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1.
Chinese Journal of Biotechnology ; (12): 1859-1873, 2022.
Article in Chinese | WPRIM | ID: wpr-927823

ABSTRACT

Leptin receptor overlapping transcript (LepROT) plays multiple roles in the regulation of immune systems. However, very little information is available about the anti-infectious mechanisms of amphibians LepROT. In this study, the cDNA sequence of the Rana dybowskii LepROT gene was determined by using RT-PCR and bioinformatics analysis. Then, the Aeromonas hydrophila (Ah) and lipopolysaccharides (LPS) infected models of R. dybowskii was constructed to obtain histopathological characteristics. Constitutive expression of LepROT mRNA and NF-κB signaling pathway were detected by real-time quantitative PCR. The full-length cDNA of LepROT gene was 396 bp and encoded 131 amino acids. Amino acid sequence analysis revealed LepROT shares 93.74% and 86.39% identity with homologues from other amphibians and mammals respectively, and the LepROT gene was quite conserved among different species. After infection, the relative expression levels of LepROT, NF-κB, IKKα and IKKβ mRNA were all significantly upregulated (P < 0.01), but showed a diverse temporal pattern of up-regulation in different tissues. Therefore, it was proposed that the LepROT gene of R. dybowskii might activate the NF-κB signaling pathway to exert anti-infectious effects, thus providing evidence for further extending the biological function of LepROT.


Subject(s)
Animals , Cloning, Molecular , DNA, Complementary , Gene Expression Profiling , Gene Expression Regulation , Mammals/metabolism , NF-kappa B/genetics , Phylogeny , RNA, Messenger/genetics , Ranidae/genetics
2.
Chinese Journal of Biotechnology ; (12): 374-385, 2022.
Article in Chinese | WPRIM | ID: wpr-927717

ABSTRACT

Phenylalaninammo-nialyase (PAL) is a key enzyme in the synthesis of methyl benzoate - a plant aroma compound. In order to understand the function of this enzyme in the formation of fragrance in the scented Rhododendron species-Rhododendron fortunei, we cloned a gene encoding this enzyme and subsequently examined the gene expression patterns and the profile of enzyme activity during development in various tissues. The full length of RhPAL gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression levels of RhPAL gene were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and the amount of phenylalanine and cinnamic acid were assayed with LC-MS. The results showed that the ORF sequence of RhPAL gene amplified from the cDNA templates of flower buds had 2 145 bp, encoding 715 amino acids, and shared 90% homology to the PAL amino acid sequences from other species. qRT-PCR analysis showed that the expression of RhPAL in petals during flowering kept in rising even until the flowers wilted. The expression of RhPAL in pistil was much higher than that in stamen, while the expression in the younger leaves was higher than in old leaves. However, the expression level was relatively lower in petal and stamen compared to that in leaves. We also measured the PAL activity by Enzyme-linked immuno sorbent assay in the petals of flowers at different flowering stages. The results showed that PAL activity reached the highest at the bud stage and then decreased gradually to the lowest when the flowers wilted, which followed a similar trend in the emission of the flower fragrance. The phenylalanine and cinnamic acid contents measured by LC-MS were highly correlated to the expression level of RhPAL in various tissues and at different flowering stages, implying that RhPAL plays an important role in the formation of the flower fragrance. This work may facilitate the breeding and improvement of new fragrant Rhododendron cultivars.


Subject(s)
Amino Acid Sequence , Cloning, Molecular , DNA, Complementary , Flowers/genetics , Rhododendron/genetics
3.
Chinese Journal of Biotechnology ; (12): 178-186, 2021.
Article in Chinese | WPRIM | ID: wpr-878552

ABSTRACT

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.


Subject(s)
Animals , Chlorocebus aethiops , Clone Cells , DNA, Complementary , Distemper Virus, Canine/genetics , Plasmids/genetics , Vero Cells
4.
Chinese Journal of Biotechnology ; (12): 2836-2844, 2021.
Article in Chinese | WPRIM | ID: wpr-887846

ABSTRACT

It has been reported that ODB genes play an important role in homologous recombination-directed DNA repair, suggesting their potential applications in plant breeding. To analyze the expression characteristics of tobacco NtODB gene, the cDNA sequence of NtODB was obtained using in silico cloning technique. The physicochemical properties, signal peptide, and advanced structures of the predicted protein were analyzed using bioinformatics tools. The results showed that the NtODB gene has a 579-bp open reading frame which encodes a protein with 192 amino acid residues. The protein NtODB is predicted to be alkaline and hydrophilic. Real-time quantitative PCR showed that NtODB was constitutively expressed in different tissues. Subcellular localization showed that NtODB was mainly expressed in cell membrane and chloroplast. These results may help us to better understand and elucidate the roles of ODB genes in the homologous recombination-directed DNA repair.


Subject(s)
Amino Acid Sequence , Base Sequence , Cloning, Molecular , Computational Biology , Computer Simulation , DNA, Complementary , Phylogeny , Plant Breeding , Tobacco/genetics
5.
Chinese Journal of Biotechnology ; (12): 2474-2482, 2021.
Article in Chinese | WPRIM | ID: wpr-887813

ABSTRACT

Spirodela polyrrhiza is a floating plant widely used in biomass utilization and eutrophication phytoremediation. It becomes a common aquatic plant everywhere with the increasingly serious eutrophication. It has been reported that S. polyrrhiza has a good effect on the remediation of eutrophication water. In order to study the absorption and transportation of phosphorus in S. polyrrhiza, we extracted RNA from S. polyrrhiza and then reverse transcribed it into cDNA, which was used as a template to amplify a specific fragment. The full-length sequence of the open reading frame (ORF) was 1 620 bp, encoding 539 amino acids, named SpPHT1;1, and the accession number in GenBank was MN720003. Bioinformatical analysis showed that SpPHT1;1 had no intron. The protein it encoded was a stable, hydrophobic protein with 11 transmembrane domains. SpPHT1;1 structure was similar to that of major facilitator superfamily (MFS) superfamily members. The cluster analysis showed that SpPHT1;1 was closely related to ZMPHT2 in maize and SBPHT1-8 in sorghum. So, it might belong to plant PHT1 family. The expression of SpPHT1;1 in leaf was significantly more than that of root under normal phosphorus condition. Low phosphorus condition could promote gene expression, and the relative expression level of SpPHT1;1 arrived at the peak at 48 h both in root and leaf. High phosphorus condition could inhibit gene expression. These results indicated that SpPHT1;1 expression would be affected by external phosphorus concentration. The results of this study are helpful for further research on the function of phosphate transporter. It also can provide theoretical basis for further development and utilization of S. polyrrhiza.


Subject(s)
Araceae/genetics , Biodegradation, Environmental , Cloning, Molecular , DNA, Complementary , Phosphate Transport Proteins/genetics
6.
Braz. j. med. biol. res ; 54(5): e10274, 2021. graf
Article in English | LILACS | ID: biblio-1153553

ABSTRACT

Prolactin (PRL) plays critical roles in regulation of biological functions with the binding of specific prolactin receptor (PRLR). Revealing the expression patterns of PRLR at different developmental stages is beneficial to better understand the role of PRL and its mechanism of action in striped hamsters. In this study, the cDNA sequence of PRLR (2866-base-pairs) was harvested from the pituitary of mature female striped hamsters (Cricetulus barabensis) that contains an 834-base-pair 5′-untranslated region (1-834 bp), a 1848-base-pair open reading frame (835-2682 bp), and a 184-base-pair 3′-untranslated region (2683-2866). The 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids. In the mature PRLR, two prolactin-binding motifs, 12 cysteines, and five potential Asn-linked glycosylation sites were detected. Our results showed that the PRLR mRNA quantity in the hypothalamus, pituitary, ovaries, or testis was developmental-stage-dependent, with the highest level at sub-adult stage and the lowest level at old stage. We also found that PRLR mRNAs were highest in pituitary, medium level in hypothalamus, and lowest in ovaries or testis. PRLR mRNAs were significantly higher in males than in females, except in the hypothalamus and pituitary from 7-week-old striped hamsters. Moreover, the PRLR mRNAs in the hypothalamus, pituitary, and ovaries or testis were positively correlated with the expression levels of GnRH in the hypothalamus. These results indicated that the PRLR has conserved domain in striped hamster, but also possesses specific character. PRLR has multiple biological functions including positively regulating reproduction in the striped hamster.


Subject(s)
Animals , Male , Female , Prolactin/genetics , Receptors, Prolactin/genetics , Receptors, Prolactin/metabolism , Pituitary Gland/metabolism , Cricetinae , Sequence Analysis , DNA, Complementary/genetics
7.
NOVA publ. cient ; 18(spe35): 43-52, jul.-dic. 2020. tab, graf
Article in Spanish | LILACS | ID: biblio-1149465

ABSTRACT

Resumen El estándar de oro actual para la detección de SARS-CoV-2, agente causal de la pandemia de neumonía atípica (COVID-19) que apareció por primera vez en la ciudad de Wuhan (provincia de Hubei, China) en diciembre de 2019 (1), es la RT-qPCR. El protocolo estándar implica la transcripción inversa de ARN de SARS-CoV-2 en cadenas de ADN complementarias (ADNc), seguida de la amplificación de regiones específicas del ADNc. Este procedimiento demanda varias horas para ser completado y deriva en que la información final del estado de la infección pueda demorar hasta 24 horas. Ante la necesidad de disminuir el riesgo de una posible propagación viral dentro de la población originada por la rápida transmisión del SARS-CoV-2, se ha buscado prevenir el contagio, la propagación nosocomial y la transmisión comunitaria posterior, a través de la identificación rápida de casos sospechosos, y predecir las posteriores ondas infecciosas de recurrencia viral. Para esto, se vienen desarrollando métodos de laboratorio rápidos o point of care testing (POCT), que disminuyen el tiempo de diagnóstico y minimizan el riesgo de contagio por parte de los operadores.


Abstract The gold test to detect SARS-CoV-2, the etiologic agent that leads to the pandemic of atypical pneumonia (COVID 2019) that first appeared in Wuhan City, Hubei Province of China in December 2019 (1), is the RT-qPCR. The standard protocol involves reverse transcription of SARS-CoV-2 RNA into complementary DNA strands (cDNA), followed by the amplification of cDNA specific regions, a procedure that takes several hours to complete and which results in the final information from the infection status can take up to 24 hours. For this reason, and due to the need to reduce the risk of possible viral spread within the population caused by the fast transmission of SARS-CoV-2, in order to prevent nosocomial spread and subsequent community transmission through the quick identification of suspected cases, and to predict the further infectious waves of viral recurrence, rapid laboratory methods or Point of Care Testing (POCT) are being developed to reduce the diagnosis time and minimize the risk of contagion by the operators. These tests are discussed below.


Subject(s)
Humans , COVID-19 , Pneumonia , DNA, Complementary , Disease Transmission, Infectious , Point-of-Care Testing
8.
Rev. cuba. hematol. inmunol. hemoter ; 36(3): e1164, jul.-set. 2020. tab, graf
Article in Spanish | LILACS, CUMED | ID: biblio-1156440

ABSTRACT

Introducción: En el Instituto de Hematología e Inmunología se realiza el estudio molecular de las leucemias mieloides agudas (LMA). Para las leucemias mieloides agudas no promielocíticas (LPM) se determinan cuatro biomarcadores: los genes de fusión RUNX1-RUNX1T1 y CBF(-MYH11, la duplicación interna en tándem del gen FLT3 (DIT FLT3) y la mutación A del gen NPM1 (NPM1-A). Objetivo: Determinar la frecuencia de estos cuatro biomarcadores, en pacientes cubanos con leucemias mieloides agudas primaria no promielocíticas. Métodos: Se incluyeron 91 pacientes entre niños y adultos, estudiados en el Instituto durante tres años desde el debut. A partir de ARN de sangre medular se obtuvo ADN complementario por transcripción inversa; se amplificaron los fragmentos correspondientes mediante la reacción en cadena de la polimerasa y el producto se analizó por electroforesis capilar. Resultados: El RUNX1-RUNX1T1 apareció en el 24,2 por ciento, fue más frecuente en los pacientes pediátricos y disminuyó significativamente con la edad. El CBFβ-MYH11 solo se encontró en adultos (4,8 por ciento). La NPM1-A con 41 por ciento fue mayoritaria entre los adultos. La DIT FLT3 se observó en el 21,6 por ciento y no mostró relación con la edad. NPM1-A y DIT FLT3 fueron las aberraciones con mayor presencia simultánea. Conclusiones: Por primera vez se describe la frecuencia de los cuatro biomarcadores moleculares en los pacientes cubanos con leucemias mieloides agudas primaria no promielocíticas; su comportamiento fue similar a lo descrito por otros autores, aunque se encontraron algunas particularidades(AU)


Introduction: At the Institute of Hematology and Immunology, the molecular study of acute myeloid leukemias (AML) is carried out. For nonpromyelocytic acute myeloid leukemias, four biomarkers are determined: the RUNX1-RUNX1T1 and CBF(-MYH11 fusion genes, the internal tandem duplication of the FLT3 gene (DIT FLT3), and the A mutation of the NPM1 gene (NPM1-A). Objective: To determine the frequency of these four biomarkers in Cuban patients with nonpromyelocytic primary acute myeloid leukemias. Methods: 91 patients were included, children and adults, who were studied at the Institute for three years from their disease debut. Complementary DNA was obtained from medullary blood RNA by reverse transcription. The corresponding fragments were amplified by polymerase chain reaction and the product was analyzed by capillary electrophoresis. Results: RUNX1-RUNX1T1 appeared in 24.2 percent; it was more frequent in pediatric patients and decreased significantly with age. CBFβ-MYH11 was found only in adults (4.8 percent). NPM1-A, accounting for 41 percent, represented the majority among adults. FLT3 DIT was observed in 21.6 por ciento and was not related to age. NPM1-A and DIT FLT3 were the disorders with the greatest concurrence. Conclusions: For the first time, the frequency of the four molecular biomarkers is described in Cuban patients with primary non-promyelocytic acute myeloid leukemias. Its characterization was similar to that described by other authors, although some peculiarities were found(AU)


Subject(s)
Humans , Biomarkers , Leukemia, Myeloid, Acute/genetics , Polymerase Chain Reaction , DNA, Complementary , Reverse Transcription , Electrophoresis, Capillary
9.
Arq. bras. med. vet. zootec. (Online) ; 72(2): 523-534, Mar./Apr. 2020. ilus, graf
Article in English | LILACS, VETINDEX | ID: biblio-1128390

ABSTRACT

Insulin-like growth factor-1 (IGF-1) is regarded as a crucial clinically significant therapeutic agent against several pathological conditions. Recently, recombinant DNA (rDNA) technology has enabled the production of many drugs of rDNA-origin including IGF-1. Securing a readily available supply of IGF-1 is invaluable to clinical research and biotechnological domains. In this work, the cloning of a full-length bovine IGF-1 cDNA and the successful expression of its cognate recombinant IGF-1 protein is reported. Single-strand cDNA was prepared from liver tissues, through the specific reverse transcription (RT) of IGF-1 mRNA. Subsequently, a PCR amplicon of ~543bp was successfully amplified. Recombinant pTARGET™ vector harboring IGF-1 insert was successfully cloned into competent E. coli JM109 cells. SDS-PAGE analysis revealed that the recombinant IGF-1 has been expressed at the expected size of 7.6kDa. The outcome provides a robust basis for transecting the recombinant pTARGETTM vector, harboring the IGF-1 cDNA insert, into mammalian cells. Optimal initial glucose concentration was found to be 10g/l with corresponding protein concentration of 6.2g/l. The proliferative biological activity crude recombinant IGF-1 protein was verified on HeLa cell lines. This is envisaged to facilitate large-scale production of recombinant IGF-1 protein, thereby enabling thorough investigation of its clinical and pharmaceutical effects.(AU)


O fator de crescimento semelhante à insulina-1 (IGF-1) é considerado um agente terapêutico clinicamente significativo contra várias condições patológicas. Recentemente, a tecnologia de DNA recombinante (rDNA) permitiu a produção de muitos medicamentos de origem rDNA, incluindo o IGF-1. Garantir um suprimento prontamente disponível de IGF-1 é inestimável para pesquisas clínicas e domínios biotecnológicos. Neste trabalho, relata-se a clonagem de um cDNA de IGF-1 bovino de comprimento total e a expressão bem-sucedida de sua proteína IGF-1 recombinante cognata. O cDNA de cadeia simples foi preparado a partir de tecidos do fígado, por meio da transcrição reversa específica (RT) do mRNA de IGF-1. Posteriormente, um amplificador de PCR de ~ 543pb foi amplificado com sucesso. O vetor pTARGET™ recombinante contendo a inserção de IGF-1 foi clonado com sucesso em células competentes E. coli JM109. A análise por SDS-PAGE revelou que o IGF-1 recombinante foi expresso no tamanho esperado de 7,6kDa. O resultado fornece uma base robusta para a transferência do vetor pTARGETTMTM recombinante, abrigando a inserção de cDNA de IGF-1 em células de mamíferos. Verificou-se que a concentração inicial ideal de glicose é 10g/L, com a concentração de proteína correspondente de 6,2g/L. A proteína IGF-1 recombinante bruta de atividade biológica proliferativa foi verificada nas linhas celulares HeLa. É previsto que isso facilite a produção da proteína IGF-1 recombinante em larga escala, permitindo, assim, uma investigação completa dos seus efeitos clínicos e farmacêuticos.(AU)


Subject(s)
Animals , Recombinant Proteins , Insulin-Like Growth Factor I/genetics , Buffaloes/genetics , Cloning, Molecular , DNA, Complementary , Escherichia coli , Real-Time Polymerase Chain Reaction/veterinary
10.
Chinese Journal of Biotechnology ; (12): 949-958, 2020.
Article in Chinese | WPRIM | ID: wpr-826881

ABSTRACT

Soybean mosaic virus (SMV), one of the major viral diseases of Pinellia ternata (Thunb.) Breit., has had a serious impact on its yield and quality. The construction of viral infectious clones is a powerful tool for reverse genetics research on viral gene function and interaction between virus and host. To clarify the molecular mechanism of SMV infection in Pinellia ternata, it is particularly important to construct the SMV full-length cDNA infectious clone. Therefore, the infectious clone of Soybean mosaic virus Shanxi Pinellia ternata isolate (SMV-SXBX) was constructed in this study by Gibson in vitro recombination system, and the healthy Pinellia ternata leaves were inoculated by Agrobacterium infiltration, further through mechanical passage and RT-PCR, confirming that the 3' end of the SMV-SXBX infectious clone had a stable infectivity when it contained 56-nt of poly(A) tail. This method is not only convenient and efficient, but also avoids the instability of SMV infectious clones in Escherichia coli. The construction of SMV full-length infectious cDNA clones laid the foundation for further study on the molecular mechanism of SMV replication and pathogenesis.


Subject(s)
DNA, Complementary , Pinellia , Virology , Plant Diseases , Virology , Potyvirus , Metabolism
12.
Gac. méd. Méx ; 155(5): 504-510, Sep.-Oct. 2019. graf
Article in English | LILACS | ID: biblio-1286551

ABSTRACT

Cancer is a multifactorial disease that constitutes a serious public health problem worldwide. Prostate cancer advanced stages are associated with the development of androgen-independent tumors and an apoptosis-resistant phenotype that progresses to metastasis. By studying androgen-independent lymphoid nodule carcinoma of the prostate (LNCaP) cells induced to apoptosis by serum elimination, we identified the activation of a non-selective cationic channel of 23pS conductance that promotes incoming Ca2+ currents, as well as apoptosis final stages. arp2cDNA was isolated and identified to be of the same cell type, and mRNA was expressed in Xenopus laevis oocytes, which was found to be associated with the activation of incoming Ca2+ currents and induction to apoptosis. cDNA, which encodes the ARP2 protein, was overexpressed in LNCaP cells and Chinese hamster ovary cells, which induced apoptosis. Our evidence suggests that protein ARP2 overexpression and transit to the cell membrane allows an increased Ca2+ incoming current that initiates the apoptosis process in epithelial-type cells whose phenotype shows resistance to programmed cell death.


Subject(s)
Humans , Animals , Male , Prostatic Neoplasms/pathology , Calcium/metabolism , Apoptosis/physiology , Apoptosis Regulatory Proteins/metabolism , Ovum/metabolism , Prostatic Neoplasms/metabolism , Xenopus laevis , RNA, Messenger/metabolism , Calcium Channels/metabolism , Cricetulus , CHO Cells , DNA, Complementary/isolation & purification , Apoptosis Regulatory Proteins/isolation & purification
13.
Electron. j. biotechnol ; 41: 48-55, sept. 2019. tab, ilus, graf
Article in English | LILACS | ID: biblio-1087162

ABSTRACT

Background: Plant gene homologs that control cell differentiation can be used as biotechnological tools to study the in vitro cell proliferation competence of tissue culture-recalcitrant species such as peppers. It has been demonstrated that SERK1 homologs enhance embryogenic competence when overexpressed in transformed tissues; therefore, cloning of a pepper SERK1 homolog was performed to further evaluate its biotechnological potential. Results: A Capsicum chinense SERK full-length cDNA (CchSERK1) was cloned and characterized at the molecular level. Its deduced amino acid sequence exhibits high identity with sequences annotated as SERK1 and predicted-SERK2 homologs in the genomes of the Capsicum annuum CM-334 and Zunla-1 varieties, respectively, and with SERK1 homologs from members of the Solanaceae family. Transcription of CchSERK1 in plant tissues, measured by quantitative RT-PCR, was higher in stems, flowers, and roots but lower in leaves and floral primordia. During seed development, CchSERK1 was transcribed in all zygotic stages, with higher expression at 14 days post anthesis. During somatic embryogenesis, CchSERK1 was transcribed at all differentiation stages, with a high increment in the heart stage and lower levels at the torpedo/cotyledonal stages. Conclusion: DNA sequence alignments and gene expression patterns suggest that CchSERK1 is the C. chinense SERK1 homolog. Significant levels of CchSERK1 transcripts were found in tissues with cell differentiation activities such as vascular axes and during the development of zygotic and somatic embryos. These results suggest that CchSERK1 might have regulatory functions in cell differentiation and could be used as a biotechnological tool to study the recalcitrance of peppers to proliferate in vitro.


Subject(s)
Capsicum/genetics , Cloning, Molecular , In Vitro Techniques , Biotechnology , Gene Expression , Cell Differentiation , Genes, Plant , DNA, Complementary/genetics , Solanaceae/genetics , Arabidopsis Proteins , Cell Proliferation , Embryonic Development , Real-Time Polymerase Chain Reaction
14.
Chinese Journal of Biotechnology ; (12): 102-113, 2019.
Article in Chinese | WPRIM | ID: wpr-771396

ABSTRACT

The biogenic monoamine 5-hydroxytryptamine (5-HT) is an ancient intracellular signaling molecule widely distributed in all animals with nervous systems, and has been implicated in principal behaviors. Tryptophan hydroxylase (TRH) induces a highly specific catalytic reaction that converts L-tryptophan (tryptophan) to 5-hydroxy-L-tryptophan (5-HTP) that is subsequently used as a substrate by aromatic L-amino acid decarboxylase (DDC) to form 5-HT. Five-HT is an ancient intracellular signaling molecule that is widely distributed in the animal kingdom and has been implicated in regulating the behaviors of animals with nervous systems. However, the role of TRH in Lepidoptera is not well understood. In this study, we cloned 1 667 bp cDNAs of Bombyx mori TRH (BmTRH), which contains a 1 632 bp open reading frame (ORF). Homology analysis revealed that BmTRH shared high amino acid identity with Homo sapiens TPH and Drosophila TRH (DmTRH). The high homology (70%) of BmTRH with DmTRH suggested that BmTRH could have a function similar to DmTRH. Gene expression analysis revealed that BmTRH was mainly expressed in head and central nervous (CNS). Moreover, immunohistochemistry and Western blotting analyses showed that BmTRH was detected only in larval nervous tissues. Taken together, our results indicate that BmTRH could likely function in the regulation of neural activities in B. mori. The transcripts of B. mori decarboxylase (BmDDC) and B. mori phenylalanine hydroxylase (BmPAH) whose proteins had TRH activity, were also expressed in the CNS tissues, indicating that unlike in Drosophila, two distinct mechanisms likely regulate 5-HT synthesis in silkworm.


Subject(s)
Amino Acid Sequence , Animals , Bombyx , Cloning, Molecular , DNA, Complementary , Insect Proteins , Phenylalanine Hydroxylase , Tryptophan Hydroxylase
15.
Article in Chinese | WPRIM | ID: wpr-777535

ABSTRACT

1-deoxy-D-xylulose-5-phosphate synthase2(DXS2) is the first key enzyme of the MEP pathway,which plays an important role in terpene biosynthesis of plants. According to the data of Swertia mussotii transcriptome, DXS2 gene(Gen Bank number MH535905) was cloned and named as Sm DXS2. The bioinformatics results showed that Sm DXS2 has no intron,with a 2 145 bp open reading frame encoding a polypeptide of 714 amino acids. They are belonging to 20 kinds of amino acids,and the most abundant amino acids include Ala,Gly and Trp. The predicted protein molecular weight was 76. 91 k Da and its theoretical isoelectric point(p I) was6. 5,which belonging to a hydrophilic protein. α-Helix and loop were the major motifs of predicted secondary structure of DXS2. The three function domains are TPP_superfamily,Transket_pyr_ superfamily and Transketolase_C superfamily,respectively. The Sm DXS2 protein shared high identity with other DXS2 proteins of plants. Phylogenetic analysis showed that Sm DXS2 protein is grouped with the gentian DXS2 protein. The recombinant protein of Sm DXS2 gene in Escherichia coli was approximately 92. 00 k Da(containing sumo-His tag protein 13 k Da),which was consistent with the anticipated size.This work will provide a foundation for further functional research of Sm DXS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.


Subject(s)
Amino Acid Sequence , Cloning, Molecular , DNA, Complementary , Genetics , Genes, Plant , Iridoids , Phylogeny , Plant Proteins , Genetics , Swertia , Genetics , Transcriptome , Transferases , Genetics
16.
Article in Chinese | WPRIM | ID: wpr-773725

ABSTRACT

Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.


Subject(s)
Cloning, Molecular , DNA, Complementary , Flavonoids , Intramolecular Lyases , Genetics , Plants, Medicinal , Genetics , Rhus , Genetics
17.
Electron. j. biotechnol ; 32: 47-54, Mar. 2018. tab, ilus, graf
Article in English | LILACS | ID: biblio-1022746

ABSTRACT

Background: Cathepsin C (CTSC) (dipeptidyl peptidase I, DPPI), is a member of the papain superfamily of cysteine proteases and involves in a variety of host reactions. However, the information of CTST in Chinese giant salamander (Andrias davidianus), an amphibian species with important evolutionary position and economic values, remained unclear. Results: The full-length salamander CTSC cDNA contained a 96 bp of 5'-UTR, a 1392 bp of ORF encoding 463 amino acids, and a 95 bp of 3'-UTR. The salamander CTSC possessed several sequence features similar to other reported CTSCs such as a signal peptide, a propeptide and a mature peptide. The active site triad of Cys, His and Asn were also found existing in salamander CTSC. Salamander CTSC mRNA was constitutively expressed in all the examined tissues with significantly variant expression level. The highest expression of CTSC was in intestine, followed with stomach, spleen, lung and brain. Following Aeromonas hydrophila infection for 12 h, salamander CTSC was significantly up-regulated in several tissues including lung, spleen, brain, kidney, heart, stomach and skin. Conclusion: CTSC plays roles in the immune response to bacterial infection, which provided valuable information for further studying the functions of CTSC in salamander.


Subject(s)
Animals , Urodela/genetics , Urodela/immunology , Gram-Negative Bacterial Infections/veterinary , Cathepsin C/immunology , Urodela/microbiology , Gram-Negative Bacterial Infections/immunology , Cloning, Molecular , Aeromonas hydrophila/physiology , Sequence Analysis , DNA, Complementary , Cathepsin C/genetics , Cathepsin C/metabolism , Reverse Transcription , Immunity, Innate/genetics
18.
Article in English | LILACS | ID: biblio-954851

ABSTRACT

Lethal factors are multifunctional oligomeric proteins found in the venomous apparatus of Scorpaeniformes fish. These toxins elicit not only an array of biological responses in vitro but also cardiovascular disorders and strong hemolytic, nociceptive and edematogenic activities in vivo. This work describes the cloning and molecular identification of two toxin subunits, denominated Sp-CTx-α and Sp-CTx-ß, from scorpionfish venom ( Scorpaena plumieri ). Methods: The primary structures were deduced after cDNA amplification by PCR with primers from conserved sequences described in Scorpaeniformes toxins. Following DNA sequencing and bioinformatic analysis, the tridimensional structures of both subunits were modeled. Results: The translated sequences (702 amino acids, each subunit) show homology with other lethal factors, while alignment between Sp-CTx-α and Sp-CTx-ß shows 54% identity. The subunits lack N-terminal signal sequences and display masses of approximately 80 kDa each. Both Sp-CTx subunits display a B30.2/SPRY domain at the C-terminal region with typically conserved motifs as described in these toxins. Secondary structure prediction identified six α-helices 18 residues long in both α and ß subunits, some of them amphiphilic with their N-terminal flanked by many basic residues, creating a cationic site associated with the cytolytic activity of these toxins. Antimicrobial potential sites were identified in Sp-CTx and share some features with other peptides presenting variable and broad-spectrum activity. A phylogenetic tree built to represent these toxins supports the proximity between scorpionfish, lionfish and stonefish. Conclusion: The study identified a putative toxin protein whose primary structure is similar to other fish toxins and with potential for production of antivenom against scorpionfish envenomation in Brazil. As a prelude to structure-function studies, we propose that the toxin is structurally related to pore-forming marine toxins.(AU)


Subject(s)
Animals , DNA, Complementary/analysis , Fish Venoms/toxicity , Peptides/analysis , Antivenins/classification , Polymerase Chain Reaction/methods , Amino Acid Sequence
19.
Braz. j. med. biol. res ; 51(8): e7044, 2018. graf
Article in English | LILACS | ID: biblio-951748

ABSTRACT

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.


Subject(s)
Animals , Bacterial Proteins/genetics , Clostridium perfringens/genetics , Sequence Analysis, RNA/methods , Genes, MDR , Drug Resistance, Multiple, Bacterial/genetics , Mass Spectrometry/methods , Bacterial Proteins/metabolism , Electrophoresis, Gel, Two-Dimensional/methods , Gene Expression Regulation, Bacterial/genetics , Genome, Bacterial/genetics , Clostridium perfringens/classification , Clostridium perfringens/drug effects , Clostridium perfringens/metabolism , DNA, Complementary , Proteome/genetics , Transcriptome/genetics , Gene Ontology
20.
Article in English | WPRIM | ID: wpr-740585

ABSTRACT

OBJECTIVES: As a first step to study the anticaries effect of ethanol alone, we investigated the effects of ethanol on the expression levels of the atpB gene and proton permeability of Streptococcus mutans in suspension cultures. METHODS: S. mutans UA159 was grown in brain heart infusion medium at either pH 4.8 or 6.8. The total extracted RNA was reverse-transcribed into cDNA using a Superscript™ First-Strand Synthesis System. The resulting cDNA and negative controls were amplified by ABI PRISM 7700 real-time PCR system with SYBR Green PCR Master Mix. For proton flux assay, bacterial suspensions were titrated to pH 4.6 with 0.5 M HCl, and then additional 0.5 M HCl was added to decrease the pH values by approximately 0.4 units. The subsequent increase in pH was monitored using a glass electrode. Ten percent (v/v) butanol was added to the suspensions at 80 min to disrupt the cell membrane. RESULTS: In a concentration-dependent manner, ethanol alone not only decreased the growth rate of S. mutans and the expression of the atpB gene but also increased the proton permeability at both pH 4.8 and 6.8. CONCLUSIONS: These findings suggest that ethanol has the potential for an anticaries ingredient. We believe that ethanol may be used together with fluoride and/or other cariostatic agents in order to develop better anticaries toothpastes and/or mouthrinses.


Subject(s)
Brain , Cariostatic Agents , Cell Membrane , DNA, Complementary , Electrodes , Ethanol , Fluorides , Gene Expression , Glass , Heart , Hydrogen-Ion Concentration , Permeability , Polymerase Chain Reaction , Protons , Real-Time Polymerase Chain Reaction , RNA , Streptococcus mutans , Streptococcus , Suspensions , Toothpastes
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