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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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Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular
acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference
genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most
and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental
conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we
propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to
normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate
category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze
acclimation in Chlamydomonas sp. ICE-L. 相似文献
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A cDNA encoding omega-3 fatty acid desaturase was isolated from developing perilla seeds and characterized. On the basis of its deduced amino acid sequence comparison, this cDNA was assumed to be a new isoform of microsomal omega-3 fatty acid desaturase gene. Accumulation of the mRNA for this cDNA showed seed-specific expression. 相似文献
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Guangting Wu Chenlin Liu Shenghao Liu Bailin Cong Xiaohang Huang 《Journal of applied phycology》2010,22(6):779-783
To study the molecular mechanism of the Antarctic sea–ice alga in adaptation to polar sea–ice environments, the RNA was prepared
for cDNA library construction of Chlamydomonas sp. ICE-L. Three different methods were tested to prepare total RNA from this psychrophilic, unicellular green alga rich
in protein and polysaccharide. Lauryl sodium sulfate- based method allowed a most effective extraction of high-quality total
RNA compared to the other methods. Total RNA extracted with this protocol was used for cDNA library construction. The recombination
rate of constructed cDNA library was 98.60%, the primary titer was 7.15 × 106 pfu, and an average sequence length was 1.2 kb. These results show that with a high-quality RNA preparation, a cDNA library
can be constructed successfully for Chlamydomonas sp. ICE-L. 相似文献
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Quan-fu Wang Yan-hua Hou Jin-lai Miao Guang-you Li 《Acta Physiologiae Plantarum》2009,31(6):1097-1102
The effect of ultraviolet-B (UV-B) radiation on Antarctic phytoplankton has become an attractive ecological issue as a result
of annual springtime ozone depletion. The effects of UV-B radiation on the growth and antioxidant enzymes were investigated
using Antarctic sea ice microalgae Chlamydomonas sp. ICE-L as the material in this study. The results demonstrated that UV-B radiation could notably inhibit the growth, especially
at high UV-B radiation intensity (70 μW cm−2). Malondialdehyde and O2
·− content in ICE-L increased rapidly in early days (1–3 days) exposed to UV-B radiation enhancement, then decreased rapidly.
In the stress of UV-B radiation enhancement, the superoxide dismutase, peroxidase and Catalase activities of 1–4 days in ICE-L
were obviously higher than those in the control, and their activities became higher at high UV-B radiation intensity (70 μW cm−2). These enzymes activity of 7 days would kept stable at low UV-B radiation intensity (35 μW cm−2), but kept high level at high UV-B radiation intensity (70 μW cm−2). However, the ascorbate peroxidase activity in ICE-L kept stable under the stress of UV-B radiation enhancement. The above
experimental results indicated that the antioxidant enzyme system played an important role in the adaptation of Antarctic
ice microalgae under the UV-B radiation change of Antarctic ecosystems. 相似文献
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The techniques of homology cloning and anchored PCR were used to clone the fucose-binding lectin (F-type lectin) gene from
Japanese sea perch (Lateolabrax Japonicus). The full-length cDNA of sea perch F-lectin (JspFL) contained a 5′ untranslated region (UTR) of 39 bp, an ORF of 933 bp
encoding a polypeptide of 310 amino acids with an estimated molecular mass of 10.82 kDa and a 3′ UTR of 332 bp. The searches
for nucleotides and protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of JspFL
was homological to the Fucose-binding lectin in other fish species. In the JspFL deduced amino acid sequence, two tandem domains
that exhibit the eel carbohydrate-recognition sequence motif were found. The temporal expressions of gene in the different
tissues were measured by real-time PCR. And the mRNA expressions of the gene were constitutively expressed in tissues including
spleen, head-kidney, liver, gill, and heart. The JspFL expression in spleen was different during the stimulated time point,
2 h later the expression level became up-regulated, and 6 h later the expression level became down-regulated. The result indicated
that JspFL was constitutive and inducible expressed and could play a critical role in the host-pathogen interaction. 相似文献
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Kim SH Kim JB Kim SY Roh KH Kim HU Lee KR Jang YS Kwon M Park JS 《Biotechnology letters》2011,33(6):1185-1193
Delta 6-fatty acid desaturase (D6DES) is used in the synthesis of polyunsaturated fatty acids (PUFAs) from microorganisms
to higher animals, including arachidonic acid (ARA) and eicosapentaenoic acid (EPA). A 1,338 bp full-length cDNA encoding
D6DES was cloned from Acanthopagrus schlegeli (AsD6DES) through degenerate- and RACE-PCR methods. A recombinant vector expressing AsD6DES (pYES-AsD6DES) was subsequently constructed and transformed into Saccharomyces cerevisiae to test the enzymatic activity of AsD6DES towards the production of n-6 and n-3 fatty acids. The exogenously expressed AsD6DES
produced γ-linolenic acid (18:3 n-6) and stearidonic acid (18:4n-3) at 26 and 36% from exogenous linoleic acid (18:2n-6) and
α-linolenic acid (18:3n-3), respectively, indicating that it is essentially a delta 6-fatty acid desaturase. 相似文献
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Expression Enhancement of a Rice Polyubiquitin Gene Promoter 总被引:11,自引:0,他引:11
An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail
repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold.
Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays
by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence
has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis
at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native
GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than
the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression
in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement
in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes. 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