CA2005597A1 - Plants having reduced lignin or lignin of altered quality - Google Patents
Plants having reduced lignin or lignin of altered qualityInfo
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- CA2005597A1 CA2005597A1 CA 2005597 CA2005597A CA2005597A1 CA 2005597 A1 CA2005597 A1 CA 2005597A1 CA 2005597 CA2005597 CA 2005597 CA 2005597 A CA2005597 A CA 2005597A CA 2005597 A1 CA2005597 A1 CA 2005597A1
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- lignin
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Abstract
PLANTS HAVING REDUCED LIGNIN OR LIGNIN OF ALTERED
QUALITY
ABSTRACT
Plants of reduced lignin content or of altered lignin composition are produced by transformation of the plants with a recombinant DNA translating to mRNA which inhibits production of protein from the normal mRNA expressed by the endogenous gene for one of the enzymes critical to the biosynthesis pathway of lignin. The inserted DNA is normally used in antisense orientation with respect to the target gene but inserts, particularly incomplete sequences, oriented in sense direction will also down-regulate the target gene. One preferred target gene is that encoding cinnamyl alcohol dehydrogenase (CAD) and this gene has been isolated from tobacco, maize, alfalfa, rape and eucalyptus. The transformed plants find commercial application as animal fodder of improved digestibility and woody species of lower lignin which facilitate production of paper pulp.
QUALITY
ABSTRACT
Plants of reduced lignin content or of altered lignin composition are produced by transformation of the plants with a recombinant DNA translating to mRNA which inhibits production of protein from the normal mRNA expressed by the endogenous gene for one of the enzymes critical to the biosynthesis pathway of lignin. The inserted DNA is normally used in antisense orientation with respect to the target gene but inserts, particularly incomplete sequences, oriented in sense direction will also down-regulate the target gene. One preferred target gene is that encoding cinnamyl alcohol dehydrogenase (CAD) and this gene has been isolated from tobacco, maize, alfalfa, rape and eucalyptus. The transformed plants find commercial application as animal fodder of improved digestibility and woody species of lower lignin which facilitate production of paper pulp.
Description
r~ r~
PS 350f;3 PLANTS HAVING REDUCED LIGNIN OR LIGNIN OF ALTERED QUALITY
_ This invention relates to crops which have lower than normal content of lignin and/or lignin of altered composition, especially crops used as animal fodder and crops such as trees used in industrial processes, for example, in papermaking and to a method, involving gene manipulation, of producing same.
Grassland farmers, as well as farmers of other fodder crops such as maize, wheat, barley, soya, bean, pea and alfalfa, face a difficult decision annually about the timing of the cutting of the crop. All grass varieties of agricultural importance suffer from the disadvantage that during the normal increase in dry matter growth, the digestibllity of the fodder decreases. The farmer, therefore, has to compromise between a lower yield of relati~ely high digestibility material and a higher yield of rather less digestible material. Another limitation is that harvesting at optimum maturity may be prevented by unfavourable weather condition~. If the decline in the digestibility could be controlled or delayed, higher yields of the more digestible material could be obtained and unfavourable weather conditions would not play such a major role in determining the quality of the crop at harvest.
Digestibility of fodder crops is determined ~ principally by the amount of lignification which occurs during the later growth stages of the plant and the degree of s~condary modification of the deposited lignin.
'~ `7 pS35063 Lignin decreases the digestibility of crop plants. Thus, plants with a reduced amount of lignin will be more efficiently used as a forage for cattle, Eor example. The yield of milk and meat would therefore increase by using reduced lignin fodder.
The effects of lignin on digestibility of animal dietary ~eeds has long been recognised.
Numerous physical and chemlcal processing methods for delignifying ligin containing biomass are known. One example of such processes is the subject o International Patent Application NQ. WO
89/09547 (published l9th October 1989) which includes a summary in some depth of other prior art in this area. It is clearly desirable that crops with reduced lignin content be available in order to obviate or mitigate the need for such post-harvest processing of crops destined for animal feed uses.
Furthermore, too large a lignin content may have an inhibitory effect on plant g~owth. Thus, a reduction of the lignification in crops such as wheat or maize may increase the grain yield.
Another industrial field in which the presence of lignin is disadvantageous is in the extraction of the cellulosic components from timber to produce wood pulp, principally for paper making. The industrial process involved in the production of paper pulp amounts, more or less, to a lignin removal process to release the cellulosic content of the woody raw material, such as tre~s and maize stalks. The process is highly energy~consuming and also results in noxious waste liquors containing the lignin dcgradation products which is both q~7 ~S35053 diffic~lt and costly to dispose of and is a major environmental pollutant. In addition, it has been suggested that residual amounts, low they may be, of lignin in paper pulp which is subsequently bleached with chlorine may result in the formation of polychlorinated biphenyls (pcs~s) which are well-recognised toxins (Chemistry in sritain, May 1989, page 458). Reduction in lignification in trees would have considerable effect on the cost of removing lignin in paper making and further reduce the already low suggested residual formation of PCB's by chlorine bleachlng.
seside cellulose and other polysaccharides, lignins are an essential component of cell walls in tissu~s like the sclerenchyma and the xylem of vascular plants. They play an important role in the conducting function of the xylem by reducing the permeability of the cell wall to water. They are also re~ponsible for ri~idity of the cell wall~
and, in woody tissues, th~y act as a bonding agent between cells, giving the plant resistance to impact, compression and bending. Finally, they are involved in mechanisms of resistance to pathogens by impedlng the penetration or the propagation o the pathogen.
Lignins are the product of polymerîsation of three primary precursors through dehyrogenation.
These precursors are the trans-coniferyl, trans-sinapyl and trans~coumaryl alcohols. The 33 monomers of these alcohols can occur in lignins in quite different proportions and with different types of linkages either to themæelves or to other molscules, thus producing different polymers.
These polymers, also known as "lignin cores", are ~S35063 always associated covalently witn hemicellulo~es.
Most lignins also contain varying amounts of aromatic carboxylic acids in ester-like combinations. Such differences in the structures of lignins are usually found in most plant species.
Dif~erences in the composition of lignins can occur in the same plant in different tissues of different ages.
The biosynthesis of lignin monomers, which is illustrated in Figure 1 of the accompanying drawings, is part of the phenylpropanoid pathway in plants, which is also responsible for the production of a wide range of other materials including the flavonoid pigments, isoflavonoids and coumarin phytoalexins, and cell division promoting dehydro-diconiferyl glucosides. In Figure l, the enzymes catalysing the various steps of the biosynthesis are indicated by the numerals 1 to 9:
these represent:
1. Phenylalanine ammonia lyase;
PS 350f;3 PLANTS HAVING REDUCED LIGNIN OR LIGNIN OF ALTERED QUALITY
_ This invention relates to crops which have lower than normal content of lignin and/or lignin of altered composition, especially crops used as animal fodder and crops such as trees used in industrial processes, for example, in papermaking and to a method, involving gene manipulation, of producing same.
Grassland farmers, as well as farmers of other fodder crops such as maize, wheat, barley, soya, bean, pea and alfalfa, face a difficult decision annually about the timing of the cutting of the crop. All grass varieties of agricultural importance suffer from the disadvantage that during the normal increase in dry matter growth, the digestibllity of the fodder decreases. The farmer, therefore, has to compromise between a lower yield of relati~ely high digestibility material and a higher yield of rather less digestible material. Another limitation is that harvesting at optimum maturity may be prevented by unfavourable weather condition~. If the decline in the digestibility could be controlled or delayed, higher yields of the more digestible material could be obtained and unfavourable weather conditions would not play such a major role in determining the quality of the crop at harvest.
Digestibility of fodder crops is determined ~ principally by the amount of lignification which occurs during the later growth stages of the plant and the degree of s~condary modification of the deposited lignin.
'~ `7 pS35063 Lignin decreases the digestibility of crop plants. Thus, plants with a reduced amount of lignin will be more efficiently used as a forage for cattle, Eor example. The yield of milk and meat would therefore increase by using reduced lignin fodder.
The effects of lignin on digestibility of animal dietary ~eeds has long been recognised.
Numerous physical and chemlcal processing methods for delignifying ligin containing biomass are known. One example of such processes is the subject o International Patent Application NQ. WO
89/09547 (published l9th October 1989) which includes a summary in some depth of other prior art in this area. It is clearly desirable that crops with reduced lignin content be available in order to obviate or mitigate the need for such post-harvest processing of crops destined for animal feed uses.
Furthermore, too large a lignin content may have an inhibitory effect on plant g~owth. Thus, a reduction of the lignification in crops such as wheat or maize may increase the grain yield.
Another industrial field in which the presence of lignin is disadvantageous is in the extraction of the cellulosic components from timber to produce wood pulp, principally for paper making. The industrial process involved in the production of paper pulp amounts, more or less, to a lignin removal process to release the cellulosic content of the woody raw material, such as tre~s and maize stalks. The process is highly energy~consuming and also results in noxious waste liquors containing the lignin dcgradation products which is both q~7 ~S35053 diffic~lt and costly to dispose of and is a major environmental pollutant. In addition, it has been suggested that residual amounts, low they may be, of lignin in paper pulp which is subsequently bleached with chlorine may result in the formation of polychlorinated biphenyls (pcs~s) which are well-recognised toxins (Chemistry in sritain, May 1989, page 458). Reduction in lignification in trees would have considerable effect on the cost of removing lignin in paper making and further reduce the already low suggested residual formation of PCB's by chlorine bleachlng.
seside cellulose and other polysaccharides, lignins are an essential component of cell walls in tissu~s like the sclerenchyma and the xylem of vascular plants. They play an important role in the conducting function of the xylem by reducing the permeability of the cell wall to water. They are also re~ponsible for ri~idity of the cell wall~
and, in woody tissues, th~y act as a bonding agent between cells, giving the plant resistance to impact, compression and bending. Finally, they are involved in mechanisms of resistance to pathogens by impedlng the penetration or the propagation o the pathogen.
Lignins are the product of polymerîsation of three primary precursors through dehyrogenation.
These precursors are the trans-coniferyl, trans-sinapyl and trans~coumaryl alcohols. The 33 monomers of these alcohols can occur in lignins in quite different proportions and with different types of linkages either to themæelves or to other molscules, thus producing different polymers.
These polymers, also known as "lignin cores", are ~S35063 always associated covalently witn hemicellulo~es.
Most lignins also contain varying amounts of aromatic carboxylic acids in ester-like combinations. Such differences in the structures of lignins are usually found in most plant species.
Dif~erences in the composition of lignins can occur in the same plant in different tissues of different ages.
The biosynthesis of lignin monomers, which is illustrated in Figure 1 of the accompanying drawings, is part of the phenylpropanoid pathway in plants, which is also responsible for the production of a wide range of other materials including the flavonoid pigments, isoflavonoids and coumarin phytoalexins, and cell division promoting dehydro-diconiferyl glucosides. In Figure l, the enzymes catalysing the various steps of the biosynthesis are indicated by the numerals 1 to 9:
these represent:
1. Phenylalanine ammonia lyase;
2. Cinnamate 4-hydroxylase;
3. p-Coumarate 3-hydroxylase;
4. Catechol-O-methyl transferase;
5. Ferulate 5-hydroxylase;
6. Hydroxycinnamate:CoA ligase;
7. Cinnamoyl:CoA reductase;
8. Cinnamyl alcohol dehyroyenase7 and, 9. Peroxidases In the first step of the pathway phenylalanine i~ deaminated under action of enzyme 1 to produce cinnamic acid. The cinnamic acid is then transformed by hydroxylation and methylation ~enzyme 2) to produce acids with different substituents on the aromatic group. The 3~7 ~S3~0~3 para-coumaric, ferulic and sinapic acids are then esterified by hydroxycinnamate: CoA ligase to produce cinnamyl-CoAs. These compounds are subsequently reduced by cinnamyl-CoA reductase (CCR) into cinnamyl-aldehydes, which are finally reduced to cinnamyl alcohols by cinnamyl alcohol dehydrogenase (CAD). Only the final two steps are specific for the biosynthesis of lignin. The cinnamyl alcohols, synthesised in the cytoplasm, are then transported to the cell wall where they - are polymerised by a peroxidase in the presence of hydrogen peroxide.
When the surface growth of the cell ceases, it is followed by a phase of wall thickening (secondary wall formation). Lignification takes place during this phase. It begins in the cell corners and extends along the middle lamella, through the primary wall and, finally, to the secondary wall. This developmentally regulated lignification is the process which, if it continues for too long, leads to excessive lignification and poor digestibility of fodder crops.
External factors can also induce qualitative and quantitative modifications in lignification.
The synthesis of new types of lignins, sometimes in tissues which are not normally lignified, may be induced by infection with pathogenic microorganisms. Lignification is stimulated by light as well as by low calcium levels, by boron and by mechanical stress.
As to the question whether suppression of lignin synthesis would have a deleterious effect on the living plant, for example one might imagine a loss of structural strength which might result in ~3~5~3~^~
~S35063 th2 plaat being unable to support itself, it has previously been shown that lignin formation in poplar trees can be inhibited by the application of a chemical inhibitor which inhibits the CAD enzyme.
S The experim2ntal results showed that it was possible to reduce the amount of lignin by ~0~
without any detected detrimental effect other than a reduction in the amount of flavonoid pigments.
~he indications are, therefore, that it is possible to reduce the amount of lignin without affecting the plant health.
An object of the present invention is to obviate or mitigate the aforesaid disadvantages.
According to the present invention there is provided a recombinant DNA comprising a plant DNA
having a promoter sequence and an insert, located 3' to the promoter and in appropriate reading frame, comprising a nucleotide sequence encod.ing a mRNA whieh is substantially homologous or complementary to mRNA encoded by an endogenous plant gene or a part thereo~ which encodes an enzyme essential to lignin biosynthesis, so that mRNA transcribed from the said insert inhibits production of the enzyme from the endogenous gene.
It i5 preferred that the insert encodes mRNA
in antisense orientation to the mRNA encoded by the said endogenous gene. However, it has been found that inserted gene sequences, particularly incompl2te sequences, even when in normal sense orientatlon, are able, by some presently not fully understood mechanism, to interfere with synthesis of normal mRNA and protein.
When the insert is used in the sense orientation a substantial part of the mRNA encoding ~S35063 the protein is desirable.
The DNA insert may be excised from the untranscribed strand of the DNA encoding the gene of interest, or, it may be synthesised.
The DNA insert may represent the whole comple~entary sequence of the gene of interest or it may be a partial sequence with sufficient degree of correspondence that the RNA transcribed from the insert has sufficient degree of correspondence to the RNA transcrihed by a normal gene that the two will bind thus preventing production of the enzyme protein. It is preferred, however, that a minimum size of insert of about 50 bases be observed when only a part of the normal plant gene is selected.
The binding of the mRNA encoded by the insert to the endogenous mRNA may take place at the stage of transcription, processing of the hnRNA, transport of the RNA to the cytoplasm or translation initiation or progression. All of these effects may be involved in the mechanism leading to reduced protein synthesis of the target enzyme.
In addition to, or as an alternative to, suppression of lignin amounts in plants, it may be advanta~eous to alter the composition. By down-regulating the activity of one or more of the enzymes in the lignin biosynthetic pathway, the proportions of the various products of enzyme transformation will be altered leading to polymer formation of compositio~ quite different from that which is normally encountered.
It is preerred that the said enzyme is cinnamyl alcohol dehydrogenase (CAD) or cinnamoyl:
CoA reductase (CCR) or catechol-O-methyl transferas~ (COMT).
~S35063 The promoter utilised in the present in~ention may be the endogenous promoter associated with the gene targetted for down-regulation or a plant promoter such as CaMV35S. preferably, the promoter is the promoter associated with the phenylalanine ammonia l~ase gene, GPAL2 or GP~L 3, sequences of which are given in Fig.6 and Fig.7 respectively.
Full details of the GPAL promoters are qiven by Cramer ~t.al. in Plant Molecular ~iology 12:
367-383 (1989) and Bevan et.al.in The EMBO Journa7, vol 8, No.7, pp 1899-1906 (1989).
Further according to the invention, there is provided a method for inhibiting lignin biosynthesis in a plant, comprising stably incorporating into the genome of the plant by transformation a recombinant DN~ comprising a plant DNA having a promoter sequence and an insert, .located 3' to the promoter and in appropriate reading frame, comprising a nucleotide sequence encoding a mRNA which is substantially homologous or complementary to mRNA encoded by an endogenous plant gene or a part thereof which encodes an enzyme essential to lignin biosynthesis, so that mRNA transcribed from the said inser.t inhibits translation of the endogenous gene to the enzyme.
The invention also provides a transformed plant possessing lower than normal ability to produce lignin characterised in that said plant has stably lncorporated within its genome a recombinant DNA of this invention.
The drawings which accompany this application are as follows:
Fig~1 is a diagram of the lignin biosynthetic pathway;
~S35063 Fig.2 shows enzyme restriction maps of clones 4A, MG10 and CAD4;
Fig.3 is an en~yme restriction map of the tobacco CAD gene;
Fig.4 illustrates the insertion of a sequence in both the sense and antisense orientation in sequence pJR1;
Fig.5 is the DNA sequence of the SR1 region shown in Fig. 3;
Fig.6 is the DNA sequence of the GPAL2 promoter:
and, Fig.7 is the DNA sequence of the GPAL3 promoter.
In a preferred embodiment, therefore, the invention comprises a recombinant DNA having a promoter sequence and, located 3' to the promoter, the insert is derived from the CAD gene, a map of which is given as Figure 3, the full DNA sequences of the region designated SR1 in Figure 3 being given in Figure 5.
Additionally, the invention provides a host transformed by the vector, preferably selected from E.coli, Agrobacterium tumefaciens and plant hosts.
preferred strain of Agrobacterium tumefaciens is strain L84404.
The invention further provides a transgenic plant which has the said antisense mRNA gene stably incorporated in its ~enome by transformation.
Several methods for transformation of plants are available: these include the use of Agrobacterium tumefaciens as a transformation vector, agroinfection, electroporation, particle bombardment and microinjection. The particular transformation method employed for incorporation of the recombinant DNA of this invention into the 3 ~51~
~S35063 genome of a plant is not particularly germane to the invention, the pre~erred method being that which, in use, produces the largest number of transformed plants. Agroinfection, electroporation and microinjection have been found to be successful in transformation of dicotyledonous plants but less so with monocots. For monocots other methods are more suitable and reference may be made to International Patent Application No. WO85/01856 and European Patent Application No. 275,069 for methods which are said to be suitable. Methods for the transformation of trees are described in European Patent Application No.0227264 and British Patent Application No. 2211204, both of which utilise infection with Agrobacterium tumefaciens.
The invention, by provision of a source of mRNA antisense to the normal RNA encoding an enzyme essential to lignin biosynthesis, thereore provides a means by which lignin synthesis in a plant may be suppressed or lignin composition altered. There is also provided, therefore, plants and plant propagating material containing the recombinant DNA of the invention. More specifically, the invention includes seeds, plants and their progeny.
Any of a number of enzymes in the li~nin biosynthetic pathway can be used as targets for down-regulation using antisense RNA. ~hese are discussed below:
Cinnamyl Alcohol Dehydrogenase (C~D~
This enzyme catalyses the reduction of cinnamyl-aldehydes into cinnamyl alcohols. C~D
has been characterised from di~ferent species:
Forsythia suspensa, soybean ~Glycine max.), spruce ~n(3~ 3~7 (Picea abies), polar (Populus euramericana) and bean ~Phaseolus vulgaris L). Except for soybean, only one form of CAD enzyme has been detected in these species. The common structure of CAD enzymes is that of a dimer of approximately 70,000 daltons with each monomer having a molecular weight of approximately 40,000 daltons. In contrast, the bean enzyme is a monomer with a molecular weiyht of 65,000 daltons, indicating that possibly a gene duplication has taken place during evolution.
Soybean has two isoenzymes, one of 43,000 daltons and one of 69,000 daltonsO The first soybean isoenzyme is specific for coniferyl alcohol while the 69,000 dalton one and all other CAD can catalyse the formation of all the cinnamyl alcohols (i.e. coniferyl, sinapyl and coumaryl alcohols).
However, the Rm of CAD for the diferent cinnamyl alcohols varies between enzymes from different species. This variation may explain the different compositions of the lignin core in different species. Lignin monomers cannot be synthesised in plants through any other biochemical pathway.
Thus CA~, as well as CCR and peroxidase, may be key enzymes in the regulation of lignification.
The utilisation of inhibitors specific for these enzymes indicates that they may regulate the quantity of lignin rather than its composition.
The ~m values for the different cinnamyl alcohols of th~ soybean CAD isoenzymes sugges~ that CAD
isoenzymes may also control the composition of lignin. The presence of Zn2~ is required for the activity of CAD, as for other alcohol dehydrogenases. The reduction of cinnamyl-aldehydes cannot be catalysed by CAD in the 5~7 ~S35063 presence of NADH ( nicotinamide adenine dinucleotide - reduced form) instead of NADPH ( nicotinamide adenine dinucleotide phosphate -reduced form).
CAD enzyme activity and transcription are stimulated by treatment of bean suspension culture cells by fungal elicitor preparations. This increased CAD activity can be regarded as a defence reaction of the plant against pathogenic microorganisms, since an increase in the activity of the enzyme may be related to the deposition of lignin in the walls of infected cells, or to the synthesis of extracellular lignin-like material and other phenolic compounds involved in the defence responses.
Cinnamoyl-CoA Reductase (CCR) This enæyme is also involved in the reduction of cinnamyl-aldehydes to cinnamyl alcohols. CCR
has been investigated in a number of species and has been purified to apparent homogeneity from spruce, soybean and poplar.
only one form of the enzyme, having a molecular weight of 36,000 daltons has been detected in theee plant species, indicating considerable evolutionary conservation. The enzyme from poplar has an equilibrium constant of 2.5 x lO 4 M. Th~ spruce enzyme gene has a value of 5.6 x 10 7 M. The enzyme exhibited the greatest affinity for feruloyl-CoA with an apparent Km f 2.55 ~m. A difference in substrate specificity was observed between the spruce and soybean enzymes-feruloyl-CoA was a good substrate for both enzyme~.
However, the soybean enzyme alone converted sinapoyl/CoA.
Peroxidase ~5'~7 ~535~63 Peroxidase is an ubiquitous enzyme found in many plant organs. There are a number o isoenzymes present in plants which are regulated in a tissue-specific and developmentally regulated manner. The cell wall bound form of this enzyme is involved in the poly~erisation of lignin monomers in the presence of hydrogen peroxide. However, the enzyme can also catalyse crosslinks between side chains of lignin polymers either to other lignin polymers or to other macromolecules such as hemicellulose. The enzyme has been purified from a number of plant sources.
Catechol-O-Methyl Transferase This enzyme catalyses the methylation of cafeic and 5-hydroxyferulic acids to ~orm ferulic and sinapic acids respectively. The diversity, biochemical activities and characters are known.
However, to achieve inhibition of lignin biosynthesis, it is possible to target other enzymes of the phenylpropanoid pathway such as phenylalanine ammonia lyase, cinnamyl CoA-ligase, chalcone synthase and others.
The down regulation of the enzymes discussed above will affect both the amount of lignin deposited and the degree of secondary modification through crosslinking either o the lignin monomers or of the lignin structures to hemicellulose, or side chain additions. Reduction in lignification referred to hecein includes both mechanisms which can be viewed as complementary rather then self exclusive.
In plants, reduced expression of a previously introduced genes, coding for chloramphenicol acetyl transferase and nopaline synthase, and o ~S35063 Z~
endogenous genes, chalcone synthase and polygalacturonase has been reported.
The present invention will now be described, by way of ill~lstration, in the following Examples.
A bean cDNA clone encoding C~D was isolated from an elicitor~induced cDNA library [Edwards et.
al.;Proc. Natl. Acad. Sci. 82 6731-6735 (1985)].
The library was screened using a nick-translated partial CAD cDNA clone, designated 4A, the sequence of which is published by Walter et.al. in Proc.
Natl. Acad. Sci. USA (1988). ~he screening was carried out under standard conditions and one clone was identified which showed a very strong hydridisation signal. This clone was named pMG10.
Agarose gel electrophoresis of the purified PstI
insert showed the length of the cDNA to be 2.2 kilobases (kb). Since the bean CAD polypeptide has a molecular weight of 65,000 daltons, the open reading frame is expected to be approximately 1770 base pairs (hp), assuming an average molecular weight of amino acids to be 110. If the ~ull length mRNA contains 5' and 3' untranslated regions of about 200 bp the total length of the complete cDNA will be approximately 2kb~ Thus pMG10 was putatively identified as a full-length C~D cDNA.
The restriction map of clone pMG10 and clone 4a were determined. This indicated that there was close homolosy of the parts of these clones (Figure 2). Common sites were ound at identical positions Ncol, ~indIII, among those enzymes tested. No sites were found for the following enzymes: AccI, AvaI, BamHI, BclI, BstEII, ClaI, DraI, E~paI, KpnI, NarI, PvuI, PvuII, SacI, SalI, Sma~, SpeI and Zho~.
~3 ~S35063 Southern blot hybridisations were carried out, using clone 4a as a probe, to identify the regions of homology between clone pMG10 and clone 4a. It was thus possible to align the maps of the two clones, showing that pMG10 presumably includes 4a (or a very closely related sequence) at its 3' end (Figure 2?.
Since the isolation of the clone pMG10, described above, the~e has been reported the isolation of a bean CAD CDNA from an elicitor induced cDNA library constructed in ~-gtll phage [Walter et.al. in Proc. Natl. Acad. Sci. USA
(1988)]. The restriction map deduced from the sequence of this clone, designated ~-CAD4, shows that it is closely related to pMG10 but not identical: most of the restriction sites found in pMG10 are also found in ~-CAD4. However, the location of some sites are different in the two clones: the HindIII site located 300bp from the 5' end of pMG10, the EcoRI site and the single BglII
site of pMG10 are not found in ~~CAD4 at identical positions. Sites for BclI, ClaI and PvuII are not present in pMG10.
The data generated from the restriction mapping and hybridisation experiments are taken as proof that pMG10 contains a CAD cDN~ clone, as most of the structural information for the CAD mRNA has been published.
This clone can now be used for the isolation of the CAD gene not only from bean but from other plant species particularly those to which this invention can be applied, for example, alfalfa, maize, brassicae, eucalyptus, pine, poplar, lolium and festuca. The gene can then be used for the ~C~ ?~ 7 ~S35063 isolation of the coding region as well as the promoter. The promoter of the CAD gene can be used to drive the expression of foreign genes in plants.
Foreign genes which can be used in this context can be genes which can inactivate virus2s, fungal or bacterial infections, as the C~D promoter will be switched on particularly after inection of plants with these agents.
ta) Screening of a ~enomic library and partial restriction mapping A genomic library of tobacco DNA ~N. tabacum var. Samsum) was constructed in ~-EMBL3. An aliquot of the library (approximately 106 pfu) was screened using the Pstl insert of pMGl~
radiolabelled by nick-translation. A total of 54 hybridising plaques were detected. Twenty of these clones were plaque purified, using standard protocols. DNA was prepared from 11 clones, designated 1, 8, 15, 22, 24, 28, 41, 46, 47 and 50.
Limited restriction maps using SalI~ EcoRI and HindIII restriction enzymes were determined for these clones. This indicated that all the clones contained the same gene. The strong cross-hybridisakion with the bean CAD cDN~ probe was taken as indication that these clones contain a tobacco CAD gene. A representative restriction map of one of the clones (clone 15) covering 11 kb is shown in Figure 3.
(b) Determination of the orientation of the_tobacco CAD ~ene in clone 15 Southern blot hybridisations were carried out using a 5' end probe (clone 4b: the subcloned 5' a'~f~'~
~S35053 Eco~I fragment of ~-CA~4) or a 3~ end probe ~clone 4a). AS both probes hybridise to the isolate DNA
from these genomic clones, the complete tobacco CAD
gene may be contained in these clones. The two probes mentioned above were used to determine the orientation of the CAD sequences within the genomic clone. The central region covering approximately 5 kb of clone 15 hybridised to these two probes delineating the tobacco CAD gene. The two external region of the clone which are located from 0 to 3 kb and from 7 to 11 kb from the 5' end of the insert do not hybridise to any of the bean CAD
cDN~s. The region extending from the EcoRI site 3 kb from the 5' end to the EcoRI site 4.3 kb from the 5' end hybridises only to the clone 4b. The reg.ion covering this 1.3 kb fragment was designated ASR1 (Figure 3). The region located between the 4.3 kb EcoRI site to the HindIII site 7kb from the 5' end hybridised only to the clone 4a. This indicates that the 5' end o the tobacco CAD gene is located in the ASR1 fragment, whereas the bulk of the coding regi3n of the gene is located in the rest of the hybridising fragment. The region between the SalI and HindIII was designated SR1 (Figure 3) (c) Detailed restriction mapping and sequence determination of the ASR1 and_SHl regions Clone 15 was restricted with ~coRI and the resulting fragments were cloned into the EcoRI site of Bluescript SK-vector. Transformants were selected on 50 ~g/ml ampicillin, and clones containing the ASR1 fragment were identified by hybridisation with the insert of clone 4b. One clone, pASR1 hybridised strongly. Restriction ~{~
enzyme digestions using HindIII and EcoRI showed that this clone contains the ASRl fragment.
A detailed restriction map of the ASP~l fragment was determined. No sites were found ~or the following enzymes: NcoI, SalI, BamHI, ClaI, ~glII, sstEII and SacI. Southern blot hybridisations to restriction digests of pASRl DNA
using clone 4a as a probe indicate that 0.25 kb EcoRI-AvaII fragment hybridises very weakly.
Since this fragment is located at the very 5' end of the hybridising region in clone 15, it may contain the 5' untranslated region including the translation start site. However,it is also possible that the weak hybridisation of this fragment to the clone 4b may be the result of low homology in this region between tobacco and bean genes. Ths ~vaII-KpnI, ~pnI-HindIII and 0.3 kb HindIII- EcoRI fragments hybridise strongly to clone 4b.
DNA sequence analysis of pASR1 insert DNA, determined using standard protocols, revealcd the following features:
The HindIII-EcoRI fragment contains a 90 base pair long intron. The exon sequences are 84%
identical to the bean CAD cDNA (from base 422 to 565 in the ~-CAD4 sequence. At the protein level, the homology is 93%. These results suggest that this region might be of importance for the functional structure of CAD~
Construction of CAD Antisense Vectors (a) Vectors based on the Tobacco CAD Gene The plasmid pASR1 (Bluescript containing the ASR1 fragment) was cut by KpnI. The 0.6 kb fragment containing the 3' end o~ ASR1 and about 50 2~ 3~
bp of the Bluescript polylinker was recovered by electroelution after separation on agarose qels, and inserted into the RpnI site of pJRl. This construct was used to transform E. coli, strain TG2. The transformants were selected by growth on kana~ycin-containing plates. 227 colonies were streaked on nitrocellulose filters, and hybridised with the radiolabelled KpnI fragment. Eight clones gave a strong signal.
Plasmid DNA was prepared from these clones.
Restriction digestions with KpnI were carried out to check the presence of the insert. Only seven of the clones contained the recombinant vector.
These were clones 1(6-6), 1(7-7), 1(8-9), 1(10~3), 2(5-13), ~(7-1) and 2(11-3)].
Since the KpnI fragment could be inserted in both orientations, it was necessary to identify the recombinant clones with this fragment in the antisense orientation. The presence of the EcoRI
site from the Bluescript polylinker introduced an asymmetry in the fragment, thus making it possible to check the orientation of the insert by a simple restriction digest with EcoRI. If the KpnI
fragment is in the antisense orientation only the CaMV 35S promoter would be excised by EcoRI
digestion, producing two fragments of approximately 0.5 and 11 kb. However, if the KpnI fragment is in the sense orientation the tobacco CAD fragment plus the CaMV 35S promoter would be excised by EcoRI. The EcoRI ragments produced in this case would be approximately 1 and 10 kb. Of the seven positive clones, two [1(6-6) and 2(5-13)l contained the insert in the sense orientation and three [
1(10-3), 2(7-1) and 2(11-3)l were identified as '7 ~S35063 antisense constructs. The two remaining clones did not cestrict with EcoRI and, therefore, remain uncharacterised. Each of the clones was shown to contain only one CAD insert fragment. The clones were renamed pCAD-ASl [1(10-3), pCAD-AS2 ~2(7-1)], pCAD-AS3 [2(11-3], pCAD-Sl ~1( 6-6)] and pCAD-.52 ~2(5-13)].
(_ Vectors based on the Bean CAD cDNA
pMG10 was restricted with NcoI, and the 1170 bp fragment covering a large portion oE the CA~
protein was isolated following agarose gel electrophoresis. This fragment was then treated with T4 polymerase, and cloned into the SmaI site of the expression vector pJR1. Ligation mixtures were transformed into suitable E. coli hosts, and the recombinant clones identified by hybridisation to radiolabelled NcoI fragment. A number of clones were isolated, characterised and found to contain the CAD fragment in either the sense or the antisense orientation. A representative clone from each group was characterised in detail and designated pBCAD.S1 (sense) and pBCAD.AS1 (antisense).
The structure of these vectors are represented in Figure 4.
The above procedure can be repeated and the CAD antisense fragments, derived either from the tobacco gene or the bean cDNA, can also be inserted into an expression vector containing the gP~L2 or gPAL3 promoter, the sequences of which are given in Figures 6 and 7. These promoters are derived from one of the phenylalanine ammonia lyas2 genes from French bean. It has been found that the gPAL2 promoter drives the expression of foreign genes ZO(~5~
~S35063 specifically in xylem tissue. The gPAL3 promoter is expressed, for example, in the pith cells, the endodermis.
TRANSFORMATION OF_OBACCO
(a) Transfer of Vectors to_Agrobacterium The antisense and sense constructs were introduced into A. tumefaciens LBA4404 by triparental mating following published procedures.
l0 The presence and integrity of the antisense constructs were checked by restriction digestion and Southern blot experiments. These results indicated that no recombination had occurred during the transfer of the vectors to Agrobacterium.
b) Tobacco Leaf Disc Transformation Tobacco (N tabaccum, variety samsum) leaf discs were transformed using well established previously published procedures. Thirty six plants containing the bean CAD antisense construct and twenty one containing the bean CAD sense construct were selected.
In addition seventeen plants containing the tobacco CAD antisense gene and twenty six containing the tobacco CAD sense gene were identified.
ANALYSIS OF TRANSFORMESD PLANTS
a) CAD ~nzyme Measurements On Tissue From Plants Plant material was used from both transformed and untransformed control plants. The material (leaf and stem1 was ground with CAD extraction buffer containing 200 mM Tris/~Cl pH 7.5, 0.5%
(w/v) PEG 6000, 5% (w/v) PVP, and 15 mM
~-mercaptoethanol (500~l). The crude homogenat*
~ ~J
Ps35063 ~2 was centrifuged and the supernatant used as source of enzyme. The assay reaction contains 10 mM
coniferyl alcohol (50~1 ), 10 mM NADP~ ( 50~1), 100 mM Tris/HCl pH 8.8 (800~1). This was incubated at 30C for 10 minutes, then enzyme extract (100~1) was added and the whole ~ixture was incubated for a for a further 10 minutes at 30c. The OD~oo was recorded against a blank supplemented with water.
Three leaf and two stem samples were taken from each plant. Assays were done in dupllcate.
Ste~ segments from control plants of the same age showed little variation in CAD enzyme activity.
Lea material also showed little variation.
Transformed plants containing the anti-sense RNA
vectors showed considerable reductions in CAD
enzyme activity. This varied between the different transformants. The maximum reduction observed was 70% in both leaf and stem.
b) Polymerase Chain Reaction to determine ~resence of antisense genes DNA was extracted from selected plants.
Oligonucleotides to sequences in the CamV promoter and nos 3' terminator were used as primers in the polymerase chain reaction (PCR). DNA from plants containing the bean CAD antisense constructs gave a product o$ ~1200 bp as expected. DN~ from plants containing the tobacco CAD antisense constructs gave a product of ~620 bp as expected. To confirm that the~e products were CAD sequences, a southern blot of these products was probed with a third oligonucleotide. The sequence of this was taken from the region of 93% homology between the bean CAD c~NA and the tobacco CAD ASR1 gene fragment.
These seguences were present in the antisense b ~
~S3~063 vectors. This oligonucleotide hybridised to all PC~ products, confirming the presence and identity of the antisense constructs.
c) Neutral Detergent Fib _ Cellulase Assay In order to detect changes in lignin-cellulase complexes, neutral detergent fibre/cellulase (NDF/C) assays were performed.
This assay determines the amount of extractable cellulose from cell preparations.
Selected plants, exhibiting at least 50%
reduction in CAD enzyme activity in both leaf and stem were grown from tissue culture to maturity in pots in a growth room ( ~10 weeks). Two control plants were grown in the same way. The plants were selfed and after seed collection the stems were harvested for NDF/C assay. Plants containing the bean CAD antisense constructs showed increased NDF/C values of two of 14 units greater than controls. Plants containing the tobacco C~D
antisense constructs showed increases of five to fourteen units above controls.
~ t~
NDF/C VALUES OF To PLANTS
__ _ P LANT ND F/C
CONTROL 1 58.6 CONTROL 2 57.5 BCAD 3 72.5 6 57.9 8 59.0 9 66.1 12 60.9 29 65.6 32 ~9.2 59 5~.5 TCAD 15 72.2 66.4 24 59.9 70.2 42 59.8 43 66.6 47 68.7 48 71.1 _ _ 63.2 d) Gsnotype Analysis Of Selected Plants Six plants were chosen for further analysis on the basis of their NDF/C values. These were BCAD
32, TCAD 15, 40, 47 and 5Q. The genotype of each of these was determined by patching out 72 seeds of each onto medium containing kanamycin (Murashige &
3'~
~S35063 Skoog salts 2.3 g/l, sucrose 15 g/l Q.8~ agar, 200~g/cm3 kanamycin). Seedlings which do not carry tha kanamycin gene bleach and die. The nu~ber of seedlings bleaching as a ratio to those not bleaching can be taken as an indicator of the numbers of genes present following the rules of segregation. We have assumed an equal number of antisense genes as kanamycin genes. An equal number of seeds was patched onto medium without kanamycin as a check on viability. In this way the genotypes of the To generation were determinsd.
~his indicated that some plants, such as T48, contains one copy of the antisense gene, whereas Tl5 contains two copies.
e) NDF/C Assay of S1 Plants Sixteen seeds from each of thsse six plants plus control were planted and grown to maturity in 7 inch pots in a glass house. The height of each plant was measured each week. Data was collected on time to flowering and growth rate for each plant. Each plant was selfed and after seed collection the lea and stem of each plant was harvested separately. The dry weight of each was recorded and then NFD/C measured. The yield of seed was recorded for each plant.
The mean NDF/C value for population of TCAD15 progeny was significantly greater than the mean NDF/C value for the control population, with individual plants having an increase of 18 units above the mean of the controls.
The other populations showed somewhat less significant increases in NDF/C values.
~ 5~7 Ps3s~63 NDF/C VALUES FOR STEM
NUMBER
_ _ .7 60.3 44.9 2 _ 63 .7 44 .7 3 50.0 50.7 q 48.3 sa.s 46. 3 _ 49.9 54.6 6 _ 55.7 54.5 7 40.7 55.1 53 .2 8 4~.4 52.~ 48.5 9 39.9 52 .2 54.1 50.0 _ 57.9 .l _ 50.1 12 44.2 _ 54.0 f) Genotype Analysis Of S1 Plants This was determined in the same way as for the To plants.
Cloning Of CAD Genes From Other Plant Species In order to generate antisense vectors for the other crop plants of interest we have isolated the CAD genes from alfalfa, maize, rape and eucalyptus.
Plasmids containing these isolated genes have been deposited in a culture collection in E.coli hosts.
PCR products have been obtained from yenomic DNA of alfalfa, rape, eucalyptus and maize uæing oligonucleotide sequences taken from the tobacco ~S35063 ~ ~a3 CAD gene sequence. The products have been sho~n to hybridisa to a third oligonucleotide, the sequence of which is present in the tobacco CAD gene.
The priming sequences are:
-5' AAT GAA AGG CTG TTC TAC AAG CTTCT
S' ATC GGT CAC AAC ~AT AAC TrrG AAT
The third sequence used as probe, CAD4 is 5' TCA GGG TCT TTA TAT CAG TTT GAA AGA AAA
These PCR products are cloned and will be fully sequenced.
Using these probes we have been able to isolate CAD genes from alfalfa, rape, eucalyptus and maize.
The plasmid pGAL2 has been deposited in Escherichia coli strain DH5 on 6th Decem~er 1988 with the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCIB 40087.
The tobacco-derived clone designated CAD
genomic clone 15 has been deposited in bacteriophage A-EMBL3 on 6th December 1988 with the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCIB 40088.
The maize-derived clone, designated pCAD;maize has been deposited in Escherichla coli, strain DH5a, on 3~ ~oJ ~g~ with the National Collection of Industrial and Marine ~acteria, Aberdeen, United Ringdom, under the Accession Number NCIB 4~
The rape-derived clone, designated pCAD;rape has been deposited in Escherichia_coli, strain D~5~, on ~ ~b~l9~ with the National Coll~ction of 3~5~
~S35063 Industrial and Marine sacteria, Aberdeen, United Kingdom, under the Accession Number NCI8 ~D~
The alfalfa-derived clone, designated pCAD;alfalfa has been deposited in Esch _ichia coli, strain DH5~, on with the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCI~ .
The eucalyptus-derived clone, designated pCAD;eucalyptus has been deposited in Escherichia coli, strain DHSa~ on 3O ~ 8~1 with the National Collection of Industrial and Marine ~acteria, Aberdeen, United Kingdom, under the Accession Number NCIB ~3O .
When the surface growth of the cell ceases, it is followed by a phase of wall thickening (secondary wall formation). Lignification takes place during this phase. It begins in the cell corners and extends along the middle lamella, through the primary wall and, finally, to the secondary wall. This developmentally regulated lignification is the process which, if it continues for too long, leads to excessive lignification and poor digestibility of fodder crops.
External factors can also induce qualitative and quantitative modifications in lignification.
The synthesis of new types of lignins, sometimes in tissues which are not normally lignified, may be induced by infection with pathogenic microorganisms. Lignification is stimulated by light as well as by low calcium levels, by boron and by mechanical stress.
As to the question whether suppression of lignin synthesis would have a deleterious effect on the living plant, for example one might imagine a loss of structural strength which might result in ~3~5~3~^~
~S35063 th2 plaat being unable to support itself, it has previously been shown that lignin formation in poplar trees can be inhibited by the application of a chemical inhibitor which inhibits the CAD enzyme.
S The experim2ntal results showed that it was possible to reduce the amount of lignin by ~0~
without any detected detrimental effect other than a reduction in the amount of flavonoid pigments.
~he indications are, therefore, that it is possible to reduce the amount of lignin without affecting the plant health.
An object of the present invention is to obviate or mitigate the aforesaid disadvantages.
According to the present invention there is provided a recombinant DNA comprising a plant DNA
having a promoter sequence and an insert, located 3' to the promoter and in appropriate reading frame, comprising a nucleotide sequence encod.ing a mRNA whieh is substantially homologous or complementary to mRNA encoded by an endogenous plant gene or a part thereo~ which encodes an enzyme essential to lignin biosynthesis, so that mRNA transcribed from the said insert inhibits production of the enzyme from the endogenous gene.
It i5 preferred that the insert encodes mRNA
in antisense orientation to the mRNA encoded by the said endogenous gene. However, it has been found that inserted gene sequences, particularly incompl2te sequences, even when in normal sense orientatlon, are able, by some presently not fully understood mechanism, to interfere with synthesis of normal mRNA and protein.
When the insert is used in the sense orientation a substantial part of the mRNA encoding ~S35063 the protein is desirable.
The DNA insert may be excised from the untranscribed strand of the DNA encoding the gene of interest, or, it may be synthesised.
The DNA insert may represent the whole comple~entary sequence of the gene of interest or it may be a partial sequence with sufficient degree of correspondence that the RNA transcribed from the insert has sufficient degree of correspondence to the RNA transcrihed by a normal gene that the two will bind thus preventing production of the enzyme protein. It is preferred, however, that a minimum size of insert of about 50 bases be observed when only a part of the normal plant gene is selected.
The binding of the mRNA encoded by the insert to the endogenous mRNA may take place at the stage of transcription, processing of the hnRNA, transport of the RNA to the cytoplasm or translation initiation or progression. All of these effects may be involved in the mechanism leading to reduced protein synthesis of the target enzyme.
In addition to, or as an alternative to, suppression of lignin amounts in plants, it may be advanta~eous to alter the composition. By down-regulating the activity of one or more of the enzymes in the lignin biosynthetic pathway, the proportions of the various products of enzyme transformation will be altered leading to polymer formation of compositio~ quite different from that which is normally encountered.
It is preerred that the said enzyme is cinnamyl alcohol dehydrogenase (CAD) or cinnamoyl:
CoA reductase (CCR) or catechol-O-methyl transferas~ (COMT).
~S35063 The promoter utilised in the present in~ention may be the endogenous promoter associated with the gene targetted for down-regulation or a plant promoter such as CaMV35S. preferably, the promoter is the promoter associated with the phenylalanine ammonia l~ase gene, GPAL2 or GP~L 3, sequences of which are given in Fig.6 and Fig.7 respectively.
Full details of the GPAL promoters are qiven by Cramer ~t.al. in Plant Molecular ~iology 12:
367-383 (1989) and Bevan et.al.in The EMBO Journa7, vol 8, No.7, pp 1899-1906 (1989).
Further according to the invention, there is provided a method for inhibiting lignin biosynthesis in a plant, comprising stably incorporating into the genome of the plant by transformation a recombinant DN~ comprising a plant DNA having a promoter sequence and an insert, .located 3' to the promoter and in appropriate reading frame, comprising a nucleotide sequence encoding a mRNA which is substantially homologous or complementary to mRNA encoded by an endogenous plant gene or a part thereof which encodes an enzyme essential to lignin biosynthesis, so that mRNA transcribed from the said inser.t inhibits translation of the endogenous gene to the enzyme.
The invention also provides a transformed plant possessing lower than normal ability to produce lignin characterised in that said plant has stably lncorporated within its genome a recombinant DNA of this invention.
The drawings which accompany this application are as follows:
Fig~1 is a diagram of the lignin biosynthetic pathway;
~S35063 Fig.2 shows enzyme restriction maps of clones 4A, MG10 and CAD4;
Fig.3 is an en~yme restriction map of the tobacco CAD gene;
Fig.4 illustrates the insertion of a sequence in both the sense and antisense orientation in sequence pJR1;
Fig.5 is the DNA sequence of the SR1 region shown in Fig. 3;
Fig.6 is the DNA sequence of the GPAL2 promoter:
and, Fig.7 is the DNA sequence of the GPAL3 promoter.
In a preferred embodiment, therefore, the invention comprises a recombinant DNA having a promoter sequence and, located 3' to the promoter, the insert is derived from the CAD gene, a map of which is given as Figure 3, the full DNA sequences of the region designated SR1 in Figure 3 being given in Figure 5.
Additionally, the invention provides a host transformed by the vector, preferably selected from E.coli, Agrobacterium tumefaciens and plant hosts.
preferred strain of Agrobacterium tumefaciens is strain L84404.
The invention further provides a transgenic plant which has the said antisense mRNA gene stably incorporated in its ~enome by transformation.
Several methods for transformation of plants are available: these include the use of Agrobacterium tumefaciens as a transformation vector, agroinfection, electroporation, particle bombardment and microinjection. The particular transformation method employed for incorporation of the recombinant DNA of this invention into the 3 ~51~
~S35063 genome of a plant is not particularly germane to the invention, the pre~erred method being that which, in use, produces the largest number of transformed plants. Agroinfection, electroporation and microinjection have been found to be successful in transformation of dicotyledonous plants but less so with monocots. For monocots other methods are more suitable and reference may be made to International Patent Application No. WO85/01856 and European Patent Application No. 275,069 for methods which are said to be suitable. Methods for the transformation of trees are described in European Patent Application No.0227264 and British Patent Application No. 2211204, both of which utilise infection with Agrobacterium tumefaciens.
The invention, by provision of a source of mRNA antisense to the normal RNA encoding an enzyme essential to lignin biosynthesis, thereore provides a means by which lignin synthesis in a plant may be suppressed or lignin composition altered. There is also provided, therefore, plants and plant propagating material containing the recombinant DNA of the invention. More specifically, the invention includes seeds, plants and their progeny.
Any of a number of enzymes in the li~nin biosynthetic pathway can be used as targets for down-regulation using antisense RNA. ~hese are discussed below:
Cinnamyl Alcohol Dehydrogenase (C~D~
This enzyme catalyses the reduction of cinnamyl-aldehydes into cinnamyl alcohols. C~D
has been characterised from di~ferent species:
Forsythia suspensa, soybean ~Glycine max.), spruce ~n(3~ 3~7 (Picea abies), polar (Populus euramericana) and bean ~Phaseolus vulgaris L). Except for soybean, only one form of CAD enzyme has been detected in these species. The common structure of CAD enzymes is that of a dimer of approximately 70,000 daltons with each monomer having a molecular weight of approximately 40,000 daltons. In contrast, the bean enzyme is a monomer with a molecular weiyht of 65,000 daltons, indicating that possibly a gene duplication has taken place during evolution.
Soybean has two isoenzymes, one of 43,000 daltons and one of 69,000 daltonsO The first soybean isoenzyme is specific for coniferyl alcohol while the 69,000 dalton one and all other CAD can catalyse the formation of all the cinnamyl alcohols (i.e. coniferyl, sinapyl and coumaryl alcohols).
However, the Rm of CAD for the diferent cinnamyl alcohols varies between enzymes from different species. This variation may explain the different compositions of the lignin core in different species. Lignin monomers cannot be synthesised in plants through any other biochemical pathway.
Thus CA~, as well as CCR and peroxidase, may be key enzymes in the regulation of lignification.
The utilisation of inhibitors specific for these enzymes indicates that they may regulate the quantity of lignin rather than its composition.
The ~m values for the different cinnamyl alcohols of th~ soybean CAD isoenzymes sugges~ that CAD
isoenzymes may also control the composition of lignin. The presence of Zn2~ is required for the activity of CAD, as for other alcohol dehydrogenases. The reduction of cinnamyl-aldehydes cannot be catalysed by CAD in the 5~7 ~S35063 presence of NADH ( nicotinamide adenine dinucleotide - reduced form) instead of NADPH ( nicotinamide adenine dinucleotide phosphate -reduced form).
CAD enzyme activity and transcription are stimulated by treatment of bean suspension culture cells by fungal elicitor preparations. This increased CAD activity can be regarded as a defence reaction of the plant against pathogenic microorganisms, since an increase in the activity of the enzyme may be related to the deposition of lignin in the walls of infected cells, or to the synthesis of extracellular lignin-like material and other phenolic compounds involved in the defence responses.
Cinnamoyl-CoA Reductase (CCR) This enæyme is also involved in the reduction of cinnamyl-aldehydes to cinnamyl alcohols. CCR
has been investigated in a number of species and has been purified to apparent homogeneity from spruce, soybean and poplar.
only one form of the enzyme, having a molecular weight of 36,000 daltons has been detected in theee plant species, indicating considerable evolutionary conservation. The enzyme from poplar has an equilibrium constant of 2.5 x lO 4 M. Th~ spruce enzyme gene has a value of 5.6 x 10 7 M. The enzyme exhibited the greatest affinity for feruloyl-CoA with an apparent Km f 2.55 ~m. A difference in substrate specificity was observed between the spruce and soybean enzymes-feruloyl-CoA was a good substrate for both enzyme~.
However, the soybean enzyme alone converted sinapoyl/CoA.
Peroxidase ~5'~7 ~535~63 Peroxidase is an ubiquitous enzyme found in many plant organs. There are a number o isoenzymes present in plants which are regulated in a tissue-specific and developmentally regulated manner. The cell wall bound form of this enzyme is involved in the poly~erisation of lignin monomers in the presence of hydrogen peroxide. However, the enzyme can also catalyse crosslinks between side chains of lignin polymers either to other lignin polymers or to other macromolecules such as hemicellulose. The enzyme has been purified from a number of plant sources.
Catechol-O-Methyl Transferase This enzyme catalyses the methylation of cafeic and 5-hydroxyferulic acids to ~orm ferulic and sinapic acids respectively. The diversity, biochemical activities and characters are known.
However, to achieve inhibition of lignin biosynthesis, it is possible to target other enzymes of the phenylpropanoid pathway such as phenylalanine ammonia lyase, cinnamyl CoA-ligase, chalcone synthase and others.
The down regulation of the enzymes discussed above will affect both the amount of lignin deposited and the degree of secondary modification through crosslinking either o the lignin monomers or of the lignin structures to hemicellulose, or side chain additions. Reduction in lignification referred to hecein includes both mechanisms which can be viewed as complementary rather then self exclusive.
In plants, reduced expression of a previously introduced genes, coding for chloramphenicol acetyl transferase and nopaline synthase, and o ~S35063 Z~
endogenous genes, chalcone synthase and polygalacturonase has been reported.
The present invention will now be described, by way of ill~lstration, in the following Examples.
A bean cDNA clone encoding C~D was isolated from an elicitor~induced cDNA library [Edwards et.
al.;Proc. Natl. Acad. Sci. 82 6731-6735 (1985)].
The library was screened using a nick-translated partial CAD cDNA clone, designated 4A, the sequence of which is published by Walter et.al. in Proc.
Natl. Acad. Sci. USA (1988). ~he screening was carried out under standard conditions and one clone was identified which showed a very strong hydridisation signal. This clone was named pMG10.
Agarose gel electrophoresis of the purified PstI
insert showed the length of the cDNA to be 2.2 kilobases (kb). Since the bean CAD polypeptide has a molecular weight of 65,000 daltons, the open reading frame is expected to be approximately 1770 base pairs (hp), assuming an average molecular weight of amino acids to be 110. If the ~ull length mRNA contains 5' and 3' untranslated regions of about 200 bp the total length of the complete cDNA will be approximately 2kb~ Thus pMG10 was putatively identified as a full-length C~D cDNA.
The restriction map of clone pMG10 and clone 4a were determined. This indicated that there was close homolosy of the parts of these clones (Figure 2). Common sites were ound at identical positions Ncol, ~indIII, among those enzymes tested. No sites were found for the following enzymes: AccI, AvaI, BamHI, BclI, BstEII, ClaI, DraI, E~paI, KpnI, NarI, PvuI, PvuII, SacI, SalI, Sma~, SpeI and Zho~.
~3 ~S35063 Southern blot hybridisations were carried out, using clone 4a as a probe, to identify the regions of homology between clone pMG10 and clone 4a. It was thus possible to align the maps of the two clones, showing that pMG10 presumably includes 4a (or a very closely related sequence) at its 3' end (Figure 2?.
Since the isolation of the clone pMG10, described above, the~e has been reported the isolation of a bean CAD CDNA from an elicitor induced cDNA library constructed in ~-gtll phage [Walter et.al. in Proc. Natl. Acad. Sci. USA
(1988)]. The restriction map deduced from the sequence of this clone, designated ~-CAD4, shows that it is closely related to pMG10 but not identical: most of the restriction sites found in pMG10 are also found in ~-CAD4. However, the location of some sites are different in the two clones: the HindIII site located 300bp from the 5' end of pMG10, the EcoRI site and the single BglII
site of pMG10 are not found in ~~CAD4 at identical positions. Sites for BclI, ClaI and PvuII are not present in pMG10.
The data generated from the restriction mapping and hybridisation experiments are taken as proof that pMG10 contains a CAD cDN~ clone, as most of the structural information for the CAD mRNA has been published.
This clone can now be used for the isolation of the CAD gene not only from bean but from other plant species particularly those to which this invention can be applied, for example, alfalfa, maize, brassicae, eucalyptus, pine, poplar, lolium and festuca. The gene can then be used for the ~C~ ?~ 7 ~S35063 isolation of the coding region as well as the promoter. The promoter of the CAD gene can be used to drive the expression of foreign genes in plants.
Foreign genes which can be used in this context can be genes which can inactivate virus2s, fungal or bacterial infections, as the C~D promoter will be switched on particularly after inection of plants with these agents.
ta) Screening of a ~enomic library and partial restriction mapping A genomic library of tobacco DNA ~N. tabacum var. Samsum) was constructed in ~-EMBL3. An aliquot of the library (approximately 106 pfu) was screened using the Pstl insert of pMGl~
radiolabelled by nick-translation. A total of 54 hybridising plaques were detected. Twenty of these clones were plaque purified, using standard protocols. DNA was prepared from 11 clones, designated 1, 8, 15, 22, 24, 28, 41, 46, 47 and 50.
Limited restriction maps using SalI~ EcoRI and HindIII restriction enzymes were determined for these clones. This indicated that all the clones contained the same gene. The strong cross-hybridisakion with the bean CAD cDN~ probe was taken as indication that these clones contain a tobacco CAD gene. A representative restriction map of one of the clones (clone 15) covering 11 kb is shown in Figure 3.
(b) Determination of the orientation of the_tobacco CAD ~ene in clone 15 Southern blot hybridisations were carried out using a 5' end probe (clone 4b: the subcloned 5' a'~f~'~
~S35053 Eco~I fragment of ~-CA~4) or a 3~ end probe ~clone 4a). AS both probes hybridise to the isolate DNA
from these genomic clones, the complete tobacco CAD
gene may be contained in these clones. The two probes mentioned above were used to determine the orientation of the CAD sequences within the genomic clone. The central region covering approximately 5 kb of clone 15 hybridised to these two probes delineating the tobacco CAD gene. The two external region of the clone which are located from 0 to 3 kb and from 7 to 11 kb from the 5' end of the insert do not hybridise to any of the bean CAD
cDN~s. The region extending from the EcoRI site 3 kb from the 5' end to the EcoRI site 4.3 kb from the 5' end hybridises only to the clone 4b. The reg.ion covering this 1.3 kb fragment was designated ASR1 (Figure 3). The region located between the 4.3 kb EcoRI site to the HindIII site 7kb from the 5' end hybridised only to the clone 4a. This indicates that the 5' end o the tobacco CAD gene is located in the ASR1 fragment, whereas the bulk of the coding regi3n of the gene is located in the rest of the hybridising fragment. The region between the SalI and HindIII was designated SR1 (Figure 3) (c) Detailed restriction mapping and sequence determination of the ASR1 and_SHl regions Clone 15 was restricted with ~coRI and the resulting fragments were cloned into the EcoRI site of Bluescript SK-vector. Transformants were selected on 50 ~g/ml ampicillin, and clones containing the ASR1 fragment were identified by hybridisation with the insert of clone 4b. One clone, pASR1 hybridised strongly. Restriction ~{~
enzyme digestions using HindIII and EcoRI showed that this clone contains the ASRl fragment.
A detailed restriction map of the ASP~l fragment was determined. No sites were found ~or the following enzymes: NcoI, SalI, BamHI, ClaI, ~glII, sstEII and SacI. Southern blot hybridisations to restriction digests of pASRl DNA
using clone 4a as a probe indicate that 0.25 kb EcoRI-AvaII fragment hybridises very weakly.
Since this fragment is located at the very 5' end of the hybridising region in clone 15, it may contain the 5' untranslated region including the translation start site. However,it is also possible that the weak hybridisation of this fragment to the clone 4b may be the result of low homology in this region between tobacco and bean genes. Ths ~vaII-KpnI, ~pnI-HindIII and 0.3 kb HindIII- EcoRI fragments hybridise strongly to clone 4b.
DNA sequence analysis of pASR1 insert DNA, determined using standard protocols, revealcd the following features:
The HindIII-EcoRI fragment contains a 90 base pair long intron. The exon sequences are 84%
identical to the bean CAD cDNA (from base 422 to 565 in the ~-CAD4 sequence. At the protein level, the homology is 93%. These results suggest that this region might be of importance for the functional structure of CAD~
Construction of CAD Antisense Vectors (a) Vectors based on the Tobacco CAD Gene The plasmid pASR1 (Bluescript containing the ASR1 fragment) was cut by KpnI. The 0.6 kb fragment containing the 3' end o~ ASR1 and about 50 2~ 3~
bp of the Bluescript polylinker was recovered by electroelution after separation on agarose qels, and inserted into the RpnI site of pJRl. This construct was used to transform E. coli, strain TG2. The transformants were selected by growth on kana~ycin-containing plates. 227 colonies were streaked on nitrocellulose filters, and hybridised with the radiolabelled KpnI fragment. Eight clones gave a strong signal.
Plasmid DNA was prepared from these clones.
Restriction digestions with KpnI were carried out to check the presence of the insert. Only seven of the clones contained the recombinant vector.
These were clones 1(6-6), 1(7-7), 1(8-9), 1(10~3), 2(5-13), ~(7-1) and 2(11-3)].
Since the KpnI fragment could be inserted in both orientations, it was necessary to identify the recombinant clones with this fragment in the antisense orientation. The presence of the EcoRI
site from the Bluescript polylinker introduced an asymmetry in the fragment, thus making it possible to check the orientation of the insert by a simple restriction digest with EcoRI. If the KpnI
fragment is in the antisense orientation only the CaMV 35S promoter would be excised by EcoRI
digestion, producing two fragments of approximately 0.5 and 11 kb. However, if the KpnI fragment is in the sense orientation the tobacco CAD fragment plus the CaMV 35S promoter would be excised by EcoRI. The EcoRI ragments produced in this case would be approximately 1 and 10 kb. Of the seven positive clones, two [1(6-6) and 2(5-13)l contained the insert in the sense orientation and three [
1(10-3), 2(7-1) and 2(11-3)l were identified as '7 ~S35063 antisense constructs. The two remaining clones did not cestrict with EcoRI and, therefore, remain uncharacterised. Each of the clones was shown to contain only one CAD insert fragment. The clones were renamed pCAD-ASl [1(10-3), pCAD-AS2 ~2(7-1)], pCAD-AS3 [2(11-3], pCAD-Sl ~1( 6-6)] and pCAD-.52 ~2(5-13)].
(_ Vectors based on the Bean CAD cDNA
pMG10 was restricted with NcoI, and the 1170 bp fragment covering a large portion oE the CA~
protein was isolated following agarose gel electrophoresis. This fragment was then treated with T4 polymerase, and cloned into the SmaI site of the expression vector pJR1. Ligation mixtures were transformed into suitable E. coli hosts, and the recombinant clones identified by hybridisation to radiolabelled NcoI fragment. A number of clones were isolated, characterised and found to contain the CAD fragment in either the sense or the antisense orientation. A representative clone from each group was characterised in detail and designated pBCAD.S1 (sense) and pBCAD.AS1 (antisense).
The structure of these vectors are represented in Figure 4.
The above procedure can be repeated and the CAD antisense fragments, derived either from the tobacco gene or the bean cDNA, can also be inserted into an expression vector containing the gP~L2 or gPAL3 promoter, the sequences of which are given in Figures 6 and 7. These promoters are derived from one of the phenylalanine ammonia lyas2 genes from French bean. It has been found that the gPAL2 promoter drives the expression of foreign genes ZO(~5~
~S35063 specifically in xylem tissue. The gPAL3 promoter is expressed, for example, in the pith cells, the endodermis.
TRANSFORMATION OF_OBACCO
(a) Transfer of Vectors to_Agrobacterium The antisense and sense constructs were introduced into A. tumefaciens LBA4404 by triparental mating following published procedures.
l0 The presence and integrity of the antisense constructs were checked by restriction digestion and Southern blot experiments. These results indicated that no recombination had occurred during the transfer of the vectors to Agrobacterium.
b) Tobacco Leaf Disc Transformation Tobacco (N tabaccum, variety samsum) leaf discs were transformed using well established previously published procedures. Thirty six plants containing the bean CAD antisense construct and twenty one containing the bean CAD sense construct were selected.
In addition seventeen plants containing the tobacco CAD antisense gene and twenty six containing the tobacco CAD sense gene were identified.
ANALYSIS OF TRANSFORMESD PLANTS
a) CAD ~nzyme Measurements On Tissue From Plants Plant material was used from both transformed and untransformed control plants. The material (leaf and stem1 was ground with CAD extraction buffer containing 200 mM Tris/~Cl pH 7.5, 0.5%
(w/v) PEG 6000, 5% (w/v) PVP, and 15 mM
~-mercaptoethanol (500~l). The crude homogenat*
~ ~J
Ps35063 ~2 was centrifuged and the supernatant used as source of enzyme. The assay reaction contains 10 mM
coniferyl alcohol (50~1 ), 10 mM NADP~ ( 50~1), 100 mM Tris/HCl pH 8.8 (800~1). This was incubated at 30C for 10 minutes, then enzyme extract (100~1) was added and the whole ~ixture was incubated for a for a further 10 minutes at 30c. The OD~oo was recorded against a blank supplemented with water.
Three leaf and two stem samples were taken from each plant. Assays were done in dupllcate.
Ste~ segments from control plants of the same age showed little variation in CAD enzyme activity.
Lea material also showed little variation.
Transformed plants containing the anti-sense RNA
vectors showed considerable reductions in CAD
enzyme activity. This varied between the different transformants. The maximum reduction observed was 70% in both leaf and stem.
b) Polymerase Chain Reaction to determine ~resence of antisense genes DNA was extracted from selected plants.
Oligonucleotides to sequences in the CamV promoter and nos 3' terminator were used as primers in the polymerase chain reaction (PCR). DNA from plants containing the bean CAD antisense constructs gave a product o$ ~1200 bp as expected. DN~ from plants containing the tobacco CAD antisense constructs gave a product of ~620 bp as expected. To confirm that the~e products were CAD sequences, a southern blot of these products was probed with a third oligonucleotide. The sequence of this was taken from the region of 93% homology between the bean CAD c~NA and the tobacco CAD ASR1 gene fragment.
These seguences were present in the antisense b ~
~S3~063 vectors. This oligonucleotide hybridised to all PC~ products, confirming the presence and identity of the antisense constructs.
c) Neutral Detergent Fib _ Cellulase Assay In order to detect changes in lignin-cellulase complexes, neutral detergent fibre/cellulase (NDF/C) assays were performed.
This assay determines the amount of extractable cellulose from cell preparations.
Selected plants, exhibiting at least 50%
reduction in CAD enzyme activity in both leaf and stem were grown from tissue culture to maturity in pots in a growth room ( ~10 weeks). Two control plants were grown in the same way. The plants were selfed and after seed collection the stems were harvested for NDF/C assay. Plants containing the bean CAD antisense constructs showed increased NDF/C values of two of 14 units greater than controls. Plants containing the tobacco C~D
antisense constructs showed increases of five to fourteen units above controls.
~ t~
NDF/C VALUES OF To PLANTS
__ _ P LANT ND F/C
CONTROL 1 58.6 CONTROL 2 57.5 BCAD 3 72.5 6 57.9 8 59.0 9 66.1 12 60.9 29 65.6 32 ~9.2 59 5~.5 TCAD 15 72.2 66.4 24 59.9 70.2 42 59.8 43 66.6 47 68.7 48 71.1 _ _ 63.2 d) Gsnotype Analysis Of Selected Plants Six plants were chosen for further analysis on the basis of their NDF/C values. These were BCAD
32, TCAD 15, 40, 47 and 5Q. The genotype of each of these was determined by patching out 72 seeds of each onto medium containing kanamycin (Murashige &
3'~
~S35063 Skoog salts 2.3 g/l, sucrose 15 g/l Q.8~ agar, 200~g/cm3 kanamycin). Seedlings which do not carry tha kanamycin gene bleach and die. The nu~ber of seedlings bleaching as a ratio to those not bleaching can be taken as an indicator of the numbers of genes present following the rules of segregation. We have assumed an equal number of antisense genes as kanamycin genes. An equal number of seeds was patched onto medium without kanamycin as a check on viability. In this way the genotypes of the To generation were determinsd.
~his indicated that some plants, such as T48, contains one copy of the antisense gene, whereas Tl5 contains two copies.
e) NDF/C Assay of S1 Plants Sixteen seeds from each of thsse six plants plus control were planted and grown to maturity in 7 inch pots in a glass house. The height of each plant was measured each week. Data was collected on time to flowering and growth rate for each plant. Each plant was selfed and after seed collection the lea and stem of each plant was harvested separately. The dry weight of each was recorded and then NFD/C measured. The yield of seed was recorded for each plant.
The mean NDF/C value for population of TCAD15 progeny was significantly greater than the mean NDF/C value for the control population, with individual plants having an increase of 18 units above the mean of the controls.
The other populations showed somewhat less significant increases in NDF/C values.
~ 5~7 Ps3s~63 NDF/C VALUES FOR STEM
NUMBER
_ _ .7 60.3 44.9 2 _ 63 .7 44 .7 3 50.0 50.7 q 48.3 sa.s 46. 3 _ 49.9 54.6 6 _ 55.7 54.5 7 40.7 55.1 53 .2 8 4~.4 52.~ 48.5 9 39.9 52 .2 54.1 50.0 _ 57.9 .l _ 50.1 12 44.2 _ 54.0 f) Genotype Analysis Of S1 Plants This was determined in the same way as for the To plants.
Cloning Of CAD Genes From Other Plant Species In order to generate antisense vectors for the other crop plants of interest we have isolated the CAD genes from alfalfa, maize, rape and eucalyptus.
Plasmids containing these isolated genes have been deposited in a culture collection in E.coli hosts.
PCR products have been obtained from yenomic DNA of alfalfa, rape, eucalyptus and maize uæing oligonucleotide sequences taken from the tobacco ~S35063 ~ ~a3 CAD gene sequence. The products have been sho~n to hybridisa to a third oligonucleotide, the sequence of which is present in the tobacco CAD gene.
The priming sequences are:
-5' AAT GAA AGG CTG TTC TAC AAG CTTCT
S' ATC GGT CAC AAC ~AT AAC TrrG AAT
The third sequence used as probe, CAD4 is 5' TCA GGG TCT TTA TAT CAG TTT GAA AGA AAA
These PCR products are cloned and will be fully sequenced.
Using these probes we have been able to isolate CAD genes from alfalfa, rape, eucalyptus and maize.
The plasmid pGAL2 has been deposited in Escherichia coli strain DH5 on 6th Decem~er 1988 with the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCIB 40087.
The tobacco-derived clone designated CAD
genomic clone 15 has been deposited in bacteriophage A-EMBL3 on 6th December 1988 with the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCIB 40088.
The maize-derived clone, designated pCAD;maize has been deposited in Escherichla coli, strain DH5a, on 3~ ~oJ ~g~ with the National Collection of Industrial and Marine ~acteria, Aberdeen, United Ringdom, under the Accession Number NCIB 4~
The rape-derived clone, designated pCAD;rape has been deposited in Escherichia_coli, strain D~5~, on ~ ~b~l9~ with the National Coll~ction of 3~5~
~S35063 Industrial and Marine sacteria, Aberdeen, United Kingdom, under the Accession Number NCI8 ~D~
The alfalfa-derived clone, designated pCAD;alfalfa has been deposited in Esch _ichia coli, strain DH5~, on with the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCI~ .
The eucalyptus-derived clone, designated pCAD;eucalyptus has been deposited in Escherichia coli, strain DHSa~ on 3O ~ 8~1 with the National Collection of Industrial and Marine ~acteria, Aberdeen, United Kingdom, under the Accession Number NCIB ~3O .
Claims (19)
1. A recombinant DNA comprising a plant DNA
having a promoter sequence and an insert, located 3' to the promoter and in appropriate reading frame, comprising a nucleotide sequence encoding a mRNA which is substantially homologous or complementary to mRNA encoded by an endogenous plant gene or a part thereof which encodes an enzyme essential to lignin biosynthesis, so that mRNA
transcribed from the said insert inhibits production of the enzyme from the endogenous gene.
having a promoter sequence and an insert, located 3' to the promoter and in appropriate reading frame, comprising a nucleotide sequence encoding a mRNA which is substantially homologous or complementary to mRNA encoded by an endogenous plant gene or a part thereof which encodes an enzyme essential to lignin biosynthesis, so that mRNA
transcribed from the said insert inhibits production of the enzyme from the endogenous gene.
2. A recombinant DNA as claimed in claim 1 wherein the insert encodes mRNA in antisense orientation to the mRNA encoded by the said endogenous gene.
3. A recombinant DNA as claimed in claim 2, in which the insert is isolated from the untranscribed strand of the DNA encoding the said endogenous gene.
4. A recombinant DNA as claimed in claim 1 wherein the insert is in the same orientation as the said endogenous gene.
5. A recombinant DNA as claimed in any preceding claim in which the insert has a minimum size of 50 bases.
6. A recombinant DNA as claimed in any preseding claim in which the said enzyme is selected from the group consisting of cinnamyl alcohol dehydrogenase (CAD), cinnamoyl: CoA reductase (CCR) and catechol-O-methyl transferase (COMT).
7. A recombinant DNA as claimed in any preceding claim in which the promoter is selected from the group consisting of CaMV35S, GPAL2,GPAL3 and endogenous plant promoter controlling expression of the endogenous CAD gene.
8. A method of inhibiting or altering lignin biosynthesis in a plant, comprising stably incorporating into the genome of the plant by transformation a recombinant DNA comprising a plant DNA having a promoter sequence and an insert, located 3' to the promoter and in appropriate reading frame, comprising a nucleotide sequence encoding a mRNA which is substantially homologous or complementary to mRNA encoded by an endogenous plant gene or a part thereof which encodes an enzyme essential to lignin biosynthesis, so that mRNA
transcribed from the said insert inhibits translation of the endogenous gene to the enzyme.
transcribed from the said insert inhibits translation of the endogenous gene to the enzyme.
9. A transformed plant possessing lower than normal ability to produce lignin characterised in that said plant has stably incorporated within its genome a recombinant DNA claimed in any of claims 1 to 6.
10. A transformed plants, as claimed in claim 9, in which the plant species is alfalfa, maize, rape, eucalyptus, poplar, lolium or festuca.
11. Tobacco CAD gene and recombinant DNA
containing same, derived from the culture desposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number 40088.
containing same, derived from the culture desposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number 40088.
12. Maize CAD gene and recombinant DNA containing same, derived from the culture deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCIB 40229, on 30 November 1989.
13. Rape CAD gene and recombinant DNA containing same, derived from the culture deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCIB 40228, on 30 November 1989
14. Alfalfa CAD gene and recombinant DNA
containing same, derived from the culture deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCIB
containing same, derived from the culture deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCIB
15. Eucalyptus CAD gene and recombinant DNA
containing same, derived from the culture deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCIB 40230, on 30 November 1989
containing same, derived from the culture deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom, under the Accession Number NCIB 40230, on 30 November 1989
16. A recombinant DNA comprising an antisense CAD
gene under control of the promoter GPAL2.
gene under control of the promoter GPAL2.
17. A recombinant DNA comprising an antisense CAD
gene under control of the promoter GPAL3.
gene under control of the promoter GPAL3.
18. A recombinant DNA comprising an antisense CAD
gene under control of the promoter CaMV35S.
gene under control of the promoter CaMV35S.
19. A recombinant DNA comprising an antisense CAD
gene under control of the endogenous plant promoter controlling the endogenous CAD gene.
gene under control of the endogenous plant promoter controlling the endogenous CAD gene.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB888829298A GB8829298D0 (en) | 1988-12-15 | 1988-12-15 | Improved fodder crops |
| GB8829298.2 | 1988-12-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2005597A1 true CA2005597A1 (en) | 1990-06-15 |
Family
ID=10648552
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2005597 Abandoned CA2005597A1 (en) | 1988-12-15 | 1989-12-14 | Plants having reduced lignin or lignin of altered quality |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2005597A1 (en) |
| GB (2) | GB8829298D0 (en) |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993005160A1 (en) * | 1991-09-10 | 1993-03-18 | Zeneca Limited | Modification of lignin synthesis in plants |
| WO1993024639A1 (en) * | 1992-05-29 | 1993-12-09 | Zeneca Limited | Expression of genes in transgenic plants |
| WO1993024638A1 (en) * | 1992-05-29 | 1993-12-09 | Zeneca Limited | Expression of genes in transgenic plants |
| WO1994008036A1 (en) * | 1992-10-06 | 1994-04-14 | The Salk Institute For Biological Studies | Methods for the modulation of lignification levels |
| US5451514A (en) * | 1991-04-26 | 1995-09-19 | Zeneca Limited | Modification of lignin synthesis in plants |
| US5886243A (en) * | 1995-11-30 | 1999-03-23 | Board Of Control Of Michigan Technological University | Genetic engineering of wood color in plants |
| US5922928A (en) * | 1995-11-30 | 1999-07-13 | Board Of Control Of Michigan Technological University | Genetic transformation and regeneration of plants |
| US5959178A (en) * | 1991-10-09 | 1999-09-28 | Imperial Chemical Industries Plc | Modification of lignin synthesis in plants |
| US5973228A (en) * | 1997-07-24 | 1999-10-26 | University Of British Columbia | Coniferin beta-glucosidase cDNA for modifying lignin content in plants |
| US6294047B1 (en) | 1999-07-30 | 2001-09-25 | Institute Of Paper | Methods for reducing fluorescence in paper-containing samples |
| US8129588B2 (en) | 2004-04-20 | 2012-03-06 | Syngenta Participations Ag | Regulatory sequences for expressing gene products in plant reproductive tissue |
| WO2013003558A1 (en) * | 2011-06-30 | 2013-01-03 | Monsanto Technology Llc | Alfalfa plant and seed corresponding to transgenic event kk 179-2 and methods for detection thereof |
| US8536406B2 (en) | 2008-04-28 | 2013-09-17 | Michigan Technological University | COMT1 gene fiber-specific promoter elements from poplar |
| US9238818B2 (en) | 2004-04-20 | 2016-01-19 | Syngenta Participations Ag | Methods and genetic constructs for modification of lignin composition of corn cobs |
| AU2013202718B2 (en) * | 2000-09-29 | 2016-05-26 | Agresearch Limited | Manipulation of plant cell walls (5) |
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-
1988
- 1988-12-15 GB GB888829298A patent/GB8829298D0/en active Pending
-
1989
- 1989-12-14 CA CA 2005597 patent/CA2005597A1/en not_active Abandoned
- 1989-12-15 GB GB898928399A patent/GB8928399D0/en active Pending
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| US5451514A (en) * | 1991-04-26 | 1995-09-19 | Zeneca Limited | Modification of lignin synthesis in plants |
| US6066780A (en) * | 1991-04-26 | 2000-05-23 | Zeneca Limited | Modification of lignin synthesis in plants |
| WO1993005160A1 (en) * | 1991-09-10 | 1993-03-18 | Zeneca Limited | Modification of lignin synthesis in plants |
| US5959178A (en) * | 1991-10-09 | 1999-09-28 | Imperial Chemical Industries Plc | Modification of lignin synthesis in plants |
| WO1993024638A1 (en) * | 1992-05-29 | 1993-12-09 | Zeneca Limited | Expression of genes in transgenic plants |
| US5633439A (en) * | 1992-05-29 | 1997-05-27 | Zeneca Limited | Expression of genes in transgenic plants using a cinnamyl alcohol dehydrogenase gene promoter |
| WO1993024639A1 (en) * | 1992-05-29 | 1993-12-09 | Zeneca Limited | Expression of genes in transgenic plants |
| WO1994008036A1 (en) * | 1992-10-06 | 1994-04-14 | The Salk Institute For Biological Studies | Methods for the modulation of lignification levels |
| US6087557A (en) * | 1992-10-06 | 2000-07-11 | The Salk Institute For Biological Studies | Methods for the modulation of lignification levels |
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| US5973228A (en) * | 1997-07-24 | 1999-10-26 | University Of British Columbia | Coniferin beta-glucosidase cDNA for modifying lignin content in plants |
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| EP3228713A1 (en) * | 2011-06-30 | 2017-10-11 | Monsanto Technology LLC | Alfalfa plant and seed corresponding to transgenic event kk 179-2 and methods for detection thereof |
| US9854778B2 (en) | 2011-06-30 | 2018-01-02 | Monsanto Technology Llc | Alfalfa plant and seed corresponding to transgenic event KK 179-2 and methods for detection thereof |
| CN103857798B (en) * | 2011-06-30 | 2018-06-15 | 孟山都技术公司 | Alfalfa plant and seed and its detection method corresponding to transgenic event KK179-2 |
| US10385355B2 (en) | 2011-06-30 | 2019-08-20 | Monsanto Technology Llc | Alfalfa plant and seed corresponding to transgenic event KK 179-2 and methods for detection thereof |
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Also Published As
| Publication number | Publication date |
|---|---|
| GB8928399D0 (en) | 1990-02-21 |
| GB8829298D0 (en) | 1989-01-25 |
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