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Cost-Effective Method of Gene Synthesis by Sequencing from Microchip-Derived Oligos for Droplet Cloning
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作者 Kimberly Wang 《Advances in Bioscience and Biotechnology》 CAS 2024年第8期474-485,共12页
Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthes... Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy. 展开更多
关键词 COST-EFFECTIVE gene Synthesis MICROCHIP Oligo Droplet cloning
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Gene Cloning and Bioinformatics Analysis of phoR Gene from Vibrio alginolyticus HY9901
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作者 Xiangyu LIU Peng ZHOU +4 位作者 Haiyun FENG Weijie ZHANG Huanying PANG Na WANG Xiaonan LU 《Asian Agricultural Research》 2024年第6期36-40,共5页
PhoR is a histidine kinase in a two-component regulatory system that regulates phosphorus metabolic pathways and undertakes the key mission of information transmission in pathogenic bacteria.The full-length phoR gene ... PhoR is a histidine kinase in a two-component regulatory system that regulates phosphorus metabolic pathways and undertakes the key mission of information transmission in pathogenic bacteria.The full-length phoR gene was successfully cloned from the Vibrio alginolyticus HY9901 strain.A comprehensive analysis of the cloned gene was conducted using bioinformatics.Sequence analysis revealed that the total length of the phoR gene(GenBank accession No.:KJ958404.1)is 1299 bp,with the coding region containing a total of 432 amino acid residues.The phylogenetic tree of PhoR revealed that it belongs to the same subclade as V.diabolicus.The SMART program was employed for the purpose of functional domain prediction,which revealed that PhoR possesses three major functional domains:PAS(amino acids 98-166),HisKA(amino acids 205-272),and HATPase_c(amino acids 317-429). 展开更多
关键词 VIBRIO ALGINOLYTICUS phoR gene gene cloning BIOINFORMATICS analysis
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Molecular Cloning of sodB Gene from Vibrio alginolyticus HY9901 and Its Bioinformatics Analysis
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作者 Shuai YANG Yingying JIANG +4 位作者 Haiyun FENG Weijie ZHANG Na WANG Xiaonan LU Huanying PANG 《Asian Agricultural Research》 2024年第7期42-47,共6页
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
关键词 Vibrio alginolyticus gene cloning sodB gene Bioinformatics analysis
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Molecular Cloning and Bioinformatics Analysis of msrA Gene from Vibrio alginolyticus Strain HY9901
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作者 Zhiqing WEI Zhihang CHEN +2 位作者 Yingzhu WEI Na WANG Huanying PANG 《Agricultural Biotechnology》 2024年第4期1-5,10,共6页
[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of m... [Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of msrA,and its bioinformatics analysis was carried out.[Results]The full length of msrA gene was 639 bp,encoding 212 amino acids,and its theoretical molecular weight was about 23729.60 Da.The protein had a stable structure,and it was hydrophobic overall.The structure of signal peptides at the N terminal of the amino acid sequence was predicted,and it was found that there was no signal peptide cleavage site and no transmembrane region.The amino acid sequence of MsrA contained multiple signal binding sites.Protein subcellular localization showed that MsrA protein was most likely located in the cytoplasm.Homology analysis showed that MsrA of V.alginolyticus had high homology with other Vibrio species,and the highest homology with V.alginolyticus.In the prediction of functional domains,MsrA had the function of methionine sulfoxide reduction.In secondary structure prediction,MsrA contained random coils at a proportion of 46.70%,which was the highest.The similarity between the tertiary structure model of MsrA and template Q87SW6.1.A was 89.15%.PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites.[Conclusions]This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress. 展开更多
关键词 Vibrio alginolyticus gene cloning MSRA Bioinformatics analysis
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The Influence of Aerial Exposure on Sea Anemones Aulactinia veratra Mucin Genes Expression Using the RNA Sequencing
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作者 Alaa Haridi 《Open Journal of Marine Science》 2024年第1期1-18,共18页
Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air duri... Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air during the low tide and produces large quantities of mucus as an external covering. The relation between low tide and mucus secretion is still unclear, and what is the role of mucin during arial exposure is not yet investigated. This study hypothesised that the mucin genes in A. veratra would have significantly high expression in response to aerial exposure. Therefore, the aim of current study was to examine and analyses the response of A. veratra mucins in response to an experiment involving three hours of aerial exposure. To achieve this, aim the RNA-sequencing and bioinformatics analyses were used to examine the expression profile of A. veratra mucin genes in response to aerial exposure. The generated results have shown that, Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra. This finding shows a significant role of mucin5B-like and mucin4-like genes in response to air stress at low tide. The data generated from this study could be used in conjunction with future mucin gene studies of sea anemones and other cnidarians to compare A. veratra mucin gene expression results across time, and to extend our understanding of mucin stress response in this phylum. 展开更多
关键词 Aulactinia veratra Mucin4-Like Mucin5B-Like CNIDARIA Mucin gene Expression RNA-Sequencing sea Anemones MUCUS Aerial Exposure
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Research Progress of Medicinal Secondary Metabolites and Gene Cloning of Dendrobium officinale 被引量:9
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作者 Ye LIANG Caihua ZOU +2 位作者 Jindu LI Shilong LI Tianzi LI 《Medicinal Plant》 CAS 2019年第3期16-18,23,共4页
Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient co... Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient content and high cost,limiting the use of D. officinale.Studying the network structure and rate-limiting steps of secondary metabolites of medicinal components of D. officinale,analyzing the secondary metabolic synthesis process,mastering the production rules of its medicinal components and carrying out gene cloning or biosynthesis,etc.are of great significance for the rational development and utilization of D. officinale resources. This paper briefly reviews the progress of the research on the secondary metabolites of D. officinale,including the detection and identification of metabolites and the identification and cloning of key metabolic enzymes. 展开更多
关键词 DENDROBIUM officinale SECONDARY METABOLITES MEDICINAL resources gene cloning
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Characterization of Caspase Gene Family Members in Spotted Sea Bass(Lateolabrax maculatus)and Their Expression Profiles in Response to Vibrio harveyi Infection
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作者 YANGLANG Arat WEN Haishen +7 位作者 MAO Xuebin TIAN Yuan WANG Lingyu LI Jinku QI Xin SRISAPOOME Prapansak LI Jifang LI Yun 《Journal of Ocean University of China》 SCIE CAS CSCD 2023年第5期1370-1382,共13页
The caspase gene family is a crucial gene cluster that regulates apoptosis which contribute to programmed cell death,cell proliferation and differentiation,and several immune responses.In our study,a complete set of 1... The caspase gene family is a crucial gene cluster that regulates apoptosis which contribute to programmed cell death,cell proliferation and differentiation,and several immune responses.In our study,a complete set of 12 caspase genes were identified in spotted sea bass Lateolabrax maculatus.These genes were divided into three subfamilies:2 inflammatory caspases(casp-1 and casp-14-like),5 apoptosis initiators(casp-2,casp-8a,casp-8b,casp-9,and casp-10),and 5 apoptosis executioners(casp-3a,casp-3b,casp-3-like,casp-6,and casp-7).Their phylogenetic relationships,synteny and gene structures were systematically analyzed.Furthermore,the relative expression profiles of the caspase family members in the liver,intestine,head kidney,and spleen were measured by q PCR after infection with Vibrio harveyi.The results showed that the overall mRNA levels of the caspase genes were dramatically increased after V.harveyi infection,and the expression patterns varied among genes and tissues.More caspase genes underwent pronounced expression changes in the head kidney and spleen than in the liver or intestine,mainly after 48 h of the challenge.Specifically,casp-3a,casp-3b,casp-3-like,casp-6,casp-7,casp-8a,casp-8b,casp-10,and casp-14-like in the head kidney,and casp-3-like,casp-6,casp-7,and casp-14-like in the spleen,were the most responsive caspase genes which may contribute significantly to immune regulation in spotted sea bass.Additionally,the apoptosis level in head kidney and spleen after infection were examined using the Caspase assay.Our study provides a systemic overview of the caspase gene family in spotted sea bass after V.harveyi infection and lays a foundation for further deciphering the biological roles of these caspase genes. 展开更多
关键词 caspase gene family spotted sea bass Vibrio harveyi gene expression APOPTOSIS
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Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913 被引量:1
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作者 ZHAO Dianli CHEN Xiulan HE Hailun SHI Mei ZHANG Yuzhong 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2007年第6期91-100,共10页
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
关键词 COLD-ADAPTED chaperone DNAJ DNAK DEEP-sea Pseudoalteromonas sp. SM9913 gene cloning sequence analysis
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Cloning and Functional Validation of Mung Bean VrPR Gene
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作者 Xiaokui Huang Yingbin Xue +3 位作者 Aaqil Khan Hanqiao Hu Naijie Feng Dianfeng Zheng 《Phyton-International Journal of Experimental Botany》 SCIE 2023年第8期2369-2382,共14页
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
关键词 Mung bean gene cloning VrPR transgenic arabidopsis functional verification
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Cloning and Bioinformatics Analysis of vscB Gene of T3SS Chaperone of Vibrio alginolyticus
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作者 Hongwei ZHENG Liangchuan CHEN +5 位作者 Haiyun FENG Yunsheng CHANG Yu DING Weijie ZHANG Huanying PANG Na WANG 《Asian Agricultural Research》 2023年第4期37-39,46,共4页
[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
关键词 Vibrio alginolyticus gene cloning vscB Bioinformatics analysis
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Defect in an immune regulator gene BrSRFR1 leads to premature leaf senescence in Chinese cabbage
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作者 Yue Xin Gengxing Song +1 位作者 Chong Tan Hui Feng 《Horticultural Plant Journal》 SCIE CAS CSCD 2024年第6期1414-1423,共10页
Leaf senescence is the final stage of leaf development, where the nutrients and energy of senescent leaves are redistributed to developing tissues or organs for plant growth, reproduction, and defense. Outer leaves ar... Leaf senescence is the final stage of leaf development, where the nutrients and energy of senescent leaves are redistributed to developing tissues or organs for plant growth, reproduction, and defense. Outer leaves are photosynthetic organs that usually senesce at the late heading stage in Chinese cabbage, and premature leaf senescence often reduces leafy head yield and quality. In this study, 11 premature leaf senescence mutants were screened from an ethyl methanesulfonate-mutagenized population of the double haploid line ‘FT' in Chinese cabbage. At the early heading stage, the mutants exhibited edge yellowing within its outer leaves, and at the mature stage, its leafy head weight decreased significantly. Genetic analysis revealed that the mutated trait of all 11 mutants corresponds to single gene recessive inheritance. Semi-diallel cross tests showed that 5 of the 11 were allelic mutants. MutMap and Kompetitive Allele Specific PCR genotyping revealed that BraA01g001400.3C was the candidate gene, which is orthologous of Arabidopsis SUPPRESSOR OF rps4-RLD 1, encoding an immune regulator, so we named it as BrSRFR1. All the BrSRFR1 in the five allelic mutants exhibited single nucleotide polymorphisms at different positions on their exons and led to premature translation termination, which confirmed that defect in BrSRFR1 led to premature leaf senescence. These results verify the role of Br SRFR1 on leaf senescence and provide a new insight into the mechanisms of leaf senescence in Chinese cabbage, which reveals a novel function of SRFR1 in plant development. 展开更多
关键词 Chinese cabbage Premature leaf senescence SRFR1 gene cloning
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Cloning and Characterization of a Family of Disease Resistance Gene Analogs from 6VS of Haynaldia villosa
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作者 KONG Fan-jing, MA You-zhi, CHEN Xiao and XIN Zhi-yong(Institute of Crop Breeding and Cultivation , Chinese Academy of Agricultural Sciences , Beijing 100081,P. R. China Open Laboratory of Saline Lake Resources and Environment of Ministryof Land and Resources , Beijing 100037 , P.R. China) 《Agricultural Sciences in China》 CAS CSCD 2003年第8期937-942,共6页
In the present study, microdissection of 6VS and the cloning of the resistance gene analogs(RGA)from them were reported. The 6VS were microdissected with needle and 10 types of resistance gene analogs were obtained by... In the present study, microdissection of 6VS and the cloning of the resistance gene analogs(RGA)from them were reported. The 6VS were microdissected with needle and 10 types of resistance gene analogs were obtained by PCR with degenerate oligonucleotide primer designed according to resistance genes. They were designated as Hvrgak1-Hvrgak10, GenBank accession numbers are AF387113-AF387121, AY040671- AY040672. Identity among RGAs was about 10-50%, and identity with cloned R gene from plants was 5-20%. Southern hybridization analysis results showed 3 RGAs, Hvrgak2, Hvrgak4, and Hvr-gak5 were linked with wheat powdery mildew resistance. These RGAs may be used as direct entrance or probes for cloning the disease resistance genes. 展开更多
关键词 6VS of Haynaldia Villosa MICRODISSECTION Resistance gene analogs(RGA) cloning
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Cloning and Identification of L-meq Gene of Marek's Disease Virus
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作者 GAO Ye ,WANG Xiao-ju,ZHENG Mei-zhu College of Chemistry,Changchun Normal University,Changchun 130032,China 《Animal Husbandry and Feed Science》 CAS 2012年第2期91-92,共2页
[Objective]To clone and identify Marek's disease virus ( MDV) serum gene. [Method]MDV genomic DNA was extracted from lymphoid tissues of the diseased chickens with latent MDV infection. The MDV L-meq gene was ampli... [Objective]To clone and identify Marek's disease virus ( MDV) serum gene. [Method]MDV genomic DNA was extracted from lymphoid tissues of the diseased chickens with latent MDV infection. The MDV L-meq gene was amplified by gradient PCR,inserted into pMD18-T vector and sequenced. The sequence was analyzed using DNAman software. [Result] The obtained sequence had 100% similarity with the published se- quence of L-meq gene,showing successful amplification of target gene. [Conclusion]The paper provides new ideas and new methods for preven- tion and treatment of Marek's disease in chickens. 展开更多
关键词 Marek's disease L-meq gene AMPLIFICATION cloning
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Molecular Cloning of a Chitinase Gene from the Ovotestis of Kuroda’s Sea Hare Aplysia kurodai
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作者 Gaku Matsunaga Syuuji Karasuda +2 位作者 Ryo Nishino Hideto Fukushima Masahiro Matsumiya 《Advances in Bioscience and Biotechnology》 2016年第1期38-46,共9页
In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a sys... In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a system for the 5’ and 3’ rapid amplification of cDNA ends, we obtained a 1352 bp chitinase gene (AkChi) from the ovotestis of A. kurodai. AkChi contains a 1263 bp open reading frame that encodes 421 amino acids. The domain structure predicted from the deduced amino acid sequence was an N-terminal signal peptide and a catalytic domain of glycoside hydrolase (GH) family 18 chitinase. A comparative analysis of the deduced amino acid sequences of AkChi with those of the acidic mammalian chitinase of the California sea hare Aplysia californica revealed the highest homology at 83%. The purified chitinase from the ovotestis was digested by trypsin, and 119 residues of digested peptides were consistent with the deduced amino acid sequence of AkChi. We used RT-PCR to evaluate the expression of AkChi in various tissues of A. kurodai, and we observed that AkChi was expressed only in the ovotestis. A phylogenetic tree analysis, performed using the amino acid sequences of AkChi and known GH family 18 chitinases, showed that AkChi was separated from the molluscan chitinases with a chitin binding domain. To our knowledge, this is the first study demonstrating the cDNA cloning of an ovotestis chitinase from a sea hare. 展开更多
关键词 CHITINASE Molecular cloning Kuroda’s sea Hare Aplysia kurodai MOLLUSC OVOTESTIS Phylogenetic Tree Analysis
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Cloning and Prokaryotic Expression of FnBP Ligand Binding Gene of Staphylococcus aureus 被引量:3
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作者 尹荣兰 杨正涛 +5 位作者 张艳晶 刘辉 刘珊 杨琦 曹永国 张乃生 《Agricultural Science & Technology》 CAS 2008年第6期43-46,共4页
[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding... [Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells. 展开更多
关键词 STAPHYLOCOCCUS aureus FNBP ligand binding gene cloning PROKARYOTIC expression
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Cloning and Bioinformatics Analysis of P23 Gene from Theileria sergenti 被引量:5
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作者 金春梅 张守发 于龙政 《Agricultural Science & Technology》 CAS 2008年第3期56-58,84,共4页
[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its g... [Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti. 展开更多
关键词 THEILERIA sergenti P23 gene cloning BIOINFORMATICS
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Cloning of Potato POTHR-1 Gene and Its Expression in Response to Infection by Phytophthora infestans and Other Abiotic Stimuli 被引量:8
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作者 田振东 柳俊 +1 位作者 谢从华 宋波涛 《Acta Botanica Sinica》 CSCD 2003年第8期959-965,共7页
A complete cDNA of potato Phytophthora infestans-induced hypersensitive response-related protein gene (POTHR-1) was cloned using rapid amplification of cDNA ends (RACE) strategy according to a fragment sequence which ... A complete cDNA of potato Phytophthora infestans-induced hypersensitive response-related protein gene (POTHR-1) was cloned using rapid amplification of cDNA ends (RACE) strategy according to a fragment sequence which we had cloned using suppression subtractive hybridization (SSH) technique. The potato POTHR-1 gene encodes a protein of 225 amino acids, which shares 81% identity with tobacco hin1 gene-enoded protein (harpin-induced protein). Southern blot revealed that there are two to three copies of POTHR-1 in potato genome. The POTHR-1 gene expression in potato leaves showed that its transcripts accumulated remarkably in leaves after 36 h inoculation with P. infestans. Mechanical wounding and jasmonic acid (JA) could induce the POTHR-1 gene expression and osmotic stress just induce a slight accumulation of POTHR-1 gene mRNA, while salicylic acid (SA) had no detectable function on the induction accumulation of POTHR-1 gene transcripts. The potato POTHR-1 gene may preferentially associate with hypersensitive response (HR) or biotic cell death during interaction between host and pathogen. 展开更多
关键词 cDNA cloning POTHR-1 gene POTATO Phytophthora infestans
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Molecular Cloning and Expression of RSSG58 Gene in Rice Sperm Cells 被引量:3
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作者 苗琛 苟小平 +4 位作者 兰利琼 鲍锦库 徐莺 王胜华 陈放 《Acta Botanica Sinica》 CSCD 2003年第2期234-241,共8页
Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive proce... Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive processes. In this study we report the rice ( Oryza sativa L.) RSSG58 gene, which was cloned from the cDNA library of rice sperm cells by using sperm cell mainly expression subtractive clone as probe. This gene encodes a putative 66.7 W polypeptide, which shows similarity to the myosin heavy chain of Arabidopsis thaliana, and consists of 579 amino acids with an isoelectric point (pI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 bp and 2 437 bp that differ only in their polyadenylation sites. Southern hybridization showed that RSSG58 has only one copy in rice genome and RSSG58 transcripts are most abundant in sperm cells, with two distinct signals. The RT-PCR analysis indicated that the transcriptions of the RSSG58 gene were various in the different development stages and tissues. The greatest accumulation of RSSG58 mRNA was detected in sperm cells, while weaker expression was detected in leaves, microspore mother cells, unicellular microspore pollen stage, two-cell stage pollens, mature pollens and pollinated ovaries. These results suggest that RSSG58 is especially abundantly expressed in rice sperm cells. 展开更多
关键词 molecular cloning RSSG58 gene sperm cell EXPRESSION RICE
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Cloning of Rabbit Bone Morphogenetic Protein 15 and Its Expression During in vitro Maturation of Rabbit Oocytes 被引量:3
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作者 尹萍 季金强 +1 位作者 李霖 丁家桐 《Zoological Research》 CAS CSCD 北大核心 2008年第6期603-607,共5页
Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15... Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation (IVM) was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits. 展开更多
关键词 RABBIT Bone morphogenetic protein 15 OOCYTE gene cloning Fluorescent quantitative RT-PCR
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Cloning and Analysis of WRKY Gene of Rice Induced by Rhizoctonia solani Kuhn 被引量:3
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作者 姜述君 马建 +4 位作者 范文艳 戴凌燕 张国庆 于涵 刘朝 《Agricultural Science & Technology》 CAS 2011年第2期191-194,共4页
[Objective] The aim was to clone the up-regulated expression gene of rice induced by Rhizoctonia solani.[Method] The EST fragment K16 obtained by suppression subtraction hybridization(SSH)was cloned and confirmed by... [Objective] The aim was to clone the up-regulated expression gene of rice induced by Rhizoctonia solani.[Method] The EST fragment K16 obtained by suppression subtraction hybridization(SSH)was cloned and confirmed by reverse transcription-polymerase chain reaction(RT-PCR).Then RT-PCR products were cloned into the PMD18-T vector and sequenced.The functions of the sequence were predicted with bioinformatics method.[Result] A 1 079 bp gene was obtained.The gene encoded a protein with 236 amino acids.The protein contains many motif sites,two WRKY domains and a C2H2 zinc finger motif.The gene showed high identities with WRKY8,WRKY24 and WRKY30 gene of rice.[Conclusion] The up-regulated expression gene induced by R.solani was representative WRKY family gene.The gene could play an important role on rice sheath blight resistance. 展开更多
关键词 RICE Rhizoctonia solani Kuhn Silico cloning WRKY gene
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