转豇豆胰蛋白酶CPTI基因棉花检测用标准分子的制备

利用PCR的方法克隆豇豆胰蛋白酶基因CPTI、基因表达调控元件35S启动子、NOS终止子以及棉花内标基因Sad1,连接到克隆载体上,构建成质粒标准分子pGB。PCR检测过程中,质粒标准分子和转基因棉花中能扩增出4个目的条带,而非转基因棉花中不能扩增出相应的条带,证明构建好的质粒标准分子能用于转基因棉花的定性检测。荧光定量PCR绘制4个目的基因片段的标准曲线,各标准曲线的相关系数均达到0.985以上,说明建立的PCR具有很好的Ct值-初始浓度相关性,达到定量分析的需求,而且荧光定量PCR反应具有很好的重复性和稳定性,可用于实际样品的定量检测。     

Marker rescue allows direct selection for recombinant plasmids in streptococci

Summary Resident deletion derivatives (Em^s or Cm^s) of the streptococcal plasmid vector pGB301 rescue antibiotic resistance genes from linearized pGB301 (Em^r, Cm^r) DNA with high frequency. Insertion of passenger DNA next to an antibiotic resistance determinant of pGB301, which is missing on the resident plasmid, forces corescue of these two plasmid domains, thus allowing direct selection for recombinant plasmids.     

Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamicin/kanamycin resistance determinant

Summary Streptococcal plasmid pGB301 is an in vivo rearranged plasmid with interesting properties and potential for the molecular cloning of genes in streptococci. Transformation of S. sanguis (Challis) with the group B streptococcal plasmid pIP501 (29.7 kb) gave rise to the deletion derivative pGB301 (9.8 kb, copy number 10) which retained the multiple resistance phenotype of its ancestor (inducible MLS-resistance, chloramphenicol resistance). Among the eight restriction endonucleases used to physically map pGB301 were four that cleaved the plasmid at single sites yielding either sticky (HpaII, KpnI) or bluntends (HpaI, HaeIII/BspRI). Passenger DNA derived from larger streptococcal plasmids (pSF351C61, 69.5 kb; pIP800, 71 kb) was successfully inserted into the HpaII site and, by blunt-end cloning, into the HaeIII/BspRI site. The gentamicin/kanamycin resistance gene of pIP800 was expressed by recombinant plasmids carrying the insert in either orientation. Insertion of passenger DNA into the HaeIII/BspRI site (but not the HpaII site) caused instability of adjacent pGB301 sequences which were frequently deleted, thereby removing the chloramphenicol resistance phenotype. The vector pGB301 has a remarkable capacity for passenger DNA (inserts up to 7 kb) and the property of instability and loss of a resistance phenotype following insertion of passenger DNA into the HaeIII/BspRI site should facilitate the identification of cloned segments of DNA when using this plasmid in molecular cloning experiments.     

Characterization of a novel TOL-like plasmid from Pseudomonas putida involved in 1,2,4-trimethylbenzene degradation.

A strain of Pseudomonas putida (TMB) was found to resemble P. putida mt-2 (PaW1) in its ability to degrade 1,2,4-trimethylbenzene, toluene, m-xylene, and p-xylene via oxidation of a methyl substituent and reaction of the meta fission pathway, but a different regulatory model is suggested. The ability of P. putida TMB to degrade these substrates was encoded by plasmid pGB (85 kilobase pairs), which showed considerable differences in size, restriction patterns, and DNA sequence from those of plasmid pWWO of strain PaW1.     

Effect of Ca2+ ions on plasmid transformation of Streptococcus lactis protoplasts.

The effects of Mg2+ and Ca2+ ions on the efficiency of the plasmid transformation of lysozyme-treated Streptococcus lactis protoplasts were compared. A 33-megadalton plasmid, pLP712, coding for lactose fermentation and a 6.5-megadalton plasmid, pGB301, coding for erythromycin and chloramphenicol resistance were used as model plasmids, and S. lactis MG1614 was the recipient. Replacing Mg2+ with Ca2+ in the transformation buffer was found to increase transformant frequency more than 10-fold with both plasmids.     

Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant.

The replication region of a 28-kilobase-pair (kbp) cryptic plasmid from Lactococcus lactis subsp. lactis biovar diacetylactis SSD207 was cloned in L. lactis subsp. lactis MG1614 by using the chloramphenicol resistance gene from the streptococcal plasmid pGB301 as a selectable marker. The resulting 8.1-kbp plasmid, designated pVS34, was characterized further with respect to host range, potential cloning sites, and location of replication gene(s). In addition to lactococci, pVS34 transformed Lactobacillus plantarum and, at a very low frequency, Staphylococcus aureus but not Escherichia coli or Bacillus subtilis. The 4.1-kbp ClaI fragment representing lactococcal DNA in pVS34 contained unique restriction sites for HindIII, EcoRI, XhoII, and HpaII, of which the last three could be used for molecular cloning. A region necessary for replication was located within a 2.5-kbp fragment flanked by the EcoRI and ClaI restriction sites. A 3.8-kbp EcoRI fragment derived from a nisin resistance plasmid, pSF01, was cloned into the EcoRI site of pVS34 to obtain a nisin-chloramphenicol double-resistance plasmid, pVS39. From this plasmid, the streptococcal chloramphenicol resistance region was subsequently eliminated. The resulting plasmid, pVS40, contains only lactococcal DNA. Potential uses for this type of a nisin resistance plasmid are discussed.     

Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant

The replication region of a 28-kilobase-pair (kbp) cryptic plasmid from Lactococcus lactis subsp. lactis biovar diacetylactis SSD207 was cloned in L. lactis subsp. lactis MG1614 by using the chloramphenicol resistance gene from the streptococcal plasmid pGB301 as a selectable marker. The resulting 8.1-kbp plasmid, designated pVS34, was characterized further with respect to host range, potential cloning sites, and location of replication gene(s). In addition to lactococci, pVS34 transformed Lactobacillus plantarum and, at a very low frequency, Staphylococcus aureus but not Escherichia coli or Bacillus subtilis. The 4.1-kbp ClaI fragment representing lactococcal DNA in pVS34 contained unique restriction sites for HindIII, EcoRI, XhoII, and HpaII, of which the last three could be used for molecular cloning. A region necessary for replication was located within a 2.5-kbp fragment flanked by the EcoRI and ClaI restriction sites. A 3.8-kbp EcoRI fragment derived from a nisin resistance plasmid, pSF01, was cloned into the EcoRI site of pVS34 to obtain a nisin-chloramphenicol double-resistance plasmid, pVS39. From this plasmid, the streptococcal chloramphenicol resistance region was subsequently eliminated. The resulting plasmid, pVS40, contains only lactococcal DNA. Potential uses for this type of a nisin resistance plasmid are discussed.     

Effect of Ca2+ ions on plasmid transformation of Streptococcus lactis protoplasts

The effects of Mg2+ and Ca2+ ions on the efficiency of the plasmid transformation of lysozyme-treated Streptococcus lactis protoplasts were compared. A 33-megadalton plasmid, pLP712, coding for lactose fermentation and a 6.5-megadalton plasmid, pGB301, coding for erythromycin and chloramphenicol resistance were used as model plasmids, and S. lactis MG1614 was the recipient. Replacing Mg2+ with Ca2+ in the transformation buffer was found to increase transformant frequency more than 10-fold with both plasmids.     

High efficiency electroporation of Lactococcus lactis subsp. lactis LM0230 with plasmid pGB301

Electroporation-mediated transformation of Lactococcus lactis with plasmid pGB301, a 9.8 kilobase pair vector (Behnke et al. 1981), has been reported by McIntyre & Harlander (1989a). Improved transformation efficiencies of 10(2)-10(3)/micrograms DNA were achieved by altering the conditions under which the bacteria were grown prior to electroporation (McIntyre & Harlander 1989b). This present investigation sought to improve still further transformation efficiencies in order to provide a reliable high frequency transformation system for Lc. lactis subsp. lactis.     

Mercury resistance determined by a self-transmissible plasmid inBacillus cereus 5

Summary Inducible mercuric reductase activity inBacillus cereus 5 was plasmid-encoded. Plasmid analysis revealed three plasmids with molecular masses of 2.6, 5.2 and 130 MDa. A mating system permitted transfer of the resistance determinant among strains ofB. cereus andB. thuringiensis. Transfer of mercury resistance fromB. cereus 5 toB. cereus 569 andB. thuringiensis occurred during mixed culture incubation on agar surfaces. The 130-MDa plasmid (pGB130) was responsible for transfer; frequencies ranged from 10^–5 to 10^–4.B. cereus 569 transconjugants inheriting pGB130 were also effective donors. High transfer frequencies and the finding that cell-free filtrates of donor cultures were ineffective in mediating transfer suggested mercury-resistance transfer was not phage-mediated. Transfer was also insensitive to DNase activity. Further evidence that pGB130 DNA carried the mercury-resistance determinant was transformation ofB. cereus 569 by electroporation with pGB130 DNA isolated fromB. cereus 5 and a mercury-resistantB. cereus 569 transconjugant. Mercury-resistant transconjugants and transformants exhibited mercuric reductase activity. Plasmid pGB130 also conferred resistance to phenylmercuric acetate.     

Development of a multifunctional and efficient conjugal plasmid for use in Streptomyces spp

A plasmid, pGB112, has recently been developed to transfer DNA from Escherichia coli to Streptomyces spp via conjugation. This technique made use of (A) E. coli replicon, (B) ampicillin (amp) resistance gene for selection in E. coli and thiostrepton (tsr) resistance gene for selection in Streptomyces, (C) a fragment of SCP2* replicon, (D) a 2.6 kb fragment of tra-cassette which consists of pIJ101 transfer gene (tra) and two ermE promoters, (E) a 0.8 kb fragment of oriT of (IncP) RK2. The results showed that this plasmid was able to transfer plasmid DNA from E. coli to Streptomyces coelicolor via conjugation, and that it could also transfer DNA between Streptomyces strains. Since this plasmid has both pBR322 and SCP2* replicons, it may provide a novel and useful method for genetic operation in E. coli and Streptomyces.An erratum to this article can be found at     

Expression of recombinant anticoagulant hirudin in the differentiated cultures of the porcine mammary epithelial cell line SI-PMEC

To express recombinant proteins in the spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) currently established in our laboratory, a chemically synthesized DNA fragment encoding the anticoagulant hirudin was used to construct a mammalian expression vector under the control of the goat beta-casein regulatory sequence. The vector, named pGB562/Hi, was transfected into the SI-PMEC cells to yield pGB562/Hi/SI-PMEC. The pGB562/Hi/SI-PMEC cells expressed recombinant hirudin only when they were differentiated into functional structures by growth on a Matrigel-coated petri dish supplemented with the lactogenic hormone prolactin. The differentiated pGB562/Hi/SI-PMEC cells produced about 0.5-0.6microg of recombinant hirudin/mg of total cellular protein. These results suggest that the established SI-PMEC cells have pharmaceutical potential to inducibly express bioactive heterogeneous proteins.     

Location of antibiotic resistance determinants, copy control, and replication functions on the double-selective streptococcal cloning vector pGB301

Summary The physical map of the streptococcal cloning vector pGB301 (9.8 kb) has been extended by localizing the cleavage sites of restriction endonucleases AvaII, AvaI, BclI, BstEII, and PvuII. The latter four enzymes cleaved pGB301 at unique sites. Insertion of chromosomal DNA from Staphylococcus aureus strain 3Ar^–m^– into the single BstEII site of pGB301 led to inactivation of the plasmid's chloramphenicol resistance determinant. Twelve deletion derivatives of pGB301 were isolated either by in vitro manipulation of pGB301 or as spontaneous deletion mutants following transformation of Challis by mixtures of recombinant plasmids. The overlapping deletions which spanned a continuous sequence of 7.7 kb ranged in size from 0.3 kb to 4.1 kb and allowed to localize the chloramphenicol and MLS-resistance determinants, the copy control function, and the replication region on the physical map of pGB301. Plasmid pGB301 together with its deletion mutants constitutes a valuable tool for further molecular cloning experiments in streptococci.     

Expression of a ribosomal protein gene in axillary buds of pea seedlings

Axillary buds of intact pea seedlings (Pisum sativum L. cv Alaska) do not grow and are said to be dormant. Decapitation of the terminal bud promotes the growth of these axillary buds, which then develop in the same manner as terminal buds. We previously showed that unique sets of proteins are expressed in dormant and growing buds. Here we describe the cloning, sequencing, and expression of a cDNA clone (pGB8) that is homologous to ribosomal protein L27 from rat. RNA corresponding to this clone increases 13-fold 3 h after decapitation, reaches a maximum enhancement of about 35-fold after 12 h, and persists at slightly reduced levels at later times. Terminal buds, root apices, and elongating internodes also contain pGB8 mRNA but fully expanded leaflets and fully elongated internodes do not. In situ hybridization analysis demonstrates that pGB8 mRNA increases in all parts of the bud within 1 h of decapitation. Under appropriate conditions, growing buds can be made to stop growing and become dormant; these buds subsequently can grow again. Therefore, buds have the capacity to undergo multiple cycles of growth and dormancy. RNA gel blots show that pGB8 expression is reduced to dormancy levels as soon as buds stop growing. However, in situ hybridization experiments show that pGB8 expression continues at growing-bud levels in the apical meristem for 2 d after it is reduced in the rest of the bud. When cultured stems containing buds are treated with indoleacetic acid at concentrations ≥10 μm, bud growth and expression of pGB8 in the buds are inhibited.     

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.     

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.     

Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium

Plasmid DNA was introduced by electroporation into Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Propionibacterium and Staphylococcus as an alternative to competent-cell or protoplast transformation. Plasmid-containing transformants were recovered in these recipients at frequencies ranging from 10(1) to 10(5) transformants micrograms-1 of pGK12. Several parameters of the protocol, including DNA concentration, voltage, plating regimen and electroporation buffers were evaluated to determine conditions that improved transformation frequencies for Lactobacillus acidophilus. Using optimized conditions, the following plasmids were introduced into L. acidophilus: pAMB1, pC194, pGB354, pGKV1, pSA3, pTRK13, pTV1 and pVA797. The ability to transfer plasmid DNA via eletroporation will greatly facilitate the application of recombinant DNA methodology and transposon technology to Gram-positive bacteria for cloning and analysis of significant genes.     

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.     

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation.

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.     

Relationship between Lactococcus lactis subsp. lactis 952, an industrial lactococcal starter culture and strains 712, ML3 and C2

Lactococcus lactis subsp. lactis 952, used as a component of a cheese starter culture, was found to be closely related to the 712/ML3/C2 group of strains. Plasmid profiles of the four isolates were very similar and factory phages isolated against strain 952 could produce plaques on all the other strains at the same titres. Treatment with mitomycin C failed to induce phage from strain 952 although homology was observed between φT712 and the chromosome of strain 952. Pulse field gel electrophoresis of the four strains revealed only minor differences in their genomic DNA. The clumping phenomenon observed during lactose transfer in strains 712 and ML3 was also evident in strain 952. The ability of strain 952 to mobilize non-conjugative vectors such as pGB301 was also examined and analysis of pGB301 transconjugants revealed that some contained novel enlarged plasmids not present in either the donor or the recipient. Preliminary characterization of these enlarged plasmids suggested the involvement of a sex factor type element in strain 952.