SI23835A - Use of macrocipines as pesticidal agents - Google Patents

Abstract

The object of the invention is to use macrocipins as pesticidal active substances for plant protection. Macrocipins are a group of proteins that are naturally present in the Macrolepiota Procera fungus. The use of these proteins for plant protection is possible in two ways. The first is the use of genetically modified plants to which the polynucleotide sequence is stably included in the genome, which encodes at least one of the proteins mentioned in the present invention. The other way is direct application of proteins to plants. In this case, the polynucleotide sequence encoding at least one of the proteins mentioned in this invention is inserted into the heterologous expression vector for the production of the recombinant protein in the bioreactor. After the cleaning process, these proteins can be applied directly to the plants that we want to protect. The polypeptides of the present invention are particularly useful for protecting against pests of agricultural plants, such as Lepidopter, Hemipter, Dipter and Coleopter insects, preferably in front of the Lupinotarsa decemlineata.

Description

SUMMARY OF THE INVENTION: USE OF MACROCIPINES AS PESTICIDAL SUBSTANCES

DESCRIPTION OF THE INVENTION

The subject of the invention is the use of macrocipins as pesticide active ingredients for plant protection. Pesticidal efficacy is demonstrated by the reduced growth and survival rate of organisms that cause damage to plant growth and / or crop production.

In the coming decades, human society will face great challenges. As the population grows and the standard of living in developing countries increases, raw material needs and competition are increasing. Increased food needs have already caused prices to rise. At the same time as the population is growing, ecological awareness is increasing, resulting in an increased need for organically grown foods. Organic food production is characterized by prohibiting the use of synthetic insecticides to protect the growth and yield of plants. However, for a sufficient supply of organically grown food, plants need to be protected from pests, for example, from insects that reduce growth and crop yield. One of the acceptable ways of protecting plants in organic farming is to use natural pesticides.

One way of protecting plants from pests is to genetically modify plants. Genetic engineering methods allow the insertion of specific DNA sections that encode plant resistance factors against pests into the plant genome. This method allows the protection of plants without the use of synthetic pesticides. The advantage of using this technology is that it lowers the cost of crop production and reduces the human exposure to the potentially harmful effects of pesticides. However, there remains a need to find suitable plant protection factors.

However, in many parts of the world, GMOs are not accepted as a source of organic food. In addition, their appearance on the market is limited by lengthy and complex legal proceedings. Nonetheless, biotechnological solutions to protect plants from pests are already widely used. The most successful and commonly used method of pest control is the use of a group of genes encoding various Bacillus thuringiensis toxins (Bt-toxins). Genetically modified crops, such as maize and cotton, into which different Cry genes encoding Bt toxins have been introduced, are grown in vast areas of many countries. Examples of patents and patent applications protecting the use of various Bttoxins are: US006100456A, US005731194A, US 20030232757A1.

The interest in finding new insecticidal agents, despite the successful protection of crops against pests by the use of Bt-toxins, has so far been increasing. Namely, Bt-toxins do not provide protection against all plant pests, and resistance to these toxins also occurs. Therefore, the need to find new, especially natural, pesticide-based plant protection products is increasing.

Fungal proteins are a rich source of diverse natural substances. One of them is a group of fungal proteins called macrocipins. It is a group of cysteine protease inhibitors isolated from the edible fungus Macrolepiota procera (Renko et al. 2010; Sabotic et al. 2007; Sabotic et al. 2009).

Macrocipins have been found to inhibit in vitro the action of many non-insect cysteine proteases (Renko et al. 2010), suggesting that they may also inhibit the activity of insect cysteine proteases in vivo and could therefore be used to inhibit their to grow. To determine the correctness of this hypothesis, we examined whether macrocipins have an inhibitory effect on proteases from the insect digestive tract. The inhibitory activity of macrocipins was found to be very weak, since they inhibited only one of the proteases tested, thus refuting the hypothesis of the use of these proteins for plant protection. The approach of protecting plants with macrocipins as inhibitors of cysteine proteases has therefore proved ineffective.

The purpose of the invention is to provide polypeptides useful as pesticidal agents for the protection of plants against pests. In addition, it is an object of the invention to provide natural ingredients for the protection of plants against pests. It is further the object of the invention to provide genetically modified plants with increased pest resistance. It is also an object of the invention to provide ingredients with biocidal activity for direct application to non-genetically modified plants.

The purpose described above was achieved by introducing polynucleotides encoding the polypeptides of claim 1, and are defined as pesticidal agents having an inhibitory effect on pest growth and survival. It is surprising that inventors have found that polypeptides encoded by the polynucleotides mentioned in this invention increase the resistance of plants to pests. The effects of pest infestation on these plants are smaller and the crop is larger, which is especially useful for the protection of agricultural plants and, due to their nature, less harmful to the environment.

The polypeptides of the present invention can be obtained in two ways. The first is by expressing the polypeptides of claim 1 directly in the plant, and the second is to apply them to the plants or parts of the plants to be protected in a recombinant form.

In one part of the invention, a polynucleotide sequence encoding at least one of the proteins of the present invention having a pesticidal action is stably incorporated into the genome of the plant to be protected against pests.

In another part of the invention, a polynucleotide sequence encoding at least one of the proteins of the present invention having a pesticidal action is inserted into a vector for heterologous expression for the production of recombinant protein in the bioreactor.

Proteins obtained in the bioreactor after the purification process can be applied directly to the plants we wish to protect. It is surprising that the inventors found that recombinant proteins remain active after application to the plant.

Definitions

In the context of the invention, the following terms have the following meanings:

Macrocipins (Macrolepiota procera cysteine jarotease inhibitors), briefly called Mcp, are cysteine protease inhibitors isolated from the Macrolepiota procera mushroom. The term macrocipin encompasses both natural and recombinant macrocipins.

A pest is an organism that adversely affects the growth or yield of agricultural plants.

Yield is defined as a measurable amount of the economic value of a crop. It can be defined in terms of quantity and / or quality.

In the context of the invention, a pesticidal active ingredient is defined as an active ingredient, especially a polypeptide, which protects plants from damage by inhibiting the growth and survival of pests.

The term heterologous in the context of the invention refers to any genetic material that is introduced into a cell or cells of an organism where that genetic material was not previously present.

In this application, the terms variants, homologs and derivatives may be used interchangeably. These terms refer to polypeptides with similar sequences and similar biological and functional properties.

The variants, homologs, and derivatives of macronuclein-encoding polynucleotides are typically those having the same function as natural macronuclein-encoding polynucleotides.

Variants, homologs and derivatives of polypeptides comprise peptides, oligopeptides, polypeptides, proteins and enzymes with amino acid substitutions, deletions and / or inclusions compared to unchanged protein and have similar biological and functional activity to unchanged proteins from which they are derived. In order to obtain such homologs, some amino acid residues in the protein need to be replaced with other amino acid residues, which have similar properties (for example, hydrophobicity, hydrophilicity, antigenicity, propensity to form or prevent the formation of alpha helices and beta surfaces). Conservative swap tables are well known and can be used by trained people to find useful swaps. Homologs can also be obtained by routine experimental techniques.

Examples of variants, homologs and derivatives are polypeptides in a sequence similar to that of macrocipin. They may have one or more conservative amino acid substitutions. The number of amino acid substitutions still useful depends on the site of the polypeptide being modified. Replacement sites can be found through routine experiments. Up to ten amino acids, preferably three to five conservative amino acid substitutions, are preferred to improve the properties of the polypeptides. having a similar side chain, for example, similar aliphatic side chains, positive or negatively charged side chains, aromatic groups on the side chains, or substitution for an amino acid that also forms hydrogen bonds. An example of the latter substitution is amino acid substitutions having side chains with a hydroxyl group. Insertions, deletions, and substitutions may not significantly alter the function of the polypeptide as a pesticide factor. The pesticidal activity of variants, homologs and derivatives can be determined by standard techniques. The activity of these should be at least 95%, preferably 99%, compared to the activity of natural macrocipin.

In addition, amino acid substitutions, deletions and insertions that do not cause changes in the three-dimensional structure or protein folding that are important for the activity of variants, homologs and derivatives are preferred. Changes in the three-dimensional structure due to amino acid substitutions, deletions, and insertions can be determined by well-known techniques such as the use of circular dichroism (CD spectra), X-ray crystallography, or NMR (nuclear magnetic resonance).

Preferably, the variants, homologs and derivatives defined above have at least 63%, more preferably at least 70% or 80%, even better 90% or even 95%, or ideally more than 99% identity with the macrocipin sequence that is defined in this document.

Variants, homologs and derivatives of polypeptides comprise peptides, oligopeptides, polypeptides, proteins and enzymes derived from natural polypeptide sequences, which, however, have some of the following changes: altered natural, glycosylated, acylated, prenylated or artificial amino acid residues that do not occur in nature . Both, however, share similar or identical functionality and / or activity. Functionality and / or activity can be determined by known methods, for example for enzymes there is a method for determining the enzymatic activity of a polypeptide by acting on a substrate. The derivatives may also have one or more non-amino acid substitutions present or additional groups present on the amino acid sequence. An example of an additional group is a reporter molecule or other ligand that is covalently or non-covalently linked to an amino acid sequence. In a part of the present invention, the ligand or reporter molecule may be linked to macrocipin, allowing for easier detection. In the second part of the present invention, unnatural amino acid residues may be added or may replace the amino acid residues present in the natural sequence.

The term plant also refers to individual parts of plants or plant tissues.

Transgenic plant 'is a plant into which at least one heterologous polynucleotide has been introduced and stably integrated into the genome.

The term vector in this document refers to a polynucleotide used to insert a gene into a target cell. There are usually nucleotide sequences in the vector that help to express the desired product or are even necessary for expression. When a vector enters a host cell, it starts to produce a protein encoded by the inserted gene. The vector typically also contains sequences important for regulating gene expression, such as enhancer sequences and promoters that lead to successful transcription of the vector-borne gene.

Description of the pictures

Figure 1. Inhibitory activity of macrocipins against major digestive cysteine proteases present in the digestion of Colorado beetle larvae. The inactivity of macrocipins to the dominant digestive cysteine proteases indicates that the mode of action of these protease inhibitors is different from that of the protease inhibitors previously used to control the Colorado beetle. Gel filtration fraction - Gel filtration fraction.

Figure 2a. Plasmid vector map of pMDC32 used to express macrocypin 4A in potato (Solanum tuberosum cv. Desiree).

Figure 2b. Schematic representation of the T-DNA region from the pMDC32 vector; RB / LB - right / left boundary sequence, Hyg ^r - hygromycin resistance encoding gene, 2x35S - CaMV 35S double promoter; nosT terminator; the region between attR1 and attR2 is replaced by a Mcp4A encoding gene by recombination.

Figure 3. Expression of macrocipin in different transgenic lines of potato (Solanum tuberosum cv. Desiree). Mcp32 Al, Mcp32 A2 and Mcp32 E2 lines were selected for further analysis (abbreviated line names are Al, A2 and E2).

Figure 4. Transmission results after evening. Coarse protein extract of tansformed plants was used. The lines Al, A2, E2, D3, E3 and E4 were tested for the presence of macrocipin. Al, A2, E2, D3, E3 and E4, coatings of protein transgenic potato lines; -K, negative control (untransformed plant); + K, positive control (recombinant macrocipin); M, scale of standard molecular weights (size in kDa).

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Figure 5. Results of the first nutritional test of Colorado beetle larvae fed with transgenic potato expressing Mcp4; gaining weight of larvae. Weight gain of Colorado potato beetle larvae fed with genetically modified wild potato (WT) and genetically modified macroscopin-expressing Al and A2 lines. The initial sample size is 20 larvae per line. The figure shows a standard error with the handles. DPI - days after hatching.

Figure 6. Results of the first nutritional test of Colorado beetle larvae fed with transgenic potato expressing Mcp4; larval survival rate. Survival rate of Colorado potato beetle larvae after feeding with genetically modified wild potato (WT) and macro -cipin-expressing Al and A2 lines. The initial sample size is 20 larvae per line. DPI - days after hatching.

Figure 7. Results of the second nutritional test of Colorado beetle larvae fed with transgenic potato expressing Mcp4; gaining weight of larvae. Weight gain of Colorado potato beetle larvae fed with genetically modified wild potato (WT) and genetically modified macrocypin-expressing Al, A2 and E2 lines. The initial sample size is 20 larvae for WT and A2 lines and 17 for Al and 14 for E2. The figure shows a standard error with the handles. DPI - days after hatching.

Figure 8. Results of a second nutritional test of Colorado beetle larvae fed with transgenic potato expressing Mcp4; larval survival rate. Survival rate of Colorado potato beetle larvae after feeding with genetically modified wild potatoes (WT) and genetically modified macrocypin-expressing Al, A2 and E2 lines. The initial sample size is 20 larvae for WT and A2 lines and 17 for Al and 14 for E2. DPI - days after hatching.

Figure 9. Results of nutritional tests of Colorado beetle larvae fed on potato leaves coated with recombinant proteins (obtained in E. coli). The larvae were fed with leaves soaked in recombinant proteins (Mcpl, Mcp3, Mcp4) and, in the control experiment, BSA (serum bovine albumin). All experiments were repeated twice. The initial number of larvae in the two experiments together was 16.

Figure 10. Nutrition testing results of Colorado beetle larvae fed on potato leaves coated with recombinant Mcpl protein (obtained from E. coli). The same results as in Figure 9 are shown, except that they only cover data for the Mcpl protein. The table also includes statistical processing with Student's T-test (*** p <0.001, ** p <0.01, * p <0.05).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to pesticide agents that are highly effective against pests, especially against crop pests. Surprisingly, some of the proteins of the macrocipin family have been found to be effective as pesticide-protecting agents. The present inventors have found, by in vivo nutritional testing, that they have an inhibitory effect on the growth and survival of Colorado beetle larvae, in particular by representatives of Mcpl and Mcp4 (see Figures 5-10).

Such an effect of macrocipins was surprising and unexpected and its action unknown. Based on the data published so far and the initial experiments shown in Figure 1, it was found that macrocypins do not inhibit the action of major cysteine proteases from the Colorado beetle digestive tract to the extent that they can inhibit growth and reduce larval survival.

The inventors have found that the pesticidal activity of macrocipins is due to the fact that macrocipins have an inhibitory effect on some proteases, such as the digestive cysteine proteases “intestines found in the digestion of Colorado beetle larvae. of macrocipins, in particular the macrocipins referred to in this application. Without being bound by the theory that macrocypins, as defined in this application, are specifically active against glycoside hydrolases, such as cellulases (glycoside of the hydrolasic family 48), have particular pesticidal activity. Thus, these macrocipins appear to have at least a dual inhibitory action against selected proteases and against glycoside hydrolases. In addition to some digestive proteases, macrozycin targets (see Table 3 in Example 2) also target glycoside hydrolase, as demonstrated by affinity chromatography with bound macrocipin, and explained in more detail in Example 2. Based on these results, it can be concluded with consideration that macrocipins have due to its dual action by inhibiting cellulases and proteases in digestion, a stronger overall effect. It is hypothesized that this multitarget inhibition allows for the surprising increase in the pesticide action efficiency of polynucleotides and polypeptides disclosed in this application.

The aforementioned theory, without being bound by it, is therefore the single most likely explanation for the superior effect of the polypeptides of the present invention, despite the fact that the experimental data shown in Example 1 indicated the non-inhibitory inactivity of macrocipins in vivo. Although not expected, proteins from the macrocipin family still showed activity against pests and were unexpectedly stable, allowing their use on or in plants. A superior effect has been shown in particular for Mcpl and Mcp4, which have been shown to be effective in inhibiting the growth and survival of Colorado beetle larvae (see Figures 5-10) in in vivo nutrition assays.

Macrocipins (Macrolepiota procera cysteine protease inhibitors) are cysteine protease inhibitors isolated from the Macrolepiota procera mushroom. These macrocipins are encoded by a gene family that is divided into five groups on the basis of sequences. Within each group, the sequence similarity of the amino acid residues is greater than 90%, and between the groups the similarity is between 75 and 86%. The amino acid sequence of macrocipins can be inferred from genomic sequence data or from cDNA sequences. Data on differences in promoter and non-coding sequences suggest that the regulation of macrocipin expression is regulated at different stages (see Sabotič et al, 2009). Macrocipins are effective protease inhibitors of the papain family (Cl family according to the Merops classification), such as papain and cysteine cathepsin endopeptidases, and also inhibit cathepsins B and H, which have both endopeptidase and exopeptidase activity. Diversity in sequences affects the inhibitory activity of macrocipins and affects the diversity of inhibitory profiles. All macrocipins have similar basic biochemical properties and are stable at high temperatures and extreme pHs.

Macrocypins are not similar to other families of protease inhibitors, so we suppose that the mechanism of inhibition of target cysteine proteases by macrocipins is unique. The crystalline structure of macrocipin revealed a motif of binding to the papain-like cysteine protease, which in a hitherto unknown way impedes access to the catalytic amino acid residues of the protease (see Renko et al, 2010). Macrocipins have a β-triperous twist, which is in the form of a tree structure with two loops in the root region, a trunk composed of a 6-stranded beta barrel and two layers of loops (6 + 3) in the canopy region. Directed mutations have shown that two cysteine cathepsin-binding loops belong to the lower layer of the canopy, while a single loop from the canopy region is responsible for the inhibition of trypsin or asparagrine endopeptidase. These loops represent a multifaceted surface that could potentially bind other classes of proteases.

It has been found that some members of the macroscopic family have extremely high activity and their use is particularly preferred. These are Mcpl, Mcp3 and Mcp4, which are described in more detail below.

Preferably, the cysteine proteases of the papain family are inhibited. Endopeptidases from this family are strongly inhibited by all the macrocipins tested. Cathepsin B, which is an exo- and endopeptidase, is weakly inhibited by Mcpl and Mcp4. Mcp3 and Mcp4 also effectively inhibit cathepsin H, which also has exo- and endopeptidase activity. In addition to cysteine proteases of the Cl family, Mcpl and Mcp3 also inhibit cysteine proteases of the C13 family, i.e., asparagine endopeptidases. The cysteine protease legumain is inhibited by all macrocipins except one representative (Mcp4), which instead weakly inhibits the serine protease trypsin.

Examples of macrocyclic polynucleotides that have proven useful in the present invention include, but are not limited to, polynucleotides ID. NO. ZAP. 1-13 (see Table 1 below). Macrocycine polynucleotides and variants thereof are suitable for use in accordance with the present invention. Variants of macrocyclic polynucleotides are typically those that have the same function as naturally occurring macrocipin polynucleotides.

Macrocycine polynucleotide variants can be prepared by methods well known in the art. For example, through directed mutagenesis, various macrocyclic polynucleic acids are obtained. A number of methods are known for targeted mutagenesis and are available to one of skill in the art, most commonly based on polymerase chain reaction amplification.

The sequences covered by the definition of the word macrocipin or macrocipin homologs are not limited to the sequences disclosed in this application (ID NO. 14-22, see Table 1 below), but also include all polypeptide-like sequences thereof with a similar twist and the like. functionality and activity and can be used in accordance with the present invention. Methods for determining the three-dimensional structure of proteins and for determining protein folding are well known to those skilled in the art and include X-ray crystallography and NMR (nuclear magnetic resonance).

Where GenBank Accession Numbers are listed, they refer to GenBank version 183.0 (accessed: 14 April 2011), which can be found at http: //www.ncbi.nlm.nih.ROv/ sites / gquerv.

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Table 1: Sequences disclosed in this application

ID. NO. ZAP./ Identification number sequences description accession numbers Of GenBank DNA / protein 1 Mcp4a (optimized sequence for expression in potatoes) DNA 2 macrocipin la gen, the entire coding sequence FJ495240.1 DNA 3 macrocipin la mRNA, the entire coding sequence FJ495239.1 DNA 4 macrocipin lb gene, complete coding sequence FJ495241.1 DNA 5 macrocipin lc mRNA, complete coding sequence FJ495242.1 DNA 6 macrocipin 2a gene, complete coding sequence FJ495243.1 DNA 7 macrocipin 2b gene, complete coding sequence FJ495244.1 DNA 8 macrocipin 3a mRNA, complete coding sequence FJ495245.1 DNA 9 macrocipin 3b gene, promoter and full coding sequence FJ495246.1 DNA 10 macrocypin 3c mRNA, complete coding sequence FJ49 5247.1 DNA 11 macrocypin 4a mRNA, complete coding sequence FJ495248.1 DNA 12 macrocipin 4b gene, complete coding sequence FJ495249.1 DNA 13 macrocipin 5a gene, complete coding sequence FJ495250.1 DNA 14 macroccipine 1 ACL99724 protein 15 macroccipine 1 ACL99725 protein 16 macroccipine 1 ACL99726 protein 17 macrocypin 3 ACL99729 protein 18 macrocypin 3 ACL99731 protein 19 macrocypin 4 ACL99727 protein 20 macrocypin 4 ACL99732 protein 21 macrocypin 4 ACL99733 protein 22 macrocypin 4 ACL99734 protein

The sequences used in accordance with the present invention are all sequences having at least 63% identity to the disclosed sequences obtained from Macrolepiota procera fungus. Less than 63% similarity to genes or cDNA sequences is too low to be useful. Preferably, the identity is at least 68% or even at least 73%.

Macrocipins or their homologs can be readily identified by routine methods well known to those skilled in the art. An example of homolog identification is by sequence alignment. Sequence alignment and comparison are well known to those skilled in the art and made possible by methods such as GAP, BESTFIT, BLAST, FASTA, TFASTA and tblastn (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&BLAST PROGRAMS = tblastn & PAGE ΤΥΡΕ = BlastSearch & SHOW DEFAULTS = on & LINK LOC = blasthome).

The tblastn 2.2.25+ algorithm available on the NCBI website is well known. This algorithm makes it possible to compare a protein sequence with a nucleotide sequence in which it checks 6 reading frames. It is useful for finding regions that encode homologous proteins in untreated nucleotide sequences. The algorithm is also useful for verifying that a protein sequence is homologous to macrocypins as described in this application. It is also useful for verifying that a gene or cDNA sequence is homologous to a gene or cDNA sequence as described in this application by comparing said predicted amino acid sequence of a gene or cDNA with a tblastn.

When comparing a potential macrocipin homologue using the tblastn algorithm, the protein sequence can be compared to a nucleotide database called a nucleotide collection (nr / nt). It is the largest database available through NCBI BLAST and includes all sequences from GenBank, RefSeq Nucleotides, EMBL (Japanese nucleotide database), DDBJ (Japanese nucleotide database) and PDB protein databases (Protein Data Bank) ). The cut off limit for the expected value of E must be set to 0.1. E is a value that describes the likelihood that sequence similarity is random.

Table 2 shows an example of alignment with the Mcp4 protein sequence (ID NO. CLOSE 20; ACL99732.1). Only the results of the comparison with previously published polynucleotides (ID no. Zap. 2-13) encoding macrocipins with at least 73% similarity to Mcp4 are shown.

Table 2: Comparison of sequence ACL99732.1 (Mcp4) with nucleotide collection (nr / nt) (threshold E <0.1) using the tblastn algorithm.

accession numbers Of GenBank ID. NO. ZAP. Description Maximum identity value E FJ495248.1 SEQID NO 11 Macrolepiota procera macrocipin 4a mRNA, total the coding sequence 100% 3e-92 FJ495249.1 SEQUE 12 Macrolepiota procera macrocipin 4a gene, promoter in the entire coding sequence 89% le-80 FJ495247.1 SEQ ID NO 10 Macrolepiota procera macrocipin 3c mRNA, total the coding sequence 82% 3e-75 FJ495245.1 SEOIDNO8 Macrolepiota procera macrocipin 3a mRNA, total the coding sequence 81% le-73 FJ495242.1 SEOIDNO5 Macrolepiota procera macrocipin lc mRNA, whole the coding sequence 80% 4e-72 FJ495239.1 SEQIDNO3 Macrolepiota procera macrocipin la mRNA, total the coding sequence 79% 9e-72 FJ495241.1 SEQIDNO4 Macrolepiota procera macrocipin lb mRNA, whole the coding sequence 79% 8e-71 FJ495244.1 SEQIDNO7 Macrolepiota procera macrocipin 2b gene, full coding sequence 78% 6e-66 FJ495243.1 SEOIDNO6 Macrolepiota procera macrocipin 2a gene, full coding sequence 74% 4e-65 FJ495246.1 SEQIDNO9 Mocrolepiota procera macrocipin 3b gene, full coding sequence 78% 9e-51

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FJ495250.1 SEQUE 13 Macrolepiota procera macrocipin sequence 5a gene, whole coding 81% 3e-47 FJ495240.1 SEQIDNO2 Macrolepiota procera macrocipin sequence la gen, the whole coding 73% 2e-41

Macrocypin polypeptides and their homologs that can be used in accordance with the present invention may also be derivatives thereof, as shown above. The term derivatives refers to peptides, oligopeptides, polypeptides, proteins, and enzymes which, when compared to natural proteins (some listed in Table 1 as ID NO. CLOSE 14-22), may involve substitutions, deletions or inclusions of natural or synthetic amino acid residues. Preferably, however, the present invention particularly protects those homologs, variants and derivatives of any of the two-head inhibitory naturally occurring macrocipins.

Macrocipin polypeptides and their homologs may also encode alternative splicing variants of macrocipin nucleic acids or genes. The term alternative splicing variant herein includes variants of nucleic acid sequences in which particular introns and / or exons have been excised, replaced, added, truncated or extended. Among such variants are those which will preserve the biological activity of the encoded protein. Such variants can be obtained by selectively selecting functional segments of the protein. Macrocipin variants obtained as alternative splicing variants can be found in nature or synthesized. Methods for the synthesis of alternative coupling variants are well known in the art.

The polypeptides of the present invention are useful for protecting plants, especially crops. Examples of plants that could be protected from pests by using these proteins are monocotyledons and dicotyledons, preferably those of the families Solanaceae, Poaceae, Fabaceae, Brassicaceae, Musaceae, Cucurbitaceae, Apiaceae, Rosaceae and Salicaceae, preferably S. tuberosum, S. Iycopersicum and 5. melongena, or from the genus Nicotiana, preferably N. tabacum or N. benthamiana.

The plants of the genus Solanum and Nicotiana, for example, N. tabacum, N. benthamiana and S. tuberosum, are the most preferred ones, as these species are among the best researched and most commonly used for protein production in plants.

The polypeptides of the present invention are useful for protection against pests of agricultural plants. The pests that can be combated with these proteins may, but need not be, selected from the insect groups of Lepidoptera, Hemiptera, Diptera and Coleoptera, preferably over the pest of the species Leptinotarsa decemlineata.

The polypeptides of the present invention have been found to be particularly useful for protection against the following pests: representatives of the Hemiptera order include pests from the families Aphididae, Aleyrodidae, Delphacidae, Psyllidae, Jassidae and Coccidae. From the family Aphididae are representatives of Aphis gossypii, Myzus persicae, Hyalopterus pruni, Lipaphis erysimi, Brevicoryne brassicae; the Delphacidae family may be pests of Nilaparvata lugens; from the family Aleyrodidae of the species Bemisia tabaci gennadius; from the Jassidae family Nephotettix bipunctatus; and from the family Coccidae species Unaspis yanonensis. Pests from the Lepidoptera order include pests from the families Noctuidae and Plutellidae. The pests of the Noctuidae family are of the genus Asparagus, Spodoptera litura Fab or Helicoverpa armigera; a pest of the family Plutellidae, but a species of Plutella xylostella.

The polypeptides of the present invention can be used to protect plants in various ways, which are described in more detail below.

In all approaches, one of the macrocycins and / or a variant, homolog or derivative thereof may be used for protection, or a combination of one or more different macrocipins and / or its variants, homologs or derivatives may also be used for protection, variants, homologs or derivatives derived from the same macrocipin. Whenever the term polypeptide or macrocipin is used throughout this document, it refers to macrocipin and / or its variant, homolog or derivative, or to a combination of different macrocipins and / or one or more of its variants, homologs or derivatives, or to two or more variants , derivatives and homologs derived from the same macrocipin or any combination, except in a context that does not permit this interpretation.

In the present invention, an intrinsic agent is used, that is, at least one polypeptide that is active as a pesticide but not added as a polypeptide but produced as such in the plant as follows: into the plant genome of the plant to be protected, a stable insert of a polynucleotide sequence encoding at least one polypeptide or any combination of the natural or modified macrocipins of the present invention. To protect this plant, an inserted polynucleotide or combination of polynucleotides, after stable integration into the genome, must be expressed, but this expression may or may not be induced (in the case of expression control by an inducible promoter). In other words, the plant itself produces a pest control agent. In this part of the invention, a polynucleotide sequence encoding at least one polypeptide of the present invention is inserted into the plant or plant genome. This allows the plant to produce the pesticide agent itself. One or more polynucleotide sequences may be used that can encode one or more polypeptides of the present invention. Thus, one polynucleotide may contain information for one or more polypeptides of the same or different types, or two or more polynucleotides encoding different polypeptides or different combinations of polypeptides may be used.

Methods for obtaining stable genetically modified plants are known to those skilled in the art. Genetically modified plants are those plants which have at least one heterologous polynucleotide sequence inserted and stably inserted into the genome.

Methods for introducing large heterologous nucleotide sequences into plant cells are well known. Those of skill in the art can choose an intake method that is appropriate for the plant we are modifying and the vector we use to aid in gene transfer. The optimal combinations of plant and vector are therefore known, or can be determined by routine experiments.

The method of gene entry into plants may be chemical, such as transfection with calcium phosphate, cyclodextrin, liposomes (lipofection), or transformation of plastids with polyethylene glycol (PEG); or non-chemical methods such as biolistically firing a gene rifle, electroporation, magnifying, and sonoporation, or virus transduction.

All of these methods or combinations of these methods are suitable for introducing the polynucleotides of the present invention into plants.

One method that allows the introduction of polinkleotides into plants is transformation by agrobacteria. Bacteria with viral expression systems may be from different groups, some examples being Agrobacterium tumefaciens, A. rhizogenes, Sinorhizobium meliloti, Rhizobium sp. and Mesorhizobium lots, and other bacteria can be used.

Plants can be transformed by vectors containing the corresponding polynucleotide sequence encoding at least one of the macrocipins, a variant thereof, a homolog or a derivative. A polynucleotide sequence encoding at least one of the macrocipins is properly coupled to one or more control sequences, at least to the promoter. The terms regulatory element, control sequence, and promoter are interchangeable throughout this document and should be understood as regulatory polynucleotide sequences that may affect the expression of the sequences with which they are ligated. The above terms also encompass transcriptional regulators of sequences derived from classic eukaryotic genomic sequences and from additional regulatory elements such as the above activation sequences, amplification sequences, silencing sequences that alter gene expression in response to a developmental or external stimulus or alter expression to tissue specificity. The term appropriately related herein refers to a functional link between a promoter sequence and a macronuclein-encoding polynucleotide sequence, meaning that this promoter sequence can initiate transcription of this polynucleotide. For the expression of a polynucleotide sequence, any promoter suitable for expression in the plant and suitable for expression of the introduced sequence may be used. The choice of promoter is not decisive. Selection of the optimal promoter is a routine task for the specialist, methods for selecting the appropriate promoters are published. Examples of suitable promoters are p35s and Act2. The promoter used can be inducible, which means that transcription is triggered or augmented in response to a developmental, chemical, environmental, or physical stimulus. The promoter may also be chemically regulated, such as promoters in which transcription is triggered by the presence or absence of molecules such as alcohol, tetracycline, steroids or metals. Also, promoters can be physically regulated. Examples of such promoters are transcription regulated by the presence or absence of light, or by low or high temperature. An example of an inducible promoter is also a stress-inducible promoter, that is, a promoter in which transcription is induced by stress factors. In addition or alternatively, the promoter may also be tissue specific, which means that it is able to preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, etc. Promoters that are capable of initiating transcription predominantly in specific tissues are referred to herein as tissue-specific. Inducible promoters may be used to trigger macrocipin transcription. Using these, macropecin expression can be switched on and off depending on the presence or absence of external stimuli and the configuration of the expression system. Using an inducible promoter, expression of pesticidal activity can be included at the command. With the use of more than one macronuclein-encoding polynucleotide, the expression of each can be regulated by inducible promoters, for example, at different times or gradually.

Thus, the expression of one or more macrocipins can be controlled by the use of different inducible promoters. In the absence of an external stimulus, expression is low or nonexistent, and with a temporally controlled external stimulus, that is, when a pest is approached or present, macrospecin expression is triggered or increased. Expression can also be included at an early stage in the development of genetically modified plants, that is, at a time when the plants are most susceptible to pest attack. By using different promoters for different macronuclei encoding polynucleotides, we can control the potency of pesticidal activity. The expression of macrocipin in an adult plant can then be reduced or stopped, allowing the plant to produce more intensively plant biomass or crop production.

Preferably, the polynucleotide encoding macrocypin or a variant thereof is appropriately linked to a constitutive promoter. The constitutive promoter is transcriptionally active among most, but not necessarily all, stages of growth and development and is expressed in all tissues.

One or more terminator sequences may also be used in a construct to be inserted into a plant. The term terminator encompasses a DNA control sequence at the end of a transcription unit that signals the processing of the 3 'end and polyadenylation of the primary transcript and the end of transcription. Additional regulatory elements may also include transcriptional and translational accelerators. Those of skill in the art will know which promotional sequences are suitable for use in the present invention. These sequences are published and can be retrieved regularly by experts in the field.

Likewise, the gene constructs present in the present invention may include sequences of origin of replication (ORI) sites that are required for maintenance and / or duplication in specific cell lines. An example is where a genetic construct must first be introduced and preserved in a bacterial cell as an episomal genetic element (i.e. a plasmid or cosmid). Preferably, the start sites are fl-ori and colEl, but other sequences may be used.

A genetic construct may, but need not be, comprise at least one gene for a selection marker. In this text, the term selection marker refers to any gene that contributes to the altered phenotype of the cell in which it is expressed. In this way, the identification and / or selection of cells that, by transfection or transformation, have received the construct with the nucleotide record described in this invention is accelerated. Suitable marker genes are well known in the art. We can choose from those that allow antibiotic or herbicide resistance, or those that allow the synthesis of new metabolites or selection through visualization. Examples of selection markers are genes that confer antibiotic resistance such as neomycin and kanamycin.

Preferably, the pMDC32 vector (see Figure 2a) is used for the preparation of the genetically modified plants, containing a macronuclein encoding a polynucleotide. The vector sequence pMDC32 was published in 2003 in Plant Physiol. (Oct; 133 (2): 462-9) published by Curtis. One version of this vector is published in GenBank under accession number FJ172534.

Preferably, the polynucleotide sequence is adapted for plant uptake according to the codon's plant use, as this increases the level and efficiency of expression. Optimization can be accomplished by using software tools available online such as GenScript, or by using molecular genetic methods known to those of skill in the art.

Heterologous sequences can also be inserted into plant cells by high-pressure bacterial culture injection. In this case, it is necessary to prepare the plant tissue, for example with mechanical abrasives (silicon carbide or carburund), to increase transfection efficiency.

In addition to the methods listed above, heterologous sequences can be introduced into plant tissues by magnifection.

Gene entry for the production of genetically modified plants may be stable or transient.

The plant cells may be part of the whole plant or only part of the plant tissue.

Transformed plants thus obtained can be propagated in many ways, such as clonal propagation or classical methods of cultivation. An example is first-generation (Tl) plants that, after self-pollination, form second-generation (T2) plants that are homozygous. These plants can be further grown using conventional methods of propagation.

The cultivation of genetically modified plants containing at least one polynucleotide encoding Mcp4 (ID NO. 19-22) or variants, homologs or derivatives thereof is preferred, as Mcp4 has the ability to inhibit cysteine and serine protease groups and glycoside hydrolase, resulting in effective protection of genetically modified plants from pests.

In another embodiment of the present invention, the polynucleotide described in the present invention is used to produce a polypeptide with pesticidal efficacy. In this part of the invention, a polynucleotide sequence encoding at least one polypeptide with pesticidal efficiency, as defined in the claims, is used to produce polypeptides in a bioreactor. The polypeptide obtained in the bioreactor can be used as a pesticide agent after the purification process. Preferably, the polynucleotide sequence is part of a vector and this vector is then used for heterologous expression of the recombinant protein in the bioreactor. In the reactor, this polypeptide is expressed and processed to produce an active and at the same time stable protein that can be used to protect against pests, especially crop pests. However, it is also possible to use other expression systems to produce the polypeptide described in the present invention. These methods are known to those skilled in the art and can be used in such a way that the polypeptide described in the present invention is obtained in an accessible and stable form.

If a vector is used to obtain the polypeptide, the vector may comprise a polynucleotide encoding at least one macrocipin or variant, homolog or derivative thereof. However, it may also contain a combination of different macrocipins and / or a combination of different variants, homologs or derivatives of macrocipin or a mixture thereof. The vector may have the same general characteristics as described for the vectors used for the production of genetically modified plants. These general features are, for example, control and regulatory sequences, marker genes, multiple cloning sites, promoters, amplification sequences or sites for initiation of duplication.

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Preferably, the host organism Escherichia coli is used for the production of heterologous recombinant proteins, since it is one of the most useful host organisms for the production of recombinant proteins and is one of the most known microorganisms from a genetic point of view.

The "pET system" (Novagen, Madison, Wl, USA) is a suitable vector system for heterologous expression in E. coli.

One of ordinary skill in the art can select the most effective combination of promoter and polynucleotide to obtain a high yield of recombinant protein.

Macrocipins can be obtained as a single polypeptide or in combination of different polypeptides. For example, using combinations of pETlla: Mcpl, pET3a: Mcp3 and pET14b: Mcp4, high yields of three different recombinant macrocipins were obtained in the bacterial expression system (see Sabotic et al., 2009).

Any modification of the protein purification process that improves the yield, processing, purification or activity of the protein may be used. An example of modification is the use of the addition of localization signal sequences or markers, such as His-tag.

The final phase of protein purification can be done in the manner described in the literature. Protein purification methods are well known to those skilled in the art and their use is routine. Macrocipins or variants, homologs or derivatives thereof may be used for protection as a single protein or in any protein blend.

Macrocipins or variants, homologs or derivatives thereof may be obtained in the form of insoluble inclusion bodies, in soluble form or as a mixture thereof. If obtained in insoluble form, it is advisable to convert them to soluble form before application to the plants.

There are several ways to get the proteins you want. Some of the methods are repeated freezing and thawing, sonication, high pressure homogenization, filtration or permeabilization with organic solvents. The method we choose depends on the sensitivity of the protein and the strength of the cells from which the protein is isolated. Macrocipins remain stable with short-term exposure to extreme pH, which takes values between pH 2 and pH 12. They are also extremely temperature-stable, maintaining their inhibitory activity at 4 ° C for several months, at room temperature for several weeks, and even at 100 for a few minutes. ° C. Macrocypins therefore have ideal properties for insulation processes, since they do not affect the reduction of their functionality and yield.

Because macrocipins are very stable, their dissolution is possible with the addition of 3M urea. Further cleaning processes may then be such as is known to those skilled in the art.

Macrocycine purification methods may include precipitation, differential dissolution, ultracentrifugation, and chromatographic methods such as size separation chromatography, (immuno) affinity chromatography, ion exchange chromatography, metal bonding by His tagging or HPLC. An example is the isolation of recombinant Mcpl and Mcp4 by size separation chromatography.

Purified macrocipin, a variant, homolog, derivative or any combination thereof, may be concentrated using lyophilization or ultrafiltration.

Purified and concentrated macrocipin or homologs thereof are readily available in the form generally used for application to plants. One or more of the macrocipins of the present invention may be mixed with excipients or dissolved and accessible in a form suitable for application to plants. Also, for the application of the pesticide active ingredient, a form that allows for custom delivery is also possible.

Preferably, the composition of the pesticidal agent containing macroccipin in dissolved form with a pH between 6 and 9 is preferred, as macrocipins are the most stable in this pH range.

The invention will now be illustrated with some examples. These examples do not limit the use of the invention but merely interpret it.

Examples

Example 1

Macrocipin action was studied by digestive protease inhibition isolates isolated from the gastrointestinal larvae of the Colorado beetle. In this way we wanted to find out what is the mode of action of macrocipin that allows pesticidal activity. Gruden et al. (1998) have shown that cysteine proteases represent a dominant proportion of digestive proteolytic activity in the digestion of Colorado beetle larvae. First, using a different substrate and different concentrations of the inhibitor, an inhibition test was performed on a coarse digestion of Colorado beetle larvae. No inhibition was detected in this test. Then, prior to the inhibition test, the digestive proteins of the Colorado potato beetle larvae were partially purified by acetone precipitation. In this case, too, the inhibitory action of macrocypin on the digestive proteases was not detected. In the third assay, however, protein extract from the digestive system of Colorado beetle larvae was fractionated by gel filtration after acetone precipitation. After fractionation, proteolytic activity on Z21 substrates was measured in each fraction

Phe-Arg-pNa, Z-Arg-Arg-pNA, and pGlu-Phe-Leu-pNA with and without the addition of single recombinant macrocipins (Mcpl, Mcp3, Mcp4).

The results of this experiment are shown in Figure 1. None of the macrocipins inhibited the activity of digestive proteases on Z-Phe-Arg-pNa and Z-Arg-Arg-pNA substrates. All macrocipins had only weak inhibitory activity for degradation of the pGlu-Phe-Leu-pNA substrate, and only at the highest inhibitor concentrations.

Example 2

In order to locate the macrospecin target in the digestion of Colorado potato beetle larvae, affinity chromatography was performed, where Mcpl was bound to the monolithic support. Homogenization produced a coarse extract of entire Colorado beetle digesters isolated from fourth-stage larvae that had been harvested in the field. The extract was then purified by centrifugation at 4 ° C for 15 min at 16,000 g and separated into fractions by size separation chromatography. Fractions containing the protein (this was determined by measuring the absorbance of the fraction at a wavelength of 280 nm) were combined and applied to affinity chromatography with macrocipin. After column washing, the bound proteins were washed with pH lowering. The washed proteins were then analyzed by SDS-PAGE gel electrophoresis, and individual protein spots were then excised from the gel. The individual spots were then digested with trypsin gel and analyzed by mass spectrometry (ESI-MS-MS). The MSDTrap XCT Ultra mass spectrometer (Agilent, USA) was used.

Results

Using macroscopic affinity chromatography, several potential target molecules from the gastrointestinal larvae of the Colorado beetle larvae to which macroscopin binds were revealed. These molecules are intestines, glycoside hydrolase from family 48, and diapause protein 1 (see Table 3 below).

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Table 3. Potential targets of macrocipin in the digestion of Colorado beetle larvae. The target molecules were determined using single spot mass spectrometry analyzed by gel electrophoresis of proteins obtained by affinity chromatography with Mcpl.

Protein identified Accession numbers Of GenBank Molecular mass glycoside hydrolase from family 48 ADU33353 73 kDa diapause protein 1 (Leptinotarsa decemlineata) CAA53691 80 kDa glycoside hydrolase from family 48 ADU33352 73 kDa digestive cysteine protease intestain (Leptinotarsa decemlineata} AAS20589 36 kDa digestive cysteine protease intestain AAS20591 36 kDa

Example 3

Binary vector preparation

Because the use of codons in potatoes is different from that of fungi, the sequence of the gene encoding Mcp4A (GenBank accession number FJ495248.1, ID no. ZAP. 11) was adapted using software on the GenScript website. A sequence (ID no. ZAP.l in Table 1) was then adapted to encode the record for Mcp4A ordered in GeneScript. This synthetic gene has already been inserted into the pDONR vector.

In a recombination reaction using the LR clonase, a DNA fragment from the pDONR vector was then transferred to the pMDC32 destination vector (Curtis and Grossniklaus 2003) according to the manufacturer's (Invitrogen) instructions. The plasmid map of the destination vector pMDC32 is shown in Figure 2. The recombination product was used to transform competent cells of E. coli bacterium with the OmniMAX ™ strain with temperature shock. Plasmids were transferred to ElectroMAX ™ Agrobacterium tumefaciens strain LBA4404 by electroporation (Invitrogen, USA).

Example 4

Plant material, growth conditions and transformation

The potatoes (Solanum tuberosum L. cv. Desiree) were vegetatively multiplied from the stem cuttings and grown in modified Murashige-Skoog medium with 3% sucrose. The plants were grown at 24 ° C and light 5000 lux with a daily night cycle of 16 hours / 8 hours.

The pMDC32 binary vector was used to obtain genetically modified potatoes. The vector was introduced into the potato by transformation with Agrobacterium tumefaciens strain LBA4404. Transformation was performed using potato slices infected with A. tumefaciens according to the method described previously (Visser, Jacobsen, Hesselingmeinders, Schans, Witholt, and Feenstra 1989). Transformed plants were collected and regenerated on selection media containing hygromycin.

Selection of genetically modified potato lines (qPCR)

In lineages grown on selection media, transgene expression was analyzed in real-time using pre-reverse transcription PCR. The results of transgene expression are shown in Figure 3. In the potato lines most expressing the gene encoding Mcp4A, the presence of the recombinant protein was further determined by Western transduction.

Transmission after evening

The Western blot method was used to detect recombinant Mcp in genetically modified plants. 200 mg of plant material per line was used for protein isolation. The plant material was homogenized using a Qiagen Tissuelyser homogenizer and resuspended in 500 μΙ buffer (50 mM Tris-HCI, pH 6.8, 15 mM DTT). After 30 minutes of incubation at 0 ° C, the samples were centrifuged and 30 μΙ of the supernatant was applied to each well of the polyacrylamide gel. Proteins were separated by gel electrophoresis in the presence of NaDS (NaDSPAGE, 12% separation gel). After electrophoresis, the proteins were transferred to the PVDF membrane (Imobillon transfer membrane). The membrane was then blocked with TBS buffer (10 mM Tris-HCI pH 7.4,150 mM NaCI) with 5% fat-free milk powder added. Incubation with primary antibodies diluted 1: 10,000 was performed overnight at 4 ° C, followed by 7 washes with TBST buffer at 25 ° C and incubation with secondary antibodies diluted 1: 20,000. The rabbit antiserum against macrocypin was used as primary antibodies, and horseradish peroxidase conjugated goat immunoglobulin as secondary antibodies. Detection was performed with a chemiluminescent detection test (Lumilight, Roche). The results of the western transfer are shown in Figure 4.

Preparation of vectors for the production of recombinant proteins in E.coli

The pET series vectors (Novagen) were used to produce recombinant macrocipins. Mcpl cDNA was cloned by ligation after restriction of the pET11 vector with Ndel and BamHI enzymes, Mcp3 after restriction of the pET3a vector with Ndel and BamHI and Mcp4 after restriction of the pET14b vector with Ncol and Ndel enzymes, so that the selected proteins were expressed without markers.

Expression of macrocypins in the form of insoluble inclusion bodies in E. coli and their isolation

Inserted expression vectors were inserted into E. coli bacterium strain BL21 (DE3). The bacteria were cultured in LB liquid medium (Luria-Bertani) supplemented with ampicillin at a concentration of 100 pg / ml at 37 ° C and 220 rpm. When attenuation (D) at 600 nm reached values between 1 and 1.2, an isopropyl thio-d-galactoside inducer at a final concentration of 0.4 mM was added to the strain transformed with the pET3a and pET14b vectors, and the strain transformed with the pETlla vector into final concentration of 1 mM. The bacteria were then grown for 6 hours. Cells were then isolated by centrifugation at 6000 g for 15 minutes and resuspended in buffer A (50 mm Tris-HCl, pH 7.5, 0.1% Triton Χ-100, 2 mM EDTA), then frozen and thawed once and sonicated at 4 ° C. The insoluble fraction was separated by centrifugation at 10,000 g for 15 minutes, redissolved in the same buffer with 3 M urea added and solubilized by stirring at 4 ° C for four hours. The remaining undissolved material was removed by centrifugation, and the supernatant was applied to a Sepharose S200 column equilibrated with Tris-HCl buffer, pH 7.5, with 0.3 M NaCl added. The fractions with inhibitory activity were combined and concentrated by ultrafiltration (Amicon UM-3).

Expression of macrocypins in soluble form

Insert expression vectors were introduced into E. coli BL21 (DE3). The bacteria were cultured at 37 ° C in liquid medium LB (Luria-Bertani) supplemented with ampicillin at a concentration of 100 pg / ml. When attenuation (D) at 600 nm reached values between 1 and 1.2, an isopropyl thio-βd-galactoside inducer at a final concentration of 1 mM was added to the strain. Expression of recombinant macrocypins in soluble form was achieved after 12 to 24 h incubation at 23 ° C and 220 rpm.

Nutritional tests

Nutritional tests with genetically modified plants were performed with the whole above-ground part of the potato plant. To maintain moisture, the stems of the cut plants were thrust into microcentrifuges containing rare agar (0.5%). Petri dishes in which the larvae were reared were kept in the dark at 28 ° C during the experiment. 14 to 20 larvae were used for each test. The larvae have already hatched on the tested potato lines, which have been replaced daily with freshly prepared plants. Wild type (WT) potatoes were used as controls. Each day, we monitored survival rates and larval mass. The results were analyzed using the Student's T-test.

Nutritional assays with recombinant proteins were performed with non-transformed potato leaves coated with recombinant proteins, and the feeds fed to the control group were coated with bovine serum albumin (BSA). The leaves were prepared by soaking them in protein solutions for a while from all sides. The larvae were kept in the dark at 28 ° C. 16 larvae were used for each test. The leaves were replaced daily with fresh ones. Each day, we monitored survival rates and larval mass. The results were analyzed using the Student's T-test.

The results of the dietary tests are shown in Figures 5-10.

Claims (14)

PATENT APPLICATIONS Use of at least one polynucleotide sequence, characterized in that it encodes at least one macrocipin or a variant thereof, for the production of a pesticide agent effective against organisms that adversely affect the growth of the plants that we wish to protect against that organism. Use according to claim 1, characterized in that the polynucleotide sequence is stably inserted into the genome of the genetically modified plant. Use according to claim 1, characterized in that the polynucleotide sequence is part of a polynucleic acid for heterologous expression of the protein in the bioreactor. Use according to claims 1 and 2, characterized in that the plant species is selected from monocotyledons or dicotyledons, preferably from the plant families Solanaceae, Poaceae, Fabaceae, Brassi caceae, Musaceae, Cucurbitac eae, Apiaceae, R osaceae and Salicaceae, preferably of the genus Solanum, preferably S. tuberosum, Solanum lycopersicum and Solanum melongena, or of the genus Nicotiana, preferably N. tabacum or N. benthamiana. Use according to any one of the preceding claims, characterized in that the pesticidal agent inhibits the growth and / or survival of organisms from the species Lepidoptera, Hemiptera, Diptera and Coleoptera, preferably where the organism is a Colorado beetle (Leptinotarsa decemlineata). Use according to any one of the preceding claims, characterized in that the polynucleotide sequence encodes at least one macrocipin indicated by identification number (ID NO. ZAP.) 1-13 or a variant, homolog or derivative thereof. Use according to claim 2, characterized in that the polynucleotide sequence encodes Mcp4 and preferably comprises at least one of the sequences indicated by identification numbers 1, 11 or 12, or variants thereof, or wherein the Mcp4 polypeptide is expressed using a polynucleotide, one of the polypeptides indicated by the identification numbers 19, 20, 21 and / or 22, or homologous thereof. Use according to claim 3, characterized in that the polynucleotide sequence encodes Mcpl and preferably comprises at least one of the sequences indicated by identification numbers 2, 3, 4 or 5 or variants thereof, or where the Mcpl polypeptide is expressed using a polynucleotide, one of the polypeptides indicated by, or having a homolog of, numbers 14, 15 or 16. Use according to claim 3, characterized in that the polypeptide obtained in the bioreactor is purified and repurposed in a form suitable for application to plants such as agricultural plants, preferably the plants mentioned in claim 4. A method for the preparation of a pest resistant plant comprising the use of at least one polynucleotide sequence according to claim 1 or a variant, homolog or derivative thereof or a mixture thereof for introduction into the genome of these plants. Method for the preparation of at least one of the polypeptides according to claim 1 or a variant, homolog or derivative thereof, or a mixture thereof, for the preparation of plants resistant to pests. A vector comprising at least one polynucleotide sequence as defined in claim 1. A bacterium comprising the vector of claim 12. A genetically modified plant, characterized in that it contains at least one stable polynucleotide sequence set forth in claim 1 in the genome.