Note: Descriptions are shown in the official language in which they were submitted.
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POLLEN SPECIFIC PROMOTER
The present invention relates to a promoter sequence which is specific for
pollen, to
constructs and transgenic plant cells and plants comprising the promoter as
well as to
methods of transforming pollen and controlling fertility in plants using this
promoter.
In order to introduce desirable genetic traits from two plants into a single
plant, such
as a variety or hybrid, cross-breeding represents the traditional approach. In
order to reliably
obtain consistent hybrids, it is necessary to ensure that the self pollination
of the parent
plants does not take place.
1o This can be achieved by ensuring that one of the parent lines is male
sterile. Various
techniques for producing male sterility are known and have been proposed in
the art. One
method involves removal of the anthers or tassels of the female parent plant,
either manually
or mechanically. This plant may then only be fertilised by pollen from the
male parent and
therefore its progeny will be hybrid. However, such a process is labour
intensive and not
1 s altogether reliable as it is possible that some female plants may miss the
detasseling process
or in some cases, the plants develop secondary tassels after detasseling has
been completed.
In addition in this system the male parent is able to self pollinate so it is
necessary to
physically separate male and female parents to allow ease of harvest of hybrid
seed. This
block planting works well for corn as the pollen is light and produced in
large quantity.
2o However, this approach is not applicable to species with heavier pollen and
in species in
which the male and female floral organs are contained within one flower e.g.
wheat, rice.
Chemical methods of preventing pollen production are also known. US Patent No.
4,801,326 describes chemicals which may be applied to the plant or soil to
prevent pollen
production. However, again such techniques are labour intensive and are not
wholly
2s reliable. It is essential that sufficient chemical is applied at the
appropriate time intervals to
ensure pollination does not occur, with the concomitant burden on the
environment. In
addition, these chemicals are very expensive.
Genetic engineering has provided an alternative way by which male sterility
can be
produced, maintained and/or subsequently restored in plants.
3o Systems have been described in which inducible promoters which allow
fertility to be
switched on or off depending upon the application or availability of an
external compound.
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For example, WO 90/08830 describes the induction of male sterility in a plant
by a cascade
of gene sequences which express a protein which disrupts pollen biosynthesis.
WO
93/18171 describes the use of a GST promoter to inducibly express chalcone
synthase (chs)
and restore fertility to a male sterile plant made sterile by "knocking out"
the endogenous
chs genes.
A different approach was described in W093/25695 (PGS). This relies upon the
use
of a tapetum specific promoter to express a pollen lethality gene, barnase, in
the tapetal cells
which are critical to pollen development thus disrupting pollen production. A
restorer gene,
barstar, can be used to restore fertility (Mariani et al. Nature Vol 357: 384-
387).
There would be advantages to expressing genes which have an impact in pollen
production in pollen directly in a controllable manner. Suitably, fertility
should be
restorable when desired.
A component of a system to genetically engineer male sterility is the
availability of a
promoter which specifically drives expression either in pollen or in tissue on
which pollen
production or development depends.
Many of the characterised genes which are specifically or highly expressed in
pollen
and germinating pollen encode proteins that are likely to play a role in cell
wall metabolism,
for example, those having homology to genes encoding enzymes involved in
pectin
degradation; polygalacturonases (SM Brown et al.., Plant Cell (1990) 2: 263-
274, SJ Tebbutt
2o et al.., Plant Mol. Biol. (1994) 25:283-299), pectate lyase (HJ Rogers et
al.:, Plant Mol Biol
20:493-502 (1992), RA Wing et al., Plant Mol Biol 14:17-28 (1989)) and pectin
methylesterase (PME) JH Mu et al.., Plant Mol Biol 25:539-544 (1994)).
Other genes highly expresed in pollen include those that encode cytoskeletal
proteins
(I. Lopez et al.., Proc. Natl, Acad Sci USA 93: 7415-7420 (1996), HJ Rogers et
al.., Plant J.
4: 875-882 (1993) and CJ Staiger et al.., Plant J. 4: 631-641 (1993)),
putative ascorbate
oxidases, a Kunitz protein inhibitor and many others whose function cannot be
inferred by
homology to known genes. The temporal expression of such genes has been
studied and
many are found to be expressed late in microsporogenesis reaching a maximum in
mature
microsporocytes. In some cases continued expression in the pollen tube has
also been
3o demonstrated (AK Kononowicz et al.., Plant Cell 4; 513-524 ( 1992)). These
genes are
referred to as "late genes". The majority of expression at this stage is from
the vegetative
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cell rather than from the generative cell and it is likely that the majority
of these 'late' genes
are transcribed from the vegetative nucleus; although this has only been
demonstrated for one
"late" gene (D. Twell, Plant J2: 887-892 (1992). A distinct class of genes
expressed in
anthers is found to have a different expression programme, being first
detectable soon after
the tetrad stage and declining in expression well before pollen maturity. It
is likely that the
major role of these 'early' genes may be during microspore differentiation and
development
rather than pollen tube growth. In addition, US Patent No. 5.086,169
(Mascarenhas)
describes the isolation of the frst pollen-specific promoter from com.
The applicants have isolated a further promoter which is specifically
expressed only
in pollen tissue. The promoter is derived from a 'late' pollen expressed gene
isolated from
maize, ZmCS.
Thus, according to a first aspect of the present invention there is provided a
recombinant nucleic acid sequence which comprises a promoter sequence of the
ZmCS gene
in maize, or a variant or fragment thereof, which acts as a promoter in
pollen.
t5 As used herein, the term "fragment" includes one or more regions of the
basic
sequence which retain promoter activity. Where the fragments comprise one or
more
regions, they may be joined together directly or they may be spaced apart by
additional
bases.
The expression "variant" with reference to the present invention means any
2o substitution of, variation of, modification of, replacement of, deletion of
or the addition of
one or more nucleotides from or to the nucleic acid sequence providing the
resultant
sequence exhibits pollen promoter expression. The term also includes sequence
that can
substantially hybridise to the nucleic acid sequence.
As used herein, the expression "ZmCS gene" refers to the gene of maize which
25 encodes a 563 amino acid sequence as described herein. A cDNA sequence
encoding this
sequence is defined in EMBL Y13285
The promoter sequence of the present invention is comprised within the clone
deposited National Collection of Industrial and Marine Bacteria as NCIMB 40915
on 26 Jan
1998. This is a Sal I fragment derived as described hereinafter. The promoter
region lies
3o within a region which consists of approximately 2kb of sequence upstream of
the
transcription start site of the ZmCS gene of maize as shown in Figure 1
hereinafter.
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According to a preferred embodiment of the present invention, there is
provided a
recombinant nucleic acid sequence which comprises a promoter sequence
comprising at least
part of the DNA sequence as shown in Figure 5 or at least part of a sequence
encoding a
promoter which has substantially similar activity to the promoter encoded by
Figure 5 or a
variant or fragment thereof.
The term "substantially similar activity" includes DNA sequences which are
complementary to and hybridise to the DNA of the present invention and which
code for a
promoter which acts in pollen. Preferably, such hybridisation occurs at, or
between, low and
high stringency conditions. In general terms, low stringency conditions can be
defined as 3 x
1o SCC at ambient temperature of between about 60°C to about
65°C, and high stringency
conditions as 0.1 x SSC at about 65°C. SSC is the name of a buffer of
O.15M NaCI, 0.01 SM
trisodium citrate. 3 x SSC is three times as strong as SSC and so on.
The pollen specific promoter of the present invention may be used to engineer
male
sterility by driving genes capable of interfering with pollen production or
viability, or to
15 express genes of interest specifically in pollen grains.
According to a second aspect of the present invention, the promoter sequence
may
form part of an expression cassette in combination with genes whose expression
in pollen,
and particularly in late pollen production, may be desirable. These include
genes which have
an impact on pollen or pollen production. Such genes may be those involved in
the control
20 of male-fertility, genes which encode insecticidal toxins (which would then
be targeted to
insect species which feed on pollen), or genes which would enhance or modify
the nutritional
value of the pollen. In addition, the promoter sequence could be used to drive
expression of
a selectable marker for use in pollen transformation. Examples of suitable
selectable marker
genes include antibiotic resistance genes such as kanamycin resistance gene,
hygromycin
25 resistance gene and the PAT resistance gene so as to enable stable
transformants to be
identified depending on the species e.g. corn, rice, wheat.
The term "expression cassette" - which is synonymous with terms such as "DNA
construct", "hybrid" and "conjugate" - includes an effect gene directly or
indirectly attached
to the regulator promoter, such as to form a cassette. An example of an
indirect attachment is
3o the provision of a suitable spacer group such as an intron sequence
intermediate the promoter
and the target gene. The DNA sequences may furthermore be on different vectors
and are
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therefore not necessarily located on the same vector. The same is true for the
term "fused" in
relation to the present invention which includes direct or indirect
attachment. Such
constructs also include plasmids and phage which are suitable for transforming
a cell of
interest.
According to a preferred embodiment, expression cassettes of the present
invention
comprise a promoter sequence as described above which is arranged to control
expression of
a gene which is deleterious to pollen development, such as genes encoding
barnase, adenine
nucleotide translocator, mutant tubulins, T-urf (as claimed in WO 97/04116) or
trehalose
phosphate phosphatase (TPP).
1o For instance, W093/25695 describes the use of the gene barnase which
encodes a
cytotoxic protein , which is under the control of a tapetum specific promoter.
Expression of
barnase in the tapetal cells disrupts these cells and leads to disruption of
pollen production.
Ribozymes are RNA molecules capable of catalysing endonucleolytic cleavage
reactions. They can catalyse reactions in trans and can be targeted to
different sequences.
15 They are therefore potential alternatives to antisense as a means of
modulating gene
expression. (Hasselhof and Gerlach (1988) Nature Vol 334: 585-591) or Wegener
et al.
(1994) Mol Gen Genet 245: (465-470) have demonstrated the generation of a
trans -dominant
mutation by expression of a ribozyme gene in plants. If required, the pollen
specific
promoter of the present invention may be used to control expression of the
ribozymes such
2o that they are specifically expressed in pollen.
Baulcombe (1997) describes a method of gene silencing in transgenic plants via
the
use of replicable viral RNA vectors (AmpliconsT"') which may also be useful as
a means of
knocking out expression of endogenous genes. This method has the advantage
that it
produces a dominant mutation i.e. is scorable in the heterozygous state and
knocks out all
25 copies of a targeted gene and may also knock out isoforms. This is a clear
advantage in
wheat which is hexaploid. Fertility could then be restored by using an
inducible promoter to
drive the expression of a functional copy of the knocked out gene. By
including the pollen
specific promoter as the elements of the AmpliconT"" vector, expression of the
gene would
then take place specifically in the pollen.
3o The use of cytotoxic or disrupter genes as means of disrupting pollen
production
requires the expression of restorer genes to regain fertility. Suitably the
construct further
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comprises a cassette comprising a nucleotide sequence which is able to
overcome the effect
of said deleterious gene, such as a restorer gene such as barstar in the case
of barnase or TPS
in the case of TPP, or a sequence which encodes a construct which is sense or
antisense to a
deleterious gene.
An alternative means of controlling expression of deleterious genes is to use
operator
sequences. Operator sequences such as lac, tet, 434 etc. may be inserted into
promoter
regions as described in WO 90/08830. Repressor molecules can then bind to
these operator
sequences and prevent transcription of the downstream gene, for example a gene
deleterious
to pollen development (Wilde et al. ( 1992) EMBO J. 11, 1251 ). Furthermore,
it is possible to
1 o engineer operator sequences with enhanced binding capacity such as the Lac
IO His mutant
as described in (Lehming et al. (1987) EMBO . 6, 3145-3153). This has a change
of amino
acid from tyrosine to histidine at position 17 thus giving tight control of
expression. Used in
combination with inducible expression of the repressor this then allows
expression of an
inactivating gene to be turned off.
15 In this way, the expression cassettes may be incorporated into expression
systems
which may be used in the control of fertility of a plant as described above.
The term "expression system" means that the system defined above can be
expressed
in an appropriate organism, tissue, cell or medium. The system may comprise
one or more
expression cassettes and may also comprise additional components that ensure
to increase
2o expression of the target gene by use of the regulator promoter.
According to a third aspect of the present invention, there is provided an
expression
system comprising
(a) a first promoter sequence which is expressed specifically in pollen;
(b) a first gene which, when expressed, disrupts pollen biogenesis. under the
control of said
25 pollen specific promoter;
(c) a second promoter sequence responsive to the presence or absence of an
exogenous
chemical inducer; and
(d) a second gene encoding an element which can inhibit either expression of
said first gene,
or can inhibit the protein coded for by said f rst gene, operably linked to
and under the
3o control of said second promoter sequence.
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Elements (a) and (b) and (c) and (d) above may be provided by one or two
individual
vectors, but preferably are contained in the same vector to ensure co-
segregation. These can
be used to transform or co-transform plant cells so as to allow the
appropriate interaction
between the elements to take place.
The second promoter sequence and the second gene provide for chemical
"switching" on and off of the first gene. Where the second promoter sequence
is responsive
to the presence of the exogenous chemical inducer, application of the chemical
inducer to
pollen or to a plant will have the effect of switching on the second gene
which thereby
counteracts the effect of the first gene. The absence of the chemical inducer
will have a
1 o similar effect where the second promoter sequence is active only in the
absence of the
chemical inducer.
Elements (c) and (d) are suitably in the form of an expression cassette
comprising a
nucleotide sequence which is able to overcome the effect of said deleterious
gene, such as a
restorer gene such as barstar in the case of barnase or TPS in the case of
TPP, or a sequence
15 which encodes a construct which is sense or antisense to a deleterious
gene, or a gene
encoding a repressor molecule in the case of operator sequences being used
operatively
interconnected with an inducible promoter.
The expression system of the present invention may further comprise a
selectable
marker, such as herbicide resistance genes or antibiotic resistance genes so
as to allow stable
2o transformants to be identified depending on the species eg corn, rice,
wheat. The presence of
a herbicide resistance gene also allows selection of male sterile progeny in a
segregating
population.
Transformation of a plant with such an expression system will result in the
production of male sterile plants and methods of producing such a plant form a
fourth aspect
25 of the present invention.
Expression systems in accordance with this embodiment of the present
invention,
wherein the gene is deleterious to viable pollen production, are useful in the
production of
hybrids but are especially useful when the male sterile line can be made
homozygous. When
"late" promoters, such as the ZmCS promoter described above, are used, because
the gene
3o products are expressed late in pollen development, primary transformants
and heterozygotes
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_g_
produce pollen which segregates I :1 for sterility i.e. 50% of the pollen is
fertile and so self
pollination leading to non-hybrid seed may occur.
In order to obtain a homozygous sterile plant, the inducible promoter must be
switched on to drive expression of the restorer gene using the appropriate
chemical such as
ethanol in the case of the AIcAlR switch or safener for the GST switch. This
then inactivates
the deleterious gene, so allowing self pollination to occur in accordance with
the model
below.
MS = Dominant male sterility
1 o Rcs = inducible restorer
Heterozygous genotype MSRcs---
Gametes MSRcs ---
~ 5 Self pollination after chemical induction gives
MSRcs ---
MSRcs MSMSRcsRcs ---MSRcs
--- MSRcs--- --- ---
All pollen from the homozygous progeny (MSMSRcsRcs) will be sterile. 50% of
the
pollen from the heterozygous progeny (MSRcs---) will be sterile. All of the
pollen from the
null (--- ---) progeny will be fertile. Staining of the pollen from these
lines with a vital stain
such as DAPI will allow identification of the plant producing 100% sterile
pollen.
Alternatively, the induction and self pollination can be repeated on this
segregating
population and the progeny analysed for segregation of sterility. Clearly all
progeny deriving
from self pollination of the homozygous line will themselves be homozygous and
male
3o sterile i.e. they will all produce 100% sterile pollen, whereas progeny
arising from self
pollination of a heterozygous line will continue to segregate for sterility.
Alternatively, if the
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gene giving rise to male sterility is linked to a gene conferring herbicide
resistance, then
progeny may be sprayed with herbicide at an early seedling stage, herbicide
tolerance will
segregate with the sterility gene. In this way, male sterile parents can be
identified and
selected for hybrid production.
Once identified, this homozygous sterile line may be used in the production of
F I
hybrids by cross pollination by an unmodified inbred male parent line. See
below.
Female parent Male parent
MSMSRcsRcs *** ***
to
Gametes MSRcs
MSRcs MSRcs
*** MSRcs*** MSRcs***
Fl
*** MSRcs*** MSRcs***
Thus, all the F 1 seed is hybrid and heterozygous for sterility, meaning that
50% of the
2o pollen from each plant is fertile. In a crop species such as corn this is
ample viable pollen to
ensure complete pollination across a field due to the sheer volume of pollen
produced by
each tassel. In wheat too this should be sufficient pollen to ensure no yield
loss as self
pollination occurs while the flower is still closed, thus reducing loss by
wind etc. In species
in which the vegetative part of the plant is harvested then reduced pollen
viability is not a
factor to be taken into account.
There is too an additional benefit to the hybrid seed producer in that should
the
farmer retain F2 seed for subsequent planting he will suffer a loss in yield
as result of loss of
heterosis. See below.
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F 1 MSRcs***
Gametes MSRcs (not viable)
MSRcs none * * * MSRcs
F2
*** none *** ***
These methods may allow for reversal of the sterility, for example in hybrid
production, by activation using an inducible promoter.
According to a fifth aspect of the present invention, there is provided a
method of
1o controlling the fertility of a plant which comprises transforming said
plant with an expression
system as described above, and when fertility is to be restored, activating
the inducible
promoter.
Suitable inducible promoters include those which are controlled by the
application of
an external chemical stimulus, such a herbicide safener. Examples of inducible
promoters
t5 include, for example, a two component system such as the alcAlalcR gene
switch promoter
system described in our published International Publication No. WO 93/21334,
the ecdysone
switch system as described in our International Publication No. WO 96/37609 or
the GST
promoter as described in published International Patent Application Nos. WO
90/08826 and
WO 93/031294, the teachings of which are incorporated herein by reference.
Such promoter
2o systems are herein referred to as "switch promoters". The switch chemicals
used in
conjunction with the switch promoters are agriculturally acceptable chemicals
making this
system particularly useful in the method of the present invention.
If the pollen specific promoter of the present invention is used to obtain
male sterility,
full restoration of fertility may not be achievable by this method as pollen
is haploid. This
25 means that only 50% of pollen produced following activation of the restorer
gene is fertile.
This may be countered by the fact that expression using the promoter of the
present invention
is highly tissue specific.
This property makes the use of the promoter of the present invention
particularly
useful in some very particular applications. For example, in some cases
transformation of
3o pollen is required. In this instance the use of the pollen specific
promoter be may highly
desirable. An example of such an application is known as MAGE LITER (male germ
line
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transformation) and is described by Stoger et al. (Plant Cell Reports 14
(1995) 273-278). In
this method, pollen is transformed by microprojectile bombardment. A pollen
specific
promoter is used to drive, for example a selectable marker gene. The fact that
the promoter
is pollen specific confers several advantages. First of all, the marker is
expressed only in the
pollen, not the rest of the plant and so the remaining plant tissue does not
contain unwanted
marker. Furthermore, having a selectable marker only in the pollen allows the
possibility of
retransforming by the usual methods and not having to have a different
selectable marker, i.e.
it allows easier gene stacking. The reasons why pollen transformation is
important are that
pollen can be made to undergo sporophytic development i.e. will give rise to
haploid and
t o doubled haploid plants. This means that homozygosity is achieved in one
step.
Alternatively, the transformed pollen can be used to pollinate wildtype plants
thus giving
seed carrying the introduced transgene, again a faster process than the
traditional
transformation route.
The expression systems of the present invention can be introduced into a plant
or
t s plant cell via any of the available methods including infection by
Agrobacterium
tumefaciens containing recombinant Ti plasmids, electroporation,
microinjection of plant
cells and protoplasts, microprojectile bombardment, bacterial bombardment,
particularly the
"fibre" or "whisker" method, and pollen tube transformation, depending upon
the particular
plant species being transformed. 'The transformed cells may then in suitable
cases be
2o regenerated into whole plants in which the new nuclear material is stably
incorporated into
the genome. Both transformed monocot and dicot plants may be obtained in this
way.
Reference may be made to the literature for full details of the known methods.
Such
methods form a further aspect of the present invention.
The method of the present invention would be useful in the production of a
wide
25 range of hybrid plants, such as wheat, rice, corn, cotton, sunflower, sugar
beet, and lettuce,
oil seed rape and tomato.
Plant cells which contain a plant gene expression system as described above,
together
with plants comprising these cells form further aspects of the invention.
According to a nineth aspect of the present invention there is provided a
replicable
3o viral DNA vector (AmpliconT"") which comprises a recombinant nucleic acid
as defined
above.
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According to a tenth aspect of the present invention, there is provided a
method of
transforming pollen cells with an expression system as described above.
The invention will now be particularly described only by way of example with
reference to the accompanying drawings in which:-
Figure 1 is a diagram showing the alignment of the ZmCS cDNA with a 2.4kb
fragment from
the 5'region of its corresponding gene. The transcriptional start point is
indicated (*), and
the putative translational start is underlined. Sa= Sal I, S=Sma I, Sp= Sph I,
H = Hind III, X =
Xho I, P = Pst I;
to
Figure 2 shows a Southern blot of maize (inbred line A188) genomic DNA. Each
lane
contains l5pg of genomic DNA digested in lane 1 with Eco RI, in lane 2 with
Hind III, and
in lane 3 with Bam HI. The Southern blot was hybridised with radiolabelled
ZmCS cDNA
probe.
t5
Figure 3 shows ethidium bromide stained gels showing the 18S RNAs of various
maize
tissue total RNAs and the northern blots of the same gels probed with the ZmCS
cDNA
probe.
(A) the gel was loaded with 10~g of total RNA from various maize tissues.
20 (B) the gel was loaded with l Opg of total RNA isolated from shoot, a
developmental staged
series of spikelets, pollen and germinating pollen.
Figure 4 shows
(A) Physical map of the ZmCS::uidA construct and junction sequence. The A
residues by *,
25 t and ~ correspond to the ZmCS transcription start point, the T to A
mutation to remove the
Hind III site (for ease of cloning) and the 3' terminus of the ZmCS promoter
region,
respectively. The uidA translation start is underlined.
(B) Histochemical analysis of GUS activity in pollen from transgenic
ZmCS::uidA tobacco
3o showing segregation of the blue staining.
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(C) Promoter activity of ZmCS in transgenic tobacco tissues from two
transgenic lines, GCS-
2( unfilled columns) and GCS-7 (filled columns). Data from each line
represents the
mean from two independent assays, normalised to a non-transgenic control. Bud
stages
are as follows: Bud-1 corresponds to buds of 5-8mm, (microspores at
meiosis/tetrad
stage), Bud-2: buds of 10-l2mm (uninucleate microspores), Bud-3: 13-lSmm,
(microspore mitosis), Bud-4: (17-22mm) early- to mid-stage binucleate
gametophyte,
Bud-5: (27-45mm) mit- to late stage binucleate gametophyte (Tebbutt et al..,
supra.).
Figure 5 shows the DNA sequence encoding the ZmCS promoter sequence in maize.
1o The underlined A is the putative transcriptional start point and the bold
and underlined ATG
is the translational start point.
Figure 6 shows an expression cassette comprising CS-barnase/barstar-nos.
EXAMPLE 1
15 ZmCS cDNA and genomic clones
Maize (inbred line A188) pollen and germinating pollen (HJ Rogers et al..,
Plant J4
(1993) 875-882) cDNA libraries were screened using cDNA probes made to pollen
and shoot
poly(A)+ RNA following standard procedures (FM Ausubel et al.., Current
protocols in
Molecular Biology, Wiley, New York (1990)). All clones, totalling 1,101, that
showed
2o hybridisation to the radiolabelled pollen cDNA but not to the radiolabelled
shoot DNA were
picked. Random colonies were picked and sequenced at the 5' end and the
sequence
compared on current databases. One clone which had an insert of 800bp showed
significant
sequence identity with known pectin methyl esterases. The pollen cDNA library
was re-
screened with this cDNA insert and the full length ZmCS clone (ZmCSc) was
identified and
25 sequenced.
A maize (inbred line B73) genomic library (8x106 plaques) was screened using a
PCR
fragment corresponding to the 5' 270bp of cDNA clone ZmCSc labelled by random
priming
(Ausubel et al.., supra). One positive clone ZmCSg was plaque purified.
Comparison of the
sequence of a 2.Skb SaII fragment of ZmCSg subcloned into pUCl9 with ZmCSc
(and
3o deposited as NCIMB 40915) showed that the two sequences overlapped and that
they were
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identical, indicating that the clones represent the same gene. A
representation of the overlap
between the 2.Skb fragment of the genomic clone with the cDNA is shown in
Figure I .
A transcriptional start point was mapped using two oligonucleotide primers
complementary to nucleotides I to 21 (5'- ACCTAGGAGAGCCTTTGCCAT-3') and 56 to
82 (5'-AGCGGGTGACGGTGGCGACCACACCGA-3') of the coding sequence (data not
shown). The products unequivocally locate the transciptional start point on
the A nucleotide
at only 1 S bases upstream of the putative ATG (Fig 1 ). Other pollen-specific
genes have
been found to have long S'-untranslated sequences (SJ Tebbutt et al.., Plant
Mol. Biol.
(1994) 25: 283-299). Thus this region of ZmCSg appears to be unusually short.
The
calculated free energy for this short 5' UTL is of 0.9kJ/mol(M. Zuker, Meths.
Enzymol.
(1989) 180: 262-288) making it unlikely that it is able to form any stable
secondary structure.
An open reading frame of 1692 by was identified and the putative ATG start
codon
(Figure 1) fits well with the consensus sequences (HA Lutcke et al.., EMBO J
6: (1987) 43
48). A putative TATAA motif (CP Joshi, Nucl. Acids Res. 15:6648-6653 (1987))
starts at
t 5 32, however no recognisable CAAAT motif is found in the 60 by upstream
from the
transciptional start (Fig. 1 ) as has been found in many other plant genes.
ZmCSc lacks a
clearly recognisable AATAAA polyadenylation site signal. This is not unusual:
30% of
plant genes lack a recognisable AATAAA motif (BD Mogen et al.., Plant Cell 2
(1990)
1261-1272). In ZmCSc, a stretch of five A residues 16 by upstream from the
poly A addition
2o site may be acting as a polyadenylation signal, although other pollen
specific transcripts have
been found to have longer than average distances between the AATAAA motif and
the
poly(A)+ addition site (Tebbutt et aL ., supra.)
EXAMPLE 2
25 Deduced amino-acid sequence of ZmCS
The predicted amino acid sequence of ZmCS (563 amino acids) was compared to
the
EMBL and GenBank databases, and revealed a high degree of homology to both
plant
(between 30.9% and 41.4%) and microbial PMEs (between 18.6% and 20.8%}. An
alignment of amino acid sequences showed conservation across both plant and
microbial
3o sequences restricted primarily to the C-terminal end of the protein which
includes four
regions likely to be the catalytic domains or active sites of the enzyme (D.
Albani et al..,
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Plant Mol Biol (1991) 16:501-513),0 Marcovic et al.., Protein Sci 1: 1288-1292
(1992)). In
vitro mutagenesis of the A. niger PME (B Duwe et al.., Biotechnol. Lefts 18:
621-626
(1996)) indicated that a histidine residue, which is conserved in ZmCS, within
the region I
may be located at the active site of the enzyme, and in A. niger is required
for enzyme
activity. However this histidine is replaced by other amino acid residues in
several PMEs of
both plant and fungal origin, suggesting that is it not essential in all PMEs.
In a comparison
of the plant PMEs, ZmCS shows a closer relationship to the P. in, flata 'late'
pollen expressed
PPE gene (JH Mu et al.., Plant Mol Biol 25: 539-544 (1994)), than to B. napuS
'early' pollen
expressed Bp 19 gene (D. Albani et al. . supra.)
f0
EXAMPLE 3
Estimation of ZmCS Gene number
A maize (inbred line A188) genomic Southern blot containing lSp,g of DNA
digested
with either BamHI, EcoRI, or Hind III was probed with radiolabelled full-
Length ZmCS
cDNA insert. Two strong hybridising bands in each lane of the blot in Figure 2
suggests the
presence of at least two similar genes in the maize genome. Several further
bands with show
a much weaker signal suggests that this gene family may also comprise several
less related
members.
2o EXAMPLE 4
Spatial and temporal expression of ZmCS
A northern blot containing lOp,g total RNA from eight maize tissues was probed
with
the cDNA ZmCSc to determine the expression programme of the gene. A transcript
of
approximately 2.Okb was detected only in pollen and germinating pollen (Figure
3(A)) ,
indicating that, within the limits of detection of this technique, expression
of this gene
appears to be restricted to these two tissues. No signal was detectable in
leaf, root, shoot,
cob, endosperm or embryo. The expression programme during spikelet development
was
also determined. Figure 3(B) shows a Northern blot containing total RNA from
0.25, 0.5 and
1.Ocm spikelets, mature pollen and germinating pollen. The ethidium bromide
stained gels
3o demonstrate the equal loadings of RNA in the lanes of each gel.
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Spikelets were staged by staining the anthers with acto-carmine, and the
anthers were
found to contain cells at the following stages: pre-meitotic sporpogenous
cells (0.25cm),
mid-prophase I (0.5 cm), maturing pollen grains ( 1.0 cm). Some overlap
between
consecutive stages is however inevitable due to the variation in the
developmental stage
between the two florets within the same spikelet. This Northern analysis shows
that ZmCS
expression is restricted to mature dehisced pollen and germinating pollen with
no detectable
expression in any other maize tissues including spikelets containing cells in
earlier stages of
microsporogenesis.
io EXAMPLE 5
Expression of ZmCS promoter/GUS constructs in transgenic plants
Transciptional fusions were made between the 5' region of ZmCSg and the
reporter
gene (3-glucuronidase (Figure 4A), and used to transform tobacco by
Agrobacterium
transformation. The construct was made as follows:- the two Sph I sites, one
within the
15 2.Skb SaII fragment which contains 2kb of 5'sequence relative to the ATG on
one within the
polylinker, were used to remove the 3' end from position -61 to +403 (Figure 1
). This was
directly replaced by and Sph I digested PCR fragment that included the region -
61 to +1,
additional restriction sites positioned at the 3' end (Bam HI, Hind III, Sal
I) and a mutation to
remove the Hind III site positioned at the transcriptional start (Figures 1
and 4A). The
20 original 5' Sal I site and the introduced 3'Sal I site were then used to
excise the ZmCS
promoter region which was cloned into the Sma I site of pGUS. The ZmCS
promoter-UID-A
transcriptional fusion was then transferred to pain 19 vector (Figure 4A) and
used for
Agrobacterium-mediated leaf disc transformation of Nicotiana tabacum var
Samsun.
Transformants were selected on kanamycin and primary transgenic plants were
regenerated,
25 two of which, positive for expression of the transgene, were taken to the
T2 generation.
Pollen grains from dehisced anthers of the transgenic plants were harvested
and
stained for GUS activity as described by J.A. Jefferson (Plant Mol Biol Rep
(1987) 5: 387-
405). Two plants were positive showing approximately SO% blue staining pollen
(Figure
4B). No blue colouration was detected in non-transgenic controls. To
investigate the
3o number of integration sites, plants from two transgenic lines were selfed,
and progeny were
scored for resistance to kanamycin. Of the progeny assessed from transgenic
plant GCS-2,
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303 were kanamycin resistant and 8 kanamycin sensitive giving a mean ratio of
38:1
indicative of at least two integration sites. The progeny of transgenic plant
GCS-7 gave a
mean ratio of kanamycin resistant to kanamycin sensitive of 3.8:1 indicative
of a single
integration site (expected ratio for one integration site is 3:1, for two,
15:1 and for three
63:1}.
Extracts were made from a range of tissues including five stages of developing
anthers, and analysed fluorimetrically fir GUS expression (Jefferson. supra.)
Figure 4(C)
shows GUS activities from two transgenic plants. Only very low levels of
expression are
detectable in tissues other than developing and mature dehisced anthers. In
tobacco the stage
l0 of bud development can be correlated with bud length (Tebutt et al..,
supra.} but this is
dependent on the growth conditions. Thus Bud-1 corresponds to microspores at
mitosis/tetrad stage; Bud-2, uninucleate microspores, Bud-3 microspore
mitosis, Bud-4, early
to mid-stage binucleate gametophyte, Bud-5 mid- to late-stage binucleate
gamteophyte.
Microspore stages as assessed by DAPI staining (data not shown) indicate that
the timing of
expression of the ZmCS promoter in tobacco agrees well with its expression in
maize based
on the Northern data (Figure3; Figure 4C). Thus both in it native environment
in maize and
in transgenic tobacco, the ZmCS promoter function late in pollen development
and is
virtually inactive before microspore mitosis. Some variation in expression can
be noted
between the two transgenic lines with the GCS-2 showing higher levels of
expression in
2o most tissues tested. Variation in expression levels are commonly found in
transgenic
populations and have been ascribed to the site of insertion of the transgene
9SLA Hobbs et
al.., Plant Mol Biol 21: 17-26 (1993)).
EXAMPLE 6
Expression of GUS in pollen driven by AIcA Inducible Promoter
A plant transformation vector comprising the Alc A promoter driving expression
of
GUS and a 35S CaMV promoter driving the expression of AIcR has been introduced
into
tobacco and tomato plants. GUS expression may be studied in all tissues before
and after
induction with ethanol as a root drench.
3o GUS staining of tomato anthers and pollen shows clear expression of GUS
after
induction. The same result is expected from pollen from other species.
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EXAMPLE 7
Preparation of CS-barnase Cassette - a dominant gametophytic male sterility
cassette.
The unique SaII site of pBluescipt SK+ (Stratagene) was replaced with a NotI
recognition site by insertion of the an oligonucleotide linker MKLINK4 (5'-
TCGATTCGGCGGCCGCCGAA-3'} into the digested SaII site. A 0.9kb, BamHI-HindIII
fragment carrying the coding region of barnase followed by a bacterial-
promoter-driven
barstar coding region, was inserted into the corresponding fragment of the
modified
pBluescript. The nos terminator on a HindIII- NotI fragment was inserted into
the
1o corresponding fragment of the resulting vector. An unwanted BamHI site was
then removed
using Stratagene's QuickChange system, following the manufacturer's
instructions and using
oIigonucleotides DAM-3A (5'-GGTCGACTCTAGAGGAACCCCGGGTACCAAGC-3')
and DAM-3S (S'- GCTTGGTACCCGGGGTTCCTCTAGAGTCGACC-3'). The resulting
plasmid (named pSK-BBN) was digested to completion with BamHI,
dephosphorylated with
is shrimp alkaline phophatase (37°C, 1 hour). A l.9kb BamHI fragment of
the CS 5' flanking
region was Iigated into this, followed by digestion with BamHI and PstI to
check for
presence and orientation of the insert, respectively. The resulting plasmid
was named pSK-
CS-BBN (Figure 6). The entire cassette is then removed as an EcoRl- Notl
fragment to a
binary plant transformation vector pVB6. The construct is then introduced into
2o Agrobacterium Tumefaciens by the freeze-thaw method. Standard techniques
are used to
introduce the DNA into tobacco.
EXAMPLE 8
Analysis of sterile transgenic plants
25 Primary transformants are selected by growth on kanamycin in tissue culture
and this
confirmed by PCR analysis. The plants are grown to maturity in the glass house
. Pollen is
collected from anthers after dehiscence and a vital stain is used to establish
whether the
pollen is fertile or sterile. 50% of the pollen is expected to be sterile.
Backcrossing these
plants with wild type plants (after anther removal) or allowing self
pollination to occur
3o results in progeny in which 50% of pollen is sterile.
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Other modifications to the present invention will be apparent to those skilled
in the
art without departing from the scope of the invention.
