Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast

In plants, the first committed step in the pathway for biosynthesis of isoprenoids is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34). Here we report for the first time the cloning of a full-length cDNA encoding HMGR (Tm–HMGR) from a taxol-producing gymnosperm, Taxus media Rehder. The full-length cDNA of Tm–HMGR (GenBank accession number: AY277740) was 2307 base pairs (bp), with a 1791-bp open reading frame (ORF) encoding a 596-amino-acid polypeptide. Bioinformatic analysis revealed that Tm–HMGR contained two trans-membrane domains and a catalytic domain, and showed high homology to other plant HMGRs. Phylogenetic analysis indicated that Tm–HMGR was more ancient than other plant HMGRs. The structural modelling showed that Tm–HMGR had the typical spatial structure of HMGRs whose catalytic domains could be folded and divided into three spatial domains, L-domain, N-domain and S-domain. Southern blot analysis revealed that Tm–HMGR belonged to a small HMGR gene family. Northern blot analysis showed that Tm–HMGR was expressed in roots, stems and needles, with higher expression in stems and needles than in roots. Functional complementation of Tm–HMGR in a HMGR-deficient mutant yeast demonstrated that Tm–HMGR mediated the biosynthesis of mevalonate and provided the general precursor for taxol biosynthesis.

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Molecular Cloning of a HMG-CoA Reductase Gene from Eucommia ulmoides Oliver

Bioscience Reports ◽

10.1007/s10540-006-9010-3 ◽

2006 ◽

Vol 26 (2) ◽

pp. 171-181 ◽

Cited By ~ 27

Author(s):

Jihong Jiang ◽

Guoyin Kai ◽

Xiaoying Cao ◽

Fengmei Chen ◽

Dongning He ◽

...

Keyword(s):

Bioinformatic Analysis ◽

Tissue Expression ◽

Full Length ◽

Eucommia Ulmoides ◽

Phylogenetic Tree Analysis ◽

Reading Frame ◽

Full Length Cdna ◽

Calculated Molecular Weight ◽

Rapid Amplification ◽

Coa Reductase

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate, which is the first committed step in the pathway for isoprenoid biosynthesis in plants. A full-length cDNA encoding HMGR (designated as EuHMGR, GenBank Accession No. AY796343) was isolated from Eucommia ulmoides by rapid amplification of cDNA ends (RACE). The full-length cDNA of EuHMGR comprises 2281 bp with a 1770-bp open reading frame (ORF) encoding a 590-amino-acid polypeptide with two trans-membrane domains revealed by bioinformatic analysis. Molecular modeling showed that EuHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. The deduced protein has an isoelectric point (pI) of 6.89 and a calculated molecular weight of about 63 kDa. Sequence comparison analysis showed that EuHMGR had highest homology to HMGR from Hevea brasiliensis. As expected, phylogenetic tree analysis indicated that EuHMGR belongs to plant HMGR group. Tissue expression pattern analysis showed that EuHMGR is strongly expressed in the leaves and stems whereas it is only poorly expressed in the roots, which implies that EuHMGR may be a constitutively expressing gene. Functional complementation of EuHMGR in HMGR-deficient mutant yeast JRY2394 demonstrated that EuHMGR mediates the mevalonate biosynthesis in yeast.

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Cloning, characterisation and expression profile of kisspeptin1 and the kisspeptin1 receptor in the hypothalamic–pituitary–ovarian axis of Chinese alligator Alligator sinensis during the reproductive cycle

Reproduction Fertility and Development ◽

10.1071/rd19332 ◽

2020 ◽

Vol 32 (8) ◽

pp. 792 ◽

Cited By ~ 1

Author(s):

Ruidong Zhang ◽

Haitao Nie ◽

Shulong Duan ◽

Peng Yan ◽

Ali Izaz ◽

...

Keyword(s):

Amino Acid ◽

Quantitative Polymerase Chain Reaction ◽

Reproductive Period ◽

Full Length ◽

Reading Frame ◽

Full Length Cdna ◽

Cdna Sequences ◽

Acid Protein ◽

Amino Acid Precursor ◽

Alligator Sinensis

Kisspeptin1 (Kiss1), a product of the Kiss1 gene, plays an important role in the regulation of reproduction in vertebrates by activating the Kiss1 receptor (Kiss1R) and its coexpression with gonadotrophin-releasing hormone (GnRH) in GnRH neurons. The purpose of this study was to clone the Kiss1 and Kiss1R genes found in the brain of Alligator sinensis and to explore their relationship with reproduction. The full-length cDNA of Kiss1 is 816bp, the open reading frame (ORF) is 417bp and the gene encodes a 138-amino acid precursor protein. The full-length cDNA of Kiss1R is 2348bp, the ORF is 1086bp and the gene encodes a 361-amino acid protein. Quantitative polymerase chain reaction showed that, except for Kiss1R expression in the hypothalamus, the expression of Kiss1 and Kiss1Rduring the reproductive period of A. sinensis was higher than that in the hypothalamus, pituitary gland and ovary during the hibernation period. The changes in GnRH2 mRNA in the hypothalamus were similar to those of GnRH1 and peaked during the reproductive period. This study confirms the existence of Kiss1 and Kiss1R in A. sinensis and the findings strongly suggest that Kiss1 and Kiss1R may participate in the regulation of GnRH secretion in the hypothalamus of alligators during the reproductive period. Furthermore, this is the first report of the full-length cDNA sequences of Kiss1 and Kiss1R in reptiles.

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Identification, Characterization, and Expression Analysis of the TaNF-YB3 Gene from Triticum aestivum

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha3926083 ◽

2011 ◽

Vol 39 (2) ◽

pp. 254

Author(s):

Xiuchun DONG ◽

Zhiyuan CHENG ◽

Shuhan CHENG ◽

Feng XU ◽

Yanrong AN ◽

...

Keyword(s):

Triticum Aestivum ◽

Full Length ◽

Day Length ◽

Reading Frame ◽

Full Length Cdna ◽

Rhythmic Expression ◽

Binding Factor ◽

A Subunit ◽

Family Gene ◽

Ccaat Box

A full-length cDNA encoding a nuclear factor-YB (NF-YB)/HAP3/CCAAT binding factor-A (CBF-A) subunit of a CCAAT-box binding complex, designated as TaNF-YB3 was isolated from Triticum aestivum. Sequence analysis indicated that the full-length cDNA was 809 bp long, including an open reading frame (ORF) of 597 bp, which encoded a deduced polypeptide of 199 amino acids and is located in chromosome 3D. The deduced protein contained conserved structural domains and showed high identity to other plant NF-YBs. TaNF-YB3 was expressed in various organs, especially in the leaves and stamens; it was also regulated by salt, mannitol, abscisic acid, wounding, and cold. Moreover, TaNF-YB3 was down-regulated by short days and vernalization, and sensitive to the transfer of day length. It was mainly induced by light and exhibited a similar diurnal rhythmic expression pattern with the CCT-domain family gene VRN2 (TaZCCT1 and TaZCCT2), but not with CO (WCO1 and TaHd1). Overall, the results suggested that TaNF-YB3, aside from having a role in regulating day length and vernalization responses, might integrate signals from other environmental stresses to perform its functions in winter wheat adaptability and development.

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Functional identification of two minor capsid proteins from Chinese wheat mosaic virus using its infectious full-length cDNA clones

Journal of General Virology ◽

10.1099/jgv.0.000532 ◽

2016 ◽

Vol 97 (9) ◽

pp. 2441-2450 ◽

Cited By ~ 9

Author(s):

Jian Yang ◽

Fen Zhang ◽

Li Xie ◽

Xi-Jiao Song ◽

Jing Li ◽

...

Keyword(s):

Mosaic Virus ◽

Full Length ◽

Capsid Proteins ◽

Cdna Clones ◽

Functional Identification ◽

Full Length Cdna ◽

Chinese Wheat

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Heat stress-induced expression of Px-pdrg and Px-aspp2 in insecticide-resistant and -susceptible Plutella xylostella

Bulletin of Entomological Research ◽

10.1017/s0007485319000543 ◽

2019 ◽

Vol 110 (2) ◽

pp. 177-184

Author(s):

Yu Wang ◽

Jing Nan Wang ◽

Xue Zhun Chen ◽

Qi Xing Hu ◽

Qi Qing Liu ◽

...

Keyword(s):

Heat Stress ◽

Plutella Xylostella ◽

P53 Protein ◽

Diamondback Moth ◽

Transcript Abundance ◽

Full Length ◽

Reading Frame ◽

Full Length Cdna ◽

Calculated Molecular Weight ◽

Apoptosis Process

Abstractp53, DNA damage regulated gene (PDRG) and apoptosis-stimulating p53 protein 2 (ASPP2) are p53-related genes which can promote apoptosis. The full-length cDNA sequence of the Px-pdrg and Px-aspp2 genes were characterized and their mRNA expression dynamics under heat stress were studied in diamondback moth (DBM) Plutella xylostella collected from Fuzhou, China. The full-length cDNA of Px-pdrg and Px-aspp2 spans 721 and 4201 bp, containing 395 and 3216 bp of the open reading frame, which encode a putative protein comprising 130 and 1072 amino acids with a calculated molecular weight of 14.58 and 118.91 kDa, respectively. As compared to 25°C, both Px-pdrg and Px-aspp2 were upregulated in chlorpyrifos-resistant (Rc) and -susceptible (Sm) strains of DBM adults and pupae under heat stress. In addition, Rc DBM showed a significantly higher expression level of Px-pdrg and Px-aspp2 in contrast to Sm DBM. The results indicate that high temperature can significantly promote apoptosis process, especially in Rc-DBM. Significant fitness cost in Rc-DBM might be associated with drastically higher transcript abundance of Px-pdrg and Px-aspp2 under the heat stress.

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Isolation, characterization and sequence analysis of a full-length cDNA clone encoding acetohydroxy acid reductoisomerase from spinach chloroplasts

Biochemical Journal ◽

10.1042/bj2770469 ◽

1991 ◽

Vol 277 (2) ◽

pp. 469-475 ◽

Cited By ~ 13

Author(s):

R Dumas ◽

M Lebrun ◽

R Douce

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Single Gene ◽

Amino Acid Sequences ◽

Full Length ◽

Open Reading Frame ◽

Amino Acid Residues ◽

Protein Precursor ◽

Reading Frame ◽

Full Length Cdna

Acetohydroxy acid reductoisomerase (AHRI), the second enzyme in the parallel isoleucine/valine-biosynthetic pathway, catalyses an unusual two-step reaction in which the substrate, either 2-acetolactate or 2-aceto-2-hydroxybutyrate, is converted via an alkyl migration and an NADPH-dependent reduction to give 2,3-dihydroxy-3-methylbutyrate or 2,3-dihydroxy-3-methylvalerate respectively. We have isolated and characterized a full-length cDNA from a lambda gt11 spinach library encoding the complete acetohydroxy acid reductoisomerase protein precursor. The 2050-nucleotide sequence contains a 1785-nucleotide open reading frame. The derived amino acid sequence indicates that the protein precursor consists of 595 amino acid residues including a presequence peptide of 72 amino acid residues. The N-terminal sequence of the first 16 amino acid residues of the purified AHRI confirms the identity of the cDNA. The derived amino acid sequence from this open reading frame shows 23% identity with the deduced amino acid sequences of the Escherichia coli and Saccharomyces cerevisiae AHRI proteins. There are two blocks of conserved amino acid residues in these three proteins. One of these is a sequence similar to the ‘fingerprint’ region of the NAD(P)H-binding site found in a large number of NAD(P)H-dependent oxidoreductases. The other, a short sequence (Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Ser-His-Gly-Phe) containing the amino acids lysine and histidine, could well be the catalytic site of the first step of the AHRI reaction. Southern-blot analysis indicated that AHRI is encoded by a single gene per haploid genome of about 7.5 kbp containing at least four introns.

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Full-Length cDNA, Prokaryotic Expression, and Antimicrobial Activity of UuHb-F-I fromUrechis unicinctus

BioMed Research International ◽

10.1155/2016/5683026 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Rongli Niu ◽

Xiang Chen

Keyword(s):

Antimicrobial Activity ◽

Prokaryotic Expression ◽

Defense Mechanism ◽

Micrococcus Luteus ◽

Antimicrobial Activities ◽

Immune Defense ◽

Full Length ◽

Urechis Unicinctus ◽

Reading Frame ◽

Full Length Cdna

Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I fromUrechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration againstMicrococcus luteuswas 2.78–4.63 μM. Our results may help elucidate the immune defense mechanism ofU. unicinctusand may provide insights into new AMPs in drug discovery.

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Isolation, Expression, and Promoter Analysis ofGbWRKY2: A Novel Transcription Factor Gene fromGinkgo biloba

International Journal of Genomics ◽

10.1155/2015/607185 ◽

2015 ◽

Vol 2015 ◽

pp. 1-17 ◽

Cited By ~ 4

Author(s):

Yong-Ling Liao ◽

Yong-Bao Shen ◽

Jie Chang ◽

Wei-Wei Zhang ◽

Shui-Yuan Cheng ◽

...

Keyword(s):

Transcription Factor ◽

Zinc Finger ◽

Stress Responses ◽

Genomic Dna ◽

Single Copy ◽

Wrky Transcription Factor ◽

Full Length ◽

Flanking Sequence ◽

Reading Frame ◽

Full Length Cdna

WRKY transcription factor is involved in multiple life activities including plant growth and development as well as biotic and abiotic responses. We identified 28WRKYgenes from transcriptome data ofGinkgo bilobaaccording to conserved WRKY domains and zinc finger structure and selected three WRKY genes, which areGbWRKY2, GbWRKY16, andGbWRKY21, for expression pattern analysis.GbWRKY2was preferentially expressed in flowers and strongly induced by methyl jasmonate. Here, we cloned the full-length cDNA and genomic DNA ofGbWRKY2. The full-length cDNA ofGbWRKY2was 1,713 bp containing a 1,014 bp open reading frame encoding a polypeptide of 337 amino acids. TheGbWRKY2genomic DNA had one intron and two exons. The deducedGbWRKY2contained one WRKY domain and one zinc finger motif.GbWRKY2was classified into Group II WRKYs. Southern blot analysis revealed thatGbWRKY2was a single copy gene inG. biloba. Manycis-acting elements related to hormone and stress responses were identified in the 1,363 bp-length 5′-flanking sequence ofGbWRKY2, including W-box, ABRE-motif, MYBCOREs, and PYRIMIDINE-boxes, revealing the molecular mechanism of upregulated expression ofGbWRKY2by hormone and stress treatments. Further functional characterizations in transiently transformed tobacco leaves allowed us to identify the region that can be considered as the minimal promoter.

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Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans.

The Journal of Cell Biology ◽

10.1083/jcb.115.6.1521 ◽

1991 ◽

Vol 115 (6) ◽

pp. 1521-1534 ◽

Cited By ~ 86

Author(s):

K W Moremen ◽

P W Robbins

Keyword(s):

Amino Acids ◽

Catalytic Domain ◽

Transmembrane Domain ◽

Full Length ◽

Open Reading Frame ◽

Cdna Clones ◽

Northern Blots ◽

Reactive Material ◽

Reading Frame ◽

High Mannose

Golgi alpha-mannosidase II (GlcNAc transferase I-dependent alpha 1,3[alpha 1,6] mannosidase, EC 3.2.1.114) catalyzes the final hydrolytic step in the N-glycan maturation pathway acting as the committed step in the conversion of high mannose to complex type structures. We have isolated overlapping clones from a murine cDNA library encoding the full length alpha-mannosidase II open reading frame and most of the 5' and 3' untranslated region. The coding sequence predicts a type II transmembrane protein with a short cytoplasmic tail (five amino acids), a single transmembrane domain (21 amino acids), and a large COOH-terminal catalytic domain (1,124 amino acids). This domain organization which is shared with the Golgi glycosyl-transferases suggests that the common structural motifs may have a functional role in Golgi enzyme function or localization. Three sets of polyadenylated clones were isolated extending 3' beyond the open reading frame by as much as 2,543 bp. Northern blots suggest that these polyadenylated clones totaling 6.1 kb in length correspond to minor message species smaller than the full length message. The largest and predominant message on Northern blots (7.5 kb) presumably extends another approximately 1.4-kb downstream beyond the longest of the isolated clones. Transient expression of the alpha-mannosidase II cDNA in COS cells resulted in 8-12-fold overexpression of enzyme activity, and the appearance of cross-reactive material in a perinuclear membrane array consistent with a Golgi localization. A region within the catalytic domain of the alpha-mannosidase II open reading frame bears a strong similarity to a corresponding sequence in the rat liver endoplasmic reticulum alpha-mannosidase and the vacuolar alpha-mannosidase of Saccharomyces cerevisiae. Partial human alpha-mannosidase II cDNA clones were also isolated and the gene was localized to human chromosome 5.

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