Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W092/0620~ PCI/EP91/018B3
~ 2089072
Title: A process for the gene manipulation of plant cells,
recombinant plasmids, recombinant bacteria, plants
T~.is i~.ventio~. .elates to a process for the gene
manipulatic of plant cells, which comprises introducing
foreign DNA into plant cells by infecting the plant cells wlth
one or more recombinant Aarobacter~um tum~aciens strains,
which contain foreign DNA to be transferred to thé plant cells
between the left- and right-hand ends of T-DNA necessary for
such a transfer and are capable of transferring this foreign
DNA to the plant cells.
The invention further relates to a recombinant plasmid
consisting of a bacterial vector plasmid and at least one DNA
insert, and to a recombinant Aarobact~rl~m tumefacie~s strain,
which contains foreign DNA to be transferred to plant cells
between the left- and right-hand ends of T-DNA necessary for
such a transfer and is capable of transferring this foreign
DNA to the plant cells.
Finally, the invention also relates to the plants
deriving from the transformed cells and to the parts
consisting of one or more cells or products thereof.
The Aarobacterium tumefaciens technology for the gene
manipulation of plants, which technology has strongly
developed and has been applied on a large scale since its
first discovery, is in principle still limited, despite all
progress made through the years, to the application in
specific, substantially dicotyl plant species, such as
tobacco, tomato, colza etc. Up to the present time a
successful transformation of monocotyl plant species has
essentially only been described for some Liliaceae, namely
~ Asparagus (Bytebier et al, Proc. Natl. Acad. Sci. USA 84,
; 5345-5349, 1987) and Narcissus ~Hooykaas et al, Nature ~11,
; -- 30 763-764, 1984), some Iridaceae (Graves et al, J. Bacteriol.
1~9, 1745-1746, 1987), some Dioscoreaceae (Schafer et al,
; Nature 3~7, 529-532, 1987), and some Gramineae, namely Zea
mays (Graves and Goldman, Plant Mol. Biol. 7, ~3-50, 1986) and
Oryza sativa (Raineri et al, Biotechnology 8, 33-38, 1990).
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W092/06205 PCT/EP9l/01883 '
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Apart from these exceptions, one has not yet succeeded in
realizing by means of the Aarobacterium ~umefaciens technology
an efficient t-~nsform~ic.. of mv..ocotyl plant species,
including the important cereals such as maize, wheat, barley,
rye, oat, rice etc. and in producing stably transformed
monocotyl plants. In a broader sense this applies to many
species of plants, not only to the cereals belonging to the
group of monocotyl plants but also to grasses such as ryegrass
and fescue, leguminous plants such as leek, onion and garlic,
ornamental plants such as tulipe, hyacinth, bromeliad, irls,
orchid etc., and other plant species such as conifer etc.
There seem to be indications that the problem is not due
to the transfer of the DNA. Thus it has been found possible to
transmit viral genomes encoded in the form of DNA by means of
Agrobacterium tumefaciens to maize (Grimsley et al,
Biotechnology 6, 185-189, 1988) and wheat (Dale et al, Seventh
International Wheat Genetics Symposium 1, 719-722, 1988).
Perhaps the problem is therefore due to the integration of the
DNA in the genome: the integration system which Aarobacterium
tumefaciens bacteria successfully use in dicotyl plants might
not or not efficiently work in monocotyl plants. Generally,
- however, it is assumed that a poor transfer of the T-DNA to
the plant is responsible for the failure of the technology in
monocotyl plants.
:
~i 25 In order yet to obtain the desired transformation in
; monocotyl plants, different alternative transformation
techniques have been conceived and developed in which no use
is made of Aarobacterium ~umefaciens bacteria for transmitting
DNA to the plant cell. Examples thereof are microinjection
with DNA, bombardment with DNA-carrying particles,
electroporation of protoplasts with DNA etc.
These techniques have been successfully employed for the
~ introduction of DNA into plant cells, but generally only a
- passing expression of the genes introduced could be observed.
As regards the use of integration systems, the selection
made so far concerns either homologous genetic recombination,
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W092/06~0~ PCT/EP91/01883
,~ 2089~72
which is a hardly efficient system and remains limited inpractice to cells ln meiosis, or transposition, which is an
efficiens s-ystem and is also active during the normal cell
cycle but has the drawback that the genes introduced are not
stable in the flrst generation and that it is necessary to
wait one or more generations before the transposon and the
genes introduced via transposition are separated by
mendelizing out.
Transposons are, in fact, genetic elements which can
- lO move within the genome of the host cells in which they are
normally present. They have virtual ends, repeated border
sequences which are in general invertedly repeated and may be
rather different in length. At the location of their insertion
into the genome a piece of host sequence is found back
directly repeated to the left and the right of the transposon
. sequence. There is generally no specific selection of the
location of insertion. In case of excision either the original
sequence is restored or a deletion occurs or part of the
sequence remains at the relevant location (insertion). In its
simplest form a transposon consists of a transposase gene
located between terminal sequences which contain transposase
binding sites and optional binding sites for other host
enzymes and terminate with the repeated border sequence. These
terminal sequences, together with the directly repeated
sequences of the target DNA to the left- and right-hand of the
border sequence, will also be referred to hereinbelow as left-
and right-hand transposon ends.
The present invention provides a process which removes
.,
the main objections of the known methods and gives a very
` ' 30 efficient and stable transformation of cells of monocotyl
.
, , plants, whlch has not been found possible so far. For this
purpose use is made according to the invention of the
~ efficient transfer system of Aqrobacterium tumefaciens
5 ~ bacteria in combination with the efficient integration system
~ 35 of transposons.
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The invention relates in a first aspect to a process for
the gene manipulation of cells of plants which, in essence,
c~nnot be transformed by Aarobacterium tumeLaciens wi~h
integration by T-DNA, with foreign DNA being introduced into
the plant cells by infecting the plant cells with one or more
recombinant Aarobacterium tumefaciens strains which contain
foreign DNA to be transferred to the plant cells between the
left- and right-hand ends of T-DNA necessary for such a
transfer and are capable of transferring this foreign DNA to
the plant cells, the foreign DNA located between the left and
right-hand ends of T-DNA comprising:
(A) a transposase gene located in an expression cassette
active in the plant cells but not between the left- and right-
hand transposon ends necessary for integration in the DNA of
the plant cells, and
(B) a recombinant transposon comprising the left- and right-
- hand transposon ends necessary for integration in the DNA of
the plant cells with an intermediate DNA fragment to be
integrated in the DNA of the plant cells but containing no D~A
fragment which can lead in the plant cells to expression of an
active transposase.
By the words "plants that, in essence, cannot be
transformed by Aarobacterium tume~aciens with integration by
T-DNA" are meant all those plant species in which
` 25 A~robacterium tumefaciens is not capable of effecting an
efficient integration of the T-DNA in the plant genome, which
~ roughly means almost all the non-dicotyl plants. The invention
is therefore eminently suited for the gene manipulatin of
monocotyl plants and other non-dicotyl plants, preference
being given according to the invention to gene manipulation of
~` plant cells of cereals such as maize, wheat, barley, rye, oat,
rice etc., grasses such as ryegrass and fescue, leguminous
j plants such as leek, onion and garlic, ornamental plants such
'` ~ as tulipe, hyacinth etc. and other plant species such as
~ 35 conifer etc.
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W092/0620~ PCT/EP91/01883
~ 208~72
According to the invention there is used a transposase
gene which is not located between the left- and right-hand
transposon ends necessary fur ir,.egra~ior, in the DNA of the
plant cells. According to the invention there is further used
- 5 a recombinant transposon containing no DNA fragment which can
lead in the plant cells to expression of an active
transposase. This means that the ecombinant transposon is an
inactive transposon. Consequently, a desirable high stability
of the transformed plants can be obtained. The drawback of
instability.normally connected with the use of transposons is,
in fact, the result of the presence of a complete (or active)
transposon, i.e. a transposon with a transposase gene located
between the transposon ends. This gene is then co-incorporated
with the transposon in the plant genome and may therefore give
rise for several generations to the undesirable instability.
According to the present invention there is used an active
transposase gene, namely in order to ensure efficient
integration of the foreign DNA located in a transposon in the
DNA of the plant cell (in the plant cell the transposase gene
leads to the formation of transposase which, in cooperation
with nuclear factors of the plant, is capable of effecting
efficient integration of the transposon in the genome) but if
this transposase gene is not within a transposon, in
conformity with the preferred embodiment, it is not itself
efficiently integrated in ~he genome in case of transformation
of non-dicotylidons, such as in particular monocotylidons.
This means that the desired stability is obtained already in
~, the first generation of plants.
The transposase gene preferably consists of a genomic
clone of the transposase gene, i.e. of genomic DNA which
comprises not only the encoding exons but also the in~rons
normally present. Although the use of a cDNA coding for ~he
transposase is also possible, practice has shown that it gives
a substantially less efficient integration of the recombinant
transposon. The use of a genomic transposase gene does not
mean that the natural promoter of the transposase gene should
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W092/06205 PCT/EPg1/01883
be used too. Practice has shown that it is very advantageous
to use, instead of the natural promoter, another promoter
active in pla..ts, in particular a promoter that can errec~
efficient e~pression in cells from which germinative cells
develop. A promoter suitable for this purpose is the known
CaMV 35S promoter. The use of the pTRl' or pTR2' promoter is
also posible and favorable if, as is also preferably done
according to the invention, transfer occurs by microinjection
with the apical meristem being mechanically damaged. As a
result, the wound-inducible regulators of gene expression for
these promoters are present in the meristematic cells. The use
of such a construct of a genomic transposase gene under
control of another promoter such as the CaMV 35S, the pTRl'
or the pTR2' promoter, therefore constitutes a highly
preferred embodiment of the invention.
According to the invention it is particularly preferred
that between the left- and right-hand transposon ends the
recombinant transposon comprises at least one foreign gene
located in an expression cassette active in the plant cells.
By this is meant any gene that is to be incorporated in the
genome of the plant for whatever reason. It may be a gene
coding for a desirable property of the plant, such as
resistance to herbicides, to pathogens, such as specific funai
and insects, or to environmental factors, or properties, such
. 25 as (in ornamental plant cultivation) a specific flower color,
flower scent, flower size etc., or properties such as (in case
of food crop) better nutritional properties, a higher yield
- etc. It may also be a gene coding for a substance one wishes
to produce on a large scale by cultivating a plant capable of
~ 30 forming the substance by means of gene manipulation. The gene
i to be introduced into the plant may therefore be a gene within
the scope of plant breedlng or a gene within the scope of
biological production of specific products ~proteins and
- enzymatic products, DNA, RNA etc.).
It is also possible that between the left- and right-
, hand transposon ends the recombinant transposon contains a
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W092/06205 PCT/EP91/01883
720~9~7~ `
promoter active in plants, instead of a foreign gene in an
expression cassette active in plants. Consequently, it becomes
possible to alter the expression behavior of specifi~ genes
and thus the phenotype.
S It is further preferred that also one or more selectable
marker genes suitable for selection of transformed bacteria
and located in an expresslon cassette active in the bacteria
are located between the left- and right-hand ends of T-DNA.
This enables selection of Aarobacterium tumefacien,s bacteria
with the desired DNA constructs. Marker genes suitable for
this purpose are known to those skilled in the art. These are
often genes the expression of which leads to a resistance tO
specific antibiotics, such as resistance to kanamycin,
chloramphenicol, ampiclllln, tetracycline, spectinomycin etc.
It is particularly preferred here that one or more
selectable marker genes suit~ble for selection of transformed
bacteria and located in an expression cassette active in
: bacteria form part of the recombinant transposon. This
preferred embodiment has the advantage that the marker genes
coupled to the foreign gene are co-incorporated in the genome
of the plant so that in cases that as a result of the
incorporation of the recombinant transposon in a gene of the
,~ host the transformed plant shows another phenotype (which is
not due to the introduced genetic information as such) or that
the foreign gene introduced proves to function differently in
later generations than in the first generations (e.g. as a
result of mutation) the recombinant transposon can be cloned
for further examination from the plant genome into bacteria
again by means of selection with the marker gene active in
bacteria.
It is further preferred that also one or more selectable
marker genes suitable for selection of transformed plant cells
` and located in an expression cassette active in the plant
cells are located between the left- and right-hand ends of
T-DNA. Also here it applies that many marker genes suitable
for this purpose are known to those skilled in the art. Thus
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W092/06205 PCT/EP91/01883
9~ 8 ~'
the resistance to, e.g., antibiotics or herbicides can be
mentioned as a property which, as ls known to those skilled in
the art, car. be used fo- a selec~ion cf ~uccessfully
transformed plants.
It is particularly preferred here that one or more
selectable marker genes suitable for selection of transformed
plant cells and located in an expression cassette active ln
the plant cells form part of the recombinant transposon. This
preferred embodiment has the advantage that the marker genes
are co-inco~porated in the genome of the plant so that also
later generations can be selected on the basis of the
properties encoded by the marker genes.
A firs~ variant of the process according to the
invention is characterized in that the plant cells are
infected with a mixture of two different recombinant ~.
tume~faclens strains, one of which comprises between the left-
and right-hand ends of T-DNA the transposase gene located in
an expression cassette active in the plant cells and the other
the recombinant transposon between the left- and right-hand
, 20 ends of T-DNA. This variant has the advantage of having a wide
; range of applications, i.e. there can always be used the same
'' Ag~,,ok~terlum strain supplying the transposase gene and only
the second strain containing the recombinant transposon must
be constructed for each new gene. In this second strain there
is used a construct in which a multiple cloning site is
located between the transposon ends so as to have a great
freedom of choice with respect to the DNA fragments to be
' ~ incorporated in the transposon.
,:;!' In practice, in this first variant according to the
''' 30 invention both the first and the second A. tumefa~,i~n~ strain
will comprise between the left- and right-hand ends of T-DNA
, at least one marker gene for selection of transformed bacteria
and at least one marker gene for selection of transformed
plant cells. It is preferred here that at least the marker
genes for selection of transformed plant cells which carry the
, two bacterial strains within the T-DNA ends are different from
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W092/06205 PCT/EP91/01883
~ , 9 20~9~72
each other so as to recognize plant cells successfully
transformed by the two strains.
.~n ltern~tivre embodlmcnt of the process according tG
the invention is characterized in that the plant cells are
` 5 infected with one recombinant ~. tumefaciens strain which
comprises between the left- and right-hand ends of T-DNA both
the transposase gene located in an e~pression cassette active
in the plant cells and the recombinant transposon. This
variant has the advantage of more chance of success of the
transformation, i.e. the desired DNA transfer and integration
will have taken place in a larger part of the treated plant
cells.
In practice, in this second variant according to the
invention the ~. tumefaciens strain will comprise between the
lS left- and right-hand ends of T-DNA at least one marker gene
for selection of transformed bacteria and at least one marker
gene for selection of transformed plant cells.
, ~ The process according to the invention is of special
~ importance to the transformation of plant cells of a monocotyl
* 20 plant species such as maize, wheat, barley, rye, oat, rice
etc. An efficient transformation of such crops has not been
found possible so far.
The invention can also be used, however, for the purpose
j of altering phenotypic characteristics of plants resulting
25 from the presence of inactive transposons in one or more genes
~ of the plant by removing such a transposon from the gene in
- which it is contained. For this purpose the invention provides
a process for the gene manipulation of cells of plants which,
in essence, cannot be transformed by Aarobacterium tumefaciens
30 with integration by T-DNA, which comprises introducing foreign
DN~ into the plant cells by infecting the plant cells with a
~ recombinant Aarobacterium tumefaciens strain which contains
r foreign DNA to be transferred to the plant cells between the
left- and right-hand ends of T-DNA necessary for such a
35 transfer and is capable of transferring this foreign DNA to
the plant cells, which process is characterized in that plant
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W092/06205 ~ PCT/EP9l/01883
cells are infected which contaln one or more inactive
transposons but no active transposase gene and that the
foreisn DNA located between the lef~.- and right~ and end~ ~f
T-DNA comprises a transposase gene located in an expression
cassette active in the plant cells, which cassette is not
located between the left- and right-hand transposon ends
necessary for integration in the DNA of the plant cells.
The present invention is not subject to special
limitations regarding the method of contacting the plant cells
to be transformed with the Aarobacterium tumefaciens bacteria.
In order to be able to obtain several generations of
transformed plants, it is particularly preferred, however,
that cells of the apical meristem in germinating seeds are
infected with the bacteria.
In connection with practical advantages, in particular a
high production per time unit and a high precision, it is
preferred that the infection is carried out by means of
needleless high-pressure injection. By this is meant a
bombardment directed to cells of the apical meristem under
high pressure of germinating seeds of the plants to be
transformed, with a liquid containing the Aarobacte~i_m
~umefaciens bacteria. Instruments suitable for such a
bombardment, such as a high-pressure '`pistol", are known per :
se.
The invention, however, is not limited to such a method
and comprises the possibility of carrying out the infection by
means of microinjection. Moreover, also other meristematic or
embryogenic tissues or cells can be used for introducing a
foreign gene.
In another aspect the invention relates to a recombinant
~`~ plasmid consisting of a bacterial vector plasmid and at least
one DNA insert, which recombinant plasmid is characterized in
that at least one DNA insert comprises a transposase gene
located in an expression cassette active in plant cells, which
expression cassette active in plant cells and filled with a
transposase gene is not located between the left- and right-
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W092/06205 PCT/EP9~/01883
~ 2~89072 ;''''`'' ;'
hand transposon ends necessary for integration in the DNA of
plant cells.
The invention further l-elatcs .o c recombinant piasmid
consisting of a bacterlal vector plasmid and at least one DNA
S insert, which recombinant plasmid is characterized in that at
least one DNA insert comprises a recombinant transposon which
contains the left- and right~hand transposon ends necessary
for integration in the DNA of plant cells but no DNA fragment
which can lead in plant cells to expression of an active
transposase.
A preferred embodiment of such a recombinant plasmid is
characterized in that between the left- and right-hand
transposon ends the recombinant transposon comprises one or
more restriction endonuclease recognition sequences suitable
as cloning site. Such a plasmid has a wide range of
applications as a carrier for genes that are to be introduced
into the genome of plants.
A special preferred embodiment of such plasmids is
characterized in that the recombinant transposon comprises
~; 20 between the left- and right-hand transposon ends at least one
foreign gene located in an expression cassette active in plant
cells.
In such recombinant plasmids it is further preferred
that the recombinant transposon comprises between the lefl-
2S and right-hand transposon ends one or more selectable marker
genes suitable for selection of transformed plant cells and
, located in an expression cassette active in plant cells, and
that the recombinant transposon comprises between the left-
and right-hand transposon ends one or more selectable marker
genes suitable for selection of transformed bacteria and
located in an expression cassette active in bacteria.
In yet another aspect the invention provides a
, recombinant A~robacterium tumefaciens strain which contains
foreign DNA to be transferred to plant cells between the left-
and right-hand ends of T-DNA necessary for such a transfer and
is capable of transferring this foreign DNA to the plant
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W092/06205 ~ PCT/EP91/01883
~ 12
cells, which strain is characterized in that a transposase
gene located in an expression cassette active in plant cells
is l^cafed between. the left- and right-hand ends of T-DN.~,
which expression cassette active in plant cells and filled
with a transposase gene is not located between the left- and
right-hand transposon ends necessary for integration in the
DNA of plant cells.
As regards this aspect, the invention also provides a
recombinant Aarobacterium tumefaciens strain which contains
foreign DNA to be transferred to plant cells between the left-
and right-hand ends of T-DNA necessary for such a transfer and
is capable of transferring this foreign DNA to the plant
cells, which strain is characterized in that between the left-
and right-hand ends of T-DNA a recombinant transposon is
located, which comprises the left- and right-hand transposon
- ends necessary for integration in the DNA of plant cells but
no DNA fragment which can lead in plant cells to expression of
an active transposase.
For such a recombinant Aarobacterium tumefaciens strain
it applies again that the recombinant transposon may comprise
between the left- and right-hand transposon ends at least one
foreign gene located in an expxession cassette active in plant
cells and/or one or more seiectable marker genes suitable for
selection of transformed plant cells and located in an
; 2S expresslon cassette active in plant cells, and/or one or more
selectable marker genes suitable for selection of transformed
bacteria and located in an expression cassette active in
; bacteria.
A special embodiment of such a recombinant Aarobacteri~m
tumefacien$ strain is characterized in that also a transposase
, gene located in an expression cassette active in plant cells
is located between the left- and right-hand ends of T-DNA,
which expression cassette active in plant cells and filled
with a transposase gene is not located between the left- and
right-hand transposon ends necessary for integration in the
; DNA of plant cells.
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W092/0620~ PCT/EP91/01883
13 2 ~8 ~ 0 72
The inventlon is finally also contained in plants and
parts consisting of one or more cells or products thereof,
which pl~nts derive from plant cells ~ransformed by ~he
process according to the invention. By parts and products of
plants is meant, e.g., the bulb, the tuber or the root, the
flowers, the seed and the meal made therefrom etc.
The invention will be further explained with reference
to the accompanying drawings, which diagrammetically show in
Fig. 1 a T-DNA construct comprising both a foreign gene
- 10 X included in an inactive transposon and a transposase gene
not located in an active transposon,
Fig. 2a a T-DNA construct comprising a foreign gene X
included in an inactive transposon,
Fig. 2b a T-DNA construct which can be used in
combination with that of Fig. 2a and comprises a transposase
gene not located in an active transposon,
Fig. 3a a T-DNA construct comprising a foreign gene X
included in an inactive transposon which comprises a marker
gene interrupted by a second inactive transposon for selecting
transformed plant cells,
Fig. 3b a T-DNA construct which can be used in
` combination with that of Fig. 3a and comprises a transposase
gene not located in an active transposon,
Fig. 4a a T-DNA construct comprising a foreign gene X
included in an inactive transposon, which foreign gene X is
;~
interrupted by a second inactive transposon,
Fig. 4b a T-DNA construct which can be used in
combination with that of Fig. 4a and comprises a transposase
gene not located in an active transposon,
Fig. 5a the structure of the recombinant plasmid pMH10,
pMH10-LTS and pMH10-DsB, respectively a plasmid having a
complete transposon, a transposase gene with only the left
transposon end and a transposon without transposase gene,
Fig. 5b an example of a T-DNA construct with plasmid
pMH10-LTS according to the invention,
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W092/0620~ PCT/EP91/01883
i 14
Fig. 6a the structure of the recombinant plasmid
pMHlODsl5GUS, ';
Fis. 6b an e~ample of a .-DNA corstruct -with this
plasmid according to the invention,
Fig. 7 the structure of the recombinant plasmid
pMHlODs34CAT,
Fig. 8 the structure of the recombinant plasmid
pMHlODsl2IAA,
Fig. 9 the structure of the recombinant plasmid
pMXllDs4IPTkan,
Fig. lO the structure of the recombinant plasmid
pUCSSAc9Dsl9, suitable for cloning foreign genes between the
transposon ends of Ac9,
Fig. lla the structure of the recombinant plasmid
pUCSS19tpnAc9P35S,
Fig. llb the structure of the recombinant plasmid
pUCSS19tpnAc9PTR1,
Fig. llc a T-DNA construct with the recombinant plasmid
pUCSS19tpnAc9P35S according to the invention.
Fig. 12a the structure of the recombinant plasmid
~ pUCSSDsl9BTKA,
- Fig. 12b a T-DNA construct with this plasmid according
to the invention,
~ Fig. 13a a cointegrate plasmid with the constructs
-` 25 pMHllDs4IPTkan and pUCtpnAc9P35S, and
Fig. 13b an example of a T-DNA construct with the
cointegrate plasmid pMHllDs4IPTkan/pUCtpnAc9P35S.
In the drawings the symbols have the following meanings:
X represents a foreign gene located in an expression
` - 30 cassette, not separately shown, active in plant cells; if the
`~ gene is interrupted by an insert, the component parts of the
gene are indicated by X' and X", respectively;
~ S1 represents a marker gene which can be used for the
,~ selection of transformed plant cells; here, too, the component
parts are indicated by S1' and S1", respectively, when the
gene is interrupted by an insert;
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W092/0620~ PCT/EP91/01883
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S2 and S3 also represent marker genes suitable for the
selection of successfully transformed plant cells, further
-efer-cd lo as plan. aelcc~lon genes;
sl and s2 represent marker genes suitable for the
selection of transformed bacteria, further referreà to as
bacteria selection genes;
tpn represents a genomic clone of a transposase gene
located in an expression cassette, not separately shown, which
can function in plant cells;
LTA and RTA represent respectively the left end of T-DNA
and the right end of T-DNA, which ends are necessary for
transferring the T-DNA by the Aarobacte~ium L~meL~s~D~
bacteria tO the plant cell;
LTS and RTS represent respectively the left end of a
transposon and the right end of an active or inactive
transposon,;
Wx represents the sequence of the waxv gene of maize
from which Ac9 was cloned;
Fig. 1 shows a T-DNA construc~ according to the
invention, one plasmid containing between the T-DNA ends LTA
and RTA both an inactive transposon having a foreign gene X
~ and a plant selection gene Sl contained therein and a
; transposase gene tpn not located on an active transposon. The
selection genes shown in Fig. 1 are facultative, for that
matter, in particular one of the bacteria selection genes and
'~ J one of the plant selection genes being dispensable, if
required. As regards the transposase gene, it is important
that it is not located within the transposon ends LTS and RTS.
This does not mean that the transposon ends must be completely
absent: often removal of (a significant portion of) one of the
transposon ends will be sufficient to prevent efficient
; incorporation of the transposase gene in the genome of the
transformed plant cells. Thus, for instance, one transposon
end could be retained in Fig. 1 beside the transposase gene
(see Fig. 5a).
. ~,
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W092/06205 PCT/EP9l/01883
~S~ 16
Figs. 2a and 2b show a preferred embodiment consisting
of an analogous construct, the elements being distributed over
.wo dl~ferent plasmids. The inactive transposon having a
foreign gene X and a plant selection gene Sl contained therein
is located on a first plasmid the T-DNA of which i5 shown in
Fig. 2a. Fig. 2b shows the T-DNA of a second plasmid which
contains a transposase gene and a plant selection gene S2. As
shown in Figs. 2a and 2b, each of the two T-DNA constructs
also contains a bacteria selection gene ~sl and s2,
respectively).
For clarity's sake, reference is made to the fact that
the present invention is in no way limited to the constructs
concretely shown. Thus only one bacteria selection gene will
easily do in the construct of Fig. 1. Also the location of the
different elements can be varied, e.g. in Fig. 1 a bacteria
- selection gene can be placed in the transposon or the location
of the plant selection genes can be altered with respect to
the other elements. Also T-DNA constructs according to the
invention may contain additional or other elements than are
shown in the drawings, e.g. one or more additional foreign
genes, one or more additional bacteria or plant selection
genes etc.
Figs. ~a ana 3b show a preferred embodiment resembling
those of Figs. 2a and 2b. An inactive transposon having a
foreign gene X and a plant selection gene S1 contained therein
is located on a first plasmid, the T-DNA of which is shown in
Fig. 3a. In this case, however, the plant selection gene S1 is
interrupted by a second inactive transposon which, if
` required, contains its own plant selection gene S3 as shown in
Fig. 3a. Fig. 3b shows the T-DNA of a second plasmid
` containing a transposase gene and a plant selection gene S2.
;- This plasmid is identical with the plasmid shown in Fig. 2b.
Figs. 9a and 9b show a preferred embodiment
corresponding to those of Figs. 3a and 3b, with the
` 35 understanding that not plant selection gene Sl but foreign
gene X is interrupted by the second inactive transposon.
~ .
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: - - ~ .. . . . .
-, - , : ~ ..
:. ~ . . . : : .
. .. . . .
W092/06205 PCT/EP91/01883
. 20~9072 el-
17
Figs. 5 through 13 show DNA constructs which will be
further illustrated by the Examples.
EXAMPLES
Example 1
Elimination of insertion mutations of sequences (Ds) derived
from transposon Ac (Activator) in maize.
10 By introducing an active transposon Ac into the genome
of maize by means of crossbreeding, the activity of this
transposon can be observed in the next generations because of
the development of mutations (insertion into a gene) or
through the elimination of a mutation owing to ~c insertion
lS (excision from a gene). In the example described here the AC
transposase gene is used for eliminatlon of insertion
mutations (in this case Ds) from genes of maize. In conformity
with the above described technology Aarobacterium is used
herein to introduce the transposase gene into cells of the
apical meristem of maize.
The plasmid pMH10 (Fig. Sa) contains the PstI clone of
~transposon Ac9 in the waxy gene of maize (Pohlman et al, 1984,
; !,Muller-Neumann et al, 1984), a ~LI-BamHI fragment or plasmid
pBR322 (Bolivar et al, 1977) with a functional QLL sequence
~. 25 for replication in gram-negative bacteria, but a non-
.,functional ~m~ and tet resistance gene and moreover a BamHI-
.PstI fragment of plasmid pR702 (Hedges and Jacobs, 1974, and
Leemans et al, 1981) containing the sequence of an ~/spc
resistance gene. The plasmid was obtained from Laboratorium
voor 5enetika RUG, Ledeganckstraat 35, B9000 Ghent, Belgium.
In this plasmid the BamHI fragment containing the pBR32
sequence of the right Ac9(wx) transposon end was replaced by a
~ mHI fragment containing the complete pUC8 plasmid (Vieira
; and Messing, 1982). The resulting plasmid is called pMHlOlLTS
(Fig. Sa). The pMHlOlLTS plasmid is transmitted by conjugation
to a E~ resistance Aarobacterium tumefaciens (A. ~m-) C58
, . .
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W092/06205 ~ ~ PCT/EP91/01883
v
18
strain containing the Ti plasmid pGV3850 (Zambryski et al,
1983), in which p3R322 is cloned as a HindIII fragment into
the T-~MA ^f pTi C58 frcm which, furthermore, .~ll HindIII
fragments were cut, except the Left ~order (LB) fragment
S (~indIII-10) and the Right Border (RB) fragment (~indIII-23)
containing the nopaline synthase gene (nos). The plasmid
pGV3850 was obtained from Laboratorium voor Genetika RUG,
Ledeganckstraat 35, B9000 Ghent, Belgium.
The method of conjugation was described by Van Haute et
al (1983). By selection of transconjugants for rif, ~/spc
and amp resistance a cointegrate was selected, pMHlOlLTS being
integrated between the borders of the T-DNA.
Maize lines obtained by inbreeding for at least ten
years were made available, in the form of seed, by the firm of
Clovis Matton, Avelgem-Kerkhove, Belgium. These lines contain
no active transposon Ac, which, inter alia, could be proved by
breaking down the DNA of young leaves with PvuII and
hybridizing it with the internal ~indIII fragment of Ac
(Ac9(wx): bp: 1270-2877) : no hybridizing band was observed
corresponding to the 2626 bp internal PvuII fragment of Ac
(Ac9(wx): bp: 719-3345); different bands were found indeed but
the smallest band e~ceeded 4 kb. Crossings with test lines for
Ac activity have never sho~n transposition phenomena in the
lines used. The lines are used for the production of
commercial hybrids.
Seeds of ten maize lines were disinfected with NaClO 2%
and then laid on sterile wet sand to germinate on sterile sand
at 25C in the dark. Three days after germination 50 nl
~~ induced A. ~m. culture were integrated with the construction
pMH10 LTS by genetic recombinantion between the borders of the
` T-DNA (Fig. 5b), injected into the apical meristem by
'~ microinjection.
'' r~ The induction medium has been described by Vernade et al
(1988). After one week at 20C the young plants were cultured
together with untreated plants in large pots in an air-
,~ conditioned greenhouse in a mixture of compost and sandy clay
1.
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W092/06205 PCT/EPg1/01883
20~9072
19
in their normal growing season (May to October). Each plant
was fertilized manually, both the plume and the ear being kept
ln small bags. Each plant was individ~aally ~.atered in ~he F^'-
The ears were harvested in September and October and after
drying the seed was stored in paper bags during winter at 6C.
The next year the plants were sown on ear lines in the soil in
the greenhouse and again fertilized manually and evaluated by
corn growers for their characteristics both during growth and
in surmaturity.
The fe,llowing improvements over the original lines were
observed in some ear lines:
- plume more branched
- 30 - 50 cm higher plants
- stronger stem
- significant increase in the number of grain rows per ear
- remains green for a longer time
- absence of fusariosis
- better anchoring by secondary roots
~ Seeds of plants having deviating features as well as of
: 20 control plants were germinated in the laboratory under sterile
conditions and DNA was prepared from the young leaves, after
~- which the plants were planted in the greenhouse in large pots,
The DNA of each plant was then broken down such as for
` `1 RFLP mapping with different restriction enzymes and hybridized
with the internal HindIII fragment of Ac9. The control plants
`~ showed the same band pattern, as regards the prominent bands,
as the original line. In plants with deviating features either
disappearance of one or more fragments hybridizing with the
DNA probe or the disappearance of a fragment and the
appearance of a new fragment with a larger molecular weight
. was observed.
;
Example 2 (comparative e~ample)
Insertion of a foreign gene into the genome of maize by using
` 35 Aarobacterium tumefaciens as transfer system and transposon Ac
~: as integration system.
!, 1
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,, : '., ' , , :
W092/0620~ ~ PCT/EP91/01883
For the purpose of~comparison an active transposon
Ac9(wx) is used here. By using the integration properties of
the transposon Ac and the transfer properties of A. ~m. a
S foreign gene located between the recogni~ion sequences of the
transposon can be incorporated in the genome of maize by means
of transposase of the same transposon.
As described, two A. tumefaciens strains are used by way
of example. The first contains the ~-glucuronidase gene of
Escheric~ coli located between the left- and right-hand
transposon ends of Ac9(wx), which, in turn, is located between
the Left and Right Border ~LB and RB) of the T-DNA on a non-
oncogenic Ti plasmid. The second contains a complete
transposon Ac9(wx) located between LB and RB of the T-DNA.
The ~. ~m. strain containing the transposon Ac9 in the
T-DNA is shown in Fig. 5a. The other A. ~m. strain was
obtained in the following manner:
Plasmid pBI 221 contains the ~-glucuronidase gene of ~. coli
under the control of the CaMV 35S promoter (Cauliflower
Mosaic Virus 35S RNA promoter) and the mRNA distal end of the
nopaline synthase gene of the Ti plasmid of ~. tum. T37. This
plasmid was available from Dr R. Jefferson, c/o AFRC
Institute, Colney Lane, Norwich NR4 7UA, U.K.
The plasmid pMH10 shown in Fig. Sa was cut with EcoRI
~; 25 and partially with ~indIII and the 10.1 ~b fragment was
~ ligated with the 3 kb ~SQRI-~1ndIII fragment of pBI 221
containing the sequence of the chimeric ~1~ gene of this last
plasmid. The resulting plasmid pMHlODsl5GUS contains the
chimeric ~i~ gene in a transcription orientation contrary to
that of the (partially) deleted transposase gene.
~m- bacteria with this plasmid, introduced in the
` manner described in Example 1, show ~-glucuronidase activity.
; The construction is shown in Figs. 6a and 6b.
i The same maize lines described in Example 1 were used
for the transformation. In three-day-old germs the apical
meristem was infected by microinjection with 50 nl of a
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W092/0620~ PCT/EP91/01883
20~9~72
21
mixture of the two above-described ~. tum. strains induced for
transfer. In a first phase some hundreds of germs were tested
afte- l o weeks ~or ~-glucuronidase activity by incubating a
piece of tissue containing the apical meristem with X-gluc
(5-bromo-4-chloro-3-indolyl-D-glucuronide). In about 7.5% of
the tissues blue staining occurred. With a dark field
microscope it was clearly visible that the cells in the tissue
cuts and not the remaining bacteria were the origin of the
purple color. In particular small cells around the vascular
; 10 bundles were.stained. With an electron microscope the
precipitate of dimeric bromo-chloro indole was found ln cells
which were removed from the place of injection up to ten cell
layers. Sporadically, cut bacteria were visible which were
'. only present in the extracellular spaces but showed no
precipitate.
Other treated germs were grown in the greenhouse in a
normal growing season, manually fertilized and the seed was
` ~ harvested as described in Example 1.
During the winter months these seeds were germinated in
!,; 20 sterile conditions and the proteins were isolated from the
. young leaves and subjected to polyacrylamide gel
electrophoresis (PAGE) under non-denaturing conditlons. Beta-
glucuronidase activity was detected by covering the gel with a
thin agarose gel containing the substrate methyl-umbelliferyl-
D-glucuronide which fluoresces in UV light if ~he ~-
glucuronidase enzyme causes release of methyl umbelliferone.
A fluorescent band was found in about 1~ of the tested
plants. DNA was prepared from leaves and pieces of roots of
these plants and, as in RFLP mapping, hybridized with a 2.9 kb
~1ndIII-EcoRI fragment containing the chimeric ~1~ gene. In
, ~ the lane of the agarose gel containing maize DNA broken down
with ~ndIII and EcoRI hybridization with the probe was found
~ on a level with the place where about 3 kb fragmen~s migrate.
-~ Of young F1 plants pieces of leaves and roots were
incubated in X-gluc as described by Jefferson (GUS gene fusion
; system user's manual, 1987). Blue stains, -ones and sectors
`~,
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' . ' '`
. ' ` ~ ~ ,.",."~.,."~
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W092/06205 ~ PCT/EPgl/01B83
22
were found in the pieces of tissues of PAGE-~-glucuronidase
positive plants: around the vascular bundles, around the
s~omata, V-shaped segments of the roots, etc.
Light microscopic and electron microscopic analyses
confirmed the expression of the ~-glucuronidase enzyme in the
plant cells.
This example clearly shows that by using an active
- transposon in combination with a chimeric transposon
containing a foreign gene but no transposase gene between the
transposon ends expression is only obtained in specific cells
still containing the chimeric transposon, while most of the
plant cells show no expression. The same phenomenon can be
observed after transformation of plants such as tobacco which
are efficiently transformed by Aarobacterium but in this case
instability occurs after transformation with a chimeric
transposon, both in the presence of an active transposon and
in the presence of a transposase gene. This shows the
; difference between plants in which Aarobacterium can
efficiently integrate foreign DNA in the plant genome and
plants in which ~arobacterium cannot.
. . .
. Example 3
Generation or mutations in barley by using a transposase gene
; and a chimeric transposon.
, .
.i In this example the mutagenic properties of a transposon
- are used by integrating a chimeric transposon containing a
plant selection gene but no transposase gene in the genome of
barley by means of a transposase gene that is not located
between transposon ends.
Use is made of two Aarobacterium strains for the
. transfer: the first strain contains the chimeric transposon
~ i between the T-DNA ends and the second strain contains the
c ' transposase gene between the T-DNA ends. This example
~`~ 35 corresponds with Figs. 3a and 3b.
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W092/0620~ PCT/EP91/01883
~ r~ 2 1~ 8 ~ 0 7 2
23
The employed plasmids are pMHlODsl5GUS and pMHlOLTS. As
described before, they are conjugated from Escherichi~ CQli to
Aqrobac~rium and integ-ation 1,. ~r'~,T35~ scle_'cd by also
adding spectinomycin to the medium.
Seeds of two- and six-rowed winter barley ear lines
~F7-F8) were supplied by the plant breeding station of the
firm of Clovis Matton, Avelgem-Kerkhove, Belgium.
Seeds of 12 ear lines were disinfected with 6~ NaClO and
the seeds laid on moist sand to germinate at 4C. Germinating
seeds were further incubated at room temperature under sterile
conditions.
In 3-5-day-old seedlings the apical meristem was
infected by microinjection with Agr3b~cterium strains
containing pMHlO~slSGUS or pMHlOLTS. Transfer was induced as
described bv Vernade e~ al (1988). Infections were conducted
both with the strain containing pMG15 (control) and with the
strain containing pMHlOLTS (control) or with both of them
(experiment). About 300 seedlings were treated per ear line: a
hundred per experiment. Further control experiments were
conducted on tobacco, for rapid tests of expression.
After 1 week at 22C the young plants were cultured
further in a greenhouse and the seed was harvested after self-
pollination.
In the next generation the plants were sown again in a
greenhouse and selected by barley growers for deviating
features.
The following variants were traced:
- difference in height of the plant during growth
- difference in time of growth
- mutations from erectant to nutant or conversely
- difference in number of grains per ear
- difference in flattening
. ,. ,-, .
. - better or poorer phytosanitary condition.
Of 37 ear lines with clear morphological or
- 35 phytosanltary differences and 110 other ear lines with less
. --.
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W092/06205 ~ ~ PCT/EP91/01883
~Q,oG9 ~ ~
24
clear deviations five ripe ears were further examined for
expression of the uidA gene in the chimexic transposon.
~ !e-r'~ hc '? plants with clear differences over the
original ear line express the ~1~ gene to a greater or lesser
degree. No ~-glucuronidase activity could be demonstrated in
the controls, except in the case of bacterial infection. Only
9 plants with less clear deviations show ~i~ expression.
In tobacco ~-glucuronidase activity was found when the
; gene was present ln the infection, with or without additional
infection with the transposase gene.
Example 4
Construction of recombinant plasmids with plant selection
~` genes or genes with industrial applications between left- and
right-hand transposon ends in the plasmid pMH10.
i, .
In this example the construction of some plant selection
genes was shown by using unique restriction sites in the
sequence of Ac9 in the plasmid pMH10.
The chloramphenicol acetyl transferase gene in an
. . expression cassette consisting of the 35S promoter of CaMV and
the nos-terminator of the T-DNA of Aarobacterium tumefaciens
is available as plasmid pCAMVCN from Pharmacia-LKB, Bromma,
Sweden. The complete chimeric gene was cut as Xbal fragment.
,:~`' 25 The plasmid pMHl0 was partially cut with ~S11 (position 1530
in Ac9Wx) and BamHl (position 4498 in Ac9Wx) and the 8.1 kb
fragment was ligated back. This plasmid, called pMHlODsB,
contains unique restriction sites in the remaining Ac9
~: sequence: ~il (five sites at position 571, 972, 1003, 1183,
30 and 1251 of AC9WX), ~hQl (at position 1125 of Ac9Wx) and Xbal
~^ (at position 1257 of AC9WX) ~Fig. 5a). The ~at expression
cassette was cloned into the unique XbaI site of Ac9. The
resulting plasmid was called piMHlODs34CAT (Fig. 7), conjugated
` to Aarobacte_ium and integrated in the plasmid pGV3850. By
-~ 35 transformatio~ of tobacco the expression of the cat gene was
:~ proved.
.~
.
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W092/06205 PCT/EP91/01883
2089072
2S
The genes iaaM and iaaH from the T-DNA of Aarobacterium
tumefaciens Ach5 (Gielen et al, 1984) were cloned from the
plasmlds pGV819 (~Q~ l clone of ~ uda_ et 31, 1986)
and pGV829 (HindIII clone of ia~H) (Budar et al, 1986). The
S complete construct with the genes iaaM and iaaH was first
cloned into pBR322 (Bolivar et al, 1977) by a threefold
ligation of the 3.6 kb pBR322 ~ I fragment, the 3.7 kb
EcoR1-ClaI fragment of pGV814 and the 2.7 kb partial
PstI-EcoR1 fragment of pGV824. From this was cloned the 6.4 kb
~I fragment in the ~iI sites (position 571 and 2881 of
Ac9Wx) of pMH10. The resulting plasmid pMHlODs12IAA (Fig. 8)
was conjugated to Aarobacterium and integrated in the plasmid
pGV3850 by additional selection with spectinomycin. The
usability of the construction was tested by transformation of
tobacco and expression of the L~ genes.
The gene ie~ from the T-DNA of Aqrobacterium tumefaciens
CS8 together wi~h the bacterial n~tII gene of transposon Tn5
of plasmid pG4L ~Inzé et al, 1989) was cloned as a BamHI
fragment in pUC19 and isolated therefrom as a partial
~hl-EcoR1 fragment of 3.7 kb and cloned into pMH11 cut with
EcoR1 and partially with SDhl. pMH11 is a derivative of pMH10
obtained by cutting in the vector plasmid of pMH10 the 0.8 kb
~hl fragment located between the str/s~c resistance gene and
the (partially deleted) ~ resistance gene, blunt ending the
ends with S1 nuclease and ligating back the vector. The PstI
clone of Ac9Wx was again incorporated in the resulting vector
in the unique ~stI site. The orientation, however, is
different from that in pMH10.
The resulting plasmid pMHllDs4IPTkan (Fig. 9) was 30 conjugated to Aarobacterium and integrated in pGV3850 by
additional selection with spectinomycin and then with
kanamycin. The usability of the construction was proved by
transformation of tobacco and expression of the gene 12~-
,~
Example 5
~'~.
~-
. ~
, .
.
W092/0620S ~ ~ PCT/EP9l/0l883
26
Construction of a recombinant transposon with a multiple
cloning site between the transposon ends.
The ~hI~ I fragment from mini-Sa, a derivative of
pR702 ~Leemans et al, 1983) containing the sm-sp adenosyl
transferase gene encoding resistance to streptomycin and
spectinomycin was blunt ended with T4 DNA polymerase and
cloned into the plasmid pUC19 (Yanisch-Perron et al, 1985),
blunt end cut with ~I. This plasmid was 4330bp and will be
referred to hereinbelow as pUCSS19.
Both orientations of the sm/sp resistance gene are
suitable. However, the work was further done with the plasmid
in which the amp resistance gene and the ~m/~ resistance gene
have the same transcription orientation. pUCSSl9 was blunt end
cut with PvuII and both the 4030bp vector fragment and the
300bp ~CS fragment were separated and stored.
. The PstI clone of Wx-Ac9 was cut from pMH10 (Fig. 5a)
; and blunt ended with T4 DNA polymerase. This fragment was
, cloned into the 4030bp ~II fragment of pUSS19. This plasmid
,;
was 8840bp and will be referred to hereinbelow as pUCSSAc9.
pUCSSAc9 was cut with NsiI and ~I and the NsiI end was
blunt ended with T4 DNA polymerase. This fragment was 4980bp
`~ and contained the ends of transposon Ac9Wx. Into this was
' cloned the 300bp ~II fragment of pUC19. One of the
orientations is shown in Fig. 10. The plasmid was 5280 bp and
will be referred to as pUCSSAcDsl9. It can be used for cloning
numerous genes with their expression cassette. It can be
'`~ conjugated to Aarobacterium with plasmid pGV3850 as described
in the first example by additional selection with
spectinomycin.
, Example 6.
Construction of recombinant plasmids containing plant
selection genes or genes with industrial applicaticns and
located between the transposon ends of pUCSSAcDsl9.
,;
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, ..
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, ' 1~ ',: . _ - ' ,, , ' ' ' ,, ' . , ., . ' , ' . ', . .
; : :. . .. . :: . . . .. . .. . .
~. . . .. . ... .. .. ... . ... .
;.................... . . . . . .
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W092/06205 PCT/EP91/01883
27 2 ~ 8 9 ~ 72
The delta endotoxin gene of the 42 MDa plasmid of
Bacillus thurin~iensis var. Berliner 1715 (Klier et al, 1982),
'he se~uence of which was published by Hofte et al, 1986, was
obtained from S.A. Solvay, Brussels, Belgium, as plasmid
pBT424. The strain is available from the Institut Pasteur in
Paris, France.
From pBT924 the BamHl~ I fragment containing the start
codon of the delta endotoxin gene and the first 1939 bp,
necessary for expression of the toxin, was cut and cloned
downstream of the pTRl' promoter of plasmid pPCV520 (Koncz and
Schell, 1986), cut with BamHl and ~glII. The correct
orientation was verified with BamHl and PstI (0.7 kb in the
correct orientation). The complete double expression cassette
pTRl'-BT~Q~ terminator gene 4/pTR2'-nptII terminator ocs was
cut again with KonI and partially with PstI as a 5.7 kb
fragment and cloned into the corresponding restriction sites
of pUCSSAcDsl9. The terminator of gene 4 contains different
stop codons in the three reading frames upstream of the
~ polyadenylation signal.
- ~ 20 The resulting plasmid (pUCSSDsl9BTKA) (~ig. 12a) was
conjugated to Aaroba~terium and integrated in pGV3850 by
- additional selection with spectinomycin as described berore
(Fig. 12b).
. Resistance to kanamycin could be shown after
transformation to tobacco.
The construct was used for making maize lines resistant
to Pyralidae, in the manner as described in Comparative
:~ Example 2, with the difference that use was made of a second
A~robacterium strain containing a transposase gene without
~ 30 transposon ends (pMHlOLTS).
,i Example 7.
- Construction of a recombinant plasmid containing the
transposase gene of Ac under control of the CaMV 35S promoter
or of the pTRl' promoter of ~grobact~ium Ach5 T-DNA.
~,
-, , ,, ,
W092/~6205 ~ ~ PCT/EP91/01883
~ 28
The plasmid pMH10 (Fig. 5a) was cut with PstI and the
4810bp Ac9 wx fragment, c~oned into pUC19, cut wit ~I. This
plasmiu was 7500bp and will be referred ~o hereinbel~w d~
pUC19Ac9wx. The work was further done with the orientation 1,
namely the one in which the BamHI sites are remotest from each
other.
The Ac9 wx fragment was cut again, as ~mHI fragment.
This fragment was partially cut with Cfr1QI and two fragments
were stored: the 3.4 kb CfrlOI fragment and the 4.2 kb BamHI-
. 10 CfrlOI fragment. The first contained a transposase genewithout a promoter and the second a transposase gene with a
promoter. The 4.2 kb fragment was cloned into pUCSS19, cut
with XmaI and BamHI. This plasmid (8.5kb) contained the normal
transposase gene of Ac but no transposon ends
(pUCSS19tpnAc9PAc9) and could be transmitted by conjugation to
Aarobacterium and integrated in the T-DNA of pGV3850 by
- additional selection of spectinomycin resistance. The 3.4 kb
fragment was cloned into pUC19, cut with ~m~I, and the
orientation was determined using the ~L~I site in the MCS and
in the transposase gene (position 1260 of Ac9Wx). This plasmid
(7.7 kb) is called pUCSS19tpnAc9cds) and may serve for cloning
new promoters upstream of the transposase gene.
;
For further use pUCSS19tpnAc9cds was taken in
orientation 1, the ~k~I fragment from this plasmid being 2.5
kb. This plasmid was cut with PstI and BamHI and the CaMV 35S
promoter fragment from pBI221, cut with Ps I and BamHI, was
cloned into it.
The resulting plasmid is called pUCSS19tpnAc9P35S
(Fig. lla). The pTRl'2' double promoter of the T-DNA of
Aarobacterium Ach5 (Velten et ~1, 1984) was obtained as a
........................................................................... .
~ BamH1-Ps~I fragment from the plasmid pPCV520 (Koncz and
; ~ Schell, 1986) and was cloned into the BamHl and PstI sites of
:. .
pUCSS19tpnAc9cds. The resulting plasmid (pUCSS19tpnAc9PTRl') -
contains the transposase gene under controi of the pTRl'
promoter (Fig. llb).
Both plasmids can be conjugated, as described before, to
'~ ~'',
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,, ; . , .. . ;. : , . , ~
W092/0620~ PCT/EP9l/01883
29 2 ~ 89 0~
Aarobacterium and integrated in pGV3850 by additional
selection for spectinomycin (Fig. llc). The use of the pTR1'
promoter has the advantage tha. in CâSe OL mic.oinject Gr; or
bombardment of Aarobacte_ium under high pressure this promoter
strongly expresses in the wounded tissue.
Example 8.
Construction of recombinant plasmids containing a chimeric
transposon and a transposase gene without transposon ends.
, Recombinants between â pMH10 derivative with a chimeric
transposon and pUC19 derivatives with a chimeric transposase
gene were selected in vivo in MC1051 bacteria after
transformation with both plasmids and selection for ampicillin
and spectinomycin, followed by alternating growth cycles with
: selection for ampicillin and then spectinomycin. This results
in cultures in which recombinant cointegrates of pMH10 and pUC
~: derivatives occur in 50% of the bacteria.
` The constructs tpnP35S and tpnPTRl' were cloned as KpnI-
~0 ~I from their respective pUCSS plasmids into pUCl9
containing the ampicillin resistance gene. The employed pMH10
derivatives were: pMHlODslSGUS, pMHlODs3qCAT, pMHlODsl2IAA and
pMHllDs4IPTkan and are all carriers of a spectinomycin
resistance gene. The cointegrate of the recombinant plasmids
pMHllDs4IPTkan and pUCSS19Ac9P35S is shown in Fig. 13 by way
. of example. These cointegrate plasmids were transmitted by
- transformation to the strain HB101 and after selection for
ampicillin and spectinomycin they were characterized by
plasmid preparation and restriction enzyme analysis. Then ~he
plasmids were transmitted by conjugation to Aarobacterium, as
~` described before.
The resulting Aqrobacterium strains are an example of
the use shown in Fig. 1.
~: The resulting recombinant AarobactQrlum strains were
tested on tobacco ror expression of respectively nopaline
' synthase (pGV3850 marker), uidA, cat, -oot ~ormation and shoot
~'
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~,~ . : . . : - . .
' ' ` ' .. : , ' :
W092/0620S ~ PCT/EP91/01883
~ 30
formation and finally for transposition, before they were used
on cereals.
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