Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 1.863
Filter
1.
Article in English | WPRIM | ID: wpr-939917

ABSTRACT

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.


Subject(s)
Cloning, Molecular , Escherichia coli/metabolism , Phenylalanine/metabolism , Phenylalanine Ammonia-Lyase/chemistry , Recombinant Proteins , Schisandra/genetics
2.
Article in Chinese | WPRIM | ID: wpr-928121

ABSTRACT

In order to explore the functions of genes of key rate-limiting enzymes chalcone isomerase(CHI) and chalcone synthase(CHS) in the biosynthesis of flavonoids in Lonicera macranthoides, this study screened and cloned the cDNA sequences of CHI and CHS genes from the transcriptome data of conventional variety and 'Xianglei' of L. macranthoides. Online bioinformatics analysis software was used to analyze the characteristics of the encoded proteins, and quantitative reverse-transcription polymerase chain reaction(qRT-PCR) to detect the expression of CHI and CHS in different parts of the varieties at different flowering stages. The content of luteo-loside was determined by high performance liquid chromatography(HPLC) and the correlation with the expression of the two genes was analyzed. The results showed that the CHI and CHS of the two varieties contained a 627 bp and 1170 bp open reading frame(ORF), respectively, and the CHI protein and CHS protein were stable, hydrophilic, and non-secretory. qRT-PCR results demonstrated that CHI and CHS of the two varieties were differentially expressed in stems and leaves at different flowering stages, particularly the key stages. Based on HPLC data, luteoloside content was in negative correlation with the relative expression of the genes. Thus, CHI and CHS might regulate the accumulation of flavonoids in L. macranthoides, and the specific functions should be further studied. This study cloned CHI and CHS in L. macranthoides and analyzed their expression for the first time, which laid a basis for investigating the molecular mechanism of the differences in flavonoids such as luteoloside in L. macranthoides and variety breeding.


Subject(s)
Acyltransferases/metabolism , Chalcone , Cloning, Molecular , Intramolecular Lyases , Lonicera/metabolism , Plant Breeding
3.
Article in Chinese | WPRIM | ID: wpr-927983

ABSTRACT

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.


Subject(s)
Amino Acid Sequence , Cloning, Molecular , Geranyltranstransferase/genetics , Pogostemon , Transcription Factors/genetics
4.
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
5.
Chinese Journal of Biotechnology ; (12): 1576-1588, 2022.
Article in Chinese | WPRIM | ID: wpr-927802

ABSTRACT

In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes LguⅠ and BbvCⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of LguⅠ and BbvCⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB-welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.


Subject(s)
Base Sequence , Cloning, Molecular , Fermentation , Plasmids/genetics , Sphingomonas/metabolism
6.
Chinese Journal of Biotechnology ; (12): 386-395, 2022.
Article in Chinese | WPRIM | ID: wpr-927718

ABSTRACT

Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate. Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0-10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20-30 ℃, and less than 60% of the activity could be retained at temperatures higher than 50 ℃. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.


Subject(s)
Ascomycota/genetics , Carboxylic Ester Hydrolases , Cloning, Molecular , Phylogeny
7.
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
8.
Chinese Journal of Biotechnology ; (12): 275-286, 2022.
Article in Chinese | WPRIM | ID: wpr-927711

ABSTRACT

This study aims to investigate the molecular mechanism of the transcription factor MYB10, which is involved in anthocyanin biosynthesis, in different colors of Ribes L. fruitification. Rapid amplification of cDNA ends (RACE) was used to clone the MYB10 genes from Ribes nigrum L. (RnMYB10), Ribes rubrum L. (RrMYB10), and Ribes album L. (RaMYB10), respectively. Phylogenetic analysis showed that RnMYB10 and RrMYB10 were evolutionarily homologous. Real-time quantitative PCR (RT-qPCR) showed that the expression of MYB10 in the fruits of Ribes nigrum L. was higher than that of Ribes rubrum L. and much higher than that of Ribes album L. The expression of RnMYB10 and RrMYB10 increased at first and then decreased as the fruit diameter increased and the fruit color deepened (the maximum expression level was reached at 75% of the fruit color change), while the expression level of RaMYB10 was very low. Overexpression of RnMYB10 and RrMYB10 in Arabidopsis thaliana resulted in purple petioles and leaves, whereas overexpression of RaMYB10 resulted in no significant color changes. This indicates that MYB10 gene plays an important role in the coloration of Ribes L. fruit.


Subject(s)
Anthocyanins , Cloning, Molecular , Fruit , Gene Expression Regulation, Plant , Phylogeny , Plant Proteins/metabolism , Ribes/genetics
9.
Article in English | WPRIM | ID: wpr-929243

ABSTRACT

Pueraria thomsonii has long been used in traditional Chinese medicine. Isoflavonoids are the principle pharmacologically active components, which are primarily observed as glycosyl-conjugates and accumulate in P. thomsonii roots. However, the molecular mechanisms underlying the glycosylation processes in (iso)flavonoid biosynthesis have not been thoroughly elucidated. In the current study, an O-glucosyltransferase (PtUGT8) was identified in the medicinal plant P. thomsonii from RNA-seq database. Biochemical assays of the recombinant PtUGT8 showed that it was able to glycosylate chalcone (isoliquiritigenin) at the 4-OH position and glycosylate isoflavones (daidzein, formononetin, and genistein) at the 7-OH or 4'-OH position, exhibiting no enzyme activity to flavonones (liquiritigenin and narigenin) in vitro. The identification of PtUGT8 may provide a useful enzyme catalyst for efficient biotransformation of isoflavones and other natural products for food or pharmacological applications.


Subject(s)
Cloning, Molecular , Genistein , Glucosyltransferases/metabolism , Isoflavones/pharmacology , Pueraria/chemistry
10.
Braz. j. biol ; 82: e239449, 2022. tab, graf
Article in English | LILACS | ID: biblio-1249271

ABSTRACT

Abstract Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.


Resumo A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.


Subject(s)
Escherichia coli/genetics , alpha-Amylases/genetics , alpha-Amylases/metabolism , Temperature , Enzyme Stability , Cloning, Molecular , Geobacillus , Hydrogen-Ion Concentration
11.
Electron J Biotechnol ; 49: 29-33, Jan. 2021. tab, ilus
Article in English | LILACS | ID: biblio-1291632

ABSTRACT

BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.


Subject(s)
Animals , Polymerase Chain Reaction/methods , Agkistrodon/genetics , Cytochromes b/genetics , Mitochondria/genetics , Snakes , Species Specificity , DNA/analysis , Cloning, Molecular , Medicine, Chinese Traditional
12.
Article in Chinese | WPRIM | ID: wpr-888138

ABSTRACT

Stolon is an important organ for reproduction and regeneration of Amana edulis. Previous analysis of transcriptome showed that MYB was one of the most active transcription factor families during the development of A. edulis stolon. In order to study the possible role of MYB transcription factors in stolon development, the authors screened out an up-regulated MYB gene named AeMYB4 was by analyzing the expression profile of MYB transcription factors. In the present study, sequence analysis demonstrated that AeMYB4 contained an open reading frame of 756 bp encoding 251 amino acids, and domain analysis revealed that the predicted amino acids sequence contained two highly conserved SANT domains and binding sites for cold stress factor CBF. By multiple sequence alignment and phylogenetic analysis, it is indicated that AeMYB4 clustered with AtMYB15 from Arabidopsis thaliana, belonging to subgroup S2 of R2 R3-MYB. And most of the transcription factors in this subfamily are related to low temperature stress. The GFP-AeMYB4 fusion protein expression vector for subcellular localization was constructed and transferred into Agrobacterium tumefaciens to infect the leaves of Nicotiana benthamiana, and the results showed the protein was located in the nucleus. To investigate the transcriptional activation, the constructed pGBKT7-AeMYB4 fusion expression vector was transferred into Y2 H Gold yeast cells, which proved that AeMYB4 was a transcription activator with strong transcriptional activity. Real-time quantitative PCR was used to detect the expression of AeMYB4 gene in three different development stages of stolon and in leaves, flowers, and bulbs of A. edulis, which indicated that AeMYB4 transcription factor was tissue-specific in expression, mainly in the stolon development stage, and that the expression was the most active in the middle stage of stolon development, suggesting that AeMYB4 gene may play an important role in stolon development. This study contributes to the further research on the function of AeMYB4 transcription factor in stolon development of A. edulis.


Subject(s)
Amino Acid Sequence , Arabidopsis/metabolism , Cloning, Molecular , Gene Expression Regulation, Plant , Humans , Phylogeny , Plant Proteins/metabolism
13.
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
14.
Chinese Journal of Biotechnology ; (12): 2813-2824, 2021.
Article in Chinese | WPRIM | ID: wpr-887844

ABSTRACT

Squalene is widely used in pharmaceutical, nutraceutical, cosmetics and other fields because of its strong antioxidative, antibacterial and anti-tumor activities. In order to produce squalene, a gene ispA encoding farnesyl pyrophosphate synthase was overexpressed in a previously engineered Escherichia coli strain capable of efficiently producing terpenoids, resulting in a chassis strain that efficiently synthesizes triterpenoids. Through phylogenetic analysis, screening, cloning and expression of squalene synthase derived from different prokaryotes, engineered E. coli strains capable of efficiently producing squalene were obtained. Among them, squalene produced by strains harboring squalene synthase derived from Thermosynechococcus elongatus and Synechococcus lividus reached (16.5±1.4) mg/g DCW ((167.1±14.3) mg/L broth) and (12.0±1.9) mg/g DCW ((121.8±19.5) mg/L broth), respectively. Compared with the first-generation strains harboring the human-derived squalene synthase, the squalene synthase derived from T. elongatus and S. lividus remarkably increased the squalene production by 3.3 times and 2.4 times, respectively, making progress toward the cost-effective heterologous production of squalene.


Subject(s)
Cloning, Molecular , Escherichia coli/genetics , Humans , Phylogeny , Squalene , Synechococcus
15.
Chinese Journal of Biotechnology ; (12): 2786-2793, 2021.
Article in Chinese | WPRIM | ID: wpr-887841

ABSTRACT

To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.


Subject(s)
Animals , Baculoviridae/genetics , CD40 Ligand/genetics , Chickens , Cloning, Molecular , Genetic Vectors/genetics , Recombinant Proteins/genetics
16.
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
17.
Article in Chinese | WPRIM | ID: wpr-921632

ABSTRACT

In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid β-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 β-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 β-oxidation in A. lancea.


Subject(s)
Amino Acid Sequence , Atractylodes/genetics , Cloning, Molecular , Coenzyme A , Escherichia coli/genetics , Phylogeny
18.
Chinese Journal of Biotechnology ; (12): 4395-4405, 2021.
Article in Chinese | WPRIM | ID: wpr-921515

ABSTRACT

To investigate the enzyme properties of the black sesame polyphenol oxidase (BsPPO), a synthesized Bsppo gene was cloned into the vector pMAL-c5x and expressed in E. coli. Subsequently, the MBP fusion label in the recombinant protein was removed by protease digestion after affinity purification. The synthesized Bsppo gene contained 1 752 bp which encodes 585 amino acids with a deduced molecular weight of 65.3 kDa. Transformation of the recombinant vector into E. coli BL21(DE3) resulted in soluble expression of the fusion protein MBP-BsPPO. The enzymatic properties of the recombinant BsPPO was investigated after MBP fusion tag excision followed by affinity purification. The results demonstrated that the optimal temperature and pH for BsPPO was 25°C and 4.0, respectively. BsPPO exhibited a good stability under low temperature and acidic environment. Low-intensity short-term light exposure increased the activity of BsPPO. Cu²⁺ could improve the activity of BsPPO while Zn²⁺ and Ca²⁺ showed the opposite effect. BsPPO could catalyze the oxidation of monophenols, diphenols, and triphenols, and exhibited good catalytic activity on l-tyrosine and vanillic acid. Moreover, BsPPO exhibited high catalytic activity on black sesame metabolites, including 2-methoxy cinnamic acid, indole-3-carboxylic acid and phloretin. These results may serve as a basis for further characterization of BsPPO.


Subject(s)
Catechol Oxidase/genetics , Cloning, Molecular , Escherichia coli/metabolism , Recombinant Proteins/genetics , Sesamum/genetics
19.
Chinese Journal of Biotechnology ; (12): 4351-4362, 2021.
Article in Chinese | WPRIM | ID: wpr-921511

ABSTRACT

To explore the function of a heat shock transcription factor gene (HSFB1) and its promoter in Amorphophallus, a 1 365 bp DNA sequence was obtained by homologous cloning from Amorphophallus albus. The gene expression level of AaHSFB1 determined by qRT-PCR indicated that AaHSFB1 gene is more sensitive to heat stress. The expression level of AaHSFB1 in roots increased followed by a decrease upon heat treatment, and the highest expression level was observed after heat treatment for 1 h. The expression level of AaHSFB1 in leaves reached the highest after heat treatment for 12 h. The expression level in bulbs did not change greatly during the heat treatment. Subcellular localization analysis showed that AaHSFB1 protein was localized in the nucleus. A 1 509 bp DNA sequence which contains the AaHSFB1 promoter was obtained by FPNI-PCR method. Bioinformatics analysis showed that the promoter contained heat stress response elements HSE and a plurality of cis-acting elements related to plant development and stress response. A prAaHSFB1::GUS fusion expression vector was constructed to further analyze the function of AaHSFB1 promoter. The expression vector was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated method, and GUS staining analysis on transgenic plants after heat treatment was performed. The results showed that AaHSFB1 promoter had very high activity in the leaves. Therefore, we speculate that AaHSFB1 may play an important role in the stress resistance of A. albus, especially when encountering heat stress.


Subject(s)
Amorphophallus/metabolism , Arabidopsis/genetics , Cloning, Molecular , Gene Expression Regulation, Plant , Plant Proteins/metabolism , Plants, Genetically Modified/genetics
20.
Article in Chinese | WPRIM | ID: wpr-879176

ABSTRACT

Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP_019237476.1) and AeCCL7(XP_019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.


Subject(s)
Boraginaceae/genetics , Cloning, Molecular , Coenzyme A , Coenzyme A Ligases/genetics , Ligases , Phylogeny
SELECTION OF CITATIONS
SEARCH DETAIL