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1.
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
2.
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
3.
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
4.
5.
The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage. 相似文献
6.
Liyuan Zhao Tiezhu Mi Yu Zhen Mingyu Li Shanying He Jing Sun Zhigang Yu 《Hydrobiologia》2009,627(1):19-30
7.
8.
低盐度可诱导鲈鱼胞浆型PEPCK基因表达 总被引:2,自引:0,他引:2
钱云霞 《中国生物化学与分子生物学报》2010,26(7):651-658
磷酸烯醇式丙酮酸羧激酶(PEPCK)催化草酰乙酸生成磷酸烯醇式丙酮酸,是糖异生途径的第1个限速酶.本研究用SMARTRACE技术从鲈鱼肝脏中分离克隆了PEPCK基因的全长cDNA序列.该基因全长2215bp,包含1个123bp的5′非翻译区和217bp的3′非翻译区,开放阅读框为1875bp,编码1个由624个氨基酸组成的蛋白质,该蛋白理论分子量为69.1kD,等电点为5.87.氨基酸序列分析表明,与其它动物的胞浆型PEPCK相似性很高,与黑鲷为94.2%,与大西洋鲑为86.4%,与人为75.9%,而与该鱼线粒体型PEPCK氨基酸同源性只有70.6%.系统发育分析显示,该蛋白首先与其它动物的cPEPCK聚成一支,然后再与鱼类的mPEPCK成簇,认为该PEPCK属于胞浆型.同时用RT-PCR分析了PEPCK基因在10个组织中的表达,结果表明只有在肝脏、消化道和肾脏有较高的表达.将鲈鱼从盐度为25的海水转入盐度为12的海水48h后,肝脏和肾脏的PEPCK基因表达有增加.实验结果表明,本实验克隆的为鲈鱼胞浆型PEPCK,低盐度可诱导其表达. 相似文献
9.
Tian Yuan Ji-Rui Gu Wen-Bo Gu Jiang Wu Shao-Rong Ge Heng Xu 《Molecular biology reports》2011,38(3):2059-2065
Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling.
In the present study, we have constructed a grass carp (Ctenopharyngodon idella) intestinal cDNA library that has over 2.3 × 105 primary clones. An expressed sequence tag (EST) of grass carp adenylosuccinate lyase (gcADSL) gene was screened from this
library. Both 5′-RACE and 3′-RACE were carried out in order to obtain the complete cDNA sequence, which contains a 1,446 bp
open reading frame encoding 482 amino acids about 54.552 kDa. The deduced amino acid sequence shares high homology with its
vertebrate counterparts, which shares 94% similarity with zebrafish, 81% with African clawed frog as well as chicken, 77%
with human and 76% with mouse. This gcADSL genomic sequence, consisted of 13 exons and 12 introns, is 8,557 bp in size. Real-time
quantitative PCR analysis revealed that the highest expression level of gcADSL was detected in muscle and the lowest in gill.
In western blotting analysis, His6-tagged gcADSL protein expressed in Escherichia coli could be recognized not only by an anti-His6-tag monoclonal antibody but also by an anti-human ADSL polyclonal antibody, indicating immunological crossreactivity occurs
between grass carp and human ADSL protein. 1,082 bp 5′-flanking region sequence was cloned and analyzed. 相似文献
10.
Shanshan Yu Hui Yang Yingmei Chai Yingying Liu Qiuxia Zhang Xinbiao Ding Qian Zhu 《Fish & shellfish immunology》2013,34(2):582-592
C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5′ untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3′ UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys61-Cys158, Cys134-Cys150) and a Ca2+/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca2+-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca2+-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR. 相似文献
11.
12.
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
13.
A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms. 相似文献
14.
Rong Huang Long-Ying Gao Ya-Ping Wang Wei Hu Qiong-Lin Guo 《Fish & shellfish immunology》2009,26(2):220-229
Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3′ and 5′ RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5′-terminal untranslated region (UTR), a 355 bp 3′-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74–96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity. 相似文献
15.
Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily that functions as a negative regulator
of skeletal muscle development and growth in mammals. However, few reports are available about the structure and function
of MSTN in teleost. Here, the MSTN gene was cloned from sea perch (Lateolabrax japonicus) by homology cloning and genomic walking. In the 4873-bp genomic sequence, three exons, two introns, and 5′ and 3′ flanking
sequences were identified. The sea perch MSTN gene encodes a 374-amino acid protein, including a signal peptide, conserved cysteine residues, and a RXXR proteolytic cleavage
domain. Expression analysis of MSTN revealed that MSTN was highly expressed in eyes, brain, and muscle; intermediately in intestine; and weakly in gill, spleen, liver, and heart.
It was demonstrated that MSTN mRNA was highly expressed in embryonic stem cell line (LJES1), but it was undetectable in several
types of somatic cell lines from sea perch, including fibroblast-like cell, epithelioid cell, and lymphocyte-like cell. Further,
it was demonstrated that the 5′ flanking region of the MSTN gene can drive the expression of green fluorescent protein (GFP) reporter gene in LJES1 cells and transgenic zebrafish (Danio rerio). This is the first report on the expression profile of MSTN gene in various types of cell cultures. 相似文献
16.
Molecular Cloning and Characterization of the Myostatin Gene in Croceine Croaker, Pseudosciaena crocea 总被引:1,自引:0,他引:1
Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. We reported
the characterization of the myostatin gene and its expression in the croceine croaker, Pseudosciaena crocea. The myostatin gene had three exons encoding 376 amino acids. The cDNA was 1,906 bp long with a 5′-UTR and 3′-UTR of 108 bp
and 667 bp, respectively. A microsatellite sequence, CA30 and CA26 separated by TA, existed in the 3′-UTR. Intron I and II were 343 bp and 758 bp in length, respectively. The deduced amino
acid sequence was highly conserved, and had more than 90% identical to shi drum, gilthead seabream, striped sea-bass, white
perch, and white bass proteins. The myostatin of croceine croaker had a putative amino terminal signal sequence (residues
1–22), a transforming growth factor-beta (TGF-β) propeptide domain (residues 41–256), a RXXR proteolytic processing site (RARR,
residues 264–267, matching the RXXR consensus site), and a TGF-β domain (residues 282–376). There were 13 conserved cysteine
residues in croceine croaker myostatin, nine of which are common to all TGF-β superfamily members. The most conserved region
of vertebrate myostatins is the TGF-β domain, which was the mature bioactive domain of the myostatin protein. The myostatin
gene was expressed not only in the skeletal muscle, but also in the other tissues. 相似文献
17.
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
18.
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
19.
Differential expression of manganese superoxide dismutase sequence variants in near isogenic lines of wheat during cold acclimation 总被引:7,自引:0,他引:7
Numerous sequence variants of wheat (Triticum aestivum L.) manganese superoxide dismutase (MnSOD) genes have been found. Quantitative real-time PCR was used to measure the expression
levels of three MnSOD genes distinguished by a variable amino acid, and three genes distinguished by sequence variation in
the 3′ untranslated region (3′ UTR), in wheat plants grown at 20°C and cold-acclimated for 1–4 weeks at 2°C. The amino acid
variants did not differ significantly in expression levels, however, differential expression of genes differing in the 3′
UTR was observed. Diploid wheat-related species also carried sequence variants of MnSOD, with differing levels of expression,
suggesting diversification of the MnSOD gene family occurred prior to the polyploidization events of hexaploid wheat. 相似文献