The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different(from Ⅰ to Ⅵ) concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase(BAD...The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different(from Ⅰ to Ⅵ) concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase(BADH: EC 1.2.1.8) activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leyrnus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH. The cDNA sequence of LcBADH was 1774bp including the open reading frame(ORF) of 1521bp(coding 506 amino acids). The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragmcnt into pET30a(+) and transformed into E. coli BL21(DE3). The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl fl-D-thiogalactoside(IPTG).展开更多
The B cells translocation gene 1 (BTG1) is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, ...The B cells translocation gene 1 (BTG1) is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, for the first time we cloned the full-length eDNA sequence of Hyriopsis schlegelii (Hs-BTG1), an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of eDNA ends (RACE) together with splicing the EST sequence from a haemocyte eDNA library, we found that Hs-BTG1 contains a 525 bp open reading frame (ORF) encoding a 174 amino-acid polypeptide, a 306 bp 5' untranslated region (5' UTR), and a 571 bp 3' UTR with a Poly(A) tail as well as a transcription termination signal (AATAAA). Homologne searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the invertebrate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas (2.03-fold), mantle (2.07-fold), kidney (2.2-fold) and haemocyte (2.5-fold) were enhanced by cadmium (Cd2+) stress, suggesting that Hs-BTG 1 may have played a significant role in H, schlegelii adaptation to adverse environmental conditions.展开更多
Summary: Rat transforming growth factor β1 (rTGFβ1) cDNA from rat lymphocytes was cloned by RT-PCR and inserted into pcDNA3 to construct an eukaryotic expression vector, which was named pcDNA3-TGFβ1. The cloned gen...Summary: Rat transforming growth factor β1 (rTGFβ1) cDNA from rat lymphocytes was cloned by RT-PCR and inserted into pcDNA3 to construct an eukaryotic expression vector, which was named pcDNA3-TGFβ1. The cloned gene was confirmed to code rat TGFβ1 by restriction enzyme analysis. pcDNA3-TGFβ1 plasmid was transfected into rat osteoblasts by using liposome-mediated gene transfer technique and the expression of TGFβ1 was detected by using irnmunohistochemical staining assay. It was found that the rat TGFβ1 expression product was obviously detectable in the transfected osteoblasts in 48 h. High expression of TGFβ1 was obtained in the rat osteoblasts in which the constructed TGFβ1 expression vector was transfected.展开更多
The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta (Guen6e) by reverse transcription polymerase chain reaction (RT-PCR) and named as HassGαq. Sequencing analysis sho...The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta (Guen6e) by reverse transcription polymerase chain reaction (RT-PCR) and named as HassGαq. Sequencing analysis showed that the fulllength of HassGαq open reading frame (ORF) is 1 062 bp, 353 amino acid residues are encoded. The predicted molecular weights (MW) and isoelectric point (PI) are 41.5 kD and 5.15, respectively. HassGαq gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside (IPTG), the GST-HassGαq fusion protein is expressed in Escherichia coil BL21, and its MW was found to be about 66 kD nearly equal to the predicted. In addition, RT-PCR analysis showed that the expressions of HassGαq are not tissue specific.展开更多
A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cD...A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.展开更多
An experiment was conducted to compare the mRNA expression difference in rice leaves and roots under drought stress and normal conditions us, ng Fluorescent Differential Display (FDD) method. One positive fragment w...An experiment was conducted to compare the mRNA expression difference in rice leaves and roots under drought stress and normal conditions us, ng Fluorescent Differential Display (FDD) method. One positive fragment was isolated by combination of the H. A. Yellow-PAGE (cont,~ined 0.1% H. A. Yellow) separation and macroarray screening methods. Compared to Arabidopsis thaliana NADPH oxidoreductase gene, it has 96% identity. The cDNA was 1423 bp, and contained a complete open reading frame of 1048 bp encoding a protein with 345 amino acid residues. Moreover, the gene expression level was higher under drought stress than that under normal conditions. The possible role of NADPH oxidoreductase gene under drought response was also discussed.展开更多
According to the sequence of the bile salt hydrolase (BSH) gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase (BSH) gen...According to the sequence of the bile salt hydrolase (BSH) gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase (BSH) gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.展开更多
Evidence has shown in mouse that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis. In the current paper, attempts were made to clone and characterize a gene encoding L...Evidence has shown in mouse that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis. In the current paper, attempts were made to clone and characterize a gene encoding Lhx8 from pig. Rapid amplification of cDNA ends (RACE) gave rise to a full-length of Lhx8 which contained 1 681 bp nucleotides, with a complete open reading frame of 885 bp, encoding a 295 amino acid polypeptide. Homology search and sequence multialignment demonstrated that the deduced pig Lhx8 protein sequence shared a high identity with Lhx8 from other mammals, including several highly conservative motifs and amino acids. The phylogenetic tree of the LIM superfamily proteins has been constructed to reveal the evolutionary relationship of various species. RT-PCR analysis showed that the Lhx8 gene was expressed in gonad and immunity tissues. In preimplantation embryos, Lhx8 mRNA expression profiling using realtime PCR revealed that its mRNA levels were highest in 4-cell stage embryos and gradually decreased until the blastocyst stage.展开更多
The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch (Lateolabrax iaponicus). The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5' untra...The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch (Lateolabrax iaponicus). The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5' untranslated regiop (UTR) of 136 bp, a 3' UTR ot 430 bp, and an ORF of 774 bp encoding a polypeptide of 258 amino acids with an estimated molecular mass of 29.31 kDa. The searches for nucleotides and protein sequence similarities with the BLAST analysis indicated that the deduced amino acid sequence of sea perch IL-1β was homological to the IL-1β in other fish species and even the mammalian. Conserved signature sequences of the IL-1β gene family were found in the sea perch IL-1β deduced amino acid sequence. Temporal expressions of the IL-1β gene in LPS or iridovirus challenged group and in control group were measured by the semi-quantitative RT-PCR. The mRNA transcripts of IL-1β could be detected in head-kidney, spleen, liver, gill and heart of the healthy individuals, and the expression level of IL-1β in head-kidney, spleen and gill was higher than that in liver and heart, but it was hard to be detected in the brain. After being stimulated by the LPS or iridovirus, the IL-1β expression in most of examined tissues was up-regulated, and also could be detected in the brain. These results indicated that the expression of sea perch IL-1β was constitutive and could be up-regulated by immune effector stimulation. Therefore the sea perch IL-1β could play a critical role in the host-pathogen interaction.展开更多
TSARG7 is a novel member of the acyltransferase family since its sequence possesses the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The porcine TSARG7 had ...TSARG7 is a novel member of the acyltransferase family since its sequence possesses the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The porcine TSARG7 had been identified by cloning in silico but had not been confirmed experimentally. The full-length mRNA of porcine TSARG7 gene was sequenced and two splice variants were discovered. The full-length cDNA of TSARG7 variant 1 was 2 513 bp and variant 2 was 2 634 bp. The putative porcine TSARG7 proteins, which were located in the cytoplasm, encoded 458 and 456 amino acids, respectively. Real-time PCR analysis showed that TSARG7 gene was expressed in various tissues, but at different levels. The expression levels of this gene were higher in the skeletal muscle, heart, and testis than that in other tissues, suggesting that the TSARG7 gene played a role in procine skeletal muscle, heart, and testis functions.展开更多
Based on the published nucleotide sequence of the apxICA of Actinobacillus pleuropneumoniae in Genbank(S4074), a pair of primers were designed. A 3 640 bp(4 687 - 8 326 bp)gene fragment was amplified by PCR from the i...Based on the published nucleotide sequence of the apxICA of Actinobacillus pleuropneumoniae in Genbank(S4074), a pair of primers were designed. A 3 640 bp(4 687 - 8 326 bp)gene fragment was amplified by PCR from the isolated strain of A. pleuropneumoniae serovar 1. Then, it was cloned into pMD18-T, identified by both restriction endonuclease and sequence analysis, and inserted into pET-28a expression vector to yield the expression plasmid. SDS-PAGE result indicated expression of apxICA in BL21 (DE3), Western blot analysis showed the protein's immunogenicity. Using the expressed protein, ELISA was established to detect serum antibody against ApxI. The feature of ELISA to detect highly virulent A. pleuropneumoniae strains infection was proved by primary clinical application.展开更多
Deoxyhypusine snyhtase (DHS) and deoxyhypusine hydroxylase (DOHH) are the two enzymes that catalyze the synthesis of hypusine within eukaryotic initiation factor 5A (eIF5A). Synthesis of hypusine is essential fo...Deoxyhypusine snyhtase (DHS) and deoxyhypusine hydroxylase (DOHH) are the two enzymes that catalyze the synthesis of hypusine within eukaryotic initiation factor 5A (eIF5A). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we described the cloning and expression of two full-length cDNAs, encoding respectively DHS-like protein and DOHH-like protein from Bombyx mori by using the methods of bioinformatics, RACE, and RT-PCR technology, named as BmDHS and BmDOHH. Sequencing results indicate that they are 1 311 and 1 874 bp in length including complete open reading frame (ORF) 1 116 and 915 bp, which encode 371 amino acids (molecular weight is about 41.11 kD and isoelectric point is 5.84) and 304 amino acids (molecular weight is about 34.30 kD and isoelectric point is 4.86), respectively. BmDHS contains only 1 exon, and BmDOHH contains 4 exons and 3 introns. The deduced amino acid sequence of BmDHS contains a deoxyhypusine synthase domain from 47 to 361 amino acid residues, and the deduced amino acid sequence of BmDOHH contains 6 E-Z type HEAT repeat domains (23-52, 54-83, 87-116, 177-206, 208-237, and 241- 270). Compared to DHS and DOHH amino acid sequences from other species, such as Homo sapiens and Drosophila melanogaster, both silkworm DHS protein and DOHH protein have more than 55% identity. The conservative regions are very similar with each other. The phylogenetic tree analysis indicated that not only DHS but also DOHH from different species has genus-specific features. The expressions of BmDHS and BmDOHH are no tissue and stage specific in our tested samples.展开更多
Both copper/zinc superoxide dismutase (SOD; Cu/Zn-SOD, SOD1) cDNA and manganese SOD (Mn-SOD, SOD2) cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE me...Both copper/zinc superoxide dismutase (SOD; Cu/Zn-SOD, SOD1) cDNA and manganese SOD (Mn-SOD, SOD2) cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE methods in this work. The SOD1 cDNA was 749 bp long and consisted of a 32-bp 5'-untranslated region (UTR), a 249-bp 3'-UTR, and a 468-bp open reading frame (ORF) encoding a 155-amino-acid protein with 16.0 kDa predicted molecular mass and 5.95 theoretical isoelectric point (p/). The SOD2 cDNA was 1687 bp long and comprised 94-bp of 5'-UTR, 912-bp 3'-UTR and 681-bp ORF encoding a 226-amino-acid protein with 25.0 kDa predicted molecular mass and 8.83 pI. The deduced amino acid sequence of SOD1 showed relatively high similarity (77.4%-87.1%) and identity (65.4%-74.4%) with the published sequences of SOD1 from other vertebrate species, whereas SOD2 protein shared slightly higher similarity (83.6%-95.6%) and identity (76.1%-88.9%) with other reported vertebrates SOD2s. Phylogenetic analysis revealed that the C. reevesii SOD1 and SOD2 were separately clustered together, and were highly conserved during evolution. Both SOD mRNA expression was detected widely in the brain, liver, muscle, kidney, gut, spleen, lung and heart at variable levels. The highest expression of the two SODs was observed in muscle, and followed in brain, liver, kidney, gut and heart, whereas low transcriptional levels were found in spleen and lung. Meanwhile, high activity of SOD 1 was kept in brain, liver, muscle, kidney and heart, and followed in gut, spleen and lung. The activities of SOD2 in brain, liver, muscle, kidney, gut and heart were significantly higher than those in spleen and lung.展开更多
A PCR-based homologous cloning strategy was used to identify an actin gene from the roots of Chinese licorice(Glycyrrhiza uralensis Fisch). Results of sequence analysis indicate that a 1137 bp cDNA with an open read...A PCR-based homologous cloning strategy was used to identify an actin gene from the roots of Chinese licorice(Glycyrrhiza uralensis Fisch). Results of sequence analysis indicate that a 1137 bp cDNA with an open reading frame encoding 377 amino acids, actin ortholog, GuActin, was successfully cloned and characterized(GenBank accession No. EU190972). Thus far, GuActin is the first actin of Chinese licorice that has been identified at a molecular level. Analysis by Northern blot shows that GuActin was expressed strongly in the roots, particularly in radicles than in stems and leaves. These results suggest that GuActin may be a member of the vegetative subfamily of the actin family.展开更多
A full-length cDNA sequence of xyloglucan endotransglycosylase gene (XET), abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromoso...A full-length cDNA sequence of xyloglucan endotransglycosylase gene (XET), abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame (ORF) encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.展开更多
The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genom...The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains: a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/(Arg+Lys) ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 (DE3) by pColdlII was also successfully observed in this study.展开更多
Background: In Sus scrofa, methionine sulfoxide reductase B3(MSRB3) is a crucial candidate gene for ear size, and an important conformational trait of pig breeds. However, challenges in MSRB3 c DNA amplification ha...Background: In Sus scrofa, methionine sulfoxide reductase B3(MSRB3) is a crucial candidate gene for ear size, and an important conformational trait of pig breeds. However, challenges in MSRB3 c DNA amplification have prevented further identification of MSRB3 allelic variants influencing pig ear size.Results: We cloned a full-length c DNA sequence of porcine MSRB3 by rapid-amplification of c DNA ends. The3,765-bp gene contained a 5'-untranslated region(UTR)(190 bp), a coding region(552 bp), and a 3'-UTR(3,016 bp) and shared 84 %, 84 %, 87 %, 86 %, and 70 % sequence identities with human, orangutan, mouse, chicken, and zebrafish,respectively. The gene encoded a 183-amino acid protein, which shared 88 %, 91 %, 89 %, 86 %, and 67 % identities with human, orangutan, mouse, chicken, and zebrafish, respectively. Tissue expression analysis using q RT-PCR revealed that MSRB3 was expressed in the heart, liver, lung, kidney, spleen, ear, muscle, fat, lymph, skeletal, and hypothalamic tissues. Three single nucleotide polymorphisms(SNPs) were identified in MSRB3: c.-735 C 〉 T in the 5' flanking region,c.2571 T 〉 C in the 3'-UTR, and a synonymous mutation of c.484 T 〉 C in the coding region. The SNPs c.-735 C 〉 T and c.2571 T 〉 C were significantly associated with ear size in a Large White × Minzhu F2 population other than in Beijing Black pigs. Subsequently, at SNP c.-735 C 〉 T, the m RNA of MSRB3 was significantly higher expressed in ears of individuals with the TT genotype(Minzhu) than those with CC(Large White).Conclusions: The porcine MSRB3 owned a 3,765-bp full-length c DNA sequence and was detected to express in ear tissue. Two SNPs of this gene were shown to be significantly associated with ear size in a Large White × Minzhu intercross population instead of Beijing Black pig population. What's more, the individuals with higher m RNA expression of MSRB3 have larger ear sizes. These results provide useful information for further functional analyses of MSRB3 influencing ear size in pigs.展开更多
Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase c DNA of Paphia textile(Pt CAT) was clo...Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase c DNA of Paphia textile(Pt CAT) was cloned using RTPCR and rapid amplification of c DNA ends(RACE). Pt CAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 k D and an estimated isoelectric point of 8.2. Sequence alignment indicated that Pt CAT contained a highly conserved catalytic signature motif(^(61)FNRERIPERVVHAKGAG^(77)), a proximal heme-ligand signature sequence(^(352)RLFSYSDP^(359)), and three catalytic amino acid residues(H^(72), N^(145), and Y^(356)). Pt CAT also contains two putative N-glycosylation sites(^(34)NKT^(36) and ^(437)NFT^(439)) and a peroxisome-targeting signal(^(511)AQL^(513)). Furthermore, Pt CAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. Pt CAT m RNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of Pt CAT m RNA: first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that Pt CAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.展开更多
[Objectives] Allene oxide synthase( AOS),the first specific enzyme in the biosynthetic pathway of jasmonate( JA),has an important role in regulating JA accumulation in plants. This study was conducted to amplify f...[Objectives] Allene oxide synthase( AOS),the first specific enzyme in the biosynthetic pathway of jasmonate( JA),has an important role in regulating JA accumulation in plants. This study was conducted to amplify full-length AOS gene from Lilium‘Siberia'( LsAOS) and to explore the spatiotemporal expression of this gene. [Methods]LsAOS was cloned by rapid amplification of c DNA ends( RACE) and analyzed bioinformatically. LsAOS expression level in nine organs/tissues and at four flowering stages was determined by quantitative PCR( q-PCR). [Results] LsAOS gene has an open reading frame of 1 542 bp,encoding a protein of 514 amino acids. The LsAOS protein sequence was more homologous with the AOS protein of Musa acuminata Colla than that in any other plant species.The LsAOS expression reached the maximum level at bud stage,and then gradually decreased over time during the flowering period. The LsAOS expression level in leaves was the highest,followed by in inner petals,roots,stems,etc. [Conclusions]JA participates in the whole process of plant development,and is closely related to stress resistance and secondary metabolism. AOS is one of the key genes in JA pathway and plays an important role in regulating the biosynthesis of JA.展开更多
2,4-dienoyl-CoA reductase 1 ( DECR1 ) is the key rate-limiting enzyme in the metabolism of polyunsaturated fatty acids. Although this protein has been studied in a variety of mammals, its role in por- cine is yet to...2,4-dienoyl-CoA reductase 1 ( DECR1 ) is the key rate-limiting enzyme in the metabolism of polyunsaturated fatty acids. Although this protein has been studied in a variety of mammals, its role in por- cine is yet to be fully elucidated. However, it is a candidate determinant/indicator of meat quality, growth traits, and carcass quality. Here, we employed RT-PCR and rapid amplification of cDNA ends (RACE) analysis to amplify the full-length cDNA of DECR1 from Mashen pig liver, and cloned it into the expression vector pET-32a+. After confirmation by sequencing and restriction analysis, the recombinant plasmid was transformed into E. coli BL21 cells. The cDNA of pig DECR1 contained 2,352 nucleotides, including a 987 bp open reading frame flanked by a 53 bp 5'-untranslated region (UTR) and a 1,312 bp 3'-UTR. The pig DECR1 coding sequence encoded 328 amino acid residues, which shared 99%, 88%, 87%, 87%, 87%, 87%, and 83% identity with those of Sus scrofa (predicted), Bos taurus, Homo sapiens, Macaca mulatta, Pan troglodytes, Equus caballus, Canis, and Mus musculus, respectively. SDS-PAGE analysis revealed that the recombinant protein was expressed and that the expression level reached its highest level after 4 h induction. Western blot analysis indicated that the molecular weight of the expressed protein was the same as that predicted, ap- proximately 35 kDa. Collectively these data provide the basis for further studies into the physiological functions and molecular mechanisms of the pig DE- CR1 gene.展开更多
基金Supported by the National Natural Science Foundation of China(Nos30590382 and 30570273)
文摘The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different(from Ⅰ to Ⅵ) concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase(BADH: EC 1.2.1.8) activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leyrnus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH. The cDNA sequence of LcBADH was 1774bp including the open reading frame(ORF) of 1521bp(coding 506 amino acids). The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragmcnt into pET30a(+) and transformed into E. coli BL21(DE3). The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl fl-D-thiogalactoside(IPTG).
基金supported by the Key Scientific and Technological Programme of Jiangxi Province,China(20121BBF60036)the Special Fund for Agro-scientific Research in the Public Interest,State Agriculture Ministry of China(200903028)+2 种基金the Science and Technology Landing Project of Jiangxi Province,China(KJLD12001)the Youth Fund of the Education Department of Jiangxi Province,China(GJJ14219)the National Natural Science Foundation of China(31160534)
文摘The B cells translocation gene 1 (BTG1) is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, for the first time we cloned the full-length eDNA sequence of Hyriopsis schlegelii (Hs-BTG1), an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of eDNA ends (RACE) together with splicing the EST sequence from a haemocyte eDNA library, we found that Hs-BTG1 contains a 525 bp open reading frame (ORF) encoding a 174 amino-acid polypeptide, a 306 bp 5' untranslated region (5' UTR), and a 571 bp 3' UTR with a Poly(A) tail as well as a transcription termination signal (AATAAA). Homologne searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the invertebrate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas (2.03-fold), mantle (2.07-fold), kidney (2.2-fold) and haemocyte (2.5-fold) were enhanced by cadmium (Cd2+) stress, suggesting that Hs-BTG 1 may have played a significant role in H, schlegelii adaptation to adverse environmental conditions.
文摘Summary: Rat transforming growth factor β1 (rTGFβ1) cDNA from rat lymphocytes was cloned by RT-PCR and inserted into pcDNA3 to construct an eukaryotic expression vector, which was named pcDNA3-TGFβ1. The cloned gene was confirmed to code rat TGFβ1 by restriction enzyme analysis. pcDNA3-TGFβ1 plasmid was transfected into rat osteoblasts by using liposome-mediated gene transfer technique and the expression of TGFβ1 was detected by using irnmunohistochemical staining assay. It was found that the rat TGFβ1 expression product was obviously detectable in the transfected osteoblasts in 48 h. High expression of TGFβ1 was obtained in the rat osteoblasts in which the constructed TGFβ1 expression vector was transfected.
文摘The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta (Guen6e) by reverse transcription polymerase chain reaction (RT-PCR) and named as HassGαq. Sequencing analysis showed that the fulllength of HassGαq open reading frame (ORF) is 1 062 bp, 353 amino acid residues are encoded. The predicted molecular weights (MW) and isoelectric point (PI) are 41.5 kD and 5.15, respectively. HassGαq gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside (IPTG), the GST-HassGαq fusion protein is expressed in Escherichia coil BL21, and its MW was found to be about 66 kD nearly equal to the predicted. In addition, RT-PCR analysis showed that the expressions of HassGαq are not tissue specific.
基金supported by the“863"Prijetof China under contract Nos 2001AA628180 and 2002AA626020.
文摘A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.
文摘An experiment was conducted to compare the mRNA expression difference in rice leaves and roots under drought stress and normal conditions us, ng Fluorescent Differential Display (FDD) method. One positive fragment was isolated by combination of the H. A. Yellow-PAGE (cont,~ined 0.1% H. A. Yellow) separation and macroarray screening methods. Compared to Arabidopsis thaliana NADPH oxidoreductase gene, it has 96% identity. The cDNA was 1423 bp, and contained a complete open reading frame of 1048 bp encoding a protein with 345 amino acid residues. Moreover, the gene expression level was higher under drought stress than that under normal conditions. The possible role of NADPH oxidoreductase gene under drought response was also discussed.
基金Supported by 863 Projects (2008AA10Z311)National Science and Technology Support Projects (2009BADB9B06)+1 种基金Started Post-doctoral Research Grant of Heilongjiang Province (LBH-Q07023)Harbin Technological Innovation of Special Funds (2007RFQXN020)
文摘According to the sequence of the bile salt hydrolase (BSH) gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase (BSH) gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.
基金supported by the High Technology Research and Development Program of China(2006AA10Z136)
文摘Evidence has shown in mouse that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis. In the current paper, attempts were made to clone and characterize a gene encoding Lhx8 from pig. Rapid amplification of cDNA ends (RACE) gave rise to a full-length of Lhx8 which contained 1 681 bp nucleotides, with a complete open reading frame of 885 bp, encoding a 295 amino acid polypeptide. Homology search and sequence multialignment demonstrated that the deduced pig Lhx8 protein sequence shared a high identity with Lhx8 from other mammals, including several highly conservative motifs and amino acids. The phylogenetic tree of the LIM superfamily proteins has been constructed to reveal the evolutionary relationship of various species. RT-PCR analysis showed that the Lhx8 gene was expressed in gonad and immunity tissues. In preimplantation embryos, Lhx8 mRNA expression profiling using realtime PCR revealed that its mRNA levels were highest in 4-cell stage embryos and gradually decreased until the blastocyst stage.
文摘The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch (Lateolabrax iaponicus). The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5' untranslated regiop (UTR) of 136 bp, a 3' UTR ot 430 bp, and an ORF of 774 bp encoding a polypeptide of 258 amino acids with an estimated molecular mass of 29.31 kDa. The searches for nucleotides and protein sequence similarities with the BLAST analysis indicated that the deduced amino acid sequence of sea perch IL-1β was homological to the IL-1β in other fish species and even the mammalian. Conserved signature sequences of the IL-1β gene family were found in the sea perch IL-1β deduced amino acid sequence. Temporal expressions of the IL-1β gene in LPS or iridovirus challenged group and in control group were measured by the semi-quantitative RT-PCR. The mRNA transcripts of IL-1β could be detected in head-kidney, spleen, liver, gill and heart of the healthy individuals, and the expression level of IL-1β in head-kidney, spleen and gill was higher than that in liver and heart, but it was hard to be detected in the brain. After being stimulated by the LPS or iridovirus, the IL-1β expression in most of examined tissues was up-regulated, and also could be detected in the brain. These results indicated that the expression of sea perch IL-1β was constitutive and could be up-regulated by immune effector stimulation. Therefore the sea perch IL-1β could play a critical role in the host-pathogen interaction.
基金supported by the National High Technology Research and Development Program of China(2006AA10Z136)
文摘TSARG7 is a novel member of the acyltransferase family since its sequence possesses the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The porcine TSARG7 had been identified by cloning in silico but had not been confirmed experimentally. The full-length mRNA of porcine TSARG7 gene was sequenced and two splice variants were discovered. The full-length cDNA of TSARG7 variant 1 was 2 513 bp and variant 2 was 2 634 bp. The putative porcine TSARG7 proteins, which were located in the cytoplasm, encoded 458 and 456 amino acids, respectively. Real-time PCR analysis showed that TSARG7 gene was expressed in various tissues, but at different levels. The expression levels of this gene were higher in the skeletal muscle, heart, and testis than that in other tissues, suggesting that the TSARG7 gene played a role in procine skeletal muscle, heart, and testis functions.
文摘Based on the published nucleotide sequence of the apxICA of Actinobacillus pleuropneumoniae in Genbank(S4074), a pair of primers were designed. A 3 640 bp(4 687 - 8 326 bp)gene fragment was amplified by PCR from the isolated strain of A. pleuropneumoniae serovar 1. Then, it was cloned into pMD18-T, identified by both restriction endonuclease and sequence analysis, and inserted into pET-28a expression vector to yield the expression plasmid. SDS-PAGE result indicated expression of apxICA in BL21 (DE3), Western blot analysis showed the protein's immunogenicity. Using the expressed protein, ELISA was established to detect serum antibody against ApxI. The feature of ELISA to detect highly virulent A. pleuropneumoniae strains infection was proved by primary clinical application.
基金funded by the 973 National Basic Research Program of China (2005CB121005)
文摘Deoxyhypusine snyhtase (DHS) and deoxyhypusine hydroxylase (DOHH) are the two enzymes that catalyze the synthesis of hypusine within eukaryotic initiation factor 5A (eIF5A). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we described the cloning and expression of two full-length cDNAs, encoding respectively DHS-like protein and DOHH-like protein from Bombyx mori by using the methods of bioinformatics, RACE, and RT-PCR technology, named as BmDHS and BmDOHH. Sequencing results indicate that they are 1 311 and 1 874 bp in length including complete open reading frame (ORF) 1 116 and 915 bp, which encode 371 amino acids (molecular weight is about 41.11 kD and isoelectric point is 5.84) and 304 amino acids (molecular weight is about 34.30 kD and isoelectric point is 4.86), respectively. BmDHS contains only 1 exon, and BmDOHH contains 4 exons and 3 introns. The deduced amino acid sequence of BmDHS contains a deoxyhypusine synthase domain from 47 to 361 amino acid residues, and the deduced amino acid sequence of BmDOHH contains 6 E-Z type HEAT repeat domains (23-52, 54-83, 87-116, 177-206, 208-237, and 241- 270). Compared to DHS and DOHH amino acid sequences from other species, such as Homo sapiens and Drosophila melanogaster, both silkworm DHS protein and DOHH protein have more than 55% identity. The conservative regions are very similar with each other. The phylogenetic tree analysis indicated that not only DHS but also DOHH from different species has genus-specific features. The expressions of BmDHS and BmDOHH are no tissue and stage specific in our tested samples.
基金supported by a grant from the National Natural Science Foundation of China (Project No. 31172383)
文摘Both copper/zinc superoxide dismutase (SOD; Cu/Zn-SOD, SOD1) cDNA and manganese SOD (Mn-SOD, SOD2) cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE methods in this work. The SOD1 cDNA was 749 bp long and consisted of a 32-bp 5'-untranslated region (UTR), a 249-bp 3'-UTR, and a 468-bp open reading frame (ORF) encoding a 155-amino-acid protein with 16.0 kDa predicted molecular mass and 5.95 theoretical isoelectric point (p/). The SOD2 cDNA was 1687 bp long and comprised 94-bp of 5'-UTR, 912-bp 3'-UTR and 681-bp ORF encoding a 226-amino-acid protein with 25.0 kDa predicted molecular mass and 8.83 pI. The deduced amino acid sequence of SOD1 showed relatively high similarity (77.4%-87.1%) and identity (65.4%-74.4%) with the published sequences of SOD1 from other vertebrate species, whereas SOD2 protein shared slightly higher similarity (83.6%-95.6%) and identity (76.1%-88.9%) with other reported vertebrates SOD2s. Phylogenetic analysis revealed that the C. reevesii SOD1 and SOD2 were separately clustered together, and were highly conserved during evolution. Both SOD mRNA expression was detected widely in the brain, liver, muscle, kidney, gut, spleen, lung and heart at variable levels. The highest expression of the two SODs was observed in muscle, and followed in brain, liver, kidney, gut and heart, whereas low transcriptional levels were found in spleen and lung. Meanwhile, high activity of SOD 1 was kept in brain, liver, muscle, kidney and heart, and followed in gut, spleen and lung. The activities of SOD2 in brain, liver, muscle, kidney, gut and heart were significantly higher than those in spleen and lung.
基金Supported by the Natural Science Foundation of Fujian Province, China(No.200804011)the Special Foundation for Young Scientists of Fujian Province, China(No.2006F3113)+1 种基金Scientific Research Foundation of Educational Committee of Fujian Province, China(No.JA07151) Technology Research and Development Program of Quanzhou’ Science and Technology Bureau, China(No.2007N6)
文摘A PCR-based homologous cloning strategy was used to identify an actin gene from the roots of Chinese licorice(Glycyrrhiza uralensis Fisch). Results of sequence analysis indicate that a 1137 bp cDNA with an open reading frame encoding 377 amino acids, actin ortholog, GuActin, was successfully cloned and characterized(GenBank accession No. EU190972). Thus far, GuActin is the first actin of Chinese licorice that has been identified at a molecular level. Analysis by Northern blot shows that GuActin was expressed strongly in the roots, particularly in radicles than in stems and leaves. These results suggest that GuActin may be a member of the vegetative subfamily of the actin family.
基金supported by the National Natural Science Foundation of China (Grant No. 30901158)the Key Project of Chinese Ministry of Education (Grant No. 104243)
文摘A full-length cDNA sequence of xyloglucan endotransglycosylase gene (XET), abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame (ORF) encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.
基金supported by the National High Technology Research and Development Program of China (Grant no.2007AA091905)the Basic Scientific Research Operation Fund of China (Grant no. GY02-2007-T11)
文摘The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains: a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/(Arg+Lys) ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 (DE3) by pColdlII was also successfully observed in this study.
基金supported by the Agricultural Science and Technology Innovation Program(ASTIP-IAS02)the National Key Technology R&D Program of China(No.2011BAD28B01)+1 种基金earmarked fund for Modern Agro-industry Technology Research SystemChinese Academy of Agricultural Sciences Foundation(No.2014ZL006)
文摘Background: In Sus scrofa, methionine sulfoxide reductase B3(MSRB3) is a crucial candidate gene for ear size, and an important conformational trait of pig breeds. However, challenges in MSRB3 c DNA amplification have prevented further identification of MSRB3 allelic variants influencing pig ear size.Results: We cloned a full-length c DNA sequence of porcine MSRB3 by rapid-amplification of c DNA ends. The3,765-bp gene contained a 5'-untranslated region(UTR)(190 bp), a coding region(552 bp), and a 3'-UTR(3,016 bp) and shared 84 %, 84 %, 87 %, 86 %, and 70 % sequence identities with human, orangutan, mouse, chicken, and zebrafish,respectively. The gene encoded a 183-amino acid protein, which shared 88 %, 91 %, 89 %, 86 %, and 67 % identities with human, orangutan, mouse, chicken, and zebrafish, respectively. Tissue expression analysis using q RT-PCR revealed that MSRB3 was expressed in the heart, liver, lung, kidney, spleen, ear, muscle, fat, lymph, skeletal, and hypothalamic tissues. Three single nucleotide polymorphisms(SNPs) were identified in MSRB3: c.-735 C 〉 T in the 5' flanking region,c.2571 T 〉 C in the 3'-UTR, and a synonymous mutation of c.484 T 〉 C in the coding region. The SNPs c.-735 C 〉 T and c.2571 T 〉 C were significantly associated with ear size in a Large White × Minzhu F2 population other than in Beijing Black pigs. Subsequently, at SNP c.-735 C 〉 T, the m RNA of MSRB3 was significantly higher expressed in ears of individuals with the TT genotype(Minzhu) than those with CC(Large White).Conclusions: The porcine MSRB3 owned a 3,765-bp full-length c DNA sequence and was detected to express in ear tissue. Two SNPs of this gene were shown to be significantly associated with ear size in a Large White × Minzhu intercross population instead of Beijing Black pig population. What's more, the individuals with higher m RNA expression of MSRB3 have larger ear sizes. These results provide useful information for further functional analyses of MSRB3 influencing ear size in pigs.
基金The National Natural Science Foundation of China under contract No.31172397the New Century Excellent Talents of Fujian Province University under contract No.JA14167the Open Research Fund Program of Fujian Provincial Key Laboratory of Marine Fishery Resources and Eco-environment under contract No.Z814041
文摘Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase c DNA of Paphia textile(Pt CAT) was cloned using RTPCR and rapid amplification of c DNA ends(RACE). Pt CAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 k D and an estimated isoelectric point of 8.2. Sequence alignment indicated that Pt CAT contained a highly conserved catalytic signature motif(^(61)FNRERIPERVVHAKGAG^(77)), a proximal heme-ligand signature sequence(^(352)RLFSYSDP^(359)), and three catalytic amino acid residues(H^(72), N^(145), and Y^(356)). Pt CAT also contains two putative N-glycosylation sites(^(34)NKT^(36) and ^(437)NFT^(439)) and a peroxisome-targeting signal(^(511)AQL^(513)). Furthermore, Pt CAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. Pt CAT m RNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of Pt CAT m RNA: first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that Pt CAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.
基金Supported by Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges in Beijing Municipality(DHT20150503)National Natural Science Foundation of China(3120164531640070)
文摘[Objectives] Allene oxide synthase( AOS),the first specific enzyme in the biosynthetic pathway of jasmonate( JA),has an important role in regulating JA accumulation in plants. This study was conducted to amplify full-length AOS gene from Lilium‘Siberia'( LsAOS) and to explore the spatiotemporal expression of this gene. [Methods]LsAOS was cloned by rapid amplification of c DNA ends( RACE) and analyzed bioinformatically. LsAOS expression level in nine organs/tissues and at four flowering stages was determined by quantitative PCR( q-PCR). [Results] LsAOS gene has an open reading frame of 1 542 bp,encoding a protein of 514 amino acids. The LsAOS protein sequence was more homologous with the AOS protein of Musa acuminata Colla than that in any other plant species.The LsAOS expression reached the maximum level at bud stage,and then gradually decreased over time during the flowering period. The LsAOS expression level in leaves was the highest,followed by in inner petals,roots,stems,etc. [Conclusions]JA participates in the whole process of plant development,and is closely related to stress resistance and secondary metabolism. AOS is one of the key genes in JA pathway and plays an important role in regulating the biosynthesis of JA.
文摘2,4-dienoyl-CoA reductase 1 ( DECR1 ) is the key rate-limiting enzyme in the metabolism of polyunsaturated fatty acids. Although this protein has been studied in a variety of mammals, its role in por- cine is yet to be fully elucidated. However, it is a candidate determinant/indicator of meat quality, growth traits, and carcass quality. Here, we employed RT-PCR and rapid amplification of cDNA ends (RACE) analysis to amplify the full-length cDNA of DECR1 from Mashen pig liver, and cloned it into the expression vector pET-32a+. After confirmation by sequencing and restriction analysis, the recombinant plasmid was transformed into E. coli BL21 cells. The cDNA of pig DECR1 contained 2,352 nucleotides, including a 987 bp open reading frame flanked by a 53 bp 5'-untranslated region (UTR) and a 1,312 bp 3'-UTR. The pig DECR1 coding sequence encoded 328 amino acid residues, which shared 99%, 88%, 87%, 87%, 87%, 87%, and 83% identity with those of Sus scrofa (predicted), Bos taurus, Homo sapiens, Macaca mulatta, Pan troglodytes, Equus caballus, Canis, and Mus musculus, respectively. SDS-PAGE analysis revealed that the recombinant protein was expressed and that the expression level reached its highest level after 4 h induction. Western blot analysis indicated that the molecular weight of the expressed protein was the same as that predicted, ap- proximately 35 kDa. Collectively these data provide the basis for further studies into the physiological functions and molecular mechanisms of the pig DE- CR1 gene.