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1.
The Cre/lox site-specific recombination controls the excision of a target DNA segment by recombination between two lox sites flanking it, mediated by the Cre recombinase. We have studied the functional expression of the Cre/lox system to excise a transgene from the rice genome. We developed transgenic plants carrying the target gene, hygromycin phosphotransferase (hpt) flanked by two lox sites and transgenic plants harboring the Cre gene. Each lox plant was crossed with each Cre plant reciprocally. In the Cre/lox hybrid plants, the Cre recombinase mediates recombination between two lox sites, resulting in excision of the hpt gene. The recombination event could be detected because it places the CaMV 35S promoter of the hpt gene adjacent to a promoterless gusA gene; as a result the gusA gene is activated and its expression could be visualized. In 73 Cre/lox hybrid plants from various crosses of T0 transgenic plants, 19 expressed GUS, and in 132 Cre/lox hybrid plants from crosses of T2 transgenic plants, 77 showed GUS expression. Molecular data proved the excision event occurred in all the GUS+ plants. Recombination occurred with high efficiency at the early germinal stage, or randomly during somatic development stages. Received. 2 April 2001 / Accepted: 29 June 2001  相似文献   

2.
To study the impact of different DNA configurations on the stability of transgene expression, a variant of the cre gene was developed. This variant allows for the highly efficient in planta removal of its own loxP-flanked coding sequence as well as other DNAs flanked by ectopic heterospecific lox sites, either lox511 or lox2272 or both, in trans. The plant intron-containing cre gene, cre INT , was configured in such a way that self-excision generated an intact hygromycin resistance selectable marker gene. In this combination, all selected transformants showed highly efficient excision. Plants obtained showed no indication of any chimerism, indicating a cell autonomous nature of the hygromycin selection during transformation and regeneration. The highly efficient concomitant removal of wildtype and heterospecific lox site-flanked DNA demonstrated that upon retransformation with the self-excising cre INT , sufficient amounts of Cre enzyme were produced prior to its removal. Plants obtained with cre INT showed much less frequently the Cre-associated phenomenon of reduced fertility than plants obtained with a continuous presence of Cre recombinase. The cre INT system has therefore advantages over systems with a continuously present Cre. The cre INT system was successfully used for removal of two chromatin boundary elements from transgene cassettes in tobacco. Analysis of plants with and without boundary elements on the same chromosomal location will contribute to a better evaluation of the role of such elements in the regulation of transgene expression in plants.  相似文献   

3.
Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic -glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F1 and F2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F1 progeny and most of the F2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.  相似文献   

4.
5.
Jia H  Pang Y  Chen X  Fang R 《Transgenic research》2006,15(3):375-384
Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus–host plant systems.  相似文献   

6.
Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either β-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.  相似文献   

7.
This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T0 plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T0 transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T0-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T2 seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T1 plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

8.
The Cre–loxP site-specific recombination system was deployed for removal of marker genes from Brassica juncea (Indian mustard). Excision frequencies, monitored by removal of nptII or gfp genes in F1 plants of crosses between LOX and CRE lines, were high in quiescent, differentiated somatic tissues but extremely poor in the meristematic regions (and consequently the germinal cells) thus preventing identification and selection of marker-free transgenic events which are devoid of both the marker gene as well as the cre gene, in F2 progeny. We show that a passage through in vitro culture of F1 leaf explants allows efficient development of marker-free transgenics in the F2 generation addressing current limitations associated with efficient use of the Cre/loxP technology for marker gene removal. N. Arumugam and Vibha Gupta have contributed equally to this work.  相似文献   

9.
The development of rapid and efficient strategies to generate selectable marker-free transgenic plants could help increase the consumer acceptance of genetically modified (GM) plants. To produce marker-free transgenic plants without conditional treatment or the genetic crossing of offspring, we have developed a rapid and convenient DNA excision method mediated by the Cre/loxP recombination system under the control of a −46 minimal CaMV 35S promoter. The results of a transient expression assay showed that −46 minimal promoter::Cre recombinase (−46::Cre) can cause the loxP-specific excision of a selectable marker, thereby connecting the 35S promoter and β-glucuronidase (GUS) reporter gene. Analysis of stable transgenic Arabidopsis plants indicated a positive correlation between loxP-specific DNA excision and GUS expression. PCR and DNA gel-blot analysis further revealed that nine of the 10 tested T1 transgenic lines carried both excised and nonexcised constructs in their genomes. In the subsequent T2 generation plants, over 30% of the individuals for each line were marker-free plants harboring the excised construct only. These results demonstrate that the −46::Cre fusion construct can be efficiently and easily utilized for producing marker-free transgenic plants.  相似文献   

10.
3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the -glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R2 pollen of R1 progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.  相似文献   

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