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1.
Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative
analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative
RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein
was successfully expressed in Escherichia coli with the molecular weight expected. 相似文献
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Xiaoying Cao Zhimin Zong Xiuyun Ju Yong Sun Chuanchao Dai Qun Liu Jihong Jiang 《Molecular biology reports》2010,37(3):1559-1567
A new full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first
committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR, GenBank Accession NO. EF062569) was 2,200 bp containing a 1,752 bp ORF encoding 583 amino acids. Bioinformatic analyzes
revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and
a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated
that at most two copies of EpHMGR gene existed in E. Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional
colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis. 相似文献
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A full-length cDNA of Rubisco activase (IBrcaI) was cloned from sweet potato (Ipomoea batatas (L.) Lam) using Rapid-Amplification of cDNA Ends (RACE). IBrcaI contains a 1,347 bp open reading frame encoding a protein of 439 amino acids. The sequence alignment of multiple Rubisco
activase genes from sweet potato and other plants showed high homology at two previously described ATP-binding sites. Western
blot analysis indicated that there are two Rubisco activase proteins in sweet potato. Expression of IBrcaI was only detected in leaves. In the 14 h light and 10 h dark photoperiods, maximal and minimal IBrcaI mRNA expression levels were detected at 8:00 in the morning and at midnight, respectively. 相似文献
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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
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Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Antarctic ice algae Chlamydomonas sp. ICE-L 总被引:1,自引:0,他引:1
Shenghao Liu Pengying Zhang Bailin Cong Chenlin Liu Xuezheng Lin Jihong Shen Xiaohang Huang 《Extremophiles : life under extreme conditions》2010,14(3):329-337
A cDNA encoding heat shock protein 70 of Antarctic ice algae Chlamydomonas sp. ICE-L (designated as CiHsp70) was identified by RT-PCR and rapid amplification of cDNA ends approaches. The full-length
cDNA of CiHsp70 was 2,232 bp, consisting of a 5′-terminal untranslated region (UTR) of 76 bp, a 3′-terminal UTR of 203 bp
with a poly (A) tail, and an open reading frame of 1,953 bp. The CiHsp70 cDNA encoded a polypeptide of 651 amino acids with
an ATPase domain of 388 amino acids, the substrate peptide binding domain of 246 amino acids and a C-terminus domain of 17
amino acids. The inducible CiHsp70 cDNA was highly homologous to other plant cytosolic Hsp70 genes and clustered together
with green algae and higher plant rather than brown algae, diatom and Cryptophyta. Antarctic ice algae were treated with different
stress conditions and messenger RNA (mRNA) expression levels of CiHsp70 were quantified by quantitative RT-PCR. The results
showed that both cold and heat shock treatments could stimulate CiHsp70 mRNA expression. Meanwhile, CiHsp70 mRNA expression
level increased 2.9-fold in response to UV-B radiation for 6 h, while the expression levels of CiHsp70 were remarkably increased
after removing the UV-B radiation and immediately providing additional 6 h visible light. Furthermore, treating with 62 or
93‰ NaCl for 2 h, CiHsp70 mRNA expression level increased 3.0- and 2.1-fold, respectively. Together, our observations revealed
that CiHsp70 as a molecular chaperone might play an important role in Antarctic ice algae Chlamydomonas sp. ICE-L acclimatizing to polar environment. 相似文献
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Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP),
which is a key precursor for diterpenes such as taxol. Herein, a full-length cDNA encoding GGPPS (designated as CgGGPPS) was cloned and characterized from hazel (Corylus avellana L. Gasaway), a taxol-producing angiosperms. The full-length cDNA of CgGGPPS was 1515 bp with a 1122 bp open reading frame (ORF) encoding a 373 amino acid polypeptide. The CgGGPPS genomic DNA sequence
was also obtained, revealing CgGGPPS gene was not interrupted by an intron. Southern blot analysis indicated that CgGGPPS belonged to a small gene family. Tissue expression pattern analysis indicated that CgGGPPS expressed the highest in leaves.
RT–PCR analysis indicated that CgGGPPS expression could be induced by exogenous methyl jasmonate acid. Furthermore, carotenoid
accumulation was observed in Escherichia coli carrying pACCAR25ΔcrtE plasmid carrying CgGGPPS. The result revealed that cDNA encoded a functional GGPP synthase. 相似文献
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Although glucose-6-phosphate isomerase (GPI) plays an important role in glycolysis of both the prokaryotes and eukaryotes,
studies on the GPI have not been involved in the halotolerant, unicellular green alga Dunaliella salina (D. salina). In this study, a 2,338 bp of full-length cDNA cloned using rapid amplification of cDNA end (RACE) technique contained an
open reading frame (ORF) of 1,980 bp encoding 660 amino acids, which has a predicted molecular weight of 73.3 kD and pI of
6.22 and shares high homology with other organisms. The cloned full-length cDNA was heterologously expressed in Escherichia coli and the recombinant GPI proteins purified using Ni-NTA His Bind column were consistent with the anticipated size of ~75 kD.
Predicted 2D and 3D structures of GPI proteins possessed potential active motifs including “GEPGTNGQHSFYQLIHQG” and “VQGFIWGINSFDQWGVELGK”,
and critical active site residues, such as Ser 241, Ser 296, Thr 298, Thr 301, Arg 358, Glu 444, His 475 and Lys 600. Real
time quantitative RT-PCR demonstrated that the expression level of the GPI gene from D. salina (DsGPI) was induced by 3.5 M NaCl with 14-fold higher than that by 1.5 M NaCl (P < 0.01), but inhibited by the light with 4-fold lower than that in the dark (P < 0.05). It is concluded that the cloned GPI gene is indeed from D. salina and may respond to salt and light. 相似文献
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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
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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
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Sebastián A. Trejo Laura M. I. López Néstor O. Caffini Claudia L. Natalucci Francesc Canals Francesc X. Avilés 《Planta》2009,230(2):319-328
Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs
to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another
one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by
RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues
belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZα
vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated
from plant latex, confirming the presence of the preprocysteine peptidase in the latex. 相似文献
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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