PL169859B1 - Method of inhibiting formation of amylopectin-type starch in potatoes - Google Patents

Abstract

Genetically engineered modification of potato for suppressing formation of amylopectin-type starch is described. The invention describes an antisense construct for inhibiting, to a varying extent, the expression of the gene coding for formation of branching enzyme (BE gene) in potato, said antisense construct comprising a tuber-specific promoter, transcription start and the first exon of the BE gene, inserted in the antisense direction. Also cells, plants, tubers, microtubers and seeds of potato comprising said antisense construct are described. Finally, amylose-type starch, both native and derivatised, derived from the potato that is modified in a genetically engineered manner, as well as a method of suppressing amylopectin formation in potato are described.

Description

The present invention relates to a method for inhibiting the formation of amylopectin-type starch in potatoes.

The modification of potato by the genetic engineering technique of the invention produces an increased amount of amylose type starch compared to the amylopectin type starch normally found in potato. This modification consists in the insertion into the potato of a gene fragment containing the start of transcription and a part of the gene coding for the formation of a branching enzyme in potato (BE gene), this incorporation in the antisense direction together with a tuber-specific promoter.

Starch in its various forms is of great importance in the food and paper industries. In the future, it will also have huge potential applications for the production of degradable polymers, for example for packaging materials. There are known many different starch products produced by modifications of native starch derived, inter alia, from corn and potatoes. Potato and maize starch compete with each other in major outlets.

In a potato tuber, starch makes up the largest proportion of the solids. About 1/4 to 1/5 of the potato starch is amylose and the remainder is amylopectin. The two starch components have different uses and therefore the possibility of producing either pure amylose or pure amylopectin is very interesting. These two starch ingredients can be made from ordinary starch; however, this requires a multi-step processing process and is consequently costly and complex.

It has now turned out that by genetic engineering it is possible to modify the potato in which the mutual proportions between the two starch components (amylose and amylopectin) in potato tubers are changed. As a result, starches are obtained of such a quality that they can compete in areas where potato starch has not been used so far. Starch from genetically engineered potatoes is of great potential importance as a food additive as it is not subject to any chemical treatment processes.

Starch synthesis

The synthesis of starch and the processes of regulating this synthesis are currently being thoroughly researched both at the level of basic research and for industrial application. Although much information has been gathered about the involvement of certain enzymes in the transformation of sucrose into starch, starch biosynthesis has not yet been elucidated. By carrying out research mainly on maize, however, it was possible to elucidate some of the synthetic pathways and to identify the enzymes involved in these reactions. The most important enzymes that synthesize starch with the subsequent production of starch grains are starch synthetase and the branching enzyme. In maize, so far it has been found

Three forms of starch synthetase have been present and tested, two of which are soluble and one is in the form of insoluble, grain bound starch. Also, the branching enzyme in maize consists of three forms, possibly encoded by three different genes.

Branching enzyme in various plant species

Starch granules contain a mixture of linear and branched molecules that make up the starch component: amylose and amylopectin. Amylopectin is formed by the interaction between starch synthetase and the branching enzyme cr-1,4-glucan and α-1,4g-glucnno-gglucosyltransferase (EC 2.4. -.- 8). The branching enzyme (BE) hydrolyzes α-1,4 linkages and synthesizes α-1,6 linkages (Mac Donald and Preiss, -985; Preiss, -988).

The endosperm of ordinary maize contains three forms of the BE protein, denoted BE I, BE Ila and BE Ilb. The amylose chain extender (ae) inhibits the activity of the BEIlb enzyme, which leads to a reduction in amylkpeUtynz content and a corresponding increase in amylose content. The endosperm of ae thus contains a different ratio of amylose to amylopeptin than that found in ordinary corn, i.e. 65:35 instead of 25:75 (De Vries Kuranda, L987).

Although the similarities between the three enzyme forms are great, each form has its own primary structure properties which make it unique. So far, genes for each form of the enzyme have not been identified, but each gene can in all probability be characterized by isolating cDN A clones for each form of the BE enzyme.

Two forms of branching enzyme (BE) have been identified in normal peas. A mutation in the r locus that produces pea ripples affects the activity of the BE enzyme, inhibiting one form of this enzyme. This results in an altered starch composition with 30% amylopectin and 70% amylose, compared to the inverse proportion found in normal round peas (Smith, 1988).

Potato branching enzyme (BE) is a mnaomeIyconzm protein, that is, it is a single form of enzyme. The molecular weight of BE from potato ranges from 79 to -03 kD depending on the cleaning method used. There are indications that potato BE should consist of many forms, but probably many of these forms are degradation products of the actual protein (Vks-Scheperkeuter, -989; Blennow and Johansson, -990).

The sequencing of the peptides of the three BE forms, separated by electrophoresis, showed such a large hkmclogy between these enzyme forms that it is assumed that they come from the same source. This assumption is supported by serological tests, as the antisera obtained from these three forms of the enzyme cross-react with each other.

Branch enzyme inhibition

By inhibiting one of the forms of the branching enzyme in maize and peas, the composition of the starch is altered in such a way that the amylose content increases strongly with the simultaneous disappearance of amylopectin production.

So far, no natural genotype with an increased amylose content has been found in the potato. However, it is possible to reduce the BE content to a variable degree, which changes the starch composition of the potato tubers towards an increase in the amylose content compared to conventional potato.

Inhibition of enzyme formation can be achieved in several ways, for example by:

- treatment with a mutagen resulting in a modification of the gene sequence coding for the production of this enzyme;

- introduction of the transposome into the gene sequence encoding the enzyme;

- genetic engineering modification consisting in modifying the expression of the gene encoding the enzyme by so-called α-anti-sense inhibition of the gene.

Fig. - shows the specific suppression of normal gene expression by hybridizing a complementary assay nucleotide to mRNA for the target gene. This non-sense nucleotide is an non-sense RNA that is tiancribed in vivo from the "reverse gene sequence" (Izant, -989).

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A wide variety of gene functions in plants have been inhibited by the antisense technique. Antisense constructs for chalcone synthetase, polygalacturonase and phospflnotricin acetyltransferase have been used to inhibit the corresponding enzymes in putenia, tomatoes, and tobacco (Van

Krol et al., 1990; Sheehy et al., 1988; Corneli:

ιωοοί

It has been found that it is possible to vary the production of amylose in potato tubers by the use of a gene antisense inhibition technique.

The subject of the invention is a method of inhibiting the formation of amylopectin-type starch in potatoes, consisting in the modification of the potato by genetic engineering by introducing into the genome of the potato tissue an antisense construct containing a tuber-specific promoter with the sequence designated as ID No. 1, the start of transcription and the first an exon of the gene encoding the formation of branching enzyme (BE gene) in potatoes, this exon being switched in the antisense direction.

According to the method of the invention, the function of the BE gene, and thus the production of amylopectin in potatoes, is inhibited to a variable extent by the use of novel antisense constructs. Such antisense constructs contain a tuber-specific promoter, an initiation of transcription, and the first exon of the gene coding for the formation of a branching enzyme (BE gene) in potato, antisense flipped.

The method according to the invention inhibits the formation of amylopectin-type starch in potatoes, whereby an increased amount of amylose-type starch is produced in the potato tubers, with a variable increase in this amount.

The method of the invention is described in more detail with reference to the accompanying drawings, in which Figure 1 shows the principle of antisense gene inhibition, Figure 2 shows the antisense constructs used in the method of the invention (according to Bevan, 1984) and Figure 3 shows the sequence of the tuber-specific promoter under the name: SEQ ID NO: 1.

Isolation of the genomic BE gene of potatoes

Based on the known peptide sequence of the BE gene product in potatoes, two synthetic oligonucleotides are partially overlapping. These two oligonucleotides (manufactured at the Institute of Cell Biology in Upsala, Sweden) are used to identify cDNA clones from the lambda gt 11 cDNA bank (prepared by Clontech, USA). These cDNA clones are used to isolate the genomic BE gene from the EMBL 3 genomic bank (manufactured at Clontech, USA).

Antisense constructs

A variable degree of increase in the amylose content in potato tubers is advantageous, therefore, various types of antisense genes are constructed that more or less inhibit expression of the BE gene in vivo. They are derived from the isolated genomic BE gene, whereby the antisense constructs contain portions of the BE gene corresponding to the sequences of the promoter region, the beginning of transcription, and the first exon.

In order to obtain both variability in amylose content as well as tissue specificity, meaning that amylopectin production should be limited to tubers only, various tuber specific promoters are conjugated to an antisense gene. In addition to the tuber own BE promoter, the following promoters are used in various combinations: 35S CaMV, Patatin 1 (obtained from Dr. M. Bevan's, UK) and the potato GBSS gene promoter.

The GBSS promoter is integrated into the potato gene encoding the formation of granule bound starch synthetase. It is the enzyme primarily responsible for the formation of amylose in potatoes.

The binary Ti plasmids pBI 121 and pBI 101 (supplied by Clontech, USA) (Fig. 2) are used as the basis for all gene constructs, which means that NPT-II and the GUS gene are selectable markers. The GUS gene is the gene encoding β-glucuronidase.

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Transforming

The resulting antisense constructs are conveniently transferred to the bacteria by the "freeze thaw" method (An. Et al., 1988). The transfer of the recombinant bacteria to the potato tissue occurs by incubation of the potato tissue with the recombinant bacteria in a suitable medium after some kind of damage to the tissue. During incubation, T-DNA from the bacteria enters the DNA of the plant host. After incubation, the bacteria are killed and the potato tissue is transferred to solid callus culture medium and incubated while growing the callus.

After a series of cultures, sprouts are formed on further suitable media, which are excised from the potato tissue.

As a first check that the antisense constructs have been transferred to the potato tissue, analysis for the presence of the marker used is performed.

The next tests are testing the expression of antisense constructs and their transfer to the potato genome by Southern and Northern hybridization methods (Maniatis et al., 1982). The number of copies of the antisense constructs that have been transferred is determined by Southern hybridization.

The expression assay at the protein level is conveniently performed on microtubers grown in vitro on transformed shoots, which allows the assay to be performed as soon as possible.

Determination of starch

The starch composition of microtubers is identical to that of conventional potato tubers, thus, the effect of antisense constructs on amylopectin production is determined in microtubers. The proportion of amylose to amylopectin is determined spectrophotometrically (e.g. by the method described by Hovenkamp-Hermelink et al., 1988).

Extraction of amylose from amylose potatoes

The extraction of amylose from so-called amylose potatoes (potatoes in which the production of amylopectin has been reduced to a varying extent by the insertion of antisense constructs) is carried out in a known manner.

Derivatization of amylose

Depending on the end-use of amylose, its physical and chemical properties are modified by derivatization. Derivatization is understood to mean the chemical, physical and enzymatic treatment of starches and combinations of these methods (modified starches).

Chemical derivatization, i.e. the chemical modification of amylose, may be carried out in various ways, e.g. by oxidation, acid hydrolysis, dextrinization, various forms of etherification such as cationization, hydroxypropylation and hydroxyethylation, various forms of esterification, e.g. with vinyl acetate, acetic anhydride or by formation of monophosphate and diphosphate derivatives either by treatment with octenyl succinate or by a combination of these methods.

Physical modification of amylose can be carried out, e.g., by drying in a tube or by extrusion.

In enzymatic derivatization, degradation (reduction in viscosity) and chemical modification of amylose are achieved by the action of available enzymatic systems.

The derivatization is carried out at different temperatures depending on the desired end product. The most common temperature range is 20-45 ° C, but temperatures up to 180 ° C are possible.

The process of the invention is described in more detail in the following examples.

Example I. Preparation of microtubers with incorporated antisense constructs

The antisense constructs (Fig. 2) are transferred to Agrobacterium tumefaciene LBA 4404 by the "freeze-thaw" method (An et al., 1988). The transfer to the potato tissue is performed following a modified recipe given by Rocha-Sosa et al. (1989).

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Leaf discs from in vitro cultivated potato plants are incubated in the dark for 2 days in liquid MS medium (Murashige and Skoog, Ł962) with the addition of 3% sucrose and 0.5% MES together with the amount of W0pl of Agrobacterium recombinant suspension per 10 ml medium. After these two days, the bacteria are killed. Leaf disks are transferred to solid callus induction medium and incubated for 4-6 weeks depending on callus growth. A solid substrate with the following composition is used:

MS medium + 3% sucrose zeatin riboside "NAA" "GA 3" antibiotic "Claforan kanamycin" Gellan mg / liter 0.02 mg / liter 0.02 mg / ihr 500 mg / liter 50 mg / liter 0 ^ 225%

The next leaf discs are transferred to a medium with a different hormone composition containing: MS + 3% sucrose "NAA" 5 mg / liter "BAP" 0.1 mg / liter "Elaforan 500 mg / liter kanamycin 50 mg / liter" Gellan 0, 25%

The leaf discs are stored in this medium for about four weeks, after which they are transferred to medium where the concentration of "Elaforan" is reduced to 250 mg / liter. If necessary, leaf discs are transferred to fresh medium every 4 to 5 weeks. After the shoots have formed, leaf discs are cut out and transferred to an identical medium.

The transfer of the anti / sense construct to leaf discs is initially confirmed by analyzing the presence of the GUS gene. Leaf extracts from regenerated suckers are analyzed for glucuronidase activity by the substrate assay method described by Jefferson et al. (L987). Activity is determined by visual evaluation.

Subsequent tests for the expression of antisense constructs and their transfer to the potato genome are carried out by the hybridization methods of Sothern and Nor ^ a, according to Maniatis et al. (L982). The number of copies of the alysense constructs that have been transferred is determined by the Soem hybridization method.

Once it has been established that the antisense constructs have been transferred into the potato genome and that they are being expressed, the determination of this expression at the protein level begins. The tests are carried out on microtubers, the growth of which on transformed shoots has been induced by cytomes. This avoids waiting for a complete potato plant with tubers to develop.

Pieces of potato vinegar stalk are cut at the nodes and placed in the modified MS medium. There, after an o-3 week incubation in the dark nécil in 1611 ^^ 1 ^ '/! m-C microtubers were formed (Bourque et al., 1987). This medium has the following composition:

MS + 6% sucrose kinetin 2.5 mg / liter 'ΌΗ ^ 2.5 mg / liter

The effect of α-sense constructs on BE gene function with respect to BE protein activity is analyzed by polyacrylamide gel electrophoresis (HoveaCamp-Hermellnk et al., L987). Starch is extracted from the microtubers and analyzed for the presence of the BE protein.

The starch composition, i.e. the proportion of amylose to amylopectin, is determined spectrophotometrically by the method of Hoveakamp-Hetmeliak et al. (-988), calculating the content of each starch component from standard graphs.

Example II. Extraction of amylose from amylose potatoes

A potato in which the main starch component is amylose, hereinafter referred to as amylose potato, modified by the genetic engineering technique according to the invention is grated to release starch from the cell walls.

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The cell walls (fibers) are separated from the fruit juice and starch on the screens of the centrifuge. Fruit juice is separated from starch in two stages: initially in hydrocyclones and then in specially constructed strip-shaped vacuum filters. This is followed by a final refining in hydrocyclones, where the remaining amount of fruit juice and fibers are separated.

The product is dried in two steps, first pre-drying on a vacuum filter and then under a flow of hot air.

Example III. Chemical derivatization of amylose

Amylose is mixed with water, used in such an amount that its concentration is 20-50%. The pH of the suspension is adjusted to 10.0-12.0 and the quaternary ammonium compound is added in such an amount to achieve a degree of substitution in the range of 0.004-0.2. The reaction temperature is kept at 20-45 ° C. After completion of the reaction, the pH is adjusted to 4-8, after which the product is washed and dried. The cationic derivative of the starch, propyl-starch 2-hydroxy-3-trimethylammonium ether, is obtained.

Example IV. Chemical derivatization of amylose

Amylose is made up of with water used in an amount such that its content in the mixture is 10-25% (by weight). The pH of the mixture is adjusted to 10.0-12.0 and a quaternary ammonium compound is added in such an amount that the degree of substitution in the final product is 0.004-0.2. The reaction temperature is kept at 20-45 ° C. After completion of the reaction, the pH is adjusted to 4-8. The final product is 2-hydroxy-3-trimethylammonium propyl starch ether.

Example 5 Chemical derivatization of amylose

Amylose is made up with water to a concentration of 20-50% (w / w), the pH of the mixture is adjusted to 5.0-12.0 and sodium hypochlorite is added in such an amount to obtain the desired viscosity of the final product. The reaction temperature is kept at 20-45 ° C. After completion of the reaction, the pH is adjusted to 4-8, after which the product is washed and dried. An oxidized starch is obtained.

Example VI. Physical derivatization of amylose

The amylose is made up with water to a concentration of 20-50% (by weight), then the slurry is fed to a heated cylinder and dried to form a starch layer.

Example VII. Chemical and physical derivatization of amylose

The amylose is chemically modified according to the processes described in Examples III-V, followed by a physical derivatization according to Example VI.

FIG. 2 Antisense constructs. Apart from RB and IB as pBIN19

^ Glucuronidase (GUS) N0S ~ t6r

Promoter

CaMV 35S

FIG. 3

AACCATCCTT CCTTTTAGCA GTGTATCAAT TTTGTAATAG AACCATGCAT 50 ACTCAATCTT AATACTAAAA TGCAACTTAA TATAGGCTAA ACCAAGTAAA 100 GTAATGTATT CAACCTTTAG AATTGTGCAT TCATAATTAG ATCTTGTTTG 150 TCGTAAAAAA TTAGAAAATA TATTTACAGT AATTTGGAAT ACAAAGCTAA 200 GGGGGAAGTA ACTAATATTC TAGTGGAGGG AGGGACCAGT ACCAGTACCT 250 AGATATTATT TTTAATTACT ATAATAATAA TTTAATTAAC ACGAGACATA 300 GGAATGTCAA GTC-GTAGCGT AGGAGGGAGT TGGTTTAGTT TTTTAGATAC 350 TAGGAGACAG AACCGGACGG CCCATTGCAA GGCCAAGTTG AAGTCCAGCC 400 GTGAATCAAC AAAGAGAGGG CCCATAATAC TGTCGATGAG CATTTCCCTA 450 TAATACAGTG TCCACAGTTG CCT7CTGCTA AGGGATAGCC ACCCGCTATT 500 CTCTTGACAC GTGTCACTGA AACCTGCTAC AAATAAGGCA GGCACCTCCT 550 CATTCTCACT CACTCACTCA CACAGCTCAA CAAGTGGTAA CTTTTACTCA S00 TCTCCTCCAA TTATTTCTGA TTTCATGCA 629

7Me

FIG. 1 5 'Gppp AUGGCAAG AAAAAAAA 3' mRNA

Antisense RNA target gene

Processing. Transcription, transport and translation

EATCGCAAGj ^

Promoter

Protein resulting from the translational process

The anti-sense construct inhibits mRNA gene expression

5 'Gppp AUGGCAAG AAAAAAAA 3' 'I I I I I

3 'UACCGUUC 5'

Antisense RNA iCTTGCCATK / f GAACGGTALMI [Antisense producing antisense gene (jRNA____

Publishing Department of the Polish Patent Office. Circulation of 90 copies. Price PLN 2.00

Claims (1)

Patent claim A method of inhibiting the formation of amylopectin-type starch in potatoes, characterized in that the potato is genetically engineered by introducing into the genome of the potato tissue an antisense construct containing a tuber-specific promoter with the sequence designated as ID No. 1, the beginning of the transcription and the first exon of the gene encoding the formation branching enzyme (BE gene) in potatoes, this exon being switched in the antisense direction.