CA2120258A1 - Dna constructs, cells and plants derived therefrom - Google Patents
Dna constructs, cells and plants derived therefromInfo
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- CA2120258A1 CA2120258A1 CA 2120258 CA2120258A CA2120258A1 CA 2120258 A1 CA2120258 A1 CA 2120258A1 CA 2120258 CA2120258 CA 2120258 CA 2120258 A CA2120258 A CA 2120258A CA 2120258 A1 CA2120258 A1 CA 2120258A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03001—Catechol oxidase (1.10.3.1), i.e. tyrosinase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
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Abstract
The invention relates to DNA construct comprising (i) a promoter functional in plants, (ii) a DNA sequence corresponding to an antisense polyphenol oxidase gene or part thereof, or a DNA sequence homologous thereto and (iii) a terminator functional in plants.
This construct provides bruising resistance in plants.
This construct provides bruising resistance in plants.
Description
W094/03607 . 2 I 2 0 2 5 8 Pcr~EPg3/olg88 D~A C0~8TRUC~ CB~5 A~D PLANT~ D2~I~ED T~BaB~RO~
800p~ o~ the i~tio~
The invention relates to novel DNA constructs~ plant cells co~taining these constructs and plants derived there~rom, as well as æeeds obtained ~rom said plants. In particular it invol~es the use of antisen~e nucleic acid technology to control gene expression in plants7 Using tissue specific promoters the control of gene expression can ~e directed to particular issues of the plant. The invention provides further, a process for obtaining plants which do not exhibit enz~matic browning of disrupted tissues.
IntroZuation Browning and discolouration causes substantial losses in a wide range of fresh and pro¢essed fruits and vegetables. In potato~s, internal bruising, also called black spot, is a physiological response of potato tubers to tissue compaction and i~ o~e of the most important problems of the potato industr~. Yearly, large amounts of the potato produc~ion are valueless for direct consumption or processing due to browning of the tubers.
i The biochemistry of browning is relatively well unders~ood in mammals and bacterial model systems. In plants, however very little is known about the biochemical processes involved in browning and the pathways responsible for discolouration in a wide variety of fruits and vegetables has not been elucidated.
In potatoes, bruising or black spot results from minor physical damage sustained by the tubers during mechanical harvest and wo 94/036~ 1 2 0 2 5 8 PCT/EP93~01988 transportation. The colour reaction results from a small number of cells within the bruise that ~ctually lyse. The grey/black colour of the bruise i5 caused by the enzymatic production of melanin, a highly polymerised phenolic compound. The enzyme polyphenol oxidase (PPO), an oxygenase, catalyses the first two oxidation reactions of the pathway and converts monophenols ~such as tyrosine) and ortho-diphenols (such as catechol) to ortho-~uinones (such as dihydroxiphenylalanine-quinone).
Autocatalytic chemical oxidation reactions conv~rt ortho-quinones into ~elanin via several reactive quinone intermediates. Although the function of plant polyphenol oxidases has not been elucidated, several possible roles of the enzyme have been postulated. These include participation in electron transport in photosynthesis and participation in the defence mechanisms of the plant against insect attack or infections. It is believed that PPO is not involved in the synthesis of phenolic compounds in healthy, intact cells. Plant PPo enzymes are nuclear-encoded copper metalloproteins localised in me~branes of plastids. In potato tubers, PPO has been localised in the rudimentary thylacoid me~branes of amyloplasts. The sub-cellular location separates the enzyme from its substrate which is primarily found in the cell vacuole. However, in disrupted cells the enzyme is mixed with substrate leading to oxidation of phenolic compounds by PPO
enzyme.
: : :
Although bruising~has been a severe pro~lem in the potato industry for many years, no progress has been made until now to prevent the formation of black spots into potato tubers through molecular technique~. The term bruising resistance, as used hereaft2r, refers to resistance against browning and discolouration upon disruption of plant cells.
We have found that bruising resistance can be obtained through interfering in the biosynthetic pathway of melanin production by the expression of an antisense PPO gene. More particularly, the production of polyphenol oxidase can be W094/0~7 ~ 2 ~ 2 0 2 5 8 PCT/EPg3/01988 reduced in specific tissues through tissue specific expres~ion of said antisense PPO gene. Since the expression of anti~nse PPO may perturb one or more of the postulated functions of PPO, especially in aerial parts of the plant, limitation of expression to tubers, by implementation of appropriate tuber specific promoters, is preferred. Furthermore, we have found that polyphenol oxidases are encoded by a family of genes comprising at least S members. We have been able to suppress the expression of genes, and particularly of all genes, expressing in tubers through the coordinate expression of only one antisense PPO gene corresponding to part or entire sequence, or homologous with one of the genes encoding polyphenol oxidase. Moreover, we have been able to produce bruising resistant plants which show a reduced sensitivity to bruising, especially potato plants of which the tubers show a reduced sensitivity to bruising.
The invention is based on the application of antisense nucleic acid technology. Although the mechanism of antisense is ~not elucidated, it~is believed that the inhibition of the expression of the target gene is obtained either through the formation of a complex of the antisense RNA and the endogenous RNA which are complementary, preventing the translation, or through~ the formation of a complex structure with the DNA
sequence, preventing the transcription.
The invention provides~a method for producing bruising resistant plants showing a reduced PPO enzyme activity and which also have been shown to have a significantly reduced ~ browning phenotype in the field.
; The invention can be used to control bruising upon disruption of tuber, vegetable, or fruit tissues in which the browning is the resuIt of PPO enzyme activity in or around the damaged plant tissue. It is understood that the invention is not restricted to potatoes but that it can also be used for the reduction of browning upon physical tissue disruption in other WOg4/03607 ~ PCT/EP93/019BX
~,120Z58 ,.1~, plants like leaf vegetables, i.e. lettuce, chicory or fruits, where ~he browning is ~he result of the expression of genes encoding PPO.
Det~iled ~es~riptio~ of the invention The present invention provides DNA constructs comprising i~
a promoter fu~ctional in plants, ii) a DNA se~uence corresponding to an antisense polyphenol oxidase ~ene or part thereof, or a DNA sequence homologous thereto and iii) a terminator functional in plants.
The term antisense polyphenol oxidase gene refers to a double stranded DNA sequence which sequence corresponds to or is homologous with the endogenous gene encoding PFO or part thereof, but which has a reversed orientation with respect to the said PPO ge~e. In a antisense DNA sequence the coding strand becomes the~template strand and vice versa.
The term homologous sequence refers to a sequence in which the aver~age nucleotide sequence homology with the contiguous gene is~ at least 65 %. ~ ~
The term DNA sequence refers to a double stranded DNA
sequence which can be~expressed in piant cells if said seguence is under:the expression control of a promoter functional in plants and having a terminator functional in plants.
The term promoter used herein refers to a transcription regulation region upstream from the coding sequence containing the regulatory se~uences required for the transcription of the adjacent coding se~uence and includes the S' non-translated :
region or so calied leader sequence of mRNA. The promoters used in DNA ~constructs according to the invention have to be functional in plant cells.
WOg4/03607 212 0 Z 5 PCT/EI93/01 ~
A further aspect of the invention comprises DNA constructs in which the promoter is a tissue specific promoter. A tissue specific promoter directs the transcription of the adjacent sequence in specific tissues of the plants. Suitable promoters for use in DNA constructs according to the invention include but are not limited to~the cauliflower mosaic virus 35S (CaMV
35S) promoter, or potato tuber specific promoters like the promoter of the ~lass I Patatin gene or preferably the granule bound starch synthase gene.
The term terminator refers to a region downstream of the coding sequence which~ directs the termination of the transcription, also call~ed the 3' non-translated region, which includes the poly-adenylation signal. Terminator sequence functional in plants are well known and described in the literature. ~
The DNA sequence ~aocording to the invention comprises at least 25~nucleotides. In~general, a suitable length of the DNA
sequence comprises lOO~to 1000, up to 2000, nucleotides. In a pre~ferred embodiment of~ the invention the DNA sequence ;comprises the entiré~ coding~sequence of the endogenous PP0 gene, or a sequence~ ho:mologous thereto, in inverted orientation.
The DNA sequence~according to the invention can be derived from~genomic DNA~or cDNA~encoding PPO. The preparation of DNA
constructs according to~ the invention is described in more detail below. The invention also provides vectors comprising the DNA construct accordlng to the invention.
The invention also includes plant cells comprising DNA
constructs according to the invention, plants derived therefrom :
and seeds of such plants. Transformed plants can be obtained using standard transformation techniques like Ti-plasmid mediated transformation,~ direct transformation methods as particle bom~ardment and the like. The preferred transformation 2 ~2 02 S ~ ` 6 technique to be used depends on the plant tissue and is known to the person skilled in the art.
The scope of the invention will be illustrated below, employing the construction of bruising resistant potatoes as an example. It is understood that similar approaches, according to the invention, can be used for obtaining other bruising resistant crops.
Browning of potato tubers upon physical damage can be r~duced when potato plants are transformed by co-cultivation with A~robacterium tumefaciens, comprising the DNA construct ac~ording to the i~vention, using internode explants and following standard protocols. In the transformed plants a reduced level of PPO activity has been measured in the tubers, whether a full length or partial sequence has been inserted in the construct in antisense orientation. The use of the tuber specific promoters preceding the DNA sequences according to the invention results in a reduction of PPO expression in the tuber but does not significontly~effect the expression of PPO genes in other tissues of the~plant.
In~order to make~DNA constructs according to the invention which will specifi~al~ly target tuber expression of PPO genes, a tuber cDNA clone bank was made. Screening this bank with a probe corresponding to the se~uence of a leaf PPO gene (Shahar T. ,: Hening N., et al., The Plant Cell, 4, 135-147) or part thereof, resulted in the isolation of several different tuber specific PPO genes. At least fiv~ different genes are expressed in the potato tuber. ~
Further sequence analysis of the different isolated genes revealed that these genes share a nucleotide homology of at least 71,7 %. Comparison of the sequences of the tuber expressed PPO genes and PPO genes expressed in leaves showed that they are diffe~ent from each other. Homology analysis with PPO genes expressed in other organisms like, bacterial, fungal W094/0~07 - 2 1 2 0 2 5 8 PCT/~P93~019~8 -or animal species revealed that.no significant homology could be found. Only conserved regions homo}ogous to ~he Cu-binding sites could be identified. From these observations the conclusion can be drawn that the PPO genes of potato and in particular those expresæed in the potato tuber are distinct from those identified in other species.
For obtaining br~ising resistance the expression of all PPO
genes ~xpressed in the tuber s~ould be substanti~lly reduced.
Depending on the potato cultivar used the expression should be reduced 5 to 50 fold. We have been able to obtain tubers according to the invention in which no PPO enzyme activity could be measured, using detection protocols as described hereafter, indicating that the expression of the occurring PPO
genes has been inhibited in these transgenic plants. Also tubers were obtained with a reduced PPO activity with respect to the activity in the tubers of the non transformed control plants. These tubers were derived from plants transformed with antisens~ constructs acrording to the invention. Consequently the invention provides DNA constructs which causes at least a reduction of the expression of the entire PPO gene family. This multi-gene inhibition can be obtained using one antisense sequence homologous to~any DNA sequence coding for PPQ. We have demonstrated that at least a reduction of PPO expression can be achieved regardless of whether tuber or leaf genes were used for the construction of the antisense PPO constructs. We have also demonstrated that the antisense sequence does ~ot have to correspond to the entire length of the coding sequence but also that partial sequences with a length of about 1200 nucleotides starting from the 5' end and including 800 nucleotides of the coding sequence, in antisense orientation, reduces the expression of the PPO genes in the tuber. Furthermore, the tubers showing a reduced PPo activity are less susceptibie to bruising. In order to determine the extent of browning one can perform standardised bruising tests in which the sensitivity to ~ruising is expressed as a Browning Index. One test is given for instance in the following examples.
WOg4/03607 . - PCT/EP93/019~
~,~ ?,oZ5~ 8 In a preferred embodiment of the invention the DNA sequence is derived from tuber~;PPo genes, and preferably from the PPO
clones isolated from the tuber cDNA clone bank which seguences are li~ted in figure la and lb.
In another preferred embodimeilt of the invention the DNA
sequenc~s in inverted orientation are preceded by a tuber specific transcription initiation region as has been set forth above. Particular criteria for the choice of a prefered tuber specific promoter which directs tuber specific expression may be summarised as follows: Expression of the antisense gene should temporally precede or at least coincide with the expression of the endogenous PPO genes. In a preferred embodiment of the invention expre~sion of the antisense gene should be in the s~me tissue location as the endogenous gene.
Furthermore, levels of expression should be at an appropriate level to obtain the antisense effect.
This invention can:be used to control bruising upon tissue disruption of tuber, vegetable or fruit tissue in which the browning is the result of PPO enzyme activity in or around the damaged plant tissue, due to cell damage. It is understood that the invention is~not restricted to potatoes but that it can also be used for the reduction browning upon bruising in other plants like leaf vegetables, i.e. Iettuce, chicory or-fruits, .
: where the brownin~ is the result of the expression of genes encoding PPO. ~
The invention will now be described further in the examples with reference to the accompanying drawings, in which:
Figure la depicts:the nucleotide sequence of a PPO tuber gene. pKG45-8. This nucleotide sequence is referred to as SEQ ID NO:1.
Insertion sites :
~ site A at the level of nucleotide 1 - site B at the level of nucleotide 1213 W~94/03607 PCT/EP93/019~8 21202~8 - site C at the~level of nucl otide 1833.
Figure lb depicts the nucleotide sequence of another P~O
tuber gene. pK~59 4. This nucleotide cequence is referred to as SEQ ID NO: 2.
Insertion sites :
- site A at the level of nucleotide 1 site E at the ~evel of nucleotide 1173 - site ~ a~ the level of nucleotide 1645A
Figure 2a is a diagra~matic repre~entation of the co~truction of differe~t recombinant vectors.
Figure 2b is a diagrammatic representation of the construction of different reco~binant DNA constructs used for the transformation of potato varieties.
Figure 3 is a ~ar diagram showing the enzyme activities of all plants transformed with antisense PPO constructs in comparison to untransformed controls. The va~iants denoted as 1, 2 and 3 are: transformants comprising CaMV 35S
promoter constructs, trahsformants comprising GBSS
promoter constructs, untransformed control, respectivily.
Figure 4 is a bar diagram showing the bruising indexes of selected plants transformed antisense PPO constructs in comparison to untransformed controls. The varian~s denoted as ~1, 2 and 3 are: transformants comprising CaMV 35S
promoter constructs, transformants comprising GBSS promoter constructs, untransformed control, respectivily.
The following examples illustrate the aspects of the invention, but does not restrict the scope of the invention.
WOg4/03607 PCT/EP93/01gBX
2,120258 ' 10 " "
Exampl~3 Example 1: I~olation, characterisation and cloning of PPO
clones from a tuber specific cDNA library In order to make antisense constructs which will specifically inhibit expression of PPO genes in the tuber; a cDNA clone bank made from tuber RNA as starting material in the vector lambda ZAPII (Stratagene Cloning systems, 11099 North ~orrey Pines Rd, La Jolla, CA 92037). The bank, comprising of 5 x 104 individual plaques, was screened with 32P labelled clone coding for a leaf PPO gene ~Shahar et al. 1992). A total of 16 positively reacting plaques were taken through three rounds of plaque-purification and were chose~ for further analysis '~
Using PCR primers directed to the termini of the lambda Z~PII vec r~cloning sites, the inserts of the 16 clones ware amplified and analyzed for their length and were typed according ~to:their~restriction sites.~ Six inserts were just ovex 2 kb in length,~ making them likely candidates for full length clones, based on work indicating that the PPO protein has a molecular weight of 4S000-59000:.
Example~2: Se ~ ence analysis and detailed characterisation of potato tuber PPO cDNA clones On the basis of the preliminary analysis of the PPO cDNA
clones, two representative cDNA clones, thought to be full-length, were chosen for sequence analysis using standard sequencing protocols ~(pKG45-8 and pKG59-4; see Figure 1).
Substrates for s ouencing were produced using the in vivo excision protocol on lambda ZAPII clones tstratagene). The remaining clones were partially sequenced in order to obtain more information about the number of individual genes being expressed in the tuber. The nucleotide sequence data revealed W0~4/03607 - PCT/EP93/Olg88 two categories of cDNA clones which, although homologous, fell into two distinct classes: Class I clones (pKG45-8; Table 1) and Class II clones (pKG59-4; Table 1). The classification of the clones given in Table 1 is based on sequence homology.
Interestingly th~ class II clones were not similar to any of the potato cDNA clones isolated and are therefore deduced to ~e uniquely expressed in tubers.
: Amongst the 16 clones the most abundant transcript was found to belong to the same gene family ~B-E) of which 10 independent cDNA clones were identified. Within this group, the B gene transcript was found most often (6 independent clones). All other transcripts (A, D and E ) were present in the cDNA bank at lower levels (,see Table 1).
;~ Tahle 1 ~: ~
Genes ~lass I (size in bp) Class II (siz~ in bp) : : A pnG45-5 (~B76 pKG45-8 (185Q)~
~: B _ ~ pKG59-4 ~1900) : pKG45-~ (1850) , ~
: pKG45-4 ~1600) _ . ~
. ~ pKG45-7 (1600) .- . ~
~: _ pKG45-9 (1400) ~:~ pKG59-1 (500) C : pKG45-11 ~1800) pKG45-3 (1500) .
D ~ pKG45-10 (1300 E pKG59-2 (800) - _ _ ,. . _ _ :
Example 3: Construction of a Ti expression-vector family for constitutive and tuber specific expression.
To achieve tuber specific expression the GBSS-G28 (Rohde et W094/03607 . PCT/EP93/01988 2~,~02~ "
al~ 1990. J. Genet. and Breed. 44: 311-315) promoter was chosen. Th~ GBSS promoter~used was i~olated from genomic DNA of the potato variety B~n~e (from seguence data of the genomic clone G28; Rohde, W. et al. 1990.) Fragments containing all sequences necessary to direct tissue specificity were isolated using PCR with standard protocols. Included in the PCR primers were restriction sites to facilitate cloning into the Ti-vect~rs.
The GBSS promoter used (from genomic clone G28; Rohde et al.
1990), contained DNA from -1184 to -8. A HindIII site ~5') and a BamHI site (3') were inserted at the termini by inclusion of the recognition sites in th~e PCR primers. This fragment was inserted into the gel purified Ti-vector (pKG1001; described below) after treatment of both fragment and vector with HindIII
and BamHI.
The CaMV 35S expression vector was constructed from the vector pBI121 (Jefferson et al. 1987. EMB0 J. 6: 3901-3907~.
The modifications;include replacement of the mutant NPTII gene in pBI121 and the deletion of the GUS coding region; the resulting vector ~pKG1001; see Figure 2a) was also the basis for the other expression vectors described below. The tuber specific promoter was inserted into pKG1001 resulting in pKG1001/GBSS containing the GBSS promoter. Figure 2a shows a diagrammatic representation of the construction of Ti-expression vectors used to make the antisense constructs~
Example 4: Cloning of antisense tuber PP0 constructs using the CaMV 35S promoter and tuber specific expression Ti-vectors Antisense constructs were made, using each of the full-length PP0 genes (from p~G45-1 and pKG5~-4). Another set of constructs were made using a shorter region around the translation initiation site (see Figure 1).
W094/03607 2 ~ 2 0 2 5 8 PCT/EP93/01988 As a general strategy for cloning PPQ genes into Ti-vectors, sequence specific PCR primers were designed against the required sites of the PP0 cDNAs ~see sequence, Figure 1, for precise location of sites). Incorporated into these primers were recognition sites for restriction enzymes to be used in the cloning (BamHI and BqlII, 5' and 3' termini respectively).
Tuber PP0 sections from pKG59-4 and pKG45-8 were inserted into the expression~ vectors described (pKGlOOl and pKGlOOl/GBSS). In these~experiments both the 5' segment and the full length sections from the two cDNAs were used (See sequence, Figure 1, for precise location of sites). Figure 4b shows a diagrammatic representation of the construction of the antisense PP0 constructs. The names and description of the 12 resulting antisense PPO clones is shown in Table 2. The table lists the promoters used and the antisense genes inserted.
Where 5' denotes the use of the fixst approximately 1200 nucIeotides of the corresponding c~NA clone.
:.:
~ :, .
Table 2 ~ ~
conse:~ct Rrn ~ter Gene pKG60 ~ -CaMV 35S~ -;p~G45-8 KG6l ~ p-CaMV 35S~ - DKG45-8/5' pKG62 - ~ ~-CaMV 35S ~- pKG5s-4 KG63 ~ P-CaMV 35S - DKG59-4/5' pKG70 ~ p-GBSS - ~KG45-8 PKG7l~ ~P-GBSS - ~KG45-8/5' pKG72 p-GBSS - ~KG59-4 pKG73 p-GBSS - pKG59-4/5' Example 5: Transformation of antisense PP0 constructs into potato plants All potato PP0 constructs were introduced into Aqrobac$erium tumefaciens strain GV3lOl via electroporation and their integrity was rechecked by restriction enzyme analysis.
W094/0~07 PCT/EPg3/01988 Subsequently, the descri~ed constructs were introduced into two lines of potato (van Gogh and Diamant) by co-cultivation using internode explants. Standard protocols were used in these procedures. Circa 50 regenerants per construct and cultivar were grown on selective media and analyzed further.
Example 6: Analysis of PPO enzyme activities in transformed tubers From a total of 1500 regenerants 882 were propagated and used to produce in vitro micro-tubers. From about 5 g of micro-tubers of each line a crude protein extract was produced with which PPO enzyme assays were performed (after the method of Flurkey, W. 1986 Plant Physiol. 8~: 614-618). Two assays were done on ea?~h transgenic line using 50 ~ catechol as a substratc for the assay in an ex~ract volume of 1 ml. Enzyme activity is expressed as the rate of change of OD at 520 nm over 1 min at 25C (slope~. Two independent repeat measurements were performed on each line and thè mean of the results were taken for a~alysis. On average 74% and 72% of antisense transformants in Diamant and van Gogh, respectively, gave lower PPO enzyme activity than the controls. 32 lines, harb~uring the antisense PPO constructs, were found to ha~e no detectable PPO
activity with this substrate. In Diamant: 7 lines with the 35S
CaMV-promoter constructs and 10 lines with the GB5S- promoter constructs showed no PPO activity. In van Gogh: 10 lines with the 35S ca?Mv- promoter constructs and 5 lines with the GBSS-promoter constructs had undetectable PPO activity levels.
No statistically significant differences were found between the different antisense PPo genes (Class I and II~ or with respect to the size of the PPo gene-sections used in the constructs.
As shown in Figure 3, both potato varieties used showed reduced means in enzyme activity in all cases where either the 35S CaMV or GBSS promoters were used. The highest average W094~03607 . . ~ 2 ~ z 5 ~ PCT/EP93/01988 suppression of PPO activity was found in lines which were transformed with constructs under CaMV 35S promoter control.
Example 7: Molecular analysis of transformants 200 of the low PPO activity potato lines were further analyzed on a molecular level. Using the Southern hybridization technique it was shown that aI1 contained T-DNA inserts of the expected sizesO Insertion copy-number ranged from 1 to l0 where 50~ had copy numbers < 3. Also PPO mRNA levels were shown to be reduced when compared to untransformed controls. Assays fsr PPO
~RNA were performed using a PCR protocol.
Example 8: Analysis of ~ruising resistance in the transformants On the basis of the data from the enzyme activity, 50 lines from each variety, including all variants of antisense-PPO
constructions r were selectQd for field trails. The selected lines represent plants with the lowest PPO enzyme activities.
In conventional breeding practice, standardised tests are carried out to determine the~ex~ent of browning in tubers from breeding lines. An index is calculated for bruising sensitivity which takes into account the level of tuber browning after subjecting the tubers o standardised mechanical damage and the specific gravity of the tubers in the test~ The resultinq Browning Index tBI) ranges from 0 to 50.
BI's of field grown tubers from the selected lines are shown in Figure 4. Statistical tests show significant differences in : the CaMV 35s promoter series and the GBSS promoter series for both varieties when compared to the mean of the controls.
The data from these trials clearly demonstrates a significant improvement of the bruising phenotype under field WOg4/03607 PCT/EP93/01988 ~o ~5~ 16 conditions as ~ direct result of the expression of the constructs provided by the invention.
Mat~ri~l a~ M~tho~
Potato plants (Solanum Tuborosum cv. van Gogh and Diamant) were grown ln ~ on MS medium (Murishige, T. and Skovg, F.
1962. Physiol. Plant 15: 473 - 497) suppleme~ted with 30g/1 sucrose.
Potato internode explants were transformed with Aqrobacterium tumefaciens (~strain GV3101; van Larebeke, N. et al. 1974. Nature 2S2: 169 - 170) containing the antisense-PPO
Ti-plasmid constructs using the co-cultivation method essentially according to protocols described (Ooms, G. 1989. et al. Theor. Appl.:Genet 73: 744 -750).
Molecular biological methods, including Southern, Northern and Western analysis, PCR and other DNA manipulations were as des*ribed by Maniatis ~:(~aniatis, et al. 1982. Molecular Cloning: A Laboratory Manual. Cold Spri~g Harbor Laboratory, Cold Spring Harbor NY.).~:
Substrates for sequencing were produced using the in vivo excision protocol on lambda ZAPII clones (Stratagene3 isolated from a sink tuber cDNA.
The bruising test and the determination of the Browning Xndex were conducted as follows: Potatoes harvested from each line, grown in separate plots, were subjected to bruising under standard conditions. 2 - 3 kg of potatoes were placed in a shaking device comprising of a wooden box with padded walls.
The box, is mechanically ag_~ated for 2 mins. After the bruising procedure, tubers are stored for 4 days at 10C ~+
1C). Subsequently the potatoes are mecl~anically peeled until 80% of the skin is removed and the degree of browning is scored W094/03607 . 21 2 0 2 ~ 8 PCT/EP93/01~88 in terms of percent of the surface area effected by discolouration. The percentages are categorised into four classes and the number of tubers in ea~h class, together with the specific gravity of the tubers is entered into the following formula from which the BI is determined:
SG x (CI+l) x (CII+2) x (CIII+3) x (CIV+4) 6 x ~CI+CII+CIII+CIV) Where SG is the specific gravity of the whole sample and CI
- CIV are the number of tubers categorised in a given class.
" ~
;~ .
~ ~ .
:~ ' ' `'.
800p~ o~ the i~tio~
The invention relates to novel DNA constructs~ plant cells co~taining these constructs and plants derived there~rom, as well as æeeds obtained ~rom said plants. In particular it invol~es the use of antisen~e nucleic acid technology to control gene expression in plants7 Using tissue specific promoters the control of gene expression can ~e directed to particular issues of the plant. The invention provides further, a process for obtaining plants which do not exhibit enz~matic browning of disrupted tissues.
IntroZuation Browning and discolouration causes substantial losses in a wide range of fresh and pro¢essed fruits and vegetables. In potato~s, internal bruising, also called black spot, is a physiological response of potato tubers to tissue compaction and i~ o~e of the most important problems of the potato industr~. Yearly, large amounts of the potato produc~ion are valueless for direct consumption or processing due to browning of the tubers.
i The biochemistry of browning is relatively well unders~ood in mammals and bacterial model systems. In plants, however very little is known about the biochemical processes involved in browning and the pathways responsible for discolouration in a wide variety of fruits and vegetables has not been elucidated.
In potatoes, bruising or black spot results from minor physical damage sustained by the tubers during mechanical harvest and wo 94/036~ 1 2 0 2 5 8 PCT/EP93~01988 transportation. The colour reaction results from a small number of cells within the bruise that ~ctually lyse. The grey/black colour of the bruise i5 caused by the enzymatic production of melanin, a highly polymerised phenolic compound. The enzyme polyphenol oxidase (PPO), an oxygenase, catalyses the first two oxidation reactions of the pathway and converts monophenols ~such as tyrosine) and ortho-diphenols (such as catechol) to ortho-~uinones (such as dihydroxiphenylalanine-quinone).
Autocatalytic chemical oxidation reactions conv~rt ortho-quinones into ~elanin via several reactive quinone intermediates. Although the function of plant polyphenol oxidases has not been elucidated, several possible roles of the enzyme have been postulated. These include participation in electron transport in photosynthesis and participation in the defence mechanisms of the plant against insect attack or infections. It is believed that PPO is not involved in the synthesis of phenolic compounds in healthy, intact cells. Plant PPo enzymes are nuclear-encoded copper metalloproteins localised in me~branes of plastids. In potato tubers, PPO has been localised in the rudimentary thylacoid me~branes of amyloplasts. The sub-cellular location separates the enzyme from its substrate which is primarily found in the cell vacuole. However, in disrupted cells the enzyme is mixed with substrate leading to oxidation of phenolic compounds by PPO
enzyme.
: : :
Although bruising~has been a severe pro~lem in the potato industry for many years, no progress has been made until now to prevent the formation of black spots into potato tubers through molecular technique~. The term bruising resistance, as used hereaft2r, refers to resistance against browning and discolouration upon disruption of plant cells.
We have found that bruising resistance can be obtained through interfering in the biosynthetic pathway of melanin production by the expression of an antisense PPO gene. More particularly, the production of polyphenol oxidase can be W094/0~7 ~ 2 ~ 2 0 2 5 8 PCT/EPg3/01988 reduced in specific tissues through tissue specific expres~ion of said antisense PPO gene. Since the expression of anti~nse PPO may perturb one or more of the postulated functions of PPO, especially in aerial parts of the plant, limitation of expression to tubers, by implementation of appropriate tuber specific promoters, is preferred. Furthermore, we have found that polyphenol oxidases are encoded by a family of genes comprising at least S members. We have been able to suppress the expression of genes, and particularly of all genes, expressing in tubers through the coordinate expression of only one antisense PPO gene corresponding to part or entire sequence, or homologous with one of the genes encoding polyphenol oxidase. Moreover, we have been able to produce bruising resistant plants which show a reduced sensitivity to bruising, especially potato plants of which the tubers show a reduced sensitivity to bruising.
The invention is based on the application of antisense nucleic acid technology. Although the mechanism of antisense is ~not elucidated, it~is believed that the inhibition of the expression of the target gene is obtained either through the formation of a complex of the antisense RNA and the endogenous RNA which are complementary, preventing the translation, or through~ the formation of a complex structure with the DNA
sequence, preventing the transcription.
The invention provides~a method for producing bruising resistant plants showing a reduced PPO enzyme activity and which also have been shown to have a significantly reduced ~ browning phenotype in the field.
; The invention can be used to control bruising upon disruption of tuber, vegetable, or fruit tissues in which the browning is the resuIt of PPO enzyme activity in or around the damaged plant tissue. It is understood that the invention is not restricted to potatoes but that it can also be used for the reduction of browning upon physical tissue disruption in other WOg4/03607 ~ PCT/EP93/019BX
~,120Z58 ,.1~, plants like leaf vegetables, i.e. lettuce, chicory or fruits, where ~he browning is ~he result of the expression of genes encoding PPO.
Det~iled ~es~riptio~ of the invention The present invention provides DNA constructs comprising i~
a promoter fu~ctional in plants, ii) a DNA se~uence corresponding to an antisense polyphenol oxidase ~ene or part thereof, or a DNA sequence homologous thereto and iii) a terminator functional in plants.
The term antisense polyphenol oxidase gene refers to a double stranded DNA sequence which sequence corresponds to or is homologous with the endogenous gene encoding PFO or part thereof, but which has a reversed orientation with respect to the said PPO ge~e. In a antisense DNA sequence the coding strand becomes the~template strand and vice versa.
The term homologous sequence refers to a sequence in which the aver~age nucleotide sequence homology with the contiguous gene is~ at least 65 %. ~ ~
The term DNA sequence refers to a double stranded DNA
sequence which can be~expressed in piant cells if said seguence is under:the expression control of a promoter functional in plants and having a terminator functional in plants.
The term promoter used herein refers to a transcription regulation region upstream from the coding sequence containing the regulatory se~uences required for the transcription of the adjacent coding se~uence and includes the S' non-translated :
region or so calied leader sequence of mRNA. The promoters used in DNA ~constructs according to the invention have to be functional in plant cells.
WOg4/03607 212 0 Z 5 PCT/EI93/01 ~
A further aspect of the invention comprises DNA constructs in which the promoter is a tissue specific promoter. A tissue specific promoter directs the transcription of the adjacent sequence in specific tissues of the plants. Suitable promoters for use in DNA constructs according to the invention include but are not limited to~the cauliflower mosaic virus 35S (CaMV
35S) promoter, or potato tuber specific promoters like the promoter of the ~lass I Patatin gene or preferably the granule bound starch synthase gene.
The term terminator refers to a region downstream of the coding sequence which~ directs the termination of the transcription, also call~ed the 3' non-translated region, which includes the poly-adenylation signal. Terminator sequence functional in plants are well known and described in the literature. ~
The DNA sequence ~aocording to the invention comprises at least 25~nucleotides. In~general, a suitable length of the DNA
sequence comprises lOO~to 1000, up to 2000, nucleotides. In a pre~ferred embodiment of~ the invention the DNA sequence ;comprises the entiré~ coding~sequence of the endogenous PP0 gene, or a sequence~ ho:mologous thereto, in inverted orientation.
The DNA sequence~according to the invention can be derived from~genomic DNA~or cDNA~encoding PPO. The preparation of DNA
constructs according to~ the invention is described in more detail below. The invention also provides vectors comprising the DNA construct accordlng to the invention.
The invention also includes plant cells comprising DNA
constructs according to the invention, plants derived therefrom :
and seeds of such plants. Transformed plants can be obtained using standard transformation techniques like Ti-plasmid mediated transformation,~ direct transformation methods as particle bom~ardment and the like. The preferred transformation 2 ~2 02 S ~ ` 6 technique to be used depends on the plant tissue and is known to the person skilled in the art.
The scope of the invention will be illustrated below, employing the construction of bruising resistant potatoes as an example. It is understood that similar approaches, according to the invention, can be used for obtaining other bruising resistant crops.
Browning of potato tubers upon physical damage can be r~duced when potato plants are transformed by co-cultivation with A~robacterium tumefaciens, comprising the DNA construct ac~ording to the i~vention, using internode explants and following standard protocols. In the transformed plants a reduced level of PPO activity has been measured in the tubers, whether a full length or partial sequence has been inserted in the construct in antisense orientation. The use of the tuber specific promoters preceding the DNA sequences according to the invention results in a reduction of PPO expression in the tuber but does not significontly~effect the expression of PPO genes in other tissues of the~plant.
In~order to make~DNA constructs according to the invention which will specifi~al~ly target tuber expression of PPO genes, a tuber cDNA clone bank was made. Screening this bank with a probe corresponding to the se~uence of a leaf PPO gene (Shahar T. ,: Hening N., et al., The Plant Cell, 4, 135-147) or part thereof, resulted in the isolation of several different tuber specific PPO genes. At least fiv~ different genes are expressed in the potato tuber. ~
Further sequence analysis of the different isolated genes revealed that these genes share a nucleotide homology of at least 71,7 %. Comparison of the sequences of the tuber expressed PPO genes and PPO genes expressed in leaves showed that they are diffe~ent from each other. Homology analysis with PPO genes expressed in other organisms like, bacterial, fungal W094/0~07 - 2 1 2 0 2 5 8 PCT/~P93~019~8 -or animal species revealed that.no significant homology could be found. Only conserved regions homo}ogous to ~he Cu-binding sites could be identified. From these observations the conclusion can be drawn that the PPO genes of potato and in particular those expresæed in the potato tuber are distinct from those identified in other species.
For obtaining br~ising resistance the expression of all PPO
genes ~xpressed in the tuber s~ould be substanti~lly reduced.
Depending on the potato cultivar used the expression should be reduced 5 to 50 fold. We have been able to obtain tubers according to the invention in which no PPO enzyme activity could be measured, using detection protocols as described hereafter, indicating that the expression of the occurring PPO
genes has been inhibited in these transgenic plants. Also tubers were obtained with a reduced PPO activity with respect to the activity in the tubers of the non transformed control plants. These tubers were derived from plants transformed with antisens~ constructs acrording to the invention. Consequently the invention provides DNA constructs which causes at least a reduction of the expression of the entire PPO gene family. This multi-gene inhibition can be obtained using one antisense sequence homologous to~any DNA sequence coding for PPQ. We have demonstrated that at least a reduction of PPO expression can be achieved regardless of whether tuber or leaf genes were used for the construction of the antisense PPO constructs. We have also demonstrated that the antisense sequence does ~ot have to correspond to the entire length of the coding sequence but also that partial sequences with a length of about 1200 nucleotides starting from the 5' end and including 800 nucleotides of the coding sequence, in antisense orientation, reduces the expression of the PPO genes in the tuber. Furthermore, the tubers showing a reduced PPo activity are less susceptibie to bruising. In order to determine the extent of browning one can perform standardised bruising tests in which the sensitivity to ~ruising is expressed as a Browning Index. One test is given for instance in the following examples.
WOg4/03607 . - PCT/EP93/019~
~,~ ?,oZ5~ 8 In a preferred embodiment of the invention the DNA sequence is derived from tuber~;PPo genes, and preferably from the PPO
clones isolated from the tuber cDNA clone bank which seguences are li~ted in figure la and lb.
In another preferred embodimeilt of the invention the DNA
sequenc~s in inverted orientation are preceded by a tuber specific transcription initiation region as has been set forth above. Particular criteria for the choice of a prefered tuber specific promoter which directs tuber specific expression may be summarised as follows: Expression of the antisense gene should temporally precede or at least coincide with the expression of the endogenous PPO genes. In a preferred embodiment of the invention expre~sion of the antisense gene should be in the s~me tissue location as the endogenous gene.
Furthermore, levels of expression should be at an appropriate level to obtain the antisense effect.
This invention can:be used to control bruising upon tissue disruption of tuber, vegetable or fruit tissue in which the browning is the result of PPO enzyme activity in or around the damaged plant tissue, due to cell damage. It is understood that the invention is~not restricted to potatoes but that it can also be used for the reduction browning upon bruising in other plants like leaf vegetables, i.e. Iettuce, chicory or-fruits, .
: where the brownin~ is the result of the expression of genes encoding PPO. ~
The invention will now be described further in the examples with reference to the accompanying drawings, in which:
Figure la depicts:the nucleotide sequence of a PPO tuber gene. pKG45-8. This nucleotide sequence is referred to as SEQ ID NO:1.
Insertion sites :
~ site A at the level of nucleotide 1 - site B at the level of nucleotide 1213 W~94/03607 PCT/EP93/019~8 21202~8 - site C at the~level of nucl otide 1833.
Figure lb depicts the nucleotide sequence of another P~O
tuber gene. pK~59 4. This nucleotide cequence is referred to as SEQ ID NO: 2.
Insertion sites :
- site A at the level of nucleotide 1 site E at the ~evel of nucleotide 1173 - site ~ a~ the level of nucleotide 1645A
Figure 2a is a diagra~matic repre~entation of the co~truction of differe~t recombinant vectors.
Figure 2b is a diagrammatic representation of the construction of different reco~binant DNA constructs used for the transformation of potato varieties.
Figure 3 is a ~ar diagram showing the enzyme activities of all plants transformed with antisense PPO constructs in comparison to untransformed controls. The va~iants denoted as 1, 2 and 3 are: transformants comprising CaMV 35S
promoter constructs, trahsformants comprising GBSS
promoter constructs, untransformed control, respectivily.
Figure 4 is a bar diagram showing the bruising indexes of selected plants transformed antisense PPO constructs in comparison to untransformed controls. The varian~s denoted as ~1, 2 and 3 are: transformants comprising CaMV 35S
promoter constructs, transformants comprising GBSS promoter constructs, untransformed control, respectivily.
The following examples illustrate the aspects of the invention, but does not restrict the scope of the invention.
WOg4/03607 PCT/EP93/01gBX
2,120258 ' 10 " "
Exampl~3 Example 1: I~olation, characterisation and cloning of PPO
clones from a tuber specific cDNA library In order to make antisense constructs which will specifically inhibit expression of PPO genes in the tuber; a cDNA clone bank made from tuber RNA as starting material in the vector lambda ZAPII (Stratagene Cloning systems, 11099 North ~orrey Pines Rd, La Jolla, CA 92037). The bank, comprising of 5 x 104 individual plaques, was screened with 32P labelled clone coding for a leaf PPO gene ~Shahar et al. 1992). A total of 16 positively reacting plaques were taken through three rounds of plaque-purification and were chose~ for further analysis '~
Using PCR primers directed to the termini of the lambda Z~PII vec r~cloning sites, the inserts of the 16 clones ware amplified and analyzed for their length and were typed according ~to:their~restriction sites.~ Six inserts were just ovex 2 kb in length,~ making them likely candidates for full length clones, based on work indicating that the PPO protein has a molecular weight of 4S000-59000:.
Example~2: Se ~ ence analysis and detailed characterisation of potato tuber PPO cDNA clones On the basis of the preliminary analysis of the PPO cDNA
clones, two representative cDNA clones, thought to be full-length, were chosen for sequence analysis using standard sequencing protocols ~(pKG45-8 and pKG59-4; see Figure 1).
Substrates for s ouencing were produced using the in vivo excision protocol on lambda ZAPII clones tstratagene). The remaining clones were partially sequenced in order to obtain more information about the number of individual genes being expressed in the tuber. The nucleotide sequence data revealed W0~4/03607 - PCT/EP93/Olg88 two categories of cDNA clones which, although homologous, fell into two distinct classes: Class I clones (pKG45-8; Table 1) and Class II clones (pKG59-4; Table 1). The classification of the clones given in Table 1 is based on sequence homology.
Interestingly th~ class II clones were not similar to any of the potato cDNA clones isolated and are therefore deduced to ~e uniquely expressed in tubers.
: Amongst the 16 clones the most abundant transcript was found to belong to the same gene family ~B-E) of which 10 independent cDNA clones were identified. Within this group, the B gene transcript was found most often (6 independent clones). All other transcripts (A, D and E ) were present in the cDNA bank at lower levels (,see Table 1).
;~ Tahle 1 ~: ~
Genes ~lass I (size in bp) Class II (siz~ in bp) : : A pnG45-5 (~B76 pKG45-8 (185Q)~
~: B _ ~ pKG59-4 ~1900) : pKG45-~ (1850) , ~
: pKG45-4 ~1600) _ . ~
. ~ pKG45-7 (1600) .- . ~
~: _ pKG45-9 (1400) ~:~ pKG59-1 (500) C : pKG45-11 ~1800) pKG45-3 (1500) .
D ~ pKG45-10 (1300 E pKG59-2 (800) - _ _ ,. . _ _ :
Example 3: Construction of a Ti expression-vector family for constitutive and tuber specific expression.
To achieve tuber specific expression the GBSS-G28 (Rohde et W094/03607 . PCT/EP93/01988 2~,~02~ "
al~ 1990. J. Genet. and Breed. 44: 311-315) promoter was chosen. Th~ GBSS promoter~used was i~olated from genomic DNA of the potato variety B~n~e (from seguence data of the genomic clone G28; Rohde, W. et al. 1990.) Fragments containing all sequences necessary to direct tissue specificity were isolated using PCR with standard protocols. Included in the PCR primers were restriction sites to facilitate cloning into the Ti-vect~rs.
The GBSS promoter used (from genomic clone G28; Rohde et al.
1990), contained DNA from -1184 to -8. A HindIII site ~5') and a BamHI site (3') were inserted at the termini by inclusion of the recognition sites in th~e PCR primers. This fragment was inserted into the gel purified Ti-vector (pKG1001; described below) after treatment of both fragment and vector with HindIII
and BamHI.
The CaMV 35S expression vector was constructed from the vector pBI121 (Jefferson et al. 1987. EMB0 J. 6: 3901-3907~.
The modifications;include replacement of the mutant NPTII gene in pBI121 and the deletion of the GUS coding region; the resulting vector ~pKG1001; see Figure 2a) was also the basis for the other expression vectors described below. The tuber specific promoter was inserted into pKG1001 resulting in pKG1001/GBSS containing the GBSS promoter. Figure 2a shows a diagrammatic representation of the construction of Ti-expression vectors used to make the antisense constructs~
Example 4: Cloning of antisense tuber PP0 constructs using the CaMV 35S promoter and tuber specific expression Ti-vectors Antisense constructs were made, using each of the full-length PP0 genes (from p~G45-1 and pKG5~-4). Another set of constructs were made using a shorter region around the translation initiation site (see Figure 1).
W094/03607 2 ~ 2 0 2 5 8 PCT/EP93/01988 As a general strategy for cloning PPQ genes into Ti-vectors, sequence specific PCR primers were designed against the required sites of the PP0 cDNAs ~see sequence, Figure 1, for precise location of sites). Incorporated into these primers were recognition sites for restriction enzymes to be used in the cloning (BamHI and BqlII, 5' and 3' termini respectively).
Tuber PP0 sections from pKG59-4 and pKG45-8 were inserted into the expression~ vectors described (pKGlOOl and pKGlOOl/GBSS). In these~experiments both the 5' segment and the full length sections from the two cDNAs were used (See sequence, Figure 1, for precise location of sites). Figure 4b shows a diagrammatic representation of the construction of the antisense PP0 constructs. The names and description of the 12 resulting antisense PPO clones is shown in Table 2. The table lists the promoters used and the antisense genes inserted.
Where 5' denotes the use of the fixst approximately 1200 nucIeotides of the corresponding c~NA clone.
:.:
~ :, .
Table 2 ~ ~
conse:~ct Rrn ~ter Gene pKG60 ~ -CaMV 35S~ -;p~G45-8 KG6l ~ p-CaMV 35S~ - DKG45-8/5' pKG62 - ~ ~-CaMV 35S ~- pKG5s-4 KG63 ~ P-CaMV 35S - DKG59-4/5' pKG70 ~ p-GBSS - ~KG45-8 PKG7l~ ~P-GBSS - ~KG45-8/5' pKG72 p-GBSS - ~KG59-4 pKG73 p-GBSS - pKG59-4/5' Example 5: Transformation of antisense PP0 constructs into potato plants All potato PP0 constructs were introduced into Aqrobac$erium tumefaciens strain GV3lOl via electroporation and their integrity was rechecked by restriction enzyme analysis.
W094/0~07 PCT/EPg3/01988 Subsequently, the descri~ed constructs were introduced into two lines of potato (van Gogh and Diamant) by co-cultivation using internode explants. Standard protocols were used in these procedures. Circa 50 regenerants per construct and cultivar were grown on selective media and analyzed further.
Example 6: Analysis of PPO enzyme activities in transformed tubers From a total of 1500 regenerants 882 were propagated and used to produce in vitro micro-tubers. From about 5 g of micro-tubers of each line a crude protein extract was produced with which PPO enzyme assays were performed (after the method of Flurkey, W. 1986 Plant Physiol. 8~: 614-618). Two assays were done on ea?~h transgenic line using 50 ~ catechol as a substratc for the assay in an ex~ract volume of 1 ml. Enzyme activity is expressed as the rate of change of OD at 520 nm over 1 min at 25C (slope~. Two independent repeat measurements were performed on each line and thè mean of the results were taken for a~alysis. On average 74% and 72% of antisense transformants in Diamant and van Gogh, respectively, gave lower PPO enzyme activity than the controls. 32 lines, harb~uring the antisense PPO constructs, were found to ha~e no detectable PPO
activity with this substrate. In Diamant: 7 lines with the 35S
CaMV-promoter constructs and 10 lines with the GB5S- promoter constructs showed no PPO activity. In van Gogh: 10 lines with the 35S ca?Mv- promoter constructs and 5 lines with the GBSS-promoter constructs had undetectable PPO activity levels.
No statistically significant differences were found between the different antisense PPo genes (Class I and II~ or with respect to the size of the PPo gene-sections used in the constructs.
As shown in Figure 3, both potato varieties used showed reduced means in enzyme activity in all cases where either the 35S CaMV or GBSS promoters were used. The highest average W094~03607 . . ~ 2 ~ z 5 ~ PCT/EP93/01988 suppression of PPO activity was found in lines which were transformed with constructs under CaMV 35S promoter control.
Example 7: Molecular analysis of transformants 200 of the low PPO activity potato lines were further analyzed on a molecular level. Using the Southern hybridization technique it was shown that aI1 contained T-DNA inserts of the expected sizesO Insertion copy-number ranged from 1 to l0 where 50~ had copy numbers < 3. Also PPO mRNA levels were shown to be reduced when compared to untransformed controls. Assays fsr PPO
~RNA were performed using a PCR protocol.
Example 8: Analysis of ~ruising resistance in the transformants On the basis of the data from the enzyme activity, 50 lines from each variety, including all variants of antisense-PPO
constructions r were selectQd for field trails. The selected lines represent plants with the lowest PPO enzyme activities.
In conventional breeding practice, standardised tests are carried out to determine the~ex~ent of browning in tubers from breeding lines. An index is calculated for bruising sensitivity which takes into account the level of tuber browning after subjecting the tubers o standardised mechanical damage and the specific gravity of the tubers in the test~ The resultinq Browning Index tBI) ranges from 0 to 50.
BI's of field grown tubers from the selected lines are shown in Figure 4. Statistical tests show significant differences in : the CaMV 35s promoter series and the GBSS promoter series for both varieties when compared to the mean of the controls.
The data from these trials clearly demonstrates a significant improvement of the bruising phenotype under field WOg4/03607 PCT/EP93/01988 ~o ~5~ 16 conditions as ~ direct result of the expression of the constructs provided by the invention.
Mat~ri~l a~ M~tho~
Potato plants (Solanum Tuborosum cv. van Gogh and Diamant) were grown ln ~ on MS medium (Murishige, T. and Skovg, F.
1962. Physiol. Plant 15: 473 - 497) suppleme~ted with 30g/1 sucrose.
Potato internode explants were transformed with Aqrobacterium tumefaciens (~strain GV3101; van Larebeke, N. et al. 1974. Nature 2S2: 169 - 170) containing the antisense-PPO
Ti-plasmid constructs using the co-cultivation method essentially according to protocols described (Ooms, G. 1989. et al. Theor. Appl.:Genet 73: 744 -750).
Molecular biological methods, including Southern, Northern and Western analysis, PCR and other DNA manipulations were as des*ribed by Maniatis ~:(~aniatis, et al. 1982. Molecular Cloning: A Laboratory Manual. Cold Spri~g Harbor Laboratory, Cold Spring Harbor NY.).~:
Substrates for sequencing were produced using the in vivo excision protocol on lambda ZAPII clones (Stratagene3 isolated from a sink tuber cDNA.
The bruising test and the determination of the Browning Xndex were conducted as follows: Potatoes harvested from each line, grown in separate plots, were subjected to bruising under standard conditions. 2 - 3 kg of potatoes were placed in a shaking device comprising of a wooden box with padded walls.
The box, is mechanically ag_~ated for 2 mins. After the bruising procedure, tubers are stored for 4 days at 10C ~+
1C). Subsequently the potatoes are mecl~anically peeled until 80% of the skin is removed and the degree of browning is scored W094/03607 . 21 2 0 2 ~ 8 PCT/EP93/01~88 in terms of percent of the surface area effected by discolouration. The percentages are categorised into four classes and the number of tubers in ea~h class, together with the specific gravity of the tubers is entered into the following formula from which the BI is determined:
SG x (CI+l) x (CII+2) x (CIII+3) x (CIV+4) 6 x ~CI+CII+CIII+CIV) Where SG is the specific gravity of the whole sample and CI
- CIV are the number of tubers categorised in a given class.
" ~
;~ .
~ ~ .
:~ ' ' `'.
Claims (20)
1. DNA construct comprising i) a promoter functional in plants, ii) a DNA sequence corresponding to an antisense polyphenol oxidase gene or part thereof, or a DNA sequence homologous thereto and iii) a terminator functional in plants.
2. DNA construct according to claim 1 in which said promoter functional in plants is a tissue specific promoter.
3. DNA construct according to claim 2 in which said tissue specific promoter is a tuber specific promoter.
4. DNA construct according to claim 3 in which said tuber specific promoter corresponds to the promoter of the granule bound starch synthase gene.
5. DNA construct according to any of claims 1 to 4 in which said DNA sequence comprises at least 25 nucleotides.
6. DNA construct according to any of claims 1 to 5 in which said DNA sequence comprises the full length coding sequence of the polyphenol oxidase gene in antisense orientation.
7. DNA construct according to any of claims 1 to 6 in which said DNA sequence corresponds to an antisense polyphenol oxidase gene endogenous in potato plant cells or part thereof, or a DNA sequence homologous thereto.
8. DNA construct according to claim 7 in which said DNA
sequence corresponds to an antisense polyphenol oxidase gene endogenous in potato tubers cells or part thereof, or a DNA
sequence homologous thereto.
sequence corresponds to an antisense polyphenol oxidase gene endogenous in potato tubers cells or part thereof, or a DNA
sequence homologous thereto.
9. DNA construct according to any of claims 1 to 8 in which the DNA sequence is derived from a gene coding for expression into polyphenol oxidase endogenous in plant cells.
10. DNA construct according to claim 9 in which the DNA
sequence is derived from cDNA corresponding to a gene coding for expression into polyphenol oxidase endogenous in potato tuber cells.
sequence is derived from cDNA corresponding to a gene coding for expression into polyphenol oxidase endogenous in potato tuber cells.
11. DNA construct according to claim 10 in which the DNA
sequence is derived from the sequence as listed in figures 1a or 1b.
sequence is derived from the sequence as listed in figures 1a or 1b.
12. Vector comprising a DNA construct according to any one of the preceding claims.
13. Bacterial cells comprising a vector according to claim 10.
14. Plant genome comprising a DNA construct according to any of claims 1 to 11.
.
.
15. Plant cells comprising a DNA construct according to any of claims 1 to 11.
16. Plant comprising plant cells according to claims 15.
17. Plant according to claim 16 in which the plant is a potato.
18. Seeds comprising a DNA construct according to any of claims 1 to 11.
19. Potato tubers comprising a DNA construct according to any of claims 1 to 11.
20. Process for obtaining plants having a reduced polyphenol oxidase activity, comprising the following steps: 1) inserting into the genome of plant cell comprising a DNA construct according to any of claims 1 to 11, 2) obtaining transformed plant cells, 3) regenerating from said transformed plant cells genetically transformed plants.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP92402199.1 | 1992-07-30 | ||
| EP92402199 | 1992-07-30 |
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| CA2120258A1 true CA2120258A1 (en) | 1994-02-17 |
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| CA 2120258 Abandoned CA2120258A1 (en) | 1992-07-30 | 1993-07-27 | Dna constructs, cells and plants derived therefrom |
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| EP (1) | EP0606454A1 (en) |
| JP (1) | JPH07503376A (en) |
| CA (1) | CA2120258A1 (en) |
| WO (1) | WO1994003607A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993015599A1 (en) * | 1992-01-31 | 1993-08-19 | Cornell Research Foundation, Inc. | Polyphenol oxidase |
| US6160204A (en) * | 1992-01-31 | 2000-12-12 | Cornell Research Foundation, Inc. | Polyphenol oxidase cDNA |
| EP0832244A4 (en) * | 1995-05-23 | 2001-10-17 | Commw Scient And Br Ind Res Or | Polyphenol oxidase genes from lettuce and banana |
| US6627794B1 (en) | 1995-05-23 | 2003-09-30 | Commonwealth Scientific And Industrial Research Organisation | Polyphenyl oxidase genes from banana |
| EP1015598A4 (en) * | 1996-02-05 | 2004-05-19 | Commw Scient Andindustrial Res | Genomic ppo clones |
| EP1555317B1 (en) | 1998-03-20 | 2011-09-28 | Commonwealth Scientific And Industrial Research Organisation | Synthetic genes and genetic constructs comprising the same |
| AUPP249298A0 (en) | 1998-03-20 | 1998-04-23 | Ag-Gene Australia Limited | Synthetic genes and genetic constructs comprising same I |
| EP2267139B1 (en) | 1998-04-08 | 2017-03-22 | Commonwealth Scientific and Industrial Research Organisation | Methods and means for obtaining modified phenotypes |
| US6423885B1 (en) | 1999-08-13 | 2002-07-23 | Commonwealth Scientific And Industrial Research Organization (Csiro) | Methods for obtaining modified phenotypes in plant cells |
| US7122719B2 (en) | 2000-11-03 | 2006-10-17 | Monsanto Technology Llc | Method of imparting disease resistance to plants by reducing polyphenol oxidase activities |
| AUPR621501A0 (en) | 2001-07-06 | 2001-08-02 | Commonwealth Scientific And Industrial Research Organisation | Delivery of ds rna |
| CA2477240C (en) | 2002-02-20 | 2012-09-25 | J.R. Simplot Company | Decreased reducing sugars in plants for the reduction of acrylamide production in heated foods |
| US7534934B2 (en) | 2002-02-20 | 2009-05-19 | J.R. Simplot Company | Precise breeding |
| DE10212892A1 (en) | 2002-03-20 | 2003-10-09 | Basf Plant Science Gmbh | Constructs and methods for regulating gene expression |
| EP2980220A1 (en) | 2005-09-20 | 2016-02-03 | BASF Plant Science GmbH | Improved methods controlling gene expression |
| US8809631B2 (en) | 2006-01-06 | 2014-08-19 | Rijk Zwaan Zaadteelt En Zaadhandel B.V. | Screening method for selecting plants that show reduced wound-induced surface discolouration and plant and plant parts thus obtained |
| KR20080093119A (en) * | 2006-01-06 | 2008-10-20 | 리즈크 즈완 자드틸트 엔 자드한델 비.브이. | Screening method for selecting plants that show a reduced wound-induced surface discolouration and plant and plant parts thus obtained |
| US9315818B2 (en) | 2006-06-07 | 2016-04-19 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Plant expression constructs and methods of utilizing same |
| WO2008083715A1 (en) * | 2007-01-08 | 2008-07-17 | Rijk Zwaan Zaadteelt En Zaadhandel B.V. | Screening method for selecting plants that show a reduced wound-induced surface discolouration and plant and plant parts thus obtained |
| EP2011389A1 (en) * | 2007-07-06 | 2009-01-07 | Rijk Zwaan Zaadteelt en Zaadhandel B.V. | Method for identifying plant material that shows a reduced discolouration, plants thus identified and use of the identified plants for the production of commercial crop plants showing a reduced discolouration |
| US8734867B2 (en) | 2007-12-28 | 2014-05-27 | Liveleaf, Inc. | Antibacterial having an extract of pomegranate combined with hydrogen peroxide |
| WO2009105870A1 (en) * | 2008-02-27 | 2009-09-03 | Okanagan Specialty Fruits Inc. | Genetically modified reduced-browning fruit-producing plant and produced fruit thereof, and method of obtaining such |
| SG10201502453SA (en) | 2009-03-04 | 2015-05-28 | Liveleaf Inc | Method and material for site activated complexing of biologic molecules |
| US9192635B2 (en) | 2011-06-24 | 2015-11-24 | Liveleaf, Inc. | Method of treating damaged mucosal or gastrointestinal tissue by administering a composition comprising a mixture of pomegranate and green tea extracts and releasably bound hydrogen peroxide |
| US8722040B2 (en) | 2011-06-24 | 2014-05-13 | Liveleaf, Inc. | Site-activated binding systems that selectively increase the bioactivity of phenolic compounds at target sites |
| WO2013184768A1 (en) | 2012-06-05 | 2013-12-12 | University Of Georgia Research Foundation, Inc. | Compositions and methods of gene silencing in plants |
| MX350970B (en) | 2012-09-24 | 2017-09-26 | Seminis Vegetable Seeds Inc | Methods and compositions for extending shelf life of plant products. |
| US10077451B2 (en) | 2012-10-18 | 2018-09-18 | Monsanto Technology Llc | Methods and compositions for plant pest control |
| WO2014081729A1 (en) * | 2012-11-20 | 2014-05-30 | J.R. Simplot Company | Tal-mediated transfer dna insertion |
| US8716351B1 (en) | 2012-12-23 | 2014-05-06 | Liveleaf, Inc. | Methods of treating gastrointestinal spasms |
| US10000767B2 (en) | 2013-01-28 | 2018-06-19 | Monsanto Technology Llc | Methods and compositions for plant pest control |
| US20140283211A1 (en) | 2013-03-14 | 2014-09-18 | Monsanto Technology Llc | Methods and Compositions for Plant Pest Control |
| CA2993801C (en) | 2013-05-02 | 2021-01-05 | J.R. Simplot Company | Potato transformation vector for silencing endogenous asparagine synthetase-1 and polyphenol oxidase-5, phosphorylase-l and dikinase r1 |
| EP3157324A1 (en) | 2014-06-17 | 2017-04-26 | J.R. Simplot Company | Potato cultivar w8 |
| US8889964B1 (en) | 2014-06-17 | 2014-11-18 | J.R. Simplot Company | Potato cultivar W8 |
| US9918441B2 (en) | 2015-05-14 | 2018-03-20 | J.R. Simplot Company | Potato cultivar V11 |
| KR20180059552A (en) | 2015-10-08 | 2018-06-04 | 제이.알.심프롯캄패니 | Potato seeds Y9 |
| EP3358970A4 (en) | 2015-10-08 | 2019-03-06 | J.R. Simplot Company | Potato cultivar x17 |
| CN105567709A (en) * | 2016-02-23 | 2016-05-11 | 浙江农林大学 | Agaricus bisporus PPO gene segment and application thereof in lowering of PPO enzyme activity |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL81737A (en) * | 1986-03-28 | 1992-11-15 | Calgene Inc | Regulation of gene expression in plant cells |
| SE467358B (en) * | 1990-12-21 | 1992-07-06 | Amylogene Hb | GENETIC CHANGE OF POTATISE BEFORE EDUCATION OF AMYLOPECT TYPE STARCH |
| AU655494B2 (en) * | 1991-07-17 | 1994-12-22 | Commonwealth Scientific And Industrial Research Organisation | Polyphenol oxidase genes |
| WO1993015599A1 (en) * | 1992-01-31 | 1993-08-19 | Cornell Research Foundation, Inc. | Polyphenol oxidase |
-
1993
- 1993-07-27 EP EP93917656A patent/EP0606454A1/en not_active Withdrawn
- 1993-07-27 JP JP6504963A patent/JPH07503376A/en active Pending
- 1993-07-27 CA CA 2120258 patent/CA2120258A1/en not_active Abandoned
- 1993-07-27 WO PCT/EP1993/001988 patent/WO1994003607A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994003607A1 (en) | 1994-02-17 |
| JPH07503376A (en) | 1995-04-13 |
| EP0606454A1 (en) | 1994-07-20 |
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