AU715462B2 - Engineered sterile plants and uses therefor - Google Patents

Description

A.

Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

(ORIGINAL)

Name of Applicant: Actual Inventor(s): Address for Service: Invention Title: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION Rebecca Louise HARCOURT William James PEACOCK Danny LLEWELLYN Simon SOUTHERTON Junko KYOZUKA Elizabeth Salisbury DENNIS DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

ENGINEERED STERILE PLANTS AND USES

THEREFOR

Details of Associated Provisional Application(s): No(s): PM9589 filed on 22 November, 1994 The following statement is a full description of this invention, including the best method of performing it known to me/us: -1- 951 122,p:\oper\ejh,PM9589.au,I 1A- ENGINEERED STERILE PLANTS AND USES THEREFOR The present invention relates generally to plants and more particularly to woody perennial plants such as eucalypts with reduced reproductive capacity. The present invention is also directed to genetic constructs useful in the generation of woody perennial plants with reduced reproductive capacity. The present invention is most particularly directed to sterile trees of the genus Eucalyptus which are useful in the establishment of wood lot plantations, in re-forestation projects and in developing modified eucalyptus trees with beneficial properties.

Bibliographic details of the publications referred to in this specification are collected at the end of the description. Sequence identity numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined after the I •bibliography.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but .ooo.i S•not the exclusion of any other element or integer or group of elements or integers.

Expanding urbanisation, the agricultural industry's requirement for more arable land and the increase in demand for wood from a range of timber-related industries are contributing to rapidly diminishing native forests. Despite the serious economic consequences of exhausting supply of this primary product, there are also serious environmental concerns commensurate with the loss of native forests. There is a need, therefore, to ensure adequate re-forestation and to establish wood lots of native trees in both national and regional parks and on private land holdings. There is also a need to develop new varieties of native trees with beneficial properties such as resistance to pest infestation or reduced frost sensitivity.

9511 22,p:\oper\ejh,PM9589.au,2 -2- Although a number of reproductive genes have been isolated from plants such as Arabidopsis and Antirrhinum, until now, analogous genes had not been cloned from eucalypts. In accordance with the present invention, the inventors have cloned a number of reproductive genes involved in floral development in woody perennials such as eucalypts. The isolation and characterization of these genes will enable the generation of a series of eucalypts and other woody perennials with reduced reproductive capacity.

Such plants will be useful in, for example, establishing wood lots for the timber and agricultural industries as well as in environmental planing. Furthermore, the availability of sterile eucalypts provides for the opportunity to introduce genes or other genetic sequences conferring new traits without the possibility that these traits are passed onto other trees in the event that the traits are deemed undesirable. The techniques developed in accordance with the present invention will allow the generation of a range of transgenic eucalypt trees exhibiting a variety of beneficial properties.

15 Accordingly, one aspect of the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a product or part, fragment, mutant, analogue or homologue thereof required for normal flower development in a woody perennial. For convenience, a part, fragment, mutant, analogue or homologue of the above-mentioned product is referred herein as a "derivative". Accordingly, the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a product or derivative thereof required for normal flower development in a woody perennial.

*o Preferably, the woody perennial is a species of the genus Eucalyptus. Examples of eucalypt trees contemplated by the present invention include but are not limited to one or more of Eucalyptus alpina, Eucalyptus annulata, Eucalyptus astringens, Eucalyptus behriana, Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus brockwayi, Eucalyptus caesia, Eucalyptus calophylla 'Rosea', Eucalyptus calycogona, Eucalyptus camaldulensis, Eucalyptus camaldulensis ssp. subcinerea, Eucalyptus campaspe, Eucalyptus camphora, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus cladocalyx 'Nana', Eucalyptus cloeziana, Eucalyptus cornuta, Eucalyptus cosmophylla, 951122,p:\oper\jhPM9589.au,3 -3- Eucalyptus crenulata, Eucalyptus crucis, Eucalyptus delegatensis, Eucalyptus desmondensis, Eucalyptus dielsii, Eucalyptus diptera, Eucalyptus diversicolor, Eucalyptus diversifolia, Eucalyptus dumosa, Eucalyptus dundasii, Eucalyptus dunnii, Eucalyptus eremophila, Eucalyptus erythrocorys, Eucalyptus erythronema, Eucalyptus ficifolia, Eucalyptus flocktoniae, Eucalyptus foecunda, Eucalyptus forrestiana, Eucalyptus gardneri, Eucalyptus gillii, Eucalyptus globulus, Eucalyptus globulus ssp globulus, Eucalyptus globulus ssp. pseudoglobulus, Eucalyptus globulus ssp maidenii, Eucalyptus globulus ssp bicostata, Eucalyptus gomphocephala, Eucalyptus gracilis, Eucalyptus grandis, Eucalyptus griffithsii, Eucalyptus grossa, Eucalyptus henrii, Eucalyptus increassata, Eucalyptus kitsoniana, Eucalyptus kondininensis, Eucalyptus kruseana, Eucalyptus lansdowneana, Eucalyptus largiflorens, Eucalyptus lehmanni, Eucalyptus leucoxylon, Eucalyptus leucoxylon (Pink) (syn. 'Rosea'), Eucalyptus lesouefii, Eucalyptus macrandra, Eucalyptus macrocarpa, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus melliodora, Eucalyptus microcarpa, Eucalyptus nicholii, Eucalyptus nitens, 15 Eucalyptus nutans, Eucalyptus obliqua, Eucalyptus occidentalis, Eucalyptus ochrophloia, Eucalyptus oleosa, Eucalyptus orbifolia, Eucalyptus ovata, Eucalyptus pauciflora, Eucalyptus parvifolia, Eucalyptus perriniana, Eucalyptus pileata, Eucalyptus pellita, Eucalyptuspilularis, Eucalyptusplatypus, Eucalyptus polyanthemos, Eucalyptus porosa, Eucalyptuspreissiana, Eucalyptus pyriformis, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus rhodantha, Eucalyptus robusta, Eucalyptus saligna, Eucalyptus salmonophloia, Eucalyptus salubris, Eucalyptus sargentii, Eucalyptus sepulcralis, Eucalyptus sideroxylon 'Rosea', Eucalyptus sieberi, Eucalyptus spathulata, Eucalyptus socialis, Eucalyptus steedmanii, Eucalyptus stoatei, Eucalyptus stricklandii, Eucalyptus tereticornis, Eucalyptus tetragona, Eucalyptus tetraptera, Eucalyptus torquata, Eucalyptus 'Torwood', Eucalyptus urnigera, Eucalyptus urophylla, Eucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo, Eucalyptus websteriana, Eucalyptus woodwardii.

The species of Eucalyptus may be selected from any or all of the above-listed species or from other species of Eucalyptus or from species related to eucalypts or which have eucalypt-like properties. Preferably, the eucalypt tree is Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cloeziana, Eucalyptus delegatensis, Eucalyptus diversicolor, Eucalpytus dunnii, Eucalyptus globulus, Eucalyptus globulus subspecies bicostata, Eucalyptus globulus 951122,p:\oper\ejhPM9589.au,4 -4subspecies globulus, Eucalyptus globulus subspecies maidenii, Eucalyptus globulus subspecies pseudoglobulus, Eucalyptus grandis, Eucalyptus henrii, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus nitens, Eucalyptus obliqua, Eucalyptus pellita, Eucalyptus pilularis, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus saligna, Eucalyptus sieberi, Eucalyptus tereticornis, Eucalyptus urophylla, Eucalyptus viminalis.

Most preferably, the eucalypt tree is Eucalyptus globulus ssp globulus or Eucalyptus nitens.

The nucleic acid is generally composed of deoxyribonucleotides and may be genomic DNA or cDNA. The nucleotide sequence of the nucleic acid molecule may correspond exactly with the nucleotide sequence of the naturally occurring gene or may contain one or more nucleotide substitutions, deletions and/or additions. Where the DNA is genomic DNA, the DNA may or may not contain introns.

15 In a particularly preferred embodiment, the reproductive gene is related in sequence and/or function to a corresponding gene in Arabidopsis and/or Antirrhinum.

Even more particularly, the reproductive gene corresponds to a gene selected from leafy (LFY), apetala 1 (API) or agamous (AG) from Arabidopsis or floricaula (FLO), squamosa (SQUA) or plena (PLE) from Antirrhinum.

o•* SIn a most preferred embodiment, the present invention provides an isolated DNA molecule selected from the following: having a sequence set forth in SEQ ID NO:1 or 3 or 5 and analogous to LFY and/or FLO; (ii) having a sequence set forth in SEQ ID NO:9 or 11 or 13 and analogous to API and/or SQUA; or (iii) having a sequence set forth in SEQ ID NO:7 or 15 and analogous to AG and/or

PLE.

951122,p:\oper\ejhPM9589.au,5 The eucalypt genes defined in (ii) and (iii) above are referred to herein as FLE, SQE and AGE, respectively.

According to preferred embodiments of the present invention, there is provided in one aspect, a DNA molecule of eucalypt origin encoding a reproductive gene and capable of hybridizing under low stringency conditions to a nucleic acid molecule having a nucleotide sequence as set forth in or complementary to SEQ ID NO: 1 or 3 or In another aspect, there is provided a DNA molecule of eucalypt origin encoding a reproductive gene and capable of hybridizing under low stringency conditions to a nucleic and molecule having a nucleotide sequence as set forth in or complementary to SEQ ID NO:9 or 11 or 13.

15 In still another aspect, there is provided a DNA molecule of eucalypt origin encoding a reproductive gene and capable of hybridizing under low stringency conditions to a nucleic and molecule having a nucleotide sequence as set forth in or complementary to SEQ ID NO:7 or For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook et al (1989) which is herein incorporated by reference where the washing steps at pages 9.52-9.57 are considered high stringency. A low stringency is defined herein as being in 0.1-0.5% w/v SDS at 37-45 0 C for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridisation, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 0.25%-0.5% w/v SDS at 45 0 C for 2-3 hours or high stringent conditions as disclosed by Sambrook et al (1989).

In an alternative embodiment, the preferred nucleic acid molecule of the present invention comprises a nucleotide sequence having at least about 45%, more preferably at least about 55%, still more preferably at least about 65%, yet still more preferably at least about 75-80% and even still more preferably at least about 85-95% nucleotide 951122,p:\oper\ejhPM9589.au,6 -6similarity to at least one of SEQ ID NO:1 or 3 or 5, SEQ ID NO:9 or 11 or 13 or SEQ ID NO:7 or The present invention also contemplates a method for identifying a gene required for normal flower development in a woody perennial, said method comprising isolating a nucleic acid molecule from said woody perennial and cloning a region of said nucleic acid molecule having at least 45% nucleotide similarity to all or a region of Arabidopsis genes LFY, API or AG or Antirrhinum genes FLO, SQUA or PLE.

Preferably, the woody perennial is a species of the genus Eucalyptus such as Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cloeziana, Eucalyptus delegatensis, Eucalyptus diversicolor, Eucalpytus dunnii, Eucalyptus globulus, Eucalyptus globulus subspecies bicostata, Eucalyptus globulus subspecies globulus, Eucalyptus globulus subspecies maidenii, Eucalyptus 15 globulus subspecies pseudoglobulus, Eucalyptus grandis, Eucalyptus henrii, Eucalyptus S' maculata, Eucalyptus marginata, Eucalyptus nitens, Eucalyptus obliqua, Eucalyptus pellita, Eucalyptus pilularis, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus saligna, Eucalyptus sieberi, Eucalyptus tereticornis, Eucalyptus urophylla, Eucalyptus viminalis. Most preferably, the species is Eucalyptus globulus ssp globulus or Eucalyptus nitens.

The nucleic acid molecules of the present invention are useful for generating antisense or ribozyme molecules or overexpression of sense molecules using a constitutive promoter or in co-suppression of the subject reproductive genes. By targeting such genes, their expression is diminished, reduced or otherwise lowered to levels which result in reproductive sterility or at least substantially reduced reproductive capacity.

According to one embodiment there is provided a nucleic acid molecule comprising at least 5 contiguous nucleotide bases capable of hybridizing to, forming a duplex with or otherwise reducing translation of target reproductive gene-specified mRNA. This embodiment of the present invention defines primers, probes and other antisense molecules capable of instituting or at least reducing translation of the reproductive gene- 951 122,p:\oper\ejhPM9589.au,7 -7specified mRNA. Although the preferred antisense molecules are at least about 10 to nucleotides in length, the present invention extends to molecules of 50-100 nucleotide bases in length or a molecule corresponding to a full length or substantially full length reproductive gene-specified mRNA.

Co-suppression involves introducing a second copy of an endogenous gene or a substantially similar copy or analogue of an endogenous gene. Over expression involves introducing a second copy of an endogenous gene or a substantially similar copy or analogue of an endogenous gene fused to a constitutive promoter.

Ribozymes are synthetic RNA molecules which possess highly specific endoribonuclease activity. In particular, they comprise a hybridizing region which is complementary in nucleotide sequence to at least part of a target RNA. Ribozymes are well described by Haseloff and Gerlach (1988) and in International Patent Application No. WO 89/05852.

15 The present invention extends to ribozymes which target reproductive gene mRNA.

o o According to this embodiment, there is provided a ribozyme comprising a hybridizing region and a catalytic region wherein the hybridizing region is capable of hybridizing to at least part of a target mRNA sequence transcribed from a reproductive gene and more particularly as defined by SEQ ID NO:1 or 3 or 5, SEQ ID NO:9 or 11 or 13 or SEQ ID NO:7 or 15, wherein said catalytic domain is capable of cleaving said target mRNA sequence to reduce or substantially inhibit reproductive capacity in a woody S. perennial and more particularly a eucalypt.

The present invention extends to genetic constructs designed to facilitate expression of the antisense molecule, co-suppression molecule, overexpression molecule or ribozyme.

Generally, the genetic construct comprises in addition to the subject nucleic acid molecule, a promoter and optionally other regulatory sequences to facilitate expression of the nucleic acid molecule. The promoter may be derived from a genomic clone encoding the reproductive gene or may be a heterologous promoter from another source.

Preferably, however, the promoter is tissue specific capable of expression substantially only in reproductive tissue for antisense, ribozyme and co-suppression molecules. For 951122,p:\oper\ejhPM9589.au,8 -8over exoression molecules the promoter is tissue non-specific, capable of expression substantially in all tissues.

In an alternative embodiment, a promoter from a genomic clone of a reproductive gene and, more particularly, the promoter from the gene corresponding to SEQ ID NO:1 or 3 or 5, SEQ ID NO:9 or 11 or 13 or SEQ ID NO:7 or 15, is operably linked to a suicide gene such as a gene encoding a toxic molecule (eg Barnase).

According to this embodiment, one aspect is directed to a genetic construct comprising a promoter or functional derivative, part, fragment, homologue or analogue thereof from a genomic clone equivalent of the nucleotide sequence defined by SEQ ID NO:1 or 3 or A second aspect of the present invention is directed to a promoter or functional 15 derivative, part, fragment, homologue or analogue thereof from a genomic clone equivalent of the nucleotide sequence defined by SEQ ID NO:9 or 11 or 13.

A third aspect of the present invention relates to a promoter or functional derivative, part, fragment, homologue or analogue thereof from a genomic clone equivalent of the nucleotide sequence defined by SEQ ID NO:7 or In accordance with these three aspects of the present invention, the genetic construct optionally further comprises a nucleic acid molecule defining an antisense molecule to o at least one of SEQ ID NO:1 or 3 or 5, SEQ ID NO:9 or 11 or 13 or SEQ ID NO:7 or 15, a sense molecule to at least one of SEQ ID NO:1 or 3 or 5, SEQ ID NO:9 or 11 or 13 or SEQ ID NO:7 or 15, or a ribozyme capable of targeting a mRNA transcript corresponding to SEQ ID NO:1 or 3 or 5, SEQ ID NO:9 or 11 or 13 or SEQ ID NO:7 or Alternatively, or in addition to, the genetic construct is multifacet and is capable of targeting at least two reproductive genes by the same or different mechanisms.

951 122,poperejhPM9589.au,9 -9- Yet another aspect of the present invention contemplates a method of minimising inflorescence in a woody perennial such as a eucalypt, said method comprising introducing into one or more cells of said woody perennial a genetic construct capable of down regulating expression of a reproductive gene in said woody perennial, regenerating a transgenic plant from said one or more cells and growing said plant for a time and under conditions sufficient to permit expression of said genetic construct.

Preferably, the woody perennial is a eucalypt, such as selected from the previously mentioned list. Preferably, the eucalyptus is Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cloeziana, Eucalyptus delegatensis, Eucalyptus diversicolor, Eucalpytus dunnii, Eucalyptus globulus, Eucalyptus globulus subspecies globulus, Eucalyptus globlulus subspecies bicostata, Eucalyptus globulus subspecies maidenii, Eucalyptus globulus subspecies pseudoglobulus, Eucalyptus grandis, Eucalyptus henrii, Eucalyptus maculata, Eucalyptus 15 marginata, Eucalyptus nitens, Eucalyptus obliqua, Eucalyptus pellita, Eucalyptus pilularis, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus saligna, Eucalyptus sieberi, Eucalyptus tereticornis, Eucalyptus urophylla, Eucalyptus viminalis. Most preferably the eucalypt is Eucalyptus globulus ssp globulus or Eucalyptus nitens.

Preferably, the reproduction gene corresponds to one or more of SEQ ID NO:1 or 3 or 5, SEQ ID NO:9 or 11 or 13 or SEQ ID NO:7 or Preferably, the transgenic plant exhibits a substantial reduction in reproductive capability.

More preferably, the plant is substantially sterile.

Still yet a further aspect of the present invention is directed to a transgenic eucalypt plant such as a eucalypt selected from the previously mentioned list wherein the plant exhibits substantially reduced reproductive sterility.

991126,p:\oper\ejh,PM9589.au,10 The present invention is further described by the following non-limiting Figures and/or Examples.

In the Figures: Figure 1 is a representation of the nucleotide sequence of genomic clone FLE2 with the predicted amino acid sequences. The positions of the PCR primers used to isolate the dene are underlined.

S* Figure 2 is a representation of the nucleotide sequence of AGEl cDNA with its predicted •amino acid sequence. The MADS box and position of the PCR primers used to isolate the gene are underlined.

S

15 Figure 3 is a representation of the nucleotide sequence of SQEl cDNA. The MADS box is underlined; the amino acids at the positions of the PCR primers used to isolate the gene •are in italics.

Figure 4 is a representation of the nucleotide sequence ofSQE2S cDNA with its predicted 20 amino acid sequence. The MADS box is single underlined.

S*

Figure 5 is a representation of the nucleotide sequence ofSQE2L cDNA with its predicted amino acid sequence. The MADS box is single underlined.

Figure 6 is a representation of nucleotide sequence of AGE2 with its predicted amino acid sequence. The MADS box is single underlined.

O//

951 122,p:\oper\ejh,PM9589.au, I11 Single and three letter abbreviations used for amino acid residues in the specification are defined in Table 1.

TABLE 1 Amino Acid Three-letter One-letter Abbreviation Symbol Alanine Ala

A

Arginine Arg

R

*0Asparagine Asn

N

Aspartic acid Asp

D

*Cysteine Cys

C

Glutamine Gin

Q

*Glutamic acid Glu

E

*Glycine Gly

G

Histidine His

H

Isoleucine le

I

Leucine Leu

L

Lysine Lys

K

Methionine Met

M

*Phenylalanine Phe

F

Proline Pro

P

Serine Ser

S

Threonine Tbr

T

Tryptophan Trp

W

Tyrosine Tyr

Y

Valine Val

V

951 122,p:\oper\ejh,PM9589.au, 12 12 A summary of SEQ ID NOs used throughout the specification is shown in Table 2.

TABLE 2 SEQ ID NO

DEFINITION

9 9 9 *99* 9 9 9 9 9 9.

9*9*u* 9 9* C 99 SEQ ID NO:1I SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 15 SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO: 10 SEQ ID NO: 11 20 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 Genomic clone of FLE Predicted amino acid sequence of FLE- 1 Genomic clone of FLE Predicted amino acid sequence of FLE-2 Genomic clone of FLE Predicted amino acid sequence of FLE-3 cDNA AGE1 clone Predicted amino acid sequence of AGE1 cDNA SQE1 clone Predicted amino acid sequence of SQEl cDNA SQE2S clone Predicted amino acid sequence of SQE2S cDNA SQE2L clone Predicted amino acid sequence of SQE2L cDNA A GE2 clone Predicted amino acid sequence of AGE2 951 1 22,p:\oper\ejh,PM9589.au, 13 13 Identification Arabidopsis and Antirrhinum genes and the names of eucalypt homologues are shown in Table 3.

TABLE 3 Arabidopsis Antirrhinum Eucalyptus Leafy (LFY) Floricaula (FLO) FLE Apetala 1 (API) Squamosa (SQUA) SQE Agamous (AG) Plena (PLE) AGE a. EXAMPLE 1 Isolation of the eucalypt genes The strategy used to isolate the eucalypt genes involved design and synthesis of polymerase chain reaction (PCR) primers to regions of amino acid homology between the Arabidopsis and Antirrhinum genes. These primers were then used to amplify from eucalypt DNA (FLE) or cDNA (AGE and SQE) short regions of DNA from the eucalypt homologue. The DNA fragments obtained were then used as probes to screen Eucalyptus globulus libraries to obtain the full length genes.

EXAMPLE 2

FLE

Three copies of FLE were isolated from a Eucalyptus globulus genomic library. Two of these copies were not functional because the sequences were interrupted by stop codons and frame shifts. These two copies were found to be two alleles at a pseudogene locus. The other copy appears to be the homologue of the LFY and FLO genes in eucalypts. The predicted protein product of FLE2 is 359 amino acids in length and 951 122,p:\oper\ejhPM9589.au,14 14shares 67% identical aa with LFY and 71% identical aa with FLO. The nucleotide sequence (SEQ ID NO:1) and the predicted amino acid sequences (SEQ ID NO:2, 4 and 6) of the genomic clone of FLE2 are shown in Figure 1.

Expression of FLE2 RNA was determined by in situ hybridization. FLE2 RNA was observed in developing floral organs in a similar manner to the expression patterns of FLORICAULA and LEAFY. In addition, FLE2 is strongly expressed in the primordia of true leaves as they develop on the flanks of apical and lateral vegetative meristems.

Expression was not detected in the centre of the vegetative apical meristem or in stems or roots. Its observed expression in vegetative and floral tissue possibly suggests a wider role for this gene in Eucalyptus development.

The promoter of the FLE2 gene was also isolated from the genomic library. One kilobase (kb) of the promoter has been sequenced and 2 kb have been used in sterility 15 constructs.

EXAMPLE 3 AGE1 and AGE2 20 Full length cDNA clones of AGE1 were isolated from a cDNA library constructed from RNA from mature flower buds. The gene encodes a protein which is 251 amino acids in length with amino acid homology between the AGE1 protein and the AG protein of about 68% and with the PLE protein, about 75%. The AG and PLE proteins contain a conserved region of 56 amino acids known as the MADS box region. This region is present in a number of different genes involved in floral development. The AGE1 gene also contains this region of 56 amino acids which is identical to that of the AG MADS box. Two of the 56 amino acids differ between PLE and AGEl. Southern blots of Eucalyptus globulus DNA probed with a fragment of AGE1 cDNA which does not contain the MADS box suggest that there is only one copy of the AGE1 gene in the eucalypt genome. The nucleotide sequence of the AGE1 cDNA and the predicted amino acid sequence are shown in Figure 2. The nucleotide sequence of another cDNA clone, AGE2, is shown in Figure 6 with its predicted amino acid sequence. AGE2 is 229 951 122,p:\oper\ejhPM9589.au,15 15 amino acids in length. It has good homology to both the AG and PLE proteins, of 62% and 64%, respectively.

The fact that both AGE1 and AGE2 were isolated from a cDNA library is a good indication that these genes are functional. An RNA probe made from AGE1 was used to screen RNA isolated from mature, medium and young flower buds, anthers, leaves, stems and roots of Eucalyptus globulus. The hybridisation pattern suggests that AGE is expressed very highly in mature flower buds and not at all in leaves, stems or roots.

The level of expression in anthers and medium flower buds is moderate. There is very low, if any, expression in young flower buds. This pattern of expression is very similar to that of AG and PLE. AGE2, however, is strongly expressed only in anthers, young flower buds and mature flower buds. It is weakly expressed in leaves.

0* In order to isolate the AGE promoter, an Eucalyptus globulus genomic DNA library was 15 screened with a fragment of AGE cDNA that does not contain the MADS region to avoid cross-hybridisation with other MADS-containing genes. A number of clones have been identified. The promoter is used to generate a series of sterility constructs in Arabidopsis and Eucalyptus.

20 EXAMPLE 4 SQE1 and SQE2 The fragment of the SQE1 and SQE2 genes amplified from cDNA was from within the MADS box. When a cDNA library constructed from RNA from young flower buds was screened with this probe, four clones were isolated. One of these genes, SQE1, contains a MADS box with 95% homology to API and 91% homology to SQUA. Sequencing of the gene has revealed 69% homology to API at the DNA level. The DNA sequence and predicted amino acid sequence of SQE1 are shown in Figure 3. Other genes isolated are designated SQE2S having a DNA sequence and predicted amino acid sequence shown in Figure 4 (SEQ ID NOs 11 and 12, respectively) and SQE2L having a DNA sequence and predicted amino acid sequence shown in Figure 5 (SEQ ID NOs 13 and 14, respectively). These genes will be investigated for their potential for use in the 951 122,p:\oper\ejhPM9589.au, 16 16sterility strategy.

The promoters of the genes may be used to generate sterility constructs.

EXAMPLE Ectopic Expression of the Eucalypt genes in Arabidopsis confirms their functional similarity to the Arabidopsis genes To examine the function of the two genes in flower development, AGE1 and AGE2 cDNAs were ectopically expressed (also known as overexpression) in Arabidopsis plants under the control of the cauliflower mosaic virus 35S promoter. More than independent transgenic plants were regenerated for each construct. The most commonly observed phenotype in the primary transformants was that the sepals developed stigmatic papillae, the petals became staminoid or were shorter than normal while the stamens and 15 carpels appeared normal or occasionally the carpels were unfused. Most of the plants, however, were sterile. 25% of 35S-AGE1 and 29% of 35S-AGE2 transformants showed this phenotype. Some plants showed a severe phenotype while others showed a weaker one. As most plants were sterile it was impossible to test the segregation of the transgene in the next generation. Of the plants that were fertile, most appeared to 20 produce a normal phenotype in the next generation, suggesting that there was very weak expression of the gene. Based on the phenotype of the primary transformants it appears that both the AGE1 and AGE2 genes from Eucalyptus are functionain Arabidopsis; that they have a similar function to the AG gene of Arabidopsis; and high levels of expression of either or both the AGE1 and AGE2 genes in transgenic Eucalyptus may lead to sterile plants. Thus, expression of flowering genes using the CaMV35S promoter to cause disruption of flowering is an alternative strategy to that of antisense, cosuppression or RNase genes.

To examine the function of the two eucalypt genes (SQEl and SQE2L) in flower development, and the Arabidopsis gene, AP1, the three cDNAs were ectopically expressed in Arabidopsis plants under the control of the CaMV35S promoter. More than independent transgenic plants were regenerated for each construct. The most 951122,p:\oper\ejh,PM9589.au,17 17commonly observed phenotype in the primary transformants was a homeotic conversion of stem meristems into flowers these plants resembled the Arabidopsis mutant terminal flowr (tfl). Development of the stem meristem, which is normally indeterminate, stops early in transgenic plants with a severe tfl-like phenotype. Instead they form an abnormal flower at the top of the stems. The terminal flower consists of the disorganised aggregation of many floral organs. 30%, 43% and 30% of the transformed plants carrying 35S-AP1, 35S-SQE1 and 35S-SQE2L, respectively, showed the tfl-like phenotype. Although the severity and the details of the phenotype varied between independent transgenic plants, the overall ranges of phenotypes observed seemed to be similar between the three different constructs.

The phenotype was analysed in detail in the T 1 generation grown on soil. The tfl-like phenotype observed in the T o generation was inherited in the next generation as a dominant Mendelian trait and it co-segregated with kanamycin resistance. Table 4 15 summarises the characteristics of six independent transgenic lines. T 1 transgenic plants S* flowered earlier compared to control plants. They formed an increased number of stems and were shorter than control plants.

These results indicate that the Eucalyptus genes, SQE1 and SQE2L have the same 20 function as the Arabidopsis AP1 gene in flower development.

951 122,p:\oper\ejhPM9589.au,18 18 TABLE 4 Comparison of flowering time, number of stems and height of main stem in transgenic (T 1 and wild type (C24) Arabidopsis plants.

Transgenic Days to Number of Height of main line flowering stems stem (cm) 35S-AP1 A15 17.0 2.0 5.2 A52 14.0 3.0 35S-SQE1 S1-23 16.0 5.5 S1-25 13.0 4.6 35S-SQE2L S2-16 12.0 1.8 2.3 S2-28 13.5 5.3 2.4 Control 19.7 1.2 18.8 S. EXAMPLE Genetic Constructs Conferring Sterility in vivo model using Arabidopsis A number of constructs are tested for their ability to prevent flowering in Arabidopsis (Table Constructs are transformed into wild-type Arabidopsis and plants regenerated.

One plant, from construct 1, independent transgenic line 1, had a phenotype very similar to that of the LEAFY mutant which exhibits female-fertility but is completely male sterile. This result strongly indicates that an antisense approach is capable of mimicking the mutant phenotypes observed in Arabidopsis. This plant was cross-pollinated and a small number of seeds collected for further analysis. One other plant from construct 1 had a similar but less pronounced phenotype. Plants from all other lines of construct 1 and the other constructs have been allowed to self-pollinate, seed from the TO plants 951122,p:\oper\ejh,PM9589.au,19 19has been collected and planted out in soil. Seed of seven independent (TO) transgenic lines were planted out from construct 1. One line (1-17) showed several plants (T1) with an altered phenotype. The TO plant that produced this seed appeared normal. One of the eleven plants from this line (1-17) has a phenotype very similar to that of the strongest allele of the LEAFY mutant which is male-sterile with very much reduced female fertility. Nine plants have phenotypes similar to the mutants of the weaker alleles of LEAFY. These eleven plants will be further analysed. These results strongly support the hypothesis that we can produce sterile plants by introducing antisense to these genes into transgenic plants.

Sterility constructs are made with antisense to LEAFY API and both LEAFY and API in the one construct. These antisense constructs are expressed by the histone promoter H4A or the CaMV35S promoter which has been observed to give high expression in young flower buds.

r rr r r r r r r r .r a r r r r ,r 1 r TABLE Constructs tested in Arabidopsis and the number of transformed plants obtained.

Promoter Gene Number of plants obtained 1. leafy antisense leafy 52 2. leafy antisense AG-MADS 84 3. 35S antisense leafy 4. FLE2 antisense FLE2 951122,p:\oper\ejh,PM9589.au,20 Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

*B

B B

S**

951 122,p:\oper\ejh,PM9589.au,21 21

REFERENCES:

Haseloff J. and Gerlach Nature 334: 5 86-594, 1988 Sambrook et al, Molecular Cloning :A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, New York, USA, 1989 *a a a a 4. a a 951 122,p:\oper\ejb,PM9589.au,22 -22- SEOUENCE LISTING GENERAL INFORMATION: APPLICANT: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION INVENTORS: HARCOURT, KYOZUKA, DENNIS, E., PEACOCK, SOUTHERTON, S. and LLEWELLYN, D.

(ii) TITLE OF INVENTION: ENGINEERED STERILE PLANTS AND USES

THEREFOR

(iii) NUMBER OF SEQUENCES: 16 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DAVIES COLLISON CAVE STREET: 1 LITTLE COLLINS STREET CITY: MELBOURNE STATE: VICTORIA S• COUNTRY: AUSTRALIA ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: AU COMPLETE FILING DATE: 21-NOV-1995 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: PM9589 FILING DATE: 22-NOV-1994 (viii) ATTORNEY/AGENT INFORMATION: NAME: HUGHES, DR E JOHN L REFERENCE/DOCKET NUMBER: EJH/EK (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: +61 3 9254 2777 TELEFAX: +61 3 9254 2770 TELEX: AA31787 9511 2 2,p:\oper\ejhPM9589.au,23 -23 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1500 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 121..453 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CCACTACGTA CGTACATACA GTGTACATTG AATATACTAA ACAGATGGAC CCATGTGTTT ATAAAACAAG GAAGCGAGAG TCCTGTACCC TTACACGCTG AAGCCATTTG AAGCGCGAGA 120 ATG GAT CCA GAA GCA TTT GCG GTT GTG GGG TTG CGA ACG ATG GGG GGA 168 Met Asp Pro Giu Ala Phe Ala Val Val Gly Leu Arg Thr Met Gly Gly 1 5 10 CTG GAG GAG CTG TTC GAG GCT TAT GGC ATA AGG TAC CTC ACG GCC TCC 216 Leu Glu Giu Leu Phe Giu Ala Tyr Gly Ile Arg Tyr Leu Thr Ala Ser 20 25 AGG ATA GCG GAA ATG GGG TTT ACG GCC AAC ACC CTC CTC GAC ATG AAG 264 Arg Ile Ala Giu Met Gly Phe Thr Ala Asn Thr Leu Leu Asp Met Lys 40 GAG GAG GAG CTC GAC GAC ATG ATG AAC TCC CTC TCC CAC ATC TTC CGC 312 Glu Glu Giu Leu Asp Asp Met Met Asn Ser Leu Ser His Ile Phe Arg 55 TGG GAC CTC CTC GTC GGC GAG CGC TAC GGC ATC AAG GCC GCC ATC CGC 360 Trp Asp Leu Leu Val Gly Glu Arg Tyr Gly Ile Lys Ala Ala Ile Arg 70 75 GCC GAG CGC CGA CGC CTC CTC GAA GCC GAT GAC CGC CGC CAC CAC CTC 408 Ala Glu Arg Arg Arg Leu Leu Giu Ala Asp Asp Arg Arg His His Leu 90 CAC TCC ACC GAC CAT GCC CTC CTC GAT GCT CTC TCC CAC CAA GGT 453 His Ser Thr Asp His Ala Leu Leu Asp Ala Leu Ser His Gin Gly 100 105 110 ACCTTAGCTC GGCCTCTTTG ATTTTTGACG TTTTGGGTCA TTATGATTAT TCCCACGCAG 513 GGCTGTCGGA GGAACAAGTG GTGCAGCACT CAGAGAAGGA TCAGCTGGGC AGGGCGGGAA 573 GCGGGGACAC GGCGGGCACG TCGTGGGGCG CCCAACAACA GAGAAAGAAG CATCGTCATC 633 GTCACCACAT CACCGCGATG AAAGGAGCGG CCACGGAAGA GGACGAGGAG GACGAGGAGG 693 AAGTGGAGGA GATGAGGAGG CAGAGGGAGC ACCCCTTCAT AGTGACGGAG CCCGGGGAGG 753 TGGCGCGTGG GAAGAAGAAC GGCCTGGACT ACCTCTTCCA TCTCTACGAC CAGTGCCGCG 813 ACTTCCTCCT CCAAGTCCAA TCCTTGGCCA AGGAGCGGGG CGAGAAATGC CCCACCAAGG 873 TCCTCCGCCT CTCTTCTTTT TATAGTCTTT CGATACAATC AAACTTCTTC GAACGTACCC 933 951 122,p:\oper\cjh,PM9589.au,24 24 AAGCACGGAT CATTCAAACA AGAAGACAAT GCAGGTGACG AACCAGGTGT TCAGGTACGC GAAGAAGGCG GGAGCAAGCT ACATAAACAA GCCGAAGATG AGGCACTATG TCCACTGCTA CGCCCTGCAC TGCCTGGACG AGCACGCCTC CAACGCCCTT CGCAAGAGCT TCAAGGAGCG CGGGGAGAAC GTCGGCGCCT GGAGGCAAGC CTGTTACCAC CCCCTGGTCA CCATCGCCGG CCGCAGGGCC GGCTGGGACA TCGACGCCAT CTTCAATGCC CACCCCCGCC TCTGCATCTG GTATGTCCCC ACCAAGCTCC GCCAGCTCTG CCATGCTCAC CGCCACTCCT CCGCCTCTGC TGCTTCCTCC GCTTCCACCT CCACCTCTGC CCCCACCGCT CACCATCTCG AACTCCCTTA CTAGTTCGTG CCCGTTCCTC ATTCTCTTGT GTTTTAGTGT CGTATCAGCG CATGATCATG AGGGAGATGA AAGTACTGTG CTGTGGTCTA TTTCTACTGC AATGAGTATT AATTGAATTT TAACGTGTAG CTGAGCTGAG CTGAGCT 993 1053 1113 1173 1233 1293 1353 1413 1473 1500 a

S

a a.

INFORMATION FOR SEQ ID NO:2: Wi SEQUENCE CHARACTERISTICS: LENGTH: 111 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE Asp Pro Glu Ala 5 Glu Glu Leu Phe Met 1 Leu TYPE: protein DESCRIPTION: SEQ ID NO:2: Phe Ala Val Val Gly Leu Arg 10 Glu Ala Tyr Gly Ile Arg Tyr 25 Gly Phe Thr Ala Asn Thr Leu Thr Met Gly Gly Leu Leu Arg Ile Ala Glu Met Glu Glu Glu Leu Asp Trp Asp Leu Leu Val Thr Ala Ser Asp Met Lys Ile Phe Arg Asp Met Met Asn Ser 55 Gly Glu Arg Tyr Gly 70 Leu Ser Ile Lys His Ala Glu Arg Arg Arg Leu Leu Glu Ala Asp 90 Asp Arg Leu Ser Ala Ala Ile Arg His His His Gin Gly 110 His Ser Thr Asp His Ala Leu Leu Asp Ala 100 105 951122,p:\oper\ejhPM9589.au,25 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1500 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 516..872 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCACTACGTA CGTACATACA GTGTACATTG AATATACTAA ACAGATGGAC CCATGTGTTT ATAAAACAAG GAAGCGAGAG TCCTGTACCC TTACACGCTG AAGCCATTTG AAGCGCGAGA 120 ATGGATCCAG AAGCATTTGC GGTTGTGGGG TTGCGAACGA TGGGGGGACT GGAGGAGCTG 180 TTCGAGGCTT ATGGCATAAG GTACCTCACG GCCTCCAGGA TAGCGGAAAT GGGGTTTACG 240 GCCAACACCC TCCTCGACAT GAAGGAGGAG GAGCTCGACG ACATGATGAA CTCCCTCTCC 300 CACATCTTCC GCTGGGACCT CCTCGTCGGC GAGCGCTACG GCATCAAGGC CGCCATCCGC 360 GCCGAGCGCC GACGCCTCCT CGAAGCCGAT GACCGCCGCC ACCACCTCCA CTCCACCGAC 420 CATGCCCTCC TCGATGCTCT CTCCCACCAA GGTACCTTAG CTCGGCCTCT TTGATTTTTG 480 ACGTTTTGGG TCATTATGAT TATTCCCACG CAGGG CTG TCG GAG GAA CAA GTG 533 Leu Ser Giu Giu Gin Val 1 GTG CAG CAC TCA GAG AAG GAT CAG CTG GGC AGG GCG GGA AGC GGG GAC 581 Val Gin His Ser Glu Lys Asp Gin Leu Gly Arg Ala Gly Ser Gly Asp 15 ACG GCG GGC ACG TCG TGG GGC GCC CAA CAA CAG AGA AAG AAG CAT CGT 629 Thr Ala Gly Thr Ser Trp Giy Ala Gin Gin Gin Arg Lys Lys His Arg 30 CAT CGT CAC CAC ATC ACC GCG ATG AAA GGA GCG GCC ACG GAA GAG GAC 677 His Arg His His Ile Thr Ala Met Lys Gly Ala Ala Thr Giu Giu Asp 45 GAG GAG GAC GAG GAG GAA GTG GAG GAG ATG AGO AGO CAG AGG GAG CAC 725 Glu Glu Asp Giu Glu Giu Val Glu Glu Met Arg Arg Gin Arg Glu His 60 65 CCC TTC ATA GTG ACG GAG CCC GG GAG GTG GCG CGT GGG AAG AAG AAC 773 Pro Phe Ile Val Thr Giu Pro Gly Glu Val Ala Arg Gly Lys Lys Asn 80 GGC CTG GAC TAC CTC TTC CAT CTC TAC GAC CAG TGC CGC GAC TTC CTC 821 Gly Leu Asp Tyr Leu Phe His Leu Tyr Asp Gin Cys Arg Asp Phe Leu 95 100 CTC CAA GTC CAA TCC TTG GCC AAG GAG CGG GGC GAG AAA TGC CCC ACC 869 Leu Gin Val Gln Ser Leu Ala Lys Giu Arg Gly Glu Lys Cys Pro Thr 105 110 115 951 122,p:\oper\ejh,PM9589.au,26 26 AAG GTCCTCCGCC ±cLTCrTCTTT TTATAGTCTT TCGATACAAT CAAACTTCTT Lys CGAACGTACC CAAGCACGGA TCATTCAAAC AACAAGACAA TGCAGGTGAC GAACCAGGTG TTCAGGTACG CGAAGAAGGC GGGAGCAAGC TACATAAACA AGCCGAAGAT GAGGCACTAT GTCCACTGCT ACGCCCTGCA CTGCCTGGAC GAGCACGCCT CCAACGCCCT TCGCAAGAGC TTCAAGGAGC GCGGGGAGAA CGTCGGCGCC TGGAGGCAAG CCTGTTACCA CCCCCTGGTC ACCATCGCCG GCCGCAGGGC CGGCTGGGAC ATCGACGCCA TCTTCAATGC CCACCCCCGC CTCTGCATCT GGTATGTCCC CACCAAGCTC CGCCAGCTCT GCCATGCTCA CCGCCACTCC TCCGCCTCTG CTGCTTCCTC CGCTTCCACC TCCACCTCTG CCCCCACCGC TCACCATCTC GAACTCCCTT ACTAGTTCGT GCCCGTTCCT CATTCTCTTG TGTTTTAGTG TCGTATCAGC GCATGATCAT GAGGGAGATG AAAGTACTGT GCTGTGGTCT ATTTCTACTG CAATGAGTAT TAATTGAATT TTAACGTGTA GCTGAGCTGA GCTGAGCT INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid TOPOLOGY: linear 922 982 1042 1102 1162 1222 1282 1342 1402 1462 1500 a. a a. (ii) MOLECULE (Xi) SEQUENCE Ser Glu Glu Gin TYPE: protein DESCRIPTION: SEQ ID NO:4: Leu 1 Arg Ala Gly Ser Gly Gin Arg Lys Lys His Ala Ala Thr Glu Glu Val Val Gin His Ser 10 Asp Thr Ala Gly Thr 25 Arg His Arg His His 40 Asp Giu Glu Asp Glu Glu Lys Asp Gin Leu Gly Ser Trp Gly Ile Thr Ala Glu Glu Val Ala Gin Gin Met Lys Gly Glu Giu Met Arg Arg Ala Arg Gin Arg Giu His 70 Gly Lys Lys Asn Pro Phe Ile Val Thr Glu Leu Phe Pro Gly Giu Gly Leu Asp His Leu Tyr Asp Ala Lys Glu Arg 110 Gin Cys Arg Asp 100 Gly Glu Lys Cys 115 Phe Leu Leu Pro Thr Lys Gin Val 105 Gin Ser Leu 951 122,p:\opr\ejhPM9589.au,27 -27- INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1500 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 968..1354 (xi) SEQUENCE DESCRIPTION: SEQ ID CCACTACGTA CGTACATACA GTGTACATTG AATATACTAA ACAGATGGAC CCATGTGTTT ATAAAACAAG GAAGCGAGAG TCCTGTACCC TTACACGCTG AAGCCATTTG AAGCGCGAGA ATGGATCCAG AAGCATTTGC TTCGAGGCTT ATGGCATAAG

S*

S

*5 S

S.

S* S

GCCAACACCC

CACATCTTCC

GCCGAGCGCC

CATGCCCTCC

ACGTTTTGGG

ACTCAGAGAA

GCGCCCAACA

CGGCCACGGA

AGCACCCCTT

ACTACCTCTT

CCAAGGAGCG

TCCTCGACAT

GCTGGGACCT

GACGCCTCCT

TCGATGCTCT

TCATTATGAT

GGATCAGCTG

GGTTGTGGGG

GTACCTCACG

GAAGGAGGAG

CCTCGTCGGC

CGAAGCCGAT

CTCCCACCAA

TATTCCCACG

GGCAGGGCGG

TTGCGAACGA TGGGGGGACT GGAGGAGCTG GCCTCCAGGA TAGCGGAAAT GGGGTTTACG GAGCTCGACG ACATGATGAA CTCCCTCTCC GAGCGCTACG GCATCAAGGC CGCCATCCGC GACCGCCGCC ACCACCTCCA CTCCACCGAC GGTACCTTAG CTCGGCCTCT TTGATTTTTG CAGGGCTGTC GGAGGAACAA GTGGTGCAGC GAAGCGGGGA CACGGCGGGC ACGTCGTGGG 180 240 300 360 420 480 540 600 660 720 780 840 900 ACAGAGAAAG AAGCATCGTC ATCGTCACCA CATCACCGCG ATGAAAGGAG AGAGGACGAG GAGGACGAGG AGGAAGTGGA GGAGATGAGG AGGCAGAGGG CATAGTGACG GAGCCCGGGG AGGTGGCGCG TGGGAAGAAG AACGGCCTGG CCATCTCTAC GACCAGTGCC GCGACTTCCT CCTCCAAGTC CAATCCTTGG GGGCGAGAAA TGCCCCACCA AGGTCCTCCG CCTCTCTTCT TTTTATAGTC TTTCGATACA ATCAAACTTC TTCGAACGTA CCCAAGCACG GATCATTCAA ACAAGAAGAC AATGCAG GTG ACG AAC CAG GTG TTC AGG TAC GCG AAG AAG GCG GGA GCA Val Thr Asn Gin Val Phe Arg Tyr Ala Lys Lys Ala Gly Ala 1 5 AGC TAC ATA AAC AAG CCG AAG ATG AGG CAC TAT GTC CAC TGC TAC GCC Ser Tyr Ile Asn Lys Pro Lys Met Arg His Tyr Val His Cys Tyr Ala 20 25 CTG CAC TGC CTG GAC GAG CAC GCC TCC AAC GOC CTT CGC AAG AGC TTC Leu His Cys Leu Asp Glu His Ala Ser Asn Ala Leu Arg Lys Ser Phe 40 AAG GAG CGC GGG GAG AAC GTC GGC GCC TGG AGG CAA GCC TGT TAC CAC Lys Glu Arg Gly Glu Asn Val Gly Ala Trp Arg Gin Ala Cys Tyr His 55 960 1009 1057 1105 1153 951122,p:\oper\ejhPM9589.au,28 -28- CCC CTG GTC ACC ATC GCC GGC CGC AGG GCC GGC TGG GAC ATC GAC GCC 1201 Pro Leu Val Thr Ile Ala Gly Arg Arg Ala Gly Trp Asp Ile Asp Ala 70 ATC TTC AAT GCC CAC CCC CGC CTC TGC ATC TGG TAT GTC CCC ACC AAG 1249 Ile Phe Asn Ala His Pro Arg Leu Cys Ile Trp Tyr Val Pro Thr Lys 85 CTC CGC CAG CTC TGC CAT GCT CAC CGC CAC TCC TCC GCC TCT GCT GCT 1297 Leu Arg Gin Leu Cys His Ala His Arg His Ser Ser Ala Ser Ala Ala 100 105 110 TCC TCC GCT TCC ACC TCC ACC TCT GCC CCC ACC GCT CAC CAT CTC GAA 1345 Ser Ser Ala Ser Thr Ser Thr Ser Ala Pro Thr Ala His His Leu Glu 115 120 125 CTC CCT TAC TAGTTCGTGC CCGTTCCTCA TTCTCTTGTG TTTTAGTGTC 1394 Leu Pro Tyr GTATCAGCGC ATGATCATGA GGGAGATGAA AGTACTGTGC TGTGGTCTAT TTCTACTGCA 1454 ATGAGTATTA ATTGAATTTT AACGTGTAGC TGAGCTGAGC TGAGCT 1500 INFORMATION FOR SEQ ID NO:6: S* SEQUENCE CHARACTERISTICS: LENGTH: 129 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Val Thr Asn Gln Val Phe Arg Tyr Ala Lys Lys Ala Gly Ala Ser Tyr S1 5 10 Ile Asn Lys Pro Lys Met Arg His Tyr Val His Cys Tyr Ala Leu His 25 Cys Leu Asp Glu His Ala Ser Asn Ala Leu Arg Lys Ser Phe Lys Glu 35 40 Arg Gly Glu Asn Val Gly Ala Trp Arg Gin Ala Cys Tyr His Pro Leu 55 Val Thr Ile Ala Gly Arg Arg Ala Gly Trp Asp Ile Asp Ala Ile Phe 70 75 Asn Ala His Pro Arg Leu Cys Ile Trp Tyr Val Pro Thr Lys Leu Arg 90 Gin Leu Cys His Ala His Arg His Ser Ser Ala Ser Ala Ala Ser Ser 100 105 110 Ala Ser Thr Ser Thr Ser Ala Pro Thr Ala His His Leu Glu Leu Pro 115 120 125 Tyr 951 122,p:\oper\ejkPM9589.au,29 -29- INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 1143 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 154..906 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CGGCCGCCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTGGT TCAAGAACAC ACCAAAGAAG AGAGACCCAT GTTAAGCTGT TAGTATCTGG AGTCTTATTC TTTTCATATT TTTCTCCAAA GCATCCTCGT CCCCAAGGTT TTTTTGCGGC AGT ATG GAG TTC CCG AGT GAA TTT Met Glu Phe Pro Ser Glu Phe 1 TCA GAG GCC TCT TCA CAG AAG AGA ATC GGG GGG AGA GGG AAA ATA GAG Ser Glu Ala Ser Ser Gin Lys Arg Ile Gly Gly Arg Gly Lys Ile Glu 10 15 S.

S

5

*SSSS*

S. S *5 S S *SS S6*5

S

S. S S *5

S.

B*

ATC AAA Ile Lys 25 CGC CGG Arg Arg CGG ATC GAG AAC ACG ACG AAC CGG CAG GTC ACC TTT TGT AAA Arg Ile Glu Asn Thr Thr Asn Arg Gin Val Thr Phe Cys Lys 30 AAC GGG TTG TTG AAG AAG GCT TAT GAG CTA TCG GTG TTG TGC Asn Gly Leu Leu Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys 45 50 120 174 222 270 318 366 414 462 510 558 GAT GCT GAA Asp Ala Glu GAA TAT GCT Glu Tyr Ala GCA AGC AGT Ala Ser Ser GTG GCG CTT ATT GTC TTC TCG AGC CGT GGC AGG CTC TAT Val Ala Leu Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr 60 65 AAC AAC AGT GTC AGA GGA ACA ATT GAG AGG TAC AAG AAA Asn Asn Ser Val Arg Gly Thr Ile Glu Arg Tyr Lys Lys 75 80 GAT TCC TCA CAT CCA CAG TCC GTT TCT GAA GTG AAC ACT Asp Ser Ser His Pro Gin Ser Val Ser Glu Val Asn Thr 95 100 CAG TTT Gin Phe 105 TAT CAG CAA GAA GCA TCC AAG CTT CGG AGA CAG ATA AGA GAA Tyr Gin Gin Glu Ala Ser Lys Leu Arg Arg Gin Ile Arg Glu 110 115

ATC

Ile 120 CAG GTC TCA AAT AGG CAT ATT CTA GGT GAG GGT ATA AGT GAT TTG Gin Val Ser Asn Arg His Ile Leu Gly Glu Gly Ile Ser Asp Leu 125 130 135 AGC TTC AAG Ser Phe Lys AGC CGA GTT Ser Arg Val GAT CTC AAG AAT CTC GAG AGC AAA TTA GAG AAA TCG ATC Asp Leu Lys Asn Leu Glu Ser Lys Leu Glu Lys Ser Ile 140 145 150 AGA TCA AAG AAG AAT GAG ATG CTT TTT GCC GAG ATT GAG Arg Ser Lys Lys Asn Glu Met Leu Phe Ala Glu Ile Glu 155 160 165 951122,pAoper\ejh,PM9589.au,30 30 TAC ATG CAG AAG AG GAA ATT GAG CTG CAA AAT GAT AAC ATG TAT CTG 702 Tyr Met Gin Lys Arg Glu Ile Glu Leu Gin Asn Asp Asn Met Tyr Leu 170 175 180 AGA GCA AAG ATA GCT GAG AAC GAG AGA OCA CAA CAG CAG CAG CAG CAA 750 Arg Ala Lys Ile Ala Glu Asn Glu Arg Ala Gin Gin Gin Gin Gin Gin 185 190 195 GGG AGT GAT CAT CAC TTC AAC ATG CCG GGA TCG TCG TCG GTG TAC GAG 798 Gly Ser Asp His His Phe Asn Met Pro Gly Ser Ser Ser Val Tyr Glu 200 205 210 215 GCG CTG CCG TCT CAG CCT GCA TAC GAT CGC AAC TTC CTG CAA GTC AAT 846 Ala Leu Pro Ser Gin Pro Ala Tyr Asp Arg Asn Phe Leu Gin Val Asn 220 225 230 GTC CTG GAA CCA AAT CAC CAA TCT TAT TCT CGA TCC GAC CAC ACT GCC 894 Val Leu Glu Pro Asn His Gin Ser Tyr Ser Arg Ser Asp His Thr Ala 235 240 245 CTC CAA CTC GTC TAATTAAAGC TCAGTTAATA TGTAGCACTA GCAACTGGAC 946 Leu Gin Leu Val 250 C. Se GTATTGGCAC TCTAGATTAT GCTTTTTAAT ATGCTAGAGT TAGTTGAAAG AGGAATCAAT 1006 GAACTGGGGA GAGACCCGCA TGATGAGTTT CGCCTAAACA GTGGAAGAAC TCTCTGATAT 1066

**S

TTCTCTTCAT CACTCTTCAA ACTCTTCTGA TGAATCGTGT TGTACAAGGG TTATTGCAAT 1126 STTTTATATAG TACTTAA 1143 INFORMATION FOR SEQ ID NO:8:

C

SEQUENCE CHARACTERISTICS: LENGTH: 251 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Glu Phe Pro Ser Glu Phe Ser Glu Ala Ser Ser Gin Lys Arg Ile 1 5 10 Gly Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Asn 25 Arg Gin Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala 40 Tyr Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe 55 Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ala Asn Asn Ser Val Arg Gly 70 75 Thr Ile Glu Arg Tyr Lys Lys Ala Ser Ser Asp Ser Ser His Pro Gin 90 951 122,p:\oper\ejkPM9589.au,31 31 Ser Val Ser Glu Val Asn Thr Gin Phe Tyr Gin Gin Glu Ala Ser Lys 100 105 110 Leu Arg Arg Gin Ile Arg Glu Ile Gin Val Ser Asn Arg His Ile Leu 115 120 125 Gly Glu Gly Ile Ser Asp Leu Ser Phe Lys Asp Leu Lys Asn Leu Glu 130 135 140 Ser Lys Leu Glu Lys Ser Ile Ser Arg Val Arg Ser Lys Lys Asn Glu 145 150 155 160 Met Leu Phe Ala Glu Ile Glu Tyr Met Gin Lys Arg Glu Ile Glu Leu 165 170 175 Gin Asn Asp Asn Met Tyr Leu Arg Ala Lys Ile Ala Glu Asn Glu Arg 180 185 190 Ala Gin Gin Gin Gin Gin Gin Gly Ser Asp His His Phe Asn Met Pro 195 200 205 Gly Ser Ser Ser Val Tyr Glu Ala Leu Pro Ser Gin Pro Ala Tyr Asp 210 215 220 Arg Asn Phe Leu Gin Val Asn Val Leu Glu Pro Asn His Gin Ser Tyr 225 230 235 240 Ser Arg Ser Asp His Thr Ala Leu Gin Leu Val O* 245 250 e* INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 1084 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA So, (ix) FEATURE: NAME/KEY: CDS LOCATION: 107..838 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TAACGCCCCA TCTCTCTTCT CTCTCTCTCT CTCTCTGTGG CTTTGTCTTT TCTTTTGTTT CTTGCCGTTT TGGCGTGTGT GTTGGGTTGT GTGAATTGGA GCGAGG ATG GGG AGG 115 Met Gly Arg 1 GGG AGG CTG CAG CTG AAG AGG ATA GAG AAC AAG ATC AAC CGA CAA GTC 163 Gly Arg Leu Gin Leu Lys Arg Ile Glu Asn Lys Ile Asn Arg Gin Val 10 ACC TTC TCC AAG AGG AGG GCG GGT CTG CTC AAG AAG GCC CAC GAG ATC 211 Thr Phe Ser Lys Arg Arg Ala Gly Leu Leu Lys Lys Ala His Glu Ile 25 30 951122,p:\oper\ejh,PM9589.au,32 -32d. TCC GIC CTC TGC GAC GCC GAG GTC GCC CTC ATC GTC TTC TCC GCC AAG Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe Ser Ala Lys 45 GGC AAG CTC TTC GAG TAC TCC ACC GAT TCC TGC ATG GAG AGA ATT CTC Gly Lys Leu Phe Giu Tyr Ser Thr Asp Ser Cys Met Glu Arg Ile Leu 60 GAA CGC TAT GAA AGA TAC TCA TAT GCG GAG CAC CAA GTT CTT GCA AGC Glu Arg Tyr Giu Arg Tyr Ser Tyr Ala Giu His Gln Val Leu Ala Ser 75 GAG ACG GAA TCG ATT GGT AGC TGG ACT TTG GAG CAT GCT AAG CTC AAG Glu Thr Glu Ser Ile Gly Ser Trp Thr Leu Giu His Ala Lys Leu Lys 90 GCC AGA CTT GAA GTT TTA CAC AGA AAT TAT AGG CAT TTC ATG GOC GAA Ala Arg Leu Giu Val Leu His Arg Asn Tyr Arg His Phe Met Giy Glu 100 105 110 115 GAT CTT GAT TCT TTG AGT CTC AAG GAC CTC CAA AAT TTG GAG CAG CAA Asp Leu Asp Ser Leu Ser Leu Lys Asp Leu Gin Asn Leu Giu Gin Gin 120 125 130 CTG GAG TCT GCT CTT AAA CAC ATA AGA TCG AGA AAG AAT CAG CTC ATG Leu Giu Ser Ala Leu Lys His Ile Arg Ser Arg Lys Asn Gin Leu Met 135 140 145 CAT GAA TCA ATC TCA GTG ATT CAG AAA AAG GAT AGG GCA TTG CAG GAG His Giu Ser Ile Ser Val Ile Gin Lys Lys Asp Arg Ala Leu Gin Glu 150 155 160 CAA AAT AAC CTG CTT ACA AAG AAA ATA AAG GAG AAG GAG AGG GCA CTA Gin Asn Asn Leu Leu Thr Lys Lys Ile Lys Giu Lys Glu Arg Ala Leu 165 170 175 GCA CAG CAA GCT CAG TGG GAG CAG CAA GAC CAT GCC CTT GAC TCA CCT Ala Gin Gin Ala Gin Trp Giu Gin Gin Asp His Ala Leu Asp Ser Pro 180 185 190 195 GTT GTT CTA CCC CAC TAC TTG CCA TCT CTC GAC ATC AAT GGC TCT TAT Val Val Leu Pro His Tyr Leu Pro Ser Leu Asp Ile Asn Gly Ser Tyr 200 205 210 CAA GCG AGA CAC AAC GGA CAC GAT GAC GGA GAG AAC CTG ACT CAG CCT Gin Ala Arg His Asn Gly His Asp Asp Gly Glu Asn Leu Thr Gin Pro 215 220 225 CGG GCT GGT ACA CTT CTT CCT CCG TGG ATC TCT GCA TCT CAA TTA AGG Arg Ala Gly Thr Leu Leu Pro Pro Trp Ile Ser Ala Ser Gin Leu Arg 230 235 240 CTT TGACATAAAA GGGAACTTCT GCTGCTATAT CTTTGATAAG CAATTCTGAA Leu GTCAAGTGAT GAATTGCATG GCATAATCTC AGGCTCTCCC CCACCCTTGT TCTAGCGAAA TAAACATGTC CCTGCTTTTT GCTGCATTCA GTACTCCTAA GTGTGGTCCT CCGTGTATGT ATAGCACCAT TGCAAATATG CATACCAATG TACATGTATG CAAAATTATA TATGGAAGTT AACACAACCA TAAAAA 259 307 355 403 451 499 547 595 643 691 739 787 835 888 948 1008 1068 1084 951 122,p:\oper\ejh,PM9589.au,33 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 244 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:iO:

S

*5SS S *SS S

S

S *5 5 Met Arg His Ser Arg 65 Leu Lys Met Glu Gin 1.45 Leu Arg Asp Gly Thr Gin Glu Ala 50 Ile Ala Leu Gly Gin 130 Leu Gin Ala Ser Ser 210 Gin Val Ile Lys Leu Ser Lys Giu 115 Gin Met Glu Leu Pro 195 Tyr Pro Gly Arg Gly Arg Phe Val Lys Arg Thr Arg Leu Glu Glu Asn 165 Gin Val Ala Ser Leu Leu Tyr 70 Giu Leu Asp Ser Ser 150 Asn Gin Leu Arg Lys Cys Phe 55 Giu Ser Glu Ser Ala i3 5 Ile Leu Ala Pro His 215 Leu Gin Leu Lys Arg Asp 40 Giu Arg Ile Vai Leu 120 Leu Ser Leu Gin His 200 Asn Arg Ala Tyr Tyr Gly Leu 105 Ser Lys Vai Thr Trp 185 Tyr Gly Arg 10 Ala Giu Ser Ser Ser 90 His Leu His Ile Lys 170 Giu Leu His Ile Gly Val Thr Tyr 75 Trp Arg Lys Ile Gln 155 Lys Gin Pro Asp Glu Leu Ala Asp Aia Thr Asn Asp Arg 140 Lys Ile Gin Ser Asp 220 Asn Leu Leu Ser Giu Leu Tyr Leu 125 Ser Lys Lys Asp Leu 205 Gly Lys Lys Ile Cys His Glu Arg Gin Arg Asp Giu His 190 Asp Glu Ile Lys Val Met Gin His His Asn Lys Arg Lys 175 Ala Ile Asn Asn Ala Phe Giu Vai Ala Phe Leu Asn Ala 160 Giu Leu Asn Leu Ala Gly 230 Thr Leu Leu Pro Pro Trp Ile Ser Ala Ser 225 Gin Leu Arg Leu 235 240 951122,p:\oper\qjhPM9589.au,34 34- INFORMATION eOX SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 1032 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 184..799 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TCGAACAGTT CAGAAAGACG GATAGATACA TACACAGAGA CATCGATTGC ATCATCTTCT CTACCTCATT GTGCGTGCGG TGCGTGCGGT GCGTGCTTTA TATAGACGCT CGCTCTTACG 120 TTGTTCTCGT CCGCCCGGTG GTGTGGTGGT TGGAGAGAGA GGGAGAGAGA GTAGAGAGAG 180 **!AAA ATG GGG AGG GGG AGG GTG CAG CTG AAG AGG ATA GAG AAC AAG ATA 228 Met Gly Arg Gly Arg Val Gin Leu Lys Arg Ile Glu Asn Lys Ile 1 5 10 AAC AGG CAA GTG ACG TTC TCG AAG CGG AGA TGC GGG CTG CTG AAG AAG 276 p.

Asn Arg Gin Val Thr Phe Ser Lys Arg Arg Cys Gly Leu Leu Lys Lys 25 SGCC CAC GAG ATC TCC GTC CTC TGC GAC GCC GAC GTC GCC CTC ATC GTC 324 Ala His Glu Ile Ser Val Leu Cys Asp Ala Asp Val Ala Leu Ile Val 40 TTC TCC ACC AAG GGC AAG CTC TTC GAG TAC GCC ACC GAC TGT TGC ATG 372 Phe Ser Thr Lys Gly Lys Leu Phe Glu Tyr Ala Thr Asp Cys Cys Met 55 *GAG AGG ATC CTC GAG CGT TAT GAG AGA TAT TCA TAT GCA GAG AGC CAG 420 Glu Arg Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Ser Gin 70 GTT CTC ACA AAC AAT GCC GAA ACC AAT GGG AAC TGG ACT TTG GAA CAT 468 Val Leu Thr Asn Asn Ala Glu Thr Asn Gly Asn Trp Thr Leu Glu His 85 90 GCA AAA CTC AAG GCC AGG ATG GAG ATT CTG CAG AAA AAT CAA AAG AAT 516 Ala Lys Leu Lys Ala Arg Met Glu Ile Leu Gln Lys Asn Gin Lys Asn 100 105 110 CTG ATG GGA GAA GAA CTC GAT TCT CTA AGC CTC AAA GAG CTT CAG AAT 564 Leu Met Gly Glu Glu Leu Asp Ser Leu Ser Leu Lys Glu Leu Gln Asn 115 120 125 TTG GAG CAT CAG CTT GAC ACT GCT CTT AAG AAC ATT CGG TCT AGA AAG 612 Leu Glu His Gln Leu Asp Thr Ala Leu Lys Asn Ile Arg Ser Arg Lys 130 135 140 ATT CAA CTC ATG TGT GAA TCC ATC TCT GAG CTT CAG AGA AAG GAT AAG 660 Ile Gin Leu Met Cys Glu Ser Ile Ser Glu Leu Gin Arg Lys Asp Lys 145 150 155 GCG CTG CAA GAA CAA AAC AAC ATG CTG GCA AAG AAG GTG AAA GAG AAG 708 Ala Leu Gln Glu Gin Asn Asn Met Leu Ala Lys Lys Val Lys Glu Lys 160 165 170 175 951122,p:\oper\ejhPM9589.au,35 35 GAG AAG GCA CTG GCA CAG CAA ACT CAA TGG GAC AAC CCG CAA GAT GAT 756 Glu Lys Ala Leu Ala Gin Gin Thr Gin Trp Asp Asn Pro Gin Asp Asp 180 185 190 GGC CTC ACC TCT TCC TCG GGG TCC TTA CCA TCC GAG TGG AAT T 799 Gly Leu Thr Ser Ser Ser Gly Ser Leu Pro Ser Glu Trp Asn 195 200 205 GAGGAGGGAG CGGCCCTCGG TCCACCTCAA CATCGTAATG CCACCCTTTT TCCTTCATGG 859 ATGCTTAGCC ACCTCCAAGC CTAATTGTCC AATTTATTTG CATGGGAACC ATATATCATT 919 ACCAATATTA TCTAATGCTT CTTATCGTAT ATATCCAGTC GATATGTAAC CATAATGTAA 979 CATGTCTTAG TACATGCATT GTGGGGATAC TTGCAAAAAC CTTGTTTTGT AGG 1032 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 205 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Gly Arg Gly Arg Val Gin Leu Lys Arg Ile Glu Asn Lye Ile Asn 1 5 10 1 Arg Gin Val Thr Phe Ser Lys Arg Arg Cys Gly Leu Leu Lye Lys Ala 20 25 His Glu Ile Ser Val Leu Cys Asp Ala Asp Val Ala Leu Ile Val Phe 35 40 Ser Thr Lys Gly Lys Leu Phe Glu Tyr Ala Thr Asp Cys Cys Met Glu 55 Arg Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Ser Gin Val 70 75 Leu Thr Asn Asn Ala Glu Thr Asn Gly Asn Trp Thr Leu Glu His Ala 90 Lys Leu Lys Ala Arg Met Glu Ile Leu Gin Lys Asn Gin Lys Asn Leu 100 105 110 Met Gly Glu Glu Leu Asp Ser Leu Ser Leu Lys Glu Leu Gin Asn Leu 115 120 125 Glu His Gin Leu Asp Thr Ala Leu Lys Asn Ile Arg Ser Arg Lys Ile 130 135 140 Gin Leu Met Cys Glu Ser Ile Ser Glu Leu Gln Arg Lys Asp Lys Ala 145 150 155 160 Leu Gin Glu Gin Asn Asn Met Leu Ala Lys Lys Val Lys Glu Lys Glu 165 170 175 Lys Ala Leu Ala Gin Gin Thr Gin Trp Asp Asn Pro Gln Asp Asp Gly 180 185 190 Leu Thr Ser Ser Ser Gly Ser Leu Pro Ser Glu Trp Asn 195 200 205 951122,p:\oper\ejhPM9589.au,36 36- INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 1070 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 184..920 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TCGAACAGTT CAGAAAGACG GATAGATACA TACACAGAGA CATCGATTGC ATCATCTTCT CTACCTCATT GTGCGTGCGG TGCGTGCGGT GCGTGCTTTA TATAGACGCT CGCTCTTACG 120 TTGTTCTCGT CCGCCCGGTG GTGTGGTGGT TGGAGAGAGA GGGAGAGAGA GTAGAGAGAG 180 AAA ATG GGG AGG GGG AGG GTG CAG CTG AAG AGG ATA GAG AAC AAG ATA 228 Met Gly Arg Gly Arg Val Gln Leu Lys Arg Ile Glu Asn Lys Ile 1 5 10 SAAC AGG CAA GTG ACG TTC TCG AAG CGG AGA TGC GGG CTG CTG AAG AAG 276 Asn Arg Gln Val Thr Phe Ser Lys Arg Arg Cys Gly Leu Leu Lys Lys 25 GCC CAC GAG ATC TCC GTC CTC TGC GAC GCC GAC GTC GCC CTC ATC GTC 324 Ala His Glu Ile Ser Val Leu Cys Asp Ala Asp Val Ala Leu Ile Val 35 40 TTC TCC ACC AAG GGC AAG CTC TTC GAG TAC GCC ACC GAC TGT TGC ATG 372 Phe Ser Thr Lys Gly Lys Leu Phe Glu Tyr Ala Thr Asp Cys Cys Met 50 55 GAG AGG ATC CTC GAG CGT TAT GAG AGA TAT TCA TAT GCA GAG AGC CAG 420 Glu Arg Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Ser Gln S. 65 70 GTT CTC ACA AAC AAT GCC GAA ACC AAT GGG AAC TGG ACT TTG GAA CAT 468 Val Leu Thr Asn Asn Ala Glu Thr Asn Gly Asn Trp Thr Leu Glu His 85 90 GCA AAA CTC AAG GCC AGG ATG GAG ATT CTG CAG AAA AAT CAA AAG AAT 516 Ala Lys Leu Lys Ala Arg Met Glu Ile Leu Gln Lys Asn Gln Lys Asn 100 105 110 CTG ATG GGA GAA GAA CTC GAT TCT CTA AGC CTC AAA GAG CTT CAG AAT 564 Leu Met Gly Glu Glu Leu Asp Ser Leu Ser Leu Lys Glu Leu Gln Asn 115 120 125 TTG GAG CAT CAG CTT GAC ACT GCT CTT AAG AAC ATT CGG TCT AGA AAG 612 Leu Glu His Gin Leu Asp Thr Ala Leu Lys Asn Ile Arg Ser Arg Lys 130 135 140 ATT CAA CTC ATG TGT GAA TCC ATC TCT GAG CTT CAG AGA AAG GAT AAG 660 Ile Gin Leu Met Cys Glu Ser Ile Ser Glu Leu Gin Arg Lys Asp Lys 145 150 155 GCG CTG CAA GAA CAA AAC AAC ATG CTG GCA AAG AAG GTG AAA GAG AAG 708 Ala Leu Gin Glu Gin Asn Asn Met Leu Ala Lys Lys Val Lys Glu Lys 160 165 170 175 951 122,p:\oper\ejh,PM9589.au,37 37 GAG AAG GCA CTG GCA CAG CAA ACT CAA TGG GAC AAC CCG CAA GAT GAT Giu Lys Ala Leu Ala Gin Gin Thr Gin Trp Asp Asn Pro Gin Asp Asp 180 185 190 GGC CTC ACC TCT TCC TCG GTT ATT CTC TCT CAA TCT TTG CAG CCT CTC Gly Leu Thr Ser Ser Ser Val Ile Leu Ser Gin Ser Leu Gin Pro Leu 195 200 205 AAC ATT GGG GGT CCT TAC CAT CCG AGT GGA ATT GAG GAG GGA GCG GCC Asn Ile Gly Gly Pro Tyr His Pro Ser Gly Ile Glu Glu Gly Ala Ala 210 215 220 CTC GGT CCA CCT CAA CAT CGT AAT GCC ACC CTT TTT CCT TCA TGG ATG Leu Gly Pro Pro Gin His Arg Asn Ala Thr Leu Phe Pro Ser Trp Met 225 230 235 CTT AGC CAC CTC CAA GAA TA ATGATCCAAT TTATTTGCAT GGGAACCATA Leu Ser His Leu Gin Glu 240 245 TATCATTACC AATATTATCT AATGCTTCTT ATCGTATATA TCCAGTCGAT ATGTAACCAT AATGTAACAT GTCTTAGTAC ATGCATTGTG GGGATACTTG CAAAAACCTT GTTTTGTAGG INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 245 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULJE TYPE: protein 756 804 852 900 950 1010 1070 a. Met 1 Arg His Ser Arg Leu Lys Met Giu Gin 145 (xi) SEQUENCE Gly Arg Gly Arg 5 Gin Val Thr Phe Giu Ile Ser Val Thr Lys Gly Lys Ile Leu Giu Arg Thr Asn Asn Ala Leu Lys Ala Arg 100 Gly Giu Giu Leu 115 His Gin Leu Asp 130 Leu Met Cys Giu Val Gin Leu Lys Ser Leu Leu Tyr 70 Giu Met Asp Thr Ser 150 Lys Cys Phe 55 Giu Thr Giu Ser Ala 135 Ile Arg Asp 40 Glu Arg Asn Ile Leu 120 Leu Ser DESCRIPTION: SEQ ID NO:l4: Arg 25 Ala Tyr Tyr Gly Leu 105 Ser Lys Glu Arg Cys Asp Ala Ser Asn 90 Gin Leu Asn Leu Ile Gly Val Thr Tyr 75 Trp Lys Lys Ile Gin 155 Giu Leu Ala Asp Ala Thr Asn Glu Arg 140 Arg Asn Leu Leu Cys Giu Leu Gin Leu 125 Ser Lys Lys Lys Ile Cys Ser Giu Lys 110 Gin Arg Asp Ile Lys Val Met Gin His Asn Aen Lys Lys Asn Ala Phe Giu Val Ala Leu Leu Ile Ala 160 951 122,p:\oper\ejhPM9589.au,38 38 Leu Gin Glu Gin Asn Asn Met Leu Ala Lys Lys Val Lys Glu Lys Glu 165 170 175 Lys Ala Leu Ala Gin Gin Thr Gin Trp Asp Asn Pro Gin Asp Asp Gly 180 185 190 Leu Thr Ser Ser Ser Val Ile Leu Ser Gin Ser Leu Gin Pro Leu Asn 195 200 205 Ile Gly Gly Pro Tyr His Pro Ser Gly Ile Glu Glu Gly Ala Ala Leu 210 215 220 Gly Pro Pro Gin His Arg Asn Ala Thr Leu Phe Pro Ser Trp Met Leu 225 230 235 240 Ser His Leu Gin Glu 245 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 997 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 38..724 too.

(xi) SEQUENCE DESCRIPTION: SEQ ID GCTTCCATCT CCAGGGAAGA GTTTTGAACT TGCGACC ATG GTT TTT CCA ACC CAA ~Met Val Phe Pro Thr Gin SS 1 GCC ACG CCC GAG GAG TCC CCG CAG AGG AAA ATG GGG AGG GGA AAG ATC 103 Ala Thr Pro Glu Glu Ser Pro Gin Arg Lys Met Gly Arg Gly Lys Ile 10 15 GAG ATC AAG CGG ATC GAG AAC ACG ACG AAT CGG CAA GTG ACT TTC TGC 151 *Glu Ile Lys Arg Ile Glu Asn Thr Thr Asn Arg Gin Val Thr Phe Cys 30 AAG CGG CGG AAT GGC CTC CTC AAG AAG GCA TAT GAA CTC TCC GTT CTT 199 Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr Glu Leu Ser Val Leu 45 TGC GAA CGC GAG GTC GCC CTC ATC GTC TTC TCC AGC CGC GGT CGC CTC 247 Cys Glu Arg Glu Val Ala Leu Ile Val Phe Ser Ser Arg Gly Arg Leu 60 65 TAT GAG TAT GCC AAC GAT AGT GTC AAA GCA ACC ATC GAG AGG TAC AAG 295 Tyr Glu Tyr Ala Asn Asp Ser Val Lys Ala Thr Ile Glu Arg Tyr Lys 80 AAG GCT TTC TCA GAT TCC TCC AGT AGC GGG ATC CGT TCT GAA GCT AAT 343 Lys Ala Phe Ser Asp Ser Ser Ser Ser Gly Ile Arg Ser Glu Ala Asn 95 100 GTT CAG TTT TAT CAG CAA GAA TCC GCA AAG TTG CAA CAA CAG ATT AAT 391 Val Gin Phe Tyr Gin Gin Glu Ser Ala Lys Leu Gin Gin Gin Ile Asn 105 110 115 951122,:\oper\cjhPM9589.au,39 r 9 -39- AAC ATG CAG AAC AAT AAC AGG CAA CTG GTG GGT GAC TCA ATT GCT GGG Asn Met Gin Asn Asn Asn Arg Gin Leu Val Gly Asp Ser Ile Ala Gly 120 125 130 ATG AAT ATG AAG GAT ATG AAG ACT ACG GAG CAA AAA CTA GAA AAA GCA Met Asn Met Lys Asp Met Lys Thr Thr Glu Gin Lys Leu Glu Lys Ala 135 140 145 150 ATC GCT AAA ATT CGC GCC AAA AAG AAT GAG CTT CTA TTT GCG GAG ATT Ile Ala Lys Ile Arg Ala Lys Lys Asn Glu Leu Leu Phe Ala Glu Ile 155 160 165 GAG TAT ATG CAA AAA AGG GAA ATT GAC CTA CAC AAC AAC AAT CAA GTT Glu Tyr Met Gin Lys Arg Glu Ile Asp Leu His Asn Asn Asn Gin Val 170 175 180 CTG CGA GCG AAG ATC GCG GAG AGC GAG AGG TCC CAA CAT GCG GAT ATG Leu Arg Ala Lys Ile Ala Glu Ser Glu Arg Ser Gin His Ala Asp Met 185 190 195 AAT TTG ATG CCG GGA GGT ACC AAC TAT GAC TTC ATG CAG CCA TCT TCT Asn Leu Met Pro Gly Gly Thr Asn Tyr Asp Phe Met Gin Pro Ser Ser 200 205 210 TCT CAG CCT TTT GAT TCC AGA AAC TAC TTC CAA GTG AAC GTG CTG Ser Gin Pro Phe Asp Ser Arg Asn Tyr Phe Gin Val Asn Val Leu 215 220 225 TAGCACCCAA CCACTGTGAG TGTGCACAAG TCTAGTATCG ATTGAAGGGA ATATAACTAG TATATGGAGC TTCTAAGTGC TTATTATGAG GTTATGATGG ACTTCATCAG ATGTTAATTA AGTAGAAGCA GTTGGCAGAT GCATCTATTA GGTGAAGGGC GTCCTCATCC TTTTGTTGAT TAGCATCCTT AGATGTCTTA AAATTTCCAC CATCTTTGTT AAGGTGTGTA TTTGGGTCCC TATGTACTAA ACTGAATATG AACTATCGAA CTA INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 229 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Val Phe Pro Thr Gin Ala Thr Pro Glu Glu Ser Pro Gln Arg Lys 1 5 10 Met Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Asn 25 Arg Gin Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala 40 Tyr Glu Leu Ser Val Leu Cys Glu Arg Glu Val Ala Leu Ile Val Phe 55 Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ala Asn Asp Ser Val Lys Ala 70 75 439 487 535 583 631 679 724 784 844 904 964 997 951 122,p:\opcr\ejh,PM9589.au,40 40 Thr Ile Leu Gly Gin 145 Leu His Ser Phe Gin 225 11ife Arg Gin Asp 130 Lys Leu Asn Gin Met 210 Val Glu Ser Gin 115 Ser Leu Phe Asn His 195 Gin Asn Tyr Ala Ile Ala Lys Glu 165 Gin Asp Ser Leu Lys Asn Asn Gly Ala 150 Ile Val Met Ser Liys Vai Asn Met 135 Ile Glu Leu Asn Ser 215 Phe Phe 105 Gin Met Lys Met Ala 185 Met Pro Asp Gin Asn Asp Arg 155 Lys Ile Gly Asp Ser Gin Asn Met 140 Ala Arg Ala Gly Ser 220 Ser Giu Arg 125 Lys Lys Glu Glu Thr 205 Arg Ser Ser 110 Gin Thr Lys Ile Ser 190 Asn Ann Ser Ala Leu Thr Asn Asp 175 Glu Tyr Tyr Gly Lys Val Glu Giu 160 Leu Arg Asp Phe a a a. A a a p a.

Claims (25)

1. An isolated nucleic acid molecule comprising a sequence of nucleotides required for normal flower development in the genus Eucalyptus and wherein said nucleotide sequence is a homologue of a gene selected from leafy (LFY) apetala 1 (API) and agomous (AG) from Arabidopsis and floricaula (FLO), squamosa (SQUA) or plena (PLE) from Antirrhinum.

2. An isolated nucleic acid molecule according to claim 1 or 2 wherein the species of Eucalyptus is selected from Eucalyptus alpina, Eucalyptus annulata, Eucalyptus astringens, Eucalyptus behriana, Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus brockwayi, Eucalyptus caesia, Eucalyptus calophylla 'Rosea', Eucalyptus calycogona, Eucalyptus camaldulensis, Eucalyptus camaldulensis ssp. subcinerea, Eucalyptus campaspe, Eucalyptus S* camphora, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus cladocalyx 'Nana', Eucalyptus cloeziana, Eucalyptus cornuta, Eucalyptus cosmophylla, Eucalyptus crenulata, Eucalyptus crucis, Eucalyptus delegatensis, Eucalyptus desmondensis, Eucalyptus dielsii, 0* 0 S* Eucalyptus diptera, Eucalyptus diversicolor, Eucalyptus diversifolia, Eucalyptus dumosa, Eucalyptus dundasii, Eucalyptus dunnii, Eucalyptus eremophila, Eucalyptus erythrocorys, Eucalyptus erythronema, Eucalyptus ficifolia, Eucalyptus flocktoniae, Eucalyptus foecunda, Eucalyptusforrestiana, Eucalyptus gardneri, Eucalyptus gillii, Eucalyptus globulus, Eucalyptus 0* globulus ssp globulus, Eucalyptus globulus ssp. pseudoglobulus, Eucalyptus globulus ssp maidenii, Eucalyptus globulus ssp bicostata, Eucalyptus gomphocephala, Eucalyptus gracilis, Eucalyptus grandis, Eucalyptus griffithsii, Eucalyptus grossa, Eucalyptus henrii, Eucalyptus increassata, Eucalyptus kitsoniana, Eucalyptus kondininensis, Eucalyptus kruseana, Eucalyptus lansdowneana, Eucalyptus largiflorens, Eucalyptus lehmanni, Eucalyptus leucoxylon, Eucalyptus leucoxylon (Pink) (syn. 'Rosea'), Eucalyptus lesouefii, Eucalyptus macrandra, Eucalyptus macrocarpa, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus melliodora, Eucalyptus microcarpa, Eucalyptus nicholii, Eucalyptus nitens, Eucalyptus nutans, Eucalyptus obliqua, Eucalyptus occidentalis, Eucalyptus ochrophloia, Eucalyptus oleosa, Eucalyptus orbifolia, Eucalyptus ovata, Eucalyptus pauciflora, Eucalyptus parvifolia, Eucalyptus perriniana, Eucalyptus pileata, Eucalyptus pellita, Eucalyptus pilularis, Eucalyptus platypus, '\Eucalyptus polyanthemos, Eucalyptus porosa, Eucalyptus preissiana, Eucalyptus pyriformis, Lif I 0' O" P:\OPER\EJH\39013-CL.217 5/8/98 -42- Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus rhodantha, Eucalyptus robusta, Eucalyptus saligna, Eucalyptus salmonophloia, Eucalyptus salubris, Eucalyptus sargentii, Eucalyptus sepulcralis, Eucalyptus sideroxylon 'Rosea', Eucalyptus sieberi, Eucalyptus spathulata, Eucalyptus socialis, Eucalyptus steedmanii, Eucalyptus stoatei, Eucalyptus stricklandii, Eucalyptus tereticornis, Eucalyptus tetragona, Eucalyptus tetraptera, Eucalyptus torquata, Eucalyptus 'Torwood', Eucalyptus urnigera, Eucalyptus urophylla, Eucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo, Eucalyptus websteriana, Eucalyptus woodwardii.

3. An isolate nucleic acid molecule according to claim 1 or 2 wherein the species of Eucalyptus is Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cloeziana, Eucalyptus delegatensis, Eucalyptus diversicolor, Eucalpytus dunnii, Eucalyptus globulus, Eucalyptus globulus subspecies bicostata, Eucalyptus se** globulus subspecies globulus, Eucalyptus globulus subspecies maidenii, Eucalyptus globulus subspecies pseudoglobulus, Eucalyptus grandis, Eucalyptus henrii, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus nitens, Eucalyptus obliqua, Eucalyptus pellita, Eucalyptus pilularis, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus saligna, Eucalyptus sieberi, Eucalyptus tereticornis, Eucalyptus urophylla, Eucalyptus viminalis.

4. An isolated nucleic acid molecule according to any one of claims 1 to 3 wherein the species of Eucalyptus is Eucalyptus globulus ssp globulus or Eucalyptus nitens.

5. An isolated nucleic acid molecule according to any one of claims 1 to 4 wherein the nucleic acid is DNA.

6. An isolated nucleic acid molecule according to any one of claims 1 to 5 wherein the nucleic acid molecule is selected from the following: comprises a nucleotide sequence set forth in SEQ ID NO: 1 or 3 or 5 and is analogous to LFY and/or FLO; comprises a nucleotide sequence set forth in SEQ ID NO:9 or 11 or 13 and is analogous to API and/or SQUA; or P:\OPER\EJH\39013-CL.217 26/11/99 -43- (iii) comprises a nucleotide sequence set forth in SEQ ID NO:7 or 15 and is analogous to AG and/or PLE.

7. An isolated DNA molecule of eucalypt origin encoding a reproductive gene and which is capable of hybridising under low stringency conditions to a nucleic acid molecule having a nucleotide sequence as set forth in or complementary to SEQ ID NO: 1 or 3 or

8. An isolated DNA molecule of eucalypt origin encoding a reproductive gene and which is capable of hybridising under low stringency conditions to a nucleic acid molecule having a nucleotide sequence as set forth in or complementary to SEQ ID NO:9 or 11 or 13. S

9. An isolated DNA molecule of eucalypt origin encoding a reproductive gene and which is capable of hybridising under low stringency conditions to a nucleic acid molecule having a nucleotide sequence as set forth in or complementary to SEQ ID NO:7 or

10. A method of minimising inflorescence in a species of a Eucalyptus, said method comprising introducing into one or more cells of said Eucaplyptus a genetic construct capable of down regulating or up regulating expression of a reproductive gene in said Eucaplyptus S: wherein said gene is a homologue of a gene selected from leafy (LFY), apetala 1 (API) and agomous (AG) from Arabidopsis and floricaula (FLO), squamosa (SQUA) and plena (PLE) from Antirrhinum, regenerating a transgeneic plant from said one or more cells and growing said plant for a time under conditions sufficient to permit expression of said genetic construct.

11. A method according to claim 10 wherein the species of Eucalyptus is selected from Eucalyptus alpina, Eucalyptus annulata, Eucalyptus astringens, Eucalyptus behriana, Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus brockwayi, Eucalyptus caesia, Eucalyptus calophylla 'Rosea', Eucalyptus calycogona, Eucalyptus camaldulensis, Eucalyptus camaldulensis ssp. subcinerea, Eucalyptus campaspe, Eucalyptus camphora, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus cladocalyx 'Nana', Eucalyptus cloeziana, i "'uLalyptus cornuta, Eucalyptus cosmophylla, Eucalyptus crenulata, Eucalyptus crucis, S Eu lyptus delegatensis, Eucalyptus desmondensis, Eucalyptus dielsii, Eucalyptus diptera, P:\qPER\EJH\39013-CL.217 26/11/99 43A Eucalyptus diversicolor, Eucalyptus diversifolia, Eucalyptus dumosa, Eucalyptus dundasii, Eucalyptus dunnii, Eucalyptus eremophila, Eucalyptus erythrocorys, Eucalyptus erythronema, .9 09 .9 0 9 9 900900 9000 0 0 9000 0000 So p. 9. 0@ 99 S 0 4 S 009S S 9 S S .9 *059 9e S S 0 99 9* B 9 9 0*00 99 9 *9 99 9 -V P:\OPER\EJH\39013-CL.217 5/8/98 -44- Eucalyptus ficifolia, Eucalyptus flocktoniae, Eucalyptus foecunda, Eucalyptus forrestiana, Eucalyptus gardneri, Eucalyptus gillii, Eucalyptus globulus, Eucalyptus globulus ssp globulus, Eucalyptus globulus ssp. pseudoglobulus, Eucalyptus globulus ssp maidenii, Eucalyptus globulus ssp bicostata, Eucalyptus gomphocephala, Eucalyptus gracilis, Eucalyptus grandis, Eucalyptus griffithsii, Eucalyptus grossa, Eucalyptus henrii, Eucalyptus increassata, Eucalyptus kitsoniana, Eucalyptus kondininensis, Eucalyptus kruseana, Eucalyptus lansdowneana, Eucalyptus largiflorens, Eucalyptus lehmanni, Eucalyptus leucoxylon, Eucalyptus leucoxylon (Pink) (syn. 'Rosea'), Eucalyptus lesouefii, Eucalyptus macrandra, Eucalyptus macrocarpa, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus melliodora, Eucalyptus microcarpa, Eucalyptus nicholii, Eucalyptus nitens, Eucalyptus nutans, Eucalyptus obliqua, Eucalyptus occidentalis, Eucalyptus ochrophloia, Eucalyptus oleosa, Eucalyptus orbifolia, Eucalyptus ovata, Eucalyptus pauciflora, Eucalyptus parvifolia, Eucalyptus perriniana, Eucalyptus pileata, Eucalyptus pellita, Eucalyptus pilularis, Eucalyptus platypus, Eucalyptus polyanthemos, Eucalyptus porosa, Eucalyptus preissiana, Eucalyptus pyriformis, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus rhodantha, Eucalyptus robusta, Eucalyptus saligna, Eucalyptus salmonophloia, Eucalyptus salubris, Eucalyptus sargentii, Eucalyptus sepulcralis, Eucalyptus sideroxylon 'Rosea', Eucalyptus sieberi, Eucalyptus spathulata, Eucalyptus socialis, Eucalyptus steedmanii, Eucalyptus stoatei, Eucalyptus stricklandii, Eucalyptus tereticornis, Eucalyptus tetragona, Eucalyptus tetraptera, Eucalyptus o9 torquata, Eucalyptus 'Torwood', Eucalyptus urnigera, Eucalyptus urophylla, Eucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo, Eucalyptus websteriana, Eucalyptus woodwardii.

12. A method according to claim 11 wherein the species of Eucalyptus is selected from Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cloeziana, Eucalyptus delegatensis, Eucalyptus diversicolor, Eucalpytus dunnii, Eucalyptus globulus, Eucalyptus globulus subspecies bicostata, Eucalyptus globulus subspecies globulus, Eucalyptus globulus subspecies maidenii, Eucalyptus globulus subspecies pseudoglobulus, Eucalyptus grandis, Eucalyptus henrii, Eucalyptus maculata, Eucalyptus i-'marginata, Eucalyptus nitens, Eucalyptus obliqua, Eucalyptus pellita, Eucalyptus pilularis, Slyptus regnans, Eucalyptus resinifera, Eucalyptus saligna, Eucalyptus sieberi, Eucalyptus P:\OPER\EJH\39013-CL.217 26/11/99 tereticornis, Eucalyptus urophylla, Eucalyptus viminalis.

13. A method according to claim 10 or 11 or 12 wherein the species of Eucalyptus is Eucalyptus globulus ssp globulus or Eucalyptus nitens.

14. A method according to any one of claims 10 to 13 wherein the reproductive gene corresponds to one or more of SEQ ID NO:1 or 3 or 5, SEQ ID NO:9 or 11 or 13 and/or SEQ ID NO:7 or

15. A method according to any one of claims 10 to 14 wherein the resulting plant is substantially sterile.

16. A transgenic Eucalyptus exhibiting down regulated expression of a reproductive gene wherein said reproductive gene is a homologue of a gene selected from leafy (LFY), apetala 1 •(API) and agomous (AG) from Arabidopsis and floricaula (FLO), squamosa (SQUA) and plena *0*

17. A transgenic plant according to claim 16 wherein said plant is substantially sterile. *0 0*

18. A transgenic plant according to claim 16 or 17 wherein the species of Eucalyptus is selected from Eucalyptus alpina, Eucalyptus annulata, Eucalyptus astringens, Eucalyptus behriana, Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus brockwayi, Eucalyptus caesia, Eucalyptus calophylla 'Rosea', Eucalyptus calycogona, Eucalyptus camaldulensis, Eucalyptus camaldulensis ssp. subcinerea, Eucalyptus campaspe, Eucalyptus camphora, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus cladocalyx 'Nana', Eucalyptus cloeziana, Eucalyptus cornuta, Eucalyptus cosmophylla, Eucalyptus crenulata, Eucalyptus crucis, Eucalyptus delegatensis, Eucalyptus desmondensis, Eucalyptus dielsii, Eucalyptus diptera, Eucalyptus diversicolor, Eucalyptus diversifolia, Eucalyptus dumosa, Eucalyptus Sdundasii, Eucalyptus dunnii, Eucalyptus eremophila, Eucalyptus erythrocorys, Eucalyptus er Otronema, Eucalyptus ficifolia, Eucalyptus flocktoniae, Eucalyptus foecunda, Eucalyptus beiaa uaytsbnhmi.Eclpu orods ualpu rcwyEclpu esa uaytsclpyl RsaEclpu ayooa uaytscmluess P:\O.PER\EJH\39013-CL.217 26/11/99 45A forrestiana, Eucalyptus gardneri, Eucalyptus gil/u, Eucalyptus globulus, Eucalyptus globulus ssp globulus, Eucalyptus globulus ssp. pseudoglobulus, Eucalyptus globulus ssp maidenii, Eucalyptus globulus ssp bicostata, Eucalyptus gomphocephala, Eucalyptus gracilis, Eucalyptus grandis, Eucalyptus grfflthsii, Eucalyptus grossa, Eucalyptus henrii, Eucalyptus increassata, 9* I I 19*1 I I. I. I I I I I I 9 I I **I*II I I I 0 II.. 00 I II 0* 0 ~7K7 y P:\OPER\EJH\39013-CL.217 5/8/98 -46- Eucalyptus kitsoniana, Eucalyptus kondininensis, Eucalyptus kruseana, Eucalyptus lansdowneana, Eucalyptus largiflorens, Eucalyptus lehmanni, Eucalyptus leucoxylon, Eucalyptus leucoxylon (Pink) (syn. 'Rosea'), Eucalyptus lesouefii, Eucalyptus macrandra, Eucalyptus macrocarpa, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus melliodora, Eucalyptus microcarpa, Eucalyptus nicholii, Eucalyptus nitens, Eucalyptus nutans, Eucalyptus obliqua, Eucalyptus occidentalis, Eucalyptus ochrophloia, Eucalyptus oleosa, Eucalyptus orbifolia, Eucalyptus ovata, Eucalyptus pauciflora, Eucalyptus parvifolia, Eucalyptus perriniana, Eucalyptus pileata, Eucalyptus pellita, Eucalyptus pilularis, Eucalyptus platypus, Eucalyptus polyanthemos, Eucalyptus porosa, Eucalyptus preissiana, Eucalyptus pyriformis, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus rhodantha, Eucalyptus robusta, Eucalyptus saligna, Eucalyptus salmonophloia, Eucalyptus salubris, Eucalyptus sargentii, Eucalyptus sepulcralis, Eucalyptus sideroxylon 'Rosea', Eucalyptus sieberi, Eucalyptus spathulata, Eucalyptus socialis, Eucalyptus steedmanii, Eucalyptus stoatei, Eucalyptus stricklandii, Eucalyptus tereticornis, Eucalyptus tetragona, Eucalyptus tetraptera, Eucalyptus torquata, Eucalyptus 'Torwood', Eucalyptus urnigera, Eucalyptus urophylla, Eucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo, Eucalyptus websteriana, Eucalyptus woodwardii.

19. A transgenic plant according to claim 18 wherein the species of Eucalyptus is selected from Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cloeziana, Eucalyptus delegatensis, Eucalyptus diversicolor, Eucalpytus dunnii, Eucalyptus globulus, Eucalyptus globulus subspecies bicostata, Eucalyptus globulus subspecies globulus, Eucalyptus globulus subspecies maidenii, Eucalyptus globulus subspecies pseudoglobulus, Eucalyptus grandis, Eucalyptus henrii, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus nitens, Eucalyptus obliqua, Eucalyptus pellita, Eucalyptus pilularis, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus saligna, Eucalyptus sieberi, Eucalyptus tereticornis, Eucalyptus urophylla, Eucalyptus viminalis.

A transgenic plant according to any one of claims 16 to 19 wherein the species of ucalyptus is Eucalyptus globulus ssp globulus or Eucalyptus nitens. r i f P:\OPER\EJH\39013-CL217 12/8/98 -47-

21. A method for identifying a gene required for normal flower development in a species of Eucalyptus, said method comprising isolating a nucleic acid molecule from said Eucalyptus and cloning a region of said nucleic acid molecule having at least 45% nucleotide similarity to all or a region of Arabidopsis genes LFY, AP1 or AG or Antirrhinium genes FLO, SQUA or PLE.

22. A method according to claim 21 wherein the species of Eucalyptus is selected from Eucalyptus alpina, Eucalyptus annulata, Eucalyptus astringens, Eucalyptus behriana, Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus brockwayi, Eucalyptus caesia, Eucalyptus calophylla 'Rosea', Eucalyptus calycogona, Eucalyptus camaldulensis, Eucalyptus camaldulensis ssp. subcinerea, Eucalyptus campaspe, Eucalyptus camphora, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus cladocalyx 'Nana', Eucalyptus cloeziana, Eucalyptus cornuta, Eucalyptus cosmophylla, Eucalyptus crenulata, Eucalyptus crucis, Eucalyptus delegatensis, Eucalyptus desmondensis, Eucalyptus dielsii, Eucalyptus diptera, Eucalyptus diversicolor, Eucalyptus diversifolia, Eucalyptus dumosa, Eucalyptus dundasii, S Eucalyptus dunnii, Eucalyptus eremophila, Eucalyptus erythrocorys, Eucalyptus erythronema, 9. Eucalyptus ficifolia, Eucalyptus flocktoniae, Eucalyptus foecunda, Eucalyptus forrestiana, Eucalyptus gardneri, Eucalyptus gillii, Eucalyptus globulus, Eucalyptus globulus ssp globulus, Eucalyptus globulus ssp. pseudoglobulus, Eucalyptus globulus ssp maidenii, Eucalyptus globulus ssp bicostata, Eucalyptus gomphocephala, Eucalyptus gracilis, Eucalyptus grandis, Eucalyptus griffithsii, Eucalyptus grossa, Eucalyptus henrii, Eucalyptus increassata, Eucalyptus kitsoniana, Eucalyptus kondininensis, Eucalyptus kruseana, Eucalyptus lansdowneana, Eucalyptus largiflorens, Eucalyptus lehmanni, Eucalyptus leucoxylon, Eucalyptus leucoxylon (Pink) (syn. 'Rosea'), Eucalyptus lesouefii, Eucalyptus macrandra, Eucalyptus macrocarpa, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus melliodora, Eucalyptus microcarpa, Eucalyptus nicholii, Eucalyptus nitens, Eucalyptus nutans, Eucalyptus obliqua, Eucalyptus occidentalis, Eucalyptus ochrophloia, Eucalyptus oleosa, Eucalyptus orbifolia, Eucalyptus ovata, Eucalyptus pauciflora, Eucalyptus parvifolia, Eucalyptus perriniana, Eucalyptus pileata, Eucalyptus pellita, Eucalyptus pilularis, Eucalyptus platypus, Eucalyptus polyanthemos, Eucalyptus porosa, Eucalyptus preissiana, Eucalyptus pyriformis, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus rhodantha, Eucalyptus robusta, SEucalyptus saligna, Eucalyptus salmonophloia, Eucalyptus salubris, Eucalyptus sargentii, P:\OPER\EJH\39013-CL.217 12/8/98 -48- Eucalyptus sepulcralis, Eucalyptus sideroxylon 'Rosea', Eucalyptus sieberi, Eucalyptus spathulata, Eucalyptus socialis, Eucalyptus steedmanii, Eucalyptus stoatei, Eucalyptus stricklandii, Eucalyptus tereticornis, Eucalyptus tetragona, Eucalyptus tetraptera, Eucalyptus torquata, Eucalyptus 'Torwood', Eucalyptus urigera, Eucalyptus urophylla, Eucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo, Eucalyptus websteriana, Eucalyptus woodwardii.

23. A method according to claim 21 or 22 wherein the species of Eucalyptus is selected from Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cloeziana, Eucalyptus delegatensis, Eucalyptus diversicolor, Eucalpytus dunnii, Eucalyptus globulus, Eucalyptus globulus subspecies bicostata, Eucalyptus globulus subspecies globulus, Eucalyptus globulus subspecies maidenii, Eucalyptus globulus subspecies pseudoglobulus, Eucalyptus grandis, Eucalyptus henrii, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus nitens, Eucalyptus obliqua, Eucalyptus pellita, Eucalyptus pilularis, :Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus saligna, Eucalyptus sieberi, Eucalyptus tereticoris, Eucalyptus urophylla, Eucalyptus viminalis.

24. A method according to any one of claims 21 to 23 wherein the species of Eucalyptus is a Eucalyptus globulus ssp globulus or Eucalyptus nitens. a.

25. An isolated nucleic acid molecule according to any one of claims 1 to 9 or a method according to any one of claims 10 to 15 or a transgenic Eucalyptus according to any one of claims 16 to 20 or a method according to any one of claims 21 to 24 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 12th day of August, 1998 COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION By Its Patent Attorneys DAVIES COLLISON CAVE 1 ^^c