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1.
R. Peerbolte F. A. Krens R. M. W. Mans M. Floor J. H. C. Hoge G. J. Wullems R. A. Schilperoort 《Plant molecular biology》1985,5(4):235-246
Summary With the DNA transformation procedure developed in our laboratory (13) several transformed tobacco SR1 tissues were obtained which, apart from selected and non-selected pTi sequences (T+), also had acquired non-selected calf thymus carrier DNA sequences (C+), being integrated in their nuclear genomes. From one such tissue (cNT4), with a shooty crown gall phenotype and expressing mannopine synthesis activity (Mas+), shoots were grafted and mature, flowering plants (gNT4) were obtained. After cross pollination with wild type SR1 tobacco pollen 49% of the seedlings obtained, had the maternal NT4-like crown gall phenotype and 51% showed wild type (SR1) features. The mannopine locus segregated independently from the locus determining the crown gall phenotype. When screened for integrated (transforming) foreign DNA sequences 97% of the NT4-like seedlings turned out to be C+T+. Most of the SR1-like seedlings, having a wild type tobacco morphology, proved to be transformed as well: roughly a 1:1:1:1 ratio as found for C+T+:C-T+: C+T:C T SR1-like seedlings. Based on the segregation of transforming sequences during meiosis a model is presented showing the integration of these sequences in three different host chromosomes. 相似文献
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Tinjuangjun P. Loc N.T. Gatehouse A.M.R. Gatehouse J.A. Christou P. 《Molecular breeding : new strategies in plant improvement》2000,6(4):391-399
We used particle bombardment to transform two elite Thai rice varieties, Khao Dawk Mali 105 (KDML105) and Supanburi 60 (SP60), with the snowdrop lectin gene gna (Galanthus nivalis agglutinin). This gene confers resistance to sap-sucking insects such as the brown planthopper (BPH; Nilaparvata lugens), which is one of the most damaging pests of rice. Traditionally, KDML105 and SP60 have been regarded as recalcitrant to transformation, and this is the first account of successful gene transfer to these varieties. By molecular analysis, we confirmed the recovery of over thirty gna-transgenic lines. GNA protein expression was characterised by western blot analysis, and we achieved expression levels of up to 0.25% total soluble protein. GNA-producing R1 transgenic plants were significantly more resistant to BPH than control plants (P<0.0001), with 37% and 42% reduction in nymphal survival for constitutive and phloem-specific expression, respectively. Transferring the gna gene to these superior rice varieties thus represents a major step forward for crop improvement in Thailand, and should help to reduce the damage caused by rice pests, and hence increase yields for this vital domestic and export market. 相似文献
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Fengjuan Li 《Bioscience, biotechnology, and biochemistry》2013,77(5):812-817
Nannochloropsis are model species for investigating biofuel production by algae. To develop them into an integrated photons-to-fuel production platform, high efficiency transformation methods are necessary. Here, we obtained the β-tubulin promoter regions of all recognized species of genus Nannochloropsis, and successfully transformed all five marine species by electroporation. In addition, the PCR amplified double stranded DNA fragments (PCR fragments) based transformation system was established in these Nannochloropsis species, which showed much higher transformation efficiency (10.7–61.2 × 10?6, 1.5–13-fold) than that of linearized plasmid based transformation. The cotransformation of N. salina using a circular plasmid containing a non-selectable GUS gene and a PCR fragment containing only a selection marker cassette was also achieved and found to be very efficient (over 50%). This simple and highly efficient transformation protocol reported in our study provided a useful tool for gene functional analysis and genetic engineering of the oleaginous Nannochloropsis species. 相似文献
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Kexuan Tang Quanan Hu Xianfen Sun Bingliang Wan Huaxiong Qi Xinggui Lu 《In vitro cellular & developmental biology. Plant》2001,37(3):334-340
Summary Mature seed-derived callus from an elite Chinese japonica rice cv. Ewan 5 was cotransformed with two plasmids, pWRG1515 and
pRSSGNAl, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. Thirty-five independent transgenic rice plants were regenerated from 177 bombarded calluses. Eighty-three
percent of the transgenic plants contained all three genes, as revealed by Southern blot analysis. Western blot analysis revealed
that 23 out of 29 gna-containing transgenic plants expressed Galanthus nivalis agglutinin (GNA) (79%) at various levels, with the highest expression being approximately 0.5% of total soluble protein.
Genetic analysis confirmed Mendelian segregation of all three transgenes (gna, hpt and gusA) in the R2 progeny. Amongst the R2 generation two independent homozygous lines were identified that expressed all three transgenes.
Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper
(Nilaparvata lugens, BPH) by decreasing the survival, overall fecundity of BPH, retarding development, and decreasing the feeding of BPH. These
BPH-resistant lines have been incorporated into a rice insect resistance breeding program. This is the first report that homozygous
transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred
enhanced resistance to BPH. 相似文献
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Irradiation of protoplasts with X-rays or ultraviolet light does not seem to influence the level of transient expression of
foreign DNA inPetunia protoplasts, whereas the number of stably transformed colonies is significantly raised. This may indicate that irradiation
influences integration and/or the expression of marker genes and does not result in enhanced uptake rates of plasmids into
protoplasts and cell nuclei. Co-transformation with plasmids carrying a gene for kanamycin resistance (neomycin phosphotransferase
II) and a gene for hygromycin resistance (hygromycin phosphotransferase) revealed that the cotransformation rates were not
stimulated by irradiation when measuring expression. Twenty-five kanamycin resistant but hygromycin sensitive colonies were
examined with Southern or slot blotting and all were found to contain the coding sequence for the hygromycinphosphotransferase
gene in their genomes. No obvious differences regarding copy number of integrated genes were observed when comparing transformed
colonies derived from irradiated and non-irradiated protoplasts. 相似文献
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Michael P. Whitehead Sarah Jane Gurr Carolyn Grieve Shiela E. Unkles David Spence Martin Ramsden James R. Kinghorn 《Gene》1990,90(2):193-198
We report the development of a homologous transformation system for Cephalosporium acremonium using the niaD gene of the nitrate assimilation (NA) pathway. Mutants in the NA pathway were selected on the basis of chlorate resistance by conventional means. Screening procedures were developed to differentiate between nitrate reductase apoprotein structural gene mutants (niaD) and molybdenum cofactor gene mutants (cnx) as wt. C. acremonium, unlike most filamentous fungi, fails to grow on minimal medium with hypoxanthine as a sole source of nitrogen. Phage clones carrying the niaD gene were isolated from a C. acremonium library constructed in λEMBL3 using the A. nidulans niaD gene as a heterologous probe. An 8.6-kb EcoRI fragment was subcloned into pUC18, and designated pSTA700. pSTA700 was able to transform stable niaD mutants to NA at a frequency of up to 40 transformants per μg DNA. Transformants were easily visible since the background growth was low and no abortives were observed. Gene replacements, single copy homologous integration and complex multiple integrations were observed. The niaD system was used to introduce unselected markers for hygromycin B resistance and benomyl resistance into C. acremonium by cotransformation. 相似文献
8.
Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns 总被引:28,自引:0,他引:28
Fu X Duc LT Fontana S Bong BB Tinjuangjun P Sudhakar D Twyman RM Christou P Kohli A 《Transgenic research》2000,9(1):11-19
Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors. 相似文献
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Segregation of genes transferred to one plant cell from two separate Agrobacterium strains 总被引:22,自引:0,他引:22
Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil bacteria which transfer DNA (T-DNA) to plant cells. Two Agrobacterium strains, each with a different T-DNA, can infect plants and give rise to transformed tissue which has markers from both T-DNAs. Although marker genes from both T-DNAs are in the tissue, definitive proof that the tissue is a cellular clone and that both T-DNAs are in a single cell is necessary to demonstrate cotransformation. We have transferred two distinguishable T-DNAs, carried on binary vectors in separate Agrobacterium rhizogenes strains, into tomato cells and have recovered hairy roots which received both T-DNAs. Continued expression of marker genes from each T-DNA in hairy roots propagated from individual root tips indicated that both T-DNAs were present in a single meristem. Also, we have transferred the two different T-DNAs, carried on identical binary vector plasmids in separate Agrobacterium tumefaciens strains, into tobacco cells and recovered plants which received both T-DNAs. Transformed plants with marker genes from each T-DNA were outcrossed to wild-type tobacco plants. Distribution of the markers in the F1 generation from three cotransformed plants of independent origin showed that both T-DNAs in the plants must have been present in the same cell and that the T-DNAs were genetically unlinked. Cotransformation of plant cells with T-DNAs from two bacterial strains and subsequent segregation of the transferred genes should be useful for altering the genetic content of higher plants. 相似文献
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