IE83282B1 - Imidazolinone resistant ahas mutants - Google Patents

Description

AMERICAN CYANAMID COMPANY, a corporation organized and existing under the Taws of the State of Maine, United States of America, of One Cyanamid PTaza, Wayne, State of New Jersey, 07470, United States of America IHIDAZOLINONE RESISTANT AHAS HUTANTS This invention relates to novel DNA sequences that encode novel variant forms of acetohydroxy acid synthase enzyme (hereinafter AHAS). The AHAS enzyme is a critical enzyme routinely produced in a variety of plants and. a broad range of microorganisms. Normal AHAS function is inhibited by imidazolinone herbicides; however, new’ AHAS enzymes encoded by the mutant DNA sequences function normally catalytically even in the presence of imidazolinone herbicides and, therefore, confer herbicide resistance upon the plant or microor- ganism containing them.

The novel DNA sequences are derived from corn and have a substitution of an amino acid at position 621 of the normal AHAS sequence. This substitution in the AHAS gene sequence results in a fully functional enzyme, but renders the enzyme specifically resistant to inhibition by a variety of imidazolinone herbicides.

The availability of these variant sequences provides a tool for transformation of different crop plants to imidazolinone herbicide resistance, as well as pro- viding novel selectable markers for use in other types of genetic transformation experiments.

BACKGROUND OF THE INVENTION The use of herbicides in agriculture is now widespread. Although there are a large number of available compounds which effectively destroy weeds, not all herbicides are capable of selectively targeting the undersirable plants over crop plants, as well as being non-toxic to animals. Often, it is necessary to settle for compounds which are simply less toxic to crop plants than to weeds. In order to overcome this problem, development of herbicide resistant crop plants has become a major focus of agricultural research.

An important aspect of development of herbi- cide-resistance is an understanding of the herbicide target, and then manipulating the affected biochemical pathway in the crop plant so that the inhibitory effect is avoided while the plant retains normal biological function. one of the first discoveries of the bio- chemical mechanism of herbicides related to a series of structurally unrelated herbicide compounds, the imi- dazolinones, the sulfonylureas and the triazolopyrimi- dines. It is now known (Shaner gt glg Elan; Physiol. lg: 545-546,1984; U.S. Patent No. 4,761,373) that each of these herbicides inhibits plant growth by inter- ference with an essential enzyme required for plant growth, acetohydroxyacid synthase (AHAS; also referred to as acetolacetate synthase, or ALS). AHAS is re- quired for the synthesis of the amino acids isoleucine, leucine and valine.

In tobacco, AHAS function is encoded by two unlinked genes, gggg and gggg. There is substantial identity between the two genes, both at the nucleotide level and amino acid level in the mature protein, al- though the N-terminal, putative transit region differs more substantially (Lee gt al, gfigg Q; 1: 1241-1248, 1988). Arabidopsis, on the other hand, has a single AHAS gene, which has also been completely sequenced (Mazur gt al.,’ Plant Phvsiol. e_5:111o-1117, 1987).

Comparisons among sequences of the AHAS genes in higher plants indicates a high level of conservation of certain regions of the sequence; specifically, there are at least 10 regions of sequence conservation. It has previously been assumed that these conserved regions are critical to the function of the enzyme, and that retention of that function is dependent upon substantial sequence conservation.

It has been recently reported (U.s. Patent No. 5,013,659) that mutants exhibiting herbicide resistance possess mutations in at least one amino acid in one or more of these conserved regions. In parti- cular, substitution of certain amino acids for the wild type amino acid at these specific sites in the AHAS protein sequence have been shown to be tolerated, and indeed result in herbicide resistance of the plant possessing this mutation, while retaining catalytic function. The mutations described therein encode either cross resistance for imidazolinones and sulfonylureas or sulfonylurea-specific resistance, but AHAS amino acid sequence.

SUMMARY OF THE INVENTION The present invention provides novel nucleic acid sequences encoding functional monocot AHAS enzymes insensitive to imidazolinone herbicides, as defined by claim 1. The Sequences in question comprise a mutation in the codon encoding , the amino acid serine at position 621 in the corn (maize) AHAS sequence, or in the corresponding position in other monocot sequences. other monocots, such as wheat, are also known to exhibit imidazolinone specific mutations (e.g., ATCC Nos. 40994-97). In corn, the wild type sequence has a serine at this position. In a preferred embodiment, the substitution is asparagine for serine, but alternate substitutions for serine include aspartic acid, glutamic acid, glutamine and tryptophane. Although the claimed sequences are originally derived from monocots, the novel sequences are useful in methods for producing imidazolinone resistant cells in any type of plant, said methods comprising transforming a target plant cell with one or more of the altered sequences provided herein. Alter- natively, mutagenesis is utilized to create mutants in plant cells or seeds containing a nucleic acid sequence encoding an imidazolinone insensitive AHAS. In the case of mutant plant cells isolated in tissue culture, plants which possess the imidazolinone resistant or insensitive trait are then regenerated. The invention thus also encompasses plant cells, bacterial cells, fungal cells, plant tissue cultures, adult plants, and plant seeds that possess a mutant nucleic acid sequence and which express functional imidazolinone resistant AHAS enzymes.

The availability of these novel herbicide resistant plants enables new methods of growing crop plants in the presence of imidazolinones. Instead of growing non-resistant plants, fields may be planted with the resistant plants produced by mutation or by transformation with the mutant sequences of the present invention, and the field routinely treated with imi- dazolinones, with no resulting damage to crop plants.

The mutant nucleic acids of the present in- vention also provide novel selectable markers for use in transformation experiments. The nucleic acid sequence encoding a resistant AHAS is linked to a second gene prior to transfer to a host cell, and the entire construct transformed into the host. Putative transformed cells are then grown in culture in the presence of inhibitory amounts of herbicide; surviving cells will have a high probability of having success- fully acquired the second gene of interest. Alter- nately, the resistant AHAS gene can be cotransformed on an independent plasmid with the gene of interest, whereby about 50% of all transformants can be expected to have received both genes.

The following definitions should be under- stood to apply throughout the specification and claims.

A "functional" or "normal" AHAS enzyme is one which is capable of catalyzing the first step in the pathway for synthesis of the essential amino acids isoleucine, leucine and valine. A "wild-type" AHAS sequence is a sequence present in an imidazolinone sensitive member of a given species. A "resistant" plant is one which produces a mutant but functional AHAS enzyme, and which is capable of reaching maturity when grown in the presence of normally inhibitory levels of imidazoli— none. The term "resistant", as used herein, is also intended to encompass "tolerant" plants, i.e., those plants which phenotypically evidence adverse, but not lethal, reactions to the imidazolinone.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: AHAS enzyme activity in 10-day old maize seedlings (corn lines A619 or XI12) in the presence of imazethapyr (Pursuit“ A) or chlorsulfuron (Oust” B). Herbicide resistant AHAS activity is calculated as percentage of AHAS activity in the absence of inhibitor. The standard error between experimets is 10%.

Figure 2: Southern analysis of genomic clones in phage EMBL3. Phages 12-1A (from W22), 12—7A, 18-8A, 12-11, and 12—17A (From x112) are digested with Xbal or Sall, separated on a 1% agarose gel, transfered onto nitrocellulose and hybridized with an AHAS CDNA fragment as probe.

Figure 3: Southern analysis of genomic DNA from corn lines XI12, XA17, QJ22, A188 and B73. The DNA is digested with xbal, separated on a 1% agarose .. 6- gel, transferred onto nitrocellulose and hybridized with an AHAS cDNA fragment as probe.

Figure 4: Restriction map of plasmid pcD8A.

The mutant AHAS gene from xI12 was subcloned as a 8kb Pstl fragment into vector pKS(+). The location and orientation of the AHAS gene is indicated by an arrow.

The restriction sites of Pstl, xhol, HindIII, xbaI and C1aI are represented by symbols.

Figure 5: Nucleotide sequencing gel of the non-coding strand (A) and the double stranded DNA sequence (B) of AHAS clones W22/4-4, B73/10-4 and XI12/8A in the region of amino acids 614 to 633. The position of the cytosine to thymidine transition is indicated by an arrow.

Figure 6: Nucleotide and deduced amino acid sequences of the XI12/8A mutant AHAS gene.

‘ Figure 7: Nucleotide sequence alignment of x112/an, B73/7-4 and W22/1A glgz genes. (*) marks the base change causing the mutation at position 621, (#) differences from the B73/7-4 sequence and (>) repre- sents silent changes.

Figure 8: Amino acid sequences and alignment of XI12/BA, B73/7-4 and W22/1A algz genes. (*) marks the mutation at position 621, (#) marks differences from the B73/7-4 sequence, and (>) represents silent changes.

DETAILED DESCRIPTION OF THE INVENTION The gene of the present invention is iso- latable from corn maize line XI12 (seed deposited with the American Type Culture Collection as Accession Number 75051), and has been inserted into plasmid pXI12/8A (deposited with the American Type Culture Collection as Accession Number 68643). It is also isolatable from any other imidazo1inone—specific herbicide resistant mutant, such as the corn line QJ22 (deposited as a cell culture with the American Type Culture Collection as Accession Number 40129), or the various wheat plants (seed deposited with the American Type Collection as Accession Numbers 40994, 40995, 40996, or 40997). A genomic DNA library is created, for example, in phage EMBL-3 with DNA from one of the imidazolinone resistant mutants, preferably one which is homozygous for the resistance trait, and is screened with a nucleic acid probe comprising all or a part of a wild-type AHAS sequence.

In maize, the AHAS gene is found at two loci, ls1 and alsz (Burr and Burr, Trends in Genetics :55-61, 1991); the homology between the two loci is 95% at the nucleotide level. The mutation in XI12 is mapped to locus glgz on chromosome 5, whereas the nonmutant gene is mapped to locus glgl on chromosome 4 (Newhouse gt alL, "Imidazolinone-resistant crops". lg The Imidazolinone Herbicides, Shaner and O'Connor (Eds.), CRC Press, Boca Raton, FL, in Press) Southern analysis identifies some clones containing the mutant alsz gene, and some containing the non-mutant algl gene. Both types are subcloned into sequencing vec- tors, and sequenced by the dideoxy sequencing method. sequencing and comparison of wild type and mutant AHAS genes shows a difference of a single nucleotide in the codon encoding the amino acid at position 621 (Figure 5). specifically, the codon AQT encoding serine in the wild type is changed to A1_\T encoding asparagine in the mutant AHAS (Figure 8). The mutant AHAS gene is otherwise similar to the wild type gene, encoding a protein having 638 amino acids, the first 40 of which constitute a transit peptide which is thought to be cleaved during transport into the chloroplast in yigg. The sequence of the algl non- mutant gene from x112 appears to be identical to the glgl gene from B73.

The mutant genes of the present invention confer resistance to imidazolinone herbicides, but not to sulfonylurea herbicides. Types of herbicides to which resistance is conferred are described for example in U.S. Patent Nos. 4,188,487; 4,201,565; 4,221,586; 4,297,128: 4,554,013: 4,608,079; 4,638,068: 4,747,301: 4,650,514; 4,698,092; 4,701,208; 4,709,036; 4,752,323; 4,772,311; and 4,798,619.

It will be understood by those skilled in the art that the nucleic acid sequence depicted in Figure 6 is not the only sequence which can be used to confer imidazolinone-specific resistance. Also contemplated are those nucleic acid sequences which encode an identical protein but which, because of the degeneracy of the genetic code, possess a different nucleotide sequence. The invention also encompasses genes encod- ing AHAS sequences in which the aforestated mutation is present, but which also encode one or more silent amino acid changes in portions of the molecule not involved with resistance or catalytic function. Also contem- plated are gene sequences from other imidazolinone resistant monocots which have a mutation in the corre- sponding region of the sequences.

For example, alterations in the gene sequence which result in the production of a chemically equi- valent amino acid at a given site are contemplated; thus, a codon for the amino acid alanine, a hydrophobic amino acid, can readily be substituted by a codon encoding another hydrophobic residue, such as glycine, or may be substituted with a more hydrophobic residue such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also he expected to produce a biologically equivalent product.

The invention also encompasses chimaeric genes, in which the substituted portion of the corn AHAS gene is recombined with unaltered portions of the normal AHAS genes from other species. Thus, throughout the speci- fication and claims, wherever the term "imidazo- linone-specific resistant corn AHAS gene" is used, it is intended to cover each of these alternate embodi- ments as well as the sequence of Figure 6.

Isolated AHAS DNA sequences of the present invention are useful to transform target crop plants, and thereby confer imidazolinone resistance. A broad range of techniques currently exist for achieving direct or indirect transformation of higher plants with exogenous DNA, and any method by which the novel se- quence can be incorporated into the host genome, and stably inherited by its progeny, is contemplated by the present invention. The imidazolinone specific resis- tance trait is inherited as a single dominant nuclear gene. The level of imidazolinone resistance is in- creased when the gene is present in a homozygous state; such corn plants, for example, have a resistance level of about 1,000 times that of a non-resistant plant.

Plants heterozygous for the trait, however, have a resistance of about 50-500 times that of a non-resistant plant.

Transformation of plant cells can be mediated by the use of vectors. A common method of achieving transformation is the use of Aqrobacterium tumefaciens to introduce a foreign gene into the target plant cell.

For example, the mutant AHAS sequence is inserted into a plasmid vector containing the flanking sequences in the Ti-plasmid T—DNA. The plasmid is then transformed into E; ggli. A triparental mating among this strain, an Agrobacterium strain containing a disarmed Ti-plasmid containing the virulence functions needed to effect transfer of the AHAS containing T-DNA sequences into the target plant chromosome, and a second EL 99;; strain containing a plasmid having sequences necessary to mobilize transfer of the AHAS construct from g; ggli to Agrobacterium is carried out. A recombinant Agrg; bacterium strain, containing the necessary sequences for plant transformation is used to infect leaf discs.

Discs are grown on selection media and successfully transformed regenerants are identified. The recovered plants are resistant to the effects of herbicide when grown in its presence. Plant viruses also provide a possible means for transfer of exogenous DNA.

Direct uptake of plant cells can also be employed. Typically, protoplasts of the target plant are placed in culture in the presence of the DNA to be transferred, and an agent which promotes the uptake of DNA by protoplast. Useful agents in this regard are polyethylene glycol or calcium phosphate.

Alternatively, DNA uptake can be stimulated by electroporation. In this method, an electrical pulse is used to open temporary pores in a protoplast cell membrane, and DNA in the surrounding solution is then drawn into the cell through the pores. similarly, microinjection can be employed to deliver the DNA directly into a cell, and preferably directly into the nucleus of the cell.

In each of the foregoing techniques, trans- formation occurs in a plant cell in culture. Subse- quent to the transformation event, plant cells must be regenerated to whole plants. Techniques for the regeneration of mature plants from callus or protoplast culture are now well known for a large number of different species (see, e.g., Handbook of Plant Cell Culture, Vols. 1-5, 1983-1989 McMillan, N.Y.) Thus, once transformation has been achieved, it is within the knowledge in the art to regenerate mature plants from the transformed plant cells.

Alternate methods are also now available which do not necessarily require the use of isolated cells, and therefore, plant regeneration techniques, to achieve transformation. These are generally referred to as "ballistic" or "particle acceleration" methods, in which DNA coated metal particles are propelled into plant cells by either a gunpowder charge (Klein gt g;L, Nature ggz: 70-73, 1987) or electrical discharge (EPO 270 356). In this manner, plant cells in culture or plant reproductive organs or cells, e.g. pollen, can be stably transformed with the DNA sequence of interest.

In certain dicots and monocots direct uptake of DNA is the preferred method of transformation. For example, in corn, the cell wall of cultured cells is digested in a buffer with one or more cell wall degrad- ing enzymes, such as cellulase, hemicellulase and pectinase, to isolate viable protoplasts. The protoplasts are washed several times to remove the enzymes, and mixed with a plasmid vector containing the gene of interest. The cells can be transformed with either PEG (e.g. 20% PEG 4000) or by electroporation.

The protoplasts are placed on a nitrocellulose filter and cultured on a medium with embedded corn cells functioning as feeder cultures. After 2-4 weeks, the cultures in the nitrocellulose filter are placed on a medium containing about 0.32 uM of the imidazolinone and maintained in the medium for 1-2 months. The nitrocellulose filters with the plant cells are trans- ferred to fresh medium with herbicides and nurse cells every two weeks. The untransformed cells cease growing and die after a few weeks.

The present invention can be applied to transformation. of virtually any type of plant, both monocot and dicot. Among the crop plants for which transformation to herbicide resistance is contemplated ..12_ are corn, wheat, rice, millet, oat, barley, sorghum, sunflower, sweet potato, alfalfa, sugar beet, Brassica species, tomato, pepper, soybean, tobacco, melon, squash, potato, peanut, pea, cotton, or cacao. The novel sequences may also be used to transform ornamen- tal species, such as rose, and woody species, such as pine and poplar.

The novel sequences of the invention also are useful as selectable markers in plant genetics studies.

For example, in plant transformation, sequences encod- ing imidazolinone resistance could be linked to a gene of interest which is to be used to transform a target imidazolinone sensitive plant cell. The construct comprising both the gene of interest and the imidazo- linone resistant sequence are introduced into the plant cell, and the plant cells are then grown in the pres- ence of an inhibitory amount of an imidazolinone.

Alternately, the second gene of interest can be cotransformed, on a separate plasmid, into the host cells. Plant cells surviving such treatment presumably have acquired the resistance gene as well as the gene of interest, and therefore, only transformants survive the selection process with the herbicide. Confirmation of successful transformation and expression of both genes can be achieved by Southern hybridization of genomic DNA, by PCR or by observation of the phenotypic expression of the genes.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES . confirmation of Whole Plant Herbicide Resistance in XI12 X112 plants are treated with herbicides at days to the V3 leaf stage (4-5 leaves, of which 3 have visible ligules). Imazethapyr is applied at rates of 2000, 500, 250, 125 and 62.5 g/ha. Chlorsulfuron is applied at 32, 16, 8, 4 and 2 g/ha. Plants are treated postemergence at a spray ‘volume of 400 1/ha. After are placed in the greenhouse for spraying, plants further observation.

XI12 plants are unaffected at all rates of however, no visible increased Thus, XI12 imazethapyr treatment; resistance to chlorsulfuron is noted. displays selective resistance to the imidazolinone at the whole plant level (See Figure 1).

The resistance in XI12 is also shown to be inherited as a single dominant allele of a nuclear gene. Heterozygous resistant XI12 are selfed, and the selfed progeny segregate in the 3 resistant:1 sus- ceptible ratio expected for a single dominant allele of a nuclear gene. In this study, the segregating seed- lings are sprayed postemergence with lethal doses of 250 g/ha) protocols described above, to establish segregation for imazethapyr (125 or following spraying resistance.

. AHAS Extraction Seeds of XI12 are sown in soil in a green- house maintained at day/night temperature of 80°C and hour photoperiod. Plants are harvested two weeks after planting. The basal portion of the shoot is used for the extraction of AHAS. powdered in liquid nitrogen and then homogenized in g of the tissue is AHAS assay buffer comprising 100 mM potassium phosphate buffer (pH 7.5) containing 10 mM pyruvate, 5 mM Mgclz, mM EDTA, 100 uM FAD (flavin adenine dinucleotide), 1 mM leucine, 10% The homogenate is centrifuged at 10,000 rpm mM valine, glycerol and 10 mM cysteine. for 10 minutes and 3 ml of the supernatant are applied onto an equilibrated Bio-Rad Econo-Desalting column (10 DG) and eluted with 4 ml AHAS assay buffer.

AHAS activity is measured by estimation of the product, acetolactate, after conversion by decarboxylation in the presence of acid to acetoin.

Standard reaction mixtures contain the enzyme in 50 mM potassium phosphate (pH 7.0) containing 100 mM sodium pyruvate, 10 mM Mgclz, 1 mM thiamine pyrophosphate, uM FAD, and appropriate concentrations of different inhibitors. This mixture is incubated at 37°C for 1 to 3 hours depending upon the experiment. At the end of this incubation period, the reaction is stopped with the addition of H2804 to make a final concentration of 0.85% H2304 in the tube. The reaction product is allowed to decarboxylate at 60°C for 15 minutes. The acetoin formed is determined by incubating with creatine (0.17%) and 1-naphthol (1.7% in 4N NaOH). The absorption of color complex formed is measured at 520 nm.

AHAS activity from B73, A619, or other wild-type maize lines is highly sensitive to inhibition by imazethapyr (PURSUIT”) with an ISO of 1 uM (See Fig- ure 1). Contrary to this observation, XI12 shows 70-80% of enzyme activity at the highest concentrations (100 pM) of PURSUIT” or ARSENAL” (imazepyr), and about 70% in the presence of SCEPTER” (imazequin). This result shows a 100-fold increase in tolerance of AHAS activity from xI12 to imazethapyr as compared to the in yitrg, AHAS activity from A619. Sensitivity of AHAS activity from the two lines to sulfonylureas gives a different picture. In the presence of OUST” (su1fo- meturon methyl), at 100 nM, AHAS activity of XI12 is only 15-20%. AHAS activity of A619 in the presence of OUST” IS 5-10%, and in the presence of PURSUIT” is -20% (See Figure 1). 3. Cloning of X112 AHAS Genes Seeds of the xI12 mutant derived from an imidazolinone resistant corn tissue culture line are planted: plants obtained therefrom appear to be segre- gating for the mutant AHAS phenotype. In order to obtain homozygous resistant seed material, a population of xI12 mutant plants are selfed. After selecting for herbicide resistance for three consecutive growing seasons, the seeds are homozygous for the mutant AHAS gene. Homozygous seeds are collected and used to grow seedlings to be used in AHAS gene isolation.

DNA is extracted from 7 days old etiolated seedlings of a homozygous X112 line. 60 g of plant tissue is powdered in liquid nitrogen, and transfered into 108 ml DNA extraction buffer (1.4 M Nacl, 2.0% Ctab (hexadecyl trimethyl ammonium bromide), 100 mM tris—Cl pH 8.0, 20 mM EDTA, 2% Mercaptoethanol) and 54 ml water. After incubation at 50-60°C for 30 minutes the suspension is extracted with an equal amount of chloroform. The DNA is precipitated by adding an equal amount of precipitation buffer (1% Ctab, 50 mM Tris-Cl pH 8.0, 10 mM EDTA). To purify the genomic DNA, a high speed centrifugation in 6.6M Cscl and ethidium bromide is performed (Ti80 rotor, 50,000 rpm, 20°C, 24 hours). The purified DNA is extracted with salt saturated Butanol and dialyzed for hours against 3 changes of 1 1 dialysis buffer (10 mM Tris-Cl Ph 8.0, 1 mM EDTA, 0.1M NaCl). The concentration of the X112 genomic DNA is determined spectrophotometrically to be 310 mg/ml. The yield is 0.93 mg.

The XI12 genomic DNA is used to create a genomic library in the phage EMBL-3. The DNA is partially digested with Mbol and the fragments are separated on a sucrose gradient to produce size range between 8 to 22 kb before cloning into the Bamfil site of EMBL-3. After obtaining 2.1 x 106 independent clones, the library is amplified once. The titer of the library is determined 9 X 1010 pfu/ml.

To obtain probes for analysis of the X112 library, a W22 (wild—type) cDNA library in lambda gt11, purchased from Clontech Inc., CA, is screened with an 800 nt BamH1 probe isolated from Arabidopsis AHAS genomic clone. The phages are plated in a density of 50,000 pfu/15 cm plate, transferred onto nitrocellulose filters, prehybridized in 6x ssc, 0.2% SDS for 2 hours and hybridized with the Arabidopsis AHAS probe in 6x SSC, 0.2% SDS overnight. one positive phage is identi- fied, further purified and used for subcloning of a 1.1 kb EcoR1 fragment. The 1.1 kb EcoRl fragment is subcloned into pGemA—4 and used as a probe to identify the x112 AHAS genes.

The XI12 genomic library is plated on 12 -cm plates (concentration of 50,000 pfu/plate) and is screened with the W22 AHAS cDNA probe. The filters are prehybridized (2 hours) and hybridized (over night) in Church buffer (0.5 M Na Phosphate, 1 mM EDTA, 1% BSA, 7% sns) at 65°C and washed at 65°C in 2x ssc, 0.2% sns and 0.3 x ssc, 0.2% SDS. 12 positive plaques are obtained from a total of 7.5 x 105 pfu screened and 5 positive clones are further purified and isolated according to Chisholm (BioTechniques 1:21-23, 1989).

Southern analysis (See Figure 2) showed that the phage clones represented two types of AHAS clones: Type-1 clones contain one large xbal (>6.5 kb) fragment hybridizing to the AHAS cDNA probe, Type-2 clones contained two 2.7 and 3.7 kb xbal fragments hybridizing to the AHAS cDNA probe. Genomic Southern of XI12 DNA demonstrated, that the xbal fragments obtained by digesting genomic DNA and by hybridizing to the AHAS cDNA probe correspond to the xbal fragments identified in the XI12 phage clones (See Figure 3). Restriction digest and Southern Analysis also demonstrate that of the 5 AHAS clones, one clone represents the mutant algz genes and four represent the alsl gene. -17..

The AHAS genes corresponding to the mutant (clone 12/8A) and the non-mutant locus located on chromosome 4 (clone 12/17A) (clone 12/8A) sequencing vector Both locus located on chromosome 5 are subcloned as a Pstl fragment or as xbal (12/17A) into the pBluescript II KSm13(+) (pKS+; .7 kb and 3.7 kb xbal fragments from phage 12/17A fragment stratagene). contain one complete copy of AHAS genes which are identified. The sequence of each is obtained by dideoxy sequencing (Pharmacia T7 sequencing Kits) using primers complementary to the AHAS coding sequence.

The methods of DNA extraction, cloning of the genomic library and screening’ of the library are as described for the X112 genomic DNA. The B73 AHAS genes are subcloned into the sequencing vector pKS+ as xbal fragments and are sequenced. The sequence is obtained by dideoxy sequencing, using primers complementary to the AHAS coding sequence as described for the SIl2 AHAS genes.

A W22 genomic library in EMBL3 purchased from CA is screened.

Clontech Inc., The phages are plated in a density of 50,000 pfu/7 inch plate, transferred and hybridized with the (prehybridization 0.5% sns, 1x °C, onto nitrocellulose filters, W22 AHAS cDNA probe described above and hybridization conditions: 6 x SSC, Denhard's 100 mg/ml calf thymus DNA, conditions: 3x x ssc, 0.2% sos for 2 hours at 65°C, and 0.3 x SSC, 0.2% SDS for 2 hours). Two positive phages (12/1A and 12/4-4) are identified and further purified. washing The W22 genomic clone 12/1A is subcloned as two 0.78 kb (pGemA—4) and 3.0 kb (pGemA-14; Sall fragments into the vector pGem-A2, and as a 6.5 kb (pCD200). The Promega) xbal fragment into the vector pKS+ coding strand sequence of the W22 AHAS gene is obtained by dideoxy sequencing of nested deletions created from subclones pGem A-14 and pGem A-4 of phage 12—1A. This sequence is used to design oligonucleotides The obtained by complementary to the AHAS non-coding strand. strand is pCD200 of the non—coding of complementary to the coding strand. sequence dideoxy sequencing clone using primers Upon complementing the sequencing of the W22 AHAS gene, primers of both DNA strands are designed and used for the sequencing of the AHAS genes isolated from the X112 and B73 genomic libraries.

. Cloning of QJ22 AHAS Genes The sequence of the gene encoding AHAS in the maize line QJ22, which is selectively resistant to imidazolinones, is also determined. A genomic library of QJ22 is prepared in an EMBL3 vector. A library of ,000 phage is screened with an 850 nucleotide SalI/ClaI fragment isolated from an AHAS clone (A-4) derived from the wild-type maize line W22. Five positive phages are picked and submitted to a second The analyzed by PCR to round of screening to partially purify the phage. partially purified phage are determine if any clones represent the QJ22 alsl gene. such clones are identified as a 3.7kb XbaI fragment with a gene specific SmaI site at position 495. The the positive clone with these characteristics. second screen indicates presence of a single The PCR product is purified using a commer- cial kit (Magic PCR Preps) from Promega, and the purified DNA is sequenced with a Taq polymerase se- quencing system "fmol", also from Promega Sequence analysis of both strands of the DNA of the QJ22 mutant AHAS shows a nucleotide transition from G to A in the codon for amino acid 621. This mutation is identical to the one seen in XIl2 and the remainder of the sequence is typical of an alsl gene.

RESULTS The sequence of the mutant AHAS genes is compared with the sequences obtained from the wild type corn lines B73 and W22 (See Figure 7). The x112 mutant gene (XI12/8A) and the QJ22 mutant gene and the wild type gene are identical except for the amino acid change at position 621, causing a single nucleotide transition from AGT to AAT (See Figure 8). The XI12 mutant XI12/8A and the wild-type B73/7-4 gene show an additional difference at position 63. on the other hand, the non-mutant XI12 AHAS gene cloned in xI12/17A is completely homologous to the corresponding B73/10-2 in the region coding for the mature AHAS protein (data not shown).

DEPOSIT OF BIOLOGICAL MATERIALS The following biological materials were deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20857, as follows: g; coli XLI Blue harboring plasmid px12/8A, deposited on July 3, 1991, Accession Number ATCC 68643 XI12 corn seed deposited on July 16, 1991, Accession Number ATCC 75051.

Claims (21)

1. A monocot nucleic acid sequence encoding a functional AHAS enzyme, which enzyme has an amino acid substitution relative to a wild—type monocot Al-lAS enzyme, which substitution confers imidazolinone-specific resistance to the enzyme.

2. The nucleic acid sequence of claim 1 in which the functional AHAS enzyme has an amino acid substitution at position 621 in maize or the corresponding substitution on monocots other than maize.

3. The sequence of Claim 2 in which the substituted amino acid is asparagine.

4. A functional monocot AHAS enzyme which has at least one amino acid substitution relative to a monocot wild-type AHAS enzyme for conferring imidazolinone-specific resistance to the enzyme.

5. A functional monocot AHAS enzyme according to Claim 4 which has one amino acid substitution.

6. The enzyme of Claim 4 in which the monocot is com and the substitution is at position 621 in the wild—type corn AHAS enzyme.

7. The enzyme of Claim 6 in which the substituted amino acid is asparagine.

8. A transformation vector comprising the nucleic acid of Claim 1.

9. A host cell comprising the nucleic acid sequence of Claim 1, or the vector of Claim 8.

10. The host cell of Claim 9 which is a plant cell or a bacterial cell. 22

11. An imidazolinone-specific resistant mature plant, or seed or pollen thereforrn, containing the nucleic acid sequence of Claim 1.

12. A method of conferring imidazolinone-specific resistance to a plant cell which comprises providing the plant cell with the nucleic acid sequence of Claim 1.

13. A method of selecting host cells successfully transformed with a gene of interest which comprises providing to prospective host cells the gene of interest linked to the nucleic acid sequence of Claim 1, or unlinked but in the presence of the nucleic acid sequence of Claim 1, growing the cells in the presence of an inhibitory amount of imidazolinone and identifying surviving cells as containing the gene of interest.

14. A nucleic acid construct comprising the sequence of Claim 1 linked to a gene encoding an agronomically useful trait.

15. A monocot nucleic acid sequence according to any one of claims 1 to 3 , substantially as described herein by way of example and/or with reference to the accompanying drawings.

16. A monocot AHAS enzyme according to any one of claims 4 to 7, substantially as described herein by way of example and/or with reference to the accompanying drawings.

17. A transformation vector according to claims 8, substantially as described herein by way of example and/or with reference to the accompanying drawings.

18. A host cell according to claim 9 or claim 10, substantially as described herein by way of example and/or with reference to the accompanying drawings.

19. An imidazolinone-specific resistant mature plant or seed or pollen according to claim 11, substantially as described herein by way of example and/or with reference to the accompanying drawings. 23

20. A method of claim 12 or claim 13, substantially as described herein by way of example and/or with reference to the accompanying drawings.

21. A nucleic acid construct according to claim 14, substantially as described herein by way of example and/or with reference to the accompanying drawings. AMERICAN CYANAMID COMPANY 17 Sheets