共查询到20条相似文献,搜索用时 31 毫秒
1.
W. Kofer H.-U. Koop G. Wanner K. Steinmüller 《Molecular genetics and genomics : MGG》1998,258(1-2):166-173
Plastids contain a NAD(P)H-plastoquinone-oxidoreductase (NDH complex) which is homologous to the eubacterial and mitochondrial NADH-ubiquinone-oxidoreductase (complex I), but the metabolic function of the enzyme is unknown. The enzyme consists of at least eleven subunits (A-K), which are all encoded on the plastid chromosome. We have mutagenized ndhC and ndhJ by insertion, and ndhK and ndhA-I by deletion and insertion, of a cassette which carried a spectinomycin resistance gene as a marker. The transformation was carried out by the polyethylene glycol-mediated plastid transformation method. Southern analysis revealed that even after repeated regeneration cycles each of the four different types of transformants had retained 1–5% of wild-type gene copies. This suggests that complete deletion of ndh genes is not compatible with viability. The transformants displayed two characteristic phenotypes: (i) they lack the rapid rise in chlorophyll fluorescence in the dark after illumination with actinic light for 5?min; in the wild-type this dark-rise reflects a transient reduction of the plastoquinone pool by reduction equivalents generated in the stroma; and (ii) transformants with defects in the ndhC-K-J operon accumulate starch, indicating inefficient oxidation of glucose via glycolysis and the oxidative pentose phosphate pathway. Both observations support the theory of chlororespiration, which postulates that the NDH complex acts as a valve to remove excess reduction equivalents in the chloroplast. 相似文献
2.
Homologous recombination and allele replacement in transformants of Fusarium fujikuroi 总被引:3,自引:0,他引:3
The ascomycete Fusarium fujikuroi could be transformed stably to hygromycin resistance only when the transforming plasmid contained a fragment of DNA from
the fungus. The transformation frequencies were roughly independent of the sequence of the particular fungal DNA fragment
used, of its size (1.8 or 6 kb), and of whether this DNA was present only once in the fungal genome or about forty times (the
genes for ribosomal RNA). The plasmid was integrated into the fungal genome by homologous recombination in the eighteen transformants
tested; ectopic integration was never observed. The carB gene of F. fujikuroi was cloned and shown to complement unpigmented mutants deficient in phytoene dehydrogenase. A mutant carB allele was prepared in vitro and used to transform wild-type protoplasts; the transformants contained a genomic duplication
and were heterozygous for carB; the mutant allele replaced the original wild-type allele when this was spontaneously lost in the transformants. This loss
was due to gene conversion in some cases and to recombination between repeated sequences in others.
Received: 5 November 1999 / Accepted: 16 March 2000 相似文献
3.
Minoru Ueda Tetsuki Kuniyoshi Hiroshi Yamamoto Kazuhiko Sugimoto Kimitsune Ishizaki Takayuki Kohchi Yoshiki Nishimura Toshiharu Shikanai 《The Plant journal : for cell and molecular biology》2012,72(4):683-693
The chloroplast NADH dehydrogenase‐like (NDH) complex mediates cyclic electron transport and chloro‐respiration and consists of five sub‐omplexes, which in angiosperms further associate with photosystem I (PSI) to form a super‐complex. In Marchantia polymorpha, 11 plastid‐encoded subunits and all the nuclear‐encoded subunits of the A, B, membrane and ferredoxin‐binding sub‐complexes are conserved. However, it is unlikely that the genome of this liverwort encodes Lhca5 and Lhca6, both of which mediate NDH–PSI super‐complex formation. It is also unlikely that the subunits of the lumen sub‐complex, PnsL1–L4, are encoded by the genome. Consistent with this in silico prediction, the results of blue‐native gel electrophoresis showed that NDH subunits were detected in a protein complex with lower molecular mass in Marchantia than the NDH–PSI super‐complex in Arabidopsis. Using the plastid transformation technique, we knocked out the ndhB gene in Marchantia. Although the wild‐type genome copies were completely segregated out, the ΔndhB lines grew like the wild‐type photoautotrophically. A post‐illumination transient increase in chlorophyll fluorescence, which reflects NDH activity in vivo in angiosperms, was absent in the thalli of the ΔndhB lines. In ruptured chloroplasts, antimycin A‐insensitive, and ferredoxin‐dependent plastoquinone reduction was impaired, suggesting that chloroplast NDH mediates similar electron transport in Marchantia and Arabidopsis, despite its possible difference in structure. As in angiosperms, linear electron transport was not strongly affected in the ΔndhB lines. However, the plastoquinone pool was slightly more reduced at low light intensity, suggesting that chloroplast NDH functions in redox balancing of the inter system, especially under low light conditions. 相似文献
4.
H. Yamazaki Y. Ohnishi K. Takeuchi N. Mori N. Shiraishi Y. Sakata H. Suzuki S. Horinouchi 《Applied microbiology and biotechnology》1999,52(3):401-409
Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic
transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet
(UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5′-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura+ transformants with 1 μg pRPPyr4 DNA and 1 × 107 viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4
sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man0∼1GlcNAc2 at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains.
Received: 1 February 1999 / Received revision: 26 February 1999 / Accepted: 20 March 1999 相似文献
5.
We have established a simple and efficient plastid transformation system for liverwort, Marchantia polymorpha L., suspension-culture cells, which are homogenous, chloroplast-rich and␣rapidly growing. Plasmid pCS31 was constructed to
integrate an aadA expression cassette for spectinomycin-resistance into the trnI–trnA intergenic region of the liverwort plastid DNA by homologous recombination. Liverwort suspension-culture cells were bombarded
with pCS31-coated gold projectiles and selected on a medium containing spectinomycin. Plastid transformants were reproducibly
isolated from the obtained spectinomycin-resistant calli. Selection on a sucrose-free medium greatly improved the efficiency
of selection of plastid transformants. Homoplasmic plastid transformant lines were established by␣successive subculturing
for 14 weeks or longer on the spectinomycin-containing medium. The plastid transformation system of liverwort suspension-culture
cells should facilitate the investigation of the fundamental genetic systems of plastid DNA, such as replication. 相似文献
6.
We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the
recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the
rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear
DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6 × 106-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 × 103 genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil
samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification
(e.g. by PCR).
Received: 20 May 1997 / Accepted: 17 October 1997 相似文献
7.
Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants 总被引:1,自引:0,他引:1
Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid
gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous
recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination
events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two
different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory
plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as
10–60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo
further, “secondary” recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance
of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events
described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco
was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended
recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid
transformants containing the desired transgene.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
8.
Y. Yang R. Karthikeyan S. E. Mack E. J. Vonarx B. A. Kunz 《Molecular & general genetics : MGG》1999,261(4-5):777-787
Genetic stability relies in part on the efficiency with which post-replicative mismatch repair (MMR) detects and corrects
DNA replication errors. In Escherichia coli, endogenous transition mispairs and insertion/deletion (ID) heterologies are corrected with similar efficiencies – but much
more efficiently than transversion mispairs – as revealed by mutation rate increases in MMR mutants. To assess the relative
efficiencies with which these mismatches are corrected in the yeast Saccharomyces cerevisiae, we examined repair of defined mismatches on heteroduplex plasmids and compared the spectra for >1000 spontaneous SUP4-o mutations arising in isogenic wild-type or MMR-deficient (pms1, mlh1, msh2) strains. Heteroduplexes containing G/T mispairs or ID heterologies were corrected more efficiently than those containing
transversion mismatches. However, the rates of single base-pair insertion/deletion were increased much more (82-fold or 34-fold,
respectively) on average than the rate of base pair substitutions (4.4-fold), with the rates for total transitions and transversions
increasing to similar extents. Thus, the relative efficiencies with which mismatches formed during DNA replication are repaired
appear to differ in prokaryotic and eukaryotic cells. In addition, our results indicate that in yeast, and probably other
eukaryotes, these efficiencies may not mirror those obtained from an analysis of heteroduplex correction.
Received: 15 November 1998 / Accepted: 4 February 1999 相似文献
9.
In photosynthetic eukaryotes, the redox state of the plastoquinone (PQ) pool is an important sensor for mechanisms that regulate
the photosynthetic electron transport. In higher plants, a multimeric nicotinamide adenine dinucleotide (phosphate) (NAD(P))H
dehydrogenase (NDH) complex and a plastid terminal oxidase (PTOX) are involved in PQ redox homeostasis in the dark. We recently
demonstrated that in the microalgae Chlamydomonas reinhardtii, which lacks the multimeric NDH complex of higher plants, non-photochemical PQ reduction is mediated by a monomeric type-II
NDH (Nda2). In this study, we further explore the nature and the importance of non-photochemical PQ reduction and oxidation
in relation to redox homeostasis in this alga by recording the ‘dark’ chlorophyll fluorescence transients of pre-illuminated
algal samples. From the observation that this fluorescence transient is modified by addition of propyl gallate, a known inhibitor
of PTOX, and in a Nda2-deficient strain we conclude that it reflects post-illumination changes in the redox state of PQ resulting
from simultaneous PTOX and Nda2 activity. We show that the post-illumination fluorescence transient can be used to monitor
changes in the relative rates of the non-photochemical PQ reduction and reoxidation in response to different physiological
situations. We study this fluorescence transient in algae acclimated to high light and in a mutant deficient in mitochondrial
respiration. Some of our observations indicate that the chlororespiratory pathway participates in redox homeostasis in C. reinhardtii. 相似文献
10.
We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were
used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed
the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration
of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied
depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes
investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation
with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to
the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation
efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation,
electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion
at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic
sites in a high proportion of transformants.
Received: 6 March 1998 / Accepted: 25 May 1998 相似文献
11.
A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische
Forschung (IGF) BAC library, consists of 10 752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100 kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA
and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid
DNA, 3.2% clones with the 180 bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA
repeat. With its extensive genome coverage, its rather uniformly sized inserts (80 kb <85% <120 kb) and low contamination
with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing.
Received: 26 November 1997 / Accepted: 19 February 1998 相似文献
12.
Vojcic L Despotovic D Martinez R Maurer KH Schwaneberg U 《Applied microbiology and biotechnology》2012,94(2):487-493
Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered
by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤103 transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent
transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional
incubation step in the competence development phase and a recovery step during the transformation procedure. In addition,
the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural
competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 105 transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA. 相似文献
13.
U. Kües J. D. Granado R. Hermann R. P. Boulianne K. Kertesz-Chaloupková M. Aebi 《Molecular & general genetics : MGG》1998,260(1):81-91
Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores)
at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various
reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation
with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore
production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores
in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12 h light/12 h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before
karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.
Received: 11 May 1998 / Accepted: 15 July 1998 相似文献
14.
N. Uematsu C. Matsuoka Y. Agemizu E. Nagoshi K. Yamamoto 《Molecular & general genetics : MGG》1999,261(3):523-529
The chromosomal tonB gene of Escherichia coli was used as a target for the detection of spontaneous deletion mutations. The deletions were isolated in both recA
+ and recA
− cells, and mutants carrying large deletions were identified because they also lacked part or all of the trp operon. The frequencies of tonB-trp deletion were 1.79 × 10−9 and 1.09 × 10−9 for recA
+ and recA
− cells, respectively. We analyzed 12 deletions from recA
+ and 10 from recA
− cells by cloning and direct sequencing. The deletions ranged in size from 5612 bp to 15142 bp for recA
+ and from 5428 bp to 13289 for recA
− cells. Three deletions from recA
+ cells and five deletions from recA
− cells were found to have occurred between short sequence repeats at the termini of the deletion, leaving one copy of the
repeat in the mutant sequence. Seven deletions from recA
+ cells and three deletions from recA
− cells did not have repeats at their termini; in these cases, the DNA sequences that are adjacent to the deletion termini
in the wild-type are characterized by short (2–4 bp) repeats. From these results, a model is presented for the generation
of deletion mutations which involves formation of an asymmetric crossover mediated by repeated sequences of 2- to 4-bp.
Received: 14 September 1998 / Accepted: 22 December 1998 相似文献
15.
H. Prokisch O. Yarden M. Dieminger M. Tropschug I. B. Barthelmess 《Molecular & general genetics : MGG》1997,256(2):104-114
The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit
of calcineurin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction
of the target function had been achieved in antisense transformants with multiple construct integrations. Induction conditions
caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the
distinctive tip-high Ca2+ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for calcineurin in the precise
regulation of apical growth, a common form of cellular proliferation. In vitro inhibition of N. crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants
to the calcineurin-specific drugs CsA and FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype
very similar to that of the cna-1 antisense mutants is consistent with this idea.
Received: 18 February 1997 / Accepted: 20 April 1997 相似文献
16.
SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential
for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of
alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region
of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for
the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues.
C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon
+ cells, but not in lon
− cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion
mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore,
the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did
not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ
protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable
in lon
+ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with,
Lon, and are necessary, but not sufficient, for degradation by Lon.
Received: 8 October 1996 / Accepted: 27 November 1996 相似文献
17.
18.
Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker 总被引:6,自引:0,他引:6
Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series
of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used
to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of
this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas.
Received: 26 October 1999 / Accepted: 28 December 1999 相似文献
19.
Sterol composition and growth of transgenic tobacco plants expressing type-1 and type-2 sterol methyltransferases 总被引:3,自引:0,他引:3
Transgenic tobacco (Nicotiana tabacum L.) plants with altered sterol composition were generated by transformation with plant cDNAs encoding type-1 and type-2 sterol
methyltransferases (SMTs; EC 2.1.1.41). For both SMT1 and SMT2 transformants, the transformation was associated with a reduction
in the level of cholesterol, a non-alkylated sterol. In SMT1 transformants a corresponding increase of alkylated sterols,
mainly 24-methyl cholesterol, was observed. On the other hand, in SMT2 transformants the level of 24-methyl cholesterol was
reduced, whereas the level of sitosterol was raised. No appreciable alteration of total sterol content was observed for either
genotype. The general phenotype of transformants was similar to that of controls, although SMT2 transformants displayed a
reduced height at anthesis. The results show that plant sterol composition can be altered by transformation with an SMT1 cDNA
without adverse effects on growth and development, and provide evidence, in planta, that SMT1 acts at the initial step in
sterol alkylation.
Received: 27 June 2000 / Accepted: 22 July 2000 相似文献
20.
Abida Yasmeen Bushra Mirza Samia Inayatullah Naila Safdar Maryam Jamil Shawkat Ali M. Fayyaz Choudhry 《Plant Molecular Biology Reporter》2009,27(1):20-28
In this study, in planta transformation of tomato (Solanum lycopersicum L.), using fruit injection and floral dip, is reported. Agrobacterium tumefaciens strain EHA 105 containing one of three constructs, i.e., pROKIIAP1GUSint (carrying the Apetala 1 [AP1] gene), pROKIILFYGUSint (carrying the LEAFY [LFY] gene), or p35SGUSint (carrying the β-glucuronidase [GUS] gene), was used for plant transformation. For fruit injection transformation, no significant effects (p > 0.05) of the construct used were observed. The highest frequency of transformation was obtained following 48-h incubation
of tomato fruit with bacterial cells harboring either one of the three constructs; transformation frequencies of 17%, 19%,
and 21% for AP1, LFY, and GUS gene constructs, respectively, were obtained. When fruit maturity was evaluated in fruit injection experiments, mature red
fruit resulted in higher frequency of transformants than immature green fruit with 40%, 35%, and 42% for AP1, LFY, and GUS gene constructs, respectively. For floral dip transformation, a higher number of transformants was obtained when the GUS gene construct was used instead of either the AP1 or LFY gene construct, thus suggesting a possible inhibitory effect of the flowering genes used. When flowers were transformed prior
to rather than following pollination, they yielded a higher transformation frequency, 12% for the LFY construct and 23% for the GUS construct (p < 0.05), although no transformant was obtained with the AP1 gene construct. All putative GUS-positive transformants were analyzed using polymerase chain reaction and confirmed for the
presence of the transgene. Compared to control plants, transgenic plants carrying either the AP1 or LFY transgene flowered earlier and showed several different morphological characters. 相似文献