CA1332718C - Expression cassette for plants - Google Patents
Expression cassette for plantsInfo
- Publication number
- CA1332718C CA1332718C CA 606866 CA606866A CA1332718C CA 1332718 C CA1332718 C CA 1332718C CA 606866 CA606866 CA 606866 CA 606866 A CA606866 A CA 606866A CA 1332718 C CA1332718 C CA 1332718C
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- dna
- expression cassette
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- plant
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Abstract
ABSTRACT
The present invention relates to an expression cassette which can express a gene at high levels; to transformed bacterial and plant cells which contain the expression cassette; to transgenic plants which are produced from plant cells transformed with the expression cassette; and to a process of producing transgenic plants which exhibit a desired trait.
The present invention relates to an expression cassette which can express a gene at high levels; to transformed bacterial and plant cells which contain the expression cassette; to transgenic plants which are produced from plant cells transformed with the expression cassette; and to a process of producing transgenic plants which exhibit a desired trait.
Description
-1- i 3 ~ 2 ~ , 4634.P CNl EXPRESSION CASSETTE FOR PLANTS
FIELD OF INVENTION
The present invention relates to an i~proved expression cassette useful for expressing genes at high levels in transformed plants.
BACKGROUND OF THE INVENTION
Application of genetic engineerirlg techniques to improve plant species can lead to plant species whlch exhibit increased resistance to pathogens, which display an increased tolerance to severe or erratic weather conditions and/or which are more nutritious. It ls well established that the charac~eristics of pathogen tolerance, weather tolerance and nutritional composition are produced by the genetic makeup of the plant. Thus, introducing genetic material into a plant can bring about preferred characteristics and, accordingly, improved plant species.
The genetic factors which efect these desirable charactistics include genes for peptide products whose presence confers desired traits as well as those whose nucleotide sequences impede the production of undesirable proteins. Plants which have antipathogenic proteins present in the cells di.splay increased resistance to pathogen challenge. ~eather tolerance can be conferred by the production of certain proteins in plants. The nutritional value oE
plants is often related to the presence oi proteins and, in particular, desirable amino acids which make them up. In addition to genes which encode peptides that effect the various charActeris~ic~, traits can be in~luenced by the presenca of RNA molacules which are not translated, Thus, tha introduction of genatic informfltion can be used to provide or supplement desirable traits in plants.
Genetic in~ormation may be introduced into plan~s which will result in an increased rasistanca to pathogenic chnllanga. Specifi-cally, ~he presence of an;~ipathogenic proteins can increasa resis-tance against viral inEection as well as act as agents agianst bacteria and other microrganisms. Furthermore, peptides which act as pestlcides, fungicides and herbicides may be introduced and ex-pressed. In addition to introducing genetic sequences which encode such protein products, resistance to the same pathogenic agents may coni`erred on a plant by introducing ~enetic information which can be transcrlbed in the plant cell into RNA that will lnhlbit translation o~ RNA encoding proteins which are necessary in pathogen challenge.
*
FIELD OF INVENTION
The present invention relates to an i~proved expression cassette useful for expressing genes at high levels in transformed plants.
BACKGROUND OF THE INVENTION
Application of genetic engineerirlg techniques to improve plant species can lead to plant species whlch exhibit increased resistance to pathogens, which display an increased tolerance to severe or erratic weather conditions and/or which are more nutritious. It ls well established that the charac~eristics of pathogen tolerance, weather tolerance and nutritional composition are produced by the genetic makeup of the plant. Thus, introducing genetic material into a plant can bring about preferred characteristics and, accordingly, improved plant species.
The genetic factors which efect these desirable charactistics include genes for peptide products whose presence confers desired traits as well as those whose nucleotide sequences impede the production of undesirable proteins. Plants which have antipathogenic proteins present in the cells di.splay increased resistance to pathogen challenge. ~eather tolerance can be conferred by the production of certain proteins in plants. The nutritional value oE
plants is often related to the presence oi proteins and, in particular, desirable amino acids which make them up. In addition to genes which encode peptides that effect the various charActeris~ic~, traits can be in~luenced by the presenca of RNA molacules which are not translated, Thus, tha introduction of genatic informfltion can be used to provide or supplement desirable traits in plants.
Genetic in~ormation may be introduced into plan~s which will result in an increased rasistanca to pathogenic chnllanga. Specifi-cally, ~he presence of an;~ipathogenic proteins can increasa resis-tance against viral inEection as well as act as agents agianst bacteria and other microrganisms. Furthermore, peptides which act as pestlcides, fungicides and herbicides may be introduced and ex-pressed. In addition to introducing genetic sequences which encode such protein products, resistance to the same pathogenic agents may coni`erred on a plant by introducing ~enetic information which can be transcrlbed in the plant cell into RNA that will lnhlbit translation o~ RNA encoding proteins which are necessary in pathogen challenge.
*
-2- ~ 3~27~
Tolerance to varying and severe climate conditions can be conferred in plants by introduction of genetic information. The presence of various proteins can provide plants with the ability to adapt to and survive under sub-optimum climatic conditions. Genes which confer traits that through evolution have developed in plants that make them more adapted to survive cold, heat, drought or flooding ior example, can be isolated. These genes can be introduced into plants which lack them and the plants will exhibit the phenotype the genes accord.
Similarly, the nutritional value of plants can be changed and increased through ~the introduction of genetic information. Genes encoding peptides and proteins comprised of amino acids which are desirable may be introduced into plants. The genes are expressed and the plant displays the improved composition accordingly.
The genes which are to be introduced into the plant may be derived from any source. Many genes which cGnfer desirable traits in one species of plants may be transferred to confer the trait in a siecond, different plant. Antipathogenlc genes may be derived from patho~ens. Genes may be synthesized. Alternatively, the genes may be identical to those already found in the plant genome where they, by providing multiple copies of the genes, will be introduced in order to supplement and enhance the trait. Accordingly, heterologous as well as endogenous genes may be introduced into plants to confer ~r supplement a desired trait.
Methods to introduce genetic material into plants are wid~ly known ~o those skilled in the ar~. Extensive work has been done with vectors of A~robacterium tumerfaciens. Additionally, genetic material has b~en introduced into the genome of plants by imp~cting plant cells with mlcropro~ectile~ coated with the ~enetic material.
When the genetlc material is introduced in~o the plant çells it can be incorporated into the plant's genome and if linked to the proper genetic regulatory elements, expressed. The expression of the introduced genes can confer trait.s on the host plant.
In order to achieve expression of introduced genetic material, the structural genes sought to be expressed must be linked to regulatory sequences. These regulatory sequences include a promoter, an initiation codon and a polyadenylation addition signal. Without these elements operably linked to the gene qought to be expressed, ~3~2711 ~
Tolerance to varying and severe climate conditions can be conferred in plants by introduction of genetic information. The presence of various proteins can provide plants with the ability to adapt to and survive under sub-optimum climatic conditions. Genes which confer traits that through evolution have developed in plants that make them more adapted to survive cold, heat, drought or flooding ior example, can be isolated. These genes can be introduced into plants which lack them and the plants will exhibit the phenotype the genes accord.
Similarly, the nutritional value of plants can be changed and increased through ~the introduction of genetic information. Genes encoding peptides and proteins comprised of amino acids which are desirable may be introduced into plants. The genes are expressed and the plant displays the improved composition accordingly.
The genes which are to be introduced into the plant may be derived from any source. Many genes which cGnfer desirable traits in one species of plants may be transferred to confer the trait in a siecond, different plant. Antipathogenlc genes may be derived from patho~ens. Genes may be synthesized. Alternatively, the genes may be identical to those already found in the plant genome where they, by providing multiple copies of the genes, will be introduced in order to supplement and enhance the trait. Accordingly, heterologous as well as endogenous genes may be introduced into plants to confer ~r supplement a desired trait.
Methods to introduce genetic material into plants are wid~ly known ~o those skilled in the ar~. Extensive work has been done with vectors of A~robacterium tumerfaciens. Additionally, genetic material has b~en introduced into the genome of plants by imp~cting plant cells with mlcropro~ectile~ coated with the ~enetic material.
When the genetlc material is introduced in~o the plant çells it can be incorporated into the plant's genome and if linked to the proper genetic regulatory elements, expressed. The expression of the introduced genes can confer trait.s on the host plant.
In order to achieve expression of introduced genetic material, the structural genes sought to be expressed must be linked to regulatory sequences. These regulatory sequences include a promoter, an initiation codon and a polyadenylation addition signal. Without these elements operably linked to the gene qought to be expressed, ~3~2711 ~
expression cflnnot take place. Expression will take place when the genetic material to be introduced is con.structed such that the gene to be expressed is downstream of the promoter and initiation codon, in proper reading frame with the initiation codon and upstream from the polyadenylation addition signal.
Higher levels of expression are often desirable and can be acheived with the additlon of genetic sequences in addition to those minimally required. Untranslated regions flanking the promoter can oiten lead to a higher level of gene expression. Similarly, un-translated regions flanking the initiation codon can also result in ahigher level of expression. In addition, the sequences flanking the poly A addition signal may lead to more efficient processing of the RNA transcribed and gene expression accordingly.
Expression of the coat protein of tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, and potato virus X in transgenic plants has resulted in plants which are resistant to infection by the re~pective virus. In order to produce such transgenic plants, the coat protein gene must be inserted into the genome of the plant.
Furthermore, the co~t protein gene must contain all the genetic control sequences necessary for the expression of the gene after lt has been incorporated into the plant genome.
The present invention relates to an improved expression cassette for introducing desired genes into plants. This cassette comprises a promoter, an AT rich 5' untranslated region, an initiation region comprislng the sequence AAXXATGG, a gene and a poly(A) adtli~lon signal which contains untranslated flanking regions, ~ccording to the present invention, genes which are introduced into plAnts wlll be expressed at high levels bacause the genetic regulatory saquences controlling expres~ion facilitate such high expresslon.
INFORMATION DISCLOSURE
European patent application EP 0 223 452 describes plants that are re~istant to viral diseases and methods for producing them. The process described comprises the steps of transforming a plant with a DNA insert comprising a promoter, a DNA sequence derived from the vlrus, and a poly(A) addition sequence.
PCT patent application PCT/US86/00514 rafers to a method of confarring resistance to a parasite to a hGst of the parasite, An et al, (1985) "New cloning vehlcles for transformation of - ~ 3 ~
higher plants", EMB0 J. 4:277-285 describe the construction of an expression plasmid which may be stably replicated in both E. coli and A. tumerfaciens.
An, G. (1986) "Development of plant promoter expresslon vectors S and their use for analysis of differential ac~ivity of nopaline synthase promoter in transformed tobacco cells", Plant Physiol.
81:86-91, reports ~ast differences in promoter activities of transferred genes within ~he same cells AS well as in independently derived cell lines.
Koæak, M. (1986) "Point mutations define a sequence flanking the AU~ initiator codon that modulates translation by sukaryotic ribo-somes", Cell 44:283-292, discloses the optimal sequence around the ATG initiator codon of the preproinsulin gane for initiation by eukaryotic ribosomes.
Loesch-Fries et al. (1987) "Expression of alfalfa mosaic virus RNA 4 in transgenic plants confers virus resistance", EMBO J
6:1845-1851, dlsclose that expression of the coat protein gene of alfalfa mosaic virus in transgenic plants confers resistance to infection by the virus.
Mazur, B. J, and Chui, C.-F. (1985) "Sequence of a genomic DNA
clone for the small subunit of ribulose bis-phosphate carboxylase-oxygenase from tobacco", Nucleic Acids Research 13:2373-2386, disclose the DNA sequence o the small subunit of ribulose bis-phosphate carboxylase-oxygenase from tobacco.
Olson, M. K. et al ~1989) "EnhancemenC oE heterologous polypeptide expression by alterations in the ribosome-binding~site sequence", J. Biotech. 9 179-190, discloses the increase in gene expression o~ heterologous genes in E. coli due ~o ~he pre~encQ of an AT-rich 5' untranslated region.
Pietræak et A~ 8~) "Expression in plants of t~o bacterial an~iblotic resistant genes after pro~oplast transformation with a new plant expression vector", Nucleic Acids Research 14:5857-5868, dis-close expression in plants of foreign genes introduced into the plant using an expression vector containing a movable expression csssette consistlng of tha Cauliflower mosaic virus 35S promoter and transcription terminator sepera~ed by a polylinker containing several unique restriction sites.
Powell-Abel et al. (1986) ~Delay of disease development in 5 ~3~7:~ 8 transgenic plants that express the tobacco mosaic vims co~t protein gene", Science 232:738-743, disclose increased resistance toward infectlo~ by tobacco mosaic virus in transgenic plants containing the coat protein gene from tobacco mosaic virus.
Tumer et al. (1987) "Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plantsn, EMBO J. 6:1181-1188, disclose transgenic tobacco and tomato plants transformed with the coat protein gene of alfalfa mosaic virus ~isplay increased resistance to infection by alfalfa mosaic virus.
SUMMAR~ OF THE INVENTION
The present invention relates to an expression eassette which can express a desired gene at high levels. The present invention relates to nn expression vector which comprlses an expression cassette. The high level expression vector of the present invention eomprises: a promoter.; a 5' untransl~ted region which is at l~ast 60% A and T; an initiation codon comprising Kozak's element; a cloning site where a desired gene may be inserted to form a functional expr~ssion unit; and a 3' untranslated re~ion which comprises a poly(A) additi~n signal and flanking sequence which yields hiKh level expression. The present invention relates to trnnsformed bacterial and plant cells which contain the expression vector, The present lnvention relates to ~ransgenic plants which ar~
produc~d rom plant cells transformed with the expreasion vector.
2S Th~ presen~ invention relates to a proaess of producin~ transg~nic plnnts with desirable traits by producing the plant~ from plan~ cells which havc been transformed with an expression vector which contnins ~ene conierring SUC~I trai~s, More particularly, the preaent in~ent.ion provide~ a D~IA plant expre0~ion cu~ette oompri~ing: (a) a promoter;
(b)a Cuoumber Mosaic Virus (CMV) coat protein gene 5' untranslated region; (c) an initiation region, the ini-tiation region comprising the sequence AAXXATGG; (d) a gene; and (e) a poly(A) addition signal, the poly(A) addition ~ignal containing untranslated flanking sequences; wherein the promoter is upstream from the (b) :
. . .
- 5a - ~ r~ g region; the (b) region i8 up~tream ~rom the initiation region; the initiation region is upstream and operably linked to the DNA molecule encoding a gene, and the gene is upstream and operably linked to the poly(A) addition aienal .
DETAILED DESCRIPTION OF THE INVENTION
Certain convention~ are used in Charts 1-9 to illu~trate plasmids and DNA fragments as follows:
(1) The single line figures represent both circular and linear double-stranded DNA.
(2) A~terisks (*) indicate that the molecule represented i3 ¢ircular. Lack of an a~terisk indicate~ that the molecule is linear.
(~) Junctions between the natural boundaries of functional component~ are indi¢ated by vertical lines along the . " .~
"t -6- ~3~ 3 horlzontal lines.
Higher levels of expression are often desirable and can be acheived with the additlon of genetic sequences in addition to those minimally required. Untranslated regions flanking the promoter can oiten lead to a higher level of gene expression. Similarly, un-translated regions flanking the initiation codon can also result in ahigher level of expression. In addition, the sequences flanking the poly A addition signal may lead to more efficient processing of the RNA transcribed and gene expression accordingly.
Expression of the coat protein of tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, and potato virus X in transgenic plants has resulted in plants which are resistant to infection by the re~pective virus. In order to produce such transgenic plants, the coat protein gene must be inserted into the genome of the plant.
Furthermore, the co~t protein gene must contain all the genetic control sequences necessary for the expression of the gene after lt has been incorporated into the plant genome.
The present invention relates to an improved expression cassette for introducing desired genes into plants. This cassette comprises a promoter, an AT rich 5' untranslated region, an initiation region comprislng the sequence AAXXATGG, a gene and a poly(A) adtli~lon signal which contains untranslated flanking regions, ~ccording to the present invention, genes which are introduced into plAnts wlll be expressed at high levels bacause the genetic regulatory saquences controlling expres~ion facilitate such high expresslon.
INFORMATION DISCLOSURE
European patent application EP 0 223 452 describes plants that are re~istant to viral diseases and methods for producing them. The process described comprises the steps of transforming a plant with a DNA insert comprising a promoter, a DNA sequence derived from the vlrus, and a poly(A) addition sequence.
PCT patent application PCT/US86/00514 rafers to a method of confarring resistance to a parasite to a hGst of the parasite, An et al, (1985) "New cloning vehlcles for transformation of - ~ 3 ~
higher plants", EMB0 J. 4:277-285 describe the construction of an expression plasmid which may be stably replicated in both E. coli and A. tumerfaciens.
An, G. (1986) "Development of plant promoter expresslon vectors S and their use for analysis of differential ac~ivity of nopaline synthase promoter in transformed tobacco cells", Plant Physiol.
81:86-91, reports ~ast differences in promoter activities of transferred genes within ~he same cells AS well as in independently derived cell lines.
Koæak, M. (1986) "Point mutations define a sequence flanking the AU~ initiator codon that modulates translation by sukaryotic ribo-somes", Cell 44:283-292, discloses the optimal sequence around the ATG initiator codon of the preproinsulin gane for initiation by eukaryotic ribosomes.
Loesch-Fries et al. (1987) "Expression of alfalfa mosaic virus RNA 4 in transgenic plants confers virus resistance", EMBO J
6:1845-1851, dlsclose that expression of the coat protein gene of alfalfa mosaic virus in transgenic plants confers resistance to infection by the virus.
Mazur, B. J, and Chui, C.-F. (1985) "Sequence of a genomic DNA
clone for the small subunit of ribulose bis-phosphate carboxylase-oxygenase from tobacco", Nucleic Acids Research 13:2373-2386, disclose the DNA sequence o the small subunit of ribulose bis-phosphate carboxylase-oxygenase from tobacco.
Olson, M. K. et al ~1989) "EnhancemenC oE heterologous polypeptide expression by alterations in the ribosome-binding~site sequence", J. Biotech. 9 179-190, discloses the increase in gene expression o~ heterologous genes in E. coli due ~o ~he pre~encQ of an AT-rich 5' untranslated region.
Pietræak et A~ 8~) "Expression in plants of t~o bacterial an~iblotic resistant genes after pro~oplast transformation with a new plant expression vector", Nucleic Acids Research 14:5857-5868, dis-close expression in plants of foreign genes introduced into the plant using an expression vector containing a movable expression csssette consistlng of tha Cauliflower mosaic virus 35S promoter and transcription terminator sepera~ed by a polylinker containing several unique restriction sites.
Powell-Abel et al. (1986) ~Delay of disease development in 5 ~3~7:~ 8 transgenic plants that express the tobacco mosaic vims co~t protein gene", Science 232:738-743, disclose increased resistance toward infectlo~ by tobacco mosaic virus in transgenic plants containing the coat protein gene from tobacco mosaic virus.
Tumer et al. (1987) "Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plantsn, EMBO J. 6:1181-1188, disclose transgenic tobacco and tomato plants transformed with the coat protein gene of alfalfa mosaic virus ~isplay increased resistance to infection by alfalfa mosaic virus.
SUMMAR~ OF THE INVENTION
The present invention relates to an expression eassette which can express a desired gene at high levels. The present invention relates to nn expression vector which comprlses an expression cassette. The high level expression vector of the present invention eomprises: a promoter.; a 5' untransl~ted region which is at l~ast 60% A and T; an initiation codon comprising Kozak's element; a cloning site where a desired gene may be inserted to form a functional expr~ssion unit; and a 3' untranslated re~ion which comprises a poly(A) additi~n signal and flanking sequence which yields hiKh level expression. The present invention relates to trnnsformed bacterial and plant cells which contain the expression vector, The present lnvention relates to ~ransgenic plants which ar~
produc~d rom plant cells transformed with the expreasion vector.
2S Th~ presen~ invention relates to a proaess of producin~ transg~nic plnnts with desirable traits by producing the plant~ from plan~ cells which havc been transformed with an expression vector which contnins ~ene conierring SUC~I trai~s, More particularly, the preaent in~ent.ion provide~ a D~IA plant expre0~ion cu~ette oompri~ing: (a) a promoter;
(b)a Cuoumber Mosaic Virus (CMV) coat protein gene 5' untranslated region; (c) an initiation region, the ini-tiation region comprising the sequence AAXXATGG; (d) a gene; and (e) a poly(A) addition signal, the poly(A) addition ~ignal containing untranslated flanking sequences; wherein the promoter is upstream from the (b) :
. . .
- 5a - ~ r~ g region; the (b) region i8 up~tream ~rom the initiation region; the initiation region is upstream and operably linked to the DNA molecule encoding a gene, and the gene is upstream and operably linked to the poly(A) addition aienal .
DETAILED DESCRIPTION OF THE INVENTION
Certain convention~ are used in Charts 1-9 to illu~trate plasmids and DNA fragments as follows:
(1) The single line figures represent both circular and linear double-stranded DNA.
(2) A~terisks (*) indicate that the molecule represented i3 ¢ircular. Lack of an a~terisk indicate~ that the molecule is linear.
(~) Junctions between the natural boundaries of functional component~ are indi¢ated by vertical lines along the . " .~
"t -6- ~3~ 3 horlzontal lines.
(4) Gene.s or functional coMponents are lndicated below the horizontal lines.
(5) Restriction sites are indicated above the horizontal lines.
(6) Distances between genes and restriction sites are not to scale. The figures show the relative positions only unless indicated otherwise.
(7) The following abbreviations are used to denote function and components:
a) Pc~ ~ CaMV35S promoter;
b) Ic ~ CMV intergenic region, the intergenic region comprising the initiation codon and AT rich 5' untranslated region;
c) SCa - CaMV35S poly(A) addition signal; and d) Nos - Nos nptII gene.
Most of the recombinant DNA methods employed in practicing the present invention are standard procedures, well known to those skilled in the art, and described in detail in, for example, European Patent Application Publication Number 223452 published November 29, 1986, ~nzymes are obtained from commercial sources and are used according to the vendor's recommendations or other variations known in the art.
General re~erences containing such standard techniques include the following: R. Wu, ed. (1979) Methods in En~ymology, Vol~ 68; J. H.
Miller ~1972) Experiments in Molecular Genetlcs; T. Mani~tis e~
~1982) Molecular Cloning: A Laboratory Manual; ~. M. Clover, ad.
~1985) DNA Clonlng Vol, II; H,G. Polites and K.R. Marotcl (19~7) "A
s~op-wise protocol ior cDNA synthesis". Biotechniques 4:514~520, S.B, Gelvin and R,A, Schilperoort, ed9, In~roduc~ion, Expression, and Analysis oP Gene Products ln Plant~, For the purposes of the present disclosure ~he following definitions Por terms used herein are meant to apply.
"Expression cassette" means fl DNA frflgment which contains a gene operably linked to regulatory sequences necessary for gene expres-sion.
"Promoter" means a promoter which is functional in the host plant.
. .; ~ ,. ~ .
p~
"Initiation region" includes the initiation codon and nucleotides flanking the inltiation codon.
"Operably linked" refers to the linking of nucleotide regions encoding specific genetic information such that the nucleotide regions are contiguous, the functionality of the region is preserved and will perform relative to the other regions as part of a functional unit.
"AT rich 5"' untranslated reglon is a nucleotide sequence is composed of at least 60~i adenine or thymine nucleotides.
"Untranslated flanking re~ion" refers to nucleotide sequences which are 3' of the termination codon and end at the poly~A) addition signal.
'~Vector" is a vehicle by means of which DNA fragments can be introduced into host organisms.
"Expression vector" is a vehicle by means of which DNA fragments that contain sufficient genetic information can be introduced into host organisms and can, therefore, be expressed by the host.
"Antipathogenic gene" is a gene which encodes a DNA sequence which is either the antisense sequence of a pathogenic gene or the 2Q antipathogenic gene encodes a peptide whose presence ln an organism confe.rs an increased resistenca to a pathogen.
To practice the present invention, the gene sought to be introduced into the plant's genetic material must be inserted into a vector containing the genetic regulatory sequences necessary to express the inser~ed gene. Accordingly, a vector must be constructod to provide tho regulatory soquences such that they will be Eunctional upon inserting a desirod geno, Whon the oxpression vector/insert construct is assemblod, it is used to transform plant cells which are then used to regonerate plan~s. Those tran~genic plants carry the gene in the oxpresslon voctor/insert construct. Tho gene is expressed in the plant and the trait which the gene influences wlll be conferred upon the plant.
In order to express the gene, the necessary genetic regulatory sequences must be provided. Both transcription and translation signals are necessary for gene expression once the gene is transferred and integrated into a plant genome. It must, therefore, be engineered to contain a plant expressible promoter, a translation initiation codon ~ATG) and a plant functional poly(A) additlon signal -8- ~33~
(AATAM) 3' of its translation termination codon.
In the present lnvention, additional genetic regulatory sequences are provided in order to obtain high levels of gene expression. As described above, an expression vector must contain a promoter, an intiation codon and a poly(A) addition signal. In order to get a higher level of expression, untranslated regions 5' and 3' to the inserted genes are provided. Furthermore, certain sequences flanking the lnitiation codon optimize express~on. The promoter used is one that ls chosen for high level expression.
A 5' untranslated region which results in higher level expres-sion of an in~erted gene is provided downstream from the promoter and upstream from the initiation codon. This region contains at least 60~ of the sequence as Adenlne and Thymine. l`here is a statistical bias for expression when such an AT rich region is located between the promoter and intiation codon. This preference is utilized in the preferred embodiment of the present invention by inclusion of an AT
rich 5' untranslated region intermediate of the promoter and initi-ation codon.
The present invention also contains a specific nucleotide sequence flanking the initiation codon. This preferred sequence, termed Kozak's element, is AAXXA~GG wherein X represents any of the four nucleotides. The presence of the ~nitiation codon following Kozak's rule results in higher level expression when used in an expression vector. In the preferred embodiment of the present invention, the small subunit of ribulose bis-phosphate carbo~ylAse~oxygenase (SS RVBISC0) contains an initiation codon in which Kozak's elemen~ is used.
Furthermore, the present invention contains n 3' untranslated region downstream from the clonl.ng ~ite ~er~ th~ co~t protein gene 30 is in~ar~ad and ups~ream rom the poly(A) addition signal. The sequenca of this 3' untranslated region res~lts in a statistical bias for protein production. The sequence promotes high level expression.
The poly(A) addition signal is found directly downstream from the 3' untranslated region and can be derived from the same source. In the preferred embodiment of the present invention, the 3' untranslated region and poly(A) addition signal are derived from CaMV 35S gene or the phaseolin seed storage protein gene.
~ he poly(A) addition signal from CaMV, nopaline synthase, _ 9 ~ o ! ~
octopine synthase, bean storage protein, and SS RUBISC0 genes are also suitable for this construction. Several promoters which function in plants are available, but the best promoters are the constitutive promoter from cauliflower mosaic virus (CaMV, a plant DNA virus) and the SS RUBISC0 gene.
Using methods well known to those having ordinary skill in the art, plant cells are transformed with the vector construct and the plant cells are induced to regenerate. The resulting plants contain the coat protein genes and produce the coat protein. The production of the protein confers upon the plant an increased resistance to infection by the virus from which the coat protein gene was derived.
In addition, the expression vector of the present invention comprises a DNA molecule comprising a promoter, an AT rich 5' untranslated region, an initiation region, a gene and a polyadenylation signal with untranslated flanking regions wherein the promoter is upstream and operably linked to the AT rich 5' untrans-lated region, the AT rich 5' untranslated region is upstream and operably linked to the initiation region which is upstream and operably linked to the antipathogenic gene which is upstream and operably linked to the poly(A) addition signal.
The DNA molecule described above may comprise a promoter derived from the Cauliflower mosaic virus CaMV 35S gene. The DNA molecule comprising the expression vector described above may contain an AT-rich 5' untranslated region is derived from the 5' untranslated region of Cucumber mosaic virus CMV coat protein gene or SS RU~ISC0 gene, Similarly, the initiation region in the ~NA mol~cule described above mAy be derived from the 5' ~mtranslated reglon of Cucumber mo3alc vlrus C~V CoAt protein gene or SS RUBISCO gene. The initia-tion region oE the DNA molecule described above comprlses the DNA
a~quence AAXXATGG. Furthermore, the DNA molecule describqd above mAy compri~e n poly(A) adclitlon slgnal derived from either the Cauli-flower mosaic virus CaMV 35S gsne; the phaselin storage protein gene, the nopalinesynthase gene, the octopine synthase gene, the bean storage protein gene or the SS RUBISC0 gene.
The expression vector described above may comprise a DNA
molecule in which the antipathogen gene is a viral pathogen coat protein gene, a viral enzyme gene, a gene derived from a host gene or an unrelated gene. Furthermore, the introduced gene may be for a ~3~7 ~ ~, peptide which acts as a pesticide, a fungicide or a herbicide when expressed in a plant. The gene may encode a peptide whose presence confers tolerance to severe or eradic cllmatlc conditlons. The gene, alternatively, may encode a proteln whose presPnce in a plant enhances the nutritional value of the plant. The present invention provides a recombinant DNA molecule which comprises any desired gene operably linked to genetic regulatory elements necessary for high expression in transformed plants. Moreover, the gene ~ay not be required to be transcribed into a peptide. The gene may encode an antisense strand to a nucleotide sequence forwhich inhibition of translation is desirable.
The expression vector may be an Agrobacterium derived binary vector. The expression vector may be used to transformed cells and transgenic plants may be produced comprising the transformed cells.
DESCRIPTION OF THE PREFERRED F~BODIMENTS
Example 1 Isolation of WMVII RNA.
Watermelon mosaic virus II tWMV II) was propagated in zucchini squash (Cucurbita pepo L) plants and RNA was isolated by the method described by Yeh and Gonsalves (Virology 143:260, 1985).
Example 2 Isolation of PRV-p RNA.
Papayn ringspot virus strain prv (PRV-p) was propagated in ~elly melon, Cucumis metuliferus tNand.) Mey. Acc. 2549 plants and RNA was isolated by the method described by Yeh and Gonsalves (Virology 143:260, 1985).
Example 3 Isolation of ZYMV RNA.
Zucchini yellow mosaic viru~ (ZYMV) was propagated in zucchini squash ~Cucurbita pepo L) plants and RNA was isolated by ~he method described by Yeh and Gonsalves (Virology 143:260, 1985).
Example 4 Syn~hesls of Doublo~strandad cDNA.
'rha procedure ~Ised to make double stranded oDNA from lsolated viral ~NA i9 the same for all viral RNA isolated above. The purified RNA was sub~ected to the cDNA synthesis protocol described by Polites and Marotti (Biotechniques 4:514, 1986) and because this RNA contains an A-rich region at its 3'-end (similar to that found for many eukaryotic mRNAs) the procedure was straight-forward. The synthesis of double stranded cDNA was also done as described by Polites and Marotti. APt~r double-stranded cDNA was synthesized, it was purified by passage through a G-100 Sephadex* column, precipitated with -trade mark , 11 ~ 3 ~
ethanol, and suspended in 20 ~1 of lOX EcoRI methylase buffer (100 mM
NaCl, 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 80 ~M S-adenosyl methio-nine, and 100 ~g/ml bovine serum albumin). An additional amount of S-adenosyl methionine (1 ~1 of a 32 mM solution) was added to the reaction mixture, followed by the addition of 1 ~1 (20 units) EcoRI
methylase. The reaction was incubated at 37C for 30 minutes and stopped by incubation at 70C for 10 minutes Then 1 ~1 (5 units) of E. coli DNA polymerase I Klenow fragment was added and incubated at 37~C for 10 minutes, followed by phenol/chloroform extraction and ethanol precipitatLon. The pelle~ was washed in 70~ ethanol, then 70% ethanol/0 3 M sodium acetate. The pellet was dri~d and re-suspended ln 8 ~1 of 0.5 ~g/~l phosphoryla~ed EcoRI llnkers (Col-laborative Research, Inc., 128 Spring St., ~exington, MA 02173). One ~1 of lOX lLgase buffer (800 mM Tris-HCl ph 8.0, 200 mM MgC12. 150 mM
DTT, 10 mM ATP) and 1 ~1 of T4 DNA ligase (4 units) were added, and the reaation was incubated overnight at 15C. The ligation reaction was stopped by incubation at 65C ~or 10 minutes. Sixty ~1 of H20, 10 ~1 of lOX EcoRI salts (900 mM Tris-HCl pH 8.0, 100 mM MgC12, 100 mM NaCl), and 10 ~1 of EcoRI (10 units/~l) were added, and the reaction was incubated at 37C for 1 hour. The reaction was stopped by phenol/chloroform and chloroform extractions. The reaction mixture was then size fractionated by passage through a Sephadex*G-100 column and the fractions containing the largest double strande~
cDNA molecules were concentrated by butanol extraations, pr~aipita~ed with ethanol, and resuspended in 10 ~1 of H20 Five ~1 of the double ~tranded cDNAs was added eo 0.5 ~g oi~ pUCl~ DNA (which h~ b~en pr~vlously treated with phosphAtase to remove the 5' phosphates), 1 ~1 o~ lOX ligase buer, and 1 ~1 of T4 llgase, and the r~action was incubated a~ 15~ for 16 hours. The resulting liga~ed pUC19~coa~
pro~oin ~ene double stranded cDN~ moleculea were trans~ormed into ~,coli hoa~ cells aa described by the manui`acturer (Bethesda Reaearch Lnboratories, Inc., Gaithersburg, MD 20877) and plated on medium containing 50 ~g/ml ampicillin, 0.04 mM IPTG, and 0.004~ X-Gal.
Bacterial colonies showing no blue color were selected for further analysis, Glones containing the 3'-region and posslbly the coat protein gene were identified by hybridi~ation against a 32P-labeled oligo-dT. Bacterial colonies showing hybridization to this probe should contain at lea~t the poly(A) re~ion of the p~rticular po~y-*Trade-mark -12- ~ 3 `~
vlrus ~enome Several of the hybridizing bacterial clones were selected and plasmid DNAs were isolated accordlng to methods known to those skilled ln the art.
Example 5 Identification of the PRV-p Coat Protein Gene.
Several of the cloned cDNAs of PVP-p RNA were sequenced by the chemical DNA sequencing method described by Maxam and Gilbert (Methods of Enzymology 65:499, 1980). Based on this information and comparative analysis with other potyviruses clone number pPRV-117 was found to contain a complete copy of the PRV-p co~t protein gene. The N-terminus of the co~t protein w~s identiiied by the locatlon of th~
dipeptide sequence Gln-Ser. The length of the PRV-p coat prot~in gene coding region is consistent with a gene encoding a protein of about 33 kDal.
Example 6 Construction of a Plant-Expressible PRV-p Coat Protein Gene Cassette with CaMV 35S Promoter and Polyadenylation Signal and CMV 5' ~ntranslated Region and Translation Initiator ATG.
Attachment of the necessary plant regulatory signals to the PRV-p coat protein gene was accomplished by constructing a translational fusion with a clone originally designed for ~he expression of the CMV
20 coat protein gene, using clone pUC1813/CPl9. Plasmid p~C1813/CPl9 is a vector suitable for agrobacterium mediated gene transfer. An ~coRI-EcoRI fragment was removed from pDH51/CPl9 and placed into the EcoR~ site of the plAsmid, pUC1813 ~available from Robert K., Department of Chemistry, Washington State University, Pullman, 25 Washington), creating plasmid pUC1813/CPl9. Th:l.s trans-lational fusion clone w~s conatructed by flrst identiPying two restriction enzyme s:ltes within clone pUC1813/CP19. One site ~Tthlll I) is located between amino acids 13 to 17 while the other si~e (~stX
I~ ia located near ~he ond of the 3'-untranslated regiorl of the CMV
aoa~ pro~ein gen~.
Addition oE ~hese specific resitriction enzyme sites to the PRV-p CoAt protein gene was accomplished by the polymerase chain reaction technique, using an instrument and Taq polymerase purchased from Perkin Elmer-Cetus, Emeryville, Ca. Specifically, two respective 5' and 3' oligomers (CGACGTCGTCAGTCCMGAATGAAGCTGTG, contaLning a Tthlll I site and (CCCACGAAAGTGGGGTGAAACAGGGTCGAGTCAG, containine ~ BstX I
.; , ;
~ ~ ?~
site), sharing at least 20 nucleotides with the PRV-p coat protein gene were used to prime synthesis and gene amplification of the coat protein gene. After synthesis, the amplified fragments were digested with Tthlll I and BstX I to expose the sites.
As shown in Chart 1, pUC1813/CPl9 is the expression vector which contains the CMV coat protein gene. Plasmld pUC1813/CP19 contains Tthlll I nnd BstX I sites.
The digested, amplified fragments are ligated into the respec-tive exposed sites of pUC1813/CPl9 and the expected new constructlon 1~ was identified using methods known to those skilled ln the art.
Poly~erase chain reaction techniques were ~sed to amplify PRV-P coat protein gene containing the TthlllI and BstXI sites. ~he plasmid pUC1813/CP19 and PRV-P coat protein gene fragments were digested with TthlllI and BstXI and ligated to each other. The resulting clone, 15 designated pUC1813/CP19-PRVexp, was subJected to nucleotide sequenc-ing to ensure tha~ the cloning and gene amplification diA not introduce any detrimental artifacts. The sequence showed no arti-facts, suggesting that this plant expression cassette should be capable of expressing a PRV-p coat protein gene which contains an additional 16 amino acids of CMV coat protein at its N-terminus.
Example 7 Construction of a Micro T DNA Plasmid Containing the Plant-expressible PRV-p Coat Protein Gene Construction.
As depicted in Chart 2, the plant expression cassette Eor the PRV-p coat protein gene was transferred into a ~uitable micro T-DNA
vector which contains the necessary Agrobacterlum T-DNA tran~Eer aignals for transfer Erom an Agrobacterium and integration into a plant genome, and a wide host-range origin o~ replication (for replication in Agrobacterium). Plasmid pUC1813/CPlg-PRVexp was digested with Hind III and ttle resulting 2.2 kb inaert Eragment contalning ~he plant axpressible cassette was removed and ligated into the Hind III site ~f the modified Agrobacterium-derived micro-vector pGA482 (modification included the addi~ion of the ~-glucuroni-da~e gene). The micro T-DNA vector, pGA482, is available from G. An, Institute of Biological Chemistry, Washington State University, Pullman, WA. The resulting plasmid was deslgnated, pGA482/G/CPl9-PRVexp and i9 shown in Chart 2. This plasmid (or derivatives thereof) waa ~ransferred into virulent or avirulent strains of Agrobacterium tumei`aciens or rhizogenes, such as A208, C58, LBA4404, ;~:' ~ ' ' . ' ~; ;''`,';'`` '.'," ''" '"';' ''' '"'';';' ' ' " " ' ' ~ .
~3~2~3 ~3 C58Z707, A4RS, A4RS(pRiB278b), and others. Strnins A208 C5~, LBA4404, and A4RS nre available from American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD. Bacterla A4RS (pRiB-278b)is ~vailable from Dr. F. Casse-Delbart, C.N.~.A., Routede Saint Cyr. F78000, Versailles, France. Strain C58Z707 is available from Dr, A.G.Hepburn, Dept. of Agronomy, University of Illinols, Urbana, IL.
After transfer of the engineered plasmld pGA482/G/CP19-PRVexp into any of the above listed Agrobacterium strains, these Agro-bacterium strains can be used to transfer and integrate within a plant genome the plant-e.xp~essible PRV-p coat protein gene contained wlthin lts T-DNA region. This transfer can be accompliQhed using the standard methods for T-DNA transfers whlch are known to those skllled ln the art, or this transfer can be accompllshed using the methods described in WO 89/5858, published June 29, 1989, entltled "Agrobacterlum Mediated Trans-formation of Germinating Plnnt Seedsn.
Example 8 Construction of a Plant-expresslon Cassette for Expreasion of Varlous Genes ln Transgenlc Plants.
In the preferred embodiment of the present invention, the followlng expression cassette was constructed to provide the neces-sary plant regulatory slgnals (which include the addition o~ a pro~oter, 5' untranslated region, translation initintion codon, and polyndenyl~tion 3ignnl) to the gene inser~s in order to ~chleve ~5 high l~vel expression of ~he lnserts. The exprs.sslon cassette may be used to expreas any genes inserted ~herein. ~ccordingly, the ~pplicability oE the expression aassetta goes beyond lts use in expr~saine co~ protein genes. Rather, the expression cassette may be used to exprass ,any desired protein in transgenlc pl~ants. ! The expression cassette is the preferred expresslon system for expresslng vlr~l co~t protein genes ln plants.
The expresslon cassette of the preferred embodiment contalns: a constltutive promoter; a 5' untranslated reglon whlch enhances gene expression; an lnitiation codon which comprlse Kozak's element; a cloning site where the gene to be expressed may be inserted ~o produce a func~ional expression unlt; and a 3' untranslated reglon whlch comprises a poly(A) additlon signal and untranslated ~lanking , . ' ''~ . ` ' '.1 ., . ~ ~.
I
regions which result in a hi~her level of expression.
More specifically, the expression cassette which is the prefer-red embodiment of the present invention consists of the cauliflower mosaic vlrus (CaMV) 35S transcript promoter, the 5'-untranslated region of cucumber mosaic virus (CMV), the CMV translation inltiation codon, and the CaMV polyadenylation signal. The construction of this expression cassette utilized the Polymerase Chain Reaction (PCR) technique to obtain correct position of the plant regulatory signals and the addition of convenient restriction enzyme sites which allow for the easy addition of a coat protein gene and the excision of the completed cassette so it can be transferred to other plasmids.
To accomplish the construction of this expression essse~te the following oligomers were synthesi~ed:
1. 5'-G MGCTTCCGGAAACCTCCTCGGATTCC-3', contains a HindIII site at its 5'-end and contains 21 bases which are identical eo 21 bases in the 5'-flanking region of CaMV.
2. 5'-GCCATGGTTGACTCGACTCAATTCTACGAC-3', contains a NcoI site at its 5'-end which contains a tr~nslation initiation codon which eonforms to Kozak's rules and has 21 bases which are identical to 21 bases in the antisense strand of the CMV S'-untranslated region.
3. 5'-GCCATGGTTGCGCTGAAATCACCAGTCTC-3', contains a NcoI site at lts 5'-end (which contains the same translstion initiation codon as oligomer 2) and has 20 bases which are identical to 20 bases in the 3'-untransla~ed region of CaMV.
4. 5'-GAAGCTTGGTACCACTGGATTTTGGTT-3', contains a HindIII ~ite at its 3'-end and has a 20 base match with the flanking ~A region 3' of the CaMV polyadenylation si~e (on the antisense strand), These ollgomers were used to amplify sequene~ eontained within the CMV expres~ion clone refarred to as pUC1813/CPl9, shown in Chart 1, and rei'erred ~o above. As depieted in Chart 3, the PCR teehni~ue was used to ampli~y Ithe gene regulatory regions in p~C1813jCP19.
Amplifieation of the 5'-flanking, CMV 5'~untranslated region, and CMV
initiation eodon (which was modified to eonform to Kozak's rul~
AAXXATGG) resulted in a fragment oi` about 400 base pairs in length and amplifieation of the CaMV 3-untranslated and flanking regions resulted in a fragment of about 200 base pairs in length. These fragments were digested with NeoI and HindIII, isolated from a polyaerylamide gel, and then ligated into HindIII digested and -16~ J i ~
phosphatase treated pUC18. The resulting clone is referred to as pl8CaMV/CMV-exp and is shown in Chart 3.
Example 9 Identification of the WMVII Coat Protein Gene The cloned l~ffVII cDNA insert from clone pWMVII-41-3.2 which was produced as described above, was sequenced by using both the chemical (Maxam and Gilbert, Methods of Enzymology 65:499, 1980) and enzymatic (Sanger e. al., Proc. Natl. Acad. Sci. U.S.A. 74:5463, 1977) meth~ds.
Based on this information and comparative analysis with other potyviruses, the nucleotide sequence of clone pWMVII-41-3.2 was found to contain a complete copy of the WMVII coat proteln gene. The N-terminus of the coat protein was suggested by the location of the dipeptide sequence Gln-Ser. The length of tha WMVII coat proteln gene coding region (281 amino acids) i9 consistent with a gene encoding a protein of about 33 kD.
Example 10 Construction of a Plant-expressible WMVII Coat Protein Gene Cassette with CaMV 35S Promoter and Polyadenylatlon Signal and CMV Intergenic Region and Translation Initiator ATG.
As depicted in Chart 4, attachment of the necessary plant regulatory signals to the WMVII coat protein gene was accomplished by using the PCR technique to amplify the ~MVII coat protein gene using oligomers which would add the necessary sites to its 5' and 3' se~uences, Followlng this amplification tha resulting fragment is digested with the appropriate restriction enzyme and cloned into the NcoI site of the abo~e described expression cassette containing plasmid, pl8CaMV/CMV-exp. Clones containing the WMVII coat protein gena insert need only be checked to determine correct orientation wlth respact with tha CaMV promotar. Howevor, to ensure that no artifacts have been lncorporatsd during the PCR ampli~ication the entire c04t protein gena region is checked by nucleotide sequence analysis.
To obtnin the amplified WMVII coat protein gene with NcoI
restriction enzyme sites on both ends the follo~iDg two oligomars were syntheqized:
1. 5'-ACCATGGTGTCTTTACAATCAGGAAAAG-3', which adds a NcoI site to the 5'-end oi the WMVII coat protain gene and retains the same ATG
translation start codon which i9 present in the expression cassette, pl8CaMV/CMV-exp.
2. 5'-ACCATGGCGACCCGAAATGCT MCTGTG-3', which adds a NcoI site ~L 3 ~
to the 3'-end of the WMVII coat protein gene, this site can be ligated into the expression cassette, pl8CaMV/CMV-exp.
The cloning of this PCR WMVII coat protein gene, using these two oligomers, into pl8CaMV/CMV-exp yields A plant expressible WMVII gene (referred to as pl8WMVII-exp) which, following transcription and translation, will generate a WMVII coat protein which is ~dentical to that derived from the WMVII coat protein gene nucleotide sequence.
However, this coat protein will differ, because of necessary genetic engineering to add the ATG initiation codon and by including the last four amino acids of the 54 kD nuclear lnclusion protein (which is adJacent to the Glu-Ser protease cleavage site); the amino acids added are Val-Ser Leu-Glu-N-ter WMVII. The addition oE these four amino acid residues should not aEfect the ability of this coat prote~n to yield plants which are resistant to WMVII infections, because the N-terminal region of potyvlrus coat proteins appear not to be well conserved for eithsr length or amino acid identity.
However, if this is found to be a problem its replacement would involve the use of a different oligomer to obtain N-terminal varia-tions of the WMVII coat protein gene. The cloned construction of the plant expressible WMVII coat protein gene is referred to as pl8WMV-II-exp, and is shown in Chart 4.
Example 11 Construction of a Micro T-DNA Plasmid Containing the Plant-expressible WMVII Coat Protein Gene Construction.
As depicted in Chart 5, the plant expression cas~ette for the WMVII coat protein gene was transi`srred in~o a suitnble micro-T-DNA
vector which contains tho necessary Agrobacterium T-DNA transfer 3i~nals (to mediated transfer from an Agrobacteri~n and intRgration into A plant ~enome) and wide-hos~ range origin o~ replication (~or replic~ion in Agrob~cterium) to form plasmid pGA482/G/CPWMVII-exp.
To construct this plasmid, plasrnid pl8WMVII-exp was digested with Hlnd III (which cuts within the polycloning sites of pUC18, well outside of the expression cassette), and an 1.8 kb fragment contain-inB the plant-expressible cassette was removed and ligated into the Hind III site of the modified Agrobacterium-derived micro-vector 3S pGA482 (modification included the addition of the ~-glucuronidase gene), The micro T-DNA vector, pGA482, is shown ln Chart 2 and available from G. An, Institute of Biological Chemistry, Washington StAte University, Pullman, WA. The resulting plasmid was designa~ed, ~ 3 ~
pGA482/G/CPWMVII-exp is shown in Chart 5. This plasmid (or deriva-tives thereof) was transferred into virulent or avirulent strains of Agrobacterium tumefaciens or rhi~ogenes, such as A208, C58, LBA4404, C58Z707, A4RS, A4RS(pRiB278b), and others. Strains A208 C58, S LBA4404, and A4RS are available from American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD. Bacteria A4RS(pRiB-27Bb)is available from Dr. F. Casse-Delbart, C.N.R.A., Routede Saint Cyr. F78000, Versailles, France. Bacterin C58Z707 is avnilable from Dr. A.G.Hepburn, Dept. of Agronomy, Unlversity of Illinois, Urbana, IL.
After transfer of the engineered plasmid pGA482/G/CPWMVII-exp into any of the above listed Agrobacterium strains, these Agrobac-terium strains can be used to transfer nnd integrate within a plant genome the plant-expressible WMVII coat protein gene contained wlthin its T-DNA region. This transfer can be accomplished using the standard methods for T-DNA transfers which are known to those skilled in the art, or this transfer can be accomplished using the methods described in WO 89/5858, published June 29, 1989, ~ n~itled "Agrobacterium Mediated Transformation of Germinating Plant Seedsn. In addition, it has recently been shown that such Agrobacteria are capabla of transferring and integrating their T-DNA
regions into the genome of soybean plants. Thus these strains could be used to transfer the plant expressible WMVII coat protein Bene in~o ~he genome of soybean to develop a soybean plant llne which is ra~ ant to inf~ction from soybean mosaic vlrus ~trains~
Exnmple 12 MicroproJectile TransEer of p~MVII exp into Plant Tias~es.
Recently an alternative approach for the trans~e~ and integra-~ion o~ DNA into n plant genome has been daveloped. This technique relies on the use of micropro~ectiles on which the DNA tplasmid form) is attached. These micropro~ectiles are accelerated to high velocit-ies and their momentum is used to penetrate plant call walls and membranes. After penetration into a plant cell the attached DNA
leaches off the microproJectile and i8 transferred to the nucleus where DNA repair enzymes integrate the "free" DNA into the plant genome. In its present form the process is entlrely random, but plant tissues which have been successfully transformed by the plasmid DNA (or part of it) can be identii`ied and cultured to homogeneity by ; :
the use of selectable marker genes (such as the bacterial neomycin phosphotransferase II gene, NPTII), or reporter genes (such as the bacterial beta-glucuronidase gene, Gus). Successful use of particle acceleration to transform plants has recently been shown for soybean and the transfer of pl8WMVII-exp into the genome could result in obtaining soybean plants which are resistant to infections from soybean mosaic virus strains.
The use of this process for the transfer of pl8WMVII-exp can be accomplished after the nddition of either plant expressible genes NPTII or Gus genes or both. Plasmids that have the nptII and Gus genes to pl8WMVII-exp are shown in Chart 6, and referred to as pl8GWMVII-exp and pl8NGWMVII-exp. In addit~on, the construction described in Example 11 can also be used for micropro~ectile transfer as it already has both the np~II and Gus genes attached to the pWMVII-exp cassette (see Chart 5). The only difficulty which ~he use of pGA482GG/cpWMVII-exp may encounter during transfer by the micro-projectile process is due to its large size, about 18~b, which may have a lower efficiency transfer and such larger plasmid generally yield less DNA during propagation.
To construct plasmid pl8GWMVii-exp, plasmid pl8WMVii-exp is digested with BamHI and ligated with a 3.0 kilobase BamHI isolated ~ragment containing the Gus gene. To construct plasmid pl8NGWMVii-exp, the plasmid pl8GWMVii-exp is digested with SmaI and ligated with a 2.4 kb isolated fragment containing the Nos-nptII gene ~enernt~d by digestion with Dral and Stul.
Example 13 IdentiPication of the ZYMV Coat Protein Gene.
The cloned ZYMV cDNA insert from clone pZYMV~15, which was cloned using the method described above, wa9 sequonced by using both the chcmical ~MAxam and Gilbert, Methods of Enzymology 65:499, 1980) 3~ flnd enzymatic ~Sanger et nl., Proc. Natl. Acad. Sci. ~.S.A. 74:5463, 1977) methods. Based on this information and comparative analysis with other potyviruses the nucleotide sequence of clone pZYNV-15 was found to contain a complete copy of the ZYMV coat protein gene. The N-terminus of the coat protein was suggested by the location of the dipeptide sequence Gln-Ser which is characteristic of cleavage sites in potyviruses (see Dougherty et al. EMB0 J. 7:1281, 1988). The length oi' the Z~MV coat protein gene coding region (280 amino acids) i9 aonsl9tent with fl gene encoding a protein of about 31.3 kD.
Example 14 Construction of a Plant-expressible ZYMV Coat Pro~ein Gene Cassette wlth CaMV 35S Promoter and Polyadenylation Signal and CMV Intergenic Region and Translation Initiator ATG.
As depicted in Ch~rt 7, attachment of the necessary plant regulatory signals to the ZYMV coat prote~n gene was accomplished by using the PCR technlque to amplify the ZYMV coat protein gene using oligomers which would add the necessary sites to its 5' and 3' sequences. Following this amplification the re~ulting fragment is digested with the appropriate restriction enzyme and cloned into the NcoI site of the above expression cassette containlng plasmid, pUC18CP-exp. Clones containing the ZYMV coat protein gene insert need only be checked to determine correct orientation with respect with the CaMV promoter. However, to ensure that no artiiacts have been incorporated during the PCR amplification the entire coat protein gene region is checked by nucleotide sequence analysis.
To obtain the amplifled ZYMV coat protein gene with NcoI
restriction enzyme sites on both ends the following two oligomers were synthesized:
1. 5'-ATCATTCCATGGGCACTCM CCM CTGTGGC-3', which adds a NcoI
site to the 5'-end of th0 ZYMV coat protein gene and retains the same ATG translation start codon which is present in the expression cassette, pUC18cpexp.
2. 5'-AGCTM CCATGGCTAAAGATATCAAATA MGCTG-3', which adds a NcoI
site to the 3'-end of the ZYMV coat proteln g~ne, this ~lte can be ligated into the expression cassette, pUCl8cpexp.
The cloning of this PCR ZYMV coat protein gene, using these two oligo~rs, into pUC18cpexp yields a plant expressible ~YMV gene ~r~fRrr~d to as pUC18cpZY~) which following eran~cription and translation will generat~ a ZYMV coat protein which i~ identical to that derived from the ZYMV coat protein Benfl nucleotide sequance.
However, this coat protein will differ, because of necessary genetic enginearing to add the ATG initiation codon followed by Gly, which is the amino acid 3' ad~acent to the Ser of the polyprotein cleavage site. The Gly amino acid residue was selected for the potentlal N-terminal amino acld because many potyvirus coat proteins have eitheran Ser, Gly, or Ala at their N-terminal. However, if this is found to be a problem its replacement would involve the use of a different oligornar to obtain a different N-terminal amino acid for the ZYMV
~e~
coat protein. The cloned construction of the plant expressible ZYMV
coat protein gene is referred to pUC18cpZYMV, and is shown in Chart 7.
Example 15 Construction of a Micro T-DNA Plasmid Containing the Plant-expressible ZYMV Coat Protein Gene Construction.
Following the teachings of Example 11 with appropriate modifica-tions, the construction of a micro T-DNA plasmid containing a plant-expressible ZYMV coat protein was constructed. Plasmid pUC18cpZYMV
(Chart 7) was digested with Hind III (which cuts within the poly-cloning sites of pUC18, well outside of the expression cassette), anda 1.6 kb fragment containing the plant-expressible cassetta was removed and ligated into the Hind III site of the micro-vector pGA482 (Chart 2). The resulting plasmid was designnted, pGA482GG/cpZYMV is shown in Chart 8.
15After transfer of the engineered plasmid pGA482GG/cpZYMV into Agrobacterium strains, the Agrobacteriu~ utrains can be used to transfer and integrate within a plant genome the plant-expressible ZYMV coat protein gene contained within $t9 T-DNA region.
Example 16 Micropro~ectile Transfer of pUC18cpZYMV into Plant Tissues, Following the teachings of Example 12, the micropro,~ectile transfer technique can be used to introduce the ZYMV coat protein gene with appropriate genetic regulatory sequences into plant tissues.
25The use of this process for the transPer of pUC18cpZ~MV can be accompllshed sPter the nddition oP either plant expres~ible genes NPTII or Gus genes or both, Plasmids thnt have the nptII and Gus genes to pUC18cpZYMV are shown in Chart 9 and referred to ns pUC18Gc-pZYMV and pUC18NGcpZYMV. In addition, the construction described in 30Examplu 15 can ~190 be used fnr microproJectile trans~er a3 it nlready ~as both the np~II and Gus genas attached to the pUC18cpZYMV
cassette (see Chart 8). The only difficulty which the use of pGA4-82GG/cpZYMV may encoun~er during transfer by the micropro~ectile process i5 due to its large size, about 18kb, which may have a lower efficiency transfer and such larger plasmid generally yield less DNA
during propagation.
To construct plasmid pUC18GCPZYMV, plasmid pUC18CPZYMV ls digested with BamHI and ligated to a 3.0 BamHI isolated fragment ~,,,,",,,"~ ",~ ,"~ "..,",~"~,:','`,:"~
-22- ~ 3 ~
which contalns the Gus gene. To construct plasmid pVC18GCPZYMV, plasmid pVC18GCPZYMV is digested with SmaI and ligated with a 2.4 kb isolated fragment containing the Nos nptII gene isolated by digestion with DraI and StuI.
: .;
r- .
-23~ 7. i~
CHARTS
pPRV117 *, I I *
IPRV-p Coat Protein Gene pUC1813/Cpl9 HindIII NcoI TthlllIBstXI NcoI HindIII
* _l I I I I I - I ~ . I , I I I - ~ * , .
I PCa I Ic ¦ ¦CMV Coat Protein Gene¦ I Sca 2~
pUC1813/Cpl9-PRVexp HlndIII NcoI TthlllI BstXI NcoI HindIII
cn I Ic ~ CMV ¦ PRV p Coat--~t- - I *
Coat Protein (16AA) gene j ~ I ' ' ` j ! ' ~ e~ 7.~ Ji ,~ ~) 5 pGA482 HindIII
I Br I I NOS I I CaMV I I BL
Gus Gene pGA482/G/CPl9-PRVexp NindIII TthlllI BstXI HindIII
¦Br¦ ¦ Nos ~ ¦PCalIcl CMV I PRV-p Coat ~ ¦sC81 ¦CaNV¦ IBLI
Coat Protein Gus 30Protein Gene Gene (16AA) ~3~7~
HindIII NcoI NcoI HindIII
1¦ Pca ¦ Ic l lSca pl8CaMV/CMV-exp HindIII NcoIHindIII
* ~ I --I ---. *
I Pca ¦ Ic ¦ I Sca ~ 3 ~
p~ 41-3.2 * I I *
¦WMVII Coat Protein¦
Gene PCR Generated Gene NcoI NcoI
¦ WMVII Coat Protein Gene¦
pl8~MVII-exp HindIII NcoI NcoI HindIII B~mHI SmaI
* I ~ I - ............... ~ 1 1 1 1 1 * .
I PCa I Ic ¦WMV Coat Protein I I Sc~ ¦
Gene ~` ~ 3~
pGA482/G/CPWMVII-exp HindIII NcoI NcoI HindIII
* I I I 11 1 1 11 ~I ~ I I *
rl I Nos ll Pca I Ic I WMVII Coat ¦¦ SCa ¦ ¦ CaMV ¦ IBL¦
Protein Gus Gene Gene : I I ' ~ ! ' pl8GWMVII-exp HindIII NcoI NcoI HindIII BamMI BamHI SmaI
PCa I Ic ¦ WMV Coat II Sca I¦ CaMV
Prote$n Gus Gene Gene pl8NGWMVII-exp HindIII NcoI NcoI HindIII Ba~HI Bam~lI
IPCal IC I~VII coatl I Sca I I CaMV ¦ ¦ No~ ¦
Protein Gu~
Gene Gen~
::~
:L3~27~ ~
pZYMV-15 * I l *
¦ZYMV Coat Protein¦
Gene ZYMV Coat Protein Gene Ncol NcoI
IZYMV Coat Protein Gene¦
pUC18CpZYMV
HindlII NcoI NcoI HindIII Ban~lI SmaI
35 * . ~ I *
PCa I Ic ¦ZYMV Co~t Protein¦¦ Sca ¦
Gene ~ 3 ~
5 pGA482/GG/cpZYMV
HindIII NcoI NcoI HindIII
Br I I Nos I Pca I Ic I ZYMV Coat ¦ ¦ SCa ¦ ~ CEIMV ¦ IBL~
Protein Gus Gene G~ne . , I ,; ~
,: ' -31- ` ~3~2~
pUC18GCpZYMV
HindIII NcoI NcoI HindIII BamHI BamHI SmaI
¦PcpIICIZYMV co~tlIScaI ¦ CaMN ¦
Protein Gus Gene Gene pUC18NGCpZYMV
~5 HindlII NcoI NcoI HindIII BamHI Ban~lI
* 11 1 -~ L 11 1 1 1 1 *
IPCal Ic ¦ZYMV Co~t 1I Sca I¦ CaMV I 1 No5 ¦
30 Proteln Gu~
Gene Gene " , , i I , .
a) Pc~ ~ CaMV35S promoter;
b) Ic ~ CMV intergenic region, the intergenic region comprising the initiation codon and AT rich 5' untranslated region;
c) SCa - CaMV35S poly(A) addition signal; and d) Nos - Nos nptII gene.
Most of the recombinant DNA methods employed in practicing the present invention are standard procedures, well known to those skilled in the art, and described in detail in, for example, European Patent Application Publication Number 223452 published November 29, 1986, ~nzymes are obtained from commercial sources and are used according to the vendor's recommendations or other variations known in the art.
General re~erences containing such standard techniques include the following: R. Wu, ed. (1979) Methods in En~ymology, Vol~ 68; J. H.
Miller ~1972) Experiments in Molecular Genetlcs; T. Mani~tis e~
~1982) Molecular Cloning: A Laboratory Manual; ~. M. Clover, ad.
~1985) DNA Clonlng Vol, II; H,G. Polites and K.R. Marotcl (19~7) "A
s~op-wise protocol ior cDNA synthesis". Biotechniques 4:514~520, S.B, Gelvin and R,A, Schilperoort, ed9, In~roduc~ion, Expression, and Analysis oP Gene Products ln Plant~, For the purposes of the present disclosure ~he following definitions Por terms used herein are meant to apply.
"Expression cassette" means fl DNA frflgment which contains a gene operably linked to regulatory sequences necessary for gene expres-sion.
"Promoter" means a promoter which is functional in the host plant.
. .; ~ ,. ~ .
p~
"Initiation region" includes the initiation codon and nucleotides flanking the inltiation codon.
"Operably linked" refers to the linking of nucleotide regions encoding specific genetic information such that the nucleotide regions are contiguous, the functionality of the region is preserved and will perform relative to the other regions as part of a functional unit.
"AT rich 5"' untranslated reglon is a nucleotide sequence is composed of at least 60~i adenine or thymine nucleotides.
"Untranslated flanking re~ion" refers to nucleotide sequences which are 3' of the termination codon and end at the poly~A) addition signal.
'~Vector" is a vehicle by means of which DNA fragments can be introduced into host organisms.
"Expression vector" is a vehicle by means of which DNA fragments that contain sufficient genetic information can be introduced into host organisms and can, therefore, be expressed by the host.
"Antipathogenic gene" is a gene which encodes a DNA sequence which is either the antisense sequence of a pathogenic gene or the 2Q antipathogenic gene encodes a peptide whose presence ln an organism confe.rs an increased resistenca to a pathogen.
To practice the present invention, the gene sought to be introduced into the plant's genetic material must be inserted into a vector containing the genetic regulatory sequences necessary to express the inser~ed gene. Accordingly, a vector must be constructod to provide tho regulatory soquences such that they will be Eunctional upon inserting a desirod geno, Whon the oxpression vector/insert construct is assemblod, it is used to transform plant cells which are then used to regonerate plan~s. Those tran~genic plants carry the gene in the oxpresslon voctor/insert construct. Tho gene is expressed in the plant and the trait which the gene influences wlll be conferred upon the plant.
In order to express the gene, the necessary genetic regulatory sequences must be provided. Both transcription and translation signals are necessary for gene expression once the gene is transferred and integrated into a plant genome. It must, therefore, be engineered to contain a plant expressible promoter, a translation initiation codon ~ATG) and a plant functional poly(A) additlon signal -8- ~33~
(AATAM) 3' of its translation termination codon.
In the present lnvention, additional genetic regulatory sequences are provided in order to obtain high levels of gene expression. As described above, an expression vector must contain a promoter, an intiation codon and a poly(A) addition signal. In order to get a higher level of expression, untranslated regions 5' and 3' to the inserted genes are provided. Furthermore, certain sequences flanking the lnitiation codon optimize express~on. The promoter used is one that ls chosen for high level expression.
A 5' untranslated region which results in higher level expres-sion of an in~erted gene is provided downstream from the promoter and upstream from the initiation codon. This region contains at least 60~ of the sequence as Adenlne and Thymine. l`here is a statistical bias for expression when such an AT rich region is located between the promoter and intiation codon. This preference is utilized in the preferred embodiment of the present invention by inclusion of an AT
rich 5' untranslated region intermediate of the promoter and initi-ation codon.
The present invention also contains a specific nucleotide sequence flanking the initiation codon. This preferred sequence, termed Kozak's element, is AAXXA~GG wherein X represents any of the four nucleotides. The presence of the ~nitiation codon following Kozak's rule results in higher level expression when used in an expression vector. In the preferred embodiment of the present invention, the small subunit of ribulose bis-phosphate carbo~ylAse~oxygenase (SS RVBISC0) contains an initiation codon in which Kozak's elemen~ is used.
Furthermore, the present invention contains n 3' untranslated region downstream from the clonl.ng ~ite ~er~ th~ co~t protein gene 30 is in~ar~ad and ups~ream rom the poly(A) addition signal. The sequenca of this 3' untranslated region res~lts in a statistical bias for protein production. The sequence promotes high level expression.
The poly(A) addition signal is found directly downstream from the 3' untranslated region and can be derived from the same source. In the preferred embodiment of the present invention, the 3' untranslated region and poly(A) addition signal are derived from CaMV 35S gene or the phaseolin seed storage protein gene.
~ he poly(A) addition signal from CaMV, nopaline synthase, _ 9 ~ o ! ~
octopine synthase, bean storage protein, and SS RUBISC0 genes are also suitable for this construction. Several promoters which function in plants are available, but the best promoters are the constitutive promoter from cauliflower mosaic virus (CaMV, a plant DNA virus) and the SS RUBISC0 gene.
Using methods well known to those having ordinary skill in the art, plant cells are transformed with the vector construct and the plant cells are induced to regenerate. The resulting plants contain the coat protein genes and produce the coat protein. The production of the protein confers upon the plant an increased resistance to infection by the virus from which the coat protein gene was derived.
In addition, the expression vector of the present invention comprises a DNA molecule comprising a promoter, an AT rich 5' untranslated region, an initiation region, a gene and a polyadenylation signal with untranslated flanking regions wherein the promoter is upstream and operably linked to the AT rich 5' untrans-lated region, the AT rich 5' untranslated region is upstream and operably linked to the initiation region which is upstream and operably linked to the antipathogenic gene which is upstream and operably linked to the poly(A) addition signal.
The DNA molecule described above may comprise a promoter derived from the Cauliflower mosaic virus CaMV 35S gene. The DNA molecule comprising the expression vector described above may contain an AT-rich 5' untranslated region is derived from the 5' untranslated region of Cucumber mosaic virus CMV coat protein gene or SS RU~ISC0 gene, Similarly, the initiation region in the ~NA mol~cule described above mAy be derived from the 5' ~mtranslated reglon of Cucumber mo3alc vlrus C~V CoAt protein gene or SS RUBISCO gene. The initia-tion region oE the DNA molecule described above comprlses the DNA
a~quence AAXXATGG. Furthermore, the DNA molecule describqd above mAy compri~e n poly(A) adclitlon slgnal derived from either the Cauli-flower mosaic virus CaMV 35S gsne; the phaselin storage protein gene, the nopalinesynthase gene, the octopine synthase gene, the bean storage protein gene or the SS RUBISC0 gene.
The expression vector described above may comprise a DNA
molecule in which the antipathogen gene is a viral pathogen coat protein gene, a viral enzyme gene, a gene derived from a host gene or an unrelated gene. Furthermore, the introduced gene may be for a ~3~7 ~ ~, peptide which acts as a pesticide, a fungicide or a herbicide when expressed in a plant. The gene may encode a peptide whose presence confers tolerance to severe or eradic cllmatlc conditlons. The gene, alternatively, may encode a proteln whose presPnce in a plant enhances the nutritional value of the plant. The present invention provides a recombinant DNA molecule which comprises any desired gene operably linked to genetic regulatory elements necessary for high expression in transformed plants. Moreover, the gene ~ay not be required to be transcribed into a peptide. The gene may encode an antisense strand to a nucleotide sequence forwhich inhibition of translation is desirable.
The expression vector may be an Agrobacterium derived binary vector. The expression vector may be used to transformed cells and transgenic plants may be produced comprising the transformed cells.
DESCRIPTION OF THE PREFERRED F~BODIMENTS
Example 1 Isolation of WMVII RNA.
Watermelon mosaic virus II tWMV II) was propagated in zucchini squash (Cucurbita pepo L) plants and RNA was isolated by the method described by Yeh and Gonsalves (Virology 143:260, 1985).
Example 2 Isolation of PRV-p RNA.
Papayn ringspot virus strain prv (PRV-p) was propagated in ~elly melon, Cucumis metuliferus tNand.) Mey. Acc. 2549 plants and RNA was isolated by the method described by Yeh and Gonsalves (Virology 143:260, 1985).
Example 3 Isolation of ZYMV RNA.
Zucchini yellow mosaic viru~ (ZYMV) was propagated in zucchini squash ~Cucurbita pepo L) plants and RNA was isolated by ~he method described by Yeh and Gonsalves (Virology 143:260, 1985).
Example 4 Syn~hesls of Doublo~strandad cDNA.
'rha procedure ~Ised to make double stranded oDNA from lsolated viral ~NA i9 the same for all viral RNA isolated above. The purified RNA was sub~ected to the cDNA synthesis protocol described by Polites and Marotti (Biotechniques 4:514, 1986) and because this RNA contains an A-rich region at its 3'-end (similar to that found for many eukaryotic mRNAs) the procedure was straight-forward. The synthesis of double stranded cDNA was also done as described by Polites and Marotti. APt~r double-stranded cDNA was synthesized, it was purified by passage through a G-100 Sephadex* column, precipitated with -trade mark , 11 ~ 3 ~
ethanol, and suspended in 20 ~1 of lOX EcoRI methylase buffer (100 mM
NaCl, 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 80 ~M S-adenosyl methio-nine, and 100 ~g/ml bovine serum albumin). An additional amount of S-adenosyl methionine (1 ~1 of a 32 mM solution) was added to the reaction mixture, followed by the addition of 1 ~1 (20 units) EcoRI
methylase. The reaction was incubated at 37C for 30 minutes and stopped by incubation at 70C for 10 minutes Then 1 ~1 (5 units) of E. coli DNA polymerase I Klenow fragment was added and incubated at 37~C for 10 minutes, followed by phenol/chloroform extraction and ethanol precipitatLon. The pelle~ was washed in 70~ ethanol, then 70% ethanol/0 3 M sodium acetate. The pellet was dri~d and re-suspended ln 8 ~1 of 0.5 ~g/~l phosphoryla~ed EcoRI llnkers (Col-laborative Research, Inc., 128 Spring St., ~exington, MA 02173). One ~1 of lOX lLgase buffer (800 mM Tris-HCl ph 8.0, 200 mM MgC12. 150 mM
DTT, 10 mM ATP) and 1 ~1 of T4 DNA ligase (4 units) were added, and the reaation was incubated overnight at 15C. The ligation reaction was stopped by incubation at 65C ~or 10 minutes. Sixty ~1 of H20, 10 ~1 of lOX EcoRI salts (900 mM Tris-HCl pH 8.0, 100 mM MgC12, 100 mM NaCl), and 10 ~1 of EcoRI (10 units/~l) were added, and the reaction was incubated at 37C for 1 hour. The reaction was stopped by phenol/chloroform and chloroform extractions. The reaction mixture was then size fractionated by passage through a Sephadex*G-100 column and the fractions containing the largest double strande~
cDNA molecules were concentrated by butanol extraations, pr~aipita~ed with ethanol, and resuspended in 10 ~1 of H20 Five ~1 of the double ~tranded cDNAs was added eo 0.5 ~g oi~ pUCl~ DNA (which h~ b~en pr~vlously treated with phosphAtase to remove the 5' phosphates), 1 ~1 o~ lOX ligase buer, and 1 ~1 of T4 llgase, and the r~action was incubated a~ 15~ for 16 hours. The resulting liga~ed pUC19~coa~
pro~oin ~ene double stranded cDN~ moleculea were trans~ormed into ~,coli hoa~ cells aa described by the manui`acturer (Bethesda Reaearch Lnboratories, Inc., Gaithersburg, MD 20877) and plated on medium containing 50 ~g/ml ampicillin, 0.04 mM IPTG, and 0.004~ X-Gal.
Bacterial colonies showing no blue color were selected for further analysis, Glones containing the 3'-region and posslbly the coat protein gene were identified by hybridi~ation against a 32P-labeled oligo-dT. Bacterial colonies showing hybridization to this probe should contain at lea~t the poly(A) re~ion of the p~rticular po~y-*Trade-mark -12- ~ 3 `~
vlrus ~enome Several of the hybridizing bacterial clones were selected and plasmid DNAs were isolated accordlng to methods known to those skilled ln the art.
Example 5 Identification of the PRV-p Coat Protein Gene.
Several of the cloned cDNAs of PVP-p RNA were sequenced by the chemical DNA sequencing method described by Maxam and Gilbert (Methods of Enzymology 65:499, 1980). Based on this information and comparative analysis with other potyviruses clone number pPRV-117 was found to contain a complete copy of the PRV-p co~t protein gene. The N-terminus of the co~t protein w~s identiiied by the locatlon of th~
dipeptide sequence Gln-Ser. The length of the PRV-p coat prot~in gene coding region is consistent with a gene encoding a protein of about 33 kDal.
Example 6 Construction of a Plant-Expressible PRV-p Coat Protein Gene Cassette with CaMV 35S Promoter and Polyadenylation Signal and CMV 5' ~ntranslated Region and Translation Initiator ATG.
Attachment of the necessary plant regulatory signals to the PRV-p coat protein gene was accomplished by constructing a translational fusion with a clone originally designed for ~he expression of the CMV
20 coat protein gene, using clone pUC1813/CPl9. Plasmid p~C1813/CPl9 is a vector suitable for agrobacterium mediated gene transfer. An ~coRI-EcoRI fragment was removed from pDH51/CPl9 and placed into the EcoR~ site of the plAsmid, pUC1813 ~available from Robert K., Department of Chemistry, Washington State University, Pullman, 25 Washington), creating plasmid pUC1813/CPl9. Th:l.s trans-lational fusion clone w~s conatructed by flrst identiPying two restriction enzyme s:ltes within clone pUC1813/CP19. One site ~Tthlll I) is located between amino acids 13 to 17 while the other si~e (~stX
I~ ia located near ~he ond of the 3'-untranslated regiorl of the CMV
aoa~ pro~ein gen~.
Addition oE ~hese specific resitriction enzyme sites to the PRV-p CoAt protein gene was accomplished by the polymerase chain reaction technique, using an instrument and Taq polymerase purchased from Perkin Elmer-Cetus, Emeryville, Ca. Specifically, two respective 5' and 3' oligomers (CGACGTCGTCAGTCCMGAATGAAGCTGTG, contaLning a Tthlll I site and (CCCACGAAAGTGGGGTGAAACAGGGTCGAGTCAG, containine ~ BstX I
.; , ;
~ ~ ?~
site), sharing at least 20 nucleotides with the PRV-p coat protein gene were used to prime synthesis and gene amplification of the coat protein gene. After synthesis, the amplified fragments were digested with Tthlll I and BstX I to expose the sites.
As shown in Chart 1, pUC1813/CPl9 is the expression vector which contains the CMV coat protein gene. Plasmld pUC1813/CP19 contains Tthlll I nnd BstX I sites.
The digested, amplified fragments are ligated into the respec-tive exposed sites of pUC1813/CPl9 and the expected new constructlon 1~ was identified using methods known to those skilled ln the art.
Poly~erase chain reaction techniques were ~sed to amplify PRV-P coat protein gene containing the TthlllI and BstXI sites. ~he plasmid pUC1813/CP19 and PRV-P coat protein gene fragments were digested with TthlllI and BstXI and ligated to each other. The resulting clone, 15 designated pUC1813/CP19-PRVexp, was subJected to nucleotide sequenc-ing to ensure tha~ the cloning and gene amplification diA not introduce any detrimental artifacts. The sequence showed no arti-facts, suggesting that this plant expression cassette should be capable of expressing a PRV-p coat protein gene which contains an additional 16 amino acids of CMV coat protein at its N-terminus.
Example 7 Construction of a Micro T DNA Plasmid Containing the Plant-expressible PRV-p Coat Protein Gene Construction.
As depicted in Chart 2, the plant expression cassette Eor the PRV-p coat protein gene was transferred into a ~uitable micro T-DNA
vector which contains the necessary Agrobacterlum T-DNA tran~Eer aignals for transfer Erom an Agrobacterium and integration into a plant genome, and a wide host-range origin o~ replication (for replication in Agrobacterium). Plasmid pUC1813/CPlg-PRVexp was digested with Hind III and ttle resulting 2.2 kb inaert Eragment contalning ~he plant axpressible cassette was removed and ligated into the Hind III site ~f the modified Agrobacterium-derived micro-vector pGA482 (modification included the addi~ion of the ~-glucuroni-da~e gene). The micro T-DNA vector, pGA482, is available from G. An, Institute of Biological Chemistry, Washington State University, Pullman, WA. The resulting plasmid was deslgnated, pGA482/G/CPl9-PRVexp and i9 shown in Chart 2. This plasmid (or derivatives thereof) waa ~ransferred into virulent or avirulent strains of Agrobacterium tumei`aciens or rhizogenes, such as A208, C58, LBA4404, ;~:' ~ ' ' . ' ~; ;''`,';'`` '.'," ''" '"';' ''' '"'';';' ' ' " " ' ' ~ .
~3~2~3 ~3 C58Z707, A4RS, A4RS(pRiB278b), and others. Strnins A208 C5~, LBA4404, and A4RS nre available from American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD. Bacterla A4RS (pRiB-278b)is ~vailable from Dr. F. Casse-Delbart, C.N.~.A., Routede Saint Cyr. F78000, Versailles, France. Strain C58Z707 is available from Dr, A.G.Hepburn, Dept. of Agronomy, University of Illinols, Urbana, IL.
After transfer of the engineered plasmld pGA482/G/CP19-PRVexp into any of the above listed Agrobacterium strains, these Agro-bacterium strains can be used to transfer and integrate within a plant genome the plant-e.xp~essible PRV-p coat protein gene contained wlthin lts T-DNA region. This transfer can be accompliQhed using the standard methods for T-DNA transfers whlch are known to those skllled ln the art, or this transfer can be accompllshed using the methods described in WO 89/5858, published June 29, 1989, entltled "Agrobacterlum Mediated Trans-formation of Germinating Plnnt Seedsn.
Example 8 Construction of a Plant-expresslon Cassette for Expreasion of Varlous Genes ln Transgenlc Plants.
In the preferred embodiment of the present invention, the followlng expression cassette was constructed to provide the neces-sary plant regulatory slgnals (which include the addition o~ a pro~oter, 5' untranslated region, translation initintion codon, and polyndenyl~tion 3ignnl) to the gene inser~s in order to ~chleve ~5 high l~vel expression of ~he lnserts. The exprs.sslon cassette may be used to expreas any genes inserted ~herein. ~ccordingly, the ~pplicability oE the expression aassetta goes beyond lts use in expr~saine co~ protein genes. Rather, the expression cassette may be used to exprass ,any desired protein in transgenlc pl~ants. ! The expression cassette is the preferred expresslon system for expresslng vlr~l co~t protein genes ln plants.
The expresslon cassette of the preferred embodiment contalns: a constltutive promoter; a 5' untranslated reglon whlch enhances gene expression; an lnitiation codon which comprlse Kozak's element; a cloning site where the gene to be expressed may be inserted ~o produce a func~ional expression unlt; and a 3' untranslated reglon whlch comprises a poly(A) additlon signal and untranslated ~lanking , . ' ''~ . ` ' '.1 ., . ~ ~.
I
regions which result in a hi~her level of expression.
More specifically, the expression cassette which is the prefer-red embodiment of the present invention consists of the cauliflower mosaic vlrus (CaMV) 35S transcript promoter, the 5'-untranslated region of cucumber mosaic virus (CMV), the CMV translation inltiation codon, and the CaMV polyadenylation signal. The construction of this expression cassette utilized the Polymerase Chain Reaction (PCR) technique to obtain correct position of the plant regulatory signals and the addition of convenient restriction enzyme sites which allow for the easy addition of a coat protein gene and the excision of the completed cassette so it can be transferred to other plasmids.
To accomplish the construction of this expression essse~te the following oligomers were synthesi~ed:
1. 5'-G MGCTTCCGGAAACCTCCTCGGATTCC-3', contains a HindIII site at its 5'-end and contains 21 bases which are identical eo 21 bases in the 5'-flanking region of CaMV.
2. 5'-GCCATGGTTGACTCGACTCAATTCTACGAC-3', contains a NcoI site at its 5'-end which contains a tr~nslation initiation codon which eonforms to Kozak's rules and has 21 bases which are identical to 21 bases in the antisense strand of the CMV S'-untranslated region.
3. 5'-GCCATGGTTGCGCTGAAATCACCAGTCTC-3', contains a NcoI site at lts 5'-end (which contains the same translstion initiation codon as oligomer 2) and has 20 bases which are identical to 20 bases in the 3'-untransla~ed region of CaMV.
4. 5'-GAAGCTTGGTACCACTGGATTTTGGTT-3', contains a HindIII ~ite at its 3'-end and has a 20 base match with the flanking ~A region 3' of the CaMV polyadenylation si~e (on the antisense strand), These ollgomers were used to amplify sequene~ eontained within the CMV expres~ion clone refarred to as pUC1813/CPl9, shown in Chart 1, and rei'erred ~o above. As depieted in Chart 3, the PCR teehni~ue was used to ampli~y Ithe gene regulatory regions in p~C1813jCP19.
Amplifieation of the 5'-flanking, CMV 5'~untranslated region, and CMV
initiation eodon (which was modified to eonform to Kozak's rul~
AAXXATGG) resulted in a fragment oi` about 400 base pairs in length and amplifieation of the CaMV 3-untranslated and flanking regions resulted in a fragment of about 200 base pairs in length. These fragments were digested with NeoI and HindIII, isolated from a polyaerylamide gel, and then ligated into HindIII digested and -16~ J i ~
phosphatase treated pUC18. The resulting clone is referred to as pl8CaMV/CMV-exp and is shown in Chart 3.
Example 9 Identification of the WMVII Coat Protein Gene The cloned l~ffVII cDNA insert from clone pWMVII-41-3.2 which was produced as described above, was sequenced by using both the chemical (Maxam and Gilbert, Methods of Enzymology 65:499, 1980) and enzymatic (Sanger e. al., Proc. Natl. Acad. Sci. U.S.A. 74:5463, 1977) meth~ds.
Based on this information and comparative analysis with other potyviruses, the nucleotide sequence of clone pWMVII-41-3.2 was found to contain a complete copy of the WMVII coat proteln gene. The N-terminus of the coat protein was suggested by the location of the dipeptide sequence Gln-Ser. The length of tha WMVII coat proteln gene coding region (281 amino acids) i9 consistent with a gene encoding a protein of about 33 kD.
Example 10 Construction of a Plant-expressible WMVII Coat Protein Gene Cassette with CaMV 35S Promoter and Polyadenylatlon Signal and CMV Intergenic Region and Translation Initiator ATG.
As depicted in Chart 4, attachment of the necessary plant regulatory signals to the WMVII coat protein gene was accomplished by using the PCR technique to amplify the ~MVII coat protein gene using oligomers which would add the necessary sites to its 5' and 3' se~uences, Followlng this amplification tha resulting fragment is digested with the appropriate restriction enzyme and cloned into the NcoI site of the abo~e described expression cassette containing plasmid, pl8CaMV/CMV-exp. Clones containing the WMVII coat protein gena insert need only be checked to determine correct orientation wlth respact with tha CaMV promotar. Howevor, to ensure that no artifacts have been lncorporatsd during the PCR ampli~ication the entire c04t protein gena region is checked by nucleotide sequence analysis.
To obtnin the amplified WMVII coat protein gene with NcoI
restriction enzyme sites on both ends the follo~iDg two oligomars were syntheqized:
1. 5'-ACCATGGTGTCTTTACAATCAGGAAAAG-3', which adds a NcoI site to the 5'-end oi the WMVII coat protain gene and retains the same ATG
translation start codon which i9 present in the expression cassette, pl8CaMV/CMV-exp.
2. 5'-ACCATGGCGACCCGAAATGCT MCTGTG-3', which adds a NcoI site ~L 3 ~
to the 3'-end of the WMVII coat protein gene, this site can be ligated into the expression cassette, pl8CaMV/CMV-exp.
The cloning of this PCR WMVII coat protein gene, using these two oligomers, into pl8CaMV/CMV-exp yields A plant expressible WMVII gene (referred to as pl8WMVII-exp) which, following transcription and translation, will generate a WMVII coat protein which is ~dentical to that derived from the WMVII coat protein gene nucleotide sequence.
However, this coat protein will differ, because of necessary genetic engineering to add the ATG initiation codon and by including the last four amino acids of the 54 kD nuclear lnclusion protein (which is adJacent to the Glu-Ser protease cleavage site); the amino acids added are Val-Ser Leu-Glu-N-ter WMVII. The addition oE these four amino acid residues should not aEfect the ability of this coat prote~n to yield plants which are resistant to WMVII infections, because the N-terminal region of potyvlrus coat proteins appear not to be well conserved for eithsr length or amino acid identity.
However, if this is found to be a problem its replacement would involve the use of a different oligomer to obtain N-terminal varia-tions of the WMVII coat protein gene. The cloned construction of the plant expressible WMVII coat protein gene is referred to as pl8WMV-II-exp, and is shown in Chart 4.
Example 11 Construction of a Micro T-DNA Plasmid Containing the Plant-expressible WMVII Coat Protein Gene Construction.
As depicted in Chart 5, the plant expression cas~ette for the WMVII coat protein gene was transi`srred in~o a suitnble micro-T-DNA
vector which contains tho necessary Agrobacterium T-DNA transfer 3i~nals (to mediated transfer from an Agrobacteri~n and intRgration into A plant ~enome) and wide-hos~ range origin o~ replication (~or replic~ion in Agrob~cterium) to form plasmid pGA482/G/CPWMVII-exp.
To construct this plasmid, plasrnid pl8WMVII-exp was digested with Hlnd III (which cuts within the polycloning sites of pUC18, well outside of the expression cassette), and an 1.8 kb fragment contain-inB the plant-expressible cassette was removed and ligated into the Hind III site of the modified Agrobacterium-derived micro-vector 3S pGA482 (modification included the addition of the ~-glucuronidase gene), The micro T-DNA vector, pGA482, is shown ln Chart 2 and available from G. An, Institute of Biological Chemistry, Washington StAte University, Pullman, WA. The resulting plasmid was designa~ed, ~ 3 ~
pGA482/G/CPWMVII-exp is shown in Chart 5. This plasmid (or deriva-tives thereof) was transferred into virulent or avirulent strains of Agrobacterium tumefaciens or rhi~ogenes, such as A208, C58, LBA4404, C58Z707, A4RS, A4RS(pRiB278b), and others. Strains A208 C58, S LBA4404, and A4RS are available from American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD. Bacteria A4RS(pRiB-27Bb)is available from Dr. F. Casse-Delbart, C.N.R.A., Routede Saint Cyr. F78000, Versailles, France. Bacterin C58Z707 is avnilable from Dr. A.G.Hepburn, Dept. of Agronomy, Unlversity of Illinois, Urbana, IL.
After transfer of the engineered plasmid pGA482/G/CPWMVII-exp into any of the above listed Agrobacterium strains, these Agrobac-terium strains can be used to transfer nnd integrate within a plant genome the plant-expressible WMVII coat protein gene contained wlthin its T-DNA region. This transfer can be accomplished using the standard methods for T-DNA transfers which are known to those skilled in the art, or this transfer can be accomplished using the methods described in WO 89/5858, published June 29, 1989, ~ n~itled "Agrobacterium Mediated Transformation of Germinating Plant Seedsn. In addition, it has recently been shown that such Agrobacteria are capabla of transferring and integrating their T-DNA
regions into the genome of soybean plants. Thus these strains could be used to transfer the plant expressible WMVII coat protein Bene in~o ~he genome of soybean to develop a soybean plant llne which is ra~ ant to inf~ction from soybean mosaic vlrus ~trains~
Exnmple 12 MicroproJectile TransEer of p~MVII exp into Plant Tias~es.
Recently an alternative approach for the trans~e~ and integra-~ion o~ DNA into n plant genome has been daveloped. This technique relies on the use of micropro~ectiles on which the DNA tplasmid form) is attached. These micropro~ectiles are accelerated to high velocit-ies and their momentum is used to penetrate plant call walls and membranes. After penetration into a plant cell the attached DNA
leaches off the microproJectile and i8 transferred to the nucleus where DNA repair enzymes integrate the "free" DNA into the plant genome. In its present form the process is entlrely random, but plant tissues which have been successfully transformed by the plasmid DNA (or part of it) can be identii`ied and cultured to homogeneity by ; :
the use of selectable marker genes (such as the bacterial neomycin phosphotransferase II gene, NPTII), or reporter genes (such as the bacterial beta-glucuronidase gene, Gus). Successful use of particle acceleration to transform plants has recently been shown for soybean and the transfer of pl8WMVII-exp into the genome could result in obtaining soybean plants which are resistant to infections from soybean mosaic virus strains.
The use of this process for the transfer of pl8WMVII-exp can be accomplished after the nddition of either plant expressible genes NPTII or Gus genes or both. Plasmids that have the nptII and Gus genes to pl8WMVII-exp are shown in Chart 6, and referred to as pl8GWMVII-exp and pl8NGWMVII-exp. In addit~on, the construction described in Example 11 can also be used for micropro~ectile transfer as it already has both the np~II and Gus genes attached to the pWMVII-exp cassette (see Chart 5). The only difficulty which ~he use of pGA482GG/cpWMVII-exp may encounter during transfer by the micro-projectile process is due to its large size, about 18~b, which may have a lower efficiency transfer and such larger plasmid generally yield less DNA during propagation.
To construct plasmid pl8GWMVii-exp, plasmid pl8WMVii-exp is digested with BamHI and ligated with a 3.0 kilobase BamHI isolated ~ragment containing the Gus gene. To construct plasmid pl8NGWMVii-exp, the plasmid pl8GWMVii-exp is digested with SmaI and ligated with a 2.4 kb isolated fragment containing the Nos-nptII gene ~enernt~d by digestion with Dral and Stul.
Example 13 IdentiPication of the ZYMV Coat Protein Gene.
The cloned ZYMV cDNA insert from clone pZYMV~15, which was cloned using the method described above, wa9 sequonced by using both the chcmical ~MAxam and Gilbert, Methods of Enzymology 65:499, 1980) 3~ flnd enzymatic ~Sanger et nl., Proc. Natl. Acad. Sci. ~.S.A. 74:5463, 1977) methods. Based on this information and comparative analysis with other potyviruses the nucleotide sequence of clone pZYNV-15 was found to contain a complete copy of the ZYMV coat protein gene. The N-terminus of the coat protein was suggested by the location of the dipeptide sequence Gln-Ser which is characteristic of cleavage sites in potyviruses (see Dougherty et al. EMB0 J. 7:1281, 1988). The length oi' the Z~MV coat protein gene coding region (280 amino acids) i9 aonsl9tent with fl gene encoding a protein of about 31.3 kD.
Example 14 Construction of a Plant-expressible ZYMV Coat Pro~ein Gene Cassette wlth CaMV 35S Promoter and Polyadenylation Signal and CMV Intergenic Region and Translation Initiator ATG.
As depicted in Ch~rt 7, attachment of the necessary plant regulatory signals to the ZYMV coat prote~n gene was accomplished by using the PCR technlque to amplify the ZYMV coat protein gene using oligomers which would add the necessary sites to its 5' and 3' sequences. Following this amplification the re~ulting fragment is digested with the appropriate restriction enzyme and cloned into the NcoI site of the above expression cassette containlng plasmid, pUC18CP-exp. Clones containing the ZYMV coat protein gene insert need only be checked to determine correct orientation with respect with the CaMV promoter. However, to ensure that no artiiacts have been incorporated during the PCR amplification the entire coat protein gene region is checked by nucleotide sequence analysis.
To obtain the amplifled ZYMV coat protein gene with NcoI
restriction enzyme sites on both ends the following two oligomers were synthesized:
1. 5'-ATCATTCCATGGGCACTCM CCM CTGTGGC-3', which adds a NcoI
site to the 5'-end of th0 ZYMV coat protein gene and retains the same ATG translation start codon which is present in the expression cassette, pUC18cpexp.
2. 5'-AGCTM CCATGGCTAAAGATATCAAATA MGCTG-3', which adds a NcoI
site to the 3'-end of the ZYMV coat proteln g~ne, this ~lte can be ligated into the expression cassette, pUCl8cpexp.
The cloning of this PCR ZYMV coat protein gene, using these two oligo~rs, into pUC18cpexp yields a plant expressible ~YMV gene ~r~fRrr~d to as pUC18cpZY~) which following eran~cription and translation will generat~ a ZYMV coat protein which i~ identical to that derived from the ZYMV coat protein Benfl nucleotide sequance.
However, this coat protein will differ, because of necessary genetic enginearing to add the ATG initiation codon followed by Gly, which is the amino acid 3' ad~acent to the Ser of the polyprotein cleavage site. The Gly amino acid residue was selected for the potentlal N-terminal amino acld because many potyvirus coat proteins have eitheran Ser, Gly, or Ala at their N-terminal. However, if this is found to be a problem its replacement would involve the use of a different oligornar to obtain a different N-terminal amino acid for the ZYMV
~e~
coat protein. The cloned construction of the plant expressible ZYMV
coat protein gene is referred to pUC18cpZYMV, and is shown in Chart 7.
Example 15 Construction of a Micro T-DNA Plasmid Containing the Plant-expressible ZYMV Coat Protein Gene Construction.
Following the teachings of Example 11 with appropriate modifica-tions, the construction of a micro T-DNA plasmid containing a plant-expressible ZYMV coat protein was constructed. Plasmid pUC18cpZYMV
(Chart 7) was digested with Hind III (which cuts within the poly-cloning sites of pUC18, well outside of the expression cassette), anda 1.6 kb fragment containing the plant-expressible cassetta was removed and ligated into the Hind III site of the micro-vector pGA482 (Chart 2). The resulting plasmid was designnted, pGA482GG/cpZYMV is shown in Chart 8.
15After transfer of the engineered plasmid pGA482GG/cpZYMV into Agrobacterium strains, the Agrobacteriu~ utrains can be used to transfer and integrate within a plant genome the plant-expressible ZYMV coat protein gene contained within $t9 T-DNA region.
Example 16 Micropro~ectile Transfer of pUC18cpZYMV into Plant Tissues, Following the teachings of Example 12, the micropro,~ectile transfer technique can be used to introduce the ZYMV coat protein gene with appropriate genetic regulatory sequences into plant tissues.
25The use of this process for the transPer of pUC18cpZ~MV can be accompllshed sPter the nddition oP either plant expres~ible genes NPTII or Gus genes or both, Plasmids thnt have the nptII and Gus genes to pUC18cpZYMV are shown in Chart 9 and referred to ns pUC18Gc-pZYMV and pUC18NGcpZYMV. In addition, the construction described in 30Examplu 15 can ~190 be used fnr microproJectile trans~er a3 it nlready ~as both the np~II and Gus genas attached to the pUC18cpZYMV
cassette (see Chart 8). The only difficulty which the use of pGA4-82GG/cpZYMV may encoun~er during transfer by the micropro~ectile process i5 due to its large size, about 18kb, which may have a lower efficiency transfer and such larger plasmid generally yield less DNA
during propagation.
To construct plasmid pUC18GCPZYMV, plasmid pUC18CPZYMV ls digested with BamHI and ligated to a 3.0 BamHI isolated fragment ~,,,,",,,"~ ",~ ,"~ "..,",~"~,:','`,:"~
-22- ~ 3 ~
which contalns the Gus gene. To construct plasmid pVC18GCPZYMV, plasmid pVC18GCPZYMV is digested with SmaI and ligated with a 2.4 kb isolated fragment containing the Nos nptII gene isolated by digestion with DraI and StuI.
: .;
r- .
-23~ 7. i~
CHARTS
pPRV117 *, I I *
IPRV-p Coat Protein Gene pUC1813/Cpl9 HindIII NcoI TthlllIBstXI NcoI HindIII
* _l I I I I I - I ~ . I , I I I - ~ * , .
I PCa I Ic ¦ ¦CMV Coat Protein Gene¦ I Sca 2~
pUC1813/Cpl9-PRVexp HlndIII NcoI TthlllI BstXI NcoI HindIII
cn I Ic ~ CMV ¦ PRV p Coat--~t- - I *
Coat Protein (16AA) gene j ~ I ' ' ` j ! ' ~ e~ 7.~ Ji ,~ ~) 5 pGA482 HindIII
I Br I I NOS I I CaMV I I BL
Gus Gene pGA482/G/CPl9-PRVexp NindIII TthlllI BstXI HindIII
¦Br¦ ¦ Nos ~ ¦PCalIcl CMV I PRV-p Coat ~ ¦sC81 ¦CaNV¦ IBLI
Coat Protein Gus 30Protein Gene Gene (16AA) ~3~7~
HindIII NcoI NcoI HindIII
1¦ Pca ¦ Ic l lSca pl8CaMV/CMV-exp HindIII NcoIHindIII
* ~ I --I ---. *
I Pca ¦ Ic ¦ I Sca ~ 3 ~
p~ 41-3.2 * I I *
¦WMVII Coat Protein¦
Gene PCR Generated Gene NcoI NcoI
¦ WMVII Coat Protein Gene¦
pl8~MVII-exp HindIII NcoI NcoI HindIII B~mHI SmaI
* I ~ I - ............... ~ 1 1 1 1 1 * .
I PCa I Ic ¦WMV Coat Protein I I Sc~ ¦
Gene ~` ~ 3~
pGA482/G/CPWMVII-exp HindIII NcoI NcoI HindIII
* I I I 11 1 1 11 ~I ~ I I *
rl I Nos ll Pca I Ic I WMVII Coat ¦¦ SCa ¦ ¦ CaMV ¦ IBL¦
Protein Gus Gene Gene : I I ' ~ ! ' pl8GWMVII-exp HindIII NcoI NcoI HindIII BamMI BamHI SmaI
PCa I Ic ¦ WMV Coat II Sca I¦ CaMV
Prote$n Gus Gene Gene pl8NGWMVII-exp HindIII NcoI NcoI HindIII Ba~HI Bam~lI
IPCal IC I~VII coatl I Sca I I CaMV ¦ ¦ No~ ¦
Protein Gu~
Gene Gen~
::~
:L3~27~ ~
pZYMV-15 * I l *
¦ZYMV Coat Protein¦
Gene ZYMV Coat Protein Gene Ncol NcoI
IZYMV Coat Protein Gene¦
pUC18CpZYMV
HindlII NcoI NcoI HindIII Ban~lI SmaI
35 * . ~ I *
PCa I Ic ¦ZYMV Co~t Protein¦¦ Sca ¦
Gene ~ 3 ~
5 pGA482/GG/cpZYMV
HindIII NcoI NcoI HindIII
Br I I Nos I Pca I Ic I ZYMV Coat ¦ ¦ SCa ¦ ~ CEIMV ¦ IBL~
Protein Gus Gene G~ne . , I ,; ~
,: ' -31- ` ~3~2~
pUC18GCpZYMV
HindIII NcoI NcoI HindIII BamHI BamHI SmaI
¦PcpIICIZYMV co~tlIScaI ¦ CaMN ¦
Protein Gus Gene Gene pUC18NGCpZYMV
~5 HindlII NcoI NcoI HindIII BamHI Ban~lI
* 11 1 -~ L 11 1 1 1 1 *
IPCal Ic ¦ZYMV Co~t 1I Sca I¦ CaMV I 1 No5 ¦
30 Proteln Gu~
Gene Gene " , , i I , .
Claims (12)
1. A DNA plant expression cassette comprising:
a) a promoter;
b) an AT rich Cucumber Mosaic Virus (CMV) coat protein gene 5' untranslated region;
c) an initiation region, said initiation region comprising the sequence AAXXATGG;
d) a DNA molecule encoding a gene; and e) a poly(A) addition signal; said poly(A) addition signal containing untranslated flanking sequences; wherein said promoter is upstream from said b) AT rich region;
said b) AT rich region is upstream from said initiation region; said initiation region is upstream and operably linked to said DNA molecule encoding a gene, and said gene is upstream and operably linked to said poly(A) addition signal.
wherein X is a DNA nucleotide selected from A, G, C and T.
a) a promoter;
b) an AT rich Cucumber Mosaic Virus (CMV) coat protein gene 5' untranslated region;
c) an initiation region, said initiation region comprising the sequence AAXXATGG;
d) a DNA molecule encoding a gene; and e) a poly(A) addition signal; said poly(A) addition signal containing untranslated flanking sequences; wherein said promoter is upstream from said b) AT rich region;
said b) AT rich region is upstream from said initiation region; said initiation region is upstream and operably linked to said DNA molecule encoding a gene, and said gene is upstream and operably linked to said poly(A) addition signal.
wherein X is a DNA nucleotide selected from A, G, C and T.
2. A DNA plant expression cassette according to Claim 1 wherein said promoter is Cauliflower Mosaic Virus (CaMV) 35S
promoter.
promoter.
3. A DNA plant expression cassette according to Claim 1 wherein said initiation region is derived from the 5' untranslated region selected from the group consisting of the 5' untranslated region of Cucumber Mosaic Virus (CMV) coat protein gene and the 5 untranslated region of SS RUBISCO gene.
4. A DNA molecule according to Claim 1 wherein said poly(A) addition signal is selected from the group consisting of: the poly(A) signal derived from Cauliflower Mosaic Virus (CaMV) 35S
gene; the poly(A) signal derived from phaseolin storage protein gene; the poly(A) signal derived from nopalinesynthase gene; the poly(A) signal derived from octopine synthase gene; the poly(A) signal derived from bean storage protein gene; and, the poly(A) signal derived from SS RUBISCO.
gene; the poly(A) signal derived from phaseolin storage protein gene; the poly(A) signal derived from nopalinesynthase gene; the poly(A) signal derived from octopine synthase gene; the poly(A) signal derived from bean storage protein gene; and, the poly(A) signal derived from SS RUBISCO.
5. A DNA plant expression cassette according to Claim 2 wherein said poly(A) addition signal is derived from Cauliflower Mosaic Virus (CaMV) 35s gene.
6. A DNA plant expression cassette according to Claim 1 wherein said gene is an antipathogenic gene.
7. A DNA plant expression cassette according to Claim 5 wherein said gene is an antipathogenic gene.
8. A DNA plant expression cassette according to Claim 6 wherein said antipathogenic gene is a coat protein gene.
9. A DNA plant expression cassette according to Claim 7 wherein said antipathogenic gene is a coat protein gene.
10. A transformed plant cell containing a DNA plant expression cassette according to Claim 1.
11. A transformed plant cell according to Claim 10 wherein said DNA plant expression cassette comprises:
a) a Cauliflower Mosaic Virus (CaMV) 35S promoter; and, b) a poly(A) addition signal derived from Cauliflower Mosaic Virus (CaMV) 35S gene.
a) a Cauliflower Mosaic Virus (CaMV) 35S promoter; and, b) a poly(A) addition signal derived from Cauliflower Mosaic Virus (CaMV) 35S gene.
12. A process for introducing a trait in a plant comprising the steps of:
a) isolating a gene whose expression in a plant confers said trait;
b) constructing a DNA plant expression cassette according to Claim 1 comprising said isolated gene;
c) transforming plant cells with said DNA plant expression cassette; and d) regenerating plants from said transformed plant cells.
a) isolating a gene whose expression in a plant confers said trait;
b) constructing a DNA plant expression cassette according to Claim 1 comprising said isolated gene;
c) transforming plant cells with said DNA plant expression cassette; and d) regenerating plants from said transformed plant cells.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US23441288A | 1988-08-19 | 1988-08-19 | |
| US07/234,412 | 1988-08-19 | ||
| US32353689A | 1989-03-14 | 1989-03-14 | |
| US07/323,536 | 1989-03-14 | ||
| US36870989A | 1989-06-19 | 1989-06-19 | |
| US07/368,709 | 1989-06-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1332718C true CA1332718C (en) | 1994-10-25 |
Family
ID=27398566
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 606866 Expired - Fee Related CA1332718C (en) | 1988-08-19 | 1989-07-27 | Expression cassette for plants |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1332718C (en) |
-
1989
- 1989-07-27 CA CA 606866 patent/CA1332718C/en not_active Expired - Fee Related
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