WO1997030163A1 - Plants having enhanced nitrogen assimilation/metabolism - Google Patents
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- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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Definitions
- the present invention relates to genetic engineering of plants to display enhanced agronomic characteristics and, in particular, the genetic engineering of plants to display enhanced agronomic characteristics by enhancing the nitrogen assimilatory and metabolism capacities of the plants.
- nitrogen In many ecosystems, both natural and agricultural, the primary productivity of plants is limited by the three primary nutrients: nitrogen, phosphorous and potassium. The most important of these three limiting nutrients is usually nitrogen.
- Nitrogen sources are often the major components in fertilizers (Hageman and Lambert, 1988, In. Corn and Corn Improvement, 3rd ed., Sprague & Dudley, American Society of Agronomy, pp. 431-461). Since nitrogen is usually the rate-limiting element in plant growth, most field crops have a fundamental dependence on inorganic nitrogenous fertilizer. The nitrogen source in fertilizer is usually ammonium nitrate, potassium nitrate, or urea. A significant percentage of the costs associated with crop production result from necessary fertilizer applications.
- Nitrogen is taken up by plants primarily as either nitrate (NO 3 ) or ammonium
- NH 4 + is the predominate form of inorganic nitrogen present and can be taken up directly by plants.
- GS glutamine synthetase
- GGAT glutamate synthase
- nitrate and ammonia can be detected in the transporting vessels (xylem and phloem), the majority of nitrogen is first assimilated into organic form (e.g., amino acids) which are then transported within the plant.
- organic form e.g., amino acids
- Glutamine, asparagine and aspartate appear to be important in determining a plant's ability to take up nitrogen, since they represent the major long-distance nitrogen transport compounds in plants and are abundant in phloem sap. Aside from their common roles as nitrogen carriers, these amino acids have somewhat different roles in plant nitrogen metabolism. Glutamine is more metabolically active and can directly donate its amide nitrogen to a large number of substrates. Because of this reactivity, glutamine is generally not used by plants to store nitrogen.
- asparagine is a more efficient compound for nitrogen transport and storage because of its higher N:C ratio. Asparagine is also more stable than glutamine and can accumulate to higher levels in vacuoles. Indeed, in plants that have high nitrogen assimilatory capacities, asparagine appears to play a dominant role in the transport and metabolism of nitrogen (Lam et al, 1995, Plant Cell 7: 887-898). Because of its relative stability, asparagine does not directly participate in nitrogen metabolism, but must be first hydrolysed by the enzyme asparaginase (ANS) to produce aspartate and ammonia which then can be utilized in the synthesis of amino acids and proteins. However, in addition to aspartate and asparagine, a number of other amino acids can act as storage compounds.
- ANS asparaginase
- the solutes that accumulate during osmotic adjustment include sugars, organic acids and amino acids, such as alanine, aspartate, proline and glycine betaine (Good and Zaplachinski, 1994, Physiol Plant 90: 9- 14; Hanson and Hitz, 1982, Annu Rev Plant Physiol 33: 163-203; Jones and Turner, 1978, Plant Physiol 61 : 122-126).
- Corn, cotton, soybean and wheat have all demonstrated osmotic adjustment during drought (Morgan, ibid.).
- One of the best characterized osmoregulatory responses is the accumulation of proline (Hanson and Hitz, ibid.).
- proline levels increase as much as 100-fold in response to osmotic stress (Voetberg and Sharp, 1991, Plant Physiol 96: 1125-1130).
- the accumulation of proline results from an increased flux of glutamate to pyrroline-5-carboxylate and proline in the proline biosynthetic pathway, as well as decreased rates of proline catabolism (Rhodes et al., 1986, Plant Physiol 82:890-903; Stewart et al., 1977, Plant Physiol. 59:930-932).
- Alanine is one of the common amino acids in plants. In Brassica leaves under normal conditions, alanine and aspartate concentrations are roughly equal and have been found to be twice that of asparagine concentrations. In comparison, glutamate levels were double that of alanine or aspartate (Good and Zaplachinski, ibid.). Alanine is synthesized by the enzyme alanine aminotransferase (AlaAT) from pyruvate and glutamate in a reversible reaction (Goodwin and Mercer, 1983, Introduction to Plant Biochemistry 2nd Ed., Pergamon Press, New York, N.Y., pp. 341-343), as shown in Figure 2.
- AlAT alanine aminotransferase
- alanine is an amino acid that is known to increase under other specific environmental conditions such as anaerobic stress (Muench and Good, 1994, Plant Mol. Biol. 24:417-427; Vanlerberge et al., 1993, Plant Physiol. 95:655-658).
- Alanine levels are known to increase substantially in root tissue under anaerobic stress. As an example, in barley roots alanine levels increase 20 fold after 24 hours of anaerobic stress.
- the alanine aminotransferase gene has also been shown to be induced by light in broom millet and when plants are recovering from nitrogen stress (Son et al., 1992, Arch Biochem Biophys 289: 262-266). Vanlerberge et al. (1993) have shown that in nitrogen starved anaerobic algae, the addition of nitrogen in the form of ammonia resulted in 93% of an N 15 label being inco ⁇ orated directly into alanine. Thus, alanine appears to be an important amino acid in stress response in plants.
- nitrate transporter genes Tsay et al. 1993. Cell 72:705; Unkles et al. 1991 PNAS 88:204), nitrate reductase (NR) and nitrite reductase (NiR) (Crawford, 1995, Plant Cell
- Glutamine synthetase (GS) and glutamate synthetase (GOGAT) have been cloned (Lam et al., ibid.; Zehnacker et al., 1992, Planta 187:266-274; Peterman and Goodman, 1991, Mol. Gen. Genet.
- Gen. Genet. 236:315-325 has reported increases in total soluble protein content in transgenic tobacco plants overexpressing an alfalfa GS gene and similar increases in total soluble protein content in transgenic tobacco plants expressing antisense RNA to a GS gene.
- this invention involves a genetic construct which comprises a nitrogen assimilation/metabolism pathway enzyme coding sequence operably associated with an inducible promoter.
- a genetic construct acts to confer to a plant or plant cell, into which it is introduced, enhanced agronomic characteristics.
- Such genetic constructs can be inserted into plant transformation vectors and/or introduced to plant cells. Transformed plants can be produced which contain the genetic construct of the present invention.
- a genetic construct adapted for expression in a plant system comprising a nitrogen assimilation/metabolism enzyme coding sequence operably associated with an inducible promoter
- a method for producing a plant comprising: fransforming a plant cell by introducing a genetic construct having a nitrogen assimilation/metabolism enzyme coding sequence operably associated with an inducible promoter; and regenerating the plant cell to a plant.
- the promoter is selected to be inducible and preferably inducible in response to a condition where it would be desirable to cause the plant to have enhanced nitrogen uptake, assimilation or use capabilities.
- suitable promoters include, but are not limited to, those which are: induced by application of sources of nitrogen; stress inducible; wound inducible or induced by application of other chemicals.
- Transgenic plants containing the genetic construct of the present invention exhibit enhanced agronomic characteristics over control plants or plants having constitutively over-expressed nitrogen assimilation/metabolism genes.
- the agronomic characteristic which is enhanced over prior art plants can include enhanced stress tolerance and or more efficient nitrogen uptake, storage or metabolism allowing the plants of the present invention to be cultivated with lower nitrogen fertilizer input and in nitrogen starved conditions or allowing faster growth, greater vegetative and/or reproductive yield.
- Plant cells, vectors and plants including the genetic construct are also provided according to the present invention.
- a genetic construct adapted for expression in a plant system comprising a coding region for alanine aminotransferase enzyme operably associated with a promoter element from Brassica turgor gene-26.
- Plant cells, vectors and plants including the genetic construct are also provided according to the present invention.
- Figure 1 Major pathways of nitrogen assimilation and metabolism in plants.
- Nitrogen assimilation occurs primarily through the activities of glutamine synthetase (GS) and glutamate synthase (GOGAT). While not indicated as such, aspartate aminotransferase also catalyses the reverse reaction. The roles of glutamate dehydrogenase (GDH) are postulated, as indicated by the dashed lines.
- Figure 2 Pathway for alanine biosynthesis by the enzyme alanine aminotransferase (AlaAT) (From Goodwin and Mercer, 1983).
- Figure 3 DNA sequence of the Brassica napus btg-26 promoter.
- Figure 4A Northern blot analysis of btg-26 expression during droughting. Total RNA (10 mg) from leaf tissue taken from control plants having 97% relative water content (97% RWC) and plants dehydrated to the % RWC's indicated, was fractionated on a 1.2% agarose formaldehyde gel and probed with btg-26 genomic DNA.
- Figure 4B Quantitative analysis of btg-26 induction. Each time point represents the mean induction determined from three independent slot blots and two Northern blots. All blots were reprobed with a cyclophilin cDNA control to correct for loading error. Induction is determined relative to the level of expression in fully hydrated plants (97%).
- Figure 4C Northern blot analysis of btg-26 expression during cold acclimation and heat shock.
- Figure 4D Northern blot analysis of btg-26 expression during salinity stress.
- the RNA was fractionated on a 1.2% agarose formaldehyde gel and probed with btg-26 genomic DNA.
- Figure 4E Northern blot analysis of btg-26 expression during exposure to abscisic acid (ABA).
- Figure 5 Nucleotide and deduced amino acid sequence of the AlaAT cDNA from barley.
- Figure 6 Plasmid construct p25.
- Figures 7 A to 7C Plasmid constructs containing the AlaAT coding region and the CaMV, btg-26 and trg-31 promoters that were used for the transformation of Brassica napus plants.
- Figure 8 Plasmid construct pCGNl 547 used in producing the overexpressed/ AlaAT or stress inducible/AlaAT transformants.
- Figure 9 Brassica napus plants grown under nitrogen starved conditions for three weeks followed by drought for 3 days.
- the plants are identified as A, B and C, as follows: Plant A is a control, wild-type plant; Plant B contains a CaMV/ AlaAT construct; and Plant C contains a btg-26/AlaAT construct.
- the present invention is directed to a genetic construct having a coding sequence of a nitrogen assimilation/metabolism enzyme operably linked to a inducible promoter.
- the promoter sequence is preferably inducible under conditions where it is desirable for plants to take-up, store and/or use nitrogen.
- Such a gene can be in purified or isolated form or introduced to an expression cassette, cloning or transformation vector for use in the transformation of plant cells or plants.
- mRNA Transcription of DNA into messanger RNA (mRNA) is regulated by a region of the gene known as the promoter.
- the promoter sequences useful in the present invention are those which are inducible and, preferably, those which regulate gene expression in response to a condition in which it would be desirable to cause a plant to assimilate and/or metabolize nitrogen.
- Many inducible promoters are known in the art and for example, include, but are not limited to, promoters induced upon the addition of nitrogen or when a plant is under nitrogen stress, those induced by specific environmental conditions, such as drought stress, osmotic stress, wound stress, heat stress, anaerobic stress, and salt stress; or promoters induced by addition of chemical agents, such as for example auxins.
- such genetic constructs When used to transform plants, such genetic constructs can provide transformed plants with enhanced agronomic characteristics and which it is postulated can assimilate and/or use nitrogen when it is most needed or beneficial.
- the promoter is a stress inducible promoter such as, for example, the inducible promoters 26g from Pisum sativum (Guerrero and Mullet, 1988, Plant Physiol, 88:401-408; Guerrero et al., 1990, Plant Mol Biol 15: 11-26), trg-31 from tobacco (Guerrero and Crossland, 1993, Plant Mol Biol 21 :929-935) or btg-26 from Brassica napus as discussed in Example 1 and shown in Figure 3 and SEQ ID NO: 1 , the plant is induced to produce the gene product upon application of a suitable stress to drive the promoter. Such a plant thereby can have increased stress tolerance, such as by enhanced osmoregulation.
- the promoter is induced by the presence of nitrate, such as, for example, the nitrate reductase promoter (Cheng et al, 1988, ibid.; Cheng et al, 1991 ,
- the plant will be induced to assimilate and/or use nitrogen upon application of a nitrogenous fertilizer.
- the promoter can be inducible under nitrogen stress conditions (Son D. et al, 1992, Plant Cell Physio, 33: 507-509) so that a plant's ability to take up and use nitrogen efficiently is increased under conditions of low nitrogen.
- the promoter can also be induced by an exogenously applied chemical such as, for example, copper (Met et al., 1993, PNAS 90:4567) or abscisic acid (Marcotte et al, 1989, Plant Cell 1 :969-976). These chemicals can be included in formulations for nitrogenous fertilizer.
- the inducible promoter useful in the present invention can be homologous or heterologous to the plant to which it is to be introduced. Multiple copies of the promoters can be used.
- the promoters may be modified, if desired, to alter their expression characteristics, for example, their expression levels and/or tissue specificity.
- the inducible promoter is selected, or ligated to another sequence, or otherwise modified, to exhibit root specific activity.
- a root specific promoter is the GS 15 as described by Hirel et al, 1992, Plant Mol Biol. 20:207-218.
- the selected wild-type or modified inducible promoter can be used as described herein.
- the coding regions of interest are those for enzymes active in the assimilation and/or metabolism of nitrogen and, preferably, those which are active in the assimilation of ammonia into amino acids or those which use the formed amino acids in biosynthetic reactions.
- these enzymes include, but are not limited to, glutamine synthetase (GS), asparagine synthetase (AS), glutamate synthase (also known as glutamate 2: oxogluturate amino transferase and GOGAT), asparaginase (ANS), glutamate dehydrogenase (GDH), aspartate aminotransferase (AspAT) and alanine aminotransferase (AlaAT) (activity shown in Figure 2).
- glutamine synthetase GS
- AS asparagine synthetase
- glutamate synthase also known as glutamate 2: oxogluturate amino transferase and GOGAT
- ANS glut
- Sequence information for these enzymes is known, for example, as follows: GS from soybean (Hirel et al, ibid); AS from pea (Tsai, F-Y and Coruzzi, G.M. 1990, EMBO J. 9: 232-332); GOGAT from tobacco (Zehnacker, C. et al, 1992. Planta 187:226-274); ANS (Casado, A. et al. 1995 Plant Physiol. 108:1321); AspAT from alfalfa (Udvardi, M. And Kahn, M. 1991. Mol. Gen.
- nitrogen assimilation/metabolism enzymes which are of interest are those for nitrogen transporters (Tsay et al. 1993. Cell 72:705 and Unkles et al. 1991 PNAS 88:204) and/or nitrate reductase (Vincentz, M. And Caboche, M. 1991 EMBO J. 10:1027-1035).
- the nitrogen assimilation/metabolism enzyme coding regions can be used in their wild-type forms or can be altered or modified in any suitable way to achieve a desired improvement.
- These coding region can be obtained from any source and can be homologous or heterologous to the plant cell into which it is to be introduced.
- the coding region is heterologous to the promoter to which it is linked, in that it is not linked to an unmodified, inducible promoter to which the coding region is naturally linked.
- the genetic construct can be modified in any suitable way to provide for expression in plants.
- the genetic construct is adapted to be transcribable and translatable in a plant system, and, for example, contains all of the necessary 5' and 3' non-translated regions including poly-adenylation sequences, start sites and termination sites which allow the coding sequence to be transcribed to mRNA (messenger ribonucleic acid) and the mRNA to be translated in the plant system. These sequences and elements can be obtained from any source.
- the gene construct can be introduced to a plant transformation or cloning vector.
- the vectors will, as is known, contain sequences suitable for inclusion in such vectors, such as one or more bacterial or plant-expressible selectable or screenable markers, for example a Kanamycin resistance gene (NPTII), ⁇ -glucuronidase (GUS) genes and/or sequences required for replication and transformation such as, for example the right and, optionally, the left T-DNA borders, where the vector is to be used in an Agrobacterium-mediated transformation system.
- NPTII Kanamycin resistance gene
- GUS ⁇ -glucuronidase
- cloning or transformation vectors are for example plasmids, cosmids and/or viral DNA or RNA. Methods for preparation of such genetic constructs and vectors are well known in the art.
- the gene construct of the present invention can be introduced to a plant cell by any useful method.
- a large number of processes are available and are well known to deliver genes to plant cells.
- One of the best known processes involves the use of Agrobacterium or similar soil bacteria as a means for introduction of the genetic construct into the plant.
- target tissues are co-cultivated with Agrobacterium which inserts the gene of interest to the plant genome.
- Such methods are well known in the art as illustrated by US Patent 4,940,838 of Schilperoort et al., Horsch et al. 1985, Science 227:1229-1231, Fisher and Guiltinan, 1995, Plant Mol. Biol. Reporter 13:278, and Bayley et al., 1992, Theoret.
- Alternative gene transfer and transformation methods useful in the present invention include, but are not limited to liposomes, electroporation or chemical-mediated uptake of free DNA, targeted microprojectiles and microinjection. These methods are well documented in the prior art.
- Cells that have been transformed with the gene construct of the present mvention can be regenerated into differentiated plants using standard nutrient media supplemented with shoot-inducing or root-inducing hormones, using methods known to those skilled in the art.
- Suitable plants for the practice of the present invention include, but are not limited to, canola, corn, rice, tobacco, soybean, cotton, alfalfa, tomato, wheat and potato. Many other plants can also be advantageously engineered with the gene of the present invention.
- Transformed plants according to the present invention can be used for breeding, crop production and the like.
- the genetic construct of the present invention can include any promoter which is inducible, a particularly preferred promoter is the btg-26 which was found to be inducible by stresses such as heat shock, drought, salinity and ABA concentration.
- the btg-26 promoter element is as depicted in Figure 3 and SEQ ID NO: 1.
- Nucleotide sequences homologous to the btg-26 promoter described herein are those nucleic acid sequences which are capable of hybridizing to the nucleic acid sequence depicted in Figure 3 (SEQ ID NO: 1) in standard hybridization assays or are homologous by sequence analysis (at least 45% of the nucleotides are identical to the sequence presented herein) using the BLAST programs of Altschul et al (1989, J. Mol. Biol. 215:403-410).
- Homologous nucleotide sequences refer to nucleotide sequences including, but not limited to, promoter elements from other plant species or genetically engineered derivatives of the promoter element according to the present invention.
- the promoter element can be engineered to alter its activity, for example its level of expression. Such engineered promoters are useful in the present invention.
- Example 1 Isolation and characterization of osmotic stress-induced promoter
- a Brassica napus (cv. Bridger) genomic DNA library (Clontech, Palo Alto, California) was screened using standard techniques (Ausubel et al., 1989, Current Protocols in Molecular Biology, Wiley, Wiley, N.Y.) with the Pisum sativum 26g cDNA (complementary deoxyribonucleic acid) clone (Guerrero et al., ibid), 32 P-labelled with a Random Primer Kit (Boehringer Mannheim, Laval, Quebec).
- sativum 26g gene (Guerrero et al., ibid) was used. Total RNA was isolated from the third leaf of whole plants that had been either watered continuously or dehydrated for four days. Using low stringency hybridization, RNA blot analysis identified a single 1.75 kb transcript that is greatly induced in draughted plants. To determine if this mRNA represents a single copy gene in B. napus, genomic DNA was digested with EcoRI, Hindlll or Bglll and analyzed by DNA blot hybridization using the P. sativum 26g cDNA. A single band was identified in each lane. It was concluded that this transcript represents a single copy, drought-induced gene in B. napus. This gene is referred to as btg-26 (Brassica turgor gene - 26).
- a B. napus genomic DNA library in EMBL-3 (Clontech, Palo Alto, California) was screened with the P. sativum 26g cDNA. From 40,000 plaques analyzed, a single positive clone was identified with an insert size of approximately 16 kb. A 4.4 kb Sail fragment containing the entire gene was subcloned.
- the promoter sequence of the btg-26 gene was determined by identification of the mRNA start site using primer extension (Ausubel, ibid.) and is shown in Figure 3 and SEQ ID NO:l. In Figure 3, the transcription start site is bolded, underlined and indicated by +1.
- the TATA box and CAAT box are in bold and double underlined. Postulated functional regions are underlined.
- the sequence of the btg-26 promoter, coding region and 3' region has been presented in Stroeher et al, (1995, Plant Mol. Biol. 27:541-551). Expression analysis of btg-26
- RNA blot analysis Induction of btg-26 expression during droughting was examined by RNA blot analysis. Potted B. napus plants were naturally dehydrated by withholding water for various lengths of time. Whole leaves were used either to determine relative water content (RWC) of individual plants or to isolate total RNA. As shown in Figures 4A and 4B, btg-26 expression is induced rapidly during water loss, reaching a six-fold increase over expression in fully hydrated plants at 81% RWC, increasing to eleven-fold induction at 63% RWC. Further decreases in RWC were associated with a decrease in total amount of btg-26 transcript. At 30% RWC expression was only 3.5-fold over fully hydrated levels. Because other physiological stresses alter intracellular water content, btg-26 expression was examined in B.
- RNA blot analysis indicated that there was no change in btg-26 expression when plants were transferred from normal growth conditions to 4°C for one day. However, plants left at 4°C for four days showed a five-fold induction in btg-26 mRNA. A similar increase was seen when plants were shifted to 40°C for two or four hours. These results are shown in Figure 4C and demonstrate that expression of btg-26 is induced during temperature stress. To examine the effect of salt stress, plants were watered to capacity one day or four days with 50 mM, 150 mM, or 450 mM NaCl.
- the level of btg-26 expression was not affected by 50 mM NaCl regardless of length of exposure.
- the plants watered with 150 mM NaCl showed a two-fold increase in btg-26 mRNA after four days.
- Exposure to 450 mM NaCl caused the most notable induction, twelve-fold after one day, dropping to four-fold after four days.
- Figure 4D for Northern blots showing these results.
- leaves were cut at the petiole and placed in a solution of 0 ⁇ M, 50 ⁇ M or lOO ⁇ M ABA (mixed isomers, Sigma), 0.02% Tween-20 and pH 5.5 for 24 hours.
- btg-26 expression was induced 2.5-fold when leaves were exposed to 100 ⁇ M ABA.
- leaves were exposed to 50 ⁇ M ABA. no induction of expression was observed.
- This step involved the production of either constitutive or stress-induced AlaAT constructs and the introduction of them into Brassica napus using Agrobacterium mediated genetic transformation.
- the approach of introducing specific sense or antisense cDNA constructs into plants to modify specific metabolic pathways has been used in a number of species and to modify a number of different pathways. (See Stitt & Sonnewald 1995 for a review; Ann. Rev. of Plant Physiol. and Plant Mol. Biol. 46:341-368).
- the AlaAT cDNA was introduced under the control of three different promoters.
- the CaMV355 promoter which has been shown to be a strong constitutive promoter in a number of different plant species; (2) the btg-26 promoter described in Example 1 and (3) the trg-31 promoter which was isolated from tobacco by Guerrero and Crossland (ibid.).
- the CaMV promoter induces the constitutive overexpression of AlaAT whereas btg-26 and trg-31 should induce over expression of AlaAT only under conditions of specific stresses, including drought stress.
- the barley AlaAT cDNA clone 3A (As shown in Figure 5 and SEQ ID NO:2 and Muench and Good, ibid) was cloned into the pT7T3-19U vector (Pharmacia Canada) and used for site directed mutagenesis using two specific primers.
- Primer 1 introduced a BamHl restriction site between nucleotides 48-53, while primer 2 was used to introduce a second BamHl restriction site between nucleotides 1558-1563 (See Figure 5).
- the 1510 bp fragment was then cloned into the vector p25 ( Figure 6) which had been cut with BamHl .
- p25 contains the double CaMV35S (Ca2) promoter, which has been shown to give high constitutive levels of expression, and the nopaline synthase (NOS) terminator inserted into the Kpnl and Pstl site of pUC19 with a BamHl, Xbal and Pvul polylinker between the CaMV and NOS region of the plasmid.
- the resulting plasmid was called pCa2/AlaAT/NOS, and is shown in Figure 7A.
- the plasmids pbtg-26/AlaAT/NOS ( Figure 7B) and ptrg-31/AlaAT/NOS ( Figure 7C) were created as follows.
- the trg-31 promoter was subcloned as a 3.0 kb Xbal /BamHl fragment into the Xbal/BamHl site of pCa2/AlaAT/NOS which had been digested with Xbal /BamHl to release only the Ca2 promoter, resulting in a 3 kb promoter fragment inserted in front of the AlaAT coding region.
- pbtg-26/AlaAT/NOS was created by inserting a BamHl site at nucleotides +9 to +14 (see Figure 3) and subcloning the 330 bp Kpnl/BamHl fragment (-320 to +10 in Figure 3) into the Kpnl/BamHl site of pCa2/AlaAT/NOS which had been digested to release the Ca2 promoter. Plasmid constructs pbtg-26/AlaAT/NOS and ptrg-31/AlaAT/NOS are shown in Figures 7B and 7C, respectively.
- Example 4 Transformation and analysis of Brassica napus plants with AlaAT constructs.
- pCGN1547 is an Agrobacterium binary vector developed by McBride and Summerfelt (1990, Plant Mol. Biol. 14:269-276).
- pCGN1547 contains the neomycin phosphotransferase II (NPTII) gene which encodes Kanamycin resistance.
- NPTII neomycin phosphotransferase II
- Transgenic Brassica plants (v. Westar) were produced using the well established cotyledon transformation and regeneration protocols as described by Moloney et al. (ibid.). Kanamycin resistant plantlets were transferred to soil and then grown. The initial generation, or primary transformants, were referred to as the TO generation and were allowed to self. Each subsequent generation was bagged to ensure selfing and referred to as the Tl , T2 generation, respectively. All putative TO transgenic plants were tested for the insertion of the Agrobacterium construct using PCR primers that amplify the NPTII gene and by testing for NPTII activity as described by Moloney et al (ibid.).
- Example 5 Analysis of transformed Brassica plants containing the AlaAT constructs
- Transgenic plants were assayed for AlaAT activity. Extractions were carried out on ice as described previously (Good and Crosby, 1989, Plant Physiol 90:1305-1309). Leaf tissue was weighed and ground with sand using a mortar and pestle in extraction buffer containing 0.1 M Tris-HCl (pH 8.5), 10 mM dithiothreitol, 15% glycerol and 10% (w/v) PVPP. The extract was clarified by centrifugation at 6,000 rpm and the supernatant was assayed for enzyme activity. AlaAT assays were performed in the alanine to pyruvate direction as described previously (Good and Crosby, ibid) using alanine to start the reaction.
- Ca2/AlaAT/NOS After transformation 20 Ca2/AlaAT/NOS, 24 btg-26/AlaAT/NOS and 21 trg- 31 /AlaAT/NOS plants were produced which appeared to be transformed, based on the amplification of an NPTII PCR product and NPT activity. AlaAT activity was measured, using the method described above, in the leaf tissue of several of these transformants. As can be seen from Table 1 , the btg-26/ AlaAT/NOS plants had AlaAT activity levels that ranged from 1.63 to 3.89 times that of the wild-type, control plants. Ca2/AlaAT/NOS plants had activity levels that ranged from 1.51 to 2.95 times that of wild-type, control plants. Western blots confirmed that the transgenic plants had elevated levels of AlaAT, based on the cross reactivity of a band with the barley AlaAT antibody.
- Tl seed from the primary transformants of the groups Ca2/AlaAT and btg- 26/ AlaAT were grown along with control, wild-type plants under normal conditions including planting at a 1 cm depth in 13 cm diameter plastic pots containing a soil and fertilizer mixture as described by Good and Maclagan (ibid.). These pots were placed in growth chambers under the following conditions: i) 16 h of 265 mmol m '2 s "1 provided by VITA-LITE U.H.O. fluorescent tubes, ii) day and night temperatures of 21°C and 15°C respectively, iii) relative humidity of 85%- 97% and iv) daily watering with 1/2 strength Hoagland's solution.
- Example 7 Growth of primary transformants under nitrogen-starved/drought conditions
- Tl seed from the primary transformants of the Ca2/ AlaAT and btg-26/AlaAT groups were grown along with control, wild-type plants for four weeks under normal conditions
- FIG. 9 shows representative plants from the three groups after the treatment at an identical time point.
- Plant A is a control, wild-type plant
- Plant B is a Ca2/ AlaAT transformed plant
- Plant C is a btg-26/AlaAT plant. It can be seen that plant C (btg-26) clearly has a faster growth rate than plants A (control) and B (Call AlaAT).
- senescing leaves are present on plants A and B while plant C has no senescing leaves.
- Example 8 Growth of secondary transformants under low nitrogen and well fertilized conditions
- T2 seed from the Tl transformants of the btg26/AlaAT/NOS plants and Ca2/AlaAT/NOS primary transformants were tested to ensure that the original Tl plant was homozygous by using PCR (Polymerase Chain Reaction). 20bp primers specific to T-DNA were used to ensure that the plant was transgenic. DNA from the plants was amplified in the presence of primers so that in a plant containing the insert, an appropriate sized band was amplified and in plants lacking the insert a band was not amplified. T2 seed from homozygous Tl transgenics were then used as described below.
- a comparitive construct was prepared, according to Example 3, by substituting the AlaAT coding region with a GUS coding region to produce a plasmid construct pbtg-
- the plants transformed with the CaMV35S promoted constructs had growth characteristics similar to those of the controls.
- able 2 Average Leaf Area & Stem Diameter (Well Fertilized)
- a genetic construct according to the present invention is used to transform tobacco.
- Transgenic tobacco plants are produced using the well established whole leaf transformation protocol as described by Fisher and Guiltinan (1995; Plant Mol. Biol. Reporter 13:278) and are selected on Kanamycin.
- the initial generation, or primary transformants are selfed to produce Tl generation and are tested to confirm insertion of the construct according to
- Transgenic plants are tested for AlaAT activity as described in Example 5.
- the Tl seed is tested to ensure homozygousity of the Tl plants as in Example 8.
- T2 seed from selfed homozygous Tl plants is grown as in Example 8 using the well fertilized protocol and nitrogen starved protocol of Example 8.
- Transgenic cotton plants are produced containing the genetic constructs of Example 3 using a whole leaf transformation protocol as described by Bayley et al. (1992; Theoret. Applied Genet 83:645). TO, Tl and T2 plants are produced and confirmed for transformation and homozygousity as in Example 9. Plants are grown and compared to control plants.
- Example 11 Transformation and analysis of maize (Zea mays)
- a 340 bp promoter fragment from the rab2S gene of rice is used (Pla et al. Plant Mol. Biol. 21:259).
- the ra >28 gene from maize has been shown to be responsive to both osmotic stress and drought. This was inserted as a blunt ended fragment into where the Kpnl/BamHl btg-26 promoter had been inserted in the AlaAT construct of Figure 7B. This resulted in a construct prab28/ AlaAT/NOS.
- the CaMV promoter construct of Figure 7A was also used. These fragments are then separately subcloned into the monocot transformation vector pSB 131 available from Clonetech (Palo Alto, Calif).
- This vector is similar to many of the commonly used dicot vectors such as pBI121 in that is carries the gene of interest along with a selectable marker.
- the selectable marker of the vector is the BAR gene which codes for Basta resistance (Ishida et al., 1996, Nature Biotechnology 14:745).
- This plasmid containing the rab28/AlaAT construct is then introduced into the Agrobacterium strain LBA4404 (readily available) by electroporation. PCR is used to confirm that Agrobacterium is transformed.
- Transgenic plants are produced by infection of immature embryos for maize inbred lines and selection is made on Basta as described by Ishida et al (ibid). Transgenic plants are tested for insertion of the gene using PCR and then for AlaAT activity as described in Example 5.
- Tl, T2 plants are produced and confirmed for transformation and homozygousity as in Example 9. T2 plants are grown and compared with controls.
- a genetic construct according to the present invention is used to transform rice.
- Example 11 The plant transformation vector and Agrobacterium strain as in Example 11 are used to obtain transgenic rice plants by infection of calli which had been derived from rice scutellum tissue. (Hirei et al. 1994 Plant Journal 6:271). Transgenic plants are tested for insertion of the gene using PCR and then for AlaAT activity as described in Example 5.
- Tl , T2 plants are produced and confirmed for transformation and homozygousity as in Example 9. T2 plants are grown and compared with controls.
- a tetracycline inducible promoter is used with a nitrogen assimilation/metabolism gene.
- a tetracycline inducible promoter system is commercially available from
- TnlO a transposable element from bacteria encoding tetracycline resistance
- Tetracycline repressor TetR
- This construct is used to transform canola, tobacco and rice as described in
- Examples 3, 4, 9 and 12. The plants are then tested for the insertion of the gene using PCR and for the induction of AlaAT activity in the presence of doxycycline. Transgenic plants are grown and after about two weeks, one group of each plant species has doxycycline included in the watering media such that the AlaAT gene is induced.
- a copper inducible promoter is used with a nitrogen assimilation/metabolism gene.
- a copper inducible promoter by Met et al. (1993 PNAS 90:4567) can be turned on and off in an inducible manner by the addition of copper to the media.
- the AlaAT cDNA is cloned into a vector under control of this copper inducible promoter. Therefore in the presence of copper alanine aminotransferase will be expressed.
- This construct is used to transform canola, tobacco and rice according to Examples
- a nitrogen inducible promoter is used with a nitrogen assimilation/metabolism gene.
- NR nitrate reductase
- Transgenic plants are grown and treated to nitrogen-starved conditions followed by application of a source of nitrate. Transgenic plants are compared with controls.
- the aspartate aminotransferase (AspAT) coding region from alfalfa (Udvardi, M. and Kahn, M., ibid) is cloned between the BamHl/BamHl sites using the construct of Figure 7B to replace the AlaAT coding region. This results in a btg-26/ AspAT/NOS construct. This construct is used to transform canola and tobacco as described in Examples 4,
- Transgenic plants are grown and treated to nitrogen starved conditions according to Example 8. Transgenic plants are compared with controls.
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- CAAGAAATTC CACTCTTTCA AGAAGATAGT GAGATCCTTG GGATACGGCG AGGAGGATCT 960
- CACGGTGTTC CATGAGGCGT TCATGTCAGA GTATCGTGAC TAAACTGGTG CAACATGTGG 1560
- GATTACATAC AACCCTCATG GGGTTTTCGT AGGCGTTCTT GGTTTTGCCC CCCCCCCT 1620
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Abstract
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AU15868/97A AU727264B2 (en) | 1996-02-14 | 1997-02-14 | Plants having enhanced nitrogen assimilation/metabolism |
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US08/599,968 US6084153A (en) | 1996-02-14 | 1996-02-14 | Plants having enhanced nitrogen assimilation/metabolism |
US08/599,968 | 1996-02-14 |
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WO2001055433A2 (en) * | 2000-01-28 | 2001-08-02 | The Governors Of The University Of Alberta | Tissue-specific expression of target genes in plants |
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WO2016162371A1 (en) | 2015-04-07 | 2016-10-13 | Basf Agrochemical Products B.V. | Use of an insecticidal carboxamide compound against pests on cultivated plants |
EP3338552A1 (en) | 2016-12-21 | 2018-06-27 | Basf Se | Use of a tetrazolinone fungicide on transgenic plants |
Also Published As
Publication number | Publication date |
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GB2325232B (en) | 2000-11-29 |
AU1586897A (en) | 1997-09-02 |
AU727264B2 (en) | 2000-12-07 |
GB2325232A (en) | 1998-11-18 |
GB9817804D0 (en) | 1998-10-14 |
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