JP2013039064A - Insect resistant protein, and insect resistant gene encoding insect resistant protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an insect resistant protein exhibiting sufficient insect resistance against insects even in a small amount, an insect resistant gene encoding the insect resistant protein, a recombinant DNA obtained by recombining the insect resistant gene, a recombinant vector containing the same, a host cell and a plant cell introduced with the recombinant vector, a transformed body obtained by introducing the recombinant vector into a host, a recovered protein recovered with them, and an insect resistant agent containing the same as an active ingredient.SOLUTION: This insect resistant protein having a specific amino acid sequence originated from a plant, desirably from the exudate of a plant of Cucurbitaceae such as Benincasa cerifera, etc., is provided by showing ≥60% homology with the amino acid sequence, or having an amino acid sequence showing ≥60% homology with phloemlectin.

Description

  The present invention relates to an insect-resistant protein, an insect-resistant gene encoding the insect-resistant protein, a recombinant DNA obtained by recombining the insect-resistant gene, a recombinant vector containing these, and a host into which the recombinant vector has been introduced Cells and plant cells, transformants comprising host cells and transformants which are descendants or clones thereof, transformed plants comprising plant cells and transformed plants which are descendants or clones thereof, and propagation materials of transformed plants Further, the present invention relates to recovered proteins recovered by these, and insecticides containing these as active ingredients.

A protein having insect resistance (hereinafter referred to as “insect-resistant protein”) is an indispensable material for introducing an insect-resistant gene into a plant and performing genetic breeding of the insect-resistant plant.
Moreover, it is also possible to use as insecticides, such as a novel agrochemical, by spraying the insect-resistant protein which was expressed and collect | recovered to microorganisms, a cultured cell, and a multicellular animal and plant individual.

  Currently, an insect-resistant protein widely used in industry includes Bt toxin (protein) produced by Bacillus thuringiensis, a Gram-positive bacterium. It is known that such Bt toxin exhibits insecticidal / insect-resistant activity at a low concentration (about 1 ppm) (see, for example, Non-Patent Document 1 or 2).

However, the Bt toxin is derived from bacteria and has a persistent resistance to use as a genetic resource for recombinants such as genetic recombination work.
In addition, with regard to Bt toxins, the emergence of resistant and resistant insects has been feared and reported, and in fact, resistant insects have emerged in the Japanese moths such as Japanese moths. From these things, discovery of plant-derived insect-resistant protein is desired.

In contrast, plant-derived insect-resistant proteins include cowpea-derived protease inhibitors (for example, see Patent Document 1 or Non-Patent Document 3), kidney bean-derived amylase inhibitors (for example, see Non-Patent Document 4), Snowdrop-derived lectins (see, for example, Patent Document 2 or Non-Patent Document 5) are known.
In addition, the present inventors have invented MLX56, which is an insect-resistant protein derived from mulberry, and have applied for a patent (see, for example, Patent Document 3).

U.S. Pat. No. 4,640,836 US Pat. No. 5,545,820 JP 2008-245640 A

Canadian Journal of Microbiology 51, 988-995 (2005) Journal of Pesticide Reform 14, 13-20 (1994) Pest Management Science 57, 57-65 (2001) Plant Physiology 107,1233-1239 (1995) Journal of InsectPhysiology 43, 727-739 (1997)

 However, the protease inhibitor described in Patent Document 1 or Non-Patent Document 3, the amylase inhibitor described in Non-Patent Document 4), and the lectin described in Patent Document 2 or Non-Patent Document 5 all have low insect resistance activity. There are drawbacks. That is, protease inhibitors exhibit insect-resistant activity only to the extent that the growth of moth larvae was halved in 2 weeks, even when added at a high concentration reaching 2% of the total protein. When amylase inhibitors are expressed in peas, they are expressed in a large amount of 2-3% of the total soluble protein of beans, and finally 70% of the weevil die on the way, but 30% grows normally. Moreover, if it is 1%, more than 80% will grow normally to an adult. Lectins exhibit insect-resistant activity only to the extent that their body weight is reduced by 20 to 50% even if the larvae are fed in a diet containing 2% of the total protein amount and a large amount of the total amount of protein in an artificial feed for one month.

On the other hand, expressing a large amount (% order) of insect-resistant protein in a plant is a burden on the plant and may adversely affect the growth of the plant.
In addition, even when spraying what is expressed in bacteria or the like, there is a drawback that a large amount of insect-resistant protein must be sprayed, and the insect-resistant effect by spraying is slow, and there is a problem in pest control.

  The present inventors have invented MLX56, which is an insect-resistant protein derived from mulberry. However, considering that insects having resistance / resistance may eventually appear, another kind of There is a need to invent new insect-resistant proteins.

  The present invention relates to an insect-resistant protein exhibiting sufficient insect resistance even in a small amount, an insect-resistant gene encoding the insect-resistant protein, a recombinant DNA obtained by recombining the insect-resistant gene, Recombinant vectors containing these, host cells and plant cells into which the recombinant vector has been introduced, transformants into which the recombinant vector has been introduced into the host, recovered proteins recovered therefrom, and resistance to these containing active ingredients The purpose is to provide an insecticide.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that Togan leaves have remarkable insect resistance (toxicity / growth inhibitory activity) against erisan compared to other Cucurbitaceae leaves. It was found that the worm property was caused by a high molecular factor (protein) contained in the exudate. In addition, exudate means the liquid which exudes from the wound of a plant, for example, an emulsion, a sieve tube exudate, etc. are mentioned.
And when the protein in the exudate of tougan was newly fractionated in order to newly identify a proteinaceous insect-resistant factor derived from another species of plant, it was found that a protein having a predetermined amino acid sequence could solve the above problem The headline and the present invention were completed.

  The present invention resides in (1) a plant-derived insect-resistant protein having an amino acid sequence having 60% or more homology with a phloem lectin.

  The present invention provides (2) a plant-derived insect-resistant protein having an amino acid sequence having 60% or more homology with the first partial amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and SEQ ID NO: 3 in the sequence listing The insect-resistant protein comprising at least one amino acid sequence selected from the group consisting of amino acid sequences having 60% or more homology with the second partial amino acid sequence shown in FIG.

  The present invention resides in (3) an insect-resistant protein derived from a plant, wherein the homology to the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is 60% or more.

  The present invention resides in (4) the insect-resistant protein according to any one of the above (1) to (3), wherein the plant is a Cucurbitaceae plant and is extracted from an exudate of the Cucurbitaceae plant.

  The present invention resides in (5) an insect resistance gene encoding the insect resistance protein according to any one of (1) to (4) above.

  The present invention resides in (6) an insect resistance gene derived from a plant, wherein the homology to the base sequence represented by SEQ ID No. 4 in the sequence listing is 60% or more.

  The present invention comprises (7) a second DNA hybridized under stringent conditions with a first DNA comprising the base sequence shown in SEQ ID NO: 4 in the sequence listing, or a base sequence complementary to the second DNA. It resides in an insect resistance gene containing the third DNA.

  The present invention resides in (8) a recombinant DNA obtained by recombining the insect resistance gene according to any one of (5) to (7) above.

  The present invention resides in (9) a recombinant vector comprising the insect resistance gene according to any one of (5) to (7) above or the recombinant DNA according to (8) above.

  The present invention resides in (10) a host cell into which the recombinant vector described in (9) has been introduced.

  The present invention resides in (11) a plant cell into which the recombinant vector according to (9) is introduced.

  The present invention resides in (12) a transformant comprising the host cell described in (10) above.

  The present invention resides in (13) a transformant which is a descendant or clone of the transformant described in (12) above.

  This invention exists in the transformed plant body containing the plant cell of (14) said (11) description.

  The present invention resides in (15) a transformed plant that is a descendant or clone of the transformed plant described in (14) above.

  The present invention resides in (16) a propagation material obtained from the transformed plant described in (14) or (15) above.

  The present invention resides in (17) a recovered protein recovered by the host cell described in (10) above.

  The present invention resides in (18) a recovered protein recovered by the plant cell described in (11) above.

  The present invention resides in (19) a recovered protein recovered by the transformant described in (12) or (13) above or the transformed plant described in (14) or (15) above.

  The present invention resides in (20) an insect-resistant agent comprising the insect-resistant protein according to any one of (1) to (4) as an active ingredient.

  The present invention resides in (21) an insect-resistant agent containing the insect-resistant gene according to any one of (5) to (7) as an active ingredient.

  The present invention resides in (22) an insect-resistant agent comprising the recovered protein according to any one of (17) to (19) as an active ingredient.

  According to the insect-resistant protein of the present invention, an extremely low concentration (for example, 0.01% per wet body of artificial feed) by having a homology of 60% or more with a phloem lectin. Even so, sufficient insect resistance can be exhibited.

  According to the insect-resistant protein of the present invention, the amino acid sequence having 60% or more homology with the first partial amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and the second partial amino acid shown in SEQ ID NO: 3 in the sequence listing By including two or more amino acid sequences selected from the group consisting of amino acid sequences having 60% or more homology with the sequences, sufficient insect resistance is exhibited even at extremely low concentrations, as described above. be able to.

  According to the insect-resistant protein of the present invention, an insect-resistant protein having a homology of 60% or more with respect to the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is more effective against insects even in a small amount. Shows sufficient insect resistance.

  The insect-resistant protein of the present invention is a cucurbitaceae plant, and if it is extracted from the exudate of the cucurbitaceae plant, it is contained as a main component in the sieve tube liquid, so that the purification is relatively It becomes easy.

  The insect resistance gene of the present invention encodes the aforementioned insect resistance protein. For example, according to an insect resistance gene having a homology of 60% or more with respect to the base sequence shown in SEQ ID NO: 4 in the sequence listing, gene introduction is carried out into organisms such as plants, microorganisms, cultured cells, multicellular animals and plants, and insects. By carrying out genetic breeding of gene insect-resistant plants, the obtained gene insect-resistant organisms exhibit sufficient insect resistance. In addition, the second DNA hybridized under stringent conditions with the recombinant DNA obtained by recombination of the insect resistance gene and the first DNA containing the base sequence shown in SEQ ID NO: 4 in the sequence listing, or the second DNA Insect-resistant genes containing a third DNA having a complementary base sequence with the same effect.

The recombinant vector of the present invention exhibits a function of carrying the above insect resistance gene or recombinant DNA to a heterologous host. Thereby, an insect resistance gene or recombinant DNA can be integrated into another gene.
For example, a recombinant vector can be introduced into a host cell or plant cell.

  The transformant of the present invention includes a host cell. Also includes its progeny or clones. For example, insect resistance can be expressed in E. coli transformed with an insect resistance gene or recombinant DNA.

  The transformed plant of the present invention contains plant cells. Also includes its progeny or clones. For example, plants transformed with insect-resistant genes or recombinant DNA can be dispensed with complicated pesticide spraying operations, and are also effective against pests that lurk inside plant tissues such as stems and are difficult to control. Can be demonstrated.

  In addition, sufficient insect resistance is exhibited even in the recovered protein recovered by the host cell, the plant cell, the transformant or the transformed plant.

  Since the propagation material of the present invention is obtained from a transformed plant body, it exhibits sufficient insect resistance.

According to the insecticide of the present invention, it is possible to easily remove insects that cause damage to human bodies, insects that inhibit plant growth, and the like.
The insect-resistant protein, the insect-resistant gene or the recovered protein is preferably used as an active ingredient of an insect-resistant agent.

FIG. 1 is a graph showing the growth inhibitory activity of the non-adsorbed fraction and the adsorbed fraction obtained in the examples of the present invention. FIG. 2 is a graph showing the growth inhibitory activity of the gel filtration active fraction in the non-adsorbed fraction obtained in the example of the present invention. FIG. 3 is a diagram showing the results of Native-PAGE electrophoresis of the gel filtration active fraction in the examples of the present invention. FIG. 4 is a graph showing the results of the insect resistance test in the examples of the present invention.

Preferred embodiments of the present invention will be described in detail.
(Insect-resistant protein)
The insect-resistant protein of the present invention has the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. The present invention includes those having homology of 60% or more, preferably 80% or more, more preferably 95% or more with the amino acid sequence.

Here, in the present invention, “insect resistance” means insecticidal properties or properties that inhibit insect growth (growth inhibitory properties).
Further, “60% or more homology with an amino acid sequence” means that 60% or more amino acids are the same sequence in the amino acid sequence. That is, in the amino acid sequence, it means that less than 60% of one or more amino acids may be converted by substitution, deletion, addition and / or insertion.

  Amino acid sequence homology can be determined using the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993) by Carlin and Arthur. Incidentally, programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990).

When an amino acid sequence is analyzed using BLASTX, parameters are set to score = 50 and wordlength = 3, for example.
When using BLAST and Gapped BLAST programs, the default parameters of each program are used. In addition, the specific method of these analysis methods is well-known (http://www.ncbi.nlm.nih.gov/).

  The insect-resistant protein of the present invention includes residues 8 to 155 and residues 156 to 303 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and a phloem lectin and Has a homology of 60% or more. That is, the insect-resistant protein of the present invention can be said to be a protein belonging to the sieve lectin family since the motif as the sieve lectin is substantially preserved in the amino acid sequence.

The amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing (hereinafter referred to as “first partial amino acid sequence”) is the 8th to 155th residues of the amino acid sequence shown in SEQ ID NO: 1, The amino acid sequence shown in No. 3 (hereinafter referred to as “second partial amino acid sequence”) is the 156th to 303rd residues of the amino acid sequence shown in SEQ ID No. 1.
Accordingly, the insect-resistant protein has a first partial amino acid sequence and a second partial amino acid sequence having 60% or more homology with the sieve lectin. In the present invention, 60% or more, preferably 80% or more, more preferably 95% or more of homology with the first partial amino acid sequence, and more preferably 60% or more with the second partial amino acid sequence, The homology is preferably 80% or more, more preferably 95% or more.

  Here, in the insect-resistant protein of the present invention, from the group consisting of an amino acid sequence having 60% or more homology with the first partial amino acid sequence and an amino acid sequence having 60% or more homology with the second partial amino acid sequence It suffices if it contains at least one selected amino acid sequence. That is, a plurality of first partial amino acid sequences may be included, only a second partial amino acid sequence may be included, or a first partial amino acid sequence and a second partial amino acid sequence may be included. . In this case, sufficient insect resistance can be exhibited even at a very low concentration.

  Note that the first partial amino acid sequence and the second partial amino acid sequence have a characteristic that a cysteine residue does not exist. Incidentally, the known sieve lectin has a cysteine residue because it is considered to interact with other molecules by a disulfide bond. For this reason, it is considered that the insect-resistant protein expresses insect resistance by a novel action mechanism. For example, chitin is one of the main components of the envelope that exists in the insect gastrointestinal tract, and it is considered that the envelope is related to insect resistance.

The insect-resistant protein of the present invention is preferably a plant-derived protein. In this case, the consumer is less resistant than bacteria.
Although it does not specifically limit as this plant, It is preferable that it is a plant which takes out an exudate.
Specific examples include cucurbitaceae, asteraceae, asteraceae, convolvulaceae, mulberry, euphorbiaceae, scallop, oleander, scallop, poppy, urushi, hypericaceae, legume, cactus, lily, etc. Plant.

Among these, it is preferable that the said plant is a Cucurbitaceae plant. That is, the insect-resistant protein is more preferably extracted from an exudate of a Cucurbitaceae plant.
Among Cucurbitaceae plants, the insect-resistant protein is more preferably one extracted from a eucalyptus Benincasa hispida exudate of Tougan leaves. In this case, since the insect-resistant protein is a major component of the exudate, purification becomes relatively easy.

  According to the insect-resistant protein, sufficient insect resistance is exhibited even at a very low concentration. Examples of insects that exhibit the insect-resistant activity of the insect-resistant protein include insects of the order Coleoptera, Lepidoptera, Diptera, Hymenoptera, Hemiptera, Straight-eye, Lepidoptera, etc. And arthropods such as ticks.

The insect-resistant protein is suitably used as an insect-resistant agent such as an insecticide, agricultural chemical, insect-proof bait.
Here, the insect-resistant bait means a bait that exhibits insect resistance by containing an insect-resistant substance in the bait and feeding the insect. That is, when the insect-resistant protein is used as an insect-resistant bait, insects that cause damage to the human body, insects that inhibit the growth of plants, and the like eat it, and thus insect growth is inhibited, or insects Will be killed, so insects can be removed easily.

In the above insect-resistant protein, another amino acid may be connected to both ends of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.
In this case, from the viewpoint of exerting excellent insect resistance, it is preferable that 0.1% by mass or more of the total amount of the protein containing the other amino acid is the amino acid sequence, and 0.2% by mass or more. More preferably, the amino acid sequence is as described above.

  The insect-resistant protein of the present invention includes a variant, a derivative encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence shown in SEQ ID NO: 1. Alleles, variants and homologues are also included.

  As a method for preparing DNA related to such an amino acid sequence, for example, site-directed mutagenesis method (Kramer, W. & Fritz, H.-J. (1987) Oligonucleotide-directed construction of mutagenesis via gapped duplex DNA Methods in Enzymology, 154: 350-367).

The insect-resistant protein of the present invention can be synthesized by a solid phase method, a liquid phase method, or a biological synthesis method.
Here, the solid phase method uses a polystyrene polymer gel bead having a diameter of about 0.1 mm whose surface is modified with an amino group as a solid phase, and from there, the amino acid chain is extended one by one by a dehydration reaction. When the target amino acid sequence is completed, the target substance is obtained by cutting out from the solid surface.
The liquid phase method is a method in which synthesis is performed in a liquid phase without fixing the protein to be synthesized to the solid phase, and purification is performed every time one amino acid residue is extended.

  Biological synthesis is a method for constructing artificial DNA that combines a translation region with the genetic code of a protein to be synthesized under the control of a promoter suitable for mass expression. It is a method of synthesizing to cultured cells. In addition, it does not necessarily use a living cell, but includes the case of using a cell-free transcription / translation system using a cell extract containing all factors involved in gene transcription and translation.

The insect-resistant protein of the present invention is purified as follows.
First, a toucan exudate from a cucurbitaceae plant is extracted and separated into a supernatant and a particle layer. The separation means is not particularly limited, and examples thereof include centrifugation and filtration.

  When performing centrifugation as the separating means, the centrifugal force is preferably 15000 to 20000 G, and is preferably rotated for 1 to 60 minutes. Then, a supernatant containing at least 5% of a desired protein (a ratio of mass (g) to volume (ml)) is obtained. In addition, it is preferable to filter the obtained supernatant with a filter with a pore diameter of 0.1 to 0.8 μm after centrifugation. Thereby, impurities such as contamination can be reliably removed.

Next, the insect-resistant protein of the present invention is extracted from the obtained supernatant. Examples of such extraction means include gel filtration chromatography, electrophoresis, ion exchange chromatography and the like.
Among these, it is preferable to use ion exchange chromatography. In the present invention, it is more preferable to use anion exchange chromatography.
And the active fraction containing an insect-resistant protein is obtained by desalting and concentrating the obtained fraction using a semipermeable membrane. The fractionation, desalting, and concentration by anion exchange chromatography may be repeated as necessary.

(Insect resistance gene)
The insect resistance gene of the present invention encodes the amino acid sequence shown in SEQ ID NO: 1 of the above insect resistance protein. That is, the insect resistance gene of the present invention is derived from a plant and has a base sequence represented by SEQ ID NO: 4 in the sequence listing. The present invention includes those having homology of 60% or more, preferably 80% or more, more preferably 95% or more with the base sequence.

Here, the term “insect resistance gene” refers to a factor that defines a genetic trait of insect resistance. Usually, they are arranged in a certain order on the chromosome.
Further, “60% or more homology with the base sequence” means that 60% or more of the base sequences are the same in the base sequence. That is, it means that in the base sequence, less than 60% of one or more bases may be converted by substitution, deletion, addition and / or insertion.

  The homology of the nucleotide sequence can be determined using the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993) by Carlin and Arthur. .

  When a base sequence is analyzed using BLASTX, parameters are set to score = 100 and wordlength = 12, for example. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.

  In the above insect-resistant gene, the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing is the N sequence identified from the purified protein using the cDNA obtained by performing reverse transcription reaction using total RNA derived from Togan petiole tissue. It is identified by PCR using a degenerate primer designed from the terminal amino acid sequence.

  According to the above insect-resistant gene, by introducing a gene into an organism such as a plant, a microorganism, a cultured cell, a multicellular animal or plant, and an insect and performing genetic breeding of the insect-resistant organism, , Exhibiting sufficient insect resistance.

  The insect resistance gene is preferably used as an insecticide such as an insecticide, an agrochemical, and insect bait. The insect-resistant bait has the same meaning as described above.

  The insect resistance gene of the present invention includes variants, derivatives, alleles, variants, and the like that have a base sequence in which one or more bases are substituted, deleted, added, and / or inserted in the base sequence shown in SEQ ID NO: 4. Homologs and the like are also included.

  In addition, methods well known to those skilled in the art for preparing DNAs having such base sequences include, for example, the site-directed mutagenesis method (Kramer, W. & Fritz, H.-J. (1987) Oligonucleotide -directed construction of mutagenesis via gapped duplex DNA. Methods in Enzymology, 154: 350-367).

  The insect resistance gene is a DNA hybridized under stringent conditions (hereinafter, referred to as “first DNA” for convenience) with a DNA comprising the base sequence shown in SEQ ID NO: 4 in the Sequence Listing. Or “having a base sequence complementary to the second DNA”. That is, by hybridizing under stringent conditions using DNA consisting of the base sequence shown in SEQ ID NO: 4 as a probe and DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 4 as a primer, A DNA comprising a base sequence having high homology with the base sequence shown in SEQ ID NO: 4 can be isolated. In the present invention, “DNA” includes genomic DNA, cDNA, and chemically synthesized DNA. Further, genomic DNA and cDNA may be prepared by a known method.

  Here, “hybridize under stringent conditions” means hybridization conditions of 6M urea, 0.4% SDS, 0.5 × SSC or stringency equivalent thereto. It should be noted that DNA with higher homology can be isolated by using conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1 × SSC. Further, under each condition, the temperature may be about 40 ° C. or higher, and if conditions with higher stringency are required, the temperature may be about 50 ° C., and further about 65 ° C.

  In place of the hybridization technique (Southern, EM (1975) Journal of Molecular Biology, 98, 503), the polymerase chain reaction (PCR) technique (Saiki, RK et al. (1985) Science, 230, 1350 -1354, Saiki, RK et al. (1988) Science, 239, 487-491).

(Recombinant DNA)
The recombinant DNA of the present invention is a DNA containing the gene of the present invention obtained by genetic engineering techniques, which is obtained by recombination of the aforementioned insect resistance gene.
For example, a part of a DNA molecule consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing is mixed with a part of another DNA molecule by cleavage and recombination.

(Recombinant vector)
The recombinant vector of the present invention contains the above-described insect resistance gene or the above-described recombinant DNA.
The recombinant vector of the present invention exhibits a function of carrying the above insect resistance gene or recombinant DNA to a heterologous host. Thereby, an insect resistance gene or recombinant DNA can be integrated into another insect resistance gene.
The recombinant vector includes E. coli. An E. coli-Agrobacterium shuttle vector or the like is used.

(Host cell)
The above recombinant vector has been introduced into the host cell of the present invention.
The host cell is not particularly limited as long as it is a cell suitable for the expression of a recombinant protein, and examples thereof include yeast, various animal cells, insect cells and the like in addition to E. coli.

A known method may be used for introducing the recombinant vector into the host cell.
For example, for introduction into E. coli, introduction methods using calcium ions (Mandel, M. & Higa, A. (1970) Journal of Molecular Biology, 53, 158-162, Hanahan, D. (1983) Journal of Molecular Biology, 166, 557-580). For example, in the case of an insect (Silkworm), a recombinant vector prepared based on piggyBac may be transformed using the method of Tamura et al. (Nat. Biotechnol. 18, 81-84, 2000).

(Plant cells)
The above recombinant vector is introduced into the plant cell of the present invention.
Plant cells include monocotyledonous and dicotyledonous cells.
Examples of monocotyledonous plants include gramineous plants and liliaceous plants.
Examples of gramineous plants include rice, wheat, barley, corn, oat, sorghum, rye, millet, sugar cane and the like.
Examples of liliaceae include leeks and asparagus.
Examples of the dicotyledonous plant include cruciferous plants, legumes, eggplants, cucurbits, convolvulaceae, roses, mulberries, mallows, and the like.
Examples of the cruciferous plants include Arabidopsis thaliana, Chinese cabbage, rapeseed, cabbage and cauliflower.
Examples of legumes include soybean, azuki bean, kidney bean, and cowpea.
Examples of solanaceous plants include tomato, eggplant, potato, tobacco, and pepper.
Examples of cucurbitaceae plants include cucumber, cucumber, melon and watermelon.
Convolvulaceae plants include morning glory, sweet potato, convolvulus and the like.
Examples of the Rosaceae plants include roses, strawberries, and apples.
Mulberry plants include mulberry, fig, rubber tree and the like.
Examples of mallows include cotton and kenaf.
In addition to cultured cells, the plant cells of the present invention include cells in the plant body. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots.

A known method may be used to introduce the recombinant vector into the plant cell.
Examples thereof include a polyethylene glycol method, electroporation, an Agrobacterium-mediated method, and a particle gun method.

(Transformant)
The transformant of the present invention contains a host cell and can be obtained by introducing the above-described recombinant vector into the host. That is, the transformant has been transformed with an insect resistance gene or recombinant DNA.

  If a transformant in which the insect-resistant gene or recombinant DNA of the present invention is introduced into the genome or a DNA that suppresses the expression of these genes is obtained, sexual or asexual reproduction can be performed from the transformant. Progeny or clones can be obtained.

(Transformed plant)
The transformed plant body contains plant cells and is produced by a known method according to the type of plant. In addition, as this plant, the monocotyledonous plant and the dicotyledonous plant which were mentioned above are used.
The transformed plant body can omit the troublesome operation of spraying agricultural chemicals, and can easily exert an insect-resistant effect against a pest that is hidden inside a plant tissue such as a stem and is difficult to remove.

  For example, as a method of obtaining a transformed plant body, a method of regenerating a plant body by introducing an insect resistance gene or recombinant DNA into protoplasts using polyethylene glycol (Datta, SK (1995) In Gene Transfer ToPlants (Potrykus I and Spangenberg Eds.) Pp66-74), a method of regenerating a plant by introducing an insect resistance gene or recombinant DNA into a protoplast by electric pulse (Toki et al. (1992) Plant Physiol. 100, 1503-1507), A method of introducing an insect-resistant gene or recombinant DNA into a cell or tissue by decompression treatment or pressure treatment and electroporation to regenerate a plant body (an electroporation method including the use of decompression treatment / pressure treatment ( Patent No. 4273231)), a method of regenerating a plant by directly introducing an insect resistance gene or recombinant DNA into a cell by a particle gun method (Christou et al. (1991) Bio / technology, 9: 957-962.) And Agrobacterium-mediated introduction of insect-resistant genes or recombinant DNA to regenerate plants (ultra-rapid monocotyl transformation method (Patent No. 3141084) ) And the like.

  Transformed plant cells can regenerate plant bodies by redifferentiation. Although the method of redifferentiation varies depending on the type of plant cell, for example, the method of Akama et al. (Plant Cell Reports 12: 7-11 (1992)) is used for Arabidopsis, and Fujimura et al. Plant Tissue Culture Lett. 2:74 (1995)).

  If a transformed plant into which the insect-resistant gene or recombinant DNA of the present invention is introduced into the genome or a DNA that suppresses the expression of these genes is obtained, sexual reproduction or asexuality can be obtained from the transformed plant. Progeny or clones can be obtained by reproduction. The transformed plants include not only plants into which insect resistance genes have been introduced, but also those into which insect resistance genes have been introduced for the preparation of insect resistance proteins.

(Breeding material)
Reproductive materials (eg, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) can be obtained from transformed plants, their progeny or clones. According to such a breeding material, it becomes possible to mass-produce transformed plants.

(Recovered protein)
The recovered protein of the present invention is recovered from the host cell, the plant cell, the transformant, and the transformed plant.
For example, a recombinant protein expressed in a host cell can be purified from the host cell or its culture supernatant by a known method, and the protein can be recovered. In addition, when the recombinant protein is expressed as a fusion protein with the above-described maltose binding protein or the like, affinity purification can be easily performed.

Microorganisms, cultured cells, multicellular animals and plants, insects, and the like, which are transformants into which the insect resistance gene of the present invention has been introduced, can be prepared and expressed in the transformants to recover the recovered protein.
Such recovered proteins are suitably used as insecticides such as insecticides, agricultural chemicals and insect baits. The insect-resistant bait has the same meaning as described above.

(Insect repellent)
The insect-resistant agent of the present invention contains the above-described insect-resistant protein, the above-described insect-resistant gene, or the above-described recovered protein as an active ingredient.

  When the insect-resistant protein is used as an active ingredient of an insect-resistant agent, a microorganism, plant, animal, or other organism containing the insect-resistant protein is roughly purified or purified, and the organism is allowed to express the insect-resistant protein. A crude product obtained by roughly purifying or purifying insect-resistant proteins by biochemical techniques is used. These crudely purified or purified products are referred to as purified insect-resistant proteins.

  The purified insect-resistant protein is used in the form of liquid, powder, granule, tablet or the like. In addition, a bulking agent, a spreading agent and the like may be appropriately added to these insecticides.

  The content of the purified insect-resistant protein contained in the insect-resistant agent may be 0.01% by mass or more with respect to the total amount of the insect-resistant agent, and may be 0.02% by mass or more from the viewpoint of certainty. preferable.

  When the insect resistance gene is used as an active ingredient of an insect resistance agent, a microorganism, plant, animal, or other organism containing the insect resistance gene is roughly purified or purified, and the organism is allowed to express the insect resistance gene. A crudely purified or purified insect resistance gene is used.

  When the recovered protein is used as an active ingredient of an insecticide, a crudely purified or purified protein recovered from host cells, plant cells, transformants, and transformed plants is used. These crudely purified or purified products are referred to as purified recovered proteins.

  The purified recovered protein is used in the form of liquid, powder, granule, tablet or the like. In addition, a bulking agent, a spreading agent and the like may be appropriately added to these insecticides.

  According to these insecticides, insects that cause damage to human bodies, insects that inhibit plant growth, and the like can be easily removed.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

(Purification of insect-resistant protein)
To a mixed solution consisting of 20 mM Tris-HCl buffer (pH 8.2), 5 mM EDTA, and 10 mM dithiothreitol, 500 μl of a cucurbitaceae leaf beetle Benincasa hispida exudate was added, and 1 ml of exudate. Got.
This exudate was centrifuged with a centrifuge (product name: KUBOTA inverter micro cooling centrifuge 1920, manufactured by Kubota Corporation). The centrifugation conditions were a rotational speed of 8000 rps, 4 ° C., and 15 minutes.
The separated supernatant was taken out and filtered through a 0.45 μm filter.

Next, the non-adsorbed fraction and the adsorbed fraction were obtained from the supernatant using anion exchange chromatography (product name: HiTrap DEAE-FF, manufactured by GE Healthcare Japan).
The obtained non-adsorbed fraction and adsorbed fraction were desalted and concentrated to obtain a purified protein.

(Bioassay)
Bioassay was performed on the obtained purified protein. That is, 0, 3.5, and 7 for the edible artificial feed for insects (trade name: L4M, manufactured by Nippon Nosan Kogyo Co., Ltd.) obtained by steaming purified protein with water 2.5 added to dry powder 1. Addition and mixing at concentrations of 0, 14 μg / 100 mg to prepare insect-resistant baits, and the insect-resistant baits were fed to the larvae that hatched erysan (a broad-ranging lepidopterous insect), and the body weight was measured two days later. .
It can be said that those having a reduced body weight of Erysan depending on the concentration of the purified protein have growth inhibitory activity.

FIG. 1 shows the growth inhibitory activity of the obtained non-adsorbed fraction and adsorbed fraction.
As shown in FIG. 1, a non-adsorbed fraction A exhibiting growth inhibitory activity and adsorbed fractions B and C were observed from the purified protein of toucan exudate.

Next, the non-adsorbed fraction A is collected and concentrated, and the non-adsorbed fraction A is further purified using gel filtration chromatography (product name: Superdex 75G, manufactured by GE Healthcare Japan) to obtain the active fraction (hereinafter referred to as “active fraction”). For convenience, it is referred to as “gel filtration active fraction”.) D was isolated. The obtained gel filtration active fraction D was desalted and concentrated, and then bioassay was performed as described above. The growth inhibitory activity of the gel filtration active fraction in the obtained non-adsorbed fraction is shown in FIG.
In addition, about 3.82 μg of gel filtration active fraction D, which is an insect-resistant protein, was purified from 1 ml of toucan sieve liquid. Thus, since the insect-resistant protein of the present invention is contained in a relatively large amount, purification is easy.

(Electrophoresis)
Next, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. For electrophoresis, 400 μl of gel filtration active fraction D was mixed with an equal volume of Native-PAGE buffer, and polyamide gel was added so as to be 12.5% (ratio of mass to volume). Native-PAGE electrophoresis was performed at room temperature (25 ° C., pH 6.8 to 8.8). The obtained results are shown in FIG. In addition, each lane of FIG. 3 is from left, size marker, tougan sieve tube liquid, DEAE-FF non-adsorbed fraction A, gel filtration active fraction D (reducing agent dithiothreitol-), gel peractive fraction D ( 50 mM dithiothreitol +).

  As shown in FIG. 3, from the behavior of the gel filtration active fraction D, the molecular weight of this gel filtration active fraction D is considered to be about 35 kDa, and even in the presence of a reducing agent (dithiothreitol), the behavior of SDS-PAGE is observed. Since no change was seen, it is considered to be a monomeric protein.

(Structural analysis)
Next, the structure of the protein in the gel filtration active fraction D was analyzed for its primary structure by internal amino acid sequence analysis and cDNA cloning using primers designed based on the results.
The revealed primary structure (SEQ ID NO: 1) was homologous to Phloem lectin of Cucurbitaceae as a result of Blast search, but as described above, the molecular weight predicted from the deduced amino acid sequence is about 35 kDa. It was not a dimer but a monomer, showed homology with Phloem lectin but had no cysteine residue, and the same sequence was repeated twice. This component is considered to have a function as a phloem lectin such as binding to N-acetylglucosamine as confirmed by Phloem lectin.

(Insect resistance test 1)
The gel filtration active fraction D was added to the above-mentioned artificial feed for edible insects (hereinafter simply referred to as “artificial feed”) and fed to Elisan, and the weight gain of Elisan was investigated. The obtained results are shown in FIG.
As shown in FIG. 4, the insect-resistant protein of the present invention showed remarkable growth inhibition and insect-resistant effects even at a low concentration of 20% (0.01% as the amount of protein) in the artificial feed. .
It was also found that this effect was prominent even after 2 days, and showed a remarkable growth inhibitory effect in a short time, such as the weight gain was reduced by about half even after 2 days.

(Insect resistance test 2)
A conventional lectin, protease inhibitor, amylase inhibitor, MLX56, and the insect-resistant protein of the present invention were added to the artificial feed and fed to Erysan, and the weight gain of Erysan was investigated. Table 1 shows the respective action concentrations for erysan.

(Table 1)

  It was found that the insect-resistant protein of the present invention exhibits growth inhibitory activity against erysan only when it is present at about 7 μg (0.007%) in 100 mg of artificial feed. This effect is 10 to 100 higher than other plant-derived insect-resistant proteins such as snowdrop lectin, which is currently being studied worldwide for practical use in agriculture (nurturing insect-resistant plants by gene transfer). The double effect was high, showing the same effect as MLX56 previously invented by the present inventors.

  One of the active factors contained in the adsorbed fraction obtained by anion exchange chromatography was also isolated. This component is considered to be about 35 kDa from the result of SDS-PAGE. About growth inhibitory activity, the activity comparable to the previous factor was shown in a small amount. As for the structure of this component, the N-terminal and internal amino acid sequences are currently being analyzed, and the primary structure will be clarified by cDNA cloning based on the information obtained in the future.

  The insect-resistant protein of the present invention can be used as a genetic material for insect-resistant genetically modified plants, particularly in agriculture. That is, an insect-resistant plant can be created by introducing and expressing the insect-resistant gene of the present invention.

By the way, although not so common in Japan and European countries, the cultivation of insect-resistant transgenic plants is increasing worldwide, especially in the new continent, Africa and China.
However, the proteins used in insect-resistant genetically modified plants are concentrated on Bt toxins derived from bacteria, and considering the emergence of resistant pests, the discovery of proteins from other plants is awaited. There are few effective insect-resistant proteins that show insect resistance. For this reason, the insect-resistant protein of the present invention has a possibility of being widely used internationally in the future.

The insect-resistant protein of the present invention has a low insect-resistant activity as compared to the Bt protein, but is a plant-derived insect-resistant protein that is less resistant to consumers than a bacterial-derived toxin that is resistant to consumers. Differentiate from Bt toxin.
The insect-resistant protein of the present invention exhibits remarkable insect resistance and insect growth inhibitory activity with a small amount of expression of about 0.01% (compared to other insect-resistant proteins). This amount can be expressed in plants with current technology and can be used for genetic breeding of insect-resistant plants.
In addition, since this protein is derived from cultivated plants (not from microorganisms) (in some countries, it has food experience in fruits and leaves), there is an advantage that there is little psychological resistance of consumers.

A: Non-adsorbed fraction B, C: Adsorbed fraction D: Gel filtration active fraction

Claims (22)

A plant-derived insect-resistant protein,
An insect-resistant protein having an amino acid sequence having a homology of 60% or more with a phloem lectin. A plant-derived insect-resistant protein,
Amino acid sequence having 60% or more homology with the first partial amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and an amino acid having 60% or more homology with the second partial amino acid sequence shown in SEQ ID NO: 3 in the sequence listing An insect-resistant protein comprising two or more amino acid sequences selected from the group consisting of sequences. A plant-derived insect-resistant protein,
An insect-resistant protein having a homology of 60% or more with respect to the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.   The insect-resistant protein according to any one of claims 1 to 3, wherein the plant is a Cucurbitaceae plant and is extracted from an exudate of the Cucurbitaceae plant.   The insect-resistant gene which codes the insect-resistant protein of any one of Claims 1-4. A plant-derived insect resistance gene,
An insect resistance gene having a homology of 60% or more with respect to the base sequence shown in SEQ ID NO: 4 in the sequence listing.   Insect resistance comprising the second DNA hybridized under stringent conditions with the first DNA comprising the base sequence shown in SEQ ID NO: 4 in the sequence listing, or the third DNA having a base sequence complementary to the second DNA gene.   A recombinant DNA obtained by recombining the insect resistance gene according to any one of claims 5 to 7.   A recombinant vector comprising the insect resistance gene according to any one of claims 5 to 7 or the recombinant DNA according to claim 8.   A host cell into which the recombinant vector according to claim 9 has been introduced.   A plant cell into which the recombinant vector according to claim 9 has been introduced.   A transformant comprising the host cell according to claim 10.   A transformant which is a descendant or clone of the transformant according to claim 12.   A transformed plant comprising the plant cell according to claim 11.   A transformed plant that is a descendant or clone of the transformed plant according to claim 14.   A propagation material obtained from the transformed plant according to claim 14 or 15.   A recovered protein recovered by the host cell according to claim 10.   A recovered protein recovered by the plant cell according to claim 11.   A recovered protein recovered by the transformant according to claim 12 or 13, or the transformed plant according to claim 14 or 15.   The insect-resistant agent which uses the insect-resistant protein of any one of Claims 1-4 as an active ingredient.   The insect-resistant agent which uses the insect-resistant gene of any one of Claims 5-7 as an active ingredient.   An insecticide comprising the recovered protein according to any one of claims 17 to 19 as an active ingredient.

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