Full cDNA Sequence Research Articles

BpAFP, a Broussonetia papyrifera latex chitinase, exhibits a dual role in resisting to both Verticillium wilt disease and lepidopterous pests, Plutella xylostella and Prodenia litura

Paper mulberry (Broussonetia papyrifera) is a fast-growing tree known for its tolerance to diverse biotic and abiotic stresses. To explore genes combating Verticillium wilt, a devasting and formidable disease damage to cotton and many economically significant crops, we purified an antifungal protein, named BpAFP, from the latex of paper mulberry. Based on peptide fingerprint, we cloned the full cDNA sequence of BpAFP and revealed that BpAFP belongs to Class I chitinases, sharing 74 % identity with B. papyrifera leaf chitinase, PMAPII. We further introduced BpAFP into Arabidopsis, tobacco, and cotton. Transgenic plants exhibited significant resistance to Verticillium wilt. Importantly, BpAFP also demonstrated insecticidal activity against herbivorous pests, Plutella xylostella, and Prodenia litura, when feeding the larvae with transgenic leaves. Our finding unveils a dual role of BpAFP in conferring resistance to both plant diseases and lepidopterous pests.

Lncrna Famlf-2 Promotes Leukemogenesis Via the DHX9/c-Myc Axis in Acute Lymphoblastic Leukemia

Long noncoding RNAs (lncRNAs), defined as the transcripts longer than 200 nt without protein-coding capacity, have been found to be aberrantly expressed in diverse human diseases including leukemia. Our previous study identified Familial acute myelogenous leukemia related factor ( FAMLF) in an acute myeloblastic leukemia (AML) pedigree. Two splice variants of FAMLF, FAMLF-1 and FAMLF-2 were detected. FAMLF-1 is upgraded in AML patients and associated with poor prognosis. However, the full cDNA sequence of FAMLF-2 is not verified and its role in leukemogenesis remain unclear. Herein, we aim to investigate the functional and mechanistic roles of FAMLF-2 in leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of FAMLF-2 was at higher levels in Acute Lymphoblastic Leukemia(ALL) cell lines when compared to AML cell lines. Bone marrow cells were obtained from de novo ALL patients. qRT-PCR showed FAMLF-2 was up-regulated in ALL patients (N=115) compared to healthy controls (N=72). The FAMLF-2 expression was positively correlated with the poor prognosis in ALL patients by Kaplan-Meier analysis. These data demonstrated that FAMLF-2 is overexpressed in ALL patients and is associated with poor prognosis. To verify the full cDNA sequence of FAMLF-2, 5‘ and 3‘ rapid amplification of the cDNA ends (RACE) was performed, and we identified the 2393 nt of FAMLF-2. FAMLF-2 is predicted to not have protein-coding potential. Further subcellular analysis showed FAMLF-2 is mainly localized in the nucleus of Jurkat cells by lncRNA FISH and subcellular fractionation assay. To further verify the role of FAMLF-2 in ALL leukemogenesis, a xenograft model was established in NCG mice by subcutaneously or intravenously injection of engineered Jurkat cells (knockdown group: shNC and sh FAMLF-2, overexpression group: pCDH and FAMLF-2). We found that the size and weight of tumors in FAMLF-2 knockdown group were significantly decreased compared with the empty vector in subcutaneous ALL xenograft tumor model. and the mice in the sh FAMLF-2 groups survived longer than those in the shNC groups in introvenous ALL xenograft tumor model (sh FAMLF-2 vs. shNC: 55 days vs. 43 days, P<0.01). These data suggested that downregulation of FAMLF-2 inhibits leukemia development. Although no significant difference was found in the overall survival of mice overexpressed FAMLF-2. We observed the accelerated central nervous system(CNS) infiltration of leukemia cells when FAMLF-2 was overexpressed (clinical score at 32th day from Vector vs. FAMLF-2: 2.7 scores vs. 1.2 scores, p < 0.01 ). In vitro study revealed knockdown of FAMLF-2 inhibited cell proliferation and promoted cell apoptosis in ALL cells, and vice versa. Taken together, our clinical, mouse model, and AML cell data demonstrated that FAMLF-2 promotes leukemogenesis. RNA sequencing was performed to study how FAMLF-2 promotes ALL development. Gene Set Enrichment Analysis (GSEA) of genes downregulated by FAMLF-2 showed “Myc targets” with the highest normalised enrichment score (NES) in Jurkat cells. C-Myc is a well-recognized transcription factor that involves in leukemogenesis. To investigate whether FAMLF-2 performed direct interaction with c-Myc, we performed RNA pull-down followed by mass spectrometry analysis. Instead of c-Myc, DExH-Box Helicase 9 (DHX9) has the highest abundance. The Western blot and RNA immunoprecipitation (RIP) assay were further performed to validated the direct interaction between FAMLF-2 and DHX9. DHX9 is an RNA helicase that participates in c-Myc mRNA stability. Inhibition of FAMLF-2 suppressed DHX9 and c-Myc expression. Reduced interaction between DHX9 protein and c-Myc mRNA were observed when FAMLF-2 were downregulated. Furthermore, RNA stability assays suggested that knockdown of FAMLF-2 leads to a declined half-life of c-Myc mRNA. Collectively, these results demonstrated that FAMLF-2 regulates the stability of c-Myc mRNA through direct binding of DHX9, thus involved in the ALL development. In summary, our study revealed the previous undefined cDNA sequence of FAMLF-2, and provides new insights of FAMLF-2 in leukemogenesis via the DHX9/c-Myc axis, which might act as a prospective prognostic biological marker in ALL.

Molecular cloning and characterization of endoplasmic reticulum stress related genes grp78 and atf6α from black seabream (Acanthopagrus schlegelii) and their expressions in response to nutritional regulation.

Glucose-regulated protein 78 (grp78) and activating transcription factor 6α (atf6α) are considered vital endoplasmic reticulum (ER) molecular chaperones and ER stress (ERS) sensors, respectively. In the present study, the full cDNA sequences of these two ERS-related genes were first cloned and characterized from black seabream (Acanthopagrus schlegelii). The grp78 cDNA sequence is 2606 base pair (bp) encoding a protein of 654 amino acids (aa). The atf6α cDNA sequence is 2168 base pair (bp) encoding a protein of 645 aa. The predicted aa sequences of A. schlegelii grp78 and atf6α indicated that the proteins contain all the structural features, which were characteristic of the two genes in other species. Tissues transcript abundance analysis revealed that the mRNAs of grp78 and atf6α were expressed in all measured tissues, but the highest expression of these two genes was all recorded in the gill followed by liver/ brain. Moreover, in vivo experiment found that fish intake of a high lipid diet (HLD) can trigger ERS by activating grp78/Grp78 and atf6α/Atf6α. However, it can be alleviated by dietary betaine supplementation, similar results were also obtained by in vitro experiment using primary hepatocytes of A. schlegelii. These findings will be beneficial for us to evaluate the regulator effects of HLD supplemented with betaine on ERS at the molecular level, and thus provide some novel insights into the functions of betaine in marine fish fed with an HLD.

Molecular cloning and tissue distribution of glucokinase and glucose-6-phosphatase catalytic subunit paralogs in largemouth bass Micropterus salmoides: Regulation by dietary starch levels and a glucose load

The dysregulation of glucose-G6P (glucose-6-phosphate) interconversion is thought to be one of the main reasons for the low glucose disposal of carnivorous fish, but is not yet well understood in largemouth bass Micropterus salmoides (LMB). In this study, the full length cDNA sequences of genes encoding glucokinase (Gck, catalyzing glucose phosphorylation) and glucose-6-phosphatase catalytic subunit (G6pc, catalyzing glucose dephosphorylation) were cloned by the RACE method from the liver of LMB. Subsequently, the distribution of g6pc and gck as well as their transcriptional regulation by dietary starch levels and a glucose load were investigated. Only one gck gene was identified, while the tandem duplication of g6pca.1 gene was named as g6pca.2 in LMB. The full cDNA sequences of g6pca.1, g6pca.2 and gck in LMB were 1585, 1813 and 2115 bp in length, encoding 478, 352 and 359 amino acids, respectively. Gck was predicted to contain two hexokinase domains, an ATP-binding domain and multiple functional sites, while G6pca.1 and G6pca.2 contained nine transmembrane helices, a PAP2 (type-2 phosphatidic acid phosphatase) domain and multiple functional amino acid sites. Both g6pca.1 and g6pca.2 were predominantly distributed in the liver and to some extent in the intraperitoneal fat, intestine and pyloric caeca, while gck was mainly transcribed in the liver and to some extent in the heart, intestine and brain. Both feeding a high starch diet and a glucose load stimulated the mRNA expression of gck in the liver of LMB. An increase of dietary starch from 9% to 14% down-regulated the transcription of g6pca.1 in the liver of LMB. However, both the mRNA levels of hepatic g6pca.1 and g6pca.2 were sharply up-regulated in LMB during 1–3 h after a glucose load. Overall, the results of this study suggested that the functions of G6pc (G6pca.1 and G6pca.2) and Gck in LMB were highly conserved in evolution. Though hepatic glucose-G6P interconversion was well regulated at the transcript level in LMB fed high starch diets, a futile cycle between glucose and G6P was induced in the liver after a glucose load.

A signal R3-type, CAPRICE-like MYB transcription factor from Dendrobium nobile controls trichome and root-hair development in Arabidopsis

The CAPRICE-like MYB transcription factors with R3 MYB motif play a central role in regulating trichome and root-hair development in plants. We identified the homologous gene of ENHANCER OF TRY AND CPC (ETC) in Arabidopsis from Dendrobium nobile Lindl with full cDNA sequence and genomic sequence (CAPRICE-LIKE MYB, DnCPL and DngCPL) respectively. Phylogenic analyses revealed a close relationship of CAPRICE-like MYB TFs between D. nobile and A. thaliana. Promoter analysis indicated that DnCPL is specifically expressed in trichome basal cells of leaf epidermis and root hairs. Overexpression of DnCPL results in the suppression of trichome formation and overproduction of root hairs. In transgenic plants overexpressing DnCPL and DngCPL, trichome formation was inhibited, moreover, no trichomes were observed in tissues of aerial parts, and root-hair differentiation was significantly enhanced by strongly repressing endogenous genes of AtCPC, AtTCL1, and AtTCL2 expression, thereby enhancing AtTRY expression. The DnCPL RNAi plants formed fewer lateral roots with a corresponding change in AtCPC, AtTCL1 and AtTCL2 expression. These results suggest that Dendrobium and Arabidopsis partially use similar transcription factors for epidermal cell differentiation and the CPC-like R3 MYB, DnCPL, may be a key common regulator of plant trichome and root-hair development. The results also provided genes and means of regulation to improve the survival ratio of artificially cultivated Dendrobium with more lateral roots.

Characterization of novel CACNA1A splice variants by RNA-sequencing in patients with episodic or congenital ataxia.

Loss of function variants in CACNA1A cause a broad spectrum of neurological disorders, including episodic ataxia, congenital or progressive ataxias, epileptic manifestations or developmental delay. Variants located on the AG/GT consensus splice sites are usually considered as responsible of splicing defects, but exonic or intronic variants located outside of the consensus splice site can also lead to abnormal splicing. We investigated the putative consequences on splicing of 11 CACNA1A variants of unknown significance (VUS) identified in patients with episodic ataxia or congenital ataxia. In silico splice predictions were performed and RNA obtained from fibroblasts was analyzed by Sanger sequencing. The presence of abnormal transcripts was confirmed in 10/11 patients, nine of them were considered as deleterious and one remained of unknown significance. Targeted next-generation RNA sequencing was done in a second step to compare the two methods. This method was successful to obtain the full cDNA sequence of CACNA1A. Despite the presence of several isoforms in the fibroblastic cells, it detected most of the abnormally spliced transcripts. In conclusion, RNA sequencing was efficient to confirm the pathogenicity of nine novel CACNA1A variants. Sanger or Next generation methods can be used depending on the facilities and organization of the laboratories.

Identification of potential functions of polo-like kinase 1 in male reproductive development of the oriental river prawn (Macrobrachium nipponense) by RNA interference analysis.

Polo-like kinase 1 (Plk1) has multiple functions in the cell cycle, including in the maturation of centrosomes during the G2/M transition, the separation of centrosomes, and the activation of cyclin-dependent kinase 1 expression and spindle assembly. In this study, we investigated the potential regulatory roles of Plk1 in the reproductive development of the male oriental river prawn (Machrobrachium nipponense). The full cDNA sequence of Mn-Plk1 was 2360 base pairs long, with an open reading frame of 1836 base pairs encoding 611 amino acids. Protein sequence alignment identified a conserved serine/threonine kinase domain and two Polo-boxes. Phylogenetic tree analysis revealed that Mn-Plk1 had the closest evolutionary distance with Plk1s of freshwater prawns and then with those of crustacean species, whereas the evolutionary distance with mollusks was much more distant. Quantitative PCR analysis predicted that Mn-Plk1 plays essential roles in the regulation of gonad development. RNA interference analysis and histological observations showed that expression of insulin-like androgenic gland hormone decreased as the expression of Mn-Plk1 decreased, and fewer than 5% of cells were sperm cells at day 14 in the dsPlk1 injected prawns. This result indicated that Plk1 positively regulated testis development in M. nipponense by affecting the expression of this hormone. Our results highlight the functions of Plk1 in M. nipponense and provide valuable information that can be applied to establish artificial techniques to regulate testis development in this species.

Molecular characterization and expression profiling oftransformer 2 and fruitless-like homologs in the black tiger shrimp, Penaeus monodon.

Transformer 2 (tra 2) and fruitless (fru) genes have been proven to play a key role in sex determination pathways in many Arthropods, including insects and crustaceans. In this study, a paralog of P. monodon tra 2 (Pmtra 2), P. monodon ovarian associated transformer 2 (PmOvtra 2) and 2 isoforms of P. monodon fruitless-like gene (Pmfru-1 and Pmfru-2) were identified and characterized. The full cDNA sequence of PmOvtra 2 consisted of 1,774 bp with the longest open reading frame (ORF) of 744 bp encoding for 247 amino acids. The PmOvtra 2 exhibited a predicted RNA-recognition motif (RRM) domain and two arginine-serine (RS) regions, suggesting its function in RNA splicing. The full cDNA sequence of Pmfru-1 consisted of 1,306 bp with 1,182 bp ORF encoding for 393 amino acids, whereas the full cDNA sequence of Pmfru-2 consisted of 1,858 bp with 1,437 bp ORF encoding 478 amino acids. The deduced amino acid sequences of Pmfru-1 and Pmfru-2 exhibited highly conserved domains of Fru proteins, including Broad-complex, Tramtrack and Bric-a-brac (BTB), and zinc finger (ZF) domains. In addition, Pmfru-1 and Pmfru-2 were suggestively originated from the same single genomic locus by genomic sequence analysis. Specifically, Pmfru pre-mRNA was alternatively spliced for Pmfru-1 and Pmfru-2 to include mutually exclusive exon 7 and exon 6, respectively. Temporal and spatial expression of PmOvtra 2, Pmfru-1, and Pmfru-2 were also investigated by qPCR. The results showed that all were expressed in early developmental stages with undifferentiated gonads starting from nauplius until postlarvae. The expression of PmOvtra 2 started at nauplius stage and gradually increased from mysis to postlarvae (PL) 1. However, the expression of Pmfru-1 was low at the nauplii stage and slightly increased from protozoea to PL5, whereas the expression of Pmfru-2 maintained a low level from nauplius to mysis and then gradually increased at the PL stages. Expressions of PmOvtra 2, Pmfru-1, and Pmfru-2 were detected in various tissues including nervous tissue, gill, heart, hepatopancreas, gut, and gonads. Interestingly, the sexually dimorphic expression of PmOvtra 2, Pmfru-1, and Pmfru-2 was demonstrated in fully developed gonads in which the ovary showed significantly higher expressions than the testis. The great difference in the expression pattern of PmOvtra 2, Pmfru-1, and Pmfru-2 in the ovary and testis suggested their roles in the female sex determination in P. monodon.

Identification and Characterization of the Pyruvate Dehydrogenase E1 Gene in the Oriental River Prawn, Macrobrachium nipponense.

Pyruvate dehydrogenase E1 (PDHE1) is thought to play essential roles in energy metabolism, and a previous study suggested that it also has potential regulatory roles in male sexual development in the oriental river prawn, Macrobrachium nipponense. In this study, we used rapid amplification of cDNA ends, quantitative polymerase chain reaction (qPCR), in situ hybridization, western blotting, RNA interference (RNAi), and histological analyses to assess the potential functions of Mn-PDHE1 in the sexual development of male M. nipponense. The full cDNA sequence of Mn-PDHE1 was 1,614 base pairs long, including a 1,077 base pair open reading frame that encodes 358 amino acids. qPCR analysis revealed the regulatory functions of PDHE1 in male sexual development in M. nipponense and in the metamorphosis process. In situ hybridization and western blot results indicated that PDHE1 was involved in testis development, and RNAi analysis showed that PDHE1 positively regulated the expression of insulin-like androgenic gland factor in M. nipponense. Compared with the cell types in the testes of control prawns, histological analysis showed that the number of sperm was dramatically lower after test subjects were injected with Mn-PDHE1 dsRNA, whereas the numbers of spermatogonia and spermatocytes were higher. Sperm constituted only 1% of cells at 14 days after injection in the RNAi group. This indicated that knockdown of the expression of PDHE1 delayed testis development. Thus, PDHE1 has positive effects on male sexual development in M. nipponense. This study highlights the functions of PDHE1 in M. nipponense and its essential roles in the regulation of testis development.

Molecular Characterization and Expression Analysis of Toll-like Receptor 9 Gene in Turbot, Scophthalmus Maximus

Toll-like receptors (TLRs) have emerged as key sensors of invading microbes by recognizing pathogen-associated molecular patterns and activating innate immune responses. TLR9 functions as a pattern-recognition receptor that recognizes unmethylated CpG motifs in bacterial and viral DNA. In this study, the full cDNA and genomic sequences of tlr9 in Scophthalmus maximus (smtlr9) were identified and characterized. The full-length cDNA of smtlr9 was of 3754 bp encoding a polypeptide of 1066 amino acid residues. The genome sequence of smtlr9 was composed of 4083 nucleotides, including three exons and two introns. The putative Smtlr9 protein was characterized by a signal peptide sequence, a leucine-rich repeat domain (LRD) containing 15 leucine-rich repeats (LRRs), a transmembrane region, and a Toll/interleukin-1 receptor (TIR) domain. The putative protein shows the highest sequence identity (36.0~73.6%) to human and fish TLR9s. Phylogenetic analysis grouped Smtlr9 with other teleost Tlr9s. The smtlr9 mRNA was constitutively expressed in all tissues examined, with the highest level in the head kidney and kidney. The smtlr9 transcription level was up-regulated by ODN2395 in the muscle, head kidney, spleen, and gills.

Differential expression and functional analysis of two short-chain alcohol dehydrogenases/reductases in Hedychium coronarium

Abstract In this study, the full cDNA sequences of HcADH2 and HcADH3 were cloned from Hedychium coronarium. The amino acid sequences encoded by them contained three most conserved motifs of short-chain alcohol dehydrogenase (ADH), namely NAD+ binding domain, TGxxx[AG]xG and active site YxxxK. The highest similarity between two genes and ADH from other plants was 70%. Phylogenetic analysis showed that they belonged to a member of the short-chain dehydrogenases/reductases 110C subfamily, but they were distinctly clustered in different clades. Real-time polymerase chain reaction analyses showed that HcADH2 was specifically expressed in bract, and it was expressed higher in no-scented Hedychium forrestii than other Hedychium species, but was undetectable in Hedychium coccineum. HcADH3 was expressed higher in the lateral petal of the flower than in other vegetative organs, and it was expressed the most in H. coronarium that is the most scented among Hedychium species, and its expression levels peaked at the half opening stage. HcADH2 and HcADH3 had almost no significant expression in leaves, but HcADH2 was expressed in response to external stimuli. The mechanical injury and methyl jasmonate (MeJA) treatment could induce expression of HcADH2 in leaves, whereas HcADH3 could have an induced expression only by MeJA. The recombinant HcADH3 protein, but not HcADH2, expressed in Escherichia coli-catalysed conversion of geraniol into citral. It was speculated that HcADH3 had an induced expression in vegetative organ of H. coronarium and took part in monoterpenoid biosynthesis in H. coronarium flowers, but the role of HcADH2 is relevant only for defensive reactions.

Identification and characterization of the vasa gene in the diamondback moth, Plutella xylostella

Vasa is an ATP-dependent RNA helicase, participating in multiple biological processes. It has been widely used as a germ cell marker and its promoter has become a key component of several genetic pest control systems. Here we present the vasa gene structure and its promoter activity in Plutella xylostella, one of the most destructive pests of cruciferous crops. Full length Pxvasa cDNA sequences were obtained, revealing 14 exons and at least 30 alternatively spliced transcripts. Inferred amino acid sequences showed nine conserved DEAD-box family protein motifs with partial exclusion from some isoforms. Real-time quantitative PCR indicated the up-regulation of Pxvasa in both female and male adults compared with other developmental stages, and the expression levels of Pxvasa were found to be much higher in adult gonads, especially ovaries, than in other tissues. The putative promoter region of Pxvasa was sequenced and several ecdysone-induced transcription factor (TF) binding sites were predicted in silico. To further analyze the promoter region, two upstream regulatory fragments of different lengths were tested as putative promoters in transient cell and embryo expression assays, one of which was subsequently utilized to drive Cas9 expression in vivo. A transgenic line was recovered and the expression patterns of Cas9 and native Pxvasa were profiled in adult tissues and eggs with RT-PCR. This work provides the foundation for further studies on the gene functions of Pxvasa as well as the potential application of its promoter in genetic manipulation of P. xylostella.

Identification of heme oxygenase-1 from golden pompano (Trachinotus ovatus) and response of Nrf2/HO-1 signaling pathway to copper-induced oxidative stress

Heme oxygenase-1(HO-1) is a stress-inducible enzyme that mediates antioxidative and cytoprotective effects to maintain cellular redox homeostasis. In the present study, the full sequence of HO-1 was cloned from golden pompano(Trachinotus ovatus) by RT-PCR and RACE-PCR. The full cDNA sequence of HO-1 was 1349 bp in length which comprised of a 726 bp open reading frame (ORF) preceded by 262 bp 5′-untranslated region (UTR), and followed by a 360 bp 3′UTR, encoding 241 amino acid residues. Phylogenetic analysis revealed that HO-1 showed highest similarity to that of Takifugu rubripes. Tissue distribution analysis showed that the expression level of HO-1 was relatively high in heart, liver and spleen. A trial was conducted to investigate the response of Nrf2/HO-1 signaling pathway to oxidative stress induced by copper. The results showed that mRNA expression of NF-E2-related nuclear factor2 (Nrf2), Kelch-like-ECH-associated protein1 (keap1), superoxide dismutase (SOD), catalase (CAT), HO-1, NAD(P)H quinone oxidoreductase 1 (NQO1) and Glutathione peroxidase (GSH-PX) all significantly increased in copper treated group than that in the control group. This work provides new insight into the molecular mechanism underlying the Nrf2/HO-1 pathway in oxidative response in T. ovatus.

Characterization of cyclin dependent kinase-7 and its differential response to grafting challenge in the black shell colored selected line of pearl oyster Pinctada fucata martensii

Cyclin dependent kinase-7 (Cdk-7) is a protein kinase associated with regulating the cell cycle, cell differentiation and proliferation, apoptosis and inflammatory response. This study characterized the full cDNA sequence of Cdk-7 in Pinctada fucata martensii (PmCdk-7). A full length sequence of 1473bp with an open reading frame (ORF) of 915bp and encodes a 304aa, 5′-UTR of 58bp and a 3′-UTR of 500bp was obtained. The construed amino acid sequence of PmCdk-7 comprised of a Serine/Threonine protein kinases, catalytic (S_TKc) domain with a protein kinases ATP-binding region signature (14-38aa) and the serine/Threonine protein kinases active-site signature (129-141aa) within the domain. Tissue distribution analysis revealed a high relative mRNA expression of PmCdk-7 within haemocytes. Following the insertion operation (grafting), the relative expression levels of PmCdk-7 in the haemocyte was expressed differentially among the studied groups; the black shell colored selected line (BS) and the control group (CG). High expression was recorded between 12 h and 5d with a peak at 3d suggesting a heightened level of DNA replication and inflammatory response during the pearl-sac formation and this expression was higher in BS than CS showcasing, the heightened immune capacity of BS to grafting operation. Immune stimulation experiment with bacterial endotoxin and a viral mimic revealed PmCdk-7 response to pathogenic stress. The results from our study showed that PmCdk-7 performs a vital function during the cell cycle by aiding DNA replication and also aid response to inflammations generated due to the incision from the grafting operation and long exposure to immune-stimulants (pathogens).

IKKε-like plays an important role in the innate immune signaling of the Pacific oyster (Crassostrea gigas)

IκB-related kinase ε (IKKε) plays a crucial role in the activation of nuclear factor κB (NF-κB) by phosphorylating inhibitor of NF-κB (IκB) and in the regulation of interferon (IFN) gene expression by phosphorylating IFN regulatory factors (IRFs). In this study, we cloned an IKKε homologue cDNA (designated as CgIKKε-like) from the Pacific oyster, Crassostrea gigas. The full 2896-bp cDNA sequence comprised a 2163-bp open reading frame (ORF) encoding 720 amino acids. CgIKKε-like is ubiquitously expressed, and its mRNA levels in hemocytes after poly I:C, V. alginolyticus, or OsHV-1 μVar challenge were analyzed by real-time PCR. Compared to that in the control, CgIKKε-like mRNA expression levels were significantly increased at 3 h and peaked at 6 h after OsHV-1 μVar challenge; no obvious changes were observed in expression levels until 24 h after either V. alginolyticus or poly I:C challenge, reaching a maximum at 24 h (p < 0.01) and then rapidly decreasing. CgIKKε-like transfection into human cell lines induced NF-κB and ISRE activation, while transfection with CgIKKε-like deletion mutants abolished NF-κB and ISRE reporter gene activation. Additionally, CgIKKε-like could interact with CgTBK1 and could form homodimers strongly, which may be critical for the immune signaling transduction. Last but not least, we found that CgIKKε-like may increase CgIκBs phosphorylation and could interact with CgIRF8. Together, these results suggest that CgIKKε-like could respond to pathogenic infection, participate in the immune signal transduction and activate NF-κB and ISRE reporter genes. Thus, CgIKKε-like could play an important role in the oyster immune system.

A family of auxin conjugate hydrolases from Solanum lycopersicum and analysis of their roles in flower pedicel abscission

BackgroundAuxin conjugates are hydrolyzed to release free auxin to ensure defined cellular auxin levels or gradients within tissues for proper development or response to environmental signals. The auxin concentration in the abscission zone (AZ) is thought to play an important role in mediating the abscission lag phase.ResultsIn this study, the full cDNA sequences of seven tomato ILR1-like SlILL genes were identified and characterized, All SlILLs were found to have auxin conjugate hydrolysis activity. The effects of different auxin conjugates on abscission identified IAA-Ile as a candidate to determine the auxin conjugate and auxin conjugate hydrolysis functions in abscission. Treatment of pedicel explants with IAA-Ile for different times showed that application before 6 h could effectively delay abscission. IAA-Ile pre-incubation for 2 h was sufficient to inhibit abscission. These results showed that there is not sufficient auxin conjugates in the AZ to inhibit abscission, and the optimal time to inhibit abscission by the application of exogenous auxin conjugates is before 6 h. Treatment with cycloheximide (CHX, a protein biosynthesis inhibitor) indicated that de novo synthesis of auxin conjugate hydrolases is also required to delay abscission. During abscission, SlILL1, 5, and 6 showed abscission-related gene expression patterns, and SlILL1, 3, 5, 6, and 7 showed increasing expression trends, which collectively might contribute to delay abscission. Silencing the expression of SlILL1, 3, 5, 6, and 7 using virus-induced gene silencing showed that SlILL1, 5, and 6 are major mediators of abscission in tomato.ConclusionsIn the process of abscission, auxin inhibition is concentration dependent, and the concentration of auxin in the AZ was regulated by hydrolyzed auxin conjugates. SlILR1, 5, and 6 play a key role in flower pedicel abscission.

Exploiting orthology and de novo transcriptome assembly to refine target sequence information

BackgroundThe ability to generate recombinant drug target proteins is important for drug discovery research as it facilitates the investigation of drug-target-interactions in vitro. To accomplish this, the target’s exact protein sequence is required. Public databases, such as Ensembl, UniProt and RefSeq, are extensive protein and nucleotide sequence repositories. However, many sequences for non-human organisms are predicted by computational pipelines and may thus be incomplete or incorrect. This could lead to misinterpreted experimental outcomes due to gaps or errors in orthologous drug target sequences. Transcriptome analysis by RNA-Seq has been established as a standard method for gene expression analysis. Apart from this common application, paired-end RNA-Seq data can also be used to obtain full coverage cDNA sequences via de novo transcriptome assembly.MethodsTo assess whether de novo transcriptome assemblies can be used to determine a protein’s sequence by searching the assembly for a known orthologous sequence, we generated 3 × 6 = 18 tissue specific assemblies (three organs: brain, kidney and liver; six species: human, mouse, rat, dog, pig and cynomolgus monkey). These assemblies and the manually curated human protein sequences from UniProtKB/Swiss-Prot were used in a reciprocal BLAST search to identify best matching hits.We automated and generalised our approach and present the a&o-tool, a workflow which exploits de novo assemblies of paired-end RNA-Seq data and orthology information for target sequence validation and refinement across related species. Furthermore, the a&o-tool extracts best hits’ sequences from a reciprocal BLAST search, translates them into protein sequences, computes a multiple sequence alignment and quantifies the refinement.ResultsFor the three human assemblies we observed a hit rate greater than 60% with 100% sequence coverage and identity. For assemblies from the other species we observed similar hit rates and coverage with highest identities for cynomolgus monkey.ConclusionsIn summary, we show how to refine protein sequences using RNA-Seq data and sequence information from closely related species. With the a&o-tool we provide a fully automated pipeline to perform refinement including cDNA translation and multiple sequence alignment for visual inspection. The major prerequisite for applying the a&o-tool is high quality sequencing data.

Molecular characterization, expression analysis, and ontogeny of complement component C9 in southern catfish (Silurus meridionalis)

The complement system plays an important role in host defense against invading microorganisms. Complement component C9 is the last component that is involved in the formation of the membrane attack complex (MAC) on the surface of target cells. In the present study, the full length C9 cDNA sequence of 1984 bp with an open reading frame (ORF) of 1809 bp was cloned from southern catfish (Silurus meridionalis). The deduced amino acid sequence showed similarity with other teleost fish. The mRNA expression of C9 was detected in the liver, spleen, stomach, intestine, and head kidney, with highest levels detected in the liver. The mRNA of C9 was first detected in the yolk syncytial layer at 34 h post fertilization (hpf) with whole mount in situ hybridization, followed by the liver at 36 h post hatching (hph). The mRNA expression of C9 was upregulated significantly in the liver, spleen, and intestine following the injection with Aeromonas hydrophila, suggesting that C9 played an important role in defense against invading pathogens in southern catfish. Therefore, these results provide important information to understand the functions of C9 during fish early development in fish.

Klf4 could be activated by Pou5f3/Stat3 to induce the expression of Nanog in Japanese flounder

Krüppel-like factor 4 (Klf4) is a zinc finger protein transcription factor of the KLF family and plays an important role in various important biological process, including cell proliferation, differentiation, embryonic development, and cancer formation. In this study, the full length Klf4 cDNA sequence of Japanese flounder (Paralichthys olivaceus) was analyzed. Phylogenetic, gene structure, and synteny analysis revealed that PoKlf4 was conserved to that of teleost. Transcript of PoKlf4 showed that its expression was high during early embryonic development but reduced when gastrulation began, thus indicating its possible role in embryonic development. Tissue expression pattern showed that PoKlf4 was expressed higher in ovaries than in testes. ISH revealed that PoKlf4 was also expressed in spermatogonium, oocytes, and oogonia and in high levels in epithelial and lamellar epithelial cells of gills. Functional characterization of PoKlf4 gene promoter provided valuable information in understanding the molecular regulatory mechanisms of PoKlf4 gene. PoKlf4 could up-regulate Nanog, a gene that prevents ES cells from differentiating and being up-regulated by Pou5f3 and Stat3 genes that could maintain ES cell self-renewal. These results suggested that PoKlf4 maintains the pluripotency of ES cells. This study provides valuable information in exploring the regulatory network of PoKlf4 and lays the foundation for further research on the function of this gene.

Molecular characterization and expression analysis of complement component C3 in southern catfish (Silurus meridionalis) and a whole mount in situ hybridization study on its ontogeny

The complement system plays an important role in protecting fish against attack by pathogens early in life. Complement component C3 is a central component in the complement system. The present work aimed to clone the full length C3 cDNA sequence of southern catfish (Silurus meridionalis), detect the tissue expression patterns of C3, investigate the ontogeny of C3 in embryo and larva, and assess the expression of C3 in response to pathogen infection. The full length C3 cDNA sequence of 5157 bp with an open reading frame (ORF) of 4938 bp was cloned from southern catfish. The deduced amino acid sequence showed similarity with other teleost fish. The mRNA expression of C3 was detected in liver, spleen, stomach, intestine, and head kidney with RT-PCR and in situ hybridization. Whole mount in situ hybridization results revealed that C3 was first expressed in the yolk syncytial layer at 34 h post fertilization (hpf), followed by the liver at 36 h post hatching (hph). When challenged with Aeromonas hydrophila, the transcripts of C3 showed a significant up-regulation in liver and spleen at 24 h. The results suggested that complement C3 played a key role in defense against invading pathogens in the early development stages of southern catfish. Therefore, these results provide important information to understand the functions of C3 during fish early development in Siluriformes.