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1.
2.
A major challenge for efficient transgene expression in Dunaliella salina is to find strong endogenous promoters to drive the transgene expression. In the present study, a novel glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) promoter was cloned and used to drive expressions of the bialaphos resistance (bar) gene and of the N-terminal fragment of human canstatin (Can-N). The results showed that the bar gene was transcribed by the GAPDH promoter and integrated into the genome of the transformants of D. salina. Furthermore, the PCR identification, Southern and western blots indicated that Can-N was expressed in transgenic D. salina, demonstrating that the promoter of the D. salina
GAPDH gene is suitable for driving expression of heterologous genes in transgenic D. salina. 相似文献
3.
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
4.
Improvement of efficiency of genetic transformation for <Emphasis Type="Italic">Dunaliella salina</Emphasis> by glass beads method 总被引:1,自引:0,他引:1
Dunaliella salina has been exploited as a new type of bioreactor due to its unique advantages. However, this bioreactor application was restricted
for absence of a high-efficiency and stable transformation method at present. In the present study, the cells of D. salina were transformed by glass beads. The results of histochemical staining revealed that the GUS gene was successfully expressed
in the positive transformants, and PCR and PCR-Southern blot analysis further demonstrated that the bar gene was integrated into the D. salina genome. Moreover, the three transformation methods, including glass beads, bombardment particle and electroporation, were
compared for screening a high-efficiency transformation method for gene engineering of D. salina. The results showed that transformation efficiency of the glass beads was the highest, approximately 102 transformants/μg DNA. It is concluded that the established glass beads method has been demonstrated to be an optimal transformation
way for D. salina. 相似文献
5.
Wang T Xue L Hou W Yang B Chai Y Ji X Wang Y 《Applied microbiology and biotechnology》2007,76(3):651-657
Nuclear matrix attachment regions (MARs) are known to bind specifically to the nuclear scaffold and are thought to influence
expression of the transgenes. In our previous studies, a new deoxyribonucleic acid fragment isolated from Dunaliella salina could bind to the nuclear matrix in vitro and had the typical characteristics of MARs. In this study, to investigate effects
of MARs on expression of transgenes in the stably transformed cells of D. salina, expression vectors with and without MARs, which contained chloramphenicol acetyltransferase (CAT) reporter gene driven by
D. salina ribulose 1,5-bisphosphate carboxylase/oxygenase promoter, were constructed and delivered, respectively, into cells of D. salina by electroporation. Twenty stably transformed colonies of D. salina were randomly picked out, and CAT gene expression was assayed. The results showed that the CAT enzyme of the colonies of
D. salina transformed with the expression vector containing MARs averaged out about 4.5-fold higher than those without MARs, while
the transgene expression variation among individuals of transformants decreased threefold. The CAT enzyme in the stably transformed
lines was not significantly proportional to the gene copy numbers, suggesting that the effects of MARs on transgene expression
may not be through increasing the transgene copy numbers. 相似文献
6.
Tian-yun Wang Chang-Qin Jing Wei-Hua Dong Jun-He Zhang Yu Zhang 《Molecular biology reports》2010,37(2):1099-1103
Previously we reported the nucleotide sequence of a 14-3-3 cDNA cloned from the unicellular green alga Dunaliella salina, however, the nucleotide sequence of this gene have not been reported so far. In the present study, the cloning and characterization
of the nucleotide sequence, the gene copy and expression were undertaken. The coding sequence of the gene was found to be
interrupted by five introns of 132, 266, 153, 152 and 625 bp, respectively. Introns 3-5 were found in conserved positions
as compared to the Chlamydomonas reinhardtii 14-3-3 gene. D. salina 14-3-3 cDNA was inserted into the prokaryotic expression plasmid pET-28 and transformed into E. coli BL21, and the recombinant expressed 14-3-3 protein was purified from E. coli and immunized the rabbit. Indirect ELISA coated with 14-3-3 illustrated that the rabbit antisera titration was 1:1.00E + 06.
Western blotting assays confirmed that prepared rabbit antibodies could recognize the recombinant 14-3-3 protein. Southern
blotting results showed that there was only one copy of the 14-3-3 present in the genome of D. salina and 14-3-3 expression did not change throughout the Dnualiella cell cycle. 相似文献
7.
Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina
Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on
the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the
14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained
a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino
acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore,
an EF hand motif characteristic for Ca2+-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics
revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein
gene is cell cycle-dependent. 相似文献
8.
Hua Xie Peirong Xu Yanlong Jia Jie Li Yumin Lu Lexun Xue 《Journal of applied phycology》2007,19(5):497-504
A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame
of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly
(A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes
of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to
be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants
and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme
assay, confirming that the cloned gene from D. salina is indeed NR. 相似文献
9.
Previous studies have demonstrated that flagella/cilia are critical organelles and play diverse roles of motility, sensory
perception and development in many eukaryotic cells. However, there is very little information available about flagella composition
in Dunaliella salina, a halotolerant, unicellular biflagellate green alga. In the present study, we used strategy of shotgun proteomics to identify
flagella proteins after flagella were released and collected from D. salina. A total of 520 groups of proteins were identified under a stringent filter condition (Xcorr ≥1.9, ≥2.2 and ≥3.75; ΔCn ≥ 0.1).
In addition to six kinds of known flagella proteins, the putative flagella proteins of D. salina identified by one or more peptides are abundant in signaling, cell division, metabolism, etc. The findings provide guidance
for further studies to elucidate the roles of these proteins in the function and assembly of this organelle in microalgae. 相似文献
10.
Rui-Yu Zhao Wei Xiao Hai-Li Cheng Ping Zhu Ke-Di Cheng 《Journal of industrial microbiology & biotechnology》2010,37(11):1171-1182
The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5′-untranslated region (UTR), 3′-UTR,
four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading
frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa.
The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species.
The recombinant protein was expressed in E. coli and detected by SDS–PAGE and western blot. GC–MS analysis showed that the wild-type GfSQS could catalyze the reaction from
farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that
the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate. 相似文献
11.
Yanyang Liu Junzhou Li Yuling Li Mengguan Wei Qingxin Cui Qilei Wang 《Molecular biology reports》2010,37(2):755-761
A full-length cDNA encoding a maize GTP-binding protein of the ADP-ribosylation factor family was cloned by suppression subtractive
hybridization and an in silico cloning approach. The cDNA was 938 bp in length and contained a complete ORF of 612 bp, which
encodes a protein of 203 amino acid residues. Its deduced amino acids sequence had an 83% identity with that of a GTP-binding
protein in rice. The gene was designated ZmArf2. The ZmArf2 gene consists of G1, G2, G3, G4 and G5 boxes, and Switch I and Switch II regions. Eight nucleotides differed and five amino
acids changed between the popcorn inbred N04 and the dent corn inbred Dan232. One changed amino acid was in the G1 box. RT-PCR
analysis showed that ZmArf2 expression increased in the early stages of endosperm development and was not tissue-specific. 相似文献
12.
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a key enzyme of the glycolytic pathway and it is related to the occurrence
of some diseases. The cDNA and the genomic sequence of GAPDH were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using the RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily. The cDNA of GAPDH cloned from the Giant Panda is 1191 bp in size, contains an open reading frame of 1002 bp encoding 333 amino acids. The genomic
sequence is 3941 bp in length and was found to possess 10 exons and 9 introns. Alignment analysis indicates that the nucleotide
sequence and the deduced amino acid sequence are highly conserved in some mammalian species, including Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupus familiaris and Bos taurus. The homologies for the nucleotide sequences of the Giant Panda GAPDH to that of these species are 90.67, 90.92, 90.62, 95.01 and 92.32% respectively, while the homologies for the amino acid
sequences are 94.93, 95.5, 95.8, 98.8 and 97.0%. Primary structure analysis revealed that the molecular weight of the putative
GAPDH protein is 35.7899 kDa with a theoretical pI of 8.21. Topology prediction showed that there is one Glyceraldehyde 3-phosphate
dehydrogenase active site, two N-glycosylation sites, four Casein kinase II phosphorylation sites, seven Protein kinase C
phosphorylation sites and eight N-myristoylation sites in the GAPDH protein of the Giant Panda. The GAPDH gene was overexpressed in E. coli BL21. The results indicated that the fusion of GAPDH with the N-terminally His-tagged form gave rise to the accumulation
of an expected 43 kDa polypeptide. The SDS-PAGE analysis also showed that the recombinant GAPDH was soluble and thus could
be used for further functional studies. 相似文献
13.
Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value. 相似文献
14.
Liang Wei Yi Cao Linhan Bai Xue Liang Tingting Deng Jing Li Dairong Qiao 《Journal of applied phycology》2007,19(1):89-94
Genes encoding proteins of the major light-harvesting complex of photosystem II (LHCII) in higher plants are well studied.
However, little is known about the corresponding genes in the green alga Dunaliella salina, although this knowledge might provide valuable information about the respective roles of each LHCII protein at the molecular
level under extreme environmental conditions. Here, we describe an additional LhcII gene from D. salina. An LhcII cDNA cloned by screening a D. salina cDNA library contains an open reading frame encoding a protein of 261 amino acids with a calculated molecular mass of 27.8 kDa.
The deduced amino acid sequence shows high homology with other LHCII proteins. Genomic DNA—obtained by PCR using a specific
primer set corresponding to the 5′ and 3′ untranslated regions—was used to determine the intron-exon structure. Short-term
changes in mRNA levels after a shift from low-light to high-light or dark conditions were analyzed by real-time quantitative
PCR, and indicated that this gene expresses different mRNA levels under different light conditions. 相似文献
15.
Hyaluronic acid production by recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> 总被引:1,自引:0,他引:1
A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would
be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at
25°C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml−1. The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified
enzyme is 6 and 40°C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The K
m and V
max values for birchwood xylan are 2.06 mg ml−1 and 0.49 mmol min−1mg−1, respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu2+ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity
is unaffected by EDTA even at a concentration of 5 mM. 相似文献
16.
Grabek-Lejko D Kurylenko OO Sibirny VA Ubiyvovk VM Penninckx M Sibirny AA 《Journal of industrial microbiology & biotechnology》2011,38(11):1853-1859
The ability of baker’s yeast Saccharomyces cerevisiae and of the thermotolerant methylotrophic yeast Hansenula polymorpha to produce ethanol during alcoholic fermentation of glucose was compared between wild-type strains and recombinant strains
possessing an elevated level of intracellular glutathione (GSH) due to overexpression of the first gene of GSH biosynthesis,
gamma-glutamylcysteine synthetase, or of the central regulatory gene of sulfur metabolism, MET4. The analyzed strains of H. polymorpha with an elevated pool of intracellular GSH were found to accumulate almost twice as much ethanol as the wild-type strain
during glucose fermentation, in contrast to GSH1-overexpressing S. cerevisiae strains, which also possessed an elevated pool of GSH. The ethanol tolerance of the GSH-overproducing strains was also determined.
For this, the wild-type strain and transformants with an elevated GSH pool were compared for their viability upon exposure
to exogenous ethanol. Unexpectedly, both S. cerevisiae and H. polymorpha transformants with a high GSH pool proved more sensitive to exogenous ethanol than the corresponding wild-type strains. 相似文献
17.
Cloning and expression study of a putative high-affinity nitrate transporter gene from Dunaliella salina 总被引:1,自引:1,他引:0
Qinghua He Dairong Qiao Qinglian Zhang Yin Li Hui Xu Liang Wei Ying Gu Yi Cao 《Journal of applied phycology》2004,16(5):395-400
A nitrate transporter gene, named DsNRT2.1 (GeneBank accession number AY621079), from Dunaliella salina has been cloned by screening a cDNA library, which was constructed with mRNAs from D. salina after 60 min treatment with 5 mM nitrate, with a 342 bp NRT2 cDNA fragment from D. salina as a probe. DsNRT2.1 exhibits sequence similarity to those known nitrate transporters of the NRT2 family. A hydrophobicity blot indicated that DsNRT2.1 belongs to the major facilitator superfamily (MFS). Northern analysis showed that an mRNA species of 1.9 kb can be rapidly induced by NO–
3, but not by NH+
4. Northern analysis also showed that NaCl could significantly increase the expression of DsNRT2.1. 相似文献
18.
Biomacromolecules import into the nucleus is a complex progress which requires the participation of several cytosolic factors,
and nuclear transport factor 2 (NTF2) is one of essential components in nuclear trafficking. Its main role is to transport
RanGDP from cytoplasm to nucleus by interacting with FxFG nucleoporin repeats. In the study a putative new gene, designated
as CcNTF2, was obtained from the moss (Conocephalum conicum) cDNA library we have constructed. The full-length cDNA sequence is 913 bp in size contains a 372 bp open reading frame (ORF)
flanked by a 195 bp 5′-untranslated sequence and a long 346 bp 3′-non-coding region, encoding 123 amino acids of 13,575.3 Da.
Part of the genomic sequence was also cloned and sequenced, which is 1,602 bp long and possesses two exons and one intron.
Alignment analysis showed that the CcNTF2 protein is high conserved among plant NTF2 and shares 81% similarity with the ones
from Arabidopsis thaliana and Brassica rapa. The expression of wild-type CcNTF2 was detected by immunoblotting of extraction of C. conicum and it indicated the putative protein is integral. Through functional expression of CcNTF2-green fluorescent protein (GFP)
in tobacco, it was demonstrated that CcNTF2 can accumulate at the nuclear rim. Site-directed mutagenesis analysis confirmed
CcNTF2 P71K has influence on the protein import into nucleus. In addition, overexpression of CcNTF2 P71K was observed to be
deleterious for the plant cell. It is the first illumination of NTF2 in moss, and our study established the primary foundation
for further research on moss NTF2. 相似文献
19.