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1.
Suttisrisung S Senapin S Withyachumnarnkul B Wongprasert K 《Journal of biosciences》2011,36(5):833-843
A legume-type lectin (L-lectin) gene of the red algae Gracilaria fisheri (GFL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of GFL was 1714 bp and contained a 1542 bp
open reading frame encoding 513 amino acids with a predicted molecular mass of 56.5 kDa. Analysis of the putative amino acid
sequence with NCBI-BLAST revealed a high homology (30–68%) with legume-type lectins (L-lectin) from Griffithsia japonica, Clavispora lusitaniae, Acyrthosiphon pisum, Tetraodon nigroviridis and Xenopus tropicalis. Phylogenetic relationship analysis showed the highest sequence identity to a glycoprotein of the red algae Griffithsia japonica (68%) (GenBank number AAM93989). Conserved Domain Database analysis detected an N-terminal carbohydrate recognition domain
(CRD), the characteristic of L-lectins, which contained two sugar binding sites and a metal binding site. The secondary structure
prediction of GFL showed a β-sheet structure, connected with turn and coil. The most abundant structural element of GFL was
the random coil, while the α-helixes were distributed at the N- and C-termini, and 21 β-sheets were distributed in the CRD.
Computer analysis of three-dimensional structure showed a common feature of L-lectins of GFL, which included an overall globular
shape that was composed of a β-sandwich of two anti-parallel β-sheets, monosaccharide binding sites, were on the top of the
structure and in proximity with a metal binding site. Northern blot analysis using a DIG-labelled probe derived from a partial
GFL sequence revealed a hybridization signal of ~1.7 kb consistent with the length of the full-length GFL cDNA identified
by RACE. No detectable band was observed from control total RNA extracted from filamentous green algae. 相似文献
2.
Weiwei Ren Lingxia Zhao Lida Zhang Yuliang Wang Lijie Cui Yueli Tang Xiaofen Sun Kexuan Tang 《Molecular biology reports》2011,38(3):1813-1819
Tocochromanols, usually known as vitamin E, play a crucial role in human and animal nutrition. The enzyme homogentisate phytyltransferase
(HPT) performs the first committed step of the vitamin E biosynthetic pathway. The full-length cDNA encoding HPT was isolated
from Lactuca sativa L. by rapid amplification of cDNA ends (RACE). The cDNA, designated as LsHPT, was 1,670 bp long containing an open reading frame (ORF) of 1,185 bp which encoded a protein of 395 amino acids. Sequence
analysis indicated that the deduced protein, named as LsHPT, shared high identity with other dicotyledonous HPTs. Real-time
fluorescent quantitative PCR (qPCR) analysis revealed that LsHPT was preferentially expressed in mature leaves compared with other tissues. When lettuce plants were subjected to drought
and high-light stress treatments, LsHPT expression was markedly increased. Expression of LsHPT in Arabidopsis showed that LsHPT could enhance the α-tocopherol biosynthesis in Arabidopsis. Transient expression of LsHPT via agroinfiltration resulted in 9-fold increase in LsHPT mRNA level and nearly 18-fold enhancement in α-tocopherol content compared with the negative controls. 相似文献
3.
Minchen Wu Junqing Wang Huimin Zhang Cunduo Tang Jinhu Gao Zhongbiao Tan 《World journal of microbiology & biotechnology》2011,27(4):831-839
The full-length cDNA gene that encodes an acidophilic endo-1,4-β- xylanase XynI of Aspergillus usamii E001 was amplified by rapid amplification of cDNA 3′ and 5′ ends (RACE) using the total RNA as template and then cloned onto
the pUCm-T vector, followed by sequencing. The cloned cDNA is 881 bp in length including 5′ and 3′ non-encoding regions, as
well as a 678 bp of open reading frame (ORF) which encodes an E001 XynI of 188 amino acid residues together with a signal
peptide of 37 amino acid residues. The homologies of E001 XynI with xylanases of Aspergillus niger, Aspergillus kawachii, Emericella nidulans and Penicillium funiculosum are 97.8, 92.0, 74.6 and 60.5%, respectively. From a BLAST search result, we concluded that E001 XynI belongs to the glycoside
hydrolase family 11. Its three-dimensional structure was predicted using programs based on that of the P. funiculosum xylanase (1TE1B) from the family 11. In addition, the complete DNA gene xynI encoding E001 XynI was cloned from the genomic DNA of A. usamii E001 by conventional PCR and ligation-mediated PCR amplification. The cloned xynI is 1,206 bp in length, composed of a promoter region, a 68 bp of intron and two exons when compared with the cDNA of E001
XynI. 相似文献
4.
5.
Persimmon is a commercially important fruit crop, and the fruit is rich in different kinds of bioactive compounds, among which
carotenoids contribute significantly to its color and nutritional value. In this study, the cDNA of phytoene desaturase gene
(PDS) was isolated by rapid amplification of cDNA ends (RACE) technique. Sequence analysis indicated that the full-length cDNA
of PDS was 2064 bp, encoding 586 amino acids and containing one open reading frame (ORF) of 1761 bp. Homology analysis showed that
DkPDS, which had been submitted in GenBank with accession number GU112527, shared high similarities of 80–86% with PDS cloned from other plants. Prediction of deduced proteins showed that there was no signal peptide and transmembrane topological structure in DkPDS. It was a hydrophilic and stable protein, and located in chloroplast. To examine the specific expression patterns of carotenogenic
genes we had cloned from persimmon, including phytoene synthase (DkPSY), DkPDS, ζ-carotene desaturase (DkZDS), lycopene β-cyclase (DkLCYB) and β-carotene hydroxylase (DkBCH), real-time quantitative PCR (Q-PCR) was performed in flesh at five different developmental stages. The results revealed
that the expression levels of DkPSY, DkPDS and DkZDS gradually increased. Nevertheless, the expression level of DkLCYB was very low and maintained relatively stable. The expression level of DkBCH was also at a low level from stage 1 to 4, and then reached the maximum at stage 5. In addition, the expression level of
DkZDS was higher than that of other genes. Carotenoid detection demonstrated that both β-cryptoxanthin and total carotenoids increased
with fruit development, and zeaxanthin had little change, but with a sudden increase in final stage. 相似文献
6.
Rameshwaram NR Karanam NK Scharf C Völker U Nadimpalli SK 《Glycoconjugate journal》2009,26(2):161-172
A new unique lectin (galactose-specific) purified from the seeds of Dolichos lablab, designated as DLL-II is a heterodimer composed of closely related subunits α and β. These were separated by SDS-PAGE and
isolated by electroelution. By ESI-MS analysis their molecular masses were found to be 30.746 kDa (α) and 28.815 kDa (β) respectively.
Both subunits were glycosylated and displayed similar amino acid composition. Using advanced mass spectrometry in combination
with de novo sequencing and database searches for the peptides derived by enzymatic and chemical cleavage of these subunits, the primary
sequence was deduced. This revealed DLL-II to be made of two polypeptide chains of 281(α) and 263(β) amino acids respectively.
The β subunit differed from the α subunit by the absence of some amino acids at the carboxy terminal end. This structural
difference suggests that possibly, the β subunit is derived from the α subunit by posttranslational proteolytic modification
at the COOH-terminus. Comparison of the DLL-II sequence to other leguminous seed lectins indicates a high degree of structural
conservation.
Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
7.
Tang Cunduo Guo Jing Wu Minchen Zhao Shunge Shi Hongling Li Jiangfang Zhang Huimin Wang Junqing 《World journal of microbiology & biotechnology》2011,27(12):2921-2929
The full-length cDNA sequence, which encodes a novel acidophilic β-mannanase (abbreviated as AuMan5A) of Aspergillus usamii YL-01-78, was amplified by 3′ and 5′ rapid amplification of cDNA ends (RACE) using the total RNA as template. The cDNA sequence
is 1,427 bp in length, including 5′ and 3′ non-coding regions and an open reading frame (ORF). The ORF encodes a 21-aa signal
peptide, a 17-aa propeptide, and a 345-aa mature peptide (AuMan5A) with the calculated M.W. of 37,614 Da and pI of 4.09 and
two putative N-glycosylation sites. Online analysis of amino acid sequence homology demonstrated that the AuMan5A belongs
to the glycoside hydrolase (GH) family 5. Its three-dimensional structure was predicted using Pred3D Web Server 1.0 based
on the crystal structure of the T. reesei RutC-30 β-mannanase (1QNO) from the GH family 5. Furthermore, the complete DNA sequence encoding the AuMan5A, designated
as Auman5A, was cloned from the genomic DNA of A. usamii YL-01-78 by the conventional PCR and pUCm-T vector-mediated PCR techniques. The cloned Auman5A is 2,168 bp in length, harboring 5′ and 3′ flanking regulatory regions and the full-length cDNA sequence in which two short
introns with 63 and 60 bp are inserted, respectively. 相似文献
8.
Using rapid amplification of cDNA ends, a full length cDNA (CjLTI) was cloned from apical buds of Caragana jubata, a plant species that grows under extreme cold. The cDNA obtained was 573 bp long consisting of an open reading frame of
351 bp encoding 116 amino acids. Homology analysis did not exhibit significant similarity with any sequence at NCBI database,
therefore it was deduced as a novel gene. Secondary structure analysis suggested that the deduced CjLTI contained 25.86% α-helices,
4.31% β-turns, 6.90% extended strands, and 62.93% random coils. The hydropathy profile suggested CjLTI to be a hydrophobic
protein having characteristic features of signal peptides at N-terminus. The gene exhibited down-regulation at 5 min of exposure
to low temperature (LT, 4 ± 3°C) followed by a strong up-regulation after 15 min and onwards. Methyl jasmonate (MJ) lead to
up-regulation of CjLTI starting at 5 min onwards. The gene exhibited up- and down-regulation of expression pattern in response to abscisic acid
(ABA) and salicylic acid (SA). Mild drought stress slightly up-regulated gene expression and at severe drought (up to 115%
reduction in leaf water potential) slight down-regulation of gene expression was observed. These results suggested CjLTI to be a LT responsive gene wherein MJ, ABA and SA pathways might be involved in regulating the gene expression. 相似文献
9.
10.
The basidiomycete Phanerochaete chrysosporium produces several β-1,3-glucanases when grown on laminarin, a β-1,3/1,6-glucan, as the sole carbon source. To characterize
one of the major unknown β-1, 3-glucanases with a molecular mass of 83 kDa, identification, cloning, and heterologous over-expression
were carried out using the total genomic information of P. chrysosporium. The cDNA encoding this enzyme included an ORF of 2337 bp and the deduced amino acid sequence contains a predicted signal
peptide of 26 amino acids and the mature protein of 752 amino acids. The amino acid sequence showed a significant similarity
with glycoside hydrolase family 55 enzymes from filamentous fungi and was named Lam55A. Since the recombinant Lam55A expressed
in the methylotrophic yeast Pichia pastoris degraded branched β-1,3/1,6-glucan as well as linear β-1,3-glucan, the kinetic features of the enzyme were compared with
those of other β-1,3-glucanases. 相似文献
11.
Bin Liu Lin Yao Wenguo Wang Jihai Gao Fang Chen Shenghua Wang Ying Xu Lin Tang Yongjiong Jia 《Molecular biology reports》2010,37(2):939-946
Phospholipase D (PLD, EC 3.1.4.4) is a key enzyme involved in phospholipid catabolism, initiating a lipolytic cascade in membrane
deterioration during senescence and stress, which was cloned from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels. The cDNA was 2,886 bp in length with a complete
open reading frame of 2,427 bp which encoded a polypeptide of 808 amino acids including a putative signal peptide of 53 amino
acid residues and a mature protein of 755 amino acids with a predicted molecular mass of 86 kD and a pI of 5.44, having two
highly conserved ‘HKD’ motifs. Phylogenetic analysis indicated the J. curcas PLD alpha (JcPLDα) showed a high similarity to other PLD alpha from plants. Semi-quantitative RT-PCR analysis revealed that it was especially abundant in root, stem, leaf, endosperm and flower, weakly in seed. And the JcPLDα was increasedly expressed in leaf undergoing environmental stress such as salt (300 mM NaCl), drought (30% PEG), cold (4°C)
and heat (50°C). The JcPLDα protein was successfully expressed in Escherichia coli and showed high enzymatic activities. Maximal activity was at pH 8 and 60°C. 相似文献
12.
In the present study, a ras-like nuclear (Ran) gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA
library of black tiger prawn (Penaeus monodon). The full-length black tiger prawn Ran (PmRan) cDNA consisted of 1140 nucleotides including an open reading frame (ORF) 648 bp, a 5′ untranslated region (5′UTR) of 117 bp
and a 3′UTR of 375 bp with a polyadenylation signal sequence “aataaa” and a poly (A) tail. The ORF encoded a peptide of 215
amino acids with molecular mass 24.6 kDa and a theoretical isoelectric point of 7.39. ScanProsite analysis indicated that
PmRan protein sequence contained a small GTPase Ran family motif. Homology analysis of the deduced amino acid sequence of the PmRan with other known Ran sequences by MatGAT software revealed that the PmRan show very high homology with the sequences of other animals (92.1–98.6% similarity, 85.6–98.1% identity). Analysis of the
tissue expression pattern of the PmRan gene showed that the PmRan mRNA was expressed in all tested tissues, including hepatopancreas, ovary, muscle, intestine, neurosecretory organ in eyestalk,
neurosecretory organ in brain, stomach, and heart, with the highest levels in ovary. Furthermore, the PmRan expression was found to be high level in the six ovarian stages of development. The results indicated PmRan might play an important role in ovarian development. 相似文献
13.
14.
The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length
cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5′ untranslated region (UTR) of 72 bp, an ORF (open reading frame)
of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3′ UTR of 288 bp. The
sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence.
The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle,
brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37°C. In ovarian maturation
stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage. 相似文献
15.
FaLin Zhou ShiGui Jiang JianHua Huang Lihua Qiu Dianchang Zhang Tiannfeng Su 《Molecular biology reports》2011,38(3):1921-1927
In present study, a QM gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn
(Penaeus monodon). The full-length black tiger prawn QM (PmQM) cDNA contained a 5′-UTR of 41 bp, an ORF of 663 bp encoding a polypeptide of
220 amino acids with molecular weight 25.5 kDa, and a 3′-UTR of 54 bp. Homology analysis of the deduced amino acid sequence
of the PmQM with other known QM sequences by MatGAT software revealed that the PmQM was high homology with other invertebrates.
A conserved signature sequence of the QM family was found in the PmQM deduced amino acid sequence. Analysis of the tissue
expression pattern of the PmQM gene showed that the PmQM mRNA was expressed in all tissues tested, with highest levels in
ovary. Furthermore, the PmQM expression was found to be different in three important ovarian stages of development. The results
indicated PmQM might play an important role in ovarian development. 相似文献
16.
The RT PCR and RACE methods were used to obtain the cDNA sequence of an APX gene of muskmelon after the leaves were induced
with powdery mildew. The cDNA length of the APX gene is 1,047 bp with a 750 bp ORF encoded a 249 amino acid and the molecular
weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns. The identity of the amino acid sequence deduced from the cDNA with the
APX family of other homologous members was about 74–97%. A Full-length of ORF was sub-cloned into prokaryotic expression vector
pET24a. The recombinant proteins had high expression level in E. coli. Analysis of expression at mRNA level showed that CmAPX exhibited highly tissue-specific patterns of expression. The mRNA level and enzyme activities assays showed that CmAPX might play an important role in the pathogenesis of powdery mildew. 相似文献
17.
18.
A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine–cytosine content
of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino
acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino
acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any
known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of
theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart
(about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded
by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type
of theta plasmid. 相似文献
19.
It is crucial to select stable references in gene expression analyses using quantitative real-time PCR (qRT-PCR). In this
work, seven frequently used reference genes, 18S, Actin, EF1α, α-tubulin, β-tubulin, Cyclophilin and Cytoplasmic ribosomal protein L2 (L2), from Bupleurum chinense DC. were evaluated as the internal control in five tissues, roots, stems, leaves, flowers and fruits, before tissue specific
gene expression assays. The results showed that β-tubulin was the most stable and reliable reference gene among the seven candidate genes in the measured tissues. The expression levels
of four genes involved in saikosaponins (the pharmacological active compounds of B. chinense) biosynthesis, HMGR, IPPI, FPS and β-AS, were assayed with β-tubulin as the internal control in the five tissues. All the four genes were expressed in the five tissues with different profiles
and HMGR in the order of roots > flowers, stems and leaves > fruits, IPPI of stems > leaves and fruits > roots and flowers, FPS of flowers > fruits > stems and roots > leaves and β-AS of roots > flowers, stems and fruits > leaves. The genes of FPS and β-AS were expressed predominantly in flowers and roots, respectively. This study may provide a suitable internal control for quantitative
gene expression assays in various tissues and give insight into the tissue expression profiles of four saikosaponins biosynthesis-involved
genes of medicinal B. chinense. 相似文献
20.
The full-length MECPS cDNA sequence (designated as Chmecps, GenBank Accession No.: DQ415658) was isolated by rapid amplification of cDNA ends (RACE) for the first time from Cephalotaxus harringtonia. The full-length cDNA of Chmecps was 1,146 bp containing a 753 bp open reading frame (ORF) encoding a polypeptide of 250 amino acids with a calculated mass
of 26.67 kDa and an isoelectric point of 9.35. Comparative and bioinformatics analyses revealed that ChMECPS showed extensive homology with MECPSs from other plant species. Phylogenetic analysis indicated ChMECPS was more ancient than other plant MECPSs. Southern hybridization analysis of the genomic DNA showed that Chmecps was a single copy gene. Tissue expression pattern analysis revealed that ChMECPS expressed strongly in root and leaf, weakly in stem. 相似文献