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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
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1051—Hexosyltransferases (2.4.1)
  • C12N9/107—1,4-Alpha-glucan branching enzyme (2.4.1.18)
• • 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
  • C12N15/8245—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 modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

• the present invention relates to genetically engi- neered modification of potato, resulting in the formation of an increasing amount of amylose-type starch as compared to amylopectin-type starch in the potato.
• the genetically engineered modification implies the insertion of a gene fragment into potato, said gene fragment comprising tran- scription start and a part of the gene coding for the for ⁇ mation of branching enzyme (BE gene) in potato, inserted in the antisense direction, together with a tuber-specific promoter.
• BE gene branching enzyme
• starch In the potato tuber, starch is the greatest part of the solid matter. About 1/4 to 1/5 of the starch in potato is amylose, while the remainder of the starch is amylo- pectin. These two components of the starch have different fields of application, and therefore the possibility of producing either pure amylose or pure amylopectin is most interesting.
• the two starch components can be produced from common starch, which requires a number of process steps and, consequently, is expensive and complicated.
• the starch granules contain a mixture of linear and branched molecules which form the starch components amy- lose and amylopectin.
• Amylopectin is produced by inter ⁇ action between starch synthase and branching enzyme, alpha-l,4-glucane; alpha-l,4-glucane-6-glucosyl trans ⁇ ferase (EC 2.4.1.18).
• Branching enzyme (BE) hydrolyses alpha-1,4 bonds and synthetises alpha-1,6 bonds (Mac Donald & Preiss, 1985; Preiss, 1988).
• Endosperm of normal maize contains three forms of BE protein, designated BE I, BE Ila and BE lib.
• the mutation amylose extender (ae) inhibits the activity of the enzyme BE lib, which results in a reduced content of amylopectin and a corresponding increase of the amylose content, ae endosoerm thus has a different DroDorticn of amvlose tc amylopectin than normal maize, viz. 65:35 instead of 25:75 (De Vries Kuranda, 1987).
• each of them has properties in its pri- mary structure which make them unique.
• the genes for each enzyme form have not been identified so far, but by isola ⁇ tion of cDNA clones for each BE form, each gene can in all probability be characterised.
• BE branching enzyme
• Branching enzyme (BE) in potato is a monomer protein, i.e. it is a single enzyme form.
• the molecular weight of potato BE varies between 79 and 103 kD, depending on the purifying process used. There are indications that potato BE should consist of several forms, but presumably several forms are degradation products from the actual protein (Vos-Scheperkeuter, 1989; Blennow & Johansson, 1990).
• the reduction of the formation of enzyme can be accomplished in several ways, e.g. by: - utagen treatment which results in a modification of the gene sequence coding for the formation of the enzyme
• Fig. 1 illustrates a specific suppression of normal gene expression in that a complementary antisense nucleo- tide is allowed to hybridise with mRNA for a target gene.
• the antisense nucleotide thus is antisense RNA which is transcribed in vivo from a "reversed” gene sequence (Izant, 1989).
• the antisense con- struct for chalcone synthase, polygalacturonase and phos- phinotricin acetyltransferase has been used to inhibit the corresponding enzyme in the plant species petunia, tomato and tobacco (Van der Krol et al, 1990; Sheehy et al, 1988; Cornelissen, 1989).
• the object of the invention is to provide a varyingly increased amylose production in potato tuber by using antisense gene inhibition.
• the function of the BE gene and, thus, the amylopectin production in potato are inhibited to a varying extent by using new antisense constructs.
• the antisense constructs according to the invention comprise a tuber-specific promoter, transcrip ⁇ tion start and the first exon of the gene coding for fcr- mation of branching enzyme (BE gene) in potato, inserted in the antisense direction.
• the invention also comprises a gene coding for forma ⁇ tion of branching enzyme in potato, the so-called BE gene.
• the invention further comprises vectors including the antisense constructs according to the invention.
• the invention comprises cells, plants, tubers, microtubers and seeds, whose genome contains the antisense constructs according to the inven ⁇ tion.
• the invention comprises amylose-type starch, both native and derivatised.
• the invention comprises a method of sup ⁇ pressing formation of amylopectin-type starch in potato, whereby the potato tubers form a varyingly increased amount of amylose-type starch.
• Fig. 1 illustrates the principle of the antisense gene inhibition
• Fig. 2 shows antisense constructs according to the invention (according to Bevan, 1984).
• oligo nucleotides Based on a known peptide sequence from the BE gene in potato, two synthetic oligo nucleotides overlapping one another are produced.
• the oligo nucleotides (produced at the Institute for Cell Biology, Uppsala, Sweden, at the applicant's request) are used for identification of cDNA clones from a cDNA library in lambda gt 11 (produced on the applicant's behalf by Clontech, USA).
• the cDNA clones are used for isolation of the genomic BE gene from a geno ⁇ mic library in EMBL 3 (produced on the applicant's behalf by Clontech, USA).
• Antisense Constructs A varying increase of the amylose content in potato tubers is desired, and therefore different types of anti- sense genes are constructed which more or less inhibit the expression of the BE gene in vivo.
• the binary Ti plasmides pBI 121 and pBI 101 are used as a basis for all gene struc- tures (Fig. 2), which means that NPT-II and the GUS gene are selection markers.
• the GUS gene is the gene which codes for beta-glucuronidase. Transformation
• the antisense constructs are transferred to bacteria, suitably by the "freeze-thawing" method (An et al, 1988).
• the transfer of the recombinant bacterium to potato tissue occurs by incubation of the potato tissue with the recom ⁇ binant bacterium in a suitable medium after some sort of damage has been inflicted upon the potato tissue. During tzhe incubation, T-DNA from the bacterium enters the DNA of the host plant. After the incubation, the bacteria are killed and the potato tissue is transferred to a solid medium for callus induction and is incubated for growth of callus.
• sprouts are formed which are cut away from the potato tissue.
• this is analysed regarding the presence of the used marker.
• the testing of the expression on protein level is suitably carried out on microtubers induced in vitro on the transformed sprouts, thus permitting the testing to be performed as quickly as possible.
• composition of the starch in microtubers is iden- tical with that of ordinary potato tubers, and therefore the effect of the antisense constructs on the amylopectin production is examined in microtubers.
• the proportion of amylose to amylopectin can be determined by a spectro- photometric method (e.g. according to Hovenkamp-Hermelink et al, 1988).
• Amylose is extracted from the so-called amylose potato (potato in which the formation of amylopectin has been suppressed to a varying extent by inserting the antisense constructs according to the invention) in a known manner. Derivatisation of Amylose
• phy ⁇ sical and chemical qualities can be modified by deriva- tisation.
• derivatisation is here meant chemical, phy ⁇ sical and enzymatic treatment and combinations thereof (modified starches) .
• the chemical derivatisation i.e. chemical modifica ⁇ tion of the amylose, can be carried out in different ways, for example by oxidation, acid hydrolysis, dextrinisation, different forms of etherification, such as cationisation, hydroxy propylation and hydroxy ethylation, different forms of esterification, for example by vinyl acetate, acetic anhydride, or by monophosphatising, diphosphatising and octenyl succination, and combinations thereof.
• amylose Physical modification of the amylose can be effected by e.g. cylinder-drying or extrusion.
• the derivatisation is effected at different tempera- tures, according to the desired end product.
• the ordinary range of temperature which is used is 20-45°C, but tempe ⁇ ratures up to 180°C are possible.
• the antisense constructs (see Fig. 2) are transferred to Agrobacterium tumefaciens LBA 4404 by the "freeze-thaw- ing" method (An et al, 1988).
• the transfer to potato tis ⁇ sue is carried out according to a modified protocol from Rocha-Sosa et al (1989).
• Leaf discs from potato plants cultured in vitro are incubated in darkness on a liquid MS-medium (Murashige _ Skoog; 1962) with 3% saccharose and 0.5% MES together with 100 ⁇ l of a suspension of recombinant Agrobacterium per 10 ml medium for two days. After these two days the bacte ⁇ ria are killed. The leaf discs are transferred to a solid medium for callus induction and incubated for 4-6 weeks, depending on the growth of callus. The solid medium is comDOsed as follows: MS + 3% saccarose
• leaf discs are transferred to a medium having a different composition of hormones, com- prising:
• the leaf discs are stored on this medium for about 4 weeks, whereupon they are transferred to a medium in which the "Claforan" concentration has been reduced to 250 mg/1. If required, the leaf discs are then moved to a fresh medium every 4 or 5 weeks. After the formation of sprouts, these are cut away from the leaf discs and trans ⁇ ferred to an identical medium.
• the condition that the antisense construct has been transferred to the leaf discs is first checked by analys ⁇ ing the presence of the GUS gene.
• Leaf extracts from the regenerated sprouts are analysed in respect of glucuroni- dase activity by means of the substrates described by Jefferson et al (1987).
• the acitivity is demonstrated by visual assessment.
• the effect of the antisense constructs on the func ⁇ tion of the BE gene in respect of the activity of the BE protein is analysed by means of electrophoresis on poly- acrylamide gel (Hovenkamp-Hermelink et al, 1987). Starch is extracted from the microtubers and analysed regarding the presence of the BE protein.
• composition of the starch i.e. the proportion of amylose to amylopectin, is determined by a spectro- photometric method according to Hovenkamp-Hermelink et al (1988), the content of each starch component being deter ⁇ mined on the basis of a standard graph.
• Example 2 Extraction of amylose from amylose potato.
• Potato whose main starch component is amylose, below called amylose potato, modified in a genetically engineer ⁇ ed manner according to the invention, is grated, thereby releasing the starch from the cell walls.
• the cell walls are separated from fruit juice and starch in centrifugal screens (centrisiler>.
• the fruit juice is separated from the starch in two steps, viz. first in hydrocyclones and subsequently in specially designed band-type vacuum filters. Then a finishing refining is carried out in hydro- cyclones in which the remainder of the fruit juice and fibres are separated.
• Amylose is sludged in water to a concentration of 20-50%.
• the pH is adjusted to 10.0-12.0 and a quatenary ammonium compound is added in such a quantity that the end product obtains a degree of substitution of 0.004-0.2.
• the reaction temperature is set at 20-45°C.
• the pH is adjusted to 4-8, whereupon the product is washed and dried. In this manner the cationic starch derivative 2-hydroxy-3-trimethyl ammonium propyl ether is obtained.
• Example 4 Chemical derivatisation of amylose Amylose is sludged in water to a water content of 10-25% by weight.
• Example 5 Chemical derivatisation of amylose Amylose is sludged in water to a concentration of 20-50% by weight. The pH is adjusted to 5.0-12.0, and sodium hypochlorite is added so that the end product obtains the desired viscosity. The reaction temperature is set at 20-45°C. When the reaction is completed, the pH is adjusted to 4-8, whereupon the end product is washed and dried. In this manner, oxidised starch is obtained.
• Example 6 Physical derivatisation of amylose
• Amylose is sludged in water to a concentration of 20-50% by weight, whereupon the sludge is applied to a heated cylinder where it is dried to a film.
• Example 7 Chemical and physical derivatisation of amylose
• Amylose is treated according to the process describ ⁇ ed in one of Examples 3-5 for chemical modification and is then further treated according to Example 6 for physi ⁇ cal derivatisation.
• SEQ ID No. 1 Sequenced molecule: genomic DNA Name: Promoter for the GBSS gene from potato Length of sequence: 629 bp

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• 1990
  • 1990-12-21 SE SE9004095A patent/SE9004095L/sv not_active IP Right Cessation
• 1991
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