AU774592B2 - Ion uptake modified plants and DNA sequences for coding same - Google Patents

Description

ION UPTAKE MODIFIED PLANTS AND DNA SEQUENCES CODING FOR SAME This invention relates to ion uptake modified plants and DNA sequences coding for same.

This invention has particular but not exclusive applications to an ion uptake modified plants and DNA sequences coding for same, such as potassium channel and transporter genes whose introduction to the plant genome modifies the degree of uptake of potassium, and for illustrative purposes reference will be made to such application. However, it is to be understood that this invention could be used in other applications, such as modifying plants to alter other osmotically driven :*movements, pH tolerance, cation nutrition, loading into the xylem or cytosolic volume control.

Abiotic stresses such as drought, cold, salinity and potassium deficiency can have harmful effects on plants. For example, large areas of land are affected by high salinity, limiting land use for agriculture and commercial forestry. In Australia .particularly, the growing of timber and pulp species, including Eucalyptus species and pines, is limited by the lack of salt tolerance of such species. Not being bound by theory, it is understood that in high salt environments there is an osmotic imbalance in plants, which leads to reduced plant growth or death. Further to osmotic imbalance, there is a reduction in essential macronutrients such as potassium. Sodium competes with potassium in transport or uptake, and in high sodium environments the amount of potassium entering the plant may be significantly impaired. Over expression of this potassium transporter may permit survival or normal growth in high sodium environments such as marginal, high salinity soils.

The present invention aims to alleviate at least one the above disadvantages and to provide ion uptake modified plants and DNA sequences coding for same which will be reliable and efficient in use. Other objects and advantages of this invention will hereinafter become apparent.

In one aspect, this invention resides broadly in a method of modifying an ion uptake characteristic of a plant and including the steps of: identifying and isolating a gene responsible for an ion uptake mechanism; transforming a plant cell with a genetic construct including said gene; and culturing said transformant to produce a plant.

2 The ion uptake characteristic may be modified to improve the plant's tolerance to abiotic stresses such as drought, low K' concentration, salinity, cold and the like. Modifying an ion uptake characteristic of a plant may improve growth.

Suitably, the gene responsible for an ion uptake mechanism is responsible for a potassium (K channel or transporter. A plant in accordance with the invention may be salt tolerant or may have improved growth in potassium deficient soils. By "salt tolerant" it is meant that a specified plant is capable of withstanding a higher level of salinity stress than the corresponding wild-type plant under typical growth parameters. Under the term K channel or transporter it is be understood to include a corresponding DNA or cDNA sequence of the gene responsible for the K .channel or transporter.

Suitably, the K' channel is an inward-rectifying K channel. The K+ channel ooooo may be identified and isolated using any suitable standard molecular biology techniques common to the art. The plant may be exposed to selected environmental conditions to induce a desired gene/s. For example, the plant may be treated with a relatively high level of NaCI to possibly induce genes that may be involved in ion transport and the salt tolerant phenotype. The RNA may be isolated and used to construct a cDNA library. The library may be screened with suitable probes to identify desired target gene/s, such as primers from consensus sequences of known potassium channels.

The cDNA of the isolated potassium channel may be introduced into a genetic construct capable of expressing the introduced DNA and being transformed, such as a transformation vector appropriate for its application. It is to be appreciated that a person skilled in the art would be able to select a suitable vector and introduce the DNA sequence of interest using protocols common to the art.

In order to initiate the expression of the gene of interest, regulatory elements such as promoters may be included in the genetic construct. The regulatory element may include a constitutive promoter, such as the 35S-CaMV promoter, to enable expression of the gene in all cell types. The regulatory element may also be an inducible promoter, such as the endogenous promoter, that may be inducible in response to specific environmental stimuli. Alternatively, the regulatory element may be a tissue specific promoter or a promoter that may be expressed during specific developmental periods.

3 The plant may be transformed using methods common to the art, including but not limited to biolistics and Agrobacterium infection mediated gene transfer.

Stable transformed plants may be selected using standard selection methods, including but not limited to the inclusion of antibiotic resistant genes for selection of transformants in antibiotic containing media. Stable transformed plants can be propagated using methods common to the art including micropropagation and breeding using judicious selection of parents. These parents may be d untransformed, transformed with the same genetic construct, mutant variations of the gene of interest or distinct genes.

Preferably, the K+ channel is isolated from Eucalyptus camaldulensis roots.

Accordingly, in a further aspect this invention resides in the isolated DNA sequence of the EcKT1 as set forth in SEQ ID No. 1. It is to be understood that the name EcKT1 and EKT1 are the same.

1. Sequence (Seg. ID No. 1): R A or G Y C or T S G or C K G or T 20 H C or T

GGTGAGAATG

AACAGGGGAA

AGAAGAAGAA

CTGTGCGGAG

CTCCAGAGAC

CCCTCGGCGC

TCCTCCCTCG

GGTTGTATAC

AGCCGAAGCC

GCTATCGATA

CTACCTACTT

CATGGTTCGC

AAAATCTCGC

ACTTTGGCGT

ATAGGAACTA

ACTGTGTTTG

TCGTAATCAT

ATGGCTTGGG

TTAACAACGG

GCTTTTCGAC

TGATTGGTAA

AAATGTCAAC

TTCTTCTSTG

GCAGAGCATG

TGTCGCTCTC

GGGAGCAGCC

GAGGAGCAAC

AT CGTCGT TA

ACTGCTTGGG

GCCGCTGTCC

TCGTCCTTAC

GTCGACGACC

CCTTGATGTC

ATT CG C CTC T

CTCCGWAGGG

TAACTACTTT

CAGTCCATTG

GACCCGGCAG

GATCCGGTAT

TTGGATATGG

ATATTCTTCA

TATGACGAAC

GAGCTCAACT

TCTGAAAACT

GAGGGTTTGA

GGTGTGCGGT

AATACAGCCY

CGCGYCGTCC

CAGGATATGG

CGTCGCCRTT

ATCATTGACA

CTTCTTCGTG

CGAAAAAGAT

ATATCGAYCA

CGGGGGCTAT

TCAGCGCCCT

GTGCTCCGGT

TGCAGGATGC

AAACTTGGAT

GTGACTTCGC

CGATCTGCAC

TGCTTTTCAA

TTGGTTGTTC

GTTGAGCACA

GTTGTGTTGT

TGCGAAACAG

CAGGAGGAGA

CGCCACCGGC

AGCTCCGCAG

GAGAATTTCC

C GAGT TC GG C

ATGTCGTCAA

GCTTACCTCG

TGCTTGGAGG

TTCCCTCCGA

GGCCTATTCA

GTTTTCGAGA

GTGCAAAACT

TTCTATTATC

GGGGAAAGCC

TTTACTGGTC

CCAGTGAATG

CCTCGGTTTG

ACGGTACGAG

AGAAAAAGAA

GCCGTGAAGA

GGGAGGGGTG

TGCAGGCCTT

ATC TTG C TGT

CTTCATCGTG

TGGTTCTTCT

TTCCTCAAGA

TGGATTCTTT

ACAAAGCCAC

TACAYGACCT

ACTTGCTCAG

ACATGCTCCG

TTGGAGAAAG

CCTCTGTGTT

TTCTCGCYGC

ATTCTCCACG

TATTACTACT

TGAGGGAGAT

ACGGCATACT

TCGCACCAGA

t

AAATTTAGGG

GTTGCCTCCT

ATAGGACAGA

CCTAAAGCCA

CGACCRGGTG

TGGTCTCAGA

TTGCAAAATG

GGATCTGCTA

AAAGTGGTGA

CTTTTCACAG

TACCACATTG

TCATGAACAA

GAGAGCATTC

CCTGCCTCTC

TGAGCCAACC

GGGAGGACAC

GCTCCTCCTT

GAAATGTGCC

AAGTTGCTAG

20 ATTTTCATGC

TCAGTCGTTA

GCTCTTCATG

CTTGGATAGA

CCAGAGATTT

25 CAATCCATAA

TTTGCCTCCT

GCCGTCCAAG

ATGTCAGCTG

TAATAAATCT

TTGTTAGTTG

CCCGAGAACT

TACTCTGCTC

CAGTTATCAG

GATGATTTTG

GGATTTTTCC

AAGTCTAGTA

TTACATGAAT

AGAAAATTCA

ATACAATACA

CGGCTACAAG

TTCTGAGGGA

TCCGATCCAG

TACTTATTCC

AATGAAAGAC

AAGCACCAAC

GTTGTTAAAA

TATTTGCGGG

TGCGGACGAA

TTCAATATAA

TCTCCTTCAG

TGATTGACGC

AGTTTATGCT

ACTGAGACGG

CTCTGCATAT

ATGGATTATG

TCTTTGGGAG

CASAAAATGG

ACGGCAGCAG

TGGTGGGGAT

TCGCTGTTTC

GGGGCAGACA

AGCAGATCAA

ARGAAACTGA

GCATCCCAAG

GAGGAGGACG

CACGTGATGG

GCCAATAAGC

TCCTGAAGTG

TTCAGGAGTT

AAAGTTTTAA

GGATGGCGAC

CCAACAGAAA

TTCCGAATCA

CGACTGTTTC

CAACACAAGG

YGTTATTCC

AGC TGC C TCA

ATCAGATGCT

CTTCGGCAAA

CATCTTACAT

GTGGCATTTC

GAGTATTTTC

AGACTTCTAC

ATGGAACTGA

GAGATTGGTG

ACGATTGAGC

TCCAGTCAAA

CACTTGAAGG

AGAGAACATG

TTGCCACATT

GGCCTAGATC

CGCAGCATCT

GTGCAGATCC

GCAATAAAGG

TGCTAATTTA

AACAGAATAA

GTTACGCTTC

TGARGATAAC

TCAACAAACC

CAGGGACACG

AATGCCKCCT

AAAAAACTGT

AGTAACTTCC

TGAGGGAGAT

CTTTTGTGGC

GGAGATGTTG

GTTGGAGATG

CCAAAAATGG

CATCTAGTCT

TGACAAGAAG

GTTTCCATGT

CATTATCTTC

AACTGTACAT

AGTTTTGCTC

TGCTCATTTG

AAGAGACTCT

TATCTTTTTT

AAATGACTTG

CTCCAAATGA

ATTCTCGTCA

ACAGCCTGTT

TACTTTGCTA

CAGCTGCTAC

TGTTGAAGAT

ACCTCAAGGA

GTGGCACACG

GAGAGGAGAC

CTAATGAATC

AAAGGAAGCG

TAATAGCCGA

GCGGTAGTGA

GTATCTGGAG

TCTGGACTTG

CAAAAAGCAA

ATTGAGATAG

GGACATCCAT

AAGAGATAGG

GCTATTAGAA

TGATGCTGCT

ACAATTCCTT

GTGCTTTTAA

ACGACCGGCT

AGGGGAAGCT

GCACGTAAGA

AGCTGAAATT

TTTTGAGCAA

GTCCAATAAG

CCACATCTTA

TTTATGTTCT

GTACTATGTA

AAAGGAACCA

TGCCTCAAGT

CGATTCTCTC

ACAATATCGT

CTCTTTCAGC

AGATGTGATC

CTGGTGCTGT

GGAGAGGCAA

CAGGCCACAG

GGCTAAATCG

GGGACCATTA

TCCAACCATG

GTCAATTGGA

RACTTGATGT

AGATAACAGT

AGAATTGCGC

GATTCAGAAG

ACCCGTGGTC

ACGTGGGTCA

CTCAAGGAGA

CGGGACCACC

TCAAGTTCCT

GGATGGACAC

AATTCTTTTT

GCGAACCCAA

TTTGGCCAAA

GTTTGGCATT

GCATTAATCA

AT C CGTG TGG

TATGCTACTC

AATTTGGGCT

GATGATGTAG

GGAAGTGAAG

GAAGTGCATA

GTCTAATGGA

TTCGCTCTTT

AGTAATCTGA

The predicted expressed amino acid sequences for D and E isoforms of the EcKT1 gene are respectively listed as set forth in Seq. ID Nos. 2 and 3.

2. Sequence (Sea. ID No. 2):

MEGLMRNRGG

NRVVQLRSFI

SIIDNVVNGF

VISIIPSELA

FVLRCAKLLC

YVTSLYWSIT

VLCGVSLSVC

VSSLDRRYRI

FAIDIVLTFF

QKISHSPLGG

VTVFAVHCAG

TLTTVGYGDL

GQEEMQAFSR

WENFLVLLVV

VAYLDKATYL

YGLFNMLRLW

CFYYLLAARN

HPVNVREMLF

DGSSQYSLAT

YTAWASPFEF

LVDDPKKIAW

RLRRVSALFS

HDPAETWMGK

DIFFMLFNLG

GILL SLGARS

GFLKKPKPPL

RYMTSWFALD

RLEKDRNYNY

AILHDGLGIR

LTAYL IGNMT

NLVVHGTSRT

GLRQKETLDS

DEYFPPNEDV

GEIGVLCYRP

QHLKDLKDPT

RGLDPNESDN

EAIKGGSEPV

DVTLPKSNGT

QQGHEEIGIL

TSNFHNSLFG

VGDVEGKLML

DHLVFLSKEV

RKFRDTIQAA

LPKAIRSSIL

ILQNEAPTDF

QLFTVRTKRL

ME SIL IDAEN

SGRTPLHIAA

VKLLAENGAN

TALHVAVSED

FQSIKETEMP

IMSAARDGEG

LPENFQELLE

KDDFANRNDK

SSFAQRNQLP

HYLFYNIVDQ

Y ILVTGAVDL

SQLLRLNRTT

MVAHGQLDLP

SKGSENCALL

LVSGDVGQFS

NIEIVKFLLD

PAIRSEPNLP

DVLLS INHNK

MARKKFGLTL

KVQ

PRLQDQMLAH

VYLFRGI SND

LVVKNGTEQP

LFNI IQSNVE

LSLCFATLRG

LMDYGADPNS

CTAAEQNNLD

RGADINKPDI

PASQEKTVDA

SANKPFVARR

LKVLTKNGAE

LCLKYRTDSE

LLFQLVSEMK

VGEAKSGD IC DGTI IMNNLL

DDLMLSQPLR

RDSEGNVPLW

LLKEI SRYGG

HGWTPRDLAD

AFGQSRPRRR

Al RVVVSCPE

IDDVAVIRDG

3. Seaiuence (Seq. ID No. 3):

MEGLMRNRGG

NRA VQLRSF I

SIIDNVVNGF

VISTIPSELA

FVLRCAKLLC

YVTSLYWSIT

NLVVHGTSRT

GLRQKETLDS

DEYFPPNEDV

GE IGVLCYRP

QHLKDLKDPT

RGLDPNESDN

EAIKGGSEPV

30 DVTLPKSNGT

QQGHEEIGIL

TSNFHNSLFG

VGDVEGKLML

DHLVFLSKEV

VLCGVSLSVC

VSSLDRRYRI

FAIDIVLTFF

QKISHSPLGG

VTVFAVHCAG

TLTTVGYGDL

RKFRDTIQAA

LPKAIRSSIL

ILQNEAPTDF

QLFTVRTKRL

ME SIL IDAEN SGRTPLH IAA

VKLLALNGAN

TALHVAVSED

FQS IKETEMP

IMSAARDGEG

LPENFQELLE

KDDFANRNDK

GQEEMQAFSR

WENFLVLLVV

VAYLDKATYL

YGLFNMLRLW

CFYYLLAARN

HPVNVREMLF

SSFAQRNQLP

HYLFYNIVDR

YILVTGAVDL

SQLLRLNRTT

MVAHGQLDLP

SKGSENCALL

LVSGDVGQFS

NIEIVKFLLD

PAIRSEPNLP

DVLLS INHNK

MARKKFGLTL

KVQ

DGSSQYSPAT

YTAWASPFEF

LVDDPKKIAW

RLRRVSALFS

HDPAETWMGK

DIFFMLFNLG

PRLQDQMLAH

VYLFRGISND

LVVKNGTEQP

LFNI IQSNVE

LSLCFATLRG

LMDYGADPNS

CTAAEQNNLD

RGADINKPDI

PASQEKTVDA

SANKPFVARP

LKVLTKNGAE

GILLSLGARS

GFLKKPKPPL

RYTTSWFALD

RLEKDRNYNY

AILHDGLGIR

LTAYL IGNMT

LCLKYRTDSE

LLFQLVSEMK

VGEAKSGDIC

DGTI IMNNLL

DNLMLSQPLR

RDSEGNVPLW

LLKEISRYGG

HGWTPRDLAD

AFGQSRPRRR

AIRVVVSCPE

IDDVAVIRDG

9 a.

Expression, or over expression of the EcKT1 gene may lead to improved nutrient uptake in marginal soils, and may also maintain a potassium balance in plants grown in high sodium chloride. Specific mutation of this channel may permit selected uptake of potassium without the inhibition by sodium ions. Accordingly, gene transfer of the EcKT1 genes into desired plants of interest may generate a salt tolerant plant or a plant which is able to grow in soils with low K' potassium content.

The isolated EcKT1 gene may be subcloned into the appropriate genetic vectors using standard molecular biological methods for expression in the desired target plant species. These cDNAs can be under the control of constitutive promoters, eg. 35S-CaMV, or tissue specific or inducible promoters including the endogenous promoter of said cDNAs. Thus, the isolation of new promoters has significant value to regulate the endogenous gene and/or other exotic genes of interest.

6 Accordingly, in a further aspect, this invention resides in the DNA sequence encoding the promoter of the EcKT1 gene as set forth in Seq. ID No. 4.

4. Sequence (Seg. ID No. 4): M=A or C R A or G W A or T S C or G Y C or T K=G or T V A, C or G H A, C or T D G or T B C, G or T X or N A, C, G or T a..

a a 0*

CGACGGCCCG

GTGGCAGCTT

TTTCTTTGAT

ATGTTACTAG

AGTGATCCAT

CATGTATAWT

TTGAAGAGTA

CATGTTTAAT

CATACAAATA

AAAAAATATT

CGATGTTTYC

CCGATTGATA

TATATTTGTA

GATATAATTG

AATTAATTTC

TTCCGATAGT

GGGCTAGTAC

CTAGCYTCTI(

CTAAATATAM

ATTTCAATTT

ATAGAGTCAC

TTTGGYAGTG

GCTGGACCAA

GGGTCAYGGT

ACAGGAGAGA

AAATGTCAAC

TTCTTCTSTG

GCAGAGCATG

GGCTGGTCTG

TTGAACCTTB

CTGATGGTCA

TCGAGATTCT

TCCCCAAKAA

TAGCGCATTG

CTCSAAACAC

GCRCTTAACG

AATTTGAGRG

AACACATGTA

CRTWCTCCTA

TGTTGTCATT

ATCATTARTG

TAGCRCGCGG

TTTTTTCTAT

TCATCAGCAC

AGTACTTTCR

TTCGGAACCT

ATAGCCAAAT

TTTTTTCCAC

ATCAAAATGG

ACTTTGATTT

TCCTGTCTTA

CGGCAGGAGT

KAGAKAGAGA

GAGCTCAACT

TCTGAAAACT

GGCTTTTTCT

GCCATTAGTA

AGAGACTGGG

CTTTTTCTCT

TTGTTGTGTT

RAAGTTAAAA

CYGTTWAATG

ARACTTTGCC

TATGTTTGAA

TATGAATAAT

GGSTGGAAGA

GGTRATTAAA

GTGGAGAAAA

AATACTTCAA

GTAGTGCATT

AACTTCTTAC

TAGTGTTCAA

GCCCCCACCC

ACAGTATACA

CTGCCACTCT

ATTCTTAGCG

TRRATCCAYA

TTTTTTATAT

GATGCACCCA

GAGATTCTTG

GTTGAGCACA

GTTGTGTTGT

GATTGTCCAT

TCGATGGTTG

ATCCATTCCC

CCGACAGTTA

AAGATATGTC

TATTTTCTTA

AGACCTTTAT

GCATCATATC

ATTATCATAA

AATAAATAAT

AACATTTTCC

TTCACAAAAA

AACACAACCC

ACATCTTATA

GAAAAACTGT

GTSGGTTTCA

CTTGGCAAGC

ACTTTTCAAT

CAACTGCGCA

AGCACTTTTA

TCTCTAAAGT

TCTGATGACC

CTTCCGATTG

TGGGAAGTAC

ATATTCTTGA

AGAAAAAGAA

GCCGTGAAGA

CCAAGTGCAC

GGATTTGCTC

GAAGAATAAG

TCGAGGGATT

TTTCTAGAGT

CCTAGTGTAC

ATGTATAAGT

ACAAATGGAT

TCATGTCTTG

AAAAAAACAG

AACCATGGCA

TGGCGGTGCT

TAATTGCAAT

AAAACTACAA

GCCGAAGATT

ACTTTGAAAA

ATCACAACTT

GATGCGACTT

TGACTATACA

GCACTTTGCA

GATTATTCTT

TCAGGAGGAT

CCGAAATGAC

ACTGTGCAAC

TGTGAGAATG

AACAGGGGAA

AGAAGAAGAA

The DNA sequence of the EcKT1 promoter as set forth in SEQ ID No. 4 contains ambiguity codes in addition to the four bases G, C, as there may be more than one sequence. A single DNA sequence of the EcKT1 may be isolated from tissues of plants which have been transformed with the EcKT1 promoter.

Accordingly, in a further aspect, this invention resides in the DNA sequence encoding the promoter of the EcKT1 gene as set forth in Seq. ID No. Sequence (Seg. ID No. S=G or C The last 3 bases (ATG) represent the protein translation initiation codon.

a a a a a

CGACGGCCCG

GTGGCAGCTT

TTTGTTTGAT

ATGTTACTAG

TAGTGATCCA

TCATGTATAA

15 CTTGAAGAGT

TCATGTTTAA

TCATACAAAT

GAAAAAATAT

GCGATGTTTC

ACCGATTGAT

TTATATTTGT

TGATATAATT

AAATTAATTT

TTTCCGATAG

AGGGCTAGTA

TC TAG C CTC T

TCTAAATATA

AATTTCAATT

AATAGAGTCA

TTTTGGCAGT

TGCTGGACCA

GGTCACGGTC

AGTAGATAGA

ACGAGCTCAA

TGTCTGAAAA

TG

GGCTGGTCTG

TTGAACCTTG

CTGATGGTCA

TCGAGATTCT

TTCCCCAAGA

TTAGCGCATT

ACTCGAAACA

TGCGCTTAAC

AAATTTGAGG

TAACACATGT

CCGTACTCCT

ATGTTGTCAT

AATCATTAGT

GTAGCGCGCG

CTTTTTTCTA

TTCATCAGCA

CAGTACTTTC

GTTCGGAACC

AATAGCCAAA

TTTTTTTCCA

CATCAAAATG

GACTTTGATT

ATCCTGTCTT

GGCAGGAGTG

GAGAGATTCT

CTGTTGAGCA

CTGTTGTGTT

GGCTTTTTCT

GCCATTAGTA

AGAGACTGGG

CTTTTTTCTT

ATTGTTGTGT

GAAAGTTAAA

CCCGTTTAAT

GAGACTTTGC

GTATGTTTGA

ATATGAATAA

AGGCTGGAAG

TGGTGATTAA

GGTGGAGAAA

GAATACTTCA

TGTAGTGCAT

CAACTTCTTA

GTAGTGTTCA

TGCCCCCACC

TACAGTATAC

CCTGCCACTC

GATTCTTAGC

TTAAATCCAT

ATTTTATATC

ATGCACCCTG

TGATATTCTT

CAAGAAAAAG

GTGCCGTGAA

GATTGTCCAT

TCGATGGTTG

ATCCATTCCC

TCCGACAGTT

TAAGATATGT

ATATTTTCTT

GAGACCTTTA

CGCATCATAT

AATTATTATA

TAATAAATAA

AAACATTTTC

ATTCACAAAA

AAACACAACC

AACATCTTAT

TGAAAAACTG

CGTGGGTTTC

ACT TGGCAAG

CACTTTTCAA

ACAACTGCGC

TAGCACTTTT

GTCTCTAAAG

ATCTGATGAC

TTCCGATTGC

GGAAGTACAC

GATGTGAGAA

AAAACAGGGG

GAAGAAGAAG

CCAAGTGCAC

GGATTTGCTC

GAAGAATAAG

ATCGAGGGAT

CTTTCTAGAG

ACCTAGTGTA

TATGTATAAG

CACAAATGGA

ACTATGTCTT

TAAAAAAACA

CAACCATGGC

ATGGCGGTGC

CTAATTGCAA

AAAAACTACA

TGCCGAAGAT

AACTTTGAAA

CATCACAACT

TGATGCGACT

ATGACTATAC

AGCACTTTGC

TGATTATTCT

CTCAGGAGGA

CGAAATGACG

TGTGCAACAC

TGAAATGTCA

AATTCTTCTS

AAGCAGAGCA

In another aspect, this invention resides in a method of regulating expression of a gene including the steps of: identifying and isolating a gene of interest; constructing an expression cassette including said gene and a promoter selected from those promoting genes responsible for an ion uptake mechanism; transforming a plant cell with said expression cassette; culturing said transformant to produce a plant.

The expression cassette may be introduced into a plant of interest by transformation in order to initiate the expression of any suitable gene of interest 8 during conditions which result in a modified ion uptake mechanism in the plant. For example, these conditions may result in the expression of the gene of interest during low soil K levels or high salinity. The promoter may be active in vascular and/or root tissues. The promoter may also be active in response to abscisic acid which is involved in plant adaptation processes to abiotic stresses.

The gene of interest may be responsible for expression of stress tolerance or insect resistance. Suitably, the promoter is the EcKT1 promoter.

In order that this invention may be more readily understood and put into practical effect, reference will now be made to the following figures and examples which illustrate preferred embodiments of the invention and wherein: FIG. 1 is a phylogenic tree of plant K channel proteins illustrating the relationship of the EcKT1 protein with other previously identified channel proteins from Arabidopsis, potato and maize. The phylogenic tree was produced with the Genetics Computer Group (GCC Inc., Madison, USA) programmes GrowTree, Distances and PileUp using neighbour-joining and Kimura Protein Distant algorithms; FIG. 2 depicts a schematic representation of a possible association of EcKT1 with the extracellular membrane; FIG. 3 depicts the EcKT1 expression in E. camaldulensis using Southern blot analysis; FIG. 4 is a graph of the EKT1 transcript levels in Eucalypts, FIG. 5 illustrates the GUS expression, representing an active EcKT1 promoter, in vascular tissue of transgenic Arabidopsis plants, and.

FIG. 6 depicts the GUS activity in root and aerial tissues of pEcKT1-GUS transgenic Arabidopsis plants.

EXAMPLE 1 The EcKT1 cDNA and promoter were isolated from Eucalyptus camaldulensis roots, which encodes a potassium channel and regulatory element respectively, using standard molecular biology techniques common to the art. E.

camaldulensis plants were treated with 75 mM NaCI to possibly induce genes which are involved in salt tolerance. RNA was isolated and a cDNA library constructed.

The EcKT1 gene was identified and isolated from this library by screening with a 9 radioactive probe generated from a PCR template using primers from a consensus sequence of known potassium channels.

EcKT1 is a low-affinity inward-rectifying K+ channel. The EcKT1 gene most closely resembles the Arabidopsis AKT1 gene in sequence databases as illustrated in FIG. 1. A schematic representation of possible association of EcKT1 with the extracellular membrane is illustrated in FIG. 2. It is to be appreciated that this diagram is an example and is not intended to represent the only possible membrane association configuration of the EcKT1 protein. The DNA sequence of two EcKT1 isoforms and the predicted protein sequences are illustrated in Seq. ID No. 1 to 3. The cDNA represents the full length sequences of about 3.0 kilobase pairs of DNA, which encode a complete and functional protein.

The EcKT1 cDNA was cloned into a yeast expression vector (pYES2) and transformed into the K+ uptake-deficient (trkl, trk 2) S. cerevisiae strain CY162.

The CY162 yeast cells were streaked on arginine-based medium supplemented with galactose, sucrose and 50 or 1 mM KCL and incubated for two days at 300C.

S. cerevisiae strain DBY 746 was used as the wild type control. As shown in Table 1, the EcKT1 gene is able to complement the growth of a K+ deficient yeast mutant allowing growth on standard yeast propagation media modified to contain a low concentration of This also confirms that the E. camaldulensis EcKT1 gene encodes a functional K+ channel.

Table 1 Wild Type CY162 pYES-EKT1 CY162 pYES KCI Growth Growth Growth 1mM KCI Growth Growth No Growth Southern blot analysis of the E. camaldulensis K+ channel gene family was performed, as illustrated in FIG. 3. The total genomic DNA (2ig) isolated from E.

camaldulensis tissue was digested separately with the DNA restriction enzymes BamH1, HinDIII or EcoR1. The fragments were separated by agarose gel electophoresis, blotted onto Qiabrane N' membrane and probed with an E. globulus K+ channel PCR fragment (an EcKT1 radioactive probe) and exposed to X-ray film for detection. Southern blot analysis using the low stringency conditions and an EcKT1 probe indicated a single gene in E. camaldulensis.

As illustrated in FIG. 4, EcKT1 expression levels in aerial and root tissue of Eucalyptus was assessed using RT-PCR. The level of EcKT1 transcript was normalised against a-tubulin levels under a limited number of PCR cycles. EcKT1 expression was induced in roots in response to Na stress (75 mM) and K starvation treatment alone and a combined treatment synergised the level of expression. There was no change in EcKT1 expression levels in aerial, non-root tissue with the Na* or K treatments alone, and a slight reduction when treated in combination. These results suggest that the EcKT1 K channel may play a role in K uptake from the environment and is responsive to K+ deficient conditions.

EXAMPLE 2 S 15 The promoter, or regulatory element, of the EcKT1 gene was cloned. This regulatory element when subcloned 5' of the marker gene encoding 3glucuronidase (GUS) drives the expression of said marker gene in tissues associated with the plant vasculature. Such expression may be valuable when it is desired to express a gene in this specific region, such as genes encoding transport, 20 channel or pore-like proteins in this region. For example, the promoter could target insect resistant genes to combat phloem feeding pests. The promoter would also be useful for specific targeting of stress tolerant genes.

The promoter of the EcKT1 gene was isolated using suitable molecular biology techniques that are common to the art. Nested PCR primers were used in a 'promoter finder' procedure to isolate the DNA sequences 5' of the coding sequence on genomic DNA clones. Three different DNA restriction enzymes were used to digest DNA that resulted in the cloning of three DNA fragments of sizes: kilobases Pvull; 0.8 Kb, Scal; and 0.5 Kb, EcoRI. The 1.5 Kb and 0.5 Kb promoters were isolated from genomic clone 1.8 and the 0.8 Kb promoter from genomic clone 3. These fragments were cloned and sequenced as indicated in SEQ. ID No. 4.

The PCR fragments were cloned into the vector pBI101. The pBI101 plasmid is readily available and is designed for cloning and testing promoters in plants using P-glucouronidase (GUS) expression. The 0.8 Kb and 0.5 Kb fragments were 11 subcloned to drive the expression of the marker gene GUS. Positive GUS staining, representing active promoter DNA fragments, was visible in the vascular tissues of transgenic Arabidopsis plants, as illustrated in FIG. The transgenic plants in FIG 5 of the patent are stained for GUS marker gene expression. This gives an indication of which tissues the EcKT1 promoter is active in. Using the same transgenic Arabidopsis plants the level of marker gene expression was quantified using a standard fluorometric assay for GUS enzyme activity.

The DNA sequence of the EcKT1 promoter according to Seq. ID No. 4 contains ambiguity codes in addition to the four bases G, C, This is because there was more than one sequence for the promoter. The DNA corresponding to the EcKT1 promoter from Agrobacterium cells containing the pEcKT1-GUS construct used to produce the transformed Arabidopsis plants was recovered and sequenced. The sequence of the promoter DNA recovered from Agrobacterium is given in SEQ ID No. 5 to top strand). There is still one ambiguity code (S G or The last 3 bases (ATG) represent the protein translation initiation codon.

This stretch of 1352 nucleotide bases is responsible for controlling the expression pattern of the GUS (1-glucuronidase) marker gene in vascular tissues •(Fig. Having a sequence without ambiguity codes helps in identifying sequence motifs, known as cis-acting elements, which are potential binding sites for regulatory trans-acting factors.

Communication between the root and the shoot forms a central part of the coordinated response of plants to drought and salinity. Recent evidence suggests that an outward-rectifying K+ channel expressed in vascular tissue of the root may have a major role in this process (Gaymard et al., Cell 94, 647-655 1998; Hetherington Current Physiology 8 R911 -R913 1998). The inward-rectifying EcKT1 K+ channel is also expressed in root vascular tissue and maybe involved in coordinating the plant's response to abiotic stresses such as drought and salinity.

The plant hormone abscisic acid (ABA) is involved in numerous physiological responses of the plant. These mediate adaptation processes to abiotic stresses, such as drought or water deficit, salt stress and in some cases mechanical stress.

ABA-induced gene expression is an important part of ABA action. An analysis of the cis-acting sequences required for ABA-induced gene expression identified ABRE (ABA responsive element) sequences in promoters of many ABA-response genes. The ABRE is similar to a family of sequences called the G-box, which also contain an ACGT core and are present in a number of gene promoters that respond to different environmental conditions. The EcKT1 promoter contains an ABRE (CACGTGGCA), which is underlined in Seq. ID No. The presence of an ABRE in the EcKT1 promoter suggests that the EcKT1 gene may be regulated in part by ABA and indicates that this gene may be involved in coordinating the response of plants to drought and salinity.

There are similarities in the expression pattern between the 1.352kb EcKT1 Eucalyptus promoter in Arabidopsis (FIG. 6) and the endogenous EcKT1 gene in Eucalyptus (FIG. 4).

:i The expression level in aerial tissue does not alter much during salt stress or potassium starvation.

S* The expression level in root tissue is significantly increased upon salt stress or potassium starvation.

The 1.352Kb EcKT1 promoter conferred potassium starvation inducibility to the GUS marker gene.

The 1.352Kb EcKT1 promoter conferred vascular tissue expression to the GUS marker gene.

It will of course be realised that while the above has been given by way of illustrative example of this invention, all such and other modifications and variations thereto as would be apparent to persons skilled in the art are deemed to fall within the broad scope and ambit of this invention as is herein set forth.

Claims (25)

1. A method of modifying an ion uptake characteristic of a plant and including the steps of: identifying and isolating a nucleotide sequence that encodes a K+ channel responsible for ion uptake in a plant; transforming a plant cell with a genetic construct including said nucleotide sequence; and culturing said transformant to produce a plant having a modified ion uptake characteristic.

2. A method according to claim 1, wherein the ion uptake characteristic is modified to improve the plant's tolerance to abiotic stresses selected from drought, low K concentration, salinity, cold and the like.

3. A method according to claim 1, wherein the ion uptake characteristic is modified to improve the plant's growth.

4. The method according to any one of Claims 1 to 3, wherein the nucleotide sequence is a gene or cDNA. The method according to any one of Claims 1 to 4, wherein the K channel is an inward-rectifying K+ channel.

6. The method according to any one of Claims 1 to 4, wherein the K channel is 25 an outward-rectifying K channel. .l

7. The method according to any one of Claims 1 to 5, wherein the nucleotide sequence responsible for the K channel is isolated from Eucalyptus camaldulensis roots.

8. The method according to Claim 6, wherein the nucleotide sequence is the EcKT1 gene. 14

9. The method according to Claim 7, wherein the isolated nucleotide sequence of EcKT1 is as set forth in SEQ ID No. 1.

10. The method according to any one of the preceding claims, wherein the genetic construct includes a promoter selected from a constitutive, tissue specific or inducible promoter.

11. The method according to claim 9, wherein the constitutive promoter is CaMV promoter.

12. The method according to Claim 9, wherein said inducible promoter is the promoter of the EcKT1 gene.

13. The method according to Claim 11, wherein the DNA sequence of the promoter is as set forth in SEQ ID No. 4.

14. The method according to Claim 11, wherein the DNA sequence of the promoter is set forth in SEQ ID No. The method according to any one of the preceding claims, wherein said plant is rendered salt tolerant.

16. The method according to any one of the preceding claims, wherein said plant 25 is rendered able to grow in soils with low K+ content.

17. A plant produced by the method of any one of claims 1 to 16.

18. The isolated DNA sequences of the EcKT1 gene as set forth in SEQ ID No. 1.

19. The DNA sequences encoding the promoter of the EcKT1 gene as set forth in SEQ. ID No. 4. The DNA sequence encoding the promoter of the EcKT1 gene as set forth in SEQ. ID No.

21. A method of regulating expression of a gene including the steps of: identifying and isolating a nucleotide sequence of interest; constructing an expression cassette including said nucleotide sequence and a promoter selected from those which regulate one or more genes encoding a K+ channel and which are activated in conditions that alter the ion uptake mechanism of a plant; transforming a plant cell with said expression cassette; culturing said transformant to produce a plant.

22. The method according to Claim 21, wherein the nucleotide sequence is a gene or cDNA. 23 The method according to Claim 21 or Claim 22, wherein the nucleotide sequence of interest expresses in response to low soil K ion levels.

24. The method according to Claim 21 or Claim 22, wherein the nucleotide 20 sequence of interest expresses in response to high levels of soil salinity. eo So

25. The method according to any one of Claims 21 to 24, wherein the nucleotide S-sequence of interest expresses in vascular tissue. 25 26. The method according to any one of Claims 21 to 25, wherein the nucleotide sequence of interests expresses in the root. see.

27. The method according to any one of Claims 21 to 24, wherein the nucleotide sequence of interest expresses in response to abscisic acid.

28. The method according to any one of Claims 21 to 25, wherein the nucleotide sequence of interest is responsible for expression of stress tolerance or insect resistance. 16

29. The method according to any one of Claims 21 to 26, wherein the promoter is the promoter of EcKTI.

30. A plant produced by the method of any one of claims 21 to 29. DATED THIS NINETEENTHDAY OF APRIL 2004 FORBIO LIMITED JACKSTOWN PTY LTD BY PIZZEYS PATENT TRADE MARK ATTORNEYS