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1.
Microb Cell Fact ; 21(1): 9, 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-35012550

RESUMO

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS's, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively.


Assuntos
Proteínas de Bactérias/metabolismo , Fusarium/enzimologia , Policetídeo Sintases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Xantonas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Vias Biossintéticas , Clonagem Molecular , Fusarium/genética , Isoquinolinas/metabolismo , Modelos Moleculares , Policetídeo Sintases/química , Policetídeo Sintases/genética , Domínios Proteicos , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética
2.
Gene ; 812: 146105, 2022 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-34896231

RESUMO

Anthocyanin accumulation is one of the remarkable physiological changes during fruit ripening. In plants, anthocyanin synthesis is regulated by MYB activators, but the MYB repressors has been recognized recently. Here, we isolated a repressor of anthocyanin synthesis, LcMYBx, from Litchi chinensis Sonn. LcMYBx encoded a typical R3-MYB protein and contained a conserved [D/E]Lx2[R/K]x3Lx6Lx3R motif for interacting with bHLH proteins. Overexpression of LcMYBx in tobacco suppressed anthocyanin accumulation resulting in faded petals from pale-pink to almost white. Gene expression analysis showed the strong down-regulation of endogenous anthocyanin structural and regulatory genes by LcMYBx overexpression. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that LcMYBx could interact with the transcription factors LcbHLH1 and LcbHLH3. Transient promoter activation assays showed that LcMYBx could inhibit the activation capacity of LcMYB1-LcbHLH3 complex for LcDFR gene. These results suggest that LcMYBx competed with LcMYB1 to LcbHLHs, thus preventing the activation of LcDFR by LcMYB1-LcbHLHs complex and negatively controlling anthocyanin biosynthesis.


Assuntos
Antocianinas/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/genética , Litchi/metabolismo , Tabaco/crescimento & desenvolvimento , Sequência de Aminoácidos , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Litchi/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Domínios Proteicos , Tabaco/genética , Tabaco/metabolismo
3.
Methods Mol Biol ; 2316: 237-242, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34845699

RESUMO

Determining the sequence identity of viroid RNAs present in symptomatic or asymptomatic plant tissues is critical to obtain knowledge of their distribution. It enables the development of tools for diagnostics and for studying the basic biology of viroids. With the advent of cDNA-based methods for cloning RNAs and cloning strategies that do not require prior knowledge of the viroid sequence, characterization of several newly discovered viroids has rapidly expanded our knowledge of these unusual pathogenic RNAs. This chapter describes two methods, using random primers or viroid-specific primers, to generate complementary DNA (cDNA) copies of viroid RNAs for subsequent cloning and sequence analysis.


Assuntos
Viroides , Clonagem Molecular , Primers do DNA , DNA Complementar/genética , RNA , RNA Viral/genética , Análise de Sequência , Viroides/genética
4.
Artigo em Inglês | MEDLINE | ID: mdl-34653596

RESUMO

Prostaglandins are a series of unsaturated fatty acids that play critical roles in regulating reproductive events. The prostaglandins endoperoxide H synthases-1/2 (PGHS-1/2; also named cyclooxygenases-1/2, COX-1/2) catalyse the commitment step in prostaglandin synthesis. However, the of the cox genes in teleosts, especially ovoviviparous teleosts, is still unclear. The aim of the present study was to determine the potential role of cox genes in mating and parturition behaviour using black rockfish (Sebastes schlegelii) as a model species. Two transcripts, cox1 and cox2, were cloned. The phylogenetic analysis results revealed that both cox genes were closely related to mammalian coxs. qPCR analyses of their tissue distribution showed that cox1 was mainly expressed in the heart in both sexes, while cox2 was mainly expressed in the testis and ovary. Detection of cox expression in samples from reproductive-related stages further showed that both cox genes may play important roles in mating and parturition processes. In situ hybridization further detected positive cox mRNA signals in the testis and ovary, where they are known to be involved in mating and parturition behaviour. These data suggest that cox1 and cox2 are crucial in inducing mating, gonad regeneration and parturition behaviour.


Assuntos
Ovoviviparidade , Perciformes , Animais , Clonagem Molecular , Feminino , Peixes/genética , Masculino , Parto , Perciformes/genética , Filogenia , Gravidez , Prostaglandina-Endoperóxido Sintases/genética , Distribuição Tecidual
5.
J Microbiol ; 60(1): 63-69, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34964943

RESUMO

In a previous study, a putative 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) was highly expressed in a mutant strain of Pyropia yezoensis, which exhibited an improved growth rate compared to its wild strain. To investigate the functional role of the putative ACMSD (Pyacmsd) of P. yezoensis, the putative Pyacmsd was cloned and expressed in Chlamydomonas reinhardtii. Recombinant C. reinhardtii cells with Pyacmsd (Cr_Pyacmsd) exhibited enhanced tolerance compared to control C. reinhardtii cells (Cr_control) under nitrogen starvation. Notably, Cr_Pyacmsd cells showed accumulation of lipids in nitrogen-enriched conditions. These results demonstrate the role of Pyacmsd in the generation of acetyl-coenzyme A. Thus, it can be used to enhance the production of biofuel using microalgae such as C. reinhardtii and increase the tolerance of other biological systems to nitrogen-deficient conditions.


Assuntos
Carboxiliases/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Expressão Gênica , Nitrogênio/metabolismo , Rodófitas/enzimologia , Carboxiliases/metabolismo , Clonagem Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodófitas/genética
6.
Colloids Surf B Biointerfaces ; 209(Pt 2): 112188, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34742021

RESUMO

Some microbial strains are ideal producers of extracellular enzymes that can be used in various industries. However, in many fields, especially in the pharmaceutical field, these enzymes need to be recovered and purified through multistep processes and tedious procedures before they can be used. The recovery process is difficult and increases the cost of enzyme production. Therefore, reducing purification steps will greatly benefit the utilization of microbial enzymes. The 35 M strain of Bacillus amyloliquefaciens, which has high extracellular protease production, was isolated from a phosphate mine. When cultured in a medium with soybean meal as the main component, the maximum activity of extracellular protease reached 16,992 U/mL. SDS-PAGE showed that there were two main proteins in the fermentation supernatant, with a paucity of other defined protein bands. Mass spectrometry and zymogram analysis showed that the two main bands were two proteases, corresponding to alkaline protease (AprM) and neutral protease (NprM), respectively. Gene cloning, sequencing, and further comparisons were used to confirm AprM and NprM correspond to these proteases from B. amyloliquefaciens. Notably, SDS-PAGE and zymogram analysis showed that NprM had obviously higher catalytic efficiency toward casein than did AprM. Strain 35 M is a promising protease producer with great potential for applications in industrial protease production. Additionally, this study demonstrates strain 35 M may be particularly well suited to use in degrading anti-nutritional factors in soybean meal, so as to improve the nutritional value of soybean meal.


Assuntos
Bacillus amyloliquefaciens , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Fermentação , Peptídeo Hidrolases/metabolismo , Soja
7.
Pestic Biochem Physiol ; 180: 104982, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34955175

RESUMO

The pulse beetle Callosobruchus maculatus causes potential damage to legume crops by infesting the seeds, leading to a reduction of total protein content. Arcelin found in the wild accessions of the common bean, is an insecticidal protein that has the potency to hamper the metabolism of the bruchid beetle. The arcelin gene from the wild accession of Phaseolus lunatus was isolated and the ORF encoding 158 amino acids was cloned in pET-45b (+) vector. The recombinant clones were transformed in BL21 STAR (DE3) pLysS cells, and the expressed arcelin was purified using Ni-NTA column. The recombinant protein was used in preparing an artificial diet, and the insecticidal activity was elucidated against the bruchid pest C. maculatus. Adult emergence and seed damage were drastically reduced in the treated groups. The response towards ingested diet by digestive enzymes involved in metabolism was elucidated through quantitative gene expression. The highest expression was observed in the aminopeptidase, followed by upregulation of alpha-amylase, glycoside hydrolase family 31 and cathepsin D-like aspartic protease, and downregulation of cathepsin L-like cysteine protease. The recombinant arcelin demonstrates effective insecticidal activity against the bruchid beetle. The changes in digestive enzymes to counteract the anti-nutritional nature of the protein were the strategies of the insect defense mechanism.


Assuntos
Besouros , Phaseolus , Animais , Clonagem Molecular , Besouros/genética , Phaseolus/genética , Proteínas de Plantas/genética , Sementes
8.
Plant Sci ; 314: 111102, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34895539

RESUMO

Caffeoyl CoA O-methyltransferases (CCoAOMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to a hydroxyl moiety of caffeoyl-CoA as part of the lignin biosynthetic pathway. CCoAOMT-like proteins also catalyze to a variety of flavonoids, coumarins, and phenylpropanoids. Several CCoAOMTs that prefer flavonoids as substrates have been characterized from liverworts. Here, we cloned two CCoAOMT genes, MpalOMT2 and MpalOMT3, from the liverwort Marchantia paleacea. MpalOMT3 has a second ATG codon downstream and the truncated version that lacks 11 amino acids was named MpalOMT3-Tr. Phylogenetic analysis placed MpalOMT3 at the root of the clade with true CCoAOMTs from vascular plants and placed MpalOMT2 between the CCoAOMT and CCoAOMT-like proteins. Recombinant OMTs methylated caffeoyl CoA, phenylpropanoids, and flavonoids containing two or three vicinal hydroxyl groups. MpalOMT3 showed higher catalytic activity for phenylpropanoids than MpalOMT2, but MpalOMT2 showed more promiscuous towards eriodictyol and myricetin. The lignin content in Arabidopsis thaliana stems increased with constitutive heterologous expression of MpalOMT3-Tr, but not MpalOMT2. Subcellular localization experiments indicated that the N-terminus of MpalOMT3 probably served as a chloroplast transit peptide and inhibited its enzymatic activity. Combining the phylogenetic analysis and functional characterization, we conclude that the liverwort M. paleacea harbors true CCoAOMT and CCoAOMT-like genes.


Assuntos
Lignina/biossíntese , Lignina/genética , Marchantia/enzimologia , Marchantia/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Clonagem Molecular , Genes de Plantas , Variação Genética , Genótipo , Filogenia
9.
Gene ; 808: 145971, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34543688

RESUMO

Trehalose is an important disaccharide that plays an important role in extreme environmental conditions. Trehalose-6-phosphate synthase (TPS) gene is the key gene for trehalose synthesis in Marsupenaeus japonicus. In this study, a TPS gene was isolated and characterized from M. japonicus. The full-length cDNA of TPS gene of M. japonicus (MjTPS) was 3308 bp, encoding 844 amino acids. The protein of the deduced MjTPS contained a glycol_transf_20 domain and a trehalose_PPase domain. The mRNA expression level of MjTPS was the highest in hepatopancreas. The further analysis found that MjTPS gene expression was up-regulated in a short time under low-salinity and high-nitrite stress, indicating that MjTPS gene had certain resistance to low-salinity and high-nitrite stress. Compared with the control group, both the expression of MjTPS and the trehalose content significantly decreased from 3 h to 24 h after MjTPS gene interference,. After RNAi, the mortality of M. japonicus increased, the expression level of MjTPS and the synthesis of downstream products decreased under low-salinity and high-nitrite stress, and what's more, the expression of immune genes PMO25, ERP, CD, HSP90, HSP70, HSP60, HMC and CLEC2 were significantly changed, implying that MjTPS might be participated in the immune response of M. japonicus. In addition, MjTPS gene silencing could affect the expression of CHI1 and CHS, suggesting that MjTPS might be involved in molting behavior of M. japonicus. These results provide new information for further studying the function of trehalose-6-phosphate synthase in shrimp.


Assuntos
Glucosiltransferases/genética , Penaeidae/genética , Sequência de Aminoácidos , Animais , China , Clonagem Molecular/métodos , Expressão Gênica , Glucosiltransferases/metabolismo , Penaeidae/metabolismo , Filogenia , Frutos do Mar , Trealose/metabolismo
10.
Chemosphere ; 288(Pt 1): 132437, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34627817

RESUMO

Cadmium (Cd), a widespread, severely toxic heavy metal, can cause serious reproductive toxicity in animals. However, the molecular pathways associated with Cd-induced effects remain unknown. In this study, we first cloned the vasa gene (Shvasa) and characterized the VASA protein (ShVASA) in Sinopotamon henanense. We then investigated the molecular mechanisms of Cd-induced reproductive toxicity. Shvasa was specifically expressed in the ovary and testis. ShVASA was abundant in early ovarian development and significantly less abundant in mature ovaries. During oogenesis, ShVASA was abundant and evenly distributed in the cytoplasm of the oogonium and previtellogenic oocytes, but gradually accumulated in the nuclear periphery of vitellogenic and mature oocytes. As Cd concentration increased, ShVASA abundance decreased gradually in proliferation-stage ovaries, and increased gradually in mature ovaries. Notably, at the small and large growth stages, ShVASA was upregulated following exposure to 14.5 mg/L Cd and downregulated following exposure to 29 mg/L Cd. In contrast to the unexposed control, ShVASA accumulated around the nuclear periphery in Cd-exposed previtellogenic oocytes and scattered gradually into the cytoplasm in Cd-exposed vitellogenic and mature oocytes. Shvasa RNA interference (RNAi) downregulated Shnanos and Shpiwi, but simultaneous Cd exposure and Shvasa RNAi significantly upregulated Shnanos and downregulated Shpiwi. These data suggested that Cd disrupted Shvasa expression and function, as well as the functions of Shnanos and Shpiwi, leading to severe reproductive toxicity in S. henanense.


Assuntos
Braquiúros , Animais , Cádmio/toxicidade , Clonagem Molecular , Feminino , Água Doce , Masculino , Reprodução
11.
Plant Biol (Stuttg) ; 24(1): 104-116, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34724309

RESUMO

The Corylus genus contains several important nut producing species and exhibits sporophytic self-incompatibility (SSI). However, the underlying molecular mechanisms of SSI in Corylus remain largely unknown. To clarify whether Corylus and Brassica share the same SSI molecular mechanism. We cloned ChaTHL1/2, ChaMLPK, ChaARC1, ChaEX70A1 genes from Ping'ou hybrid hazelnut using RACE techniques and tested the interaction between the ChaARC1 and ChaSRK1/2. We also examined the pistil-pollen interactions using scanning electron microscopy. We found no differences in the stigma surface within 1 h after compatible or incompatible pollination. Compatible pollen tubes penetrated the stigma surface, while incompatible pollen did not penetrate the stigma 4 h after pollination. Bioinformatics analysis revealed that ChaTHL1/2, ChaMLPK, ChaARC1 and ChaEX70A1 have corresponding functional domains. Quantitative real-time PCR (qRT-PCR) analysis showed that ChaTHL1/2, ChaMLPK, ChaARC1 and ChaEX70A1 were not regularly expressed in compatible or incompatible pollination. Furthermore, the expression patterns of ARC1, THL1/2, MLPK and Exo70A1 were quite distinct between Corylus and Brassica. According to yeast two-hybrid assays, ChaSRK1/2 did not interact with ChaARC1, confirming that the SRK-ARC1 signalling pathway implicated in the SSI response of Brassica was not conserved in Corylus. These results further reinforce the conclusion that, notwithstanding the similarity of the genetic basis, the SSI mechanism of Corylus does not conform in many respects with that of Brassica. Our findings could be helpful to better explore the potential mechanism of SSI system in Corylus.


Assuntos
Corylus , Clonagem Molecular , Corylus/genética , Flores/genética , Proteínas de Plantas/genética , Pólen/genética , Saccharomyces cerevisiae
12.
Ann Lab Med ; 42(2): 196-202, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635613

RESUMO

Background: Identifying the causal pathogen of encephalitis remains a clinical challenge. A 50-year-old man without a history of neurological disease was referred to our department for the evaluation of an intracranial lesion observed on brain magnetic resonance imaging (MRI) scans, and the pathology results suggested protozoal infection. We identified the species responsible for encephalitis using thymine-adenine (TA) cloning, suitable for routine clinical practice. Methods: We extracted DNA from a paraffin-embedded brain biopsy sample and performed TA cloning using two universal eukaryotic primers targeting the V4-5 and V9 regions of the 18S rRNA gene. The recombinant plasmids were extracted, and the inserted amplicons were identified by Sanger sequencing and a homology search of sequences in the National Center for Biotechnology Information Basic Local Alignment Search Tool. Results: The infection was confirmed to be caused by the free-living amoeba Balamuthia mandrillaris. Two of 41 colonies recombinant with 18S V4-5 primers and 35 of 63 colonies recombinant with the 18S V9 primer contained B. mandrillaris genes; all other colonies contained human genes. Pathogen-specific PCR ruled out Entamoeba histolytica, Naegleria fowleri, Acanthamoeba spp., and Toxoplasma gondii infections. Conclusions: This is the first report of B. mandrillaris-induced encephalitis in Korea based on molecular identification. TA cloning with the 18S rRNA gene is a feasible and affordable diagnostic tool for the detection of infectious agents of unknown etiology.


Assuntos
Balamuthia mandrillaris , Encefalite , Adenina , Balamuthia mandrillaris/genética , Clonagem Molecular , Encefalite/diagnóstico , Eucariotos , Humanos , Masculino , Pessoa de Meia-Idade , Timina
13.
Gene ; 807: 145960, 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-34509581

RESUMO

Opsin is a fellow of the G protein-coupled receptors (GPCRs) superfamily. It can be divided into visual and non-visual opsin according to whether it is directly involved in visual imaging. Opsin plays an important role in visual image formation and the regulation of non-image forming functions such as circadian entrainment in the growth, development and evolution of fish. Crimson snapper belongs to Perciforme mainly found in the Indo-West Pacific and the South China Sea. It is one of the most influential economic fishes in the South China Sea. In order to study the existence and expression of opsin gene in Crimson snapper, we sequenced the genome and tissue sample transcriptome of Crimson snapper. In this study, 32 opsin genes were identified from the genome of Crimson snapper. The length of these genes ranged from 1061 bp to 86203 bp and were distributed on 15 different chromosomes. The analysis of opsin gene family of Crimson snapper showed that the sws2 had two extra copies as compared with that of Zebrafish. Domain and motif analysis revealed that all the 32 opsin genes have seven-(pass)-transmembrane domain receptors (7TM receptors) each, and the opsin family contained 10 common motifs. The expression level of opsin gene, confirmed by RT-qPCR, was analyzed by using nine tissues transcriptome databases of Crimson snapper. The results showed that almost all opsin genes were highly expressed in the retina and brain, except opn7a and opn7b which were expressed in intestine and red skin, and almost no expression in other tissues. Our results provide a comprehensive basic knowledge for the opsin gene family of Crimson snapper, which has significance for the study of the function of opsin in Lutjanidaes.


Assuntos
Opsinas/genética , Perciformes/genética , Animais , Sequência de Bases/genética , China , Clonagem Molecular/métodos , Doenças dos Peixes/genética , Expressão Gênica/genética , Opsinas/metabolismo , Perciformes/metabolismo , Receptores Acoplados a Proteínas G/genética , Transcriptoma/genética
14.
BMC Plant Biol ; 21(1): 595, 2021 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-34915842

RESUMO

Sweet potato (Ipomoea batatas (L.) Lam.) is a good source of carbohydrates, an excellent raw material for starch-based industries, and a strong candidate for biofuel production due to its high starch content. However, the molecular basis of starch biosynthesis and accumulation in sweet potato is still insufficiently understood. Glucose-6-phosphate/phosphate translocators (GPTs) mediate the import of glucose-6-phosphate (Glc6P) into plastids for starch synthesis. Here, we report the isolation of a GPT-encoding gene, IbG6PPT1, from sweet potato and the identification of two additional IbG6PPT1 gene copies in the sweet potato genome. IbG6PPT1 encodes a chloroplast membrane-localized GPT belonging to the GPT1 group and highly expressed in storage root of sweet potato. Heterologous expression of IbG6PPT1 resulted in increased starch content in the leaves, root tips, and seeds and soluble sugar in seeds of Arabidopsis thaliana, but a reduction in soluble sugar in the leaves. These findings suggested that IbG6PPT1 might play a critical role in the distribution of carbon sources in source and sink and the accumulation of carbohydrates in storage tissues and would be a good candidate gene for controlling critical starch properties in sweet potato.


Assuntos
Antiporters/isolamento & purificação , Glucose-6-Fosfato/metabolismo , Ipomoea batatas/química , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Antiporters/química , Antiporters/genética , Antiporters/metabolismo , Cloroplastos/química , Clonagem Molecular , Genes de Plantas , Ipomoea batatas/genética , Ipomoea batatas/metabolismo , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Conformação Proteica , Amido/metabolismo , Açúcares/metabolismo
15.
Parasit Vectors ; 14(1): 612, 2021 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-34930413

RESUMO

BACKGROUND: It is well established that ecdysteroid hormones play an important role in arthropod development and reproduction, mediated by ecdysteroid receptors. Ticks are obligate hematophagous arthropods and vectors of pathogens. The salivary gland plays an essential role in tick growth and reproduction and in the transmission of pathogens to vertebrate hosts. During tick development, the salivary gland undergoes degeneration triggered by ecdysteroid hormones and activated by apoptosis. However, it is unknown how the ecdysteroid receptor and apoptosis regulate salivary gland degeneration. Here, we report the functional ecdysteroid receptor (a heterodimer of the ecdysone receptor [EcR] and ultraspiracle [USP]) isolated from the salivary gland of the tick Rhipicephalus haemaphysaloides and explore the molecular mechanism of ecdysteroid receptor regulation of salivary gland degeneration. METHODS: The full length of RhEcR and RhUSP open reading frames (ORFs) was obtained from the transcriptome. The RhEcR and RhUSP proteins were expressed in a bacterial heterologous system, Escherichia coli. Polyclonal antibodies were produced against synthetic peptides and were able to recognize recombinant and native proteins. Quantitative real-time PCR and western blot were used to detect the distribution of RhEcR, RhUSP, and RhCaspases in the R. haemaphysaloides organs. A proteomics approach was used to analyze the expression profiles of the ecdysteroid receptors, RhCaspases, and other proteins. To analyze the function of the ecdysteroid receptor, RNA interference (RNAi) was used to silence the genes in adult female ticks. Finally, the interaction of RhEcR and RhUSP was identified by heterologous co-expression assays in HEK293T cells. RESULTS: We identified the functional ecdysone receptor (RhEcR/RhUSP) of 20-hydroxyecdysone from the salivary gland of the tick R. haemaphysaloides. The RhEcR and RhUSP genes have three and two isoforms, respectively, and belong to a nuclear receptor family but with variable N-terminal A/B domains. The RhEcR gene silencing inhibited blood-feeding, blocked engorgement, and restrained salivary gland degeneration, showing the biological role of the RhEcR gene in ticks. In the ecdysteroid signaling pathway, RhEcR silencing inhibited salivary gland degeneration by suppressing caspase-dependent apoptosis. The heterologous expression in mammalian HEK293T cells showed that RhEcR1 interacts with RhUSP1 and induces caspase-dependent apoptosis. CONCLUSIONS: These data show that RhEcR has an essential role in tick physiology and represents a putative target for the control of ticks and tick-borne diseases.


Assuntos
Apoptose/fisiologia , Regulação da Expressão Gênica/fisiologia , Receptores de Esteroides/metabolismo , Rhipicephalus/metabolismo , Glândulas Salivares/fisiologia , Animais , Clonagem Molecular , Comportamento Alimentar , Feminino , Células HEK293 , Humanos , Fases de Leitura Aberta , Interferência de RNA , RNA de Cadeia Dupla , Receptores de Esteroides/genética
16.
Parasit Vectors ; 14(1): 611, 2021 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-34930417

RESUMO

BACKGROUND: Trehalose-6-phosphate phosphatase (TPP6) is a key enzyme in the trehalose biosynthesis pathway. The accumulation of TPP6 inside the body is harmful to the pathogen, but almost nothing is currently known about the function of TPP6 from Haemonchus contortus (CRE-GOB-1). METHODS: The H. contortus CRE-GOB-1 (HcGOB) gene was cloned and recombinant protein of GOB (rHcGOB) was expressed; transcription of the HcGOB gene at different developmental stages of H. contortus was then studied. The spatial expression pattern of the HcGOB gene in adult female and male worms was determined by both quantitative real-time PCR (qPCR) and immunofluorescence. The binding of the rHcGOB protein to goat PBMCs was assessed by immunofluorescence assay. The immunomodulatory impacts of rHcGOB on cell proliferation, nitric oxide generation and cytokine secretion were assessed by co-culture of rHcGOB protein with goat PBMCs. RESULTS: The HcGOB protein was transcribed in eggs, infective third-stage larvae (iL3s) and adults of H. contortus, with the highest transcript levels found in the egg stage. The transcript levels were significantly elevated in iL3s after manual desheathing. HcGOB was widely distributed in adult worms where it was mainly localized in the gut and gonads. rHcGOB was observed to bind to PBMCs and also to be recognized by sera collected from a goat infected with H. contortus. rHcGOB significantly activated the interleukin-10/transforming growth factor ß/signal transducer and activator of transcription 3 (IL-10/TGF-ß/STAT3) pathway in PBMCs while suppressing the transcription and expression of IL-4 and IL-17. CONCLUSIONS: These results suggest that the HcGOB gene plays an important role in the development, parasitism and reproduction of H. contortus. The rHcGOB protein affected the immunomodulatory function of PBMCs in the in vitro study, suggesting that this protein would be a promising vaccine target.


Assuntos
Haemonchus/enzimologia , Leucócitos Mononucleares/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Proliferação de Células , Clonagem Molecular , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Regulação Enzimológica da Expressão Gênica , Cabras , Haemonchus/classificação , Haemonchus/genética , Masculino , Monoéster Fosfórico Hidrolases/genética , Filogenia , Conformação Proteica , Ratos , Reprodutibilidade dos Testes
17.
Microb Cell Fact ; 20(1): 232, 2021 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-34963459

RESUMO

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.


Assuntos
Carboidratos/química , Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Álcool Desidrogenase/biossíntese , Álcool Desidrogenase/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Proteína Morfogenética Óssea 7/biossíntese , Proteína Morfogenética Óssea 7/isolamento & purificação , Proteínas de Transporte/biossíntese , Proteínas de Transporte/isolamento & purificação , Clonagem Molecular , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/isolamento & purificação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Humanos , Hidrolases/biossíntese , Hidrolases/isolamento & purificação , Corpos de Inclusão/metabolismo , Lipase/biossíntese , Lipase/isolamento & purificação , Proteínas Ligantes de Maltose , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/isolamento & purificação
18.
PLoS One ; 16(12): e0256562, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34936645

RESUMO

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Engenharia Metabólica/métodos , Pectobacterium carotovorum/enzimologia , Poligalacturonase/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Ácidos Hexurônicos/metabolismo , Pectinas/metabolismo , Pectobacterium carotovorum/genética , Poligalacturonase/genética , Cloreto de Potássio/metabolismo , Regiões Promotoras Genéticas
19.
Zhongguo Zhong Yao Za Zhi ; 46(19): 4950-4958, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34738389

RESUMO

In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid ß-oxidation pathway of Atractylodes lancea was cloned. Meanwhile, bioinformatics analysis, prokaryotic expression and gene expression analysis were carried out, which laid a foundation for the study of fatty acid ß-oxidation mechanism of A. lancea. The full-length sequence of the gene was cloned by RT-PCR with the specific primers designed according to the sequence information of KAT gene in the transcriptomic data of A. lancea and designated as AIKAT(GenBank accession number MW665111). The results showed that the open reading frame(ORF) of AIKAT was 1 323 bp, encoding 440 amino acid. The deduced protein had a theoretical molecular weight of 46 344.36 and an isoelectric point of 8.92. AIKAT was predicted to be a stable alkaline protein without transmembrane segment. The secondary structure of AIKAT was predicted to be mainly composed of α-helix. The tertiary structure of AIKAT protein was predicted by homology modeling method. Homologous alignment revealed that AIKAT shared high sequence identity with the KAT proteins(AaKAT2, CcKAT2, RgKAT and AtKAT, respectively) of Artemisia annua, Cynara cardunculus var. scolymus, Rehmannia glutinosa and Arabidopsis thaliana. The phylogenetic analysis showed that AIKAT clustered with CcKAT2, confirming the homology of 3-ketoacyl-CoA thiolase genes in Compositae. The prokaryotic expression vector pET-32 a-AIKAT was constructed and transformed into Escherichia coli BL21(DE3) for protein expression. The target protein was successfully expressed as a soluble protein of about 64 kDa. A real-time quantitative PCR analysis was performed to profile the AIKAT expression in different tissues of A. lancea. The results demonstrated that the expression level of AIKAT was the highest in rhizome, followed by that in leaves and stems. In this study, the full-length cDNA of AIKAT was cloned and expressed in E. coli BL21(DE3), and qRT-PCR showed the differential expression of this gene in different tissues, which laid a foundation for further research on the molecular mechanism of fatty acid ß-oxidation in A. lancea.


Assuntos
Atractylodes , Sequência de Aminoácidos , Atractylodes/genética , Clonagem Molecular , Coenzima A , Escherichia coli/genética , Filogenia
20.
PLoS One ; 16(11): e0257089, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34793485

RESUMO

Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.


Assuntos
Chlamydomonas reinhardtii , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes , Glicoproteína da Espícula de Coronavírus , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Clonagem Molecular , Humanos , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/isolamento & purificação
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