Patent Application
20080050819
(e) Northern blot analysis of dao1 mRNA levels from six D-serine- and D-alanine-resistant lines and wild-type plants. Ten μg total RNA was loaded per lane and separated on an agarose gel. Ethidium bromide-stained total RNA bands are shown as loading controls. (f) DAAO activity in six transgenic lines and wild type. A unit of DAAO activity is defined as the turnover of one micromole of substrate per minute. Bars represent means ±s.e.m., n=3
FIG. 6+7 Demonstration of broad applicability of the selection system. D-serine is imposing toxic effects on a variety of different plant species both monocotyledonous and dicotyledonous plants. Effects are demonstrated for popular (
FIG.: 8 Preferred basic construct and method
FIG.: 9 Construct mediating marker excision via induced homologous recombination The DNA construct introduced into the plant genome by utilizing the negative selection marker function of the dual-function marker is comprising: A first expression cassette for the dual-function marker (DAAO) under control of a promoter functional in plants (P1) and a second expression cassette for an agronomically valuable trait (TRAIT) also under control of (preferably a different) promoter functional in plants (P2). The first expression cassette is flanked by homology sequences A and A′ which allow for homologous recombination between each other, being arranged in form of a directed repeat. Within the DNA construct there is at least one (preferably—as depicted here—two) recognition sequences (RS) (cleavage sites) for a sequence specific endonuclease (RS1, RS2). The two sequences may be different (i.e., for different endonucleases) or—preferably—identical. Cleavage at said recognition sequences (RS1 and RS2) is initiated by the corresponding endonuclease (1.) resulting in double-strand breaks, which are “repaired” by homologous recombination between the homologous end-sequences (probably supported by the cellular DNA repair mechanism). The resulting genome still comprises the second expression cassette for the trait but lacks the first expression cassette for the dual-function marker. Selection is performed making use of the counter-selection function of the dual function marker (2.) e.g., employing D-isoleucine or D-valine. Thereby plant cells or plants are selected comprising the second expression cassette but lacking the first expression cassette.
FIG.: 11 D-amino acids are applicable by spraying procedure DAAO transgenic plants grown on soil photographed after selection by spraying with (1) D-alanine and (2) D-serine, and wild-type plants sprayed with (3) D-alanine and (4) D-serine. Eight seeds per plot and treatment were sown on soil, and grown for 7 d after germination before applying the selective treatment, which consisted of spraying with aqueous 50 mM solutions of D-alanine or D-serine with 0.05% Tween 80 on three consecutive days.
FIG.: 12 Alignment of the catalytic site of various D-amino acid oxidases
FIG.: 13 Vector map of construct daaoSceITetOn (Seq ID NO: 15) (length: 12466 bp)
FIG.: 14 Vector map of construct daaoNit-PRecombination (Seq ID NO: 16) (length: 12539 bp)
The chemical synthesis of oligonucleotides can be effected for example in the known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The cloning steps carried out for the purposes of the pre-sent invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, the transfer of nucleic acids to nitrocellulose and nylon membranes, the linkage of DNA fragments, the transformation of E. coli cells, bacterial cultures, the propagation of phages and the sequence analysis of recombinant DNA are carried out as described by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules were sequenced using an ALF Express laser fluorescence DNA sequencer (Pharmacia, Sweden) following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).
DNA and RNA manipulation were done using standard techniques.
The yeast R. gracilis was grown in liquid culture containing 30 mM D-alanine to induce dao1, the gene encoding DAAO. Total RNA was isolated from the yeast and used for cDNA synthesis. The PCR primers
were used to amplify the dao1 gene from the cDNA template by PCR. The PCR fragments were sub-cloned into the pGEM-T Easy vector (Promega) and subsequently ligated into the BamHI site of the CaMV 35S expression cassette of the binary vector pPCV702kana17 giving pPCV702:dao1. The vectors were subjected to restriction analysis and sequencing to check that they contained the correct constructs.
A. thaliana plants (ecotype Col-0) were grown in soil until they flowered. Agrobacterium tumefaciens (strain GV3101:pMP110 RK) transformed with the construct of interest was grown in 500 mL in liquid YEB medium (5 g/L Beef extract, 1 g/L Yeast Extract (Duchefa), 5 g/L Peptone (Duchefa), 5 g/L sucrose (Duchefa), 0.49 g/L MgSO4 (Merck)) until the culture reached an OD600 0.8-1.0. The bacterial cells were harvested by centrifugation (15 minutes, 5,000 rpm) and resuspended in 500 mL infiltration solution (5% sucrose, 0.05% SILWET L-77 [distributed by Lehle seeds, Cat. No. VIS-02]).
Flowering A. thaliana plants were then transformed by the floral dip method (Clough S J & Bent A F (1998) Plant J. 16, 735-743 (1998) with the transgenic Agrobacterium tumefaciens strain carrying the vector described above by dipping for 10-20 seconds into the Agrobacterium solution. Afterwards the plants were kept in the greenhouse until seeds could be harvested. Transgenic seeds were selected by plating surface sterilized seeds on growth medium A (4.4 g/L MS salts [Sigma-Aldrich], 0.5 g/L MES [Duchefa]; 8 g/L Plant Agar [Duchefa]) supplemented with 50 mg/L kanamycin for plants carrying the nptII resistance marker, or 0.3 to 30 mM D-amino acids (as described below) for plants comprising the dual-function marker of the invention. Surviving plants were transferred to soil and grown in the greenhouse.
Lines containing a single T-DNA insertion locus were selected by statistical analysis of T-DNA segregation in the T2 population that germinated on kanamycin or D-amino acid-containing medium. Plants with a single locus of inserted T-DNA were grown and self-fertilized. Homozygous T3 seed stocks were then identified by analyzing T-DNA segregation in T3 progenies and confirmed to be expressing the introduced gene by northern blot analyses.
Agrobacterium tumefaciens strain GV3101 transformed with the plasmid of interest was grown in 50 mL YEB medium (see Example 4a) at 28° C. overnight. The Agrobacterium solution is mixed with liquid co-cultivation medium (double concentrated MSB5 salts (Duchefa), 30 g/L sucrose (Duchefa), 3.75 mg/l BAP (6-benzylamino purine, Duchefa), 0.5 g/l MES (Duchefa), 0.5 mg/l GA3 (Gibberellic Acid, Duchefa); pH5.2) until OD600 of 0.5 is reached. Petiols of 4 days old seedlings of Brassica napus cv. Westar grown on growth medium B (MSB5 salts (Duchefa), 3% sucrose (Duchefa), 0.8% oxoidagar (Oxoid GmbH); pH 5, 8) are cut. Petiols are dipped for 2-3 seconds in the Agrobacterium solution and afterwards put into solid medium for co-cultivation (co-cultivation medium supplemented with 1.6% Oxoidagar). The co-cultivation lasts 3 days (at 24° C. and ˜50 μMol/m2s light intensity). Afterwards petiols are transferred to co-cultivation medium supplemented with the appropriate selection agent (18 mg/L kanamycin (Duchefa) for plants comprising the nptII marker kanamycin for plants carrying the nptII resistance marker, or 0.3 to 30 mM D-amino acids; as described below) for plants comprising the dual-function marker of the invention) and 300 mg/L Timetin (Duchefa)
Transformed petioles are incubated on the selection medium for four weeks at 24° C. This step is repeated until shoots appear. Shoots are transferred to A6 medium (MS salts (Sigma Aldrich), 20 g/L sucrose, 100 mg/L myo-inositol (Duchefa), 40 mg/L adeninesulfate (Sigma Aldrich), 500 mg/L MES, 0.0025 mg/L BAP (Sigma), 5 g/L oxoidagar (Oxoid GmbH), 150 mg/L timetin (Duchefa), 0.1 mg/L IBA (indol butyric acid, Duchefa); pH 5, 8) supplemented with the appropriate selection agent (18 mg/L kanamycin (Duchefa) for plants comprising the nptII marker kanamycin for plants carrying the nptII resistance marker, or 0.3 to 30 mM D-amino acids; as described below) until they elongated. Elongated shoots are cultivated in A7 medium (A6 medium without BAP) for rooting. Rooted plants are transferred to soil and grown in the greenhouse.
T1 seeds of transgenic Arabidopsis plants were surface-sterilized and sown in Petri plates that were sealed with gas-permeable tape. The growth medium was half strength MS19 with 0.5% (wt/vol) sucrose and 0.8% (wt/vol) agar, plus 3 mM D-alanine, 3 mM D-serine or 50 μg/ml kanamycin as the selective agent. Plants were grown for 5 d after germination with a 16 h photoperiod at 24° C. To evaluate the selection efficiency on different substrates, 2,074, 1,914 and 1,810 T1 seeds were sown on D-alanine-, D-serine- and kanamycin-selective plates, respectively, and the number of surviving seedlings was counted (44, 32 and 43, respectively).
To evaluate the toxic action of 3-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate, wild-type plants were sown on two sets of half strength MS agar plates, each containing one of the compounds in a range of concentrations (0.01-10 mM). Plants were slightly affected by 3-methyl-2-oxopentanoate at 0.1 mM, and total growth inhibition was observed at 1 mM. For 3-methyl-2-oxobutanoate, 5 mM was required for complete inhibition. Further, several attempts were made to probe the nature of D-serine's toxicity. In accordance with studies on E. coli, wildtype it was tried to rescue A. thaliana grown on lethal concentrations of D-serine through amendments with five potential inhibitors of D-serine toxicity (L-serine, calcium-pantothenate, β-alanine, leucine and threonine) added both separately and in combinations in a very wide range of concentrations (0.001-50 μg ml-1), without any success.
Soluble proteins were extracted by shaking 0.1 g samples of plant material that had been finely pulverized in a 1.5 ml Eppendorf tube in 1 ml of 0.1 M potassium phosphate buffer, pH 8. DAAO activity was then assayed as follows. Reaction mixtures were pre-pared containing 2,120 μl of 0.1 M potassium phosphate buffer, pH 8, 80 μl of crude protein extract and 100 μl of 0.3 M D-alanine. The samples were incubated for 2 h at 30° C. The enzyme activity was then assessed, by measuring the increase in absorbance at 220 nm (E=1.090 M−1 cm−1) associated with the conversion of D-alanine to pyruvate, after transferring the test tubes to boiling water for 10 min to stop the reaction. In control reactions, D-alanine was added immediately before boiling. One unit of DAAO activity is defined as the turnover of one micromole of substrate per minute, and activity was expressed per gram plant biomass (fresh weight). The breakdown of D-isoleucine and D-valine in DAAO incubations, and the associated production of 3-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate, were analyzed by high-performance liquid chromatography. In other respects the reactions were carried out as described above.
The qualification of the DAAO enzyme as a dual-function selection marker was demonstrated by testing germinated T1 seeds on different selective media. The T-DNA contained both 35S:dao1 and pNos:nptII, allowing D-amino acid and kanamycin selection to be compared in the same lot of seeds.
T1 seeds were sown on medium containing kanamycin (50 μg/ml), D-alanine (3 mM) or D-serine (3 mM), and the transformation frequencies found on the different selective media were 2.37%, 2.12% and 1.67%, respectively (
Further studies using lower concentrations corroborated this conclusion, and efficient selection using D-serine was achieved on concentrations lower than 1 mM (
Selection of seedlings on media containing D-alanine or D-serine was very rapid compared to selection on kanamycin. These D-amino acids inhibited growth of wild-type plants immediately after the cotyledons of wild-type plants had emerged. Therefore, transformants could be distinguished from non-transformed plants directly after germination. The difference between wild-type and transgenic plants after D-amino acid selection was unambiguous, with no intermediate phenotypes. In contrast, intermediate phenotypes are common when kanamycin resistance is used as a selection marker (
Transgenic plants grown under D-alanine and D-serine selection conditions developed normally. Early development of transgenic plants from line 3:7, 10:7 and 13:4 was compared with that of wild-type plants by cultivation on vertical agar plates. No differences in biomass, number of leaves, root length or root architecture were detected for the different sets of plants. Furthermore, soil-cultivated wild-type and transgenic plants (line 10:7) showed no differences in the total number of rosette leaves, number of inflorescences and number of siliqua after 4 weeks of growth.
Also, the phenotypes of 17 individual T1 lines, which were picked for T-DNA segregation, were studied and found indistinguishable from that of wild type when grown on soil. A problem sometimes encountered after selection on antibiotics is the growth lag displayed by transformants. This phenomenon is explained as an inhibitory effect of the antibiotic on the transgenic plants (Lindsey K & Gallois P (1990) J. Exp. Bot 41, 529-536). However, unlike seedlings picked from antibiotic selection plates, transgenic seedlings picked from D-amino acid selection plates and transferred to soil were not hampered in their growth and development, even temporarily. A possible reason for this difference is that the DAAO scavenging of D-amino acids may effectively remove the D-amino acid in the plants. Furthermore, D-alanine and D-serine may merely provide additional growth substrates, because their catabolic products are carbon and nitrogen compounds that are central compounds in plant metabolism. Quantification of dao1 mRNA from six independent D-alanine- and D-serine-resistant lines showed a range of different expression levels (
Therefore, plants containing the construct were tested as described above on two sets of media, one containing D-isoleucine and the other containing D-valine at various concentrations, to assess whether DAAO could also be used as a counter-selection marker. Unambiguous counter-selection selection was achieved when seeds were sown on either D-isoleucine or D-valine at concentrations greater than 10 mM (
Thirteen individual lines expressing DAAO were tested for their response to D-isoleucine and all of them were effectively killed, whereas wild-type plants grew well, with no sign of toxicity. Similar results were obtained for D-valine, although this compound was found to have a moderately negative effect on wild-type plants at higher concentrations (
Presumably endogenous transaminase may be specific for the L-enantiomer, so the corresponding D-enantiomer is not metabolized in wildtype plants, but only in plants expressing DAAO. The negative effects of L-isoleucine (but not of the D-form) observed on wildtype plants, supports this speculation. Incubation of cell-free extracts from dao1 transgenic line 10:7 with D-isoleucine and D-valine resulted in 15-fold and 7-fold increases in production of 3-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate, respectively, compared to extracts of wild-type plants. Further, 3-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate impaired growth of A. thaliana, corroborating the suggestion that these compounds, or products of their metabolism, are responsible for the negative effects of D-isoleucine and D-valine on the transgenic plants.
The toxicity of some D-amino acids on organisms is not well understood, and has only occasionally been studied in plants (Gamburg K Z & Rekoslavskaya N I (1991) Fiziologiya Rastenii 38, 1236-1246). Apart from A. thaliana, we have also tested the susceptibility of other plant species to D-serine, including poplar, tobacco, barley, maize, tomato and spruce. We found all tested species susceptible to D-serine at concentrations similar to those shown to be toxic for A. thaliana. A proposed mechanism for D-serine toxicity in bacteria is competitive inhibition of a-alanine coupling to pantoic acid, thus inhibiting formation of pantothenic acid (Cosloy S D & McFall E (1973) J. Bacteriol. 114, 685-694). It is possible to alleviate D-serine toxicity in D-serine-sensitive strains of Escherichia coli by providing pantothenic acid or â-alanine in the medium, but D-serine toxicity in A. thaliana could not be mitigated using these compounds. A second putative cause of D-amino acid toxicity is through competitive binding to tRNA. Knockout studies of the gene encoding D-Tyr-tRNATyr deacylase in E coli have shown that the toxicity of D-tyrosine increases in the absence of deacylase activity (Soutourina J et al. (1996) J. Biol. Chem. 274, 19109-19114), indicating that D-amino acids interfere at the tRNA level. Genes similar to that encoding bacterial deacylase have also been identified in A. thaliana (Soutourina J et al. (1996) J. Biol. Chem. 274, 19109-19114), corroborating the possibility that the mode of toxic action of D-amino acids might be through competitive binding to tRNA.
Two expression constructs are constructed for carrying out the present invention (SEQ ID NO: 15, 16). The backbone of both plasmid constructs (pSUN derivative) contains origins for the propagation in E coli as well as in Agrobacterium and an aadA expression cassette (conferring spectinomycin and streptomycin resistance) to select for transgenic bacteria cells. The sequences for constructing the DNA constructs are amplified incorporating the appropriate restriction sites for subsequent cloning by PCR. Cloning was done by standard methods as described above. The sequence of the constructs is verified by DNA sequence analysis.
The first DNA construct (SEQ ID NO: 16) comprises an expression cassette for the D-amino acid oxidase (DAAO) from Rhodotorula gracilis under control of the Arabidopsis thaliana Nitrilase promoter. The DAAO cassette is flanked by a direct repeat of the 35S terminator functioning both as transcription terminator of the DAAO expression cassette and as homology sequences.
Further comprised is a expression cassette for the β-glucuronidase which may function as a substitute for an agronomically valuable trait under control of the Arabidopsis sTPT promoter (i.e. TPT promoter truncated version, WO 03/006660; SEQ ID NO: 27 cited therein), and the CaMV 35S terminator.
The second DNA (SEQ ID NO: 15) comprises an expression cassette for the D-amino acid oxidase (DAAO) from Rhodotorula gracilis under control of the Arabidopsis thaliana Nitrilase promoter. The DNA construct further comprises a Tet on expression system. This allows for induced expression of the I-Sce-I homing endonuclease which is placed under control of a Tet-regulatable promoter. The system further requires expression of the Tet-repressor, which is realized under control of the constitutive ptXA promoter from Pisum sativa.
Both the sequences encoding the DAAO cassette, the I-Sce-I expression cassette, and the rtTA expression cassette (for the reverse tetracycline responsive repressor) are flanked by a direct repeat of the 35S terminator functioning both as transcription terminator of the I-Sce-I expression cassette and as homology sequences.
Arabidopsis thaliana plants are transformed as described above with a mixture of DNA construct I (binary vector SEQ ID NO: 16) and a second binary vector comprising a GFP (green fluorescence protein) expression cassette. In a first selection process transgenic plants are selected comprising both constructs by employing D-alanine mediated selection. 3 mM and 30 mM D-alanine are used.
D-alanine resistant plants comprising the first DNA construct (detectable by GUS staining) also comprising the gfp gene (as assessed by green fluorescence) are isolated and crossed with wild-type plants. Resulting seeds are used for a second counter-selection process, wherein said seeds are germinated on D-isoleucine comprising medium (comprising either 3 mM or 30 mM D-isoleucine). D-isoleucine resistant plants—comprising the gfp gene—can be easily selected.
Arabidopsis thaliana plants are transformed as described above with a mixture of DNA construct I (binary vector SEQ ID NO: 16). In a first selection process transgenic plants are selected comprising construct I by employing D-alanine mediated selection. 3 mM and 30 mM D-alanine are used.
D-alanine resistant plants comprising the first DNA construct (detectable by GUS staining) are isolated and crossed with a transgenic master plant comprising a transgenic expression cassette for the I-Sce-I homing endonuclease under control of a constitutive promoter (as described in WO 03/004659). Resulting seeds are used for a second counter-selection process, wherein said seeds are germinated on D-isoleucine comprising medium (comprising either 3 mM or 30 mM D-isoleucine). D-isoleucine resistant plants still comprising the GUS-expression cassette can be easily selected.
Arabidopsis thaliana plants are transformed as described above with a mixture of DNA construct II (binary vector SEQ ID NO: 15). In a first selection process transgenic plants are selected comprising construct II by employing D-alanine mediated selection. 3 mM and 30 mM D-alanine are used.
D-alanine resistant plant cells comprising the DNA construct II are isolated and further cultivated on medium lacking D-alanine. Doxycycline (Sigma; 1 to 5 μg/ml) is added for induction of the marker excision process and cells are incubated for 24 to 48 h on said induction medium. Subsequently cells are further incubated for 3 to 5 days on medium lacking the inducer and D-amino acids (to allow for reduction of DAAO protein levels from prior expression). The resulting cells are used for a second counter-selection process, wherein said cells are further selected on D-isoleucine comprising medium (comprising either 3 mM or 30 mM D-isoleucine). Selected D-isoleucine resistant cells are regenerated into fertile plants and assessed for their transgenic status. By PCR mediated analysis it can be demonstrated that the region flanked by the 35S terminator sequences was accurately excised from the plant genome deleting both the I-SceI expression cassett, the DAAO expression cassette, and the rtTA expression cassette (for the reverse tetracycline responsive repressor)
| Number | Date | Country | Kind |
|---|---|---|---|
| 04006358.8 | Mar 2004 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP05/02734 | 3/15/2005 | WO | 00 | 9/15/2006 |
