. The full-length cDNA encoding the group 4 allergen of the house dust mite species Dermatophagoides farinae (Der f 4) was generated through degenerate primer-based PCR, 5' RACE, and 3' RACE, and the cDNA fragment was cloned into an expression vector for nucleotide sequencing. Following codon optimization and removal of the signal peptide sequence, the mature gene fragment was subcloned into pET-28b (+) and transfected into E.coli BL21 cells for expression. The recombinant protein was purified by nickel affinity chromatography, identified by SDS-PAGE, Western blotting, and MALDI-TOF, and tested by ELISA for IgE reactivity with sera from individuals with asthma. Bioinformatics analyses were used to identify features of Der f 4. SDS-PAGE and Western blotting of the codon-optimized expression product showed a specific band. The mature recombinant Der f 4 was characterized as a stable and hydrophilic 57.9-kDa protein, and its secondary structure comprised alpha helix (25.3%), extended strand (22.51%), and random coils (52.19%). The structure of the recombinant protein was consistent with that of α-amylase. Among 27 pediatric asthma patients, 40.74% exhibited reactivity to rDer f 4 by ELISA. This initial cloning and characterization of the Der f 4 allergen serves as a foundation for future studies into the clinical importance and application of this protein for house dust mite allergy.

Characterization of Toll-like receptor gene expression in goldfish (Carassius auratus) during Dactylogyrus intermedius infection

Toll-like receptors (TLRs), the first and best understood innate immune receptors, play a notable role in the innate immune system by sensing pathogenic agents and initiating appropriate immune responses. However, studies about the roles of fish TLRs in response to the infection of the ectoparasitic monogenean Dactylogyrus intermedius have been surprisingly vacant. In the present study, cDNA fragments of five members of TLRs family in goldfish (Carassius auratus) were cloned and the expression patterns of nine TLRs in five tissues at different time points during D. intermedius infection were subsequently investigated. We found that the expressions of TLR4, TLR5, TLR20 and TLR22 were significantly elevated after infection at some time points, of which the transcription of TLR5 was progressively increased nearly in all tissues, whereas the mRNA levels of other TLRs (TLR2, 3, 7, 9 and 21) were down-regulated or showed no significant change compared with the control at most time points. Additionally, this paper was also conducted to explore the expression of above TLRs after re-infected with D. intermedius. The results showed a significant upregulation of TLR4, TLR5 and TLR22 in all tested tissues at these two time points, especially the levels of TLR4 and TLR22 expression, were even higher comparing with the first infection. Besides, tissue-specific expression analysis revealed that spleen featured the highest expressions of almost all the TLR-encoding genes among detected tissues. The informations obtained here could be helpful towards understanding the functions of TLRs in response to parasitic infection in goldfish and provide new insights for the development of preventive and therapeutic approaches against D. intermedius infection.

Characterization of the gene encoding component C3 of the complement system from the spider Loxosceles laeta venom glands: Phylogenetic implications

A transcriptome analysis of the venom glands of the spider Loxosceles laeta, performed by our group, in a previous study (Fernandes-Pedrosa et al., 2008), revealed a transcript with a sequence similar to the human complement component C3. Here we present the analysis of this transcript. cDNA fragments encoding the C3 homologue (Lox-C3) were amplified from total RNA isolated from the venom glands of L. laeta by RACE-PCR. Lox-C3 is a 5178 bps cDNA sequence encoding a 190kDa protein, with a domain configuration similar to human C3. Multiple alignments of C3-like proteins revealed two processing sites, suggesting that Lox-C3 is composed of three chains. Furthermore, the amino acids consensus sequences for the thioester was found, in addition to putative sequences responsible for FB binding. The phylogenetic analysis showed that Lox-C3 belongs to the same group as two C3 isoforms from the spider Hasarius adansoni (Family Salcitidae), showing 53% homology with these. This is the first characterization of a Loxosceles cDNA sequence encoding a human C3 homologue, and this finding, together with our previous finding of the expression of a FB-like molecule, suggests that this spider species also has a complement system. This work will help to improve our understanding of the innate immune system in these spiders and the ancestral structure of C3.

649. Developing FGFR4 Chimeric Antigen Receptor CAR T Cell Therapy Against Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in pediatrics with an annual incidence of 4.5 cases per 1 million. Patients with high-risk metastatic disease have dismal prognosis and newer treatments are needed. We identified, fibroblast growth factor receptor 4 (FGFR4) as an overexpressed cell surface protein in RMS by mRNA expression analysis. Furthermore, activating mutations in FGFR4 are associated with metastatic disease. FGFR4 protein overexpression in RMS provides a specific target for immune-based therapy of RMS. We are developing T cells genetically modified to express chimeric antigen receptors (CARs) that target FGFR4.To verify FGFR4 RNA expression at the protein level we performed both immunohistochemistry (IHC) and electrochemillumescence (ECL) ELISA assays. Using IHC analysis we measured FGFR4 protein levels on tissue microarrays (TMA) of normal tissue and primary tumor from RMS patients, increased staining for FGFR4 protein on RMS primary tumors, compared to normal tissues was demonstrated. FGFR4 expression measured using ECL ELISA assay, shows a range of 300 - 800pg FGFR4 per 1mg of total lysate in RMS cell lines. The range for normal tissues was 30 - 40 pg/mg for all tissues with the exception of liver, which expressed 70 pg/mg. A single-chain variable fragment (scFv) cDNA library derived from a human B cells was screened, and clones that showed binding to recombinant FGFR4 extracellular domain (ECD) selected. We identified ten specific human anti-FGFR4 scFv binders. The scFvs were cloned into prokaryotic expression vector containing the human IgG1 Fc region. Anti-FGFR4 scFv-Fc were expressed in 293FT cells by transient transfection and purified using Protein A affinity chromatography. The binding of scFv-Fcs to recombinant FGFR4 ECD was verified by ELISA. scFv-Fc binders were then assayed for binding to cell surface FGFR4 on RMS cell lines using flow cytometry. Anti-FGFR4 scFv-Fc bound to 293T cells transfected to express FGFR4 but not 293T control cells. Anti-FGFR4 scFv-Fc also bound FGFR4 on three RMS cell lines. The first two anti-FGFR4 scFv binder sequences evaluated, M410 and M412, were used to make short (S, extracellular scFv only) and long format (L, scFv with a CH2CH3 domain of IgG1) CAR constructs. Activated T cells were transduced with lentiviral CAR expression vector (LV) encoding M410-L, M412-S and M412-L CAR constructs and cell surface expression of FGFR4 CAR on transduced T cells was measured using flow cytometry. The M410 and M412 FGFR4 CARs, both short and long constructs, were tested for cell-mediated cytotoxicity against RMS cell lines. M410-L showed higher cytotoxic activity compared to M412-L. M412-S showed greater cytotoxic activity compared to M412-L CAR. Thus, overall CAR structure format may be important for its functional activity. The remaining scFv will be further analyzed in various CAR formats for its functional activity including cytotoxicity and interferon-gamma production. In summary, the overexpression of FGFR4 protein in RMS versus normal cell lines demonstrates that FGFR4 may be a suitable target for immune-based therapy. FGFR4 CAR-T cell therapy offers the potential of a novel therapeutic intervention for high-risk, refractory and relapsed RMS.

Northwest Innovation pauses permitting for planned Tacoma methanol project

Toll-like receptors (TLRs), the first and best understood innate immune receptors, play a notable role in the innate immune system by sensing pathogenic agents and initiating appropriate immune responses. However, studies about the roles of fish TLRs in response to the infection of the ectoparasitic monogenean Dactylogyrus intermedius have been surprisingly vacant. In the present study, cDNA fragments of five members of TLRs family in goldfish (Carassius auratus) were cloned and the expression patterns of nine TLRs in five tissues at different time points during D. intermedius infection were subsequently investigated. We found that the expressions of TLR4, TLR5, TLR20 and TLR22 were significantly elevated after infection at some time points, of which the transcription of TLR5 was progressively increased nearly in all tissues, whereas the mRNA levels of other TLRs (TLR2, 3, 7, 9 and 21) were down-regulated or showed no significant change compared with the control at most time points. Additionally, this paper was also conducted to explore the expression of above TLRs after re-infected with D. intermedius. The results showed a significant upregulation of TLR4, TLR5 and TLR22 in all tested tissues at these two time points, especially the levels of TLR4 and TLR22 expression, were even higher comparing with the first infection. Besides, tissue-specific expression analysis revealed that spleen featured the highest expressions of almost all the TLR-encoding genes among detected tissues. The informations obtained here could be helpful towards understanding the functions of TLRs in response to parasitic infection in goldfish and provide new insights for the development of preventive and therapeutic approaches against D. intermedius infection.

Identification and characterisation of the immune response properties of Lampetra japonica BLNK.

B cell linker protein (BLNK) is a central linker protein involved in B cell signal transduction in jawed vertebrates. In a previous study, we have reported the identification of a BLNK homolog named Lj-BLNK in lampreys. In this study, a 336 bp cDNA fragment encoding the Lj-BLNK Src homology 2 (SH2) domain was cloned into the vector pET-28a(+) and overexpressed in Escherichia coli BL21. The recombinant fragment of Lj-BLNK (rLj-BLNK) was purifiedby His-Bind affinity chromatography, and polyclonal antibodies against rLj-BLNK were raised in male New Zealand rabbits. Fluorescenceactivated cell sorting (FACS) analysisrevealed that Lj-BLNK was expressed in approximately 48% of the lymphocyte-like cells of control lampreys, and a significant increase in Lj-BLNK expression was observed in lampreys stimulated with lipopolysaccharide (LPS). Western blotting analysis showed that variable lymphocyte receptor B (VLRB) and Lj-BLNKwere distributed in the same immune-relevant tissues, and the levels of both were upregulated in supraneural myeloid bodies and lymphocyte-like cells after LPS stimulation. Immunofluorescence demonstrated that Lj-BLNK was localized in VLRB+ lymphocyte-like cells. These results indicate that the Lj-BLNK protein identified in lampreys might play an important role in the VLRB-mediated adaptive immune response.

Computational characterization and in vivo expression of squalene synthase gene in different tissues of Artemisia annua L. plants

Artemisinin, a sesquiterpene lactone endoperoxide isolated from aerial parts of Artemisia annua L. plants is a safe and potent therapeutic agent against malaria causing drug-resistant parasite strains of Plasmodium sp. Squalene synthase (SQS) is a membrane-bound enzyme catalyzing the first enzymatic step of sterol biosynthetic pathway in eukaryotes. It competes with ADS, the rate limiting enzyme of artemisinin biosynthesis pathway in A. annua L. for the precursor FPP, the product of mevalonate/alternate pathway. Hence, SQS limits artemisinin biosynthesis in A. annua L. plants. In this study, full length cDNA fragment encoding AaSQS enzyme from the leaves of A. annua L. plants was cloned and analyzed. Computational characterization was performed for basic structural and functional information. The physico-chemical properties were also studied. The putative conserved domains in corresponding sequences were searched. The multiple sequence alignment of AaSQS revealed the important motifs and identified the residue substitution in each motif. The homology modeling of AaSQS enzyme was done for the prediction of 3D-structure. Molecular docking of one substrate and two co-factors with AaSQS was performed and the docked structures showed binding affinity for motif 2 of AaSQS. Our analysis further revealed that 29 residues of motif 2 might be important for catalytic/functional activity of AaSQS enzyme. Relative ontogenetic expression analysis of SqS gene employing quantitative real-time PCR showed that the expression level of AaSqS was higher in Callus (C) followed by leaves (L), stem (S) and hairy roots (HR) that was well correlated with the squalene content in these tissues. This study thus, may provide a platform to understand and use the predicted information of AaSQS in modulating the production of artemisinin in A. annua L. plants.

Over-expression of poplar transcription factor ERF76 gene confers salt tolerance in transgenic tobacco

Ethylene response factors (ERFs) belong to a large plant-specific transcription factor family, which play a significant role in plant development and stress responses. Poplar ERF76 gene, a member of ERF TF family, can be up-regulated in response to salt stress, osmotic stress, and ABA treatment. The ERF76 protein was confirmed to be targeted preferentially in the nucleus of onion cell by particle bombardment. In order to understand the functions of ERF76 gene in salt stress response, we conducted temporal and spatial expression analysis of ERF76 gene in poplar. Then the ERF76 cDNA fragment containing an ORF was cloned from di-haploid Populus simonii×P. nigra and transferred into tobacco (Nicotiana tobacum) genome by Agrobacterium-mediated leaf disc method. Under salt stress, transgenic tobacco over-expressing ERF76 gene showed a significant increase in seed germination rate, plant height, root length, and fresh weight, as well as in relative water content (RWC), superoxide dismutase (SOD) activity, peroxidase (POD) activity, and proline content, compared to control tobacco lines. In contrast, transgenic tobacco lines displayed a decrease in malondialdehyde (MDA) accumulation, relative electrical conductivity (REC) and reactive oxygen species (ROS) accumulation in response to salt stress, compared to control tobacco lines. Over all, the results indicated that ERF76 gene plays a critical role in salt tolerance in transgenic tobacco.

Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples

BackgroundBRCA1 and BRCA2 are the two principal tumour suppressor genes associated with inherited high risk of breast and ovarian cancer. Genetic testing of BRCA1/2 will often reveal one or more...

Read-through transcripts in normal human lung parenchyma are down-regulated in lung adenocarcinoma

Read-through transcripts result from the continuous transcription of adjacent, similarly oriented genes, with the splicing out of the intergenic region. They have been found in several neoplastic and normal tissues, but their pathophysiological significance is unclear. We used high-throughput sequencing of cDNA fragments (RNA-Seq) to identify read-through transcripts in the non-involved lung tissue of 64 surgically treated lung adenocarcinoma patients. A total of 52 distinct read-through species was identified, with 24 patients having at least one read-through event, up to a maximum of 17 such transcripts in one patient. Sanger sequencing validated 28 of these transcripts and identified an additional 15, for a total of 43 distinct read-through events involving 35 gene pairs. Expression levels of 10 validated read-through transcripts were measured by quantitative PCR in pairs of matched non-involved lung tissue and lung adenocarcinoma tissue from 45 patients. Higher expression levels were observed in normal lung tissue than in the tumor counterpart, with median relative quantification ratios between normal and tumor varying from 1.90 to 7.78; the difference was statistically significant (P < 0.001, Wilcoxon's signed-rank test for paired samples) for eight transcripts: ELAVL1–TIMM44, FAM162B–ZUFSP, IFNAR2–IL10RB, INMT–FAM188B, KIAA1841–C2orf74, NFATC3–PLA2G15, SIRPB1–SIRPD, and SHANK3–ACR. This report documents the presence of read-through transcripts in apparently normal lung tissue, with inter-individual differences in patterns and abundance. It also shows their down-regulation in tumors, suggesting that these chimeric transcripts may function as tumor suppressors in lung tissue.

Prediction of VIGS efficiency by the Sfold program and its reliability analysis in Gossypium hirsutum

Genetic transformation in some plant species, including cotton (Gossypium hirsutum), is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing (VIGS) by double-stranded RNAs can serve as a reverse-genetics tool to determine gene function. However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different cDNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA (siRNA) in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of siRNA in target sequence: the disruption energy of the target (ΔGdisruption), the differential stability of siRNA duplex ends (DSSE), and the internal stability at positions 9–14 of the siRNA antisense strand (AIS), which are calculated by Sfold program (http://sfold.wadsworth.org). We find that the siRNAs with low ΔGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, GhPDS, GhCLA1, GhAOS1, and GhCXE1 via choosing different target sequences for each gene. Therefore, the Sfold program can be used to analyze target sequences when carrying out VIGS design to increase gene-silencing effects in plants.

Expression of cDNA of the gene for the capsid protein VP2 of German cockroach densovirus in the transgenic strain of Drosophila melanogaster

Transgenic strains of Drosophila melanogaster capable of expressing a cDNA fragment corresponding to open reading frame (ORF) of the gene for the German cockroach densonucleosis virus capsid protein VP2 (ORF VP2) in specific tissues and at a certain stage of development depending on the type of chosen driver strains (GAL-UAS system) were obtained. The ORF VP2 transcription was examined at the imago stage after crossing the obtained transgenic Drosophila with the driver line expressing the inducer protein (GAL4) under control of actin promoter (the ORF VP2 expression is induced in all tissues of the first-generation Drosophila). It was demonstrated that the greater part of transcribed foreign RNA was represented by three spliced variants in which RNA fragments either between nucleotides 137 and 353 or between nucleotides 609 and 1925 were excised; the third spliced variant was represented by RNA lacking both introns. Using the next-generation sequencing (NGS) technique, the proportion of unspliced form relative to spliced variants of the analyzed RNA was assessed. It was shown that the ratio of unspliced form to the identified spliced variants of the analyzed RNA was approximately 1:6. It is suggested that splicing of viral RNA foreign to Drosophila can be a sort of defense mechanism preventing the large-scale production of the capsid protein, potentially hazardous to the host organism.

Expression and functional analysis of Fas ligand‐like protein from silkworm, Bombyx mori

In the silkworm, Bombyx mori, the degradation of larval tissues is an essential process for generating raw materials to build an adult body. It has been shown that most of the larval tissues disappear in the pupal stage upon activation of Bmcaspase dependent apoptosis. As an approach to identify the ligand responsible for initiating the death pathway, we searched Silk Base database and cloned a candidate cDNA fragment encoding a protein sequence similar to the mammalian Fas ligand in the Pichia Pastoris yeast expression system. The expressed recombinant protein was purified using Ni‐agarose affinity column and Sephacryl S‐300 gel filtration column. While the gel filtration column chromatography gave a molecular weight of 108,000, the subunit molecular weight was 38,000 as estimated by using SDS‐PAGE under reducing condition, indicating that the protein was purified as a homo‐trimer. To verify its capability of inducing apoptosis, the recombinant protein was injected into the larvae of V6 (6th day after entering fifth instar) at various amounts. All larvae died within 24 hours at the dosage of 16 ng or more protein. Inside the dead bodies, the lysis of the larval tissues was observed and a remarkable elevation of Bmcaspase activity was detected; Ac‐DEVD‐MCA, an artificial fluorescent substrate, was used. Western blot analysis revealed the occurrence of BmJNK phosphorylation in the same tissues. Our results clearly showed that the purified recombinant protein is able to induce Bmcaspase‐dependent apoptosis that causes disappearance of larval tissues, possibly through BmJNK signaling pathway.

Heterologous expression and characterisation of the Aspergillus aspartic protease involved in the hydrolysis and decolorisation of red-pigmented proteins.

Aspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorises red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires the high-level production and efficient preparation of the recombinant enzyme. The genomic DNA and cDNA fragments encoding the protease were cloned from strain MK82 and sequenced. Phylogenetic analysis of PepA_MK82 and comparisons with previously reported fungal aspartic proteases showed that PepA_MK 82 clusters with different groups of these enzymes. Heterologous expression of PepA_MK82 in Pichia pastoris yielded preparations of higher purity than obtained with an Escherichia coli expression system. Total protease activity in a 100-mL culture of the P. pastoris transformant was 14 times higher than that from an equivalent culture of A. repense MK82. The recombinant PepA_MK82 was easily obtained via acetone precipitation; the final recovery was 83%. PepA_MK82 and its recombinant had similar characteristics in terms of their optimal pH, thermostability, and decolorisation activity. The recombinant was also able to decolorise flaked, dried bonito and to bleach a blood-stained cloth. Given its ability to hydrolyse and decolorise red-pigmented proteins, recombinant PepA_MK8 can be exploited in the food industry and as a stain-removal agent in laundry applications. © 2016 Society of Chemical Industry.

Overexpression of a synthetic insect–plant geranyl pyrophosphate synthase gene in Camelina sativa alters plant growth and terpene biosynthesis

A novel plastidial homodimeric insect-plant geranyl pyrophosphate synthase gene is synthesized from three different cDNA origins. Its overexpression in Camelina sativa effectively alters plant development and terpenoid metabolism. Geranyl pyrophosphate synthase (GPPS) converts one isopentenyl pyrophosphate and dimethylallyl pyrophosphate to GPP. Here, we report a synthetic insect-plant GPPS gene and effects of its overexpression on plant growth and terpenoid metabolism of Camelina sativa. We synthesized a 1353-bp cDNA, namely PTP-MpGPPS. This synthetic cDNA was composed of a 1086-bp cDNA fragment encoding a small GPPS isomer of the aphid Myzus persicae (Mp), 240-bp Arabidopsis thaliana cDNA fragment encoding a plastidial transit peptide (PTP), and a 27-bp short cDNA fragment encoding a human influenza hemagglutinin tag peptide. Structural modeling showed that the deduced protein was a homodimeric prenyltransferase. Confocal microscopy analysis demonstrated that the PTP-MpGPPS fused with green florescent protein was localized in the plastids. The synthetic PTP-MpGPPS cDNA driven by 2×35S promoters was introduced into Camelina (Camelina sativa) by Agrobacterium-mediated transformation and its overexpression in transgenic plants were demonstrated by western blot. T2 and T3 progeny of transgenic plants developed larger leaves, grew more and longer internodes, and flowered earlier than wild-type plants. Metabolic analysis showed that the levels of beta-amyrin and campesterol were higher in tissues of transgenic plants than in those of wild-type plants. Fast isoprene sensor analysis demonstrated that transgenic Camelina plants emitted significantly less isoprene than wild-type plants. In addition, transcriptional analyses revealed that the expression levels of gibberellic acid and brassinosteroids-responsive genes were higher in transgenic plants than in wild-type plants. Taken together, these data demonstrated that this novel synthetic insect-plant GPPS cDNA was effective to improve growth traits and alter terpenoid metabolism of Camelina.

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates.

Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.

Molecular characterization of a novel nitric oxide synthase gene from Portunus trituberculatus and the roles of NO/O2(-)- generating and antioxidant systems in host immune responses to Hematodinium.

Increasing evidences have established that the nitric oxide synthase (NOS) and NADPH oxidase (NOX) play important roles in host defense system by catalyzing the production of nitrogen oxide (NO) and superoxide anions (O2(-)), respectively. While, there are limited studies to explore the roles of NOS/NOX enzymes in crustacean immunity, and no studies as yet were attempted to elucidate their functions in host immune responses to parasites. In the present study, we cloned a full-length cDNA of NOS and two partial cDNA fragments of NOX and GPx from the economic valuable crab Portunus trituberculatus. The full-length cDNA of NOS was 4002 bp in length that encoded 1203 amino acids containing motifs of the NOS protein and conserved domains. The phylogenetic analysis showed that the NOS protein sequence was clustered together with those of crustacean species in the phylogenetic tree. All of the three novel genes showed high mRNA transcripts in the immune-related tissues (e.g. hemocytes, hepatopancreas) of P. trituberculatus. Striking fluctuation in the transcripts of the critical NO/O2(-)- generating/scavenging related genes (NOS, NOX, CuZnSOD, CAT, GPx) as well as in the enzymatic activities of NOS, NOX, SOD, CAT and GPx were observed in the hemocytes and hepatopancreas of P. trituberculatus post challenged with the parasitic dinoflagellate Hematodinium, indicating that the NO/O2(-)- generating and the antioxidant systems played vital roles in the crustacean innate immunity against the parasitic intrusion. The results indicated a novel respect of the host-parasite interaction between the crab host and the parasitic dinoflagellate Hematodinium.

Transgenic poplar overexpressing the endogenous transcription factorERF76gene improves salinity tolerance

The ethylene response factor (ERF) family is one of the largest plant-specific transcription factor families, playing an important role in plant development and response to stresses. The ERF76 gene is a member of the poplar ERF transcription factor gene family. First, we validated that the ERF76 gene expressed in leaf and root tissues is responsive to salinity stress. We then successfully cloned the ERF76 cDNA fragment containing an open reading frame from di-haploid Populus simonii × Populus nigra and proved that ERF76 protein is targeted to the nucleus. Finally, we transferred the gene into the same poplar clone by the Agrobacterium-mediated leaf disc method. Using both RNA-Seq and reverse transcription-quantitative polymerase chain reaction, we validated that expression level of ERF76 is significantly higher in transgenic plants than that in the nontransgenic control. Using RNA-Seq data, we have identified 375 genes that are differentially expressed between the transgenic plants and the control under salt treatment. Among the differentially expressed genes, 16 are transcription factor genes and 45 are stress-related genes, both of which are upregulated significantly in transgenic plants, compared with the control. Under salt stress, the transgenic plants showed significant increases in plant height, root length, fresh weight, and abscisic acid (ABA) and gibberellin (GA) concentration compared with the control, suggesting that overexpression of ERF76 in transgenic poplar upregulated the expression of stress-related genes and increased the ability of ABA and GA biosynthesis, which resulted in stronger tolerance to salt stress.

A new antimicrobial protein from the anterior midgut of Triatoma infestans mediates Trypanosoma cruzi establishment by controlling the microbiota.

The Reduviid Triatoma infestans is a vector for the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. The parasite must address the defense molecules and microbiota that colonize the anterior midgut of T.infestans. To obtain insight into T.cruzi - microbiota interactions in triatomine insects, we characterized a new antimicrobial product from the anterior midgut of T.infestans (TiAP) that may be involved in these relationships. The TiAP DNA fragment was cloned and expressed in a bacterial system, and the effect of the protein on bacteria and T.cruzi was evaluated by RNAi, qPCR and antimicrobial experiments. The number of T.cruzi in T.infestans anterior midguts was significantly lower in TiAP knockdown insects than in unsilenced groups. We also verified that the amount of bacteria in silenced T.infestans is approximately 600-fold higher than in unsilenced insects by qPCR. The 327-bp cDNA fragment that encodes mature TiAP was cloned into the pET-14b vector and expressed fused to a His-tag in Escherichia coli C43. The recombinant protein (rTiAP) was purified using an Ni-NTA column, followed by a HiTrap SP column. According to a trypanocidal assay, rTiAP did not interfere with the viability of T.cruzi trypomastigotes. Moreover, in antimicrobial experiments using E.coli and Micrococcus luteus, the protein was only bacteriostatic for Gram-negative bacteria. The data indicate that infection by T.cruzi increases the expression of TiAP to modulate the microbiota. The inhibition of microbiota growth by TiAP is important for parasite establishment in the T.infestans anterior midgut.