Cloning and Expression Analysis of a Squalene Epoxidase Gene from Ginkgo biloba

Squalene epoxidase is a key enzyme involved in triterpene saponins biosynthetic pathways in plants. In this study, the SE gene was isolated from Ginkgo biloba by RT-PCR, which designated as GbSE, GenBank accession number: KY751713. The length of GbSE gene is 1646 bp, contains an open reading frame of 1617 bp encoding 538 amino acids. The theoretical molecular weight and pI of GbSE protein was 58.3 kDa and 8.35, respectively. Sequence multiple alignment found that GbSE protein had high homology with SE proteins from other plants, including several highly conservative motifs and amino acids. Phylogenetic tree analysis showed that Ginkgo biloba SE belonged to the gymnospermous group, and was closely related to the SE protein of Picea sitchensis. A study of gene expression analysis indicated that the GbSE gene was highly expressed in stems, low expressed in fruits. The cDNA sequence of GbSE gene was cloned from G. biloba and the expression level of this gene was analyzed. This work can provide some theoretical basis for the research on the molecular mechanism of triterpenoid saponin biosynthesis.

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The Molecular Cloning and Expression Analysis of a CYP71 Gene in Ginkgo biloba L.

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha44110214 ◽

2016 ◽

Vol 44 (1) ◽

pp. 77-84 ◽

Cited By ~ 2

Author(s):

Xinliang LIU ◽

Fuliang CAO ◽

Jinfeng CAI ◽

Huanli WANG

Keyword(s):

Molecular Weight ◽

Expression Analysis ◽

Ginkgo Biloba ◽

Abiotic Stresses ◽

Coli Strain ◽

Cloning And Expression ◽

Cytochrome P450 Monooxygenases ◽

Biotic And Abiotic Stresses ◽

Reading Frame ◽

P450 Monooxygenases

Cytochrome P450 monooxygenases (CYPs) are a group of redox proteins that catalyze various oxidative reactions in plant secondary metabolism. To explore the function of the CYP71 gene in Ginkgo biloba under biotic and abiotic stresses, a full-length CYP gene, designated GbCYP71, was first isolated and characterized from leaves of G. biloba. It contained a 1512-bp open reading frame (ORF) encoding 503 amino-acid-deduced polypeptide whose theoretical molecular weight was 56.9 kDa. The genomic DNA sequence of GbCYP71 contained two exons and one intron. The cDNA of GbCYP71 was subcloned in a pET-32a vector and then transformed into E. coli strain BL21 (DE3). A protein with a molecular weight of 76.4 kDa was subsequently identified and found to be consistent with the above theoretical value. Transient expression analysis revealed that the GbCYP71 protein may be located in the G. biloba cell cytoplasm. GbCYP71 was expressed in almost all ginkgo tissues, including leaves, stamens, gynoecia, stems and, preferentially, roots. Expression-profiling analyses revealed that GbCYP71 can be induced by salinity stress and phytohormone signals, including salicylic acid, abscisic acid, methyl jasmonate and ethephon, but is repressed by heat and cold stresses. These results indicate that GbCYP71 mainly functions in responding to biotic and abiotic stresses.

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Molecular Cloning and Expression Analysis of a MADS-Box Gene (GbMADS2) from Ginkgo biloba

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha4319760 ◽

2015 ◽

Vol 43 (1) ◽

pp. 19-24 ◽

Cited By ~ 3

Author(s):

Xiaohui WANG ◽

Junhuan CHENG ◽

Feng XU ◽

Xingxiang LI ◽

Weiwei ZHANG ◽

...

Keyword(s):

Expression Analysis ◽

Ginkgo Biloba ◽

Flower Development ◽

Time Course ◽

Male Flower ◽

Cloning And Expression ◽

Pcr Analysis ◽

Mads Box ◽

Typical Structure ◽

Mads Box Gene

As a kind of transcription factors gene family, MADS-box genes play an important role in plant development processes. To find genes involved in the floral transition of Ginkgo biloba, a MADS-box gene, designated as GbMADS2, was cloned from G. biloba based on EST sequences by RT-PCR. Sequence analysis results showed that the cDNA sequence of GbMADS2 contained a 663 bp length ORF encoding 221 amino acids protein, which displayed typical structure of plant MADS-box protein including MADS, I, and K domains and C terminus. The sequence of GbMADS2 protein was highly homologous to those of MADS-box proteins from other plant species with the highest homologous to AGAMOUS (CyAG) from Cycas revoluta. The phylogenetic tree analysis revealed that GbMADS2 belonged to AGAMOUS clade genes. Real-time PCR analysis indicated that expression levels of GbMADS2 gene in female and male flower were significantly higher than those in root, stem, and leaves, and that GbMADS2 expression level increased along with time of flower development. The spatial and time-course expression profile of GbMADS2 implied that GbMADS2 might be involved in development of reproductive organs. The isolation and expression analysis of GbMADS2 provided basis for further studying the molecular mechanism of flower development in G. biloba.

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Molecular Cloning and Expression Analysis of a AGAMOUS-like 66 Gene (GbAGL66) in Ginkgo biloba

Biotechnology(Faisalabad) ◽

10.3923/biotech.2017.100.107 ◽

2017 ◽

Vol 16 (3) ◽

pp. 100-107 ◽

Cited By ~ 1

Author(s):

Jinshuang Dou ◽

Lanlan Wang ◽

Jiaping Yan ◽

Mingyue Fu ◽

Xian Zhang ◽

...

Keyword(s):

Molecular Cloning ◽

Expression Analysis ◽

Ginkgo Biloba ◽

Cloning And Expression

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Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3080405 ◽

1995 ◽

Vol 308 (2) ◽

pp. 405-409 ◽

Cited By ~ 17

Author(s):

Y Chiba ◽

T Midorikawa ◽

E Ichishima

Keyword(s):

Amino Acids ◽

Cell Extract ◽

Catalytic Triad ◽

Site Directed Mutagenesis ◽

Cloning And Expression ◽

Yeast Cells ◽

Directed Mutagenesis ◽

Serine Carboxypeptidase ◽

Reading Frame ◽

Sds Page

Carboxypeptidase from Aspergillus saitoi removes acidic, neutral and basic amino acids as well as proline from the C-terminal position at pH 2-5. cpdS, a cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. Analysis of the 1816-nucleotide sequence revealed a single open reading frame coding for 523 amino acids. When A. saitoi carboxypeptidase cDNA was expressed in yeast cells, carboxypeptidase activity was detected in the cell extract and was immunostained with a 72 kDa protein with polyclonal anti-(A. saitoi carboxypeptidase) serum. The recombinant enzyme treated with glycopeptidase F migrated with an apparent molecular mass of 60 kDa on SDS/PAGE, which was the same as that of the de-N-glycosylated carboxypeptidase from A. saitoi. Site-directed mutagenesis of the cpdS indicated that Ser-153, Asp-357 and His-436 residues were essential for the enzymic catalysis. It can be concluded that A. saitoi carboxypeptidase has a catalytic triad comprising Asp-His-Ser and is a member of serine carboxypeptidase family (EC 3.4.16.1).

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Molecular cloning and expression analysis of a WRKY transcription factor gene, GbWRKY20, from Ginkgo biloba

Plant Signaling & Behavior ◽

10.1080/15592324.2021.1930442 ◽

2021 ◽

pp. 1930442

Author(s):

Tingting Zhou ◽

Xiaoming Yang ◽

Guibin Wang ◽

Fuliang Cao

Keyword(s):

Transcription Factor ◽

Molecular Cloning ◽

Expression Analysis ◽

Ginkgo Biloba ◽

Wrky Transcription Factor ◽

Cloning And Expression ◽

Transcription Factor Gene ◽

Factor Gene

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The Molecular Cloning and Expression Analysis of a CYP71 Gene in Ginkgo biloba L.

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha.44.1.10214 ◽

2016 ◽

Vol 44 (1) ◽

Author(s):

Xinliang LIU ◽

Fuliang CAO ◽

Jinfeng CAI ◽

Huanli WANG

Keyword(s):

Molecular Cloning ◽

Expression Analysis ◽

Ginkgo Biloba ◽

Cloning And Expression

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Key Enzymes of Triterpenoid Saponin Biosynthesis and the Induction of Their Activities and Gene Expressions in Plants

Natural Product Communications ◽

10.1177/1934578x1000500736 ◽

2010 ◽

Vol 5 (7) ◽

pp. 1934578X1000500 ◽

Cited By ~ 4

Author(s):

Chang Ling Zhao ◽

Xiu Ming Cui ◽

Yan Ping Chen ◽

Quan Liang

Keyword(s):

Medicinal Plants ◽

Higher Plants ◽

Squalene Epoxidase ◽

Signal Pathways ◽

Triterpenoid Saponin ◽

Active Components ◽

Gene Expressions ◽

Triterpenoid Saponins ◽

Key Enzymes ◽

Saponin Biosynthesis

Triterpenoid saponins are one of the key active components of many medicinal plants. The biosynthetic pathway of triterpenoid saponins in higher plants and a lot of experimental results both indicated that the key enzymes involved in triterpenoid saponin synthesis are squalene synthase (SS), squalene epoxidase (SE), lupeol synthase (LS), dammarenediol synthase (DS), β-amyrin synthase (β-AS), cytochrome P450-dependent monooxygenase (PDMO), and glycosyltransferase (GT). The activities and coding genes of the key enzymes could be induced by a range of factors in various plant species. However, the effects of the factors on the content and composition of the triterpenoid saponins in specific plants are not certainly coincident, and different factors appear to induce the gene expressions of the key enzymes by different signal pathways and at different levels. This paper could provide a reference for strengthening the triterpenoid saponin-synthesizing capability of specific medicinal plants at enzyme and/or gene expression levels in order to improve the plants’ commercial values.

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Cloning and Expression of Mycobacterium tuberculosis and Mycobacterium leprae Dihydropteroate Synthase in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.181.21.6814-6821.1999 ◽

1999 ◽

Vol 181 (21) ◽

pp. 6814-6821 ◽

Cited By ~ 41

Author(s):

Vanida Nopponpunth ◽

Worachart Sirawaraporn ◽

Patricia J. Greene ◽

Daniel V. Santi

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Mycobacterium Tuberculosis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Mycobacterium Leprae ◽

Open Reading Frame ◽

Cloning And Expression ◽

Reading Frame ◽

Dihydropteroate Synthase

ABSTRACT The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosisdihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain ofEscherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. lepraedihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from bothM. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.

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Cloning and expression of an airway epithelial 12-lipoxygenase

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1992.262.2.l198 ◽

1992 ◽

Vol 262 (2) ◽

pp. L198-L207 ◽

Cited By ~ 3

Author(s):

N. De Marzo ◽

D. L. Sloane ◽

S. Dicharry ◽

E. Highland ◽

E. Sigal

Keyword(s):

Amino Acids ◽

Untranslated Region ◽

Polyclonal Antibodies ◽

Cloning And Expression ◽

Tracheal Epithelium ◽

Airway Epithelial ◽

Reading Frame ◽

Catalytic Function ◽

Airway Function ◽

12 Lipoxygenase

Arachidonate 12-lipoxygenase generates metabolites that may regulate airway function. To further characterize this enzyme, we isolated a cDNA corresponding to 12-lipoxygenase from a bovine tracheal epithelium cDNA library using human reticulocyte 15-lipoxygenase cDNA as a probe. The resulting 2.9-kb cDNA, the identity of which was confirmed by expression of active catalytic function in Escherichia coli has a 2.0-kb open reading frame encoding a protein of 75,000 kDa and includes 5 bp of 5'-untranslated region and 0.9 kb of 3'-untranslated region. On Northern blots, the 12-lipoxygenase cDNA hybridized to one band (3.5 kb) of bovine tracheal epithelium RNA. Polyclonal antibodies that recognize human tracheal 15-lipoxygenase cross-reacted on immunoblots to the expressed bovine tracheal 12-lipoxygenase. Further, the deduced amino acid sequence is 86% identical (93% similar) to human 15-lipoxygenase but 64% identical to human platelet 12-lipoxygenase, suggesting that the bovine tracheal enzyme is the homologue of the human 15-lipoxygenase. This is the first sequence of an epithelial lipoxygenase from any species. A comparison of the bovine sequence with other lipoxygenase sequences shows that there are only four amino acids which are conserved differences between a 12-lipoxygenase and a 15-lipoxygenase. We hypothesize that these four amino acids may be responsible for the positional specificity of the enzyme.

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