Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02224736 1997-12-16
PCT/GB96/01675
W O 97/04116
INHIBITION OF CELL RESPIRATION AND PRODUCTION OF MALE STERILE PLANTS
The present invention relates to a method of producing male sterile plants by use of a
gene, which is expressible in plants and inhibits an essential cell function. hence disrupting full
- e~cpression of a selected plant characteristic.
Our International Patent Application No. WO 90/08831 describes and claims the
disruption of respiration using a variety of disrupter genes which we refer to also as
"pollen-inactivating genes".
The ability of such inactivating genes to function in this way varies and, therefore,
there is a need for further improved gene sequences so that appropriate selection for specific
u applications may be made.
An object of this invention is to provide genes for use in inhibiting gene expression.
According to the present invention there is provided a method of inhibiting geneexpression in a target plant tissue comprising stably transforming a plant cell of a type from
which a whole plant may be regenerated with a gene construct carrying a tissue-specific or a
development-specific promoter which operates in the cells of the target plant tissue and a
disrupter gene encoding a protein which is capable, when e~cpressed, of inhibiting respiration
in the cells of the said target tissue resulting in death of the cells characterised in that the said
disrupter gene is selected from the group consisting of the T-urfl 3 gene, genes encoding an a
- or ,~-tubulin, short sense co-suppression of two essenti~l maize cell cycle genes. cdc25 and
2u replication origin activator (ROA) and a short sense construct to the adenine nucleotide
translocator (ANT) of the inner mitochondrial membrane.
Down regulation of gene activity due to short sense co-suppression is described in our
International Patent Application No.WO 90/08299.
The a- or ~-tubulin genes act as disrupters by de-stabilizing microtubule arrays in
plant cells. hence inhibiting essential microtubule function in the said target tissue resulting in
death of the cells.
The use of short sense co-suppression of two essential maize cell cycle genes, cdc25
and replication origin activator (ROA) disrupts cell division and hence provide a ~rowth
defect in the targeted organ or tissue.
Preferably the promoter is an anther- and/or tapetum-specific promoter or a
pollen-specific promoter, so that on e~pression of the said disrupter protein therein the
re_enerated plant is in male sterile. More preferably the said anther and/or tapetum-specific
CA 02224736 1997-12-16
W O 97/04116 PCT/GB96/01675 -2-
promoter was isolated using the cDNA sequences shown in Figure 1 or 2 or 3 of the
accompanying drawings and using the techniques described in our International Patent
Application No. WO 90/08826.
Plasmids cont~ininv the DNA sequences shown in Figures 1, 2 and 3 have been
deposited under the terms of the Budapest Treaty, details being as follows:
Plasmid pMS10 in an ~scherichia coli strain RR1 host, cont~ininv the gene sequence
shown in Figure I herewith, and deposited with the National Collection of Industrial &
Marine Bacteria on 9th January 1989 under the Accession Number NCIB 40090.
Plasmid pMS 14 in an ~scherichia coli strain DH5a host, cont~ining the gene
sequence shown in Figure 2 herewith, and deposited with the National Collection of
Industrial & Marine Bacteria on 9th January 1989 under the Accession Number NCIB 40099.
Plasmid pMS 18 in an ~scherichia coli strain RR1 host, Cont~inin~ the gene sequence
shown in Figure 3 herewith, and deposited with the National Collection of Industrial &
Marine Bacteria on 9th January 1989 under the Accession Number NCIB 40100.
The isolation and characterisation of these gene sequences of this invention aredescribed in full in WO 93/01294.
Other promoters may also be used, for example a promoter such as the tapetum
specific MFS14 promoter.
The present invention also provides a plant having stably incorporated in its genome
2u by transformation a gene construct carrying a gene construct carrying a tissue-specific or a
development-specific promoter which operates in the cells of the target plant tissue and a
disrupter gene encoding a protein which is capable, when expressed, of inhibiting an essenti~l
cell function such as respiration, microtubule arrays or cell division in the cells of the said
target tissue resulting in death of the cells.
The invention also provides a plant, particularly a monocotyledonous plant, and more
particularly a corn plant, having stably incorporated within its genome a gene construct
carrying a tissue-specific promoter which operates in the cells of the said target tissue and a
disrupter gene encoding a protein which is capable of inhibiting an essenti~l cell function such
as respiration or microtubules in the said cells of the said target tissue resulting in death of the
3() cells characterised in that the said disrupter gene is selected from the T-urfl3 gene, a short
sense construct of the adenine nucleotide translocator, genes encoding an a- or ,~-tubulin and
short sense down-regulation of the essential cell cycle genes, cdc25 and ROA.
CA 02224736 1997-12-16
PCT/GB96/01675
W O 97/04116
These gene constructs may be used as a means of inhibiting cell growth in a range of
organisms from simple unicells to complex multicellular org~nicmC such as plants and animals.
Bv the use of tissue- or cell-specific promoters, particular cells or tissue may be targeted and
- destroyed within complex org~nicmC. One particular application intended for this invention is
in the destruction of cells essential for male flower development, leading to male sterility.
The invention therefore provides a method of preventing or inhibiting growth anddevelopment of plant cells based on gene constructs which inhibit an essenti~l cell function
such as respiration or microtubules. The technique has wide application in a number of crops
where inhibition of particular cells or tissue is required.
Of particular interest is the inhibition of male fertility in maize for the production of Fl
hybrids in si~l. The concept of inhibition of mitochondrial function as a mecll~nicm for male
sterility arises from some previous research on T-type cytoplasmic male sterilitv in maize
(cms-T) which has shown an association between the male sterile phenotype and
mitochondrial dysfuction. Although a direct causal relationship has yet to be established
15 between mitochondrial dysfunction and cms-T, an increasing body of evidence suggests that
fully functional mitochondria, particularly in the tapetal cells, are essential. This is particularly
critical during microsporogenesis since the metabolic dem~n(ls placed on the tapetal cells
results in a 40-fold increase in mitochondrial number.
Thus we provide a number of negative mutations which act upon mitochondria to
2() inhibit functional respiration. When specifically expressed in maize anther tissue these
mutations will result in a male sterile phenotype.
We also use ~ yres~ion of a- or ~-tubulin genes to disrupt cell function. Duringnormal cell life, expression of tubulin genes is closely regulated by their endogenous
promoters and closely matches the requirements of cells for these proteins which are
2~ polymerised and assembled into microtubules during growth and development of the plant. By
expressing tubulin genes in an unre_ulated fashion using non-tubulin promoters in a particular
tissue or stage of development~ the equilibrium between free tubulin monomers and those
polmerised in microtubules is disrupted resulting in instability of the microtubule complex and
cellular dysfuntion. When expressed in the tapetum or other cells of the anther this latter
3() effect will cause the plants to be sterile.
CA 02224736 1997-12-16
W O 97/04116 PCT/GB96/01675
-4-
We also propose the use of short sense down-regulation of essenti~l cell cvcle genes,
eg cdc25 and ROA. When expressed in the tapetum or other cells of the anther this latter
effect will cause the plants to be sterile.
The method employed for transformation of the plant cells is not especially germane
to this invention and any method suitable for the target plant may be employed. Transgenic
plants are obtained by regeneration from the transformed cells. Numerous transformation
procedures are known from the literature such as agluinfe~;Lion usingAgrobac~erium
one~aci~ns or its Ti plasmid, electroporation, microinjection of plant cells and protoplasts,
microprojectile transformation and pollen tube transformation, to mention but a few.
o Reference may be made to the literature for full details of the known methods.
The development and testing of these gene constructs as disrupters of mitochondrial
function in the unicellular organism, yeast, will now be described. A mech~ni~m by which
these gene constructs may be used to inhibit plant cell growth and di~rel~liation in
transformed plants will also be described. The object of these procedures is to use yeast as a
I j model system for the identification and optimisation of gene constructs for ~:Apl essing
proteins which disrupt mitochondrial function. Plant cells will then be transformed with the
selected constructs and whole plants regenerated therefrom.
The accompanying drawings are as follows:
Figure I shows the DNA sequence of an anther-specific cDNA, carried by plasmid
2~ pMS 10,
Figure 2 shows the DNA sequence of a tapetum-specific cDNA, carried by plasmid
pMS I 4;
Figure 3 shows the DNA sequence of an anther-specific cDNA, carried by plasmid
pMS18;
Figure 4 shows the sequence of the T-urfl3 gene (SEQ ID NO I ) with the primers
Turf-l (SEQ ID NO 2) and Turf-2R (SEQ ID NO 3) underlined;
Figure 5 shows DNA encoding the 59 amino acid region from the ATP-2 gene of
Ni~olinia plumbaginifolia ((SEQ ID NOS 4 and 5) with primers PREB-IB (SEQ ID NO 6)
and PREB-R (SEQ ID NO 7) shown;
3() Figure 6 shows the cleavage site of the pre-,~ sequence;
Figure 7 is a map of vector pCaMVIIN,
Figure 8 is a map of vector RMS 17;
CA 02224736 1997-12-16
PCT/GB96/01675
WO 97/04116
Figure 9 is a map of vector pIE109;
Figure 10 shows the MFS14 promoter sequence (SEQ ID NO 8) with the following
features:
position 2198 transcription start CCT"A"CAA (concensus CTC"A"TCA)
position 2167 ATCCATT (possible TATA box motif)
position 2141 CCAT (possible CAAT box motif)
position 2233 cdna start CAC"A"CAG
position 2295 translation start GCAACAATGGCG (concensus TAAACAATGGCT),
Figure 11 is a map of vector RMS I l;
Figure 12 is a map of vector pMANT3
Figure 13 illustrates the contruction of vectors for maize cell line transformation: and
Figure 14 is a plot showing numbers of transformants produced in the various
different experiments.
The invention will now be illustrated by the following Examples.
Example I
Construction of the maize transformation vector. RMS 17.
We used the PCR to amplify the T-urfl3 gene from cms-T maize (line RW33:TMS)
and the mitochondrial targeting sequence, pre-~, from Nic-7~ianu plumbuYinolia DNA
samples from plant material were prepared using the method described by Edwards e~ al
2() (Nucliec Acids Research 1991, 19, 1349).
The complete T-urfl3 gene was amplified in the PCR using primers turf-l
(5'ATCGGATCCATGATCACTACTTTCTTAAACCTTCCT-3', SEQ ID NO 2) and turf-
2R (5'TAGTCTAGATCACGGTACTTGTACGCTATCGGT-3', SEQ ID NO 3) designed
from sequence information provided by Dewey et al ( 1986, Cell, 44, 429-449). The PCR
2~ conditions were 35 cycles of denaturing at 94~C for 0.8 min, annealing at 65~C for I min and
extension at 72~C for 2.5 mins. To aid subsequent cloning the PCR primers were designed
such that they introduce unique BamHI and XbaI restriction sites at the 5' and 3' ends of the
gene respectively. The position of these primers relative to the T-urfl 3 gene sequence is
shown in Figure 4.
3() Similarly the 59 amino region from the ATP2 gene of ~lico~iana plumbaginifolia
which encodes the functional pre-~ mitrochondrial targeting sequence was amplified in the
PCR using the primers PREB-IB
(5'ATCGGTACCGCCATGGCTTCTCGGAGGCTTCTCGCCT-3', SEQ ID NO 6) and
PREB-R (5'ATCGGATCCCGCTGCGGAGGTAGCGTA-3', SEQ ID NO 7) designed using
3~ sequence information provided from Boutry et al (1987, Nature, 328, 341). The PCR
CA 02224736 l997-l2-l6
W O 97/04116 PCT/GB96/01675
-6-
conditions were as described above except that the annealing temperature was reduced to
60~C. To aid subsequent cloning PBEB-IB and PREB-2R were designed such that theyintroduce unique ~nl and BamHI restriction sites at the 5' and 3' ends of the amplified
fragment respectively. The position of these primers relative to the ATP2 gene is shown in
Fi_ure 5.
Following amplification the pre-B PCR fragment was digested with ~nI and BamHI
to generate cohesive ends and cloned into the corresponding sites of the vector pUC 18 to
give plasmid pPB I . The TURF- 13 PCR product was then digested with BamHI and XZ~aI
and cloned into the corresponding sites in pPB 1 to give plasmid pPB2. In pPB2 the pre-~
lo sequence is fused in frame with the T-urfl3 gene so that following e~y,ession in a plant cell
the full product will be transported to mitochondria. Cleavage of the pre-~ sequence at the
predicted site between residues 55-56 will release the T-urfl3 protein which includes at its
NH~-terminus an additional 4 residues from the pre-,B sequence (Figure 6).
The pre-,B/T-urfl 3 gene fusion in pBB2 removed by digestion with the enzymes K~n I
ls and Sal I, blunted-ended and cloned into the plasmid pCAMVIlN (Figure 7) which was
digested with BamHI and blunt-ended to give pPB3. This cloning step places the pre-~/T-
urfl 3 gene fusion under transcriptional control of the CAMV 35S promoter. The Ad~lI intron
is present in this construct to boost ~,uression levels in corn cells (Mascarenhas ct al., 1990.
Plant Mol. Biol., 15, 913-920) and the nos 3' sequence provides a polyA addition site. To
produce the final vector, RMS17 (Figure 8) the PAT selection c~csette from plE109 (Figure
9) which allows in vitro selection of transformed corn cells on bialaphos was introduced as an
EcoRI fragment into the unique l~coRl site of pPB3.
Example 2
Transformation of BMS corn cells with RMS 17.
The objective ofthis experiment was to show that expression ofthe pre,B/TU~-13
gene construct in cultured BMS corn cells results in a reduction in cell viability as measured
by the establishment of transgenic calli following transformation. The RMS 17 vector (Figure
8) was introduced into cultured BMS cells using a silcon carbide fibre-mediated
transformation technique as follows:
Preparation of silicon carbide whiskers
Dry whiskers were alwavs handled in a fume cabinet, to prevent inhalation and
possible lung damage. These whiskers may be carcinogenic as they have similar properties to
CA 02224736 1997-12-16
W O 97/04116 PCT/GB96/01675
asbestos. The Silar SC-9 whiskers were provided by the Advanced Composite Material
Corporation Greer, South Carolina,USA. The sterile whisker suspensions were prepared in
advance as follows. Approximately 50mg of whiskers were deposited into a pre-weighed 1.5
ml Eppendorf tube, which was capped and reweighed to determine the weight of thewhiskers. The cap of the tube was perforated with a syrin e needle and covered with a
double layer of aluminium foil. The tube was autoclaved (121~C, 15psi, for 20 minutes) and
dried. Fresh whisker suspensions were prepared for each experiment, as it had been reported
that the level of DNA transformation when using fresh suspensions was higher than that of
older suspensions. A 5% (weight/volume) whisker suspension was prepared using sterile
u deionised water. This was vortexed for a few seconds to suspend the whiskers immediately
before use.
DNA transformation into cells
All procedures were carried out in a laminar air flow cabinet under aseptic
conditions. The DNA was transformed into the cells using the following approach. Specific
modifications to this method are indicated in the text.
Cell and whisker suspensions were pipetted using cut down Gilson pipette tips. 100~
of fresh BMS medium (see appendix 1 ) was measured into a sterile Eppendorf tube. To this
was added 40ul ofthe 5% (w/v) whisker suspension and 25~1 (Im~!ml) ofthe plasmid DNA,
which was vortexed at top speed for 60 seconds using a desktop vortex unit (Vortex Genie 2
2u Scientific industries, Inc). Immediately after this period of vortexing, 500~11 of the cell
suspension was added ie 250,~1 of packed cells. The Eppendorf tube was then capped and
vortexed at top speed for 60 seconds in an upright position. The same procedure was used to
transform the other cell lines.
Three controls were included in this experiment. Two positive control vectors were
25 pPG3 which contains the PAT selection cassette alone and RMS 15, which is identical to
RMS 17 except that the T-urfl 3 gene is replaced by the mitochondrial uncoupling protein
gene UCP, which jhas no effect on cultured BMS cells. The pre-,~ targeting sequence is
present in both constructs. A negative control, which should completely prevent
establishment of transgenic calli, was provided by RMS 13. which is identical to RMS 17
3u except that the pre~/T-urfl3 gene fusion is replaced by the cytotoxic ribonuclease gene,
barnase.
The mean numbers of transgenic calli established in this e,~pe~ ent are shown inTable 1.
CA 02224736 1997-12-16
W O 97/04116 PCT/G B96/01675
- 8 -
TABLE I
No. of transgenic calli per replicate
Vector Mean 1 2 3
pPG3 ~ 44 33 39
RMS13 0 0 0 0
RMS 15 40 30 43 38
RMS17 12 13 23 16
These data show that relative to the two positive controls, pPG3 and RMS 15, expression of
the preB/T-urfl 3 gene fusion results in a signficant decrease (p~5% or better) in the
establishment of transgenic calli. This suggests that targeting T-urfl 3 protein to mitochondria
has a deleterious effect on these cells, presumably due impartment of mitochondrial function.
Expression of the cytotoxic ribonuclease, barnase, completely abolishes the establishment of
transformed calli.
o Example 3
Construction of the maize transformation vector RMS 11
RMS11 is a transformation vector in which e~p-ession ofthe pre-,B/T-urfl3 gene
fusion is controlled by the maize tapteum promoter, MFS14. The sequence ofthe MFS14
promoter and untranslated leader region from position -2198 to +97 is shown in Figure 10. In
this way, expression of the T-urfl 3 protein is limited to the cells producing pollen and not
throu_hout the whole plant.
To construct RMS 11, the K~n I - SalI fragment from pPB2 cont~ining the pre-~/T-urfl 3 gene fusion was blunt-ended and ligated into the blunt ended BamHI site of plasmid
pSC9 to yield pPB4. In pPB4 the pre-,~/T-urfl3 gene fusion is now positioned between the -
152 to +97 MFS14 promoter fragment and the nos 3' polyadenylation sequence. Thiscomplete cassette was removed from pPB4 by digestion with Sacl and EcoR~ and cloned into
the corresponding sites of pSC7 to give plasmid pB5. pSC7 contains the - 153 to -5800 region
of the MFS 14 promoter so that the introduction of the SacI - EcoRI fragment from pPB4
recreates the full 5.8 kb MFS14 promoter.
2j RMS I I (Figure 11) was completed by introduction of the PAT in vitro selection
cassette from p IE 109 into the unique EcoF'I site of pPB5.
CA 02224736 1997-12-16
W O 97/04116 PCT/G B96/01675
g
Example 4
Transformation of maize cells with RMS I I bv particle bombardment to provide stably
transformed~ male sterile plants
The maize transformation vector, RMS 1 1 was used to transform regenerable maize5 cell cultures by particle bombardment.
Culture Material
Friable embryoyenic Type II callus was initiated from immature zygotic embryos
e,xcised from either greenhouse or filed grown A188 plants 10-12 days after pollination with
pollen from the inbred B73. The medium used for callus initiateion and maintenance was
u based onN6 medillm as modified by Armstrong and Green. Specifically, the medium
contained 6mM L-proline, 2% (w/v) sucrose, 2 mg/l 2,4-dichlorophenoxy- acetic acid (2,4-D)
and 3% (w/v) Gelrite (Trade Mark, Caroline Biological Supply) at pH 6Ø Callus was grown
for 4-4 weeks prior to suspension culture initiation. Suspension cultures were initiated in a
MS-based liquid medium cont~ininv 100 mg/l myo-inositol, 2 mg/l 2,4-D, 2 mg/l
1~ I-naphthalPn~cetic acid (NAA), 6mM proline, 200 mg/l casein hydrolysate (Difco
Laboratories), 35 (w/v sucrose and 5% (v/v) coconut water (Difco Laboratories) at pH 6Ø
Cell suspensions were m~int~ined in theis me~iurn in 125 ml Erlenmeyer flasks at 28~C in the
dark on a gyrating shaker at 125 rpm. Suspension were subcultured every 3.5 days by
addition of 3ml packed volume of cells and 10 ml culture medium to 20 ml of fresh culture
2u medium. The suspension cultures were typically 6 months to one year old at the time of
bombardment. Suspension cultures recovered from cryopreservation were used in some
transformations .
Microprojectile Bolllbaldlllelll
Cell suspensions were sieved through a 1.0 mm and then a 0.5mm screen. A packed
25 volume of 0.2 ml of the cells which passed through the sieves was then suspended in 5 ml of
suspension medium and evenly distributed on to a Whatman No.4 filter paper disc l,ia vacuum
filtration using a 4.7 cm microanalysis holder. Precipitation of supercoiled plasmid DNA on
to tungsten particles and bombardment using the DuPont PDS-1000 Biolistics (Trade Mark)
apparatus were essenti~ly as described by the manufacturers. Target plates were bombarded
3() once.
CA 02224736 1997-12-16
W O 97/04116 PCT/GB96/01675
- 10-
Transformant selection and plant re~eneration
Following bombardment, each filter disc (with cells) was tlansr~lled to N6 basedmedium cont~ining 100 mg/l myo-inositol, 2 mg/l 2,4-D, 3% (w/v) sucrose, and 0.3% (w/v)
Gelrite at pH 6Ø For selection using the NPTII gene, this medium was supplemented with
5 200 mgtl kanamvcin sulphate. The filter discs were transferred to fresh medium cont~ining
the selection agent after seven and again after 14 days. The suspension was divided into two
equal aliquots and each was evenly plated over 20 ml of solidified media cont~ining the
selective agent and 3% (w/v) Gelrite in 100 x 20 mm Petri dishes. After 2-5 weeks, rapidly
growing, putatively transformed calli were removed and transferred to the surface of fresh
1() selection me~ m Plants were renerated by transferring tissue to MS based medium
cont~ining I g/l myo-inositol, 1 mg/l NAA, 6% (w/v) sucrose, and 3% (w/v) Gelrite at pH
6Ø After 2-3 weeks. the tissue was transferred to MS media cont~ininu 0.25 mg/l NAA, and
3% (w/v) sucrose and placed in the light, where embryo ge~nh,dlion occurred. Plants were
then grown in half-strength MS based mer~ m cont~ in~ 500 mg/l myo-inositol, 3% (w/v)
sucrose and 0.3% Gelrite at pH 6.0 for approximately 1-2 weeks priro to transfer to the
greenhouse.
After transfer to the glasshouse, plants within each independently transformed clone
were tested for the presence ofthe MFS14/pre-B/T-urfl3 gene construct using the PCR.
DNA was extracted from small leaf samples using the technique described by Edwards e~ al
2() ( 1991, Nucleic Acids Research, 19, 1349) and used in the PCR with the primers, 14-SA (5'-
AGACGCTGAGCTCAAGGACGTGA-3' SEQ ID NO 9)and turf-2R (see Example I for the
sequence of this primer).
At flowering the plants within each independently transformed clone were assessed in
the glasshouse using a visual scale developed for CMS lines and described in Table 2. Plants
scoring 4 and below are functionally sterile. The accu~ ted sterility scores for each of the
independent PCR positive clones is shown in Table 3 and compared to a maize line which was
generated by bombardment with RMS 1 1 but which is PCR negative for the MFS 14/pre-B/T-
urfl3 gene construct.
Sterile plants were backcrossed with pollen from fertile, non-transgenic BE70 plants.
the progeny seeds arising from one of these crosses (clone YK23, plant 5 x BE70) were
planted in the glasshouse and allowed to flower. The presence of the MFS 1 4/pre-B/T-urfl 3
gene construct as assessed by PCR and PAT test (the latter determines whether the selectable
CA 02224736 1997-12-16
WO 97/04116 PCT/GB96/01675
marker used inthe transformation process is present) and the fertility scores of the plants are
shown in Table 4. Plants which were PCR negative were rogued from the glasshouse prior to
flowering. Plants I and 2 had an inconclusive PCR test and were kept until flowering. Plant
12 was kept as a control. As can be seen in Table 4, 6 ofthe pro_eny plants were sterile and
s this sterilitv correlated with the presence of the transgene as assessed either by PCR or PAT
testin_ This is consistent the the presence of a sin_le transgenic locus imparting sterility.
TABLE 2
Anther Classification (After L.M. Josephson)
Class 0 No anthers exerted
Class I Less than half of the anthers exerted and all were small, dry and hard with
no pollen shed.
Class 2 Most of anthers exerted but all were small. dary and hard with no pollen
shed.
Class 3 Partially fertile anthers exerted with some pollen shed, proportion of
anthers exerted was highly variable.
Class 4 Slightly abnormal anthers with approximately 75 to 100 percent exertion.
Class S Normal anthers and fully fertile.
1~ * Class 4 through 5 tassels were considered fertile.
TABLE 3
CLONE NO. OF PLANTS FERTILITY SCORE P.C.R.
WK23 2 0 +
3 3 +
WK23 2 0 +
YK23 3 0 +
4 +
YE23 4 5
CA 02224736 1997-12-16
PCT/GB96/01675
W O 97/04116 -12-
TABLE 4
PLA PCR TEST PAT TEST FERTILITY SCORE
NT
YK23/51 1 +/ O
YK23/5/2 +/- + o
YK23/5/3 - ROGUED
YK23/514 - ROGUED
YEC23/5/5 NO GERMINATION
YK23/516 + +
YK23/5/7 + + O
YK2315/8 - ROGUED
YK23/5/9 + + O
YK23/5/ 10 - ROGUED
YK23/5/1 1 + + O
Y~C23/5/12 - - 5
Example 5
Construction of the maize tran~rn,dlion vector pRMS-23
We have tested whether e~ression of a short-sense construct from the maize adenine
nucleotide translocator (ANT) gene will give rise to a defect in the growth of maize cells. A
fragment of the maize ANT was isolated using the PCR and primers designed from the
sequence ofthe maize gene published by Bathgate et al (1989, Eur. J. Biochem., 83, 303-
310).
o The fragment of theANT gene was amplified in the PCR using primers MANT- 1(5'-
ATGCCCGGGCTTGCAATGTCTGTTAGCGGTGGCATCA-3', SEQ ID NO 10) and
MANT-2RB (5-ATGCCCGGGCGATGGGGTAAGATGCAAGACCA-3', SEQ ID NO 11).
The PCR conditions were 35 cycles of denaturing at 94~C for 0.8 min, ~nn~linv at 65~C for
1 min and extension at 72~C for 2.5 mins. To aid subsequent cloning the PCR primers were
designed such that they introduce unique SmaI restriction sites at the 5' and 3' ends of the
gene. The sequence of the maize ANT gene was published in Eur. J. Biochem. ( 1989) 183,
303-310. The MANT-1 primer sequence appears at the bevinninv ofthe coding sequence of
the gene and the MANT-2R primer sequence is near the end of the gene.
CA 02224736 1997-12-16
WO 97/04116 PCT/GB96/01675
Following PCR which produced a DNA fragment of the predicted size of 1050 bp, the
DNA was digested withSmaI and subcloned into theS~nal site of pUC18 to give pMANTI.
Subsequently the nos 3' polyadenylation signal sequence was introduced 3' to the ANT gene
as a SacI- l~coRI fragment into the corresponding sites in pMANTI to yield pMANT2. A
/~indIII - Ba~nHI fragment carrying the CaMV 35s promoter and ADH lintron from
pCaMVI~N (Figure S) was introduced into the corresponding sites of pMANT2 to yield
pMANT 3. pRMS-23 (Figure 12) was completed by introduction ofthe PAT in vitro
selection cassette
from pIE 109 (Figure 7) into the unique ~coR~ site of pMANT3 .
1~ Example 6
Transformation of BMS corn cells with pRMS-23
The objective of this experiment was to show that expression of pRMS-23 in cultured
BMS corn cells results in a reduction in cell viability as measured by the establichm~nt of
transgenic calli following transformation in two separate experiments. The vector DNAs
1~ were introduced into cultured BMS cells using the silcon carbide fibre transformation
techniqiue as described in Example 2.
Following transformation with pRMS23 the mean numbers of transgenic calli
established were determined relative a positive control pPG3 wihich contains the in vi~ro
selection cassette alone (Table 5). These data show that expression of a short sense adeneine
2() nucleotide translocator gene results in a significant decrease in the establishment of transgenic
calli.
TABLE 5
No of transgenic calli per replicate
2 3 Mean
Experiment I
pPG3 16 20 22 19
pRMS-23 3 4 3 3
Experiment 2
pPG3 49 48 40 46
pRMS-23 10 15 23 16
CA 02224736 1997-12-16
W O 97/04116 PCT/G B96/01675
- 14 -
Example 7
Construction of the maize tran~r~l ,llation vectors. pTBR and pTBS
We tested whether un-regul~ted expression of a-tubulin genes will give rise to adefect in the growth of maize cells. Two constructs cont~ining the coding sequence from a-
tubulin cDNAs isolated from two biotypes of Eleusine indica were prepared. pTBR (Figure
13) contains the a-tubulin cDNA from a dinitroaniline resistant biotype of Eleusine indica
cloned as a blunt-ended h'inf I fragment into the blunt-ended BamHI site of pCaMVIlN
(Figure 7). pTBS (Figure 13) contains the a-tubulin cDNA from a dinitroaniline sensitive
biotype cloned exactly as described for pTBR.
IU Example 8
Transformation of BMS corn cells with pTBR and pTBS.
The objective of this experiment was to show that e.~l es~ion of pTBR and pTBS in
cultured BMS corn cells results in a reduction in cell viability as measured by the
establishment oftransgenic calli following Lldllsrollllation. The vector DNAs were introduced
15 into cultured BMS cells using the silcon carbide fibre transformation techniqiue as described
in Example 2.
Following transformation with pTBR and pTBS the mean numbers of transgenic calliestablished were determined relative a positive control pPG3 wihich contains the in ~,itro
selection cassette alone (Figure 14). These data show that non-regul~ted e~,ression of an a-
20 tubulin gene from either biotype of Eleusine indica results in a significant decrease in theestablishment of transgenic calli.
CA 02224736 l997-l2-l6
W O 97/04116 PCT/G B96/01675
1~
SEQUENCE LISTING
(l) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: ZENECA LIMITED
(B) STREET: 15 Stanhope Gate
(C) CITY: London
I() (E) COUNTRY: UK
(F) POSTAL CODE (ZIP): WlY 6LN
(ii) TITLE OF INVENTION: Production of Male Sterile Plants
(iil) NUMBER OF SEQUENCES: 11
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 357 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
3~ (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: T-urfl3 gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
I ()
ATCGGATCCA TGATCACTAC TTTCTTAAAC CTTCCTCCCT .TGATCAAGG TTTGGTATTT 60
TTCGGTTCTA TTTTTATTTT TTTTTTGTGC ATATTATTGA TAAAGGGATA TCTCCGTAAA 120
~5 ATGGATGATT CCTATTTGGC TCAACTCTCC GAGTTAGCCA ACCACAATAG AGTGGAAGCG 180
GCAAAAGCGG GCCACGTGGC CCTGCATGAG CTATCCTTCT CGTGGTTGAG GGGGGTTCAA 240
ATTAGGGTGA GGACCTTACC TATACAACGG AATGAAGGAG GGGGTCGAAG CAACGACCAA 300
TCCACTCTCT CTAAGCCTAA GTATTCCTCA ATGACCGATA GCGTACAAGT ACCGTGA 357
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
6~
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Turf-l primer
G5
CA 02224736 1997-12-16
W O 97/04116 PCT/GB96/01675
-16-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
5 ATCGGATCCA TGATCACTAC TTTCTTAAAC CTTCCT 36
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(Dl TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Turf-2R primer
2U
ixi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TAGTCTAGAT CACGGTACTT GTACGCTATC GGT 33
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ATP-2 gene of Nicotinia plumbaginifolia
(ix) FEATURE:
4() (A) NAME/KEY: CDS
(B) LOCATION:l..267
(D) OTHER INFORMATION:~codon_start= l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
ATG GCT TCT CGG AGG CTT CTC GCC TCT CTC CTC CGT CAA TCG GCT CAA 48
Met Ala Ser Arg Arg Leu Leu Ala Ser Leu Leu Arg Gln Ser Ala Gln
l 5 l0 15
CGT GGC GGC GGT CTA ATT TCC CGA TCG TCA GGA AAC TCC ATC CCT AAA 96
Arg Gly Gly Gly Leu Ile Ser Arg Ser Ser Gly Asn Ser Ile Pro Lys
20 25 30
55 TCC GCT TCA CGC GCC TCT TCA CGC GCA TCC CCT AAG GGA TTC CTC TTA 144
Ser Ala Ser Arg Ala Ser Ser Arg Ala Ser Pro Lys Gly Phe Leu Leu
35 40 45
AAC CGC GCC GTA CAG TAC GCT ACC TCC GCA GCG GCA CCG GCA TCT CAG 192
60 Asn Arg Ala Val Gln Tyr Ala Thr Ser Ala Ala Ala Pro Ala Ser Gln
50 55 60
CCA TCA ACA CCA CCA AAG TCC GCC AGT GAA CCG TCC GGA AAA ATT ACC 240
CA 02224736 1997-12-16
PCT/GB96/01675
W O 97/04116
-17-
Pro Ser Thr Pro Pro Lys Ser Ala Ser Glu Pro Ser Gly Lys Ile Thr
65 70 75 80
GAT GAG TTC ACC GGC GCT GGT TCG ATC GG 269
5 Asp Glu Phe Thr Gly Ala Gly Ser Ile
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 89 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Ala Ser Arg Arg Leu Leu Ala Ser Leu Leu Arg Gln Ser Ala Gln
20 l 5 l0 15
Arg Gly Gly Gly Leu Ile Ser Arg Ser Ser Gly Asn Ser Ile Pro Lys
Z5 30
Ser Ala Ser Arg Ala Ser Ser Arg Ala Ser Pro Lys Gly Phe Leu Leu
Asn Arg Ala Val Gln Tyr Ala Thr Ser Ala Ala Ala Pro Ala Ser Gln
Pro Ser Thr Pro Pro Lys Ser Ala Ser Glu Pro Ser Gly Lys Ile Thr
65 70 75 80
Asp Glu Phe Thr Gly Ala Gly Ser Ile
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~5 (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: PREB-IB primer
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ATCGGTACCG CCATGGCTTC TCGGAGGCTT CTCGCCT 37
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
- -
CA 02224736 1997-12-16
W O 97/04116 PCT/GB96/01675
-18-
ivi) ORIGINAL SOURCE:
(A) ORGANISM: PREB-2R primer
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
ATCGvALCCC GCTGCGGAGG TAGCGTA 27
l() !2) INFORMATION FOR SEQ ID NO: 9:
i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2285 base pairs
(B) TYPE: nucleic acld
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
2() (vi) ORIGINAL SOURCE:
(A) ORGANISM: MFSl4 Promoter
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
AAAAGCGTAC CAGTAAGGGA TAAAGAAAAT AAACAAACAC GAAATGCTTC CCCATCGGCC 60
AATTCGCCTA GGTGTGCTAG GAACTGGCCT ATATGTTCGT GTGTGCTTCT CCTATTTTCA 120
3~
CCAGAAAACT TAGAAAACTC TGGTATCCTT GCCCCTTGTG GATATGGGAC AATGTCAAAC l80
CGGTGATCAT ATGGCTTCTG ATATGATTGC CCCACTCTAT CCACACCAAC TCCGAGTCTA 240
TCTCAAAATA GTTTAGCCAT CTCTTCCCTA ATTTTCTACA TTGCACTCGG TGGCAGACCA 300
CCGGACCCTA GGCTGTGGGG TTCATTCGGT CGGGCATTGT TATGCCGACC TTCTTGCCAT 360
GACCGATTGA TAATGTTGAT CGGCCTGTGA TCATATGGCG TGTTGTGGGT TAAATATGTA 420
I()
GGGGGCAGAA CATACTGCCG TTGTGGTATG TAATAATTTG GTGCATAGTG TGCGACAGTA 480
GGTTCTGTGT ATGTGTATCC GATATGTCCG GTGGTACATC TGAACTGGCC GGTTGTGTTA 540
GCTATTATTG GGGCGCCACG CGTAGCCCTG GTGCGGCCCG GACTATCCGG CAGAGAAAGC 600
CGACGGTCTG TGTAAGGGCC GAACTATCCA GACAAAAGCT CGGACGGTCC GACCGTGTAG 660
AGGGCCGTCG ATCTGCCAAG CAAGGACGAT GGTGATGGTA TTTGCCCTGG ATATGAGTTC 720
5~
ATCAACATAC CATATAATGG ATGGGGCTGC AAATCCCCAT TTGTCGCCGA TGTATTAGAC 780
ATAAATATCA TGTTACTAGT TTCATATGAT GGAAAACTAG GAGCAACAGA CTTCTCCAAC 840
~5 ATACACGTTA ATTTTCTAAT TGGTTCTTCT AACCCTCTAA TCTAATGCTT CATTTGATTA 900
TGCAAATGGT CTACATACTG TTTAATAGAT TGGATGTCGT CGGGTTTACT TACGTTAGGG 960
ACTTGAAGCG AAGATAGAAG AGATGTGACG TCGGTATCGC ATGTTTGACA ACTTTCTGGT l020
6()
GACGATCCAC CATGTATTGT GACAAGAATT TCTCCTTCGT TTGACACATG TAGTCCTCGT 1080
ATTGTTGTTG CTCATCGGTC GTCGGACTCT TAATAGCCGG CTTTAGGATA TTGTCCGGGG 1140
(5 AGATATCGGT GTGATCTTTA GAACCGCCAT TTGATGGCCT GAGTTTTAGT AGATCTAGAC 1200
CA 02224736 1997-12-16
PCT/GB96/01 675
WO 97/04116
- 19-
ACATTTCCCC AACGGAGTCG CCAAAAAGTG TGTTGGCGCC GATCCAGGCG CGAAACACTG 1260
GAGATGGACC GTTTGGCGGT GTTCTCCGGG TGAGGACGGT CCGCGACCTG GGTCCAGCAG 1320
-
- CGACTCTCCT CTACGTGTGT CCGGACGGTC CGTCGTCTGG GGCTCGGACG GTCGCGATGG 1380
CGCAGAG5GT CTTCTTCTTC GCAGCCGACC TAGATCTCGC CTCCCGGGAG GGACCGTCGG 1440
GGAGGAGAGA TTGTAGGGTG TGTCTTGGCG TCGACAGGCC ACACAATACG CCTCTAGTCG 1500
ACGTAGAGCC GAAGAGAGGT GAAGGATTGA GGTAGAAGGA GGCTAAACTT GGGCTAAACT 1560
AGAACTACTG CTAATGCATA AGGTAAAAAC GAGAAGTGGA CTTCATTTGA TCGATTGTGG 1620
AAGTAATCTG ACTGTAGCCC TTTATCTATA TAAAGGGGAG GTATGGACCC GTTACAAGCC 1680
GTTTTCCGAG CTAATCTCAC GGTTTTAGTT AATAAATCCT GCGAGAAACT CGGAACTCTA 1740
2~ ACTGATTCTA CTCATGCGCG AACCATTCGT GCGCCACCGC TGCCCGTCCC GCGATCGCTC 1800
AGTTAACCCT GTGTTGTGCG CTGTGATTTG GTGGCATATA AAACCACATT TGCAATAAAA 1860
ATTTGTAGGG ATTTAACATA CCAAGTGCTG CGAAAGGAAT CGTTTTCGGA GGACCCAAAA 1920
TTAAAGAGGC AGATGCTAGA GCTCGTCCAG CTCAGCGCTG AGCACCTGTG TTGTCTTCCT 1980
CGTCCACGCC GGCGGAGATG AACGGCAACA AAGGCGGAAA GGCCGAGACG CTGAGCTCAA 2040
GGACGTGACA CCGCGCGTAC CTCGCGTTCA GTTGGCTCAC ACAACAGCAG CTCGCTCGCC 2100
CCAAGCTCCC GCGTCCTGAT CCGTAGGTGA GCCATGCAAA GGTCGCCGCG CGCCCTGATC 2160
CATTGCACCC TTCAAAGCTC GAACCTACAA ATAGCGTGCA CCAGGCATCC TGGCCACACC 2220
CACACAGCAA GCCAGCAGAG CAGAAAGCAG CCGCAGCCCC AGCCCCCACA AAGACGAAGG 2280
CAACA 2285
It) (2) INFORMATI..~ FOR SEQ ID NO: 9:
(i) SEQ YNCE CHARACTERISTICS:
(A LENGTH: 23 base pairs
(E) TYPE: nucleic acid
'; (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: 14-SA Primer
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
AGACGCTGAG CTCAAGGACG TGA 23
(2) INFORMATION FOR SEQ ID NO: 10:
6~
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02224736 1997-12-16
PCT/G B96/01675
W O 97/04116
~o
(i ) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: MANT-i Primer
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO: l0:
1()
ATGCCCGGGC TTGCAATGTC TGTTAGCGGT GGCATCA 37
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base palrs
(B) TYPE: nucleic acld
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
2()
! ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: MANT-2RB Primer
(xij SEQUENCE DESCRIPTION: SEQ ID NO: ll:
3() ATGCCCGGGC GATGGGGTAA GATGCAAGAC CA 32
