Note: Descriptions are shown in the official language in which they were submitted.
CA 022~94~6 1998-12-30 ~AU 9 7 / ~ 1
RECEIlIED ~
REGULATION OF GENE EXPRESSION IN EUKARYOTES
rhis invention relates to a method for regulation of gene expression in
eukaryotes.
This invention has particular but not exclusive application to regulation of highly
specific expression in target organs or functions of plantg, and for illustrative purposes
5 reference will be made to such application. However, it is to be understood that this
invention could be used in other applications, such as regulation of expression of other
eukaryotic genes such as regulation of specific expression in target organs and
functions of yeasts and animals.
Eukaryotic organisms of commercial benefit including plants, animals and lower
10 organisms such as yeasts sometimes divert growth resources into non commercial
structures, or express other genes of economic detriment. Other commercial species
may benefit from the introduction of char~cteristics which would enhance commercial
worth.
For example, the development of reproductive structures on forest trees
15 represents a significant burden on the resources of the trees, the reproductive effort
occurring at the expense of wood growth. Seeds from mature trees produce an
undergrowth of saplings which must be removed periodically to prevent competition
with the tree crop for soil resources and to minimize the risk of damaging fire.Austl ~lian Patent Specification No. 86224/91 discloses a method of enhancing
20 vegetative growth in a plant. This disclosure exemplifies a method wherein a
substantially tissue-specific promoter for reproductive-structure specific genes was
selected on the basis of the early appearance of the gene product and specificity of
expression in both male and female reproductive buds. Several genes and promoters
were isolated and charact6rised, each gene was modified by fusing a portion of the
25 gene including the tissue-specific promoter of the gene with a structural gene for a
ribonuclease.
Whilst the transformants exhibit a lack of reproductive structures, promoter
leakage tends to result in a low level of accumulation of the lethal ribonucle~se in non
target tissues, resulting in reduced growth and thus reduced co"~mercial potential.
Gene expression promoters are to be generally regarded as falling in one of
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CA 022~94~6 1998-12-30
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RECEIyE~ ~ ' ~ 1 97
three broad cl~sses Constitutive proi"oters continuously express the gene in alltissues. Devc'opmental promoters express the gene in specific tissues or in tissues
at specific stages. Inducible pro,noters respond by switching expression on or off in
response to the presence of metabolites, foreign compounds or other stimuli such as
5 light, heat or pressure.
However, the inserted promoters are often leaky and the preferred strong
promoters may deleteriously express in other tissues with insufficient inhibition to
prevent death of non target cells, which may adversely affect commercially important
non-target tissues. Promoter leakage cannot be predi~ted and, for example, where10 the transformants such as trees have a long growth to first flowering, the resulting
gene product activity in non target cells may negate or reduce the advantage of flower
suppression. The generally preferred strong promoters limit the promoter choices.
It is known that the expression of a lethal gene product such as Barnase under
the control of a substantially flower-tissue specific promoter may be rendered more
15 tissue specific by constitutive coexpression of an inhibitor (B or "Barstar") for the gene
product. The action of the expressed gene product in the target cell will occur where
the inhibition is insufficient to suppress gene product action, and non target cell
Barnase activity arising out of a degree of non-specificity of the promoter or promoter
leakage is substantially suppr~ssed.
The present invention aims to substantially alleviate at least one of the above
disadvantages and to provide a method for regulation of eukaryotic gene expression
which will be reliable and efficient in use. Other objects and advantages of this
invention will hereinafter become apparent.
With the foregoing and other objects in view, this invention in one aspect
25 resides broadly in a method of regulating a eukaryotically active gene, comprising
llansfo""ing a cell with construct expressing a modlJ'~tor gene product regulating the
eukaryotically active gene or its product and a further gene product regulating said
modulator gene or its product, the pro,noters of two of said gene, modulator gene and
further genes being selected from inducible pr~"~oters and developmental pro,noters
30 for the same or complementary tissues.
IFFA/Au
CA 022~94~6 1998-12-30
~EC.i'lED 2 ' :,J ~'~37
The eukaryotically active gene may comprise a native gene to the organism of
interest or may compri~e an introduced gene. The gene may be promoted by its
" - ~ . noter or comprise a heterologous construct with the gene. The
eukaryotically active gene may code for an expression product of any selected cellular
5 function. For example the gene may code for an active polypeptide including enzymes
such as a ribonuclease having a lethal effect on a target tissue or a peroxidaseconferring nematode resistance on root tissue. The gene may code for an enablingproduct for pathways for production of pigments and dyes such as anthocyanin, aninsecticidal compound such as Bt toxin, or fragrances.
Expression of reporter genes under control of inducible promoters may display
exact location of target cells in target tissues without any leakage to surrounded cells.
This may be very useful for investigation of primary steps of interactions between
pathogens and insects and plant cells. The GUS, luc, anthocyanin gene and GFP may
comprise candidates as reporters. Colourimetric or fluorescent analysis may be made
15 of micropropagated materials before commercialisation.
As used herein, the expression "lethal gene" is taken to mean a gene or genes
which encode a peptide or antisense or ribozyme or other non peptide which
significantly disrupts a target cell leading thereby to the death of the target cell. The
invention will be described hereinafter with reference to embodiments comprising a
20 eukaryotically effective lethal gene.
The lethal gene may be any gene or combination of genes which encode a
peptide or antisense or ribozyme which significantly disrupt a target cell leading
thereby to the death of the target cell. For example, the lethal gene may be selected
from those that encode ribonucle~ses such as Barnase from B. amyloliquefaciens~
25 RNase T1 from A. aryzae. bovine RNase A, RNase I and RNase H from E. coli or a
set of plant RNases (family of S-proteins). Alternatively, the lethal gene may be
selected from nucleases such as the family of resl,iction endonucleases, enzymesincluding glucanases such as 1 ~-~-glucanases or 1 -3-13-glucanases, ubiquitins, acid
pyrophosphatases and inhibitors of plant cell wall synthesis.
Preferably, the lethal gene is an RNase. A chimaeric lethal gene may be
~ME~ , S~ E~
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CA 022~94~6 1998-12-30
u 9 - I 9 0 ' '
.
constructed including the coding region of Barnase from B. amyloliquefaciens under
the contro~ of upstream (promoter~ sequences of genes expressed in the male and
female pal L;~ productive organs at the early stages of development.
Restriction of expression of a lethal gene in target cells during first events of
5 pathogen-host plant interaction in target cells may be achieved. For example,
expression of Barnase or other toxins like 13-glucanase or chitinase in attacked cells
will lead to necrosis of these cells which will prevent spreading of infection to
surrounding cells. The critical stage in this artificial hypersensitive reaction project is
blocking of possible promoter leakage in surrounding cells.
Expression of bacterial salicylate-hydroxylate gene may reduce level of salicylic
acid which is responsible for systemic expression of cassettes under control of
pathogen-inducible promoter in all plants. For example, a pathogen-inducible promoter
may be isolated from barley which is slrongly expressed after inoculation with powdery
mildew Eresify graminis (Mouradov et al., 1993). The genomic clone of this gene was
15 isolated and characterised (Mouradov et al., 1994). This promoter region may be fused
to cassettes including a lethal or other functional gene.
Three eucalypt genes designated EGM1, 2 & 3 with homology to other plant
MADS-box genes have been cloned and sequenced. Phylogenetic analysis has been
performed to examine the relatedness of the eucalypt genes to other MADS-box
20 genes. This revealed that EGM2 is likely to be homologous to the GLOBOSA MADS-
box gene group which are involved in develop" ,ent of petals and slai"ens, EGM3 and
EGM1 both fall in the AGL2 group of MADS-box genes. These genes are most
commonly expressed in the inner three whorls of the flower (petals, stamens and
carpels).
The pattem of expression of all three EGM genes has been chara~;terised.
Expression of these genes was not detected in any vegetative tissue. The EGM1
gene is expressed in the petals, stamens and the carpel of eucalypt flowers. EGM3
is expressed in the floral meristem and in sepals. EGM2 is expressed in petals and
stamens. The EGM3 and EGM2 genes have been shown by Southern analysis to be
30 single genes. EGM1 is also likely to be a single gene.
The promoter regions of all three of these genes may be used in sterility gene
~M~ n,-n S~
, i,
CA 022~94~6 1998-12-30 '
C C i i
constructs and may be isolated from a eucalypt genomic library and engineered into
lethal gene constructs.
The eukaryotica:' ~~' ~ne, modulator gene and further gene and their
respective promoters may comprise a transformation cassette with which the target
5 organism may be transformed by any known means. The promoters of the
eukaryotically active gene, modulator gene and further gene or genes are selected in
combination such that the specificity of expression of the eukaryotically active gene is
enhanced. For example, the eukaryotically active gene may be promoted by a
promoter which is inducible or developmental whereas the modulator gene may be
10 constitutive. Specificity and the usefulness of even weak promoters may then be
enhanced by promoting the further gene or genes with a promoter with the same
specificity as that used to promote the eukaryotically active gene.
Alternatively, a tissue specific promoter for the eukaryotically active gene maybe inhibited by a constitutively-promoted inhibitor gene productj which in turn is
15 controlled by an inducibly-promoted further gene product.
In a yet further alternative, the eukaryotically active gene product may be
constitutively expressed subject to the modulating effect of a modulator gene
expressing under the control of a tissue specific promoter, the modulator gene or
product being itself controlled by the expression of a further gene under the control of
20 a promoter specific for a different or complementary tissue. By this means for
example, a constitutively-promoted gene conferring insect resistance may be localized
in its effect to exclude the seeds by expressing a gene inhibitor under the control of a
seed specific promoter, the gene inhibitor itself being controlled by a further gene
product expressed under the control of a promoter specific for non-seed tissues.In a further aspect, this invention resides broadly in a method of regulating
expression of a eukaryotically active gene, comprising transforming a cell with a
transformation cassette comprising said eukaryotically active gene, a modulator gene
expressing a product regulating the eukaryotically active gene or its product, and a
further gene expressing a product regulating said modulator gene or its product, the
30 promoters of two of said gene, modulator gene and further genes being selected from
inducible
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CA 022~94~6 1998-12-30 _ ' .
promoters and developmental promoters for the same or complementary tissues.
In a further aspect this invention resides broadly in a transformation cassette
comprising a eukaryotically acti~ modulator gene expressing a product
regulating the eukaryotically active gene or its product, and a further gene expressing
5 a product regulating said modulator gene or its product, the promoters of two of said
gene, modulator gene and further genes being selected from inducible pro"~oter~ and
developmental promoters for the same or complementary tissues.
In a yet further aspect this invention resides broadly in a method of
transformation of a propagatable eukaryotic cell with an expression cassette including:
a lethal gene expressing a cytotoxic peptide in a target tissue under the control
of a promoter substantially specific to said target tissue;
an modulator gene constitutively expressing an inhibitor of the production or
action of said cytotoxic peptide, and
a further gene under the control of said substalltially specific promoter and
15 functioning to block said inhibitor or modulator gene.
In a further aspect, this invention resides broadly in an expression cassette
including:
a gene expressing a peptide cytotoxic to a target tissue under the control of aninducible promoter substantially specific to said target tissue;
an inhibitor gene expressing an inhibitor of the production or action of said
cytotoxic peptide, and
a l epressor gene under the control of said inducible promoter and functioning
to block said inhibitor or inhibitor gene.
In a further aspect, this invention relates to primers for PCR isolation of the
25 Barnase and Barstar genes from an extract of B. amyloliquefaciens. designated P1
and P2 (for Barstar) and P3 and P4 (for Barnase) and comprising:
P1 5' GCG MT TCC GCA CAT GM AAA AGC 3'
EcoRI
P2 5' GCA AGC TTA AGA MG TAT GAT GGT G3'
Hindlll
n SHEET
~r .~.~i~U
CA 022~94~6 1998-12-30
~Cr/AU 9 7 / O 0 0 8 9
R~CEIY~ 1997
P3 5' GCC CAT GGC ACA GGT TAT CM CAC G3'
Ncol ~
P4 5' CGG GTA CCT TAT CTG ATT TTT GTA A3'
Kpnl
The cytotoxic effect of the lethal gene expression product may be increased by
coupling a localisation signal sequence to the construct. For example, the cytotoxic
effect of the preferred RNase may be increased by targeting into the nucleus where
most of the pre-RNAs and mature RNAs exist in non-protected form. For this purpose
10 the coding region of, for example, the Barnase gene may be modified by fusion of a
nuclear localisation signal (NLS) sequence to the RNase gene.
Accordingly in a further aspect this invention provides a set of PCR primers forfusing the NLS sequence from VirD2 gene of A. tumefaciens to the Barstar gene and
comprising:
15 P3 5' GCC CAT GGC ACA GGT TAT CM CAC G3'
Ncol
BD1 5' CCT TAC AAAMT CAG A GT CCT TTC AAA GCG TCC G3'
3' end of Bamase 5' end of NLS of ~IrD2
BD2 5' CGG ACG CTT TGA MG GAC TCT GAT TTT TGT AAA G3'
5' end of NLS of VirD2 3' end of Bamase
D23 5'CGGGTACCTATCTCCTATTTCCCCCAGG3'
Kpnl
The tissue-specific or developmental promoter may be selected from those
tissue specific promoters that are controlling genes in plant reproductive organs during
25 the early stages of flower development. These genes are maintained in the repr~ssed
state in all plant organs during the non-flowering period and are induced substantially
in reproductive organs during the flowering period.
The develG~,,nental promoter can be isolated by any known means. For
example,
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R~- C~ O ~ 'J ~'~0''1 1997
mRNA only present during the development of the target tissue may be identified and
isolated, cDNA from these specific mRNAs prepared and used to probe the genomic~library, followed by identifying the portion of the plant gen~ '~ J~ntains promoter
region of these genes. The tissue-specific promoters may be homologous (native) or
5 heterologous (foreign) in origin. For plant reproductive organ tissues, the promoters
may be selected from those that are expressed in cells of different floral tissues in both
male and female organs, or only in specific tissues such as sepals, petals, ovary, style,
stigma, ovule, corola and others. For the generation of the sterile plants, promoters
which express in tissues at early stages of both male and female organ development
10 are preferable.
For example, the gene family encoding ho"leolic MADS-box genes which play
an important role in flower development express at the early stages of the developing
floral primordia. We have isolated MADS genes from P. radiata which displays strong
homology to the Arabidopsis homeotic MADS-box genes known as the AGAMOU~
15 AGL-2 and AGL-4 genes. These genes express in young floral primordia and are not
expressed in the inflorescence meristem.
The gene encoding an inhibitor of the lethal gene product may comprise a gene
repressing expression of the lethal gene, a gene transcribing an antisense RNA, or
may comprise a gene expressing a protein C;3p~t''Q of deactivating the lethal protein.
20 The inhibitor gene will generally be under the expression control of a constitutive
promoter which expresses the inhibitor in non target tissues, although it is envisaged
that a packaged operator-repressor system may be used.
For exa"~p'e, a Barnase gene/reproductive organ-tissue specific promoter
system may be assori~ted with an anti-Barnase (B* or Barstar) gene/promoter system.
In the alternative, negative regulation in expression of bacteriophage lambda
N gene and SOS system in E. coli could be used in ex~., ession cassettes. Expression
of the bacteriophage lambda N gene is tightly regulated via the P' promoter and
conl,ols the level of expression from this proi"oter. This system may be used toprovide the requisite negative control of the expression of an inhibitor gene in the
30 targettissue.
The SOS system is regulated by the interplay of two proteins. A transcriptional
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CA 0 2 2 ~ 9 4 ~ 6 19 9 8 - 12 - 3 0 ~,~ . . ~
RECEIIJED 2 ~ NOV 1997
g
repressor (LexA) inhibits the initiation of transcription through specific binding to its
operator targets. A RecA protein blocks LexA p-otein and thus prevents inhibition at
its specific site. This would be a one component lethal gene sys~ not
require a toxin blocking gene.
The repressor gene under the control of the inducible promoter may be selected
to repress transcription of the inhibitor gene, translation of the RNA transcript or action
of a peptide product thereof. Preferably, the repressor gene codes for a peptideproduct which directly represses expression of the inhibitor gene. Accordingly, it is
preferred that the repressor gene and inhibitor gene be adapted to form a specific
10 repressor/operator couple.
For example, the E. coli laclq gene under control of the same tissue specific
promoter as the lethal gene construct may be used to repress the action of the
repressor gene promoter modified by insertion of a lac operator sequence.
The repressor gene promoter may be a modified 35S-RNA-CaMV promoter
15 having at least one lac operator between the promoter and coding region of the
repressor. In the selection of the modified 35S-RNA-CaMV promoter exemplified
hereinafter, the present inventors have developed primers for amplification of the
modified 35S-RNA-CaMV sequences.
Accordingly, in a further aspect this invention provides primers for PCR
20 amplification of modified 35S-RNA-CaMVsequences, designated 35S1 and 35S2 and comprising:
35S1 5' GCC TCG AGC ATG GTG GAG CAC GAC AC3'
Xhol
35S2 5' GCG TCG ACT CTC CM ATG AAA TGA AC3'
Sall
For the introduction of lac operators into both the 3' and 5' region of the
modified 35S-RNA-CaMV promoter, the sequence may be amplified by PCR primers:
OP1 5' GCC TCG AGC TTT GTG AGC GGA TM C3'
Xhol
ND~ S,-~EET
CA 022~94~6 1998-12-30
-' ~7/00~ 9
~ J 1 ~ _
binding protein (repressor, laclq) and the target promoter sequence (operator) is
understood in great detail. Expression of the lac operon is under the strong and ti~h~
negative control from the laclq repressor. Purified repressor is a tetramer, formed ~rom
four identical subunits, each with 360 amino acids (MW 38,350 kDa). The operator
5 sequence constitutes 35 base pairs, including 28 base pairs of symmetrical sequence.
For the maximum repression of the expression of the inhibitor gene, the
repressor protein is preferably targeted into the nucleus of the target cells. This may
be achieved by fusion of the nuclear localisation signal sequence to the C-end of the
repressor.
In a further aspect this invention relates to a set of primers suitable for isolation
of the laclq gene and having suitable restriction enzyme cleavage sites for cloning into
the plant vectors, the two primers corresponding to the 5' and 3' ends of the gene and
designated AM 1 and AM 2:
AM 1 5'GCT CTA GAC CAT GGA ACC AGT MC GTT ATA 3'
Xba I
AM 2 5'GCG GTA CCT CAC TGC CCG CTT TC3'
Kpnl
In a yet further aspect of the present invention, there is provided a set of PCR
20 primers for use in fusing the NLS sequence to the 3' end of the laclq gene and
Cor"prisin9:
AM 1 5'GCT CTA GAC CAT GGA ACC AGT MC GTT ATA 3'
Xba I
,,..~,~..,
CA 022~94~6 1998-12-30
r - C; ' ~ ~ 2 'I It ~ 337
1 1
LD 1 5' CTG GM AGC GGG CAG GTC CTT TCA MG CGT CCG 3'
3' end of laclq 5' end of NLS in VirD2 gene
LD2 5' CGG ACG CTT TGA MG GAC CTG CCC GCT TTC CAG 3'
5' end of NLS in VirD2 3' end of laclq
D 23 5' CGG GTA CCT ATC TCC TAT TTC CCC CAG G3'
Kpnl
If desired, the method of the present invention may be a reversible method. For
example, the gene cascade may use molecular elements which will allow the reversal
of the engineered sterility to fertility thus permitting seed production. The reversal
10 mechanism may consists of placing an anti-repressor gene under the control of a
chemically switchable promoter. When, for example, restoration of fertility is required
plants may be sprayed or otherwise administered a chemical which induces expression
of anti-repressor RNA which will block inhibition of expressiGn of the modified inhibitor
gene. Accumulation of the inhibitor product in floral organs will inhibit lethal gene
15 function, thus allowing normal flower formation.
For example, for the lac/laclq system described, a chemical may be sprayed on
the plant which induces expression of anti-laclq RNA which will block inhibition of
expression of a modified Barstar gene. Accumulation of the Barstar product in floral
organs will inhibit lethal gene function, thus allowing normal flower formation. The
20 promoter region of the maize glula~l ,.Gne-S-transferase (GST ll) gene could be used
for chemically-induced expression. Expression of the GST ll promoter can be
switched on by chemicals such as dichlG~a",in (N,N-diallyl-2,2-dichloracetamide), or
fluorazole (benzyl-2-chloro-4-trifluoro",eth~1-5-thi~sl~ carboxylate).
In the case of Eucalyptus. the EGM3 promoter, comprising the upstream
25 sequences of the EGM3 gene, may be used to express the lethal gene specifically in
eucalypt floral tissue. Using this promoter, the cytotoxic Barnase gene may be
specifically expressed in the floral meristem at a very early stage of development. The
inhibitor Barstar may be expressed under the control of 35S-RNA-CaMV promoter
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~'AlJ
CA 022~94~6 1998 -12 - 30 ~ /A~r ~ 9
.
12
carrying the lac-operator sequences.
The E. coli laclq gene under the control of the EGM3 promoter could be used
as repressor gene to prevent ex~.r~ssion of Barstar in the floral meristem tissue during
flowering initiation. During flowering, the EGM3 promoter would express Barnase and
5 laclq in the floral meristem while the modified 35S-RNA-CaMV promoterwould express
Barstar in all tissue except the floral meristem, giving protection from promoter
leakage. During non-flowering period, the 35S-RNA-CaMV promoter would express
Barstar generally, giving protection from leakage.
Nematodes cause annual losses well in excess of US$100 billion to world
10 agriculture. The most important pathogens by far are the nematodes Meloidogyne
incognita and Meloidogyne javanica which attack a wide range of plants. Control
measures include use of hazardous chemicals, cultural practices such as rotation and
the use of resistant varieties in a limited range of crops. The advantages of
engineering resistance to nematodes include no requirement for specific cultural15 practices and reduced environmental risks.
The lethal gene/inhibitorlrepressorapproach can be used to eng.neer resistance
to nematodes. The ideal site to activate a plant defence against nematodes is in the
feeding cells in the vascular region of the plant root. In several plant-nematode
interactions, gene promoters are being identified by differential screening which could
20 be used to express the Barnase gene specifically at the site of nematode feeding.
Recently two genes (Lemmi9 and LemmilO) have been identified that are expressed
at high levels in giant cell feeding sites in tomato roots infected with Meloidogyne
- incognita. (Van der Eycken et al., (1996) Plant Journal 9, 45-54).
Expression of a lethal gene in feeding cells would rapidly kill the cell and disrupt
25 the reeding process which is very critical particularly for the development of the
females. Expression of the inhibitor gene in non-target tissue would be required to
prevent damage to other parts of the root due to promoter leakage. This is particularly
important since the pro,noter which shows high level expression in the giant feeding
cell is also expressed at lower levels in non target tissue. Expression of the repressor
30 gene under the feeding site promoter should block the expression of the inhibitor gene
sufficiently
~r~A~Au
CA 022~94~6 1998-12-30 ' ~
R'C~ o 2 0 jl~Oy 'j9
13
in the target tissue to allow the lethal gene to destroy the cell.
in order that this invention may be more readily understood and put into
practical effect, reference will now be made to the following Examples and the
accompanying drawings which illustrate preferred embodiments of the invention and
5 wherein:
FIG. 1 represents the construction of the Barnase/NLS gene localised lethal
gene system;
FIG. 2 represents constructs including lac operon- modified 35S-RNA-CaMV
operator/Barstar inhibitor gene system;
FIG. 3 represents the construction of laclq/NLS localised repressor gene
system;
FIG. 4 represents the mode of action of BarnaselBarstar:lacJlaclq casc~de in
accordance with the present invention;
FIG. S represents an alternative casc~de Barnase:op/LexA/RecA in accordance
with the present invention;
FIG. 6 represents the mode of action of a reversible
Barnase/Barstar:lac/laclq/antisense-laclq cascade in accordance with the
present invention;
FIG. 7 is a diagrammatic illustration of the control cassette and modes of action
of the lethal gene/NLS system of FIG. 1;
FIG. 8 is expression of PrMADS1, PrMADS2, PrMADS3, PrFL1 and PrCON1
genes in reproductive and vegetali~e tissues of P. radiata.
FIG. 9 is expression of PrMADS3 gene in male and female cones.
Fig. 10 is the gel mobility shift assay of PrMADS1, PrMADS2, and PrMADS3.
FIG 11 is the nucleotiJe sequence of the EGM1 promoter;
FIG. 12 is the nucleotide sequence of the EGM2 promoter;
FIG. 13 is the nucleotide sequence of the promoter of the eucalypt floral-
specific MADS gene EGM3;
Fig. 14 is a graphical representalion of NGIlllerll blot of total RNA extracted
from Eucalypt tissue probed with EGM1, EGM2 and EGM3 cDNA;
Fig. 15 is a graphical representation of No, ll ,er" blots of total RNA extracted
from Eucalypt tissue probed with EGM2 cDNA (6 hour exposure) and EGM3
cDNA;
.
CA 0 2 2 ~ 9 4 ~ 6 19 9 8 - 12 - 3 0 ~ CI'!.~U 9 / (~
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14
Fig. 16 is an alignment of the predicted protein sequence of the MADS-box
region of s~veral plant MADS-box genes;
FIG.17 is a phylogenetic tree showing relatedness of several plant MADS-box
genes;
FIG.18 is a Southern blot of Eucalyptus grandis DNA digested with EcoRI and
HinDIII and then probed with the EGM3 and EGM2 genes;
FIG. 19 is a Northern blot of total RNA extracted from eucalypt tissue probed
with the EGM3 cDNA (6 hour exposure).;
FIG. 20 is a diagrammatic illustration of plasmid 7 prom Barstar;
FIG. 21 is a diagrammatic illustration of plasmid pBR Barnase prom Barstar
containing V1-promoters;
FIG. 22 is a diagrammatic illustration of a plasmid containing V2-promoters;
FIG. 23 is a diagrammatic representation of an agarose separation of a Pst1
digest of an EGM3 double bin. Given that the EcoRI cloning site is at base
6772 in EGM3 bin the predicted fragment sizes from the map for this digestion
are4.9 4.8 4.4 3.3 1.9 1.1 and0.6kb.
FIG. 24 is a diagrammatic represenlalion of two gel exposures of a Pst1 digest
of an EGM3 bin showing the other possible orientations of the cloned insert.
The sizes of fray")ents expected from this digest are 7.4 4.9 4.4 1.1 0.7 and
0.6 kb.
FIG. 25 is a plasmid diagram of EGM3 double bin. The fragment containing
EGM3 pfo,noter driving laclqNls and the fagment containing 35S pomoterOP
driving Barstar can be cloned in either orientalion.
FIG 26 is a plasmid diagram of EGM3 V1 sense.
FIG 27 is a plas",id diagram of plasmid V1 (Barnase-Barstar).
FIG. 28 is a plasmid V2 (laclqNLS-35s promoterOp-Barstar).
FIG. 29 is a representation of a pro"~oter finder strategy;
FIG. 30 is the nucleotide sequence of PrMADS2 promoter
FIG. 31 is the nucleotide sequence of PrMADS3 cDNA;
FIG. 32 is the amino acid sequence of PrMADS3 protein;
FIG. 33 is the nucleotide sequence of PrMADS promoter;
CA 022~94~6 1998-12-30
.~?EC~ F1~ 2 ~3 ?!r~ 97
FIG. 34 is the nuclPot,de sequence of PrFL1 cDNA clone;
;5 IS the amino acid sequence of PrFL1 protein;
FIG. 36 is the nucleotide sequence of PrFL1 promoter; and
FIG. 37 is the nucleotide sequence of PrCon1 gene (partial).
5 EXAMPLE 1
A chimaeric reproductive organ-specific expression cassette was constructed
including a reproductive organ-specific promoter and Barnase gene from B
amvloliauefaciens. under the positive control of a gene encoding an inhibitor ofBarnase, Barstar (B*), from B. amyloliquefaciens under control of the modified
10 constitutive promoter (35S RNA CaMV) with introduced lac-operator sequence, and
under the negative control of the E. coli laclq gene under control of the same
reproductive organ-specific promoter as used to express the lethal gene as per FIG.
4.
The reproductive organ specific promoter comprises the upstream sequences
15 of genes expressed in reproductive organs at the early stages of flower development
as described hereinafter. The cytotoxic effect of the Barnase was increased by
targeting it into the nucleus where most of the pre-RNAs and mature RNAs exist in
non-protecled form. For this purpose the coding region of the B gene was modified
by fusion of the nuclear localisation signal (NLS) sequence from VirD2 gene of A.
20 tumefaciens strain C58 to the 3'-region of the B gene, as illusl,ated in FIG. 1.
The inhibitor Barstar (B*) gene portion vectors carrying lac-operator sequences
in 5'-, 3'- and 5'- plus 3'- regions of the 35S-RNA-CaMV promoter region were
constructed as per FIG.2.
The E.coli laclq gene under control of the same reproductive organ-specific
25 promoter as used to express the lethal gene has its laclq protein targeted into the
nucleus by fusion of the NLS from VirD2 gene of A. tumefaciens to the C-end of the
laclq
iJ
CA 022~94~6 1998-12-30 ,~,,,! ~ r ? / ~ O C ~ ~
. .; . , . , , ,: - ,
16
protein as per FIG. 3.
EXAMPLE 2
An ail~ .,rnaeric reproductive organ-specific expression cassette was
constructed as per FIG. 5, wherein the reproductive organ-specific promoter and
5 Barnase gene is under the positive transcriptional control of the LexA product under
control of 35S-RNA-CaMV promoter, and under the negative control of the RecA gene
product under control of the same reproductive organ-specific promoter as used to
express the lethal gene, as per FIG. 5.
E~(AMPLE 3
A reversible system was constructed substantially in accordance with Example
1. In addition a chemically switchable promoter from the maize glutathione-S-
transferase (GST ll) gene regulating an antisense laclq RNA gene was included. This
allowed seed production as per FIG. 6. Restoralion of fertility may be thus achieved
by spraying with an inducer such as dichloramin (N,N-diallyl-2,2-dichloracetamide) or
15 fluorazole(benzyl-2-chloro-4-trifluoromethyl-5-thiazole-carboxylate~ This chemical
induced expression of anti-laclq RNA blocks inhibition of expression of modified 35S-
RNA-CaMV-B* gene. Accumulation of the B* in floral organs will inhibit lethal gene
function, thus allowing normal flower formation.
E)(AMPLE 4
Modification and in-~. l;on of the laclq repressor gene into plant
expression vectors.
The pRT99GUS vector was d;gested with Xbal and Kpnl to release the coding
region of the GUS gene and become a suitable vector for expression in plants.
The laclq, epressor is available on almost all con " "ercial E. coli strains. Some
25 commercial plasmids also contain the repressor gene. In order to express this gene
in plants, the prokaryotic translational initiation codon (GTG) was changed into ATG.
In orderto isolate the laclqgene two ~cri",ers co"esponding to the 5' and 3' ends of the
gene were designed (AM 1 and AM 2). Both primers have suitable restriction enzyme
cleavage sites for cloning into the plant vectors.~0 AM 1 5'GCT CTA GAC CAT GGA ACC AGT MC GTT ATA 3'
Xba I
~.~A~ E~ S~JEET
U
CA 022~94~6 1998-12-30 rcr/Au 9 7 / O 0 0 8 9
REC~I~IED 2 ~ N~Y 1937
17
AM 2 5'GCG GTA CCT CAC TGC CCG CTT TC3'
Kpn I
After amplificati~r ~ i5 ~~" fr~gment was digested with Xbal and Kpnl and
ligated into the pBR-35SGUS vector digested with same enzymes in order to replace
5 the GUS gene (pBR-35Slaclq).
In order to fuse the NLS sequence to the 3' end of the laclq gene several PCR
were performed.
Primers:
AM 1 as mentioned above.
10 LD 1 5' CTG GM AGC GGG CAG GTC CTT TCA MG CGT CCG 3'
3' end of laclq 5' end of NLS in VirD2 gene
LD2 5' CGG ACG CTT TGA MG GAC CTG CCC GCT TTC CAG 3'
5'endofNLSinVirD2 3'endoflaclq
D 23 5' CGG GTA CCT ATC TCC TAT TTC CCC CAG G3'
Kpnl ~
Final PCR fragment containing NLS from VirD2 gene from A. tumefaciens fused
to the coding region of the laclq gene was digested with Xba I and Kpnl and ligated
into the plant expression vector pBR322-35SGUS digested with the same enzymes
(pBR-35Slaclq-NLS). This vector was ~,ansfor"~ed from the XL1 Blue into the JC
20 8697(recA-) E. coli strain in order to eliminate possible homologous recombination.
EXAMPLE 5
Barnase and Bar~tar genes.
These genes were issl~ted by PCR from the crude extract of the
B. amyloliquefaciens using P1 and P2 (for Barstar) and P3 and P4 (for Barnase)
25 primers.
P1 5' GCG MT TCC GCA CAT GM AAA AGC 3'
EcoRI
~9 ~ J~
) r ~
CA 0 2 2 5 9 4 5 6 19 9 8 - 12 - 3 0 ~ J ~
. .
18
P2 5' GCA AGC TTA AGA MG TAT GAT GGT G3'
Hindlll
P3 5' GCC CAT GGC AC ''~ ~~ i CAACAC G3'
Ncoi
5 P4 5' CGG GTA CCT TAT CTG ATT TTT GTA A3'
Kpnl
The P1-P2 PCR fragment (Barstar) was digested with EcoRI and Hindlll and
ligated into the Bluescript SK vector digested with same enzymes (BS-B*). The
BamHI-Hindlll fragment of the BS-B~ vectorwas introduced into the protein expression
10 vector pQE32 (pQE-B~).
The P3-P4 PCR fragment (Barnase) was ligated into the PCR cloning vector
p/GEM-T (pGEM-T-B). In order to fuse NLS sequence to the 3' end of the B gene
several PCR were performed.
Primers:
15 P3 5' GCC CAT GGC ACA GGT TAT CM CAC G3'
Ncol
BD1 5'CCTTACAAAMTCAGAGTCCTTTCAAAGCGTCCG3'
3' end of Barnase 5' end of NLS of VirD2
BD2 5' CGG ACG CTT TGA MG GAC TCT GAT TTT TGT AAA G3'
5' end of NLS of VirD2 3' end of Barnase
D 23 5' CGG GTA CCT ATC TCC TAT TTC CCC CAG G3'
Kpnl
Final PCR r,~g,nentcontaining NLSfrom~lrD2genefromA. tumefaciensfused
to the coding region of the Barnase gene was ligated into the PCR cloning vector
25 pGEM-T (pGEM-T-B-NLS).
EXAMPLE 6
Introduction of lac-o~)eratorsequence into the 35S-RNA-CaMV promoter.
5~1? ~
,SJ
CA 022~94~6 1998-12-30 PCr/AU 9 7 / O O 0 8 9
R~--i Y c3 2 ~ . i33?
19
The commercial plasmid pQE32 contains the ideal combination of two lac-
operators (operators I and ll). As a first step the Hindlll fragment from,the pRT99GUS
vector was introduced into the Bluescript SK ~~'~ he EcoRI-Sall fragment
of the A 1 vector carrying the GUS gene fused into the 35S-RNA-CaMV promoter and5 terminator regions was introduced into the pQE32 vector digested with the same enzymes.
The resulting vector contains 35S-RNA-CaMV promoter with two lac operons
in the upstream region (Op+ 35S-GUS).
In order to introduce the lac operator between the 35S-RNA-CaMV promoter
10 and coding region of the GUS gene initially a BamHI-Sall fragment of the A1 vector
carrying promoterless GUS gene was ligated into the pUC19 vector digested with
same enzymes (pUC19-promoterGUS). In the next step the pUC19-promoterGUS
vector was digested with EcoRI and Sa/l enzymes and ligated into the pQE32 vector
digested with the same enzymes (pQE-promoterGUS). The 35S-RNA-CaMV promoter
15 region was amplified by PCR using the 35S1 and 35S2 primers, corresponding to the
5' and 3'-region of the 35S-RNA-CaMV promoter.
35S1 5' GCC TCG AGC ATG GTG GAG CAC GAC AC3'
Xhol
35S2 5' GCG TCG ACT CTC CM ATG AAA TGA AC3'
Sall
The PCR fragment was digested with Xhol and Satl and introduced in to the
pQE-promoterGUS digested with Xhol. Clones containing the promoter in the correct
orientation were s~l~cted and resulting plasmid (35S- GUS) was transformed from the
XL1 Blue into the JC 8697 (recA-) E. coli strains in order to eliminate possible25 homologous recombination.
In order to introduce lac-operators into both the 5'- and 3'-region of the 35S-
RNA-CaMV promoter the operator-35S promoter region of the -35S-GUS vector was
amplified by PCR using the OP1 and 35S2 primers, corresponding to the 5'- and 3'-
region of the -35S promoter.
OP1 5' GCC TCG AGC TTT GTG AGC GGA TM C3'
Xhol
c~ S.~
CA 022~94~6 1998-12-30
The PCR fragment was digested with Xhol and Sall and introduced into the
pQE-promoterGUS digested with Xhol. Clones containing the promoter in the cerrect
orientation were selected and resulting plasmid ( -35S-pro" ,~ ~ "~as transformed
from the XL1 Blue into the JC 8697 (recA-) E. coli strains in order to eliminate possible
5 homologous recombination.
Barstar gene was cloned into the pQE-promoterGUS vector after digestion of
both with EcoRl+Kpnl.
One difficulty with the tissue ablation approach is the effect of promoter leakage
or expression in other tissues. To overcome this we have developed a control system
10 (gene cascade) whereby expression of a lethal gene(s) is countered in non-target
tissues (FIG. 7). Two different vectors carrying Barnase (V1) and Barstar plus
repressor (laclq) (V2) parts of these cascade were designed.
In order to clone promoter regions into the vectors V1 and V2 they have been
amplified using sets of primers with Sall restriction sites on the ends for V1 (Sa/l
15 primers) and EcoRI restriction site for V2 (EcoRI primers). In order to check the
orientation of the promoters in V1 and V2 vectors an EcoRI restriction site was
designed in the forward Sall primer downstream from Sall site and a Sall site was
designed in the EcoRI primer downstream from EcoRI:
Sall primers:
20 PrMADS2 promoter
Forward primer:
5' CCGGTCGACGMTTCCGACAGTGGAGTCCACAAAGAAAGATGCG3'
san EcoRI
Reverse primer: 5' CCGGTCGACTTCTTTCCTTCTTTTCTTTCTGC 3'
Sall
PrMADS3 womoter:
J~n ~
: ~ . i J
CA 022~94~6 1998-12-30
WO 97/30162 PCI/AU97/00089
Forward primer: 5' ACGCGTCGACGMTTCAAGA I I I CAAATCAGTCC 3'
Sall EcoRI
Reverse primer: 5' ACGCGTCGACCMGATCCCTCTGCTTCTTCACC 3'
Sall
5 PrFL1 Promoter:
Forward
primer: 5' ACGCGTCGACGMTTCGMCTTCTGGMTMGCTGC 3'
Sall EcoRI
Reverse primer: 5' ACGCGTCGACTTCATCTTACGTCACGCGAGG 3'
1 0 Sall
EcoRI primers:
PrMADS2 promoter:
Forward primer:
5' CCGGMTTCGTCGACCGACAGTGGAGTCCACAAAGAAAGATGCG3'
EcoRI Sa/l
Reverse primer: 5' CCGGMTTCTTC I I I CCTTC I I I I C I I I CTGC 3'
EcoRI
PrMADS3 promoter:
Forward primer: 5' CGGMTTCGTCGACMGAl I I CAAATCAGTCC 3'
EcoRI Sa/l
Reverse primer: 5' GCGMTTCCMGATCCCTCTGCTTCTTCACC 3'
EcoRI
PrFL1 promoter:
Forward primer:
25 5' CGGMTTCGTCGACGMCTTCTGGMTMGCTGC 3'
EcoRI Sall
SU~a 1 l l UTE SHEET (RULE 26)
CA 022~94~6 1998-12-30
U 3 7 / O O O ~ 9
RE~EIYED 2 G i''JaY ~997
22
Reverse primer: 5' GCGMTTCTTCATCTTACGTCACGCGAGG 3'
EcoRI
PCR fragments of Sall primers were digested with Sall restriction enzyme and
introduced into the V1 vector digested with Sall. The orientation of promoters was
5 checked using digestion with EcoRI enzyme.
PCR fragments of EcoRI primers were digested with EcoRI restriction enzyme
and introduced into the V1 vector digested with EcoRI. The orientation of promoters
was checked using digestion with Sall enzyme. Maps of the resulting plasmids areshown in FIGS. 20 and 21.
These vectors were linearised with Hindlll enzyme and introduced into the
Bin19 binary vector digested with Hindlll. Colonies carrying modified Bin 19 vectors
were selected on plates with kanamycin and ampicillin antibiotics. As a next step
Bin19-V1-promoter and Bin19 V2-promoter vectors were transformed into several
agrobacterium strains. Arabidopsis thaliana. Eucalyptus grandis and Pinus radiata
15 embryos and explants were co-transformed with Agrobacterivm strains.
The Hindlll fragment from the plasmid pRT99GUS (Topfer et a/. Nucleic Acids
Research (1988) 16 (17): 8725) was cloned into the Hindlll site of pBR 322. Thisinsertion resulted in two plasmids corresponding to the insert being cloned in both
orientations. The inserted region contains the Cauliflower Mosaic Virus (CaMV) 35S
20 RNA promoter, beta glucuronidase (GUS) gene, and CaMV 35S terminator region.
These plasmids were called pBrGUS1 and pBrGUS2 and provided the basis for the
lethal gene constructs. The orientation of the insertion event was checked with a
BamHI digestion: pBRGUS 2 results in BamHI fragments of 2.5kb and 4.4kb,
pBRGUS 1 in fragments of 6.1kb and 0.8kb.
The DNA encoding the laclq nuclear localisation signal peptide was amplified
from the plasmid pGEM laclqNLS by PCR using the 5' primer lacl (this has an EcoR1
site) and the 3' primer D23 which has a Kpnl site. The amplified DNA fragment was
restricted with EcoRI and Kpnl and cloned into pBRGUS 2 cut with the same enzymes.
The resulting plasmid was called pBRLac. The pBRLac plasmid was then cut with
30 Sphl and run on an agarose gel to allow the purification of the plasmid fragment
containing
~MEN
~JC'''~
CA 022~94~6 1998-12-30
. . , / I ~ '~ J
- ' ~ 7
23
the laclq gene, ampicillin resistance gene, and origin of, ~plication away from the small
Sphl fragment, which contained restriction sites that we wished to remove. The
resulting plasmid was called pBRLac BH-.
The second stage in the plasmid construction was the preparation for cloning
5 of the Barstar gene under the regulation of the modified CaMV plus lac operator
promoter. This was amplified from the plasmid p35S-op-Barstar EcoRI- which
contained the promoter/gene sequence which had been modified to remove the EcoRIsite between the promoter and coding sequence. This modification was accomplished
by cutting the 35S-op-Barstar plasmid with EcoRI, carrying out a blunting reaction with
10 T4 DNA polymerase, and then religating the blunted plasmid. The PCR was carried
out using the 5' primer pQE-F and the 3' primer Bar-3'. The PCR fragment was cutwith Xhol and Kpnl. This was cloned into the 35S-op-Barstar EcoRI- plasmid also cut
with Xhol and Kpnl, and from which the unwanted gene had been purified away by gel
electrophoresis. This enabled the removal of res,triction sites at the three prime end
15 of the Barstar coding region. This plasmid was called p35S-op-Barstar EcoRI-2.
The Barstar gene was then cut out of the p35S-op-Barstar EcoRI-2 plasmid with
Xhol and Sall and cloned into pBRLac BH- cut with Sall. The Barstar gene could go
into the Sall site in either orientation but only one orientation was found. Theorientation of the insert was ascertained by a Kpnl digest. Only a 1 kb band was seen
20 which was indicative of the orientation of the insert seen in plasmid pBRLac Op
Barstar 1. The resulting plasmid was called V2.
The plasmid was then ready for the cloning of, in this case, a flower specific
promoter, into the unique EcoRI cloning site 5 prime of the laclq gene. The EGM3promoter from the Eucalyptus MADS gene EGM3 was used forthis purpose. This was
25 cut from plasmid pEGM3 with EcoRI. This promoter was cloned in both orien~aLions,
and the resulting plasmids were called V2 EGM3 sense and V2 EGM3 antisense. The
plasmid V2 EGM3 sense was used as a source of the EGM3 regulated laclq and
CaMVop Barstar genes for the plant transformation vector. The orientation of theEGM3 promoterwas determined by an Xbal PSt1 digest. The presence of 2.3 and 0.9
30 kb bands was indicative of the correct orientation.
'4~Ei~ -D 5'1
CA 0 2 2 ~ 9 4 ~ 6 19 9 8 - 12 - 3 0 ' i ~J o ~3 9
R E u ;'~ 2 ~ ' n '1 1~97
24
The second construct V1, containing the promoterless Barnase gene, was
constructed using pBrGUS 2 as the starting point. A Bamase gene was amplified
from genomic Bacillus amyloliquefaciens using the primers Barnase 5 prime Sal, and
Barnase 3 prime Kpn. The resulting PCR product was restricted with Kpnl and San
5 as was pBrGus 1 and a ligation reaction performed. The plasmids from the resulting
colonies were used as templates for sequencing using the amplification primers.
Plasmid SB4 was found to contain a Barnase fragment of the same sequence as the
B. amyloliquefaciens Barnase gene and this called pBRBarnase and used for further
constructions.
Previous work using Barnase as a lethal gene in plants had shown that the anti-
toxin gene for Barstar was required to be present and expressed from the same
plasmid as the Barnase before a promoter region could be cloned 5 prime of the
Barnase gene (Paul et al. 1992 Plant Molecular Biology 19: 611-622). This was
achieved by using a plasmid that contained the Barstar gene and promoter from B.15 amyloliquefaciens in cis with the Barnase sequence. To this end the promoter plus
Barstar DNA region was amplified from the B. amyloliquefaciens genomic DNA usingprimers 5 prime Barstar promoter and Bar 3 prime. The PCR fragment was restricted
with Kpnl as was pGEM 3f and a ligation reaction performed. The resulting white
colonies were screened for inserts. The DNA from colony 7 was used for seqencing20 and found to contain the Barstar promoter plus Barstar sequence as in the genomic
B. amyloliquefaci~ns DNA. This plasmid was called 7 prom Barstar, as shown in
Figure 20.
The 7 prom Barstar plasmid was res(i~:ted with Kpnl as was pBR Bamase and
the promoter Barnase fragment was cloned into the Kpnl site of the PBR Barnase
25 DNA. This resulted in a plasmid known as V1. The EGM3 promoter was cut out ofpEGM3 using EcoRI, blunted using DNA polymerase I (Klenow fragment) and cloned
into the unique Sall cloning site of the V1 DNA which had also been restricted and
blunted. The resulting plasmid with the proi"oter inserted in the correct orientation
was called EGM3 Vl sense (FIG. 26).
The EM3 V1 sense DNA was linearised with Hindlll and cloned into the plant
~ ~ ~c~ _
CA 022~94~6 1998-12-30
R~Cr IVEO ~ a~7
transformation vector Bin 19 also linearised with Hindlll. This resulted in two plasmids,
EGM3 V1 Bin 1 and 2, corresponding to the two orientations of insertion. In orientation
2, the two EcoRI site present in EGM3 V1 Bin are approximately 80 bp appart. TheEGM3 V1 Bin 2 was cut with EcoRI and blunted using DNA polymerase I (Klenow
5 fragment). The EGM3 promoter laclq gene, and the CaMV 35S op Barstar genes
were cut out of EGM3 V2 sense using Aatll and Hindlll. This fragment was also
blunted and ligated into the EcoRI cut and blunted EGM3 V1 Bin 2 to create the
plasmids EGM3 Double Bin I and ll (FIG. 25), again corresponding to the two possible
orientations. These plasmids were used for transformation of plants via A
1 0 Tumefaciens.
The procedure was then repeated with further Eucalyptus MADS promoters.
These were EGM2 long and short, and a shorter version of EGM3.
Primers.
Bamase 5 prime Sal
15 5' CCGTCGACATGGCACAGGTTATCM 3'
Sall
Barnase 3 prime Kpn
5' CGGGTACCTTATCTGA ~ GTAAAGG 3'
Kpnl
20 Five prime Barstar pr~",oter
5' CGGGTACCGTCCAATCTGCAGCCGTCCGA 3'
Kpnl
Bar 3'
5' GCGGTACCTTAAGAAAGTATGATGGTG 3'
Kpnl
D23
5' CGGGTACCTATCTCCTATTTCCCCCACG 3'
Kpnl
~rr i ' l !
CA 022~94~6 1998-12-30 ~C. i.U 9 , ~ J J
26
pQE-F
TATCACGAGGCCCTTTC 3'
lacl
5' GCGMTTCMCATGGMCCAGTMCGTTATA 3'
EcoRI
EXAMPLE 7
Isolation of reproductive organ-specific promoters
Five cone-specific genes displaying strong homology to Arabidopsis thaliana and
Anthirrhinum majus floral meristem and organ identity genes were isolated from
10 P. radiata cDNA library prepared from immature female and male cones. Three of
them, PrMADS1, 2 and 3, belong to the family of MADS-box genes showing homology
to Arabido~sis AGL-2, AGL-4 and AGL-6 genes and dal1 gene from another non-
angiosperm, Picea abies (Norway spruce), respectively. The PrFL1 gene is the pine
ortholog of Arabidopsis Leafy (Ll~) and Floricaula (Flo ) gene from Anthirrhinum. The
15 PrCon1 shows strong homology to Arabidopsis CONSTANS (co) gene. To elucidate
the function of these genes we took the approach of characterising their expression
pattern in male and female cones during dirreren~ stages of cone development.
A significantly lower level of expression was detected in vegetative tissues:
vegetative buds, needles, stem and roots. In situ hybridisation showed that expression
20 of these genes is detectabl~ only in reproductive tissue cells.
Expression analysis revealed that all five genes show different pattems of
expression in different stages of develo,l~menl of male and female cones (FIGS. 8 and
9). PrMADS1, 2 and 3 genes are cone-specific: expression of both genes was
restricted to reproductive organ primordium tissues. No detectable expression of25 these genes was observed in vegetative tissues: vegeta~i~e buds, needles, stems,
roots. For PrFL1 and PrCon1 low dete~table expression was observed in vegetativebuds.
In reproductive organs low level of expression of both genes was detected at early
stages of cone development (5 mg cones) which was increased during cone
30 development (50 mg cones). In male cones expression of both genes was restricted
.S: " ;~
CA 022~94~6 1998-12-30 _j~_ ,, -.; 1 ~
RECE~VED 2 0 NO'~ ~997
27
to microsporangium containing primary sporogenous cells. In female cones expression
of both genes w_~"estricted to premature ovules.
To characterise MADS proteins as DNA-binding proteins in vitro, we expressed
both proteins in E. coli and characterised their DNA-binding properties. PrMADS1,2,3
5 proteins are sequence-specific DNA-binding proteins. Their DNA-binding consensus
sequence is similar to that of the AGAMOUS protein. All three proteins bind a DNA
sequence matching the consensus sequence of CArG box
TT(A/T)CC(AlT)(A/t)2(T/A)NNGG(-G)(A/T)2 (oligo A) for PrMADS1 and PrMADS2)
(FIG. 31). Mutation of these consensus sequences (oligo B) significantly decreases
10 their binding of PrMADS 1,2 or 3 proteins. Competition with non-radioactive oligos did
not decrease binding of any of the proteins to the CArG consensus. This indicates
that we are dealing with specific DNA-protein interactions.
Upstream sequences were isolated using a 'Promoter finder' strategy (FIG. 29).
A special adaptor was ligated to the ends of DNA r,agi"ents generated by digestion
15 of genomic DNA from P. radiata with EcoRV, Scal, Dral, Pvull and Sspl separately.
The enzymes used were selected because they have six-base recognition sites and
generate blunt ends. Following adaptor ligation, these DNA fragments were used as
a template for PCR using first adaptor primers AP1, AP2 and gene-specific primers
GSP-1 ,2.
The sequences of adaptor (first sequence), adaptor-primers and polymerase
blocking primer shown below. It is noted that adaptor primer AP1 corresponds to
bases 1 to 22 of 22 of the adaptor, adaptor primer AP2 corresponds to bases 13 to 3 1,
and the pol~",erase block corresponds to bases 41 to 48.
Adaptor-
25 5'GTMTACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGG-CTGGT-3'
Polymerase block:
AP13'-NH2-CCCGACCA-P04-5'
AP2
Adaptor primer 1, (AP1):5'-GTMTACGACTCACTATAGGGC-3'
N~-D ~,t.-~
CA 022~94~6 1998-12-30
WO 97/30162 PCTIAU97/00089
28
Adaptor Primer 2 (AP2): 5'-ACTATAGGGCACGCGTGGT-3'
The presence of the amine group on the 3' end of the lower strand blocks
poly",erase catalysed extension from free adaptom~l-lecules that have not been ligated
thus preventing the yeneralion of the primer binding site unless a defined gene specific
5 primer extends a DNA strand opposite the upper strand of the adaptor.
Primary PCR was performed using Avanlage nh Polymerase Mix (Clontech) and
adaptor primer AP1 and GSP-1 for PrMADS2 3 and PrFL1 genes.
GSP-1 sequences:
PrMADS2: 5' CGGCGCTTCCGMCTCATAGAG I I I I CCTC 3'
10 PrMADS3: 5'TTAGCGCCACTTCGGCATCGCACAGC 3'
PrFL1: 5' CMGGGACTTCAAATCC I I I CTCCCATTCATGG 3'.
PCR two step cycle parameters:
7 cycles: 94~C 25 sec
72~C 4 min
15 32 cycles: 94~C 25 sec
67~C 4 min
67~C for an additional 4 min after the final cycle.
The 1 ml of primary PCR was used in secondary PCR using the same cycling
parameters and a second set of GSP primers with AP2 adaptor-primer.
.
20 GSP-2 primers sequences:
SUBSTITUTE SHEET (RULE 26)
CA 022~94~6 1998-12-30
U 9 ~ / 0
29 ' ~
PrMADS2: 5' CGCCI I I I I CAGCAGACCATTCCGGC 3'
PRMADS3: 5' CAGC~ ;GTTTCCGGCGCTTCG 3'
~ . . - . .
PrFL ,. 5' CGTCCATGGTCCTTGTTAAAGACAGTTGTTGTTGG 3'
The 1-2 kb PCR fragments were cloned into the TA-type cloning vector and
5 sequenced. Sequences of cDNAs, proteins and promoter regions for PrMADS2,
PrMADS3 and PrFL1 are shown in FIGS 30, 33 & 36.
E)~AMPLE 8
Introduction of the chimaeric DNA sequences into the plants
Chimaeric DNA sequences were co-transferred into the plant tissue using the
10 A. tumefaciens system. Transformantswere retained by growing on selective medium.
Presence of two plasmids was proved using standard molecular biology techniques
(Southern, northern hybridizations, PCR). Plasmid A has Bamase gene and plasmid
B has Barstar gene under control of plant promoters. This creates an additional
selection for co-transformation.
The vector system designed involves a cascade of genes expressing under the
positive control of the reproductive organ-specific pro"~oters and negative control of
the E. coli lactose (lac) repressor-operdtor system. During the non-flowering period,
in the absence of the repressor protein, the operator sequence will permit constitutive
expression of the Barstar gene or antisense RNA in all organs. Because of possible
20 leakage of the reproductive organ-specific promoter, low level of the lethal gene
product will exist in dirferent plant organs and cells.
Accumulation of the Barstar protein in cytoplas,n or antisense RNA in nuclei of
these cells will block the cytotoxic effect of the lethal gene product. At the early stages
of flowering, expression of the B (or other RNases) and laclq genes in the appropriate
25 cells of the reproductive organ will dra",d~ically increase. Accumulation of the laclq
protein in the nucleus of these reproductive organ cells will block expression of the
Barstar gene (or antisense RNA) via the lac repr~ssor-operdtor interaction which will
~7E~D~ Si~
I r ~ .~ J~i.l
CA 022~94~6 1998-12-30
RECEIVEO 2 0 ~Y ~q7
increase the level of lethal gene products in the target reproductive organ cells.
EXAMPLE 9
Three Eucalypt genes (EGM1, 2 & 3) with homology to other plant MADS-box
genes have been cloned and sequenced (FIGS. 10-13 and 16). Phylogenetic analysis5 has been performed to examine the relatedness of the eucalypt genes to other MADS-
box g~nes (FIG. 17). This revealed that EGM2 is likely to be homologous to the
GLOBOSA MADS-box gene group which are involved in development of petals and
stamens. EGM3 and EGM1 both fall in the AGL2 group of MADS-box genes. These
genes are most commonly expressed in the inner three whorls of the flower (petals,
10 stamens and carpels) but their function in flower development is yet to be determined.
The pattern of expression of all three EGM genes has been characterised
(FIGS. 14 and 15). Expression of these genes was not detected in any vegetative
tissue. The EGM1 gene is expressed in the petals, sta",ens and the carpel of eucaly~t
flowers. EGM3 is expressed in the floral meristent and in sepals. EGM2 is expressed
15 in petals and stamens. The EGM3 and EGM2 genes have been shown by Southem
analysis (FIG. 18) to be single genes. EGM1 is also likely to be a single gene.
The promoter regions of all three of these genes may be used in sterility gene
constructs and have been isolated from a eucalypt genomic library. The EGM3
promoter has been engineered into lethal gene constructs.
20 Northern blots were performed on a range of tissues in order to determine the tissue
specificity of expression of the three EGM MADS-box genes. Tissue used for isolation
of RNA from roots, seedlings stems shoots leaves and mature flowers was obtainedfrom Eucalyptus grandis plants. Tissue for isolation of RNA from floral tissues
including receptacles,- petals, sta,nens, carpels, and styles was collected from E.
25 globulus flowers. This species was used bec~use it produces very large flowers,
making collection of surfic;ent amounts of RNA much easier. Ten micrograms of total
RNA was used in the blots which included the dirferent floral tissues and 20
micrograms was used in blots of E. grandis RNA which included the vegetative tissue.
Blots were probed with the three EGM genes which had been digested with
30 appropriate restriction enzymes to remove the MADS-box.
~.~.I-ND~
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CA 022~94~6 1998-12-30
rcr/Au 9 7 / 0 0 0 8 9
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31
The northern blots indicated that all three EGM genes are expressed at a high
level in eucalypt flowers. When northerns were exposed to film for long periods, weak
expression of the EGM2 ___ r r~ genes was detected in vegetative tissue. The
EGM3 gene was specifically expressed in floral tissue. Within the flower, the EGM2
5 gene was obse~ed to be expressed in stamens and petals. The EGM3 gene is
expressed in receptacles, petals, stamens, carpels and styles.
Example 10
The first part (component 1) of the cascade contains the reporter GUS gene
under control of floral meristem identity gene promoter (ie. LEAFY from ArabidoDsis
10 thaliana) with lac-operator (LEAFY-op). The second construct (component 2) contains
the repressor laclq gene under control of maize GST-27 gene promoter. This
promoter is up-regulated by treatment of plants with the safeners, dichlormid and R-
29148. The Arabidopsis plants were co-transforrrled with two components.
An alternative strategy is for two transgenic ArabidoDsis lines carrying
15 components 1 and 2 separately to be crossed. Expression from component 1 alone
produces blue flowers after staining with X-gal. Chemically-inducible induction of
expression from component two, in plants expressing component one, eliminated the
blue colour seen in flowers that were not chemically induced.
Example 11
The first component of the c~sc~de contains the Bamase gene under control
of floral meii~tei" identity gene promoter (ie. LEAFY from ArabidoDsis thaliana) with
lac-operator. The second cGi"ponent contains the repressor laclq gene under control
of maize GST-27 gene pro" ,oter. ArabidoDsis plants were co-transformed with the two
components. In non induced plants expression of only co",ponent one produces
25 sterile Arabidopsis plants. Induction of expression from component two, in plants
expressing component one, stopped expression from LEAFY-op promoter and
restored the fertility in initially sterile Arabidopsis plants.
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CA 022~94~6 1998-12-30
R~rEl'~EO 2 !~,!3'~ ~997
32
Example 12
A number of abbreviations are used in the following-text. These are in common
use in the field of plant tissue culture.
BA: benzyladenine also known as 6-benzylaminopurine.
5 IBA: indole-3-butyric acid.
NAA: 1 -naphthaleneacetic acid
TDZ: thidiazuron also known as 1-phenyl-3-~1,2,3-thiadiazol-5-yl] urea.
The following is a detailed description of the prefer,ed steps for producing transgenic
Eucalyptus from shoot and seedling explant material.
10 Shoot explants
1. Subculture shoots monthly on solid KG medium containing 0.2mM BA. Keep in lowlight (16 hour photoperiod, 100-350 Lux or 1-8 mmolm~2s ' PAR) and 22.5~C.
2. Use whole shoots 3-4 weeks after subculture.
3. Remove lower leaves leaving the top 4-6 leaves.
15 4. Wound leaves 5 to 6 times with a needle (e.g. 25G) and place whole shoots in an
Agrobacterium suspension (approximately 1X108 cfu ml-') containing 10-100mM
acetosyringone for 10 minutes to 2 hours. An Agrob~.,1e,ium suspension with an
optical density of 1.0 at 600nm diluted 1/20 is approximately 1X10~ cfu mL-'. Best
results when wound near base of leaf. Allernalively, instead of leaving the wounded
20 shoots in an Agrobacterium suspension for 1 hour, the shoots can be vacuum-
infiltrated with the Agrobacterium for 10-30 mins at 40-100 Kpa. The shoots do not
need to be wounded for the vacuum infiltration procedure. However, callus formation
is greater when wounding is effected with a needle rather than by vacuum-infiltration.
5. Blot shoots between sterile filter papers and insert shoots vertically into KG medium
25 containing 0.2mM BA. Co-culture for 2 days in the dark.
6. Transfer shoots (still upright) to KG medium containing 0.2mM BA and 200 mgL~'
A 5 ~
CA 022~94~6 1998-12-30
WO 97130162 PCT/AU97/00089
33
cefotaxime. Keep in low light for 5 days (16 hour photoperiod, 100-350 Lux or 1-8
mmolm~2s~' PAR).
7. Excise 4~ upper leaves and place them, adaxial face up, on solid callus induction
medium G22 containing 2mM BA, 2.5mM NM, 200 mgL~1 cefotaxime and 5-10 mgL'
5 geneticin. Incubate for 2 weeks in the dark.
8. Transfer explants to G22 medium containing 2mM BA, 2.5mM NM,1.OmM TDZ, 200
mgL-' cefot~i~lle and 15 mg.L~' geneticin. Subculture every 2 weeks in the dark. Brown
phenolic compounds are produced when incubated in the light.
9. After 6 weeks Ll dl ~srer explants to shoot induction medium GBA (i.e. G22 co- Itaining
10 5mM BA and 0.5mM NM) containing 200 mgL~' cefotaxime and 15 mgl~' geneticin.
Leave in dark for 5~ days then move into light (16 hour photoperiod,100-350 Lux or
1-8 mmolm-2s ' PAR). Subculture every 2 weeks.
10. After 8-10 weeks on GBA with 200 mgL~' cerol~il "e and 15 mgL~' genetici" tr~nsfer
pieces of callus with buds and callus formed on the original explants to GBA with 200
15 mgL~' cefotaxime and 30 mgL~l geneticin. Subculture every 2 weeks.
11. After 4~ weeks on this medium place regenerated shoots in liquid KG medium
containing 0.01 mM BA, 50 mgL~' cefotaxime and 5 mgL' geneticin for 2 weeks thenlransrer to medium with higher geneticin (10 mgL ') for 24 weeks. The liquid cultures
are shaken at 100-120 rpm with 8 ml of liquid in a 70 ml container.
20 12. Transfer surviving shoots and callus to solid medium (KG containing 200 mgL-'
ce~ota~ill,e and 10 mgL-' geneticin but with no hormones) and higher light intensity (16
hour photoperiod, 450 Lux or 10 mmolm-2s-' PAR). Subculture every 2 weeks.
13. Assay putative ll ansrormed shoots for marker gene(s) activity.
14. Regenerate plants from the confirmed positive shoot material. Induce rooti,)s~ by
25 transferring shoots onto KG containing 10 mgL-' geneticin but with no ho(lllones.
However, if there is no rooting after three weeks then move to the same medium
containing IBA 0.2 mgL~' (9.8 mM).
The protocol detailed above will take between 1 and 8 months from
transformation to regenerated shoots.
SlJ~ l UTE SHEET (RULE 26)
CA 022~94~6 1998-12-30 DCr~AU 9 7 / O O 0 8 9
34
Seedling explants
1. Disinfest seeds and germinate on KG containln~ v., mvl BA.
2. Using 10-12 day old seedlings, remove roots and place them in an overnight-grown
Agrobacterium suspension (approximately 1 X108 cfu ml~') containing 50mM
5 acetosyringone. An Agrobacterium suspension with an optical density of 1.0 at 600nm
diluted 1/20 is approximately 1X108 cfu mL~' . Wound the cotyledons and hypocotyl by
gently stabbing with a 30 gauge syringe needle under a dissecting microscope.
Incubate for 1 hour then remove the seedlings from the suspension and blot them
between sterile filter papers to remove excess liquid. Instead of leaving the wounded
10 cotyledons in an Agrobacterium suspension for 1 hour, the cotyledons can be vacuum-
infiltrated with the Agrobacterium for 20 mins at 95 KPa (28mm Hg). The cotyledons
do not need to be wounded for the vacuum infiltration procedure. However, the
hypocotyls require wounding even when using va~uum ir,ril~,dlion.
3. Co-cultivate on KG medium (containing 0.2 mM BA) for 2 days in the dark making
15 sure that the seedlings are standing upright in the medium.
4. Transfer the seedlings (still upright) to KG medium (containing 0.2mM BA) andcontaining 200 mgL~' cefotaxime. Continue incubation in low light (16 hour photoperiod,
100-350 Lux or 1-8 mmolm~2s~' PAR) for 5 days.
5. Excise the hypocotyls and cotyledons. Transfer the hypocotyls to callus induction
20 medium G22 containing 0.5mM BA, 1.0mM NM, 1.0mM TDZ, 200 mgL-' cefotaxime
and 10 mgL~' geneticin. Transferthe cotyledons to G22 medium containing 1.OmM BA,
1.0mM NM, 0.3mM TDZ, 200 mgL~' cef~taxime and 10 mgL~' geneticin. Continue
incubation in the dark for 2 weeks.
6. Transfer explants to the same medium containing 15 mg. L~' geneticin and 200 mg. L~
25 ' cefotaxime. Continue dark incubation. Subculture every two weeks until about 6
weeks have elapsed.
7. After 6 weeks, transfer to shoot induction medium GBA (i.e. G22 containing 5mM
BA and 0.5mM NM) containing 15 mgL~' geneticin. Leave the cultures in the dark for
5-7 days then transfer them to the light (16 hour photoperiod, 100-350 Lux or 1-8
30 mmolm~2s~'
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CA 022~94~6 1998-12-30
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8. After about 10 weeks on the GBA with 15 mgL~' geneticin and 2Qn mgL~'
cefotaxime, a number of explants will have produced s~ ese shoots are
excised and transferred to KG medium (containing 0.2mM BA) with 30 mgL~' geneticin
5 and 200 mgL ' cefotaxime. Meanwhile, the callus that has also formed on the original
explants is subcultured back to GBA with 15 mgL~' geneticin and 200 mgL~' cefotaxime
and left for a further month, by which time more shoots may develop which can then
be transferred to KG medium (containing 0.2mM BA) with 30 mgL~' geneticin and 200
mgL~' cefotaxime.
10 9. All shoots and callus are now transferred to fresh KG medium (containing 0.2mM
BA) with 30 mgL~' geneticin and 200 mgL~' cefotaxime for another month or so.
10. After 4-6 weeks on this medium place regenerated shoots in liquid KG medium
containing 0.01 mM BA, 50 mgL~' cefotaxime and 5 mgL-' geneticin for 2 weeks then
transfer to medium with higher geneticin (10 mgL ') for 2~ weeks.
15 11. Transfer surviving shoots and callus to solid medium (KG containing 200 mgL~'
cefotaxime and 10 mgL~' geneticin but with no hormones) and higher light intensity (16
hour photoperiod, 450 Lux or 10 mmolm~2s~' PAR). Subculture every 2 weeks.
12. Assay putative transformed shoots for marker gene(s) activity.
13. Regenerate plants from the conr,rllled positive shoot material. Induce rooting by
20 transferring shoots onto KG cGnlaining 10 mgL-' geneticin but with no hormones.
However, if there is no rooting after three weeks then move to the same medium
containing IBA 0.2 mgL-' (9.8 mM).
Seedlings of 12 days old or younger are best for transformation but seedlings
up to 20 days old can be used for regeneration.
Shoot regeneration frequencies for non-transformed cotyledon and hypocotyl
explants without sele_tion are approximately 80%.
Experience has shown that removal of cefotaxime from the medium, even after
3 months, will result in rapid overgrowth by Agrob~cterium. The concentration ofcefotaxime varies between liquid and solid media.
Shoot material grew faster in liquid medium and formed more shoots when
~7END~D ~i~F~T
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CA 022~94~6 1998-12-30
PCr~ J ~3 r / t3 ~3 '
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36
compared to shoots grown on solid medium. This liquid selection step has the
advantages of reducing false positives by increasing selection pressure, reduc~ng
residual Agrobacterium and increasing the amount of shoot tissue COill, ~ dgrown cultures.
The regenerated rooted transgenic plants are then moved to a soil based
medium ~nd grown into trees of a size and form suitable for planting. Clones can be
micropropagated by tissue culture propagation techniques and grown into trees of a
size and form suitable for planting.
The media used in this study were G22, GBA and KG as described by Laine
10 and David (1994). However, various basal media, including MS, B5 and P24, have
been tested and found to support shoot regeneration. The plant growth regulator
regimes were generally different from those of Laine and David (1994), which were in
the combination of 1-3 mM BA, 0.05-2 mM TDZ and 0.5-2.5 mM NM for callus
induction from leaves; 1-3 mM BA, 0.05-1 mM TDZ and 0.5-2.5 mM NM for
15 cotyledons; and 1 -3 mM BA, 0.05-2 mM TDZ and 0.5-2.5 mM NM for hypocotyls. The
differentiation medium was generally a GBA medium, which was a G22 but
supplemented with 2.5 - 5 mM BA and 0.5 mM NM (Laine and David, 1994). A KG
medium containing 0.2 mM BA is generally used as subculture medium for clone
materials. All media were solidified with 0.25% Gelrite or Phytagel. pH was adjusted
20 to 5.7 - 5.8 using potassium hydroxide before autoclaving for 15 minutes at 121~C.
A p, efer, ed method for the preparation of Agrobacterium inoculum is described
below.
1. Streak out the Ag,obacterium strain containing the construct onto plates withselection, eg YEP + rifampicin (50 mg/L) + kanamycin (100 mg/L) for LBA4404,
25 EHA105 and AGL1 cGnlaining the pBin GUSINT construct.
2. Incubate plate at 280~C for 2 days.
3. Pick a single colony into YEP broth with sele_tion and grow on shaker overnight at
280~C.
4. Use the overnight culture to inoculate (1% inoculum) fresh medium with selection
........ .
CA 022~94~6 1998-12-30
~ r !~ / r 3 ~ '
37
and grow on shaker overnight at 280CC. This additional step will produce a more
consistent inoculum for plant transformation.
5. Harvest Agrobacterium. wash and resuspend in tissue culture medium. Dilute tC a, .
appropriate concentration ready for transformation.
5 6. Streak out the resuspended Agrobacterium on to lactose yeast medium plate and
incubate plate at 280~C for 2 days. Do a Benedict's test (Bernaerts and Deley, 1963)
on the colonies to confirm they are Agrobâcterium.
This strategy will give good thick cultures of Agrobacterium on the day of
transformation. A culture grown overnight which was inoculated from a plate or
10 glycerol stock will give variable results.
An AGL1 culture grown as described above and diluted to an optical density
(600nm) of 0.98 had a viable count of 2.37 x 109 cfu/ml.
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
15 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 claimed in the claims appended hereto.
~MEND~3 S~r-~T
IP~t~ll
