Molecular cloning remains a cornerstone technique in genetic engineering and synthetic biology.In this study,we conducted a systematic comparative analysis between the classical cloning method and the Golden Gate asse...Molecular cloning remains a cornerstone technique in genetic engineering and synthetic biology.In this study,we conducted a systematic comparative analysis between the classical cloning method and the Golden Gate assembly technique,utilizing Escherichia coli as the model organism.Through polymerase chain reaction(PCR)amplification,restriction enzyme digestion,ligation,transformation,and Sanger sequencing,we assessed the operational efficiency and cloning fidelity of both strategies.Our results demonstrated that Golden Gate assembly,leveraging type IIS restriction enzymes and simultaneous ligation,significantly enhanced cloning efficiency and precision,particularly for seamless multi-fragment assembly.In contrast,the classical cloning approach maintained certain advantages in simplicity and robustness for specific experimental conditions.Challenges encountered during transformation and sequencing highlighted the critical impact of technical accuracy on experimental outcomes.This study underscores the importance of selecting appropriate cloning methodologies tailored to experimental objectives and laboratory capabilities,providing a foundation for optimized molecular cloning workflows in future synthetic biology and biotechnology applications.展开更多
The no-cloning theorem has sparked considerable interest in achieving high-fidelity approximate quantum cloning.Most of the previous studies mainly focused on the cloning of single particle states,and cloning schemes ...The no-cloning theorem has sparked considerable interest in achieving high-fidelity approximate quantum cloning.Most of the previous studies mainly focused on the cloning of single particle states,and cloning schemes used there are incapable of cloning quantum entangled states in multipartite systems.Few schemes were proposed for cloning multiparticle states,which consume more entanglement resources with loss of qubits,and the fidelity of the cloned state is relatively low.In this paper,cloning schemes for bipartite and tripartite entangled states based on photonic quantum walk and entanglement swapping are proposed.The results show that according to the proposed schemes,two high-fidelity(up to 0.75)cloned states can be obtained with less quantum resource consumption.Because of the simple cloning steps,few quantum resources and high fidelity,these schemes are both efficient and feasible.Moreover,this cloning machine eliminates the need for tracing out cloning machine,thereby minimizing resource waste.展开更多
[Objectives]This study was conducted to investigate the functional characteristics of the trxB gene in Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the trxB gene sequence of V.alginol...[Objectives]This study was conducted to investigate the functional characteristics of the trxB gene in Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the trxB gene sequence of V.alginolyticus for PCR cloning of its full-length sequence.Systematic bioinformatics analyses were conducted to predict the physicochemical properties,secondary structure,and tertiary structure of the encoded protein.[Results]The trxB gene is 960 bp in length,encoding 319 amino acid residues.The deduced protein has a predicted molecular weight of 34.32 kDa and an isoelectric point(pI)of 4.77.Analysis of the amino acid sequence revealed a distinct signal peptide cleavage site at the N-terminus,with no transmembrane domains.The functional sites are as follows:1 N-glycosylation site,1 cAMP-and cGMP-dependent protein kinase phosphorylation site,4 protein kinase C phosphorylation sites,7 casein kinase II phosphorylation sites,1 tyrosine kinase phosphorylation site,11 N-myristoylation sites,1 prenyl group binding site,3 microbody C-terminal targeting signal sites,and 1 xanthine nucleotide-disulfide oxidoreductase class II active site.Subcellular localization prediction indicated the highest probability(44.4%)for endoplasmic reticulum localization.The TrxB amino acid sequence of V.alginolyticus shares 97.2%-98.4%homology with other Vibrio species,and they were clustered within the same subgroup.Secondary structure prediction showed proportions of random coils(31.97%),alpha-helices(31.66%),extended strands(25.08%),and beta turns(11.29%).The tertiary structure model exhibited 88.68%similarity to template 5vt3.1.A.[Conclusions]This study elucidated the characterization of the TrxB protein in V.alginolyticus,laying a theoretical foundation for the development of outer membrane protein subunit vaccines against this pathogen.展开更多
The 12-oxophytodienoate reductase(OPR),a flavin mononucleotide-dependent oxidoreductase,regulates plant responses to stress conditions such as heavy metals,drought,saline-alkali,pests,and diseases by participating in ...The 12-oxophytodienoate reductase(OPR),a flavin mononucleotide-dependent oxidoreductase,regulates plant responses to stress conditions such as heavy metals,drought,saline-alkali,pests,and diseases by participating in the synthesis of plant hormones such as jasmonic acid(JA).In this study,homologous cloning was employed to obtain the Brassica napus‘Zhongshuang 11’OPR1 gene(BnOPR1).Bioinformatics analysis was conducted for the deduced protein BnOPR1,and a plant expression vector for BnOPR1 was constructed.The bioinformatics analysis revealed that BnOPR1 was 1125 bp in length,encoding 375 amino acid residues.The deduced protein had a molecular weight of 41.604 kDa,an isoelectric point of 6.05,a molecular formula of C1853H2855N511O550S16,an aliphatic index of 35.47,a lipophilicity index of 74.87,and an instability index of 39.49.It had 49 phosphorylation sites and lacked transmembrane domains and signal peptides.The phylogenetic analysis indicated that BnOPR1 had a close relationship with the OPR1 protein from Brassica rapa since they shared the same clade,while it had a distant relationship with the OPR1 protein from Raphanus sativus.In this paper,the expression vector for BnOPR1 was successfully constructed by seamless cloning and named pBnOPR1.The findings laid a foundation for further studying the roles of BnOPR1 in the response to antimony stress.展开更多
[Objectives] To analyze the function of cobQ gene from Vibrio alginolyticus strain HY9901,and to provide a reference for exploring the possible mechanism of cobQ gene from V.alginolyticus.[Methods] A pair of primers w...[Objectives] To analyze the function of cobQ gene from Vibrio alginolyticus strain HY9901,and to provide a reference for exploring the possible mechanism of cobQ gene from V.alginolyticus.[Methods] A pair of primers were designed based on the sequence of the V.alginolyticus cobQ gene and used to amplify the full-length gene by PCR.[Results] The PCR amplification results indicated that the cobQ gene has a full length of 780 bp,encoding 259 amino acid residues.The deduced amino acid sequence predicts a molecular weight of approximately 28.83 kD and an isoelectric point of 9.21.Sequence analysis revealed no N-terminal signal peptide cleavage site,suggesting the absence of both a signal peptide and transmembrane regions in this protein.The amino acid sequence contains 2 N-terminal myristoylation sites,1 N-glycosylation site,1 glycosaminoglycan attachment site,4 microbody C-terminal targeting signal sites,3 casein kinase II phosphorylation sites,and 4 protein kinase C phosphorylation sites.Subcellular localization prediction showed that the CobQ protein is primarily localized in the cytoplasm(65.2%probability).Homology analysis demonstrated that the amino acid sequence of the cobQ gene from V.alginolyticus shares up to 99%homology with other Vibrio species,clustering within the same subclade as Vibrio parahaemolyticus,indicating close phylogenetic relationships.Secondary structure prediction revealed proportions ofα-helices,random coils,and extended strands as 44.40%,36.68%,and 18.92%,respectively.The tertiary structure model exhibited 87.62%similarity to the template A0A165XBE1.1.[Conclusions] In this study,the V.alginolyticus cobq gene was successfully cloned and its sequence was analyzed by bioinformatics.It is expected to lay a foundation for the subsequent study of the regulatory mechanism of its protein on the virulence of V.alginolyticus.展开更多
Heading date is one of the most important agronomic traits that directly affect rice yield and determines the regional adaptability in specific growing environments.As a short-day plant,rice can grow under long-day(LD...Heading date is one of the most important agronomic traits that directly affect rice yield and determines the regional adaptability in specific growing environments.As a short-day plant,rice can grow under long-day(LD)conditions due to the synergistic regulation of many photosensitive genes.Using a set of chromosome segment substitution lines(CSSLs)with the indica cultivar Huanghuazhan(HHZ)as the recipient parent and Basmati Surkh 89-15(BAS)as the donor parent,we identified a QTL locus.展开更多
In this paper, we derive the explicit transformations of the optimal 1→3, 4, 5 phase-covariant cloning in three dimensions, and then generalize them to the cases of 1 → M = 3n, 3n ＋ 1, 3n ＋ 2 （n ≥ 1 integer） cl...In this paper, we derive the explicit transformations of the optimal 1→3, 4, 5 phase-covariant cloning in three dimensions, and then generalize them to the cases of 1 → M = 3n, 3n ＋ 1, 3n ＋ 2 （n ≥ 1 integer） cloning. The clone fidelities are coincident with the theoretical bounds found.展开更多
Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is...Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. Transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.展开更多
[Objective] The study aimed to clone interleukin-2(IL-2) gene from Sichuan white goose. [Method] Based on the IL-2 gene of duck accessed in GenBank, a pair of primers was designed for cloning IL-2 gene from total RNA ...[Objective] The study aimed to clone interleukin-2(IL-2) gene from Sichuan white goose. [Method] Based on the IL-2 gene of duck accessed in GenBank, a pair of primers was designed for cloning IL-2 gene from total RNA of peripheral blood lymphocytes of Sichuan white goose stimulated by ConA via RT-PCR technology. The yielded fragment was sequenced for bioinformatics analysis. [Result] The full length of IL-2 gene of Sichuan white goose is 468 bp that contains a 441 bp open reading frame(ORF), encoding 146 amino acid residues. Bioinformatics analysis shows that the amino acid sequence of IL-2 gene of Sichuan white goose contains four phosphorylation sites, a glycosylation site and a signal peptide with 21 amino acid residues. Homologies of IL-2 nucleotide sequence and amino acid sequence between Sichuan white goose and duck, chicken, turkey are 92.7%, 77.5%, 78.2% and 85.8%, 65.5%, 64.1%, respectively. By contrast IL-2 nucleotide sequence and amino acid sequence between Sichuan white goose and mammalian and rodents such as human, monkey, rat, bovine, horse, pig, cat, mouse, rabbit and deer, are all less than 45% and 28%, respectively. [Conclusion] The IL-2 gene of Sichuan white goose has closer genetic relationship with those of chicken and duck.展开更多
To investigate the expression characteristics of sex related gene of FTZ-F 1 in the half-smooth tongue-sole （Cynoglossus semilaevis）, the homologue FTZ-F1 （hsFTZ-F1） full-length cDNA was isolated from the testis b...To investigate the expression characteristics of sex related gene of FTZ-F 1 in the half-smooth tongue-sole （Cynoglossus semilaevis）, the homologue FTZ-F1 （hsFTZ-F1） full-length cDNA was isolated from the testis by homologous cloning, and the cDNA included the open reading frame and a 66bp 5＇-UTR, along with a 1619bp 3＇-UTR, encoding a predicted 485 amino acid protein. Sequence, tissue distribution and phylogenic analyses of the FTZ-F1 showed that the hsFTZ-F1 belonged to SF-1/Ad4BP group. The hsFTZ-F1 transcripts were highly abundant in the gonads, kidneys, brain and head-kidneys, but weakly in other tissues. However, the expression level in the brain and head-kidney of female was highly abundant than in the male. The hsFTZ-F1 expression was highly abundant in the embryo than in the larvae, which suggested that the hsFTZ-F1 may be involved in the organogenesis in the tongue sole.展开更多
A lectin protein(SFL) with molecular weight about 32 kD which markedly agglutinated rabbit and human red blood cells was purified from the roots of Sophora flavescens Ait. This protein, and apparently inhibited the gr...A lectin protein(SFL) with molecular weight about 32 kD which markedly agglutinated rabbit and human red blood cells was purified from the roots of Sophora flavescens Ait. This protein, and apparently inhibited the growth of Fusarium vasinfectum Atk., Gibberella saubinetii (Mont.) Sacc., and Piricularia oryzae Cav. A set of degenerate PCR primer was synthesized according to the N-terminal sequence of the purified protein. The full-length cDNA coding the lectin was cloned by RT-PCR and 5'-RACE and sequenced (GenBank AF285121). The deduced amino acid sequence indicates that a preprotein with 284 amino acid residues is firstly translated and then processed to a mature protein with 254 amino acids. A N-Glycosylation site is the Asn 182 residue.展开更多
Curcin, a ribosome-inactivating protein with a molecular weight of about 28.2 kD, which strongly inhibits the protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about (0.19 +/- 0.01) nmol/L, ...Curcin, a ribosome-inactivating protein with a molecular weight of about 28.2 kD, which strongly inhibits the protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about (0.19 +/- 0.01) nmol/L, was purified from the seeds of Jatropha curcas L. The protein has the activity of rRNA N-glycosidase. Degenerate primers were designed based on the N-terminal partial sequence from purified curcin. The full-length curcin cDNA by RT-PCR and 5'-RACE was cloned. The deduced amino acids sequence indicates that a preprotein with 20 amino acid residues is first translated and then processed to a mature protein with 251 amino acids. The deduced amino acids sequence shares homology of 33% and 57% to those of type I ribosome-inactivating proteins (RIPs) and A chain of type II RIPs, respectively. The sequence encoding mature curcin was integrated into the pQE-30 vector for expression in Escherichia coli strain M15 (pREP4). The purified recombinant curcin was able to inhibit protein synthesis in rabbit reticulocyte lysate system.展开更多
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
The cDNA encoding a variant of α bungarotoxin was cloned from the venom glands of Bungarus multicinctus by RT PCR.The deduced protein precursor contained a 21 amino acid signal peptide and a following 74 amino ...The cDNA encoding a variant of α bungarotoxin was cloned from the venom glands of Bungarus multicinctus by RT PCR.The deduced protein precursor contained a 21 amino acid signal peptide and a following 74 amino acid mature protein.The signal peptide is very similar to those of short chain neurotoxins,κ neurotoxins and cardiotoxins.The amino acid sequence of the mature protein is identical to α bungarotoxin (V31),a minor variant of α bungarotoxin identified by protein sequencing technique.Furthermore,the cDNA encoding the deletion precursor of α bungarotoxin was also cloned.By use of pMAL p2,the variant was overexpressed in E coli as a soluble fusion protein and purified by sepharose 6B amylose affinity chromatography,which was confirmed by western blotting with the antisera agai nst α bungarotoxin.The recombinant variant was achieved after digestion by factor X a.It displayed about 1/6 in vivo toxicity of natural α bungarotoxin.The successful cloning and functional expression of α bungarotoxin provided a basis for the future study of structure function of long neurotoxins.展开更多
[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
A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 3...A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 39%a identical to that of squalene synthases from Arabidopsis thaliana, tobacco, human and yeast, respectively. The AaSQS genomic DNA has a complex organization containing 14 exons and 13 introns. Full-length or C-terminal truncated cDNA was subcloned into prokaryotic expression vector pET30a and the constructed plasmid was introduced to Escherichia coli strain BL21 (DE3) for induced overexpression. No squalene synthase protein with expected molecular mass was observed in E. cola containing the putative full-length squalene synthase cDNA, however, overexpression in E. coli was achieved by truncating 30 amino acids of hydrophobic region at the carboxy terminus.展开更多
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
[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
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
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
文摘Molecular cloning remains a cornerstone technique in genetic engineering and synthetic biology.In this study,we conducted a systematic comparative analysis between the classical cloning method and the Golden Gate assembly technique,utilizing Escherichia coli as the model organism.Through polymerase chain reaction(PCR)amplification,restriction enzyme digestion,ligation,transformation,and Sanger sequencing,we assessed the operational efficiency and cloning fidelity of both strategies.Our results demonstrated that Golden Gate assembly,leveraging type IIS restriction enzymes and simultaneous ligation,significantly enhanced cloning efficiency and precision,particularly for seamless multi-fragment assembly.In contrast,the classical cloning approach maintained certain advantages in simplicity and robustness for specific experimental conditions.Challenges encountered during transformation and sequencing highlighted the critical impact of technical accuracy on experimental outcomes.This study underscores the importance of selecting appropriate cloning methodologies tailored to experimental objectives and laboratory capabilities,providing a foundation for optimized molecular cloning workflows in future synthetic biology and biotechnology applications.
文摘The no-cloning theorem has sparked considerable interest in achieving high-fidelity approximate quantum cloning.Most of the previous studies mainly focused on the cloning of single particle states,and cloning schemes used there are incapable of cloning quantum entangled states in multipartite systems.Few schemes were proposed for cloning multiparticle states,which consume more entanglement resources with loss of qubits,and the fidelity of the cloned state is relatively low.In this paper,cloning schemes for bipartite and tripartite entangled states based on photonic quantum walk and entanglement swapping are proposed.The results show that according to the proposed schemes,two high-fidelity(up to 0.75)cloned states can be obtained with less quantum resource consumption.Because of the simple cloning steps,few quantum resources and high fidelity,these schemes are both efficient and feasible.Moreover,this cloning machine eliminates the need for tracing out cloning machine,thereby minimizing resource waste.
基金Supported by Natural Science Foundation of Guangdong Province(2025A1515011061)Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean University+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[Objectives]This study was conducted to investigate the functional characteristics of the trxB gene in Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the trxB gene sequence of V.alginolyticus for PCR cloning of its full-length sequence.Systematic bioinformatics analyses were conducted to predict the physicochemical properties,secondary structure,and tertiary structure of the encoded protein.[Results]The trxB gene is 960 bp in length,encoding 319 amino acid residues.The deduced protein has a predicted molecular weight of 34.32 kDa and an isoelectric point(pI)of 4.77.Analysis of the amino acid sequence revealed a distinct signal peptide cleavage site at the N-terminus,with no transmembrane domains.The functional sites are as follows:1 N-glycosylation site,1 cAMP-and cGMP-dependent protein kinase phosphorylation site,4 protein kinase C phosphorylation sites,7 casein kinase II phosphorylation sites,1 tyrosine kinase phosphorylation site,11 N-myristoylation sites,1 prenyl group binding site,3 microbody C-terminal targeting signal sites,and 1 xanthine nucleotide-disulfide oxidoreductase class II active site.Subcellular localization prediction indicated the highest probability(44.4%)for endoplasmic reticulum localization.The TrxB amino acid sequence of V.alginolyticus shares 97.2%-98.4%homology with other Vibrio species,and they were clustered within the same subgroup.Secondary structure prediction showed proportions of random coils(31.97%),alpha-helices(31.66%),extended strands(25.08%),and beta turns(11.29%).The tertiary structure model exhibited 88.68%similarity to template 5vt3.1.A.[Conclusions]This study elucidated the characterization of the TrxB protein in V.alginolyticus,laying a theoretical foundation for the development of outer membrane protein subunit vaccines against this pathogen.
文摘The 12-oxophytodienoate reductase(OPR),a flavin mononucleotide-dependent oxidoreductase,regulates plant responses to stress conditions such as heavy metals,drought,saline-alkali,pests,and diseases by participating in the synthesis of plant hormones such as jasmonic acid(JA).In this study,homologous cloning was employed to obtain the Brassica napus‘Zhongshuang 11’OPR1 gene(BnOPR1).Bioinformatics analysis was conducted for the deduced protein BnOPR1,and a plant expression vector for BnOPR1 was constructed.The bioinformatics analysis revealed that BnOPR1 was 1125 bp in length,encoding 375 amino acid residues.The deduced protein had a molecular weight of 41.604 kDa,an isoelectric point of 6.05,a molecular formula of C1853H2855N511O550S16,an aliphatic index of 35.47,a lipophilicity index of 74.87,and an instability index of 39.49.It had 49 phosphorylation sites and lacked transmembrane domains and signal peptides.The phylogenetic analysis indicated that BnOPR1 had a close relationship with the OPR1 protein from Brassica rapa since they shared the same clade,while it had a distant relationship with the OPR1 protein from Raphanus sativus.In this paper,the expression vector for BnOPR1 was successfully constructed by seamless cloning and named pBnOPR1.The findings laid a foundation for further studying the roles of BnOPR1 in the response to antimony stress.
基金Natural Science Foundation of Guangdong Province(2025A1515011061)Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean University+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[Objectives] To analyze the function of cobQ gene from Vibrio alginolyticus strain HY9901,and to provide a reference for exploring the possible mechanism of cobQ gene from V.alginolyticus.[Methods] A pair of primers were designed based on the sequence of the V.alginolyticus cobQ gene and used to amplify the full-length gene by PCR.[Results] The PCR amplification results indicated that the cobQ gene has a full length of 780 bp,encoding 259 amino acid residues.The deduced amino acid sequence predicts a molecular weight of approximately 28.83 kD and an isoelectric point of 9.21.Sequence analysis revealed no N-terminal signal peptide cleavage site,suggesting the absence of both a signal peptide and transmembrane regions in this protein.The amino acid sequence contains 2 N-terminal myristoylation sites,1 N-glycosylation site,1 glycosaminoglycan attachment site,4 microbody C-terminal targeting signal sites,3 casein kinase II phosphorylation sites,and 4 protein kinase C phosphorylation sites.Subcellular localization prediction showed that the CobQ protein is primarily localized in the cytoplasm(65.2%probability).Homology analysis demonstrated that the amino acid sequence of the cobQ gene from V.alginolyticus shares up to 99%homology with other Vibrio species,clustering within the same subclade as Vibrio parahaemolyticus,indicating close phylogenetic relationships.Secondary structure prediction revealed proportions ofα-helices,random coils,and extended strands as 44.40%,36.68%,and 18.92%,respectively.The tertiary structure model exhibited 87.62%similarity to the template A0A165XBE1.1.[Conclusions] In this study,the V.alginolyticus cobq gene was successfully cloned and its sequence was analyzed by bioinformatics.It is expected to lay a foundation for the subsequent study of the regulatory mechanism of its protein on the virulence of V.alginolyticus.
基金supported by the Zhejiang Provincial Natural Science Foundation of China(Grant Nos.LZ24C130004 and LQ24C130008)。
文摘Heading date is one of the most important agronomic traits that directly affect rice yield and determines the regional adaptability in specific growing environments.As a short-day plant,rice can grow under long-day(LD)conditions due to the synergistic regulation of many photosensitive genes.Using a set of chromosome segment substitution lines(CSSLs)with the indica cultivar Huanghuazhan(HHZ)as the recipient parent and Basmati Surkh 89-15(BAS)as the donor parent,we identified a QTL locus.
基金supported by the National Natural Science Foundation of China(Grant Nos.11074002,61073048,and 11104057)the Natural Science Foundationof the Education Department of Anhui Province,China(Grant Nos.KJ2010ZD08 and KJ2012A245)the Postgraduate Program of Huainan NormalUniversity of China
文摘In this paper, we derive the explicit transformations of the optimal 1→3, 4, 5 phase-covariant cloning in three dimensions, and then generalize them to the cases of 1 → M = 3n, 3n ＋ 1, 3n ＋ 2 （n ≥ 1 integer） cloning. The clone fidelities are coincident with the theoretical bounds found.
文摘Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. Transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.
文摘[Objective] The study aimed to clone interleukin-2(IL-2) gene from Sichuan white goose. [Method] Based on the IL-2 gene of duck accessed in GenBank, a pair of primers was designed for cloning IL-2 gene from total RNA of peripheral blood lymphocytes of Sichuan white goose stimulated by ConA via RT-PCR technology. The yielded fragment was sequenced for bioinformatics analysis. [Result] The full length of IL-2 gene of Sichuan white goose is 468 bp that contains a 441 bp open reading frame(ORF), encoding 146 amino acid residues. Bioinformatics analysis shows that the amino acid sequence of IL-2 gene of Sichuan white goose contains four phosphorylation sites, a glycosylation site and a signal peptide with 21 amino acid residues. Homologies of IL-2 nucleotide sequence and amino acid sequence between Sichuan white goose and duck, chicken, turkey are 92.7%, 77.5%, 78.2% and 85.8%, 65.5%, 64.1%, respectively. By contrast IL-2 nucleotide sequence and amino acid sequence between Sichuan white goose and mammalian and rodents such as human, monkey, rat, bovine, horse, pig, cat, mouse, rabbit and deer, are all less than 45% and 28%, respectively. [Conclusion] The IL-2 gene of Sichuan white goose has closer genetic relationship with those of chicken and duck.
基金Supported by grants from State 863 High-Technology R&D Project of China(2006AA10A403)Shandong Genetic Improvement Key Project for Agricultural OrganismDoctor Initial Funding of Guangdong Ocean University(0712103)
文摘To investigate the expression characteristics of sex related gene of FTZ-F 1 in the half-smooth tongue-sole （Cynoglossus semilaevis）, the homologue FTZ-F1 （hsFTZ-F1） full-length cDNA was isolated from the testis by homologous cloning, and the cDNA included the open reading frame and a 66bp 5＇-UTR, along with a 1619bp 3＇-UTR, encoding a predicted 485 amino acid protein. Sequence, tissue distribution and phylogenic analyses of the FTZ-F1 showed that the hsFTZ-F1 belonged to SF-1/Ad4BP group. The hsFTZ-F1 transcripts were highly abundant in the gonads, kidneys, brain and head-kidneys, but weakly in other tissues. However, the expression level in the brain and head-kidney of female was highly abundant than in the male. The hsFTZ-F1 expression was highly abundant in the embryo than in the larvae, which suggested that the hsFTZ-F1 may be involved in the organogenesis in the tongue sole.
文摘A lectin protein(SFL) with molecular weight about 32 kD which markedly agglutinated rabbit and human red blood cells was purified from the roots of Sophora flavescens Ait. This protein, and apparently inhibited the growth of Fusarium vasinfectum Atk., Gibberella saubinetii (Mont.) Sacc., and Piricularia oryzae Cav. A set of degenerate PCR primer was synthesized according to the N-terminal sequence of the purified protein. The full-length cDNA coding the lectin was cloned by RT-PCR and 5'-RACE and sequenced (GenBank AF285121). The deduced amino acid sequence indicates that a preprotein with 284 amino acid residues is firstly translated and then processed to a mature protein with 254 amino acids. A N-Glycosylation site is the Asn 182 residue.
文摘Curcin, a ribosome-inactivating protein with a molecular weight of about 28.2 kD, which strongly inhibits the protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about (0.19 +/- 0.01) nmol/L, was purified from the seeds of Jatropha curcas L. The protein has the activity of rRNA N-glycosidase. Degenerate primers were designed based on the N-terminal partial sequence from purified curcin. The full-length curcin cDNA by RT-PCR and 5'-RACE was cloned. The deduced amino acids sequence indicates that a preprotein with 20 amino acid residues is first translated and then processed to a mature protein with 251 amino acids. The deduced amino acids sequence shares homology of 33% and 57% to those of type I ribosome-inactivating proteins (RIPs) and A chain of type II RIPs, respectively. The sequence encoding mature curcin was integrated into the pQE-30 vector for expression in Escherichia coli strain M15 (pREP4). The purified recombinant curcin was able to inhibit protein synthesis in rabbit reticulocyte lysate system.
文摘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.
基金the"95"great program of Chinese Academy of Sciences! (KY95 1-A1-3 0 1-0 2 )
文摘The cDNA encoding a variant of α bungarotoxin was cloned from the venom glands of Bungarus multicinctus by RT PCR.The deduced protein precursor contained a 21 amino acid signal peptide and a following 74 amino acid mature protein.The signal peptide is very similar to those of short chain neurotoxins,κ neurotoxins and cardiotoxins.The amino acid sequence of the mature protein is identical to α bungarotoxin (V31),a minor variant of α bungarotoxin identified by protein sequencing technique.Furthermore,the cDNA encoding the deletion precursor of α bungarotoxin was also cloned.By use of pMAL p2,the variant was overexpressed in E coli as a soluble fusion protein and purified by sepharose 6B amylose affinity chromatography,which was confirmed by western blotting with the antisera agai nst α bungarotoxin.The recombinant variant was achieved after digestion by factor X a.It displayed about 1/6 in vivo toxicity of natural α bungarotoxin.The successful cloning and functional expression of α bungarotoxin provided a basis for the future study of structure function of long neurotoxins.
基金Supported by National Natural Science Foundation of China(30771596)Ph.D.Programs Foundation of Ministry of Education of China(20060183010)~~
文摘[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.
文摘A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 39%a identical to that of squalene synthases from Arabidopsis thaliana, tobacco, human and yeast, respectively. The AaSQS genomic DNA has a complex organization containing 14 exons and 13 introns. Full-length or C-terminal truncated cDNA was subcloned into prokaryotic expression vector pET30a and the constructed plasmid was introduced to Escherichia coli strain BL21 (DE3) for induced overexpression. No squalene synthase protein with expected molecular mass was observed in E. cola containing the putative full-length squalene synthase cDNA, however, overexpression in E. coli was achieved by truncating 30 amino acids of hydrophobic region at the carboxy terminus.
文摘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.
文摘[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.
文摘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.
文摘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 .
