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
Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR (GenBank accession ...Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR (GenBank accession Nos DQ640312, DQ504163 ). The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.展开更多
White-brown complex (WBC) transporters, also called half-size ATP binding cassette G (ABCG) transporters, are involved in many biological processes, including seed development; however, the WBC transporters in gra...White-brown complex (WBC) transporters, also called half-size ATP binding cassette G (ABCG) transporters, are involved in many biological processes, including seed development; however, the WBC transporters in grapevines received less attention to date. To reveal the molecular characteristics of WBCs and the connection between WBCs and agronomic traits of stenospermocarpic (seedless) grapevine, we carried out a genomic census and analysis of ovule-associated expression for VvWBC genes in grapevine. We identified 30 VvWBC genes and cloned full-length complementary DNAs (cDNAs) for 20 of these. The tissue or organ-specific expression analysis showed that several VvWBCs exhibited distinct expression patterns with some showing tissue specificity. Twelve VvWBC genes were found to be expressed in the developing ovules. Moreover, the results of quantitative real-time PCR (qRT-PCR) suggested that four of twelve ovule-expressed VvWBCs have distinct expression profiles during the development of ovules between seeded and stenospermocarpic grapevines. These four genes might be involved in ovule abortion. Meanwhile, chromosome mapping, multiple sequence alignments, exon/intron structure analyses and synteny analyses were preformed on VvWBC genes. Our experiments provide a new perspective on the mechanism of stenospermocarpic seedlessness and put forward a framework for further study of WBC transporters.展开更多
To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was a...To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.展开更多
Primers and probes were established according to the sequences of the alpha-amylase genes of Bacillus. halodurans C-125, Therrnus sp. IM6501, B. stearothermophilus ET-1, and B, acidopullulytics. Primers were designed ...Primers and probes were established according to the sequences of the alpha-amylase genes of Bacillus. halodurans C-125, Therrnus sp. IM6501, B. stearothermophilus ET-1, and B, acidopullulytics. Primers were designed and a 0.2 kb DNA fragment was amplified, the fragment was successfully used for the detection of the amylase Ⅱ gene in a 2 842 bp region from Bacillus halodurans strain 38C1-1.展开更多
Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indic...Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.展开更多
For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids w...For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids were encoded.Bioinformatics analysis showed that the VrPR protein belonged to the NADB Rossmann superfamily,which was one of the non-transmembrane hydrophilic proteins.VrPR was assumed to have 44 amino acid phosphorylation sites and be contained in chloroplasts.The VrPR secondary structure comprised of random coil,αhelix,βangle,and extended chain,all of which were quite compatible with the anticipated tertiary structure.Moreover,analysis of the phylogenetic tree indicated that the soybean PR(Glyma.12G222200)and VrPR were closely related.Furthermore,chlorophyll content in leaves is markedly increased in Arabidopsis when VrPR is overexpressed.Ourfindings will serve as a reference for more functional studies on the PR genes in mung bean.展开更多
[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The ...[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.展开更多
Fructose-1,6-bisphosphatase(FBPase) is one of the key enzymes in Calvin circle and starch biosynthesis. In this study, the full-length of cpFBPase gene from Pyropia haitanensis was cloned by using rapid amplificatio...Fructose-1,6-bisphosphatase(FBPase) is one of the key enzymes in Calvin circle and starch biosynthesis. In this study, the full-length of cpFBPase gene from Pyropia haitanensis was cloned by using rapid amplification of cDNA ends(RACE) technology. The nucleotide sequence of PhcpFBPase consists of 1 400 bp, including a 5′ untranslated region(UTR) of 92 bp, a 3′?UTR of 69 bp, and an open reading frame(ORF) of 1 236 bp, which can be translated into a 412-amino-acid putative peptides with a molecular weight of 44.3 kDa and a theoretical pI of 5.23. Multiple sequence alignment indicated that the protein belonged to the chloroplast FBPase enzyme. Phylogenetic analysis showed that the protein assembled with the cpFBPase of a thermal tolerant unicellular red micro-algae Galdieria sulphuraria. Expression patterns analyzed by qRT-PCR revealed that the expression of PhcpFBPase gene in the thallus phage was 7-fold higher than in the conchocelis phage, which suggested the different mechanisms of inorganic carbon utilization among the different life phages of P. haitanensis. And the different response modes of PhcpFBPase mRNA levels to high temperature and desiccation stress indicated that PhcpFBPase played an important role in responsing to abiotic stress.展开更多
A total of 117 agar-decomposing cultures were isolated from coastal seawater around Qingdao, China. The phenotypic and agarolytic features of an agarolytic isolate, QM38, were investigated. The strain was gram negativ...A total of 117 agar-decomposing cultures were isolated from coastal seawater around Qingdao, China. The phenotypic and agarolytic features of an agarolytic isolate, QM38, were investigated. The strain was gram negative, strictly aerobic, curved rod and polar flagellum. On the basis of several phenotypic characters, biochemical and morphological characters and phylogenetic analysis of the gene coding for the 16S rRNA, the strain was identified as Agarivorans albus strain QM38. This strain can liquefy the agar on the solid agar plate. An excellular agarase activity was determined in liquid culture. The enzyme exhibited maximal activity at 40 ℃, pH 7.6. Its activity was greatly affected by different concentrations of agarose. The highest activity 32 U/ml was achieved in the culture supernatant. The hydrolytic product was analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). After complete hydrolysis of agarose, a series of agaro-oligosaccharides were produced. The main products of the enzymes were oligosaccharides in the degree of polymerization (DP) of 2, 4, 6 and 8. Three genes agaD01, agaD02 and agaD03, encodingβ-agarases, had been cloned from genomic DNA of Agarivorans albus strain QM38. The open reading frame of agaDOl, consisted of 2 988 bp, and shared 95.5%-98.9% identity to the β-agarase genes of some strains of Vibrio and Agarivorans. Gene agaD02 comprised 2 868 bp and encoded a 955- amino-acid protein. It showed 97.4% and 98.7~0 identity to the β-agarase genes of strain Vibrio sp. PO-303 and strain Vibrio sp. JT0107, respectively. Only partial sequence of agaD03 gene has been cloned. It showed 96.5% identity to β-agarase gene (agaB) of Pseudoalteromonas sp. CY24, and shared 96.8% identity to β-agarase-c gene of Vibrio sp. PO-303.展开更多
Pathogenesis-related proteins (PRs) play many important roles in plant defense response against pathogen attack. To better understand the molecular mechanism of PR genes involved in wheat adult plant resistance (AP...Pathogenesis-related proteins (PRs) play many important roles in plant defense response against pathogen attack. To better understand the molecular mechanism of PR genes involved in wheat adult plant resistance (APR) to stripe rust, based on a differentially expressed transcribed derived fragment (TDF), a novel PR gene from wheat cv. Xingzi 9104 infected by the Puccinia striiformis Westend f. sp. tritici Erikss. pathotype CY32, which was highly similar to the maize ZmPRIO gene and designated as TaPRIO, was identified using in silico cloning and RT-PCR method. This novel TaPRIO gene was predicted to encode a 160-amino acid protein with a deduced molecular weight of 17.06 kDa and an isoelectronic point (pI) of 5.19. An amino acid sequence analysis of TaPR10 demonstrated the presence of a typical conserved domain of pathogenesis related protein Bet v I family. Multiple alignment analysis based on the amino acids encoded by 10 different PRIO genes from maize (Zea mays), rice (Oryza sativa), broomcorn (Sorghum bicolor), and wheat (Triticum aestivum) indicated that PR proteins of class 10 was conserved among the 4 plant species with about 80% similarity. DNA sequence of TaPRIO suggested the presence of one 84-bp intron with the splicing sites of GT-AT bi-nucleotide sequence between 188 and 271 bp. Using a real-time quantitative RT-PCR (qRT-PCR), expression profiles of TaPRIO revealed that at the adult-plant stage, TaPRIO transcript was up-regulated as early as 12 h post-inoculation (hpi), with the occurrence of maximum induction at 24 hpi. At the seedling stage, TaPRIO was also slightly induced 18 hpi. However, the transcript amount was relatively lower than that of the adult-plant stage. Taken together, these results suggest that TaPRIO may participate in wheat defense response of APR to stripe rust.展开更多
Ethylene response factors (ERFs) play important roles in response to plant biotic and abiotic stresses. In this study, a gene encoding a putative AP2/ERF domain-containing protein was isolated by screening a SSH cDN...Ethylene response factors (ERFs) play important roles in response to plant biotic and abiotic stresses. In this study, a gene encoding a putative AP2/ERF domain-containing protein was isolated by screening a SSH cDNA library from rice and designated as Oryza sativa AP2/ERF-like protein (OsAP2LP) gene. OsAP2LP is 1491 bp in length, interrupted by seven introns, and encodes a putative protein of 348 amino acids. Temporal and spatial expression analysis showed that the OsAP2LP gene was preferentially expressed in roots, panicles, mature embryos and seeds in rice. Real-time quantitative PCR analysis indicated that the expression levels of the OsAP2LP gene were increased under the treatments of drought and gibberellin but decreased under the treatments of low temperature, salt, abscisic acid (ABA) and zeatin. Taken together, these results suggest that OsAP2LP might be involved in stress responses, and probably plays roles as a transcription regulator when plants response to cold, salt and drought stresses through ABA and gibberellin pathways.展开更多
Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase (SAD) is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of ...Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase (SAD) is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame (ORF). Analysis in the BLAST on NCBI shows that Jatropha curcas SAD (JSAD) gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD (RSAD) is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.展开更多
Wuschel-related homeobox (WOX) genes play essential, specific, and sometimes redundant roles in plant embryo development, shoot and root meristem maintenance, and plant development. Though much information was quick...Wuschel-related homeobox (WOX) genes play essential, specific, and sometimes redundant roles in plant embryo development, shoot and root meristem maintenance, and plant development. Though much information was quickly gained with members of the WOX gene family of Arabidopsis, monocotyledonous crops, and gymnospermous conifers, little is known about perennial woody plants. In this study, we isolated the first WOXgene family member from grape (Vitis vinifera L. cv. Cabernet Sauvignon), and named it VvWOX4 based on its characteristic domains and phylogenetic analysis. The identity of VvWOX4 was validated by MALDI-TOF MS and Western blot with polyclonal antibody against Arabidopsis thaliana Wuschel. Functional analysis showed that VvWOX4 markedly increased shoot primordia structures when overexpressed under CaMV 35S promoter in tobacco. A different expression pattern was found for VvVOX4 compared with AtWUCHEL and its expression was detected in unique organs of grapevines. Besides the expression in the vegetative shoot apical meristem (SAM) of grape shoot tips, VvWOX4 is expressed in dormant winter buds, inflorescence, young leaves, and tendril tips, but not in root tips. In young leaves, the expression of VvWOX4 is strongly upregulated by wounding, and also by plant growth regulators such as 2 mg L-1 2,4-D, 1 mg L-1 NAA and 1 mg L-1 BAP treatments, while downregulation was monitored by 1 mg L^-1 IBA treatment, and there was no response to 0.5 mg L-1 GA3 treatment. Together, our results revealed the first member of grape WOX gene family and indicated different roles and regulation of VvWOX4 in the perennial woody crop grapevine.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
Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA vp3. Restriction enzyme digestion and sequencing analysis revea...Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE TM mediated transfection in vitro with pcDNA vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L 02, and RT PCR was performed afterward. The results of RT PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment.展开更多
The purpose of this study is to find a new gene resource for the researches of molecular breeding of Rosaceae plants disease-resistance. Pyrus ussuriensis Maxim is used as a starting material to clone the full-length ...The purpose of this study is to find a new gene resource for the researches of molecular breeding of Rosaceae plants disease-resistance. Pyrus ussuriensis Maxim is used as a starting material to clone the full-length cDNA of NPR1(nonexpressor of pathogenesis- related genes 1) which is a key regulator in SA (salicylic acid)-mediated systemic acquired resistance (SAR) by homologous cloning and RACE techniques. The length of the cDNA sequence was 1 767 bp, the ORF was 1 761 bp, it coded 586 amino acids, pi=5.58, the relative molecular weight was 65.009 ku, contained 19 kinds of amino acids, and had full BTB/POZ and ANK domains. Compared the homology of NPR1 gene in GenBank database, the homology with Pyrus pyrifolia, Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Oryza sativa, Helianthus annuus were 98%, 62%, 68%, 65%, 57%, 63%. The homology offunctional area were 99%, 78%, 82%, 79%, 74%, 77%. This NPR1 gene was considered as homologic gene of Pyrus ussuriensis Maxim and named PumNPR1.展开更多
We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequenc...We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from GenBank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector pNZ8148 and yielding vector pNZ8148-BSH, pNZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol· min^-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.展开更多
To provide materials used in investigating the relationship between amino acid compositions of silk-like protein, structure, and functions, especially the biological functions, the motif genes encoding the silk fibroi...To provide materials used in investigating the relationship between amino acid compositions of silk-like protein, structure, and functions, especially the biological functions, the motif genes encoding the silk fibroin amorphous domain, SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG(F) were designed and extended using a "head-to-tail" construction strategy. The designed genes were cloned into PSLFA1180FA and multimerized to form structures containing a two-timer, a four-timer, an eight-timer, and a twelve-timer. All the resulting plasmids were digested using the restriction enzyme BamHI and the double-enzymes BglII/HindIII. Restriction enzyme analysis and DNA sequencing revealed the motif was successfully cloned into PSLFA1180FA and multimerized to form a twelve-timer without gene deletion or mutation.展开更多
It was found that the marine yeast strain YF07b could secrete a large amount of killer toxin against a pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculcttus. The marine yeast strain ...It was found that the marine yeast strain YF07b could secrete a large amount of killer toxin against a pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculcttus. The marine yeast strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The gene encoding killer toxin in the marine yeast strain YF07b was amplified by PCR technology. After sequencing, the results show that an open reading frame, consisting of 1 281 bp, encoded a presumed protein of 427 amino acids. The sequence of the cloned gene was found to have 99% match with that of the gene encoding killer toxin in Pichia anomalas strain K. A signal peptide including 17 amino acids appeared in the N-terminal domain of the killer toxin. Therefore, the mature protein consisted of 410 amino acids, its molecular mass was estimated to be 47.4 ku and its isoelctronic point was 4.5.展开更多
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The work was supported by the Hi-Tech Research and Development Program of China under contract Nos 2006AA09Z414 and 2007AA091903;the China Ocean Mineral Resources R & D Association under contract No. DYXM - 115 - 02 - 2 - 6;the National Natural Science Foundation of China under contract No. Z2004D02;the Natural Science Foundation of Shandong Province of China under contract No. Z2004D02;the Foundation for Young Excellent Scientists in Shandong Province of China under contract No. 2006BS02002;the Program for New Century Excellent Talents in University under contract No. NCET - 06 - 0578.
文摘Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR (GenBank accession Nos DQ640312, DQ504163 ). The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.
基金supported by the National Natural Science Foundation of China (31372023)the earmarked fund for China Agricultural Research System (CARS-30-yz-7)
文摘White-brown complex (WBC) transporters, also called half-size ATP binding cassette G (ABCG) transporters, are involved in many biological processes, including seed development; however, the WBC transporters in grapevines received less attention to date. To reveal the molecular characteristics of WBCs and the connection between WBCs and agronomic traits of stenospermocarpic (seedless) grapevine, we carried out a genomic census and analysis of ovule-associated expression for VvWBC genes in grapevine. We identified 30 VvWBC genes and cloned full-length complementary DNAs (cDNAs) for 20 of these. The tissue or organ-specific expression analysis showed that several VvWBCs exhibited distinct expression patterns with some showing tissue specificity. Twelve VvWBC genes were found to be expressed in the developing ovules. Moreover, the results of quantitative real-time PCR (qRT-PCR) suggested that four of twelve ovule-expressed VvWBCs have distinct expression profiles during the development of ovules between seeded and stenospermocarpic grapevines. These four genes might be involved in ovule abortion. Meanwhile, chromosome mapping, multiple sequence alignments, exon/intron structure analyses and synteny analyses were preformed on VvWBC genes. Our experiments provide a new perspective on the mechanism of stenospermocarpic seedlessness and put forward a framework for further study of WBC transporters.
文摘To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
基金Natural Science Foundation of Heilongjiang Province (C9912)
文摘Primers and probes were established according to the sequences of the alpha-amylase genes of Bacillus. halodurans C-125, Therrnus sp. IM6501, B. stearothermophilus ET-1, and B, acidopullulytics. Primers were designed and a 0.2 kb DNA fragment was amplified, the fragment was successfully used for the detection of the amylase Ⅱ gene in a 2 842 bp region from Bacillus halodurans strain 38C1-1.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityNational Natural Science Foundation of China(32073015)+1 种基金Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2024007)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.
基金This research was funded by National Natural Science Foundation of China,Grant Number(31871576)National Keypoint Research and Invention Program of the Thirteenth,Grant Number(2019YFD1002205)The APC was funded by National Keypoint Research and Invention Program of the Thirteenth.
文摘For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids were encoded.Bioinformatics analysis showed that the VrPR protein belonged to the NADB Rossmann superfamily,which was one of the non-transmembrane hydrophilic proteins.VrPR was assumed to have 44 amino acid phosphorylation sites and be contained in chloroplasts.The VrPR secondary structure comprised of random coil,αhelix,βangle,and extended chain,all of which were quite compatible with the anticipated tertiary structure.Moreover,analysis of the phylogenetic tree indicated that the soybean PR(Glyma.12G222200)and VrPR were closely related.Furthermore,chlorophyll content in leaves is markedly increased in Arabidopsis when VrPR is overexpressed.Ourfindings will serve as a reference for more functional studies on the PR genes in mung bean.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityGrants from Shenzhen Science and Technology Project(JCYJ20190813104207152)+3 种基金National Natural Science Foundation of China(32073015)Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2022005)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.
基金The National Natural Science Foundation of China under contract Nos 41176151 and 41276177the National High Technology Research&Development Program of China under contract No.2012AA100811the Funds for Distinguished Young Scientists of Fujian Province of China under contract No.2010J06016
文摘Fructose-1,6-bisphosphatase(FBPase) is one of the key enzymes in Calvin circle and starch biosynthesis. In this study, the full-length of cpFBPase gene from Pyropia haitanensis was cloned by using rapid amplification of cDNA ends(RACE) technology. The nucleotide sequence of PhcpFBPase consists of 1 400 bp, including a 5′ untranslated region(UTR) of 92 bp, a 3′?UTR of 69 bp, and an open reading frame(ORF) of 1 236 bp, which can be translated into a 412-amino-acid putative peptides with a molecular weight of 44.3 kDa and a theoretical pI of 5.23. Multiple sequence alignment indicated that the protein belonged to the chloroplast FBPase enzyme. Phylogenetic analysis showed that the protein assembled with the cpFBPase of a thermal tolerant unicellular red micro-algae Galdieria sulphuraria. Expression patterns analyzed by qRT-PCR revealed that the expression of PhcpFBPase gene in the thallus phage was 7-fold higher than in the conchocelis phage, which suggested the different mechanisms of inorganic carbon utilization among the different life phages of P. haitanensis. And the different response modes of PhcpFBPase mRNA levels to high temperature and desiccation stress indicated that PhcpFBPase played an important role in responsing to abiotic stress.
基金Shandong Provincial Natural Science Foundation,China under contract No. ZR2009EQ009Independent Innovation Foundation of Shandong University (IIFSDU)Key Lab of Marine Bioactive Substance and Modern Analytical Technique,SOA,China under contract No. MBSMAT-2009-07
文摘A total of 117 agar-decomposing cultures were isolated from coastal seawater around Qingdao, China. The phenotypic and agarolytic features of an agarolytic isolate, QM38, were investigated. The strain was gram negative, strictly aerobic, curved rod and polar flagellum. On the basis of several phenotypic characters, biochemical and morphological characters and phylogenetic analysis of the gene coding for the 16S rRNA, the strain was identified as Agarivorans albus strain QM38. This strain can liquefy the agar on the solid agar plate. An excellular agarase activity was determined in liquid culture. The enzyme exhibited maximal activity at 40 ℃, pH 7.6. Its activity was greatly affected by different concentrations of agarose. The highest activity 32 U/ml was achieved in the culture supernatant. The hydrolytic product was analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). After complete hydrolysis of agarose, a series of agaro-oligosaccharides were produced. The main products of the enzymes were oligosaccharides in the degree of polymerization (DP) of 2, 4, 6 and 8. Three genes agaD01, agaD02 and agaD03, encodingβ-agarases, had been cloned from genomic DNA of Agarivorans albus strain QM38. The open reading frame of agaDOl, consisted of 2 988 bp, and shared 95.5%-98.9% identity to the β-agarase genes of some strains of Vibrio and Agarivorans. Gene agaD02 comprised 2 868 bp and encoded a 955- amino-acid protein. It showed 97.4% and 98.7~0 identity to the β-agarase genes of strain Vibrio sp. PO-303 and strain Vibrio sp. JT0107, respectively. Only partial sequence of agaD03 gene has been cloned. It showed 96.5% identity to β-agarase gene (agaB) of Pseudoalteromonas sp. CY24, and shared 96.8% identity to β-agarase-c gene of Vibrio sp. PO-303.
基金supported by grants from the National Basic Research Program of China (2006CB708208,2006CB101901)the Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education of China (IRT0558)+1 种基金the National Natural Science Foundation of China (30930064)the 111Project from the Ministry of Education of China(B07049)
文摘Pathogenesis-related proteins (PRs) play many important roles in plant defense response against pathogen attack. To better understand the molecular mechanism of PR genes involved in wheat adult plant resistance (APR) to stripe rust, based on a differentially expressed transcribed derived fragment (TDF), a novel PR gene from wheat cv. Xingzi 9104 infected by the Puccinia striiformis Westend f. sp. tritici Erikss. pathotype CY32, which was highly similar to the maize ZmPRIO gene and designated as TaPRIO, was identified using in silico cloning and RT-PCR method. This novel TaPRIO gene was predicted to encode a 160-amino acid protein with a deduced molecular weight of 17.06 kDa and an isoelectronic point (pI) of 5.19. An amino acid sequence analysis of TaPR10 demonstrated the presence of a typical conserved domain of pathogenesis related protein Bet v I family. Multiple alignment analysis based on the amino acids encoded by 10 different PRIO genes from maize (Zea mays), rice (Oryza sativa), broomcorn (Sorghum bicolor), and wheat (Triticum aestivum) indicated that PR proteins of class 10 was conserved among the 4 plant species with about 80% similarity. DNA sequence of TaPRIO suggested the presence of one 84-bp intron with the splicing sites of GT-AT bi-nucleotide sequence between 188 and 271 bp. Using a real-time quantitative RT-PCR (qRT-PCR), expression profiles of TaPRIO revealed that at the adult-plant stage, TaPRIO transcript was up-regulated as early as 12 h post-inoculation (hpi), with the occurrence of maximum induction at 24 hpi. At the seedling stage, TaPRIO was also slightly induced 18 hpi. However, the transcript amount was relatively lower than that of the adult-plant stage. Taken together, these results suggest that TaPRIO may participate in wheat defense response of APR to stripe rust.
基金supported by the National Natural Science Foundation of China (Grant Nos.30770132 and 30570103)
文摘Ethylene response factors (ERFs) play important roles in response to plant biotic and abiotic stresses. In this study, a gene encoding a putative AP2/ERF domain-containing protein was isolated by screening a SSH cDNA library from rice and designated as Oryza sativa AP2/ERF-like protein (OsAP2LP) gene. OsAP2LP is 1491 bp in length, interrupted by seven introns, and encodes a putative protein of 348 amino acids. Temporal and spatial expression analysis showed that the OsAP2LP gene was preferentially expressed in roots, panicles, mature embryos and seeds in rice. Real-time quantitative PCR analysis indicated that the expression levels of the OsAP2LP gene were increased under the treatments of drought and gibberellin but decreased under the treatments of low temperature, salt, abscisic acid (ABA) and zeatin. Taken together, these results suggest that OsAP2LP might be involved in stress responses, and probably plays roles as a transcription regulator when plants response to cold, salt and drought stresses through ABA and gibberellin pathways.
基金Project supported by"Tenth Five Years"Key Program of the State Science and Technology Commission in China(Grant Nos.2002BA901A15,2004BA411B01)
文摘Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase (SAD) is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame (ORF). Analysis in the BLAST on NCBI shows that Jatropha curcas SAD (JSAD) gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD (RSAD) is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.
基金financially supported by the National Natural Science Foundation of China (30471212,30500347)the Beijing Natural Science Foundation(5992011)
文摘Wuschel-related homeobox (WOX) genes play essential, specific, and sometimes redundant roles in plant embryo development, shoot and root meristem maintenance, and plant development. Though much information was quickly gained with members of the WOX gene family of Arabidopsis, monocotyledonous crops, and gymnospermous conifers, little is known about perennial woody plants. In this study, we isolated the first WOXgene family member from grape (Vitis vinifera L. cv. Cabernet Sauvignon), and named it VvWOX4 based on its characteristic domains and phylogenetic analysis. The identity of VvWOX4 was validated by MALDI-TOF MS and Western blot with polyclonal antibody against Arabidopsis thaliana Wuschel. Functional analysis showed that VvWOX4 markedly increased shoot primordia structures when overexpressed under CaMV 35S promoter in tobacco. A different expression pattern was found for VvVOX4 compared with AtWUCHEL and its expression was detected in unique organs of grapevines. Besides the expression in the vegetative shoot apical meristem (SAM) of grape shoot tips, VvWOX4 is expressed in dormant winter buds, inflorescence, young leaves, and tendril tips, but not in root tips. In young leaves, the expression of VvWOX4 is strongly upregulated by wounding, and also by plant growth regulators such as 2 mg L-1 2,4-D, 1 mg L-1 NAA and 1 mg L-1 BAP treatments, while downregulation was monitored by 1 mg L^-1 IBA treatment, and there was no response to 0.5 mg L-1 GA3 treatment. Together, our results revealed the first member of grape WOX gene family and indicated different roles and regulation of VvWOX4 in the perennial woody crop grapevine.
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ThisprojectwassupportedbyagrantfromtheHubeiNaturalScienceFoundation (No .2 0 0 2ABA0 0 4 )
文摘Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE TM mediated transfection in vitro with pcDNA vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L 02, and RT PCR was performed afterward. The results of RT PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment.
文摘The purpose of this study is to find a new gene resource for the researches of molecular breeding of Rosaceae plants disease-resistance. Pyrus ussuriensis Maxim is used as a starting material to clone the full-length cDNA of NPR1(nonexpressor of pathogenesis- related genes 1) which is a key regulator in SA (salicylic acid)-mediated systemic acquired resistance (SAR) by homologous cloning and RACE techniques. The length of the cDNA sequence was 1 767 bp, the ORF was 1 761 bp, it coded 586 amino acids, pi=5.58, the relative molecular weight was 65.009 ku, contained 19 kinds of amino acids, and had full BTB/POZ and ANK domains. Compared the homology of NPR1 gene in GenBank database, the homology with Pyrus pyrifolia, Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Oryza sativa, Helianthus annuus were 98%, 62%, 68%, 65%, 57%, 63%. The homology offunctional area were 99%, 78%, 82%, 79%, 74%, 77%. This NPR1 gene was considered as homologic gene of Pyrus ussuriensis Maxim and named PumNPR1.
基金Supported by the National Natural Science Fund Project(31171657)Heilongjiang Province Natural Fund Project(ZD201207)Heilongjiang Province Postdoctoral Special Funds(LBH-Q13133)
文摘We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from GenBank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector pNZ8148 and yielding vector pNZ8148-BSH, pNZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol· min^-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.
基金National Natural Science Foundation of China(No. 51173125)Natural Science Foundations of Jiangsu Province of China(No. BK2010253,No. BK2012633)+2 种基金College Natural Science Research Project of Jiangsu Province of China(No. 12KJA43004)Science and Technology Plan Foundation of Suzhou of China(No. ZXS2012002)Priority Academic Program Development of Jiangsu Higher Education Institutions,China
文摘To provide materials used in investigating the relationship between amino acid compositions of silk-like protein, structure, and functions, especially the biological functions, the motif genes encoding the silk fibroin amorphous domain, SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG(F) were designed and extended using a "head-to-tail" construction strategy. The designed genes were cloned into PSLFA1180FA and multimerized to form structures containing a two-timer, a four-timer, an eight-timer, and a twelve-timer. All the resulting plasmids were digested using the restriction enzyme BamHI and the double-enzymes BglII/HindIII. Restriction enzyme analysis and DNA sequencing revealed the motif was successfully cloned into PSLFA1180FA and multimerized to form a twelve-timer without gene deletion or mutation.
基金This research was supported by the National Natural Science Foundation of China under contract No.30670058
文摘It was found that the marine yeast strain YF07b could secrete a large amount of killer toxin against a pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculcttus. The marine yeast strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The gene encoding killer toxin in the marine yeast strain YF07b was amplified by PCR technology. After sequencing, the results show that an open reading frame, consisting of 1 281 bp, encoded a presumed protein of 427 amino acids. The sequence of the cloned gene was found to have 99% match with that of the gene encoding killer toxin in Pichia anomalas strain K. A signal peptide including 17 amino acids appeared in the N-terminal domain of the killer toxin. Therefore, the mature protein consisted of 410 amino acids, its molecular mass was estimated to be 47.4 ku and its isoelctronic point was 4.5.