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1.
2.
Transient expression is an easy, rapid and powerful technique for producing proteins of interest in plants. Recombinational
cloning is highly efficient but has disadvantages, including complicated, time consuming cloning procedures and expensive
enzymes for large-scale gene cloning. To overcome these limitations, we developed new ligationindependent cloning (LIC) vectors
derived from binary vectors including tobacco mosaic virus (pJL-TRBO), potato virus X (pGR106) and the pBI121 vector-based
pMBP1. LIC vectors were modified to enable directional cloning of PCR products without restriction enzyme digestion or ligation
reactions. In addition, the ccdB gene, which encodes a potent cell-killing protein, was introduced between the two LIC adapter sites in the pJL-LIC, pGR-LIC,
and pMBP-LIC vectors for the efficient selection of recombinant clones. This new vector does not require restriction enzymes,
alkaline phosphatase, or DNA ligase for cloning. To clone, the three LIC vectors are digested with SnaBI and treated with T4 DNA polymerase, which includes 3′ to 5′ exonuclease activity in the presence of only one dNTP (dGTP
for the inserts and dCTP for the vector). To make recombinants, the vector plasmid and the insert PCR fragment were annealed
at room temperature for 20 min prior to transformation into the host. Bacterial transformation was accomplished with 100%
efficiency. To validate the new LIC vector systems, we were used to coexpressed the Phytophthora AVR and potato resistance (R) genes in N. benthamiana by infiltration of Agrobacterium. Coexpressed AVR and R genes in N. benthamiana induced the typical hypersensitive cell death resulting from in vivo interaction of the two proteins. These LIC vectors could be efficiently used for high-throughput cloning and laboratory-scale
in planta expression. These vectors could provide a powerful tool for high-throughput transient expression assays for functional genomic
studies in plants. 相似文献
3.
Hu Xuejun Zhang Zhichao Bao Yongming Yang Qing An Lijia 《Plant Molecular Biology Reporter》2002,20(2):189-189
An expeditious method is described for constructing T-vectors containing complementary 3′-thymidine overhangs. A T-vector
was developed by cloning a 90-bpEam 1105 I cassette containing 2Eam 1105 I restriction sites into a modified pUC119 vector. TheEam 1105 I cassette was generated by PCR with 2 specific primers containing different recognition sequences ofEam 1105 I. The recombinant vector was easily converted into a T-vector by digestion of the plasmid withEam 1105 I. The cloning efficiency of the PCR product was approximately 90%. The method described here is a simple way to construct
a variety of T-vectors. 相似文献
4.
A high-yield method was developed for producing a TA-cloning vector suitable for blue/white colony selection from a unique
parent plasmid containing a dual lacZ gene system through a one-step restriction enzyme digestion, which creates a single-base 3′-overhang. The dual lacZ gene system was realized by inserting an inner lacZ gene between two single-base 3′-overhangs, creating restriction enzyme sites within the reading-frame-adjusted outer lacZ gene sequence in the parent plasmid. The proposed method overcomes problems, such as the inefficient digestion frequently
observed when generating a TA-cloning vector and the difficulty of purifying TA-cloning vectors from the digestion mixture,
while maintaining the applicability of blue/white colony selection. Moreover, the use of TA-cloning vector prepared by the
proposed method can provide the distinguish tool of transformants carrying the cloning product from those carrying contaminating
parent plasmids, recircularized plasmids derived from incompletely digested parent plasmid fragments, or intra-molecularly
ligated TA-cloning vectors derived from T-overhangs missing TA-cloning vectors (instability of the T-overhangs is another
important consideration when designing TA-cloning vectors) by making all colonies except those carrying the cloning product
appear blue during blue/white colony selection. 相似文献
5.
We describe a method to produce site-directed mutations anywhere within cDNA by assembling mutagenized PCR fragments in proper
orientation using lambda integration in an extension of Gateway technology to yield a full-length mutated gene. This process
exploits the directionality of lambda insertion sequences ensuring integration and directionality of PCR product into a cloning
vector. The process requires only two sequential integration steps to yield a mutagenized expression vector. Mutagenized vasodilator
associated phosphoprotein (VASP) was produced by generating two PCR fragments representing the upstream and downstream portions
of the gene, substituting alanine or glutamate residues for VASP serine239. The upstream PCR was engineered with attB1 lambda
integration sequences at the 5′ region and attB2 at the 3′ region of the downstream fragment to ensure correct orientation.
The desired mutation was encoded by the forward primer of fragment 2. The reverse primer of the fragment 1 was phosphorylated
for subsequent ligation. Vent polymerase provided sequence accuracy and blunt-ended product. The first integration into a
donor vector, catalyzed by BP Clonase II created a linear product circularized by blunt end ligation, yielding hundreds of
entry vectors containing the mutagenized VASP. A second integration into destination vector yielded plasmid expressing mutant
VASP upon transfection. 相似文献
6.
A 1330-bp DNA sequence with two XcmI cassettes was inserted into pUC18 to construct an efficient XcmI T-vector parent plasmid, pYEMF. The large size of the inserted DNA fragment improved T-vector cleavage efficiency, and guaranteed
good separation of the molecular components after restriction digestion. The pYEMF-T-vector generated from parent plasmid
pYEMF permits blue/white colony screening; cloning efficiency analysis showed that most white colonies (>75%) were putative
transformants which carried the cloning product. The sequence analysis and design approach presented here will facilitate
applications in the fields of molecular biology and genetic engineering. 相似文献
7.
A pair of bifunctional expression vectors, pBL-WZX and pHY-WZX, for Escherichia coli and Bacillus licheniformis was constructed to express interesting genes in a secretory manner. The vectors contain an expression cassette consisted
of the promoter and signal peptide region of B. licheniformis amyL as well as an artificial multiple cloning site and a terminator and utilize kanamycin-resistance and/or tetracycline-resistance
for selection in both B. licheniformis and E. coli. Both vectors contain a part of 3′ terminal fragment of B. licheniformis amyL. The 5′-terminal or 3′-terminal fragment of B. licheniformis amyL can cause the integration and amplification of expression cassette in the chromosome of B. licheniformis under a kanamycin-selection pressure. pBL-WZX is an integrational vector while pHY-WZX is free one for B. licheniformis. Both vectors were succeeded in secretory expression of manL in both B. licheniformis and E. coli. 相似文献
8.
pUCPCR1 总被引:4,自引:0,他引:4
Erik de Vries 《Molecular biotechnology》1998,10(3):273-274
The mutiple cloning site of pUC19 was replaced by a multiple cloning site possessing a double Xcm1 restriction site. Digestion
withXcmI gives a linear vector with a single 3′-overhanging T-residue at both ends. This provides the easiest way of creating a vector
in which PCR fragments produced byTaq polymerase can be directly cloned without further modifications. 相似文献
9.
Complete nucleotide sequence of plasmid pGP2 from Acetobacter estunensis GP2 was identified after initial cloning of EcoRI fragment followed by preparation of deletion derivatives. Its size was
defined to 2,797 bp and several sites for several restriction enzymes were revealed by DNA sequencing. Sequence analysis predicts
three putative open reading frames (ORFs). ORF1 shows significant identity with the bacterial excinuclease α-subunit, ORF2 is a putative replication protein with low similarity with other Acetobacter plasmid’s replication proteins, and ORF3 encodes a class B acid phosphatase/phosphotransferase. The replication module comprises
a DnaA box like sequence, direct repeats, a potential prokaryotic promoter and a rep gene. The rep module is similar with
several θ-replicating, iteron-containing modules from plasmids, suggesting pGP2 replication may follow the same course. Any phenotypic
character determinant gene is absent in pGP2, suggesting this plasmid to be cryptic. However, a pGP2 derivative plasmid, containing
the putative pGP2 rep region, can replicate and is stably maintained in Acetobacter and Escherichia coli strains; it can also carry foreign DNA fragments. Thus, pGP2-X could serve as a cloning shuttle vector between these bacteria.
Prepared deletion derivatives of plasmid pGP2 suggested that Rep protein is essential for plasmid replication in host bacteria.
In its natural host, A. estunensis GP2, pGP2 maintains a four-times lower copy number than in E. coli. 相似文献
10.
Lei-ming You Jun Luo Ai-ping Wang Gai-ping Zhang Hai-bo Weng Ya-nan Guo Yun-chao Liu Qiao-mu Li Man Teng 《Molecular biology reports》2010,37(6):2757-2765
An efficient vector, designated as pCAGX, was designed for direct cloning and enhanced expression of PCR-amplified ORFs in
mammalian cells. It relied on the well-known TA-cloning principle, and utilized the CMV enhancer/chicken β-actin/rabbit β-globin
(CAG) hybrid promoter instead of the classical CMV promoter to drive more efficient transgene expression in wider host cells.
The specially designed cassette under CAG hybrid promoter contained two tandemly arrayed XcmI sites which were spaced by an additional EcoRV site. For direct cloning and expressing PCR-amplified ORFs, the T-vector was prepared by further digesting the EcoRV-linearized pCAGX with XcmI to produce T tails on both 3′-ends, which could efficiently minimize the non-recombinant background of T-vector and eliminate
the necessity of selective marker genes such as LacZ that allowed blue/white screening. Various PCR fragments in length were
prepared to verify the cloning efficiency by ligation with this vector, and GFP gene expression under control of the CAG hybrid
promoter in different host cells was assayed by flow cytometry. The results indicated that this vector was higher efficient,
especially suitable for cloning and expressing a number of interesting ORFs in parallel, and higher-level transgene expression
in different mammalian cells was obtained than the reported vectors using the CMV promoter. 相似文献
11.
Zhongbiao Tan Jianfang Li Minchen Wu Cunduo Tang Huimin Zhang Junqing Wang 《World journal of microbiology & biotechnology》2011,27(12):2767-2774
A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI,
was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut+) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His+, Mut+). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration
of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular
weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that
(10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at
a broad pH range of 7.0–10.5 and at a temperature of 30°C or below. 相似文献
12.
Godány A Bukovská G Farkasovská J Brnáková Z Dmitriev A Tkáciková E Ayele T Mikula I 《Folia microbiologica》2004,49(3):307-314
Characterization of classic type II restriction-modification systems (RMS) (restriction endonucleases and modification methyltransferases)
was carried out in isolates ofStaphylococcus aureus andStreptococcus agalactiae obtained from clinical material. Among the 100 isolates ofS. aureus two different RMS type II were detected. The first was expressed in isolates 32 and 33 (Sau32 I andSau33 I); the targeting sequence was determined as 5′-GGN CC-3′ (Sau96 I isoschizomer). The second was found in isolates no. 90, 93, 96*, and 98 (Sau90 I,Sau93 I,Sau96* I,Sau98 I) and enzymes recognized sequence 5′-CTY RAG-3′ (SmlI isoschizomer). Analysis of 40 isolates ofS. agalactiae revealed only one RMS; it was detected in two isolates (no. 16 and 23;Sag16 I andSag23 I). Restriction endonuclease expressed by these isolates cleaved DNA in sequence 5′-CTG CA/G-3′ (PstI isoschizomer). In RMS-positiveS. aureus andS. agalactiae isolates plasmid DNA capable of replication inEscherichia coli andBacillus subtilis was also detected and isolated.
This research was supported by VEGA grant of theSlovak Academy of Sciences no. 2/2059/22 and grant no. 2003 SP27/0208E 02/028/0E02 of theMinistry of Agriculture of the Slovak Republic. 相似文献
13.
Rieske protein gene in the Pacific oyster Crassostrea gigas was obtained by in silico cloning for the first time, and its expression profiles and subcellular localization were determined,
respectively. The full-length cDNA of Cgisp is 985 bp in length and contains a 5′- and 3′-untranslated regions of 35 and 161 bp, respectively, with an open reading frame
of 786 bp encoding a protein of 262 amino acids. The predicted molecular weight of 30 kDa of Cgisp protein was verified by prokaryotic expression. Conserved Rieske [2Fe–2S] cluster binding sites and highly matched-pair tertiary
structure with 3CWB_E (Gallus gallus) were revealed by homologous analysis and molecular modeling. Eleven putative SNP sites and two conserved hexapeptide sequences,
box I (THLGC) and II (PCHGS), were detected by multiple alignments. Real-time PCR analysis showed that Cgisp is expressed in a wide range of tissues, with adductor muscle exhibiting the top expression level, suggesting its biological
function of energy transduction. The GFP tagging Cgisp indicated a mitochondrial localization, further confirming its physiological
function. 相似文献
14.
V. S. Dedkov 《Applied Biochemistry and Microbiology》2010,46(9):849-853
Optimum conditions for the activity of the new DNA methylase in cell lysate were determined. Methylation of DNAs of bacteriophages
λ and T7 and plasmid pBR322 (dcm+) in the 5′-Cm5CWGG-3′ region blocked M.AjnI activity. The specificity of M.AjnI was determined using λ DNA methylated by this enzyme as well as computer modeling and data on the sensitivity of restriction
endonucleases Mval, HinfI, and BstMAI to methylation. 相似文献
15.
Cloning and expression of the ilvB gene of Escherichia coli K-12 总被引:12,自引:0,他引:12
Thomas Newman Philip Friden Ann Sutton Martin Freundlich 《Molecular & general genetics : MGG》1982,186(3):378-384
Summary A plasmid containing theilvB operon, which codes for acetohydroxy acid synthase I ofEscherichia coli K-12, was isolated using a ligated mixture of DNA from plasmid pBR322 and F'ilvB4 treated with endonucleaseSalI. A shortened derivative of this plasmid was isolated by cloning a 3.4 kb bacterial fragment into plasmid pKEN005 to yield plasmid pTCN12. The orientation of theilvB operon relative to plasmid genes was determined by restriction enzyme mapping. Measurement of the level of the product of theilvB gene, acetohydroxy acid synthase I, indicated that plasmid pTCN12 contained a functionalilvB promoter and control region. The DNA from this plasmid was used as a probe to show that the rate of synthesis ofilvB mRNA was proportional to the levels of acetohydroxy acid synthase I. 相似文献
16.
Chung DH Huddleston JR Farkas J Westpheling J 《Journal of industrial microbiology & biotechnology》2011,38(11):1867-1877
Potent HaeIII-like DNA restriction activity was detected in cell-free extracts of Caldicellulosiruptor bescii DSM 6725 using plasmid DNA isolated from Escherichia coli as substrate. Incubation of the plasmid DNA in vitro with HaeIII methyltransferase protected it from cleavage by HaeIII nuclease
as well as cell-free extracts of C. bescii. The gene encoding the putative restriction enzyme was cloned and expressed in E. coli with a His-tag at the C-terminus. The purified protein was 38 kDa as predicted by the 981-bp nucleic acid sequence, was optimally
active at temperatures between 75°C and 85°C, and was stable for more than 1 week when stored at 35°C. The cleavage sequence
was determined to be 5′-GG/CC-3′, indicating that CbeI is an isoschizomer of HaeIII. A search of the C. bescii genome sequence revealed the presence of both a HaeIII-like restriction endonuclease (Athe 2438) and DNA methyltransferase
(Athe 2437). Preliminary analysis of other Caldicellulosiruptor species suggested that this restriction/modification activity is widespread in this genus. A phylogenetic analysis based
on sequence alignment and conserved motif searches identified features of CbeI distinct from other members of this group and
classified CbeI as a member of a novel subfamily of HaeIII-like enzymes. 相似文献
17.
A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction
endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs
formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers
for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up
to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and
downstream regions flanking known sequences within the plant genome. 相似文献
18.
A potentially new thermotolerant B. licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and
compared with the type strain B. licheniformis ATCC 10716, producer of bacitracin. Studies on DNA base composition (G + C content) and DNA reassociation revealed that the
two strains show around 76% homology. Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding
for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern. Additionally, a different restriction
digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE. The I89 strain holds a 7.6-kb plasmid not present in the reference strain. The existence of various
unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and
expression vector.
Received: 29 June 1999 / Accepted: 1 September 1999 相似文献
19.
Cassettes for seed-specific expression tested in transformed embryogenic cultures of soybean 总被引:4,自引:0,他引:4
Soybean (Glycine max [L.] Merrill) lectin is a seed protein that accumulates in protein bodies of cotyledons during seed development. We have
constructed two expression cassettes containing the 5′ and 3′ regions of the soybean lectin gene connected by aNot I restriction site. One vector also contains the 32 amino acid signal sequence. Using polymerase chain reaction (PCR), the
coding region of the β-glucuronidase (uidA) gene was inserted into theNot I site of each vector. We tested the function of the expression cassettes in transformed embryogenic cultures of soybean.
Development-specific GUS expression was observed in developing somatic embryos transformed with the chimeric lectin promoter-GUS
constructs as determined by histochemical assays. Our data indicate that these cassettes could be used to drive expression
of foreign genes to modify embryo-specific traits of soybean as protein quality or quantity in the seed. 相似文献
20.
Chandrakant B. Jagtap Pradeep Kumar 《World journal of microbiology & biotechnology》2008,24(3):435-439
Marine Bacillus strain NM21 isolated from hydrocarbon-contaminated site at Naval Harbour, Mumbai grows on high-speed diesel as a source of
carbon and energy. This bacterium harbours four plasmids in it. The smallest plasmid, pNM214 was digested with EcoRI enzyme and cloned in pUC19 vector. The clone Om4 containing largest insert of >3.5 kb was sequenced by primer walking. DNA
sequence analysis showed this fragment to be homologous to replication initiation protein (rep) gene and dso (double strand origin) of different plasmids from Bacillus subtilis and Bacillus pumilus species. The putative rep gene sequence of pNM214 showed 74.3–91.6% DNA identity to B. subtilis plasmids (pTA1015, pTA1060 and pTA1040) and 86.3% to 88.9% DNA identity to B. pumilus plasmids (pPL7065, pPL10 and pSH1452). The translated amino acid sequence of rep shows that it contains all the three conserved
motifs present in the Rep protein of pC194 family of plasmids. DNA sequence comparison of putative dso of pNM214 with other bacillus plasmids belonging to pC194 group shows that it contains highly conserved nick site sequence
5′-TCTTTTCTTATCTTGATA-3′ and surrounding inverted repeats. Thus, it indicates that pNM214 to be a rolling circle replicating
plasmid belonging to the pC194 group. The presence of rep and dso like sequences in the sequenced EcoRI fragment indicate that the cloned fragment contain putative primary replicon of pNM214. 相似文献