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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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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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Establishment of Fluorescence Quantitative RT-PCR Assay for Detection of Equine Arteritis Virus
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作者 Wang Keke Liang Xinxin +7 位作者 Jiang Gangqiang Long Zhixin Hudusi Aierken Liu Zhiling Wang Yan Wu Xiaowei Xiao Yuanyuan Bai Meihua 《Animal Husbandry and Feed Science》 CAS 2024年第1期21-25,共5页
[Objective]The paper was to establish a fluorescence quantitative RT-PCR method for detection of equine arteritis virus(EAV).[Method]Primers and probes were developed for the EAV ORF7 gene sequence,and the reaction sy... [Objective]The paper was to establish a fluorescence quantitative RT-PCR method for detection of equine arteritis virus(EAV).[Method]Primers and probes were developed for the EAV ORF7 gene sequence,and the reaction system was optimized.Standard curves were established,leading to the initial development of the EAV fluorescence quantitative RT-PCR assay.The accuracy,specificity,and sensitivity of this method were subsequently evaluated.[Result]The EAV fluorescence quantitative RT-PCR assay demonstrated optimal performance at an annealing temperature of 61 C,with a final concentration of primer and probe set at 0.6μmol/L.The plasmid standard demonstrated a strong linear correlation with Ct values within the range of 1.6×10^(7)-1.6×10^(2)copies/μL.The equation of the standard curve was determined to be y=-2.68x+32.88,with an R^(2) value of 0.9927.Consequently,the EAV fluorescence quantitative RT-PCR assay was successfully established.The methodology employed was effective in detecting EAV,Theileria equi,equine herpesvirus-1(EHV-1),equine herpesvirus-4(EHV-4),and equine influenza virus(EIV).The findings indicated that the method was specifically capable of detecting EAV,while the other pathogens tested yielded negative results.The method demonstrated a high degree of specificity.It was employed to detect the standard plasmid cRNA synthesized through in vitro transcription following a 10-fold dilution.The results indicated that the minimum detection limit of the method was 1.6×10^(2) copies/μL,and it exhibited high sensitivity.The coefficient of variation,both within and between groups,was maintained at 1.8%,indicating good reproducibility.In this study,the fluorescence quantitative RT-PCR assay developed was utilized alongside the EAV fluorescence quantitative RT-PCR assay established by previous researchers to analyze a total of 234 clinical samples.Both methods yielded a positive detection rate of 14.1%,and the coincidence rate between the two techniques was found to be 100%.[Conclusion]The fluorescence quantitative RT-PCR assay developed in this study offers a novel approach and concept for the prevention and control of equine viral arteritis(EVA). 展开更多
关键词 Equine arteritis virus(EAV) ORF7 gene Fluorescence quantitative rt-pcr
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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 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 of Potato POTHR-1 Gene and Its Expression in Response to Infection by Phytophthora infestans and Other Abiotic Stimuli 被引量:7
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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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Study on the Cloning and Isolation of sus scrofa GPX2 Gene by RACE Method 被引量:2
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作者 赵华 周继昌 +2 位作者 李俊刚 赵莹 王康宁 《Agricultural Science & Technology》 CAS 2008年第1期24-28,共5页
[Objective] Using molecular biotechnology to clone the sus scrofa GPX2 gene. [Method] Using total RNA of sus scrofa duodenum as template, degenerated primer pairs were designed according to the homology alignment anal... [Objective] Using molecular biotechnology to clone the sus scrofa GPX2 gene. [Method] Using total RNA of sus scrofa duodenum as template, degenerated primer pairs were designed according to the homology alignment analysis of GPX2 gene of human, rat, mouse, dog and cattle. A sus scrofa GPX2 gene sequence of 330 bp was obtained by RT-PCR application method. Primes were designed respectively according to the known sequence, sus scrofa GPX2 gene was isolated and cloned by 3-RACE and 5-RACE method and analyzed the gene sequence. [Result] A mRNA sequence of 924 bp was successfully cloned and isolated in this research. This sequence contained complete 3'end and had higher sequence homology with human,mouse,cattle and dog GPX2 gene, and there was codon called TGA which encoding Sec on the position of No. 114-116 gene. [Conclusion] Sequence alignment analysis showed that the cloned gene was sus scrofa GPX2 gene ( NCBI GenBank database, the sequence number was D098982). 展开更多
关键词 gene clone sus scrofa GPX2 RACE rt-pcr
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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 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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Cloning, Characterization and Chromosome Localization of Two Powdery Mildew Resistance-Related Gene Sequences from Wheat 被引量:4
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作者 于玲 牛吉山 +3 位作者 马正强 陈佩度 齐莉莉 刘大钧 《Acta Botanica Sinica》 CSCD 2002年第12期1438-1444,共7页
Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe gramin... Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe graminis , and degenerate primers designed based on the conserved amino acid sequences of known plant disease_resistance genes. The cDNA sequences encoding cyclophilin_like and H +_ATPase_like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H +_ATPases isolated from other plants. Thus they were designated as Ta_Cyp and Ta_MAH . The obvious expression differences could be observed between wheat_ H. villosa 6VS/6AL translocation line and susceptible wheat cultivar 'Yangmai 5', implying that the two genes may be related with the resistance of wheat_ H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta_Cyp gene and one copy of the Ta_MAH gene. Chinese Spring nulli_tetrasomic line analysis located the Ta_Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta_Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa , amphiploid of Triticum durum _ H. villosa , wheat_ H. villosa 6VS/6AL translocation line and 'Yangmai 5', suggesting that Ta_Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa . 展开更多
关键词 cloning wheat_ Haynaldia villosa 6VS/6AL translocation line cyclophilin gene H +_ATPase gene
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Molecular Cloning and Sequence Analysis of Class Ⅱ Chitinase Gene in Leymus chinensis 被引量:5
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作者 金华 安晓雯 姜国斌 《Agricultural Science & Technology》 CAS 2009年第4期96-100,共5页
[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the ... [ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the biological function and application of chitinasa gene. [ Method] cDNA library of Leymus chinensis leaves were constructed, and its DNA sequence was determined or analyzed, while the homology of chitinasa gene and amino acid sequence was compared with that in GenBank. [ Result] One full length cDNA fragment with length of 996 bp was cloned from cDNA library of Leymus chinensis leaves. The length of ORF was 768 bp encoding 225 amino acids (GenBank accession number: EU344908). The encoding products lacked CBD and C-terminal extension region from the view of structure, but had structural characters of Class Ⅱ chitinase gene, which indicated that amino acid sequence had high homology compared with Class Ⅱ chitinase gene of rye and wheat. The constructed recombinant vector pQE-LcChi2 could express a protein of 27 kD through induction, which was consistent with the deduced encoding product of pQE-LcChi2 gene. [ Conclusion] LcChl2 gene is an expression gene, which can express in E. coll. 展开更多
关键词 Leymus chinensis Chitinase gene cloning Sequence analysis
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Cloning and Expression of a Profilin Gene from Rapeseed 被引量:4
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作者 叶秋 李旭锋 +3 位作者 徐莺 王劲 林娟 陈放 《Acta Botanica Sinica》 CSCD 2001年第7期727-730,共4页
Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA clone (designated Repro) encoding profilin gene was isolated from rapeseed ( Brassica napus L. cv. canadian Tween) using RT-PC... Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA clone (designated Repro) encoding profilin gene was isolated from rapeseed ( Brassica napus L. cv. canadian Tween) using RT-PCR technique. Sequence analysis showed 82% similarity to Zea mays L. ZmPro3, 85% to Arabidopsis AthPRF1, 82% to Nicotiana tabacum L. NTPRO, 81% to Oryza sativa L. profilin A. A new full-length cDNA was obtained by 5'-RACE and 3'-RACE techniques. Sequence analysis showed that the size of full-length cDNA is 672 bp which contains a major open reading frame of 134 amino, acids, 5' and 3' untranslated regions and a long Poly (A) tail. Northern blot analysis showed that the profilin gene is a pollen and anther specific gene. 展开更多
关键词 profilin gene 3 '-RACE 5 '-RACE rapeseed pollen rt-pcr
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Cloning and Bioinformatics Analysis of Lfcin Gene of Datong Yak 被引量:2
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作者 裴杰 阎萍 +6 位作者 姬国红 冯瑞林 梁春年 郭宪 曾玉峰 包鹏甲 褚敏 《Agricultural Science & Technology》 CAS 2009年第3期50-54,共5页
[Objective] This study was to clone Lfcin gene from Datong yak, so as to provide reference for applying this gene in feed industry and breeding industry. [Method] Using PCR technology, the lactoferricin(Lfcin)-encodin... [Objective] This study was to clone Lfcin gene from Datong yak, so as to provide reference for applying this gene in feed industry and breeding industry. [Method] Using PCR technology, the lactoferricin(Lfcin)-encoding gene was obtained from genome of Datong yak; then it was cloned into pGEM-T easy vector, and then sequenced; the sequencing results were subsequently aligned with the sequences of dairy cow accessed in GenBank. Moreover, amino acid sequences of Lfcin gene from various species including yak, dairy cow, human and mouse were used for sequence alignment and phylogenesis analysis. [Result] The second exon of lactoferrin(LF) from Datong yak, which is 778 bp in length, was obtained, within which the coding region of Lfcin gene is 75 bp (25 amino acid residues); sequence analysis showed that there is discrepancy of eleven bases between Datong yak and dairy cow; Lfcin proteins from various species shared high homeology, of which that from Datong yak and dairy cow were completely identical; phylogenesis analysis showed that cladogram based on Lfcin was consistent with species evolutionary law. [Conclusion] This study laid a foundation for the prokaryotic or eukaryotic expression of Lfcin gene and further understanding the activity of Lfcin protein. 展开更多
关键词 YAK Lfcin gene CLONE BIOINFORMATICS
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Cloning and Bioinformatic Analysis of Cinnamoyl-CoA Reductase Gene (CCR) from Pennisetum purpureum 被引量:2
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作者 朱琼华 张向前 +4 位作者 霍松 陈慧 李有涵 唐然 解新明 《Agricultural Science & Technology》 CAS 2012年第2期284-291,306,共9页
[Objective] The aim was to clone the cDNA and DNA sequences of the CCR (Cinnamoyl-CoA reductase) gene which involves in lignin biosynthesis, from Pennisetum purpureum, and to make comprehensive analysis on these seq... [Objective] The aim was to clone the cDNA and DNA sequences of the CCR (Cinnamoyl-CoA reductase) gene which involves in lignin biosynthesis, from Pennisetum purpureum, and to make comprehensive analysis on these sequences. [Method] CCR sequences were cloned from P. purpureum by using conventional RT-PCR and RACE (Rapid Amplification of cDNA Ends) methods; and the bioinformatic analyses of the CCR were conducted by means of NCBI, ProtParam ProtScale, TMHMM, TargetP, SignalP, Pfam20.0, Prosite, Swiss-Model, ClustalW2, DNAman, DNAstar and MEGA5. [Result] The cloned PpCCR (P. purpureum CCR) cDNA sequence was 1 316 bp, including a 1 110 bp ORF and 206 bp 3’-UTR. The cloned DNA sequence from PpCCR was 6 133 bp in full-length, containing five exons and four introns. Bioinformatic analysis indicated that PpCCR encoded a polypeptide of 369 amino acids, the secondary structure of which was primarily composed of random coil and α-helix, belonging to NAD-dependent epimerase/dehydratase family, and its co-factor binding sites and substrate binding sites were highly conserved. [Conclusion] DNA and cDNA sequences of CCR gene were obtained from P. purpureum, which had the typical characteristics of other homologous genes. The obtained bioinformatic data provided theoretical references for the further analysis of CCR and better application of P. purpureum in the future. 展开更多
关键词 Pennisetum purpureum Cinnamoyl-CoA reductase gene clone Bioinformatic analysis
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