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
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
PGIP gene was obtained from Brassica oleracea L. var. alboglabra, named BoPGIP2. The full length of BoPGIP2 gene is 1 102 bp and the exon is 993 bp which encodes a protein of 330 amino acids with a predicted molecular...PGIP gene was obtained from Brassica oleracea L. var. alboglabra, named BoPGIP2. The full length of BoPGIP2 gene is 1 102 bp and the exon is 993 bp which encodes a protein of 330 amino acids with a predicted molecular mass of 37.1 kDa, interrupted by one intron of 95 bp in, length. Sequence analysis revealed that it has five potential N-giycosylation sites, two protein kinase C phosphrylation sites, five casin kinase Ⅱ phosphrylation sites and four N-myristoylation sites. The amino acids sequences alignment confirmed that ^145 LRR stucture was highly conserved in all aligned PGIP sequences.展开更多
Insect pheromone-binding proteins （PBPs） play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors （ORs）. However, the PBPs of the orien...Insect pheromone-binding proteins （PBPs） play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors （ORs）. However, the PBPs of the oriental fruit moth, Grapholita molesta, an important destructive pest of stone fruits worldwide, are not well characterized. In this study, two new putative PBP genes, GmolPBP2 and GmolPBP3, were identiifed from G. molesta antennae. The deduced amino-acid sequences of these two putative PBP genes are characteristic of the odorant binding protein family, containing six conserved cysteine residues. The genomic DNA sequence of each gene contained two introns. However, the lengths and positions of the introns differed. RT-PCR analyses revealed that the two GmolPBP genes are only expressed in the antennae of female and male moths. Quantitative real-time PCR indicated that the transcription levels of GmolPBP2 are far greater than those of GmolPBP3 in both female and male antennae. GmolPBP3 showed higher transcription levels in female antennae than in male antennae, while GmolPBP2 showed similar transcription levels in both female and male antennae. The transcript levels of both genes were signiifcantly different in premating and post-coitum individuals, implying that mating affects the process of sex pheromone reception. To better understand the functions, two GmolPBPs were expressed in Escherichia coli, and the ligand binding assays were conducted. Results showed that GmolPBP2 has strong binding afifnities to two sex pheromone components, E8-12：Ac and Z8-12：Ac, as well as weaker binding afifnities to Z8-12：OH and 12：OH. GmolPBP2 also bound some ordinary odor molecules. However, the afifnity of GmolPBP3 to both sex pheromones and ordinary odor molecules was very weak. These results show that GmolPBP2 plays the main role in pheromone discrimination and recognition in the oriental fruit moth.展开更多
Phenylalanine ammonia-lyase(PAL),which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid,is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropa...Phenylalanine ammonia-lyase(PAL),which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid,is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes.Schisandra chinensis,a woody vine plant belonging to the family of Magnoliaceae,is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity.However,the functional role of PAL in the biosynthesis of lignan is relatively limited,compared with those in lignin and flavonoids biosynthesis.Therefore,it is essential to clone and characterize the PAL genes from this valuable medicinal plant.In this study,molecular cloning and characterization of three PAL genes(ScPAL1−3)from S.chinensis was carried out.ScPALs were cloned using RACE PCR.The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis.In order to determine their catalytic activity,recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli(BL21-DE3),followed by Ni-affinity purification.The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds.The optimal temperature,pH value and effects of different metal ions were determined.Vmax,Kcat and Km values were determined under the optimal conditions.The expression of three ScPALs in different tissues was also determined.Our work provided essential information for the function of ScPALs.展开更多
A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 3...A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame, encoding 130 amino acids. The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids. The molecular mass of the deduced mature peptide is 12. 3 ku. It is highly cationic with a theoretical isoelectric point of 8.5. The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus （ Litopenaeus ） setferus. Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues. Capillary elec- trophoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection. The level of Crustin mRNA began to restore at 72 h post-challenge. This indicated that Crustin gene might play an important role when shrimps are infected by bacterial pathogen.展开更多
The ultrastructure of porcine kidney(PK)-15 cells was examined after lipofectamine-aided transfection of the molecular clone of the P1 agent.PK-15 cells transfected with the tandem dimer of the P1molecular DNA clone h...The ultrastructure of porcine kidney(PK)-15 cells was examined after lipofectamine-aided transfection of the molecular clone of the P1 agent.PK-15 cells transfected with the tandem dimer of the P1molecular DNA clone had numbers of intracytoplasmic inclusions,and a few cells had intranuclear inclusions.Intracytoplasmic inclusions were round to oval and 0.1-0.3μm in diameter,and intranuclear inclusions,which were more electron dense,were of two general types:the first were round and small(0.1μm approximately)and the second were hexagonal and larger(0.4-0.8μm in diameter).Cells transfected with the tandem dimer of the P1 molecular DNA clone tested positive for P1 DNA at passage 5.This is the first report that the P1 molecular clone has infectivity in vitro and it will provide fundamental materials for further study of the biological characterization of P1.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.
基金Supported by School High-level Talent Starting Fund of Qingdao Agriculture University:Studies on Clone and Evolution of PGIPGene from Brassica crops(630745)~~
文摘PGIP gene was obtained from Brassica oleracea L. var. alboglabra, named BoPGIP2. The full length of BoPGIP2 gene is 1 102 bp and the exon is 993 bp which encodes a protein of 330 amino acids with a predicted molecular mass of 37.1 kDa, interrupted by one intron of 95 bp in, length. Sequence analysis revealed that it has five potential N-giycosylation sites, two protein kinase C phosphrylation sites, five casin kinase Ⅱ phosphrylation sites and four N-myristoylation sites. The amino acids sequences alignment confirmed that ^145 LRR stucture was highly conserved in all aligned PGIP sequences.
基金supported by the National Natural Science Foundation of China (31272043)the the Special Fund for Agro-Scientific Research in the Public Interest, China (201103024)
文摘Insect pheromone-binding proteins （PBPs） play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors （ORs）. However, the PBPs of the oriental fruit moth, Grapholita molesta, an important destructive pest of stone fruits worldwide, are not well characterized. In this study, two new putative PBP genes, GmolPBP2 and GmolPBP3, were identiifed from G. molesta antennae. The deduced amino-acid sequences of these two putative PBP genes are characteristic of the odorant binding protein family, containing six conserved cysteine residues. The genomic DNA sequence of each gene contained two introns. However, the lengths and positions of the introns differed. RT-PCR analyses revealed that the two GmolPBP genes are only expressed in the antennae of female and male moths. Quantitative real-time PCR indicated that the transcription levels of GmolPBP2 are far greater than those of GmolPBP3 in both female and male antennae. GmolPBP3 showed higher transcription levels in female antennae than in male antennae, while GmolPBP2 showed similar transcription levels in both female and male antennae. The transcript levels of both genes were signiifcantly different in premating and post-coitum individuals, implying that mating affects the process of sex pheromone reception. To better understand the functions, two GmolPBPs were expressed in Escherichia coli, and the ligand binding assays were conducted. Results showed that GmolPBP2 has strong binding afifnities to two sex pheromone components, E8-12：Ac and Z8-12：Ac, as well as weaker binding afifnities to Z8-12：OH and 12：OH. GmolPBP2 also bound some ordinary odor molecules. However, the afifnity of GmolPBP3 to both sex pheromones and ordinary odor molecules was very weak. These results show that GmolPBP2 plays the main role in pheromone discrimination and recognition in the oriental fruit moth.
基金supported by the National Key R&D Program of China(No.2017YFC1701200).
文摘Phenylalanine ammonia-lyase(PAL),which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid,is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes.Schisandra chinensis,a woody vine plant belonging to the family of Magnoliaceae,is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity.However,the functional role of PAL in the biosynthesis of lignan is relatively limited,compared with those in lignin and flavonoids biosynthesis.Therefore,it is essential to clone and characterize the PAL genes from this valuable medicinal plant.In this study,molecular cloning and characterization of three PAL genes(ScPAL1−3)from S.chinensis was carried out.ScPALs were cloned using RACE PCR.The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis.In order to determine their catalytic activity,recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli(BL21-DE3),followed by Ni-affinity purification.The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds.The optimal temperature,pH value and effects of different metal ions were determined.Vmax,Kcat and Km values were determined under the optimal conditions.The expression of three ScPALs in different tissues was also determined.Our work provided essential information for the function of ScPALs.
基金The Major State Basic Research Development Program of China under contract No2006CB101804the Natural Science Foundationof Hebei Province under contract NoC2008000596
文摘A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame, encoding 130 amino acids. The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids. The molecular mass of the deduced mature peptide is 12. 3 ku. It is highly cationic with a theoretical isoelectric point of 8.5. The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus （ Litopenaeus ） setferus. Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues. Capillary elec- trophoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection. The level of Crustin mRNA began to restore at 72 h post-challenge. This indicated that Crustin gene might play an important role when shrimps are infected by bacterial pathogen.
基金supported by the State Key Basic Research Project(973 project)of China(Grant No.2007CB116308)Planned Projects for Postdoctoral Research Funds of Jiangsu Province,China(Grant No.5910602)Postdoctoral Funds of Jiangsu Academy of Agricultural Sciences,China(Grant No.6510501)
文摘The ultrastructure of porcine kidney(PK)-15 cells was examined after lipofectamine-aided transfection of the molecular clone of the P1 agent.PK-15 cells transfected with the tandem dimer of the P1molecular DNA clone had numbers of intracytoplasmic inclusions,and a few cells had intranuclear inclusions.Intracytoplasmic inclusions were round to oval and 0.1-0.3μm in diameter,and intranuclear inclusions,which were more electron dense,were of two general types:the first were round and small(0.1μm approximately)and the second were hexagonal and larger(0.4-0.8μm in diameter).Cells transfected with the tandem dimer of the P1 molecular DNA clone tested positive for P1 DNA at passage 5.This is the first report that the P1 molecular clone has infectivity in vitro and it will provide fundamental materials for further study of the biological characterization of P1.
