JP5769145B2 - Bt toxin resistance gene derived from Lepidoptera insects and use thereof - Google Patents

Description

The present invention relates to a gene derived from a lepidopteran insect exhibiting resistance to an insecticidal protein (hereinafter referred to as “Cry toxin”) produced by Bacillus thuringiensis , and use thereof.

  Cry toxins have been utilized as microbial insecticides and in the production of pest resistant recombinant crops. However, in recent years, an individual that shows resistance to Cry toxins has emerged in the diamondback moth, an important pest of cruciferous vegetables, which has become a major problem. For this reason, studies on the development mechanism of Cry toxin resistance in pests have been conducted, and in some lepidopterous insects, resistance genes against Cry toxin have been elucidated.

  For example, a gene with recessive resistance to Cry1Ac was identified in false American tobacco moth, and this gene was found to encode a cadherin-like protein that is considered to be a receptor for Cry toxin (Non-patent Document 1). In addition, it has been reported that the same gene functions as a resistance gene in cotton beetle and giant tobacco (Non-patent Document 2, Non-patent Document 3). These genes are used for diagnosis of pest Cry toxin resistance.

  On the other hand, for the diamondback moth, which has developed resistance to Cry toxin, it has been reported that the resistance gene is not a conventionally known receptor protein of Cry toxin (cadherin-like protein, aminopeptidase N, etc.). (Non-Patent Document 4). In addition, it is estimated that even silkworms with known resistance lines against Cry1Ab have developed their resistance by mutation of genes different from those of the receptor candidate proteins (Non-patent Document 5). In addition, it has been reported that the resistance of Cry1Ab toxin-resistant cultivars is controlled by a recessive main gene seated in the 15th linkage group of the silkworm molecular gene map (Non-patent Document 6).

  As described above, in lepidopterous insects such as diamondback moth and silkworm, genes other than the gene encoding a receptor candidate protein of Cry toxin are considered to be involved in resistance to Cry toxin. Yes still not clear.

Gahan, L.J. et al., (2001) Science, 293, 857-860. Morin, S., et al., (2003) Proc. Natl. Acad. Sci. USA, 100, 5004-5009. Xu, X. Et al., (2005) Appl.Environ. Microbiol. 71 (2), 948-954. Baxter, S.W. et al., (2008) Insect Biochem. Mol. Biol., 38, 125-135. Hara, W. et al., (2005) Biotechnology of Bacillus thuringiensis, Edited by N.D.Binh, R.J.Akhurst and D.H.Dean, Science and Technics Publishing House, Vietnam, Proceedings. Kazuhisa Miyamoto (2008) Sericultural and Insect Biotech The Japanese Sericultural Society 77 (3), 195-203

  The present invention has been made in view of such circumstances, and an object thereof is to identify a novel gene derived from a lepidopteran insect involved in resistance to Cry toxins. A further object of the present invention is to breed lepidopteran insects that are resistant to insecticidal proteins using the identified resistance genes, and to identify insecticidal proteins using the identified resistance genes as indicators. The purpose is to conduct genetic diagnosis of lepidopterous insects that are resistant to the disease.

The present inventors have used the KAIKObase gene prediction model (KAIKObase ver.2.1.0, http://sgp.dna.affrc.go.jp/KAIKObase) for the genes seated in the 15th linkage group of the silkworm molecular gene map. Six predicted gene regions related to Cry toxin resistance estimated from /) were found. As a result of analyzing gene expression by PCR for these predicted genes, it was confirmed that the four genes BGIBMGA007735 , BGIBMGA007793 , BGIBMGA007736 , and BGIBMGA007792 were expressed in the midgut tissue (FIG. 1). In addition, cDNA base sequence analysis revealed that BGIBMGA007736 , BGIBMGA007792 , and BGIBMGA007793 are one gene, and revealed that there are actually two genes ( BGIBMGA007735 and BGIBMGA007792-93 ). did. In resistant strains and susceptible strains, the nucleotide sequence analysis of BGIBMGA007735 and BGIBMGA007792-93, although differences in nucleotide sequence in BGIBMGA007735 was observed in ORF in BGIBMGA007792-93, the nucleotide sequence 39 bases, in the amino acid sequence 13 Residue differences were observed (FIGS. 2 and 3). Comparison of the amino acid sequences of the BGIBMGA007792-93 protein in the resistant strain (7 strains) and the sensitive strain (10 strains), the insertion of Tyr (tyrosine) at position 234 was found as a sequence common to the resistant strains. (Figure 4). In order to confirm that the BGIBMGA007792-93 gene is a Bt resistance gene, the present inventors conducted an introduction experiment of a sensitive BGIBMGA007792-93 gene to a resistant silkworm strain. As a result, it was found that a resistant silkworm strain into which a sensitive BGIBMGA007792-93 gene was introduced acquired sensitivity to Cry toxin (Tables 4 and 5).

From the above, the present inventors have found that the BGIBMGA007792-93 gene is the main body of the Cry toxin resistance gene, and by using the gene, breeding of lepidopteran insects that are resistant to Cry toxin, As a result, it was found that lepidopterous insects exhibiting resistance to the disease can be efficiently diagnosed, and the present invention has been completed.

  More specifically, the present invention provides the following inventions.

[1] The DNA according to any one of the following (a) to (e), which encodes a protein having an activity that imparts sensitivity to an insecticidal protein produced by Bacillus thuringiensis to lepidopterous insects.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19
(C) In the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20, one or more amino acids are substituted, deleted, added, and / or inserted. DNA encoding a protein consisting of an amino acid sequence
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19
(E) DNA comprising a nucleotide sequence having a homology of 90% or more with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19
[2] The DNA according to any one of the following (a) to (e), which encodes a protein having an activity that imparts resistance to insecticidal proteins produced by Bacillus thuringiensis to lepidopterous insects.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 22, 24, 26, 28, 30, 32 or 34
(B) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 21, 23, 25, 27, 29, 31 or 33
(C) 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 of SEQ ID NO: 22, 24, 26, 28, 30, 32 or 34 DNA to encode
(D) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 21, 23, 25, 27, 29, 31 or 33
(E) DNA comprising a nucleotide sequence having a homology of 90% or more with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 21, 23, 25, 27, 29, 31 or 33
[3] The DNA according to any one of the following (a) to (c), which encodes a protein having an activity that imparts resistance to insecticidal proteins produced by Bacillus thuringiensis to lepidopterous insects.
(A) DNA encoding a double-stranded RNA complementary to the transcription product of DNA according to [1]
(B) DNA encoding an antisense RNA complementary to the transcription product of DNA according to [1]
(C) DNA encoding an RNA having ribozyme activity that specifically cleaves the transcript of the DNA according to [1]
[4] A vector comprising the DNA according to any one of [1] to [3].

  [5] Lepidoptera insect cells into which the DNA according to any one of [1] to [3] is introduced.

  [6] A lepidopteran insect comprising the cell according to [5].

  [7] An egg, offspring, or clone of the lepidopterous insect according to [6].

  [8] A method for producing a lepidopteran insect imparted with resistance to an insecticidal protein produced by Bacillus thuringiensis, which suppresses the expression or function of the DNA according to [1] in a lepidopteran insect .

  [9] A lepidopteran insect having a resistance to an insecticidal protein produced by Bacillus thuringiensis, wherein the lepidopteran insect comprises the step of introducing the DNA according to [2] or [3] into the lepidopteran insect. Production method.

  [10] A drug for imparting resistance to insecticidal proteins produced by Bacillus thuringiensis to lepidopteran insects, comprising the DNA according to [2] or [3], or a vector into which the DNA is inserted.

  [11] A method for determining sensitivity or resistance to an insecticidal protein produced by Bacillus thuringiensis in lepidopterous insects, wherein the nucleotide sequence of DNA according to [1] or [2] in a test lepidopteran insect A method characterized by analyzing.

[12] A method for breeding lepidopteran insects resistant to insecticidal proteins produced by Bacillus thuringiensis,
(A) mating a lepidopteran insect strain resistant to an insecticidal protein produced by Bacillus thuringiensis with any lepidopteran insect strain;
(B) determining the sensitivity or resistance to the insecticidal protein produced by Bacillus thuringiensis in the individual obtained by the mating in step (a) by the method according to [11], and (c) Bacillus Selecting a strain determined to have resistance to an insecticidal protein produced by thuringiensis.

According to the present invention, the BGIBMGA007792-93 gene that determines sensitivity and resistance to Cry toxin has been identified, and the chromosomal location and structure of the gene have been elucidated. The silkworm BGIBMGA007792-93 gene found by the present invention is characterized by a remarkable difference of several hundred to several thousand times in sensitivity to Cry toxin depending on the presence or absence of Tyr insertion at position 234 of the encoded protein. It has special properties. By using the identified BGIBMGA007792-93 gene, it is possible to easily determine the sensitivity and resistance of Lepidoptera insects. Further, by using the BGIBMGA007792-93 gene as a marker, it is possible to efficiently breed silkworm strains resistant to the insecticidal protein. By imparting resistance to silkworms in this way, it becomes possible to control insects by spraying insecticides containing Cry toxins on mulberry fields even during the breeding period of silkworms.

In addition, by using the BGIBMGA007792-93 gene, it is possible to elucidate the mechanism of resistance development in lepidopterous insects and to predict the development of resistance. When the frequency of resistance-type genes is high in pests, it is possible to prevent the development of insecticide resistance in the pests by changing the type of insecticidal protein contained in the pesticide used.

The expression of six candidate genes for Cry toxin resistance, 1 ( BGIBMGA007735 ), 2 ( BGIBMGA007793 ), 3 ( BGIBMGA007736 ), 4 ( BGIBMGA007792 ), 5 ( BGIBMGA007791 ), 6 ( BGIBMGA007737 ), was analyzed by RT-PCR. It is a photograph which shows a result. In the figure, “M” indicates a 100 bp DNA ladder marker. It is the figure which compared the base sequence of the BGIBMGA007792-93 gene in the resistant system | strain (branch number 2 (C2_R)) of silkworm, and a sensitivity system | strain (Rin_S). FIG. 2 is a continuation diagram of FIG. 2-1. It is the figure which compared the amino acid sequence of the protein which the BGIBMGA007792-93 gene codes in a silkworm resistant strain (C2_R) and a sensitivity strain (Rin_S). It is the figure which compared the amino acid sequence of the protein which the BGIBMGA007792-93 gene of a silkworm resistant system (7 systems) and a sensitivity system (10 systems) encodes. There is a common insertion of Tyr at position 234 in the resistance system. FIG. 4 is a continuation of FIG. 4-1. FIG. 4 is a continuation diagram of FIG. FIG. 4 is a continuation diagram of FIG. 4-3. FIG. 4 is a continuation diagram of FIG. 4-4. Fig. 6 is a continuation of Fig. 4-5. It is a photograph which shows the result of having analyzed the tissue specificity of the expression of BGIBMGA007792-93 gene in a silkworm by RT-PCR method. It is a figure which shows the effector construct used for preparation of a UAS-Bt-r system | strain. The BGIBMGA007792-93 gene derived from Rin_S is inserted downstream of UAS. It is a figure which shows the activator construct used for preparation of BmA3_GAL4 system | strain. It is a figure which shows the PCR primer for detecting the presence or absence of insertion of Tyr in the 234th position of the protein which BGIBMGA007792-93 gene codes. It is a graph which shows the result of having detected the expression of endogenous and exogenous BGIBMGA007792-93 gene in an original strain, SS16-1 strain, and SS16-3 strain.

<Sensitive and resistant DNA against Cry toxin>
The present invention relates to a DNA encoding a protein having an activity that confers sensitivity to Cry toxin to lepidopterous insects (hereinafter referred to as “sensitive DNA”), and an activity that imparts resistance to Cry toxin to lepidopterous insects DNA encoding a protein having the following (hereinafter referred to as “resistant DNA”) is provided.

The “Lepidoptera insect” from which the DNA of the present invention is derived is not particularly limited as long as it retains the BGIBMGA007792-93 gene (the silkworm-derived gene or a homologous gene thereof) that determines the sensitivity and resistance to the Cry toxin. Preferable are silkworms and lepidopterous pests. In addition, the “Cry toxin” in the present invention is a Cry toxin that can exhibit sensitivity and resistance by the function of the protein encoded by the BGIBMGA007792-93 gene (gene derived from silkworm or a homologous gene thereof). There is no restriction | limiting in particular, For example, Cry1Ab toxin and Cry1Ac toxin are mentioned. It is known that many silkworm strains show similar sensitivity and resistance to Cry1Ab toxin and Cry1Ac toxin (Kazuhisa Miyamoto (2004) About mutations in Cry1A type δ-endotoxin sensitivity in silkworm geographical varieties , Proceedings of the 6th Symposium on Insect Pathology, p.27.).

Silkworm susceptibility lines to Cry toxins identified by the present inventors: Yoshi N (Yo_S), Bagdat (Bag_S), No. 65 (N65_S), European No. 12 (E12_S), Annan (An_S), Cambouge (multiple) ) (CaM_S), Mysole (My_S), Pure Mysole (PMy_S), Watsuki (Rin_S), 912-derived (e21_S) base sequences of BGIBMGA007792-93 cDNA are shown in SEQ ID NOs: 1, 3, 5, 7, 9 11, 13, 15, 17, and 19 show the amino acid sequences of the proteins encoded by these DNAs in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, respectively.

  One embodiment of the sensitive DNA of the present invention is a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 (typically Is DNA containing the coding region of the base sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19).

As shown in this example, when comparing the amino acid sequences of the proteins encoded by the BGIBMGA007792-93 cDNA of the sensitive strain, Yo_S, N65_S and E12_S have the same amino acid sequence, and An_S and CaM_S have the same amino acid sequence. PMy_S and Rin_S have the same amino acid sequence. Comparing Bag_S and Yo_S, the 57th place is Phe for the former and Ser for the latter. Comparing Bag_S and An_S, the 15th place is Lys and the latter is Tyr, the 45th place is Ile and the latter is Tyr, and the 343rd place (345 in Figure 4) is Leu and the latter is Phe, 452 (454 in Fig. 4), the former is Gln and the latter is Ser, and 486 (48th in Fig. 4) is Ser, the latter is Thr, and 494 (in Fig. 4) 496) is Leu and the latter is His, and 554 (in Fig. 4 is 556), the former is Glu and the latter is Asp, and 676 (in Fig. 4 is 678), the former is Lys and the latter is Asn, 757th (759th in Fig. 4) is Ala and the latter is Val, and 767th (769th in Fig. 4) is Met and the latter is Thr, and 804th (in Fig. 4) The first is Tyr and the latter is Phe, and the 922th (924th in Fig. 4) is Phe and the latter is Val, and the 970th (972th in Fig. 4) is Glu and the latter is Gln, 1140 (1142 in Fig. 4) is Asp for the former and Glu for the latter. Comparing An_S and PMy_S, 494th (496th in FIG. 4) is His for the former and Leu for the latter. Comparing Bag_S and e21_S, there is an insertion of Ile at position 66 of e21_S. However, both are sensitive DNAs.

In the current state of the art, those skilled in the art will be able to identify sensitive DNA in specific susceptibility strains (eg Yo_S, Bag_S, N65_S, E12_S, An_S, CaM_S, My_S, PMy_S, Rin_S, e21_S (from 912)). When base sequence information is obtained, it is possible to modify the base sequence and obtain a DNA that is also sensitive, although the encoded amino acid sequence is different. Also in nature, it is possible that the amino acid sequence of the encoded protein is mutated due to the mutation of the base sequence. Therefore, the present invention replaces one or more amino acids in the amino acid sequence of BGIBMGA007792-93 protein (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 , 20) in these sensitive strains. , Including deletion, addition, and / or insertion, and DNA encoding a protein having an activity of conferring susceptibility to lepidopteran insects against Cry toxins. The term “plurality” as used herein refers to the number of amino acid modifications within a range in which the modified BGIBMGA007792-93 protein maintains the activity of conferring susceptibility to lepidopteran insects against Cry toxins, usually within 50 amino acids, preferably 30 It is within amino acids, more preferably within 10 amino acids (eg, within 5 amino acids, within 3 amino acids, 2 amino acids).

Furthermore, in the current state of the art, a person skilled in the art can detect a sensitive type from a specific susceptible strain (for example, Yo_S, Bag_S, N65_S, E12_S, An_S, CaM_S, My_S, PMy_S, Rin_S, e21_S (from 912)). When DNA is obtained, it is possible to obtain DNA encoding a homologous gene that is also sensitive from other lepidopteran insect strains using the base sequence information of the sensitive DNA. Therefore, the present invention is DNA that hybridizes under stringent conditions with BGIBMGA007792-93 DNA (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 ) in these sensitive strains. In addition, it also includes DNA encoding a protein having an activity that imparts sensitivity to Cry toxins to lepidopterous insects.

  Whether the thus obtained mutant DNA or homologous DNA encodes a protein having an activity that confers sensitivity to Cry toxins in lepidopterous insects is determined by, for example, applying a test DNA to a resistance strain to Cry toxin described later. It can be determined by introducing and testing whether the resistant strain acquires sensitivity to Cry toxin (see Example 3).

In addition, the resistance lines identified by the present inventors to the Cry toxin of silkworms Day 1 (J1_R), Yellow Float (Ki_R), Red Branch (Be_R), Branch 2 (C2_R), Branch 7 (C7_R) ), C Zhejiang (Cs_R), N15 (derived from No. 342, N15_R) BGIBMGA007792-93 cDNA base sequence is SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, respectively, and the proteins encoded by these DNAs Are shown in SEQ ID NOs: 22, 24, 26, 28, 30, 32, and 34, respectively.

  One embodiment of the resistant DNA of the present invention is a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 22, 24, 26, 28, 30, 32, 34 (typically, SEQ ID NO: 21, 23, 25, 27, 29, 31, and 33).

As shown in this Example, when the amino acid sequences of the proteins encoded by the BGIBMGA007792-93 cDNAs of the resistant strain and the sensitive strain are compared, the resistance strains commonly have positions 234 (position 235 in FIG. 4). ) Tyr insertion was observed. This mutation is thought to confer resistance to Cry toxins on the individual. In the current state of the art, those skilled in the art will recognize that BGIBMGA007792-93 DNA of a specific susceptible strain (eg Yo_S, Bag_S, N65_S, E12_S, An_S, CaM_S, My_S, PMy_S, Rin_S, e21_S (from 912)). It is possible to modify such a base sequence so as to confer resistance to the Cry toxin of the encoded protein. In addition, in the base sequence of BGIBMGA007792-93 DNA of a specific resistant strain (eg, J1_R, Ki_R, Be_R, C2_R, C7_R, Cs_R, N15_R (derived from No. 342), the resistance of the encoded protein to Cry toxin is maintained. In nature, it is possible that the amino acid sequence of the encoded protein is mutated due to the mutation of the base sequence.In fact, the C2_R BGIBMGA007792-93 cDNA is Comparing the amino acid sequence of the encoded protein with the amino acid sequence of the protein encoded by BGIBMGA007792-93 cDNA from the other resistance strains above, the 15th position is Thr and the latter is Lys. Accordingly, the present invention relates to the amino acid sequence of the BGIBMGA007792-93 protein (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 16) in these susceptible and resistant lines. 18, 20, 22, 24, 26, 28, 30, 32, 34) consisting of a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted. It also includes DNA encoding a protein having an activity that confers resistance to Cry toxin, where “multiple” means that the modified BGIBMGA007792-93 protein confers resistance to Cry toxin to lepidopteran insects. The number of amino acid modifications in a range having a value of generally within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (eg, within 5 amino acids, within 3 amino acids, 2 amino acids). in BGIBMGA007792-93 DNA nucleotide sequence of the system, performing modifications such as resistance is maintained against the Cry toxin protein its encoding If, modification sites, it is preferable that the portion other than the position 234 Tyr.

Furthermore, in the current state of the art, one of ordinary skill in the art can obtain BGIBMGA007792-93 DNA from specific resistive strains (eg, J1_R, Ki_R, Be_R, C2_R, C7_R, Cs_R, N15_R (from No. 342)). In this case, DNA encoding the resistant homologous gene can be obtained from other lepidopterous insect species using the nucleotide sequence information of the DNA. Therefore, the present invention is a DNA that hybridizes under stringent conditions with BGIBMGA007792-93 DNA (SEQ ID NO: 21, 23, 25, 27, 29, 31, 33 ) in a resistant strain, DNA encoding a protein having activity conferring resistance to Cry toxins is included.

  Whether or not the mutant DNA or homologous DNA thus obtained encodes a protein having an activity that imparts resistance to Cry toxins to lepidopterous insects is determined by preparing an individual that holds the test DNA in homology. Can be determined by testing whether or not has resistance to Cry toxins.

  The sensitive DNA of the present invention is a drug for imparting sensitivity to Cry toxin to lepidopterous insects in the sense that introduction thereof can impart sensitivity to Cry toxin to lepidopteran insects, The resistance-type DNA of the present invention is a drug for imparting resistance to Cry toxin to lepidopterous insects in the sense that introduction thereof can impart resistance to Cry toxin to lepidopterous insects. It is.

  In addition, artificial mutations introduced into DNA to produce the above-mentioned mutant DNA are, for example, site-directed mutagenesis method (Kramer, W. & Fritz, HJ., Methods Enzymol). 154: 350-367, 1987).

  Moreover, as a method for isolating the above-mentioned homologous gene, for example, hybridization technology (Southern, EM, Journal of Molecular Biology, 98: 503, 1975) or polymerase chain reaction (PCR) technology (Saiki, RK, et al. Science, 230: 1350-1354, 1985, Saiki, RK et al. Science, 239: 487-491, 1988). In order to isolate DNA encoding a homologous gene, a hybridization reaction is usually carried out under stringent conditions. Examples of stringent hybridization conditions include 6M urea, 0.4% SDS, 0.5xSSC conditions, or equivalent stringency hybridization conditions. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1xSSC. The isolated DNA is at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99%) at the nucleic acid level or amino acid sequence level. And the like). Sequence homology can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). Yes. When analyzing a base sequence by BLASTN, parameters are set to score = 100 and wordlength = 12, for example. When analyzing an amino acid sequence by BLASTX, parameters are set to, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

The DNA encoding the BGIBMGA007792-93 protein of the present invention is not particularly limited in form, and includes cDNA, genomic DNA, and chemically synthesized DNA. Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means. For example, genomic DNA is extracted from lepidopterous insects, a genomic library (as vectors, plasmids, phages, cosmids, fosmids, BACs, PACs, etc. can be used) is developed, Base of BGIBMGA007792-93 gene (for example, DNA described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 ) It can be prepared by colony hybridization or plaque hybridization using a probe prepared based on the sequence. It is also possible to prepare a primer specific to the BGIBMGA007792-93 gene and perform PCR using this primer. In addition, for example, cDNA is synthesized based on mRNA extracted from lepidopterous insects, and inserted into a vector such as λZAP to create a cDNA library. It can be prepared by performing hybridization or plaque hybridization, or by performing PCR.

<DNA used to suppress the expression of the sensitive BGIBMGA007792-93 gene in Lepidoptera insects>
The present invention also provides a DNA used to suppress the expression of the sensitive BGIBMGA007792-93 gene of Lepidoptera insects. By introducing these DNAs, it is possible to confer resistance to Cry toxins to lepidopterous insects. In this sense, the DNA used to suppress the expression of the sensitive BGIBMGA007792-93 gene in lepidopterous insects is a drug for conferring resistance to Cry toxins in lepidopterous insects. Here, “suppression of BGIBMGA007792-93 gene expression” includes both suppression of gene transcription and suppression of protein translation. Further, “suppression of expression” includes not only complete cessation of expression but also decrease of expression.

One embodiment of the DNA used to suppress the expression of the sensitive BGIBMGA007792-93 gene of lepidopterous insects encodes a dsRNA (double-stranded RNA) complementary to the above-described sensitive DNA transcript of the present invention. DNA. A phenomenon called RNAi (RNA interference), in which the expression of the introduced foreign gene and target endogenous gene is both suppressed by introducing dsRNA having the same or similar sequence to the target gene into the cell. Can cause. When dsRNA of about 40 to several hundred base pairs is introduced into a cell, an RNase III-like nuclease called Dicer having a helicase domain is converted to about 21 to 23 base pairs from the 3 ′ end in the presence of ATP. Each one is cut out to generate siRNA (short interference RNA). A specific protein binds to this siRNA to form a nuclease complex (RISC: RNA-induced silencing complex). This complex recognizes and binds to the same sequence as siRNA, and cleaves the transcript (mRNA) of the target gene at the center of the siRNA by RNaseIII-like enzyme activity. In addition to this pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. There is also a possibility that this dsRNA becomes Dicer's substrate again to generate new siRNA and amplify the action.

  The DNA encoding the dsRNA of the present invention includes an antisense DNA encoding an antisense RNA for any region of a target gene transcription product (mRNA), and a sense DNA encoding a sense RNA for any region of the mRNA And antisense RNA and sense RNA can be expressed from the antisense DNA and the sense DNA, respectively. Moreover, dsRNA can be produced from these antisense RNA and sense RNA.

  In the case where the dsRNA expression system of the present invention is held in a vector or the like, there are a case where antisense RNA and sense RNA are expressed from the same vector, and a case where antisense RNA and sense RNA are expressed from different vectors, respectively. is there. The antisense RNA and sense RNA are expressed from the same vector. For example, an antisense RNA expression cassette in which a promoter capable of expressing a short RNA such as polIII is linked upstream of the antisense DNA and the sense DNA. And sense RNA expression cassettes are constructed, and these cassettes are inserted into the vector in the same direction or in the opposite direction.

  It is also possible to construct an expression system in which antisense DNA and sense DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided so that antisense RNA and sense RNA can be expressed from each strand on both sides thereof. A promoter is provided oppositely. In this case, in order to avoid adding extra sequences downstream of the sense RNA and antisense RNA, a terminator is added to the 3 'end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. Further, in this palindromic style expression system, the two promoter types are preferably different.

  In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, antisense RNA expression in which a promoter capable of expressing a short RNA such as polIII is linked upstream of antisense DNA and sense DNA, respectively. A cassette and a sense RNA expression cassette are constructed, and these cassettes are held in different vectors.

The dsRNA used in the present invention is preferably siRNA. “SiRNA” means a double-stranded RNA consisting of short strands in a range that is not toxic in cells. The chain length is not particularly limited as long as the expression of the target BGIBMGA007792-93 gene can be suppressed and it does not show toxicity. The strand length of dsRNA is, for example, 15 to 500 base pairs.

  As the DNA encoding the dsRNA of the present invention, an appropriate sequence (preferably an intron sequence) is inserted between inverted repeats of the target sequence, and a double-stranded RNA having a hairpin structure (self-complementary 'hairpin' RNA (hpRNA )) (Smith, NA, et al. Nature, 407: 319, 2000, Wesley, SV et al. Plant J. 27: 581, 2001, Piccin, A. et al. Nucleic Acids Res. 29 : E55, 2001) can also be used.

The DNA encoding the dsRNA of the present invention need not be completely identical to the base sequence of the target BGIBMGA007792-93 gene, but is at least 70% or more, preferably 80% or more, more preferably 90% or more (for example, 95% %, 96%, 97%, 98%, 99% or more). Sequence identity can be determined by the technique described above (BLAST program).

  The part of the double-stranded RNA in which the RNAs in dsRNA are paired is not limited to a perfect pair, but mismatch (corresponding base is not complementary), bulge (no base corresponding to one strand) ) Or the like may include an unpaired portion. In the present invention, both bulges and mismatches may be included in the double-stranded RNA region where RNAs in dsRNA pair with each other.

Another embodiment of the DNA used for suppressing the expression of the sensitive BGIBMGA007792-93 gene of the lepidopterous insect is a DNA encoding an antisense RNA complementary to the above-mentioned sensitive DNA transcript (antisense). DNA). Antisense DNA suppresses target gene expression by inhibiting transcription initiation by triplex formation, suppressing transcription by hybridization with a site where an open loop structure is locally created by RNA polymerase, Inhibition of transcription by hybridization with certain RNA, suppression of splicing by hybridization at the junction of intron and exon, suppression of splicing by hybridization with spliceosome formation site, suppression of transition from nucleus to cytoplasm by hybridization with mRNA , Splicing suppression by hybridization with capping site and poly (A) addition site, translation initiation suppression by hybridization with translation initiation factor binding site, translation suppression by hybridization with ribosome binding site near initiation codon, translation of mRNA Area or poly Outgrowth inhibitory peptide chains by the formation of a hybrid with over arm binding sites, and the like gene silencing and the like by hybridization with interaction site between a nucleic acid and protein. They inhibit transcription, splicing, or translation processes and suppress target gene expression (Hirashima and Inoue, "Neurochemistry Experiment Course 2, Nucleic Acid IV Gene Replication and Expression", edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin , pp.319-347, 1993). The antisense DNA used in the present invention may suppress the expression of the target BGIBMGA007792-93 gene by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the target gene is designed, it is considered effective for inhibiting translation of the gene. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. As described above, a DNA containing an antisense sequence of a non-translated region as well as a translated region of a gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.

Antisense DNA is based on the sequence information of the sensitive DNA of the present invention (for example, DNA consisting of the nucleotide sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19). Or by the phosphorothioate method (Stein, Nucleic Acids Res., 16: 3209-3221, 1988). The prepared DNA can be introduced into lepidopteran insects by a known method described later. The sequence of the antisense DNA is preferably complementary to the endogenous sensitive BGIBMGA007792-93 gene transcript of the Lepidoptera insect, but it is completely complementary as long as it can effectively inhibit gene expression. It does not have to be. The transcribed RNA preferably has a complementarity of 90% or more (eg, 95%, 96%, 97%, 98%, 99% or more) to the transcript of the target gene. In order to effectively inhibit the expression of the target gene, the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.

Another embodiment of the DNA used to suppress the expression of the sensitive BGIBMGA007792-93 gene of lepidopterous insects is a DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcript of the sensitive DNA of the present invention. is there. Some ribozymes have a group I intron type or a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 35: 2191, 1990).

  For example, the self-cleaving domain of hammerhead ribozyme cleaves 3 ′ of C15 of G13U14C15, but it is important for U14 to form a base pair with A at position 9, and the base at position 15 is In addition to C, it has been shown to be cleaved by A or U (Koizumi et. Al., FEBS Lett. 228: 225, 1988). If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. (Koizumi et.al., FEBS Lett. 239: 285, 1988, Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 35: 2191, 1990, Koizumi et.al., Nucleic. Acids. Res. 17: 7059, 1989).

Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, Nature 323: 349, 1986). It has been shown that this ribozyme can also be designed to cause target-specific RNA cleavage (Kikuchi and Sasaki, Nucleic Acids Res. 19: 6751, 1992, Hiroshi Kikuchi, Chemistry and Biology 30: 112, 1992). The ribozyme designed to cleave the target is linked to a promoter such as the cauliflower mosaic virus 35S promoter and a transcription termination sequence so that it is transcribed in lepidopteran insect cells. Such structural units can be arranged in tandem so that multiple sites in the target gene can be cleaved to further increase the effect (Yuyama et al., Biochem. Biophys. Res. Commun. 186: 1271, 1992 ). Using such a ribozyme, the transcription product of the target BGIBMGA007792-93 gene can be specifically cleaved to suppress the expression of the gene.

<Vector, transformed lepidopterous insect cell, transformed lepidopterous insect plant>
The present invention also introduces a vector containing the DNA of the present invention (sensitive DNA, resistant DNA, DNA for suppressing the expression of the BGIBMGA007792-93 gene), the DNA of the present invention or a vector containing the same. Lepidoptera insect cells and lepidopteran insects containing the cells are provided.

  As lepidopteran insect cells into which the vector of the present invention is introduced, the sensitivity and resistance to Cry toxins in the insect cells can be modified by introducing the BGIBMGA007792-93 gene (a silkworm-derived gene or its corresponding gene). Although there will be no restriction | limiting in particular if it is a thing, For example, a silkworm cell can be mentioned.

  As the vector of the present invention, for example, an autonomously replicable vector or a vector capable of homologous recombination in a chromosome can be used. When introducing the DNA of the present invention into a silkworm egg, for example, a method of injecting a transposon into a silkworm early egg as a vector (Tamura, T. et al., (2000) Nature Biotechnology 18: 81-84 ). For example, a transposon together with a vector in which the above DNA is inserted between the transposon inverted terminal repeats (Handler AM. Et al., (1998) Proc. Natl. Acad. Sci. USA 95 (13): 7520-5) A vector having a DNA encoding a transferase (helper vector) is introduced into a silkworm egg. Examples of the helper vector include, but are not limited to, pHA3PIG (Tamura, T. et al., (2000) Nature Biotechnology 18, 81-84). As the transposon in the present invention, piggyBac is preferable, but is not limited to this, and mariner, minos, etc. can also be used (Shimizu, K. et al., (2000) Insect Mol. Biol., 9, 277-281; Wang W. et al., (2000) Insect Mol Biol 9 (2): 145-55). In the present invention, a transformed silkworm can also be produced by using a baculovirus vector (Yamao, M. et al., (1999) Genes Dev 13: 511-516).

  Vectors can be introduced into silkworm cells by, for example, calcium phosphate precipitation method, electric pulse perforation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofectamine method ( GIBCO-BRL) and a known method such as a microinjection method.

  The DNA of the present invention can be introduced into lepidopterous insects as exogenous DNA, but can also be introduced into lepidopteran insects by crossing with strains having the DNA of the present invention. “Introduction” in the present invention includes both forms.

  Once a lepidopteran insect in which the DNA of the present invention is introduced into a chromosome is obtained, an egg, offspring, or clone can be obtained from the insect and the lepidopteran insect can be propagated on the basis thereof. is there. The present invention includes lepidopterous insect cells into which the DNA of the present invention has been introduced, lepidopterous insects containing the cells, eggs, offspring and clones of the lepidopterous insects.

<Method of producing lepidopteran insects with resistance to Cry toxin>
The present invention also provides a lepidopteran insect imparted with resistance to Cry toxin, characterized by suppressing the expression or function of the sensitive DNA of the present invention (sensitive BGIBMGA007792-93 gene). Provide a production method. In the present invention, `` conferring '' resistance to Cry toxins to lepidopterous insects is not only to give resistance to Cry toxins to strains that have no resistance to Cry toxins, but already to certain Cry toxins. It is intended to include further increasing resistance to Cry toxins in strains that are resistant to toxins.

One embodiment for suppressing the expression or function of the sensitive DNA of the present invention in lepidopterous insects is to introduce the above-described resistant DNA of the present invention into the lepidopteran insect. Since resistance to Cry toxins in lepidopteran insects is a recessive gene, both BGIBMGA007792-93 alleles in individuals are usually resistant to confer resistance traits to Cry toxins in lepidopteran insects. It needs to be DNA. Thereby, only resistant DNA is expressed in the individual, and resistance to Cry toxin can be imparted to lepidopteran insects. Introduction of the resistant DNA of the present invention into the lepidopteran insect chromosome can be performed, for example, by mating or homologous recombination. Instead of introducing resistant DNA, a specific DNA sequence may be introduced into sensitive DNA on the lepidopteran insect chromosome to destroy its function.

Another embodiment for suppressing the expression or function of the sensitive DNA of the present invention in lepidopterous insects is to introduce the DNA for suppressing the expression of the BGIBMGA007792-93 gene of the present invention into the lepidopterous insect. It is. As a result, no sensitive translation product is produced from the sensitive DNA in the individual, and thus resistance to Cry toxins can be imparted to lepidopteran insects.

  Other embodiments for suppressing the expression or function of the sensitive DNA of the present invention in lepidopterous insects include, for example, binding to a drug that suppresses the expression of sensitive DNA or a sensitive translation product and suppressing the function. It is also possible to use drugs that do this.

<Method for determining sensitivity or resistance to Cry toxin in lepidopterous insects>
The present invention also provides a method for determining sensitivity or resistance to Cry toxins in Lepidoptera insects. One embodiment of the determination method of the present invention is a method characterized by analyzing the base sequence of the BGIBMGA007792-93 gene in Lepidoptera insects.

In analyzing the base sequence of the BGIBMGA007792-93 gene, an amplification product obtained by amplifying the BGIBMGA007792-93 gene by PCR can be used. In case of carrying out the PCR, primers used are not limited as long as it can specifically amplify the BGIBMGA007792-93 gene, BGIBMGA007792-93 gene sequence information (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33).

The “control base sequence” to be compared with the base sequence of the BGIBMGA007792-93 gene in the lepidopteran insect to be tested is typically the base sequence of the BGIBMGA007792-93 gene in a sensitive or resistant strain. The determined nucleotide sequence of the BGIBMGA007792-93 gene and the nucleotide sequence in the sensitive strain (eg, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 ) or the nucleotide sequence in the resistant strain (For example, SEQ ID NO: 21, 23, 25, 27, 29, 31, 33 ), whether the BGIBMGA007792-93 gene in the lepidopteran insect to be tested is resistant or sensitive Can be evaluated. In particular, the presence or absence of insertion of Tyr at position 234 in the protein encoded by the BGIBMGA007792-93 gene is a preferable index for such evaluation.

By performing PCR using a primer pair (see FIG. 8) containing a primer capable of detecting the presence or absence of insertion of Tyr at position 234 in the protein encoded by the BGIBMGA007792-93 gene, BGIBMGA007792- By analyzing the base sequence of 93 genes, it is possible to easily determine the sensitivity or resistance to Cry toxin in lepidopteran insects.

Whether or not the base sequence of the BGIBMGA007792-93 gene in the lepidopteran insect to be tested differs from the base sequence of the control can be analyzed indirectly by various methods other than the determination of the direct base sequence described above. it can. Examples of such methods include PCR-SSCP (single-strand conformation polymorphism) method, RFLP method using restriction fragment length polymorphism (RFLP), Examples include PCR-RFLP, denaturant gradient gel electrophoresis (DGGE), allele specific oligonucleotide (ASO) hybridization, and ribonuclease A mismatch cleavage.

  The base sequence can be determined by a conventional method such as the dideoxy method or the Maxam-Gilbert method. In determining the base sequence, a commercially available sequence kit and sequencer can be used. Furthermore, the base sequence can also be determined by a large-scale high-speed sequencing method using a device generally called a next-generation sequencer.

<Method of breeding lepidopterous insects resistant to Cry toxin>
The present invention also provides a method for breeding lepidopteran insects resistant to Cry toxins. The breeding method of the present invention comprises the steps of (a) mating a lepidopteran insect line resistant to Cry toxin with an arbitrary lepidopteran insect line, (b) sensitivity or resistance to Cry toxin in an individual obtained by the crossing And (c) selecting a strain determined to have resistance to Cry toxin.

  Examples of `` arbitrary lepidopteran insect lines '' to be crossed with Cry toxin-resistant lepidopteran insect lines include, for example, susceptible strains, and individuals obtained by mating between susceptible lines and resistant lines. Not limited. By using the breeding method of the present invention, it becomes possible to select lepidopteran insects resistant to Cry toxin at an early stage, and it is possible to grow a strain having a trait resistant to Cry toxin in a shorter period of time than before. Can be done.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and a comparative example, this invention is not limited to a following example.

[Example 1] Prediction of gene in chromosome region where resistance gene sits and confirmation of expression A silkworm resistant line (C2_R) and a sensitive line (Rin_S) were crossed to produce a hybrid (F1: sensitive). Then, the F1 male and the female of the resistant strain (C2_R) are mated to produce a backcross (BC1: a mixture of resistant and sensitive individuals), and this BC1 individual is fed with Cry1Ab toxin to resist Individuals were selected and the genomic DNA of each individual was analyzed. The present inventors performed linkage analysis using 1365 individuals of resistant BC1 (Japanese Patent Laid-Open No. 2007-202554), and performed gene prediction and expression confirmation within the 82 kbp range in which the resistance gene is predicted to sit. . When gene prediction was performed using the gene prediction model in KAIKObase ver.2.1.0 (http://sgp.dna.affrc.go.jp/KAIKObase/), six genes were found in the 82 kbp region ( Table 1).

For three genes, BGIBMGA007735 , BGIBMGA007793 , and BGIBMGA007792 , homologous mRNA base sequence data were reported in other species. On the other hand, with the remaining three predictive genes ( BGIBMGA007736 , BGIBMGA007791 , BGIBMGA007737 ), no homologous mRNA nucleotide sequence data were reported in other species.

Mid-gut tissues of 5th instar larvae were collected from the resistant strain (C2_R) and the sensitive strain (Rin_S), total RNA was extracted, and cDNA synthesis was performed. Primers were synthesized based on the predicted gene sequences, and expression analysis of 6 predicted genes was performed by RT-PCR method. As a result, no expression was detected in the two resistance candidate genes, 5 ( BGIBMGA007791 ) and 4 ( BGIBMGA007737 ). On the other hand, the remaining four resistance candidate genes, 1 ( BGIBMGA007735 ), 2 ( BGIBMGA007793 ), 3 ( BGIBMGA007736 ), and 4 ( BGIBMGA007792 ), were confirmed to be expressed in the midgut tissue (FIG. 1).

Therefore, next, with respect to BGIBMGA007735 , BGIBMGA007793 , BGIBMGA007736 , and BGIBMGA007792 , gene fragments were amplified by RT-PCR, 3′-RACE, and 5′-RACE, and the nucleotide sequences were determined. As a result, it was found that the three genes BGIBMGA007793 , BGIBMGA007736 , and BGIBMGA007792 are part of the same gene. BGIBMGA007736 is a short gene candidate in the intron part of the genes encoded by BGIBMGA007792 and BGIBMGA007793 and was not included in the actual mature mRNA. Therefore, the existence of two genes ( BGIBMGA007735 and BGIBMGA007792-93 ) became clear. Comparison of the base sequences of the regions coding for the BGIBMGA007735 and BGIBMGA007792-93 proteins in the resistant strain (C2_R) and the sensitive strain (Rin_S) reveals the sequence inside the marker in BGIBMGA007735 . There was no difference. In BGIBMGA007792-93 , the full length of the ORF was clarified, and a difference of 39 bases in the base sequence and 13 residues in the amino acid sequence was seen (FIGS. 2 and 3). From the above, it was suggested that the BGIBMGA007792-93 gene may be a Bt resistance gene.

[Example 2] Comparison of deduced amino acid sequences in resistant and susceptible lines of resistance candidate genes cDNA was prepared from the midgut tissue of resistant lines (7 lines) and sensitive lines (10 lines), and BGIBMGA007792-93 The nucleotide sequence of the ORF region of the gene encoding this protein was determined. The deduced amino acid sequences were compared to examine whether there were mutations common to resistant strains. The seven resistant lines investigated were J1_R, Ki_R, Be_R, C2_R, C7_R, Cs_R, and N15_R (derived from No. 342). The susceptibility strains examined were Yo_S (Japanese strain), Bag_S, N65_S, E12_S, An_S, CaM_S, My_S, PMy_S (strains closely related to Rin_S), Rin_S (strain used in this study), e21_S (No.912) 10 origin). Comparing 17 lines with 7 resistant lines and 10 sensitive lines, the insertion of Tyr (tyrosine) at position 234 (position 235 in FIG. 4) was found as a sequence common to the resistant lines (FIG. 4). ).

[Example 3] Demonstration of candidate resistance gene using transgenic silkworm Since the resistance gene is a recessive gene, it is expected that resistance is caused by the loss of function of the gene. In order to prove this, an experiment (rescue experiment) was performed in which the resistant silkworm strain was returned to sensitivity by introducing a sensitive BGIBMGA007792-93 gene into the resistant silkworm strain.
(I) In producing a transgenic silkworm, it is important to select a tissue in which the gene is forcibly expressed. Therefore, first, the RT-PCR method was used to investigate in which tissue the BGIBMGA007792-93 gene was expressed. The tissues examined were: amg: anterior midgut, mmg: midgut, pmg: posterior midgut, afb: chest fat pad, pfb: abdominal fat pad, sg: silk gland, mt: Malpighi duct, hc: blood cell, te: testis, ov: ovary, int: hull. As a result, expression was confirmed only in the midgut (FIG. 5). Regarding the midgut, the expression of the BGIBMGA007792-93 gene was investigated in each of the three sites: amg: anterior midgut, mmg: midgut, pmg: posterior midgut. Was confirmed.
(Ii) In producing a transgenic silkworm, it is desirable that the original silkworm strain has a resistant BGIBMGA007792-93 gene, has white eyes and eggshells, and is non-dormant. Therefore, next, silkworm lines “white / c” and “w1-pnd” were crossed to create a silkworm line having all the traits. White / c is a line with white eyes and eggshells and a dormant trait, and there are individuals with resistant and sensitive genes. W1-pnd is a non-dormant strain with white eyes and eggshell, and has a resistance-type gene.
(Iii) A UAS-Bt system (SS16-1, SS16-3) was produced. First, a susceptible silkworm (606 strain) BGIBMGA007792-93 gene was ligated downstream of the UAS sequence activated by GAL4 and incorporated into a piggyBac transposon vector (FIG. 6). This vector was injected into the silkworm strain (resistant / non-dormant w1-pnd strain) prepared in (ii). The UAS-Bt-r line was maintained in cross with a resistant / dormant white / c line. A sensitive BGIBMGA007792-93 gene is located downstream of the UAS sequence, and when the UAS sequence is activated by GAL4 (ie, when the effector construct functions), EGFP is expressed by the eye at the time of the egg.
(Iv) A line expressing GAL4 in the midgut was prepared. First, an activator construct (FIG. 7) was introduced into a silkworm strain in the same manner as in the production of the UAS-Bt strain to create a resistant / dormant BmA3_GAL4 strain (52-2). When the activator construct functions, DsRed2 is expressed visually during the egg season, and GAL4 is expressed in the midgut of the larvae.

Of the F1 obtained by crossing the GAL4 and UAS-Bt lines expressing the midgut, only individuals with both the GAL4 and UAS-Bt genes express UAS-Bt in the midgut. By DsRed2), they were selected at the egg stage, and the larvae molted at the age of 2 were subjected to bioassay.
(V) In the bioassay, 15 μl of a 4-fold serially diluted Cry1Ab toxin solution was applied to the back of a mulberry leaf hollowed to a diameter of 14 mm. The 2nd instar larvae immediately after molting were reared on the mulberry leaves one by two for two days and further on untreated mulberry leaves for two days to investigate the number of dead insects. The test used 24 2nd instar larvae per concentration group. The breeding container was a 24-well plate for cultured cells, and the temperature was 25 ° C. LC ₅₀ was calculated from mortality by the probit method.

Table 2 shows the LC ₅₀ of Cry1Ab toxin against the resistant line (C2_R) and the sensitive line (Rin_S) that have been used in the analysis of Cry1Ab toxin resistance gene so far. The LC ₅₀ value shows a large value is resistance, and it is sensitive to death at a low concentration. The Rin_S strain has a recessive resistance candidate gene (dominant susceptibility candidate gene) cloned to create a construct for UAS-Bt strain creation. Of the lines created as UAS-Bt lines, two lines, SS16-1 and SS16-3, were used in this study.

In the table, 1) indicates the 50% lethal concentration as the toxin protein concentration per 1 cm ^{2 of} mulberry leaves. 2) shows the 95% confidence limit.

Table 3 shows LC in non-dormant and dormant lines of white egg mutations used for the production and maintenance of transgenic silkworms, and resistant / dormantant BmA3_GAL4 lines (52-2) that express GAL4 in the midgut. ₅₀ was shown. Note that the white / c and w1-pnd lines are not pure lines and the genetic background is not uniform, so the resistance strength is considered to vary to some extent. All strains are resistant to Cry1Ab toxin.

  In the table, 1) is a dormant strain that has white eyes and eggshells and is resistant to Cry1Ab toxin.The strain used to maintain the prepared strain is 2), and the Bt gene downstream of the UAS sequence is resistant to Cry1Ab toxin. The strains injected with the constructs 3) are white / c-derived strains that express the transcription factor Gal4 that binds to UAS in the midgut.

F1 (52-2 × SS16-3 and 52-2 × SS16-1) 2nd instar larvae obtained by crossing the midgut-expressing GAL4 line with the UAS-Bt line have the same or better sensitivity than the Rin_R line. (Table 4; first and third rows of data rows). On the other hand, white / c × SS16-1 and white / c × SS16-3 (Table 4; 2nd and 4th lines; no GAL4) used as controls showed resistance. Similar results were obtained in tests using 4th instar larvae (Table 5). In addition, 52-2 × white / C does not have the BGIBMGA007792-93 gene and thus exhibits resistance. These results indicate that the silkworm changes to susceptibility by expressing the sensitive BGIBMGA007792-93 gene in the resistant silkworm, and that the BGIBMGA007792-93 gene is the main body of the Cry1Ab toxin resistance gene. Has been demonstrated.

In the table, 1) is a dominant sensitive BGIBMGA007792-93 gene forced expression line, 2) is a dominant sensitive BGIBMGA007792-93 gene introduced, but the expression is suppressed, 3) is The lines expressing only the GAL4 gene in the midgut are shown.

[Example 4] Quantification of the expression level in the midgut tissue of the sensitive BGIBMGA007792-93 gene introduced into the resistant strain Quantification of whether the introduced gene is expressed in the silkworm used in the bioassay in Example 3 Verification was performed using PCR. In the BGIBMGA007792-93 gene, it has been suggested that the cause of resistance and sensitivity is due to a difference of one amino acid (FIG. 4). However, since the sequences of other parts are extremely conserved, it is difficult to distinguish and quantify the expression levels of both in this part. Therefore, in silkworms introduced with BGIBMGA007792-93 gene sensitive type, in order to distinguish the expression of BGIBMGA007792-93 gene sensitive type introduced with BGIBMGA007792-93 gene resistant type which is originally expressed in the same individual A primer utilizing a single nucleotide polymorphism near the 3 ′ end of mRNA was designed (FIG. 8, SEQ ID NOs: 35 to 38).

  First, gene expression levels of Rin_S sensitive lines and C2_R resistant lines, which are the original lines, were measured with this primer (FIG. 9, original line). As a result, each expression level was measurable, and even when both were quantified, data was obtained that would have quantified each gene quantity. Therefore, the midgut of the 4th instar larva of the transgenic line was dissected, mRNA was extracted, cDNA was prepared, and the expression level was quantified (relative quantification with respect to the ribosomal protein gene). Both SS16-1 and SS16-3 lines were used for quantification.

As a result, in the progeny obtained by crossing these sensitive BGIBMGA007792-93 genes with the GAL4 line (Fig. 9, SS16-1 and SS16-3 A), the introduced gene (Fig. 9, shaded) However, the expression level was equal to or higher than that of the original resistance gene, and it was proved that the introduced gene was reliably expressed in the midgut of the silkworm larvae. In addition, in both SS16-1 and SS16-3 lines (B and E) not crossed with GAL4 lines, it was also observed that the susceptibility genes introduced in some individuals were slightly expressed, From the above bioassay results, it is considered that this level of expression does not significantly affect the assay results.

From the above, it was proved that the BGIBMGA007792-93 gene is involved in the toxic expression of Cry toxin in silkworms.

The BGIBMGA007792-93 gene found by the present invention can be used for breeding a Cry toxin-resistant silkworm practical strain in the sericulture industry. By using this gene as a marker, the breeding period of such silkworm lines can be shortened. If the Cry toxin resistant silkworm strain is used, even if the mulberry leaves in the mulberry field are contaminated by the drift of insecticides mainly composed of the Cry toxin sprayed around the mulberry field to control lepidopterous pests, It can prevent damage to silkworms. Until now, insecticides could not be applied to mulberry gardens during the breeding period of silkworms, but by applying resistance to Cry toxins to silkworms, it is possible to control lepidopterous insects in mulberry gardens using insecticides even during the breeding period.

Also, in the field of pest control, the BGIBMGA007792-93 gene found by the present invention is used to elucidate the mechanism of Cry toxin resistance development in pests including lepidoptera and to predict the development of Cry toxin resistance in pest populations, etc. Available to: Comparing the DNA sequence information of genes that show homology with the found genes between resistant and susceptible pests, and if there is a relationship between resistance and DNA sequence information, the mechanism of resistance development It leads to elucidation. Pest species whose resistance development mechanism has not been elucidated at present include Cry1Ac-resistant diamondback moths (Cruciferae pests), Cry1Ab-resistant and Cry1F-resistant European corn borers (corn pests), etc., BGIBMGA007792 The -93 gene may contribute to elucidation of the resistance mechanism of these insects. In addition, if the resistance of a certain lepidopteran pest is due to the mutation of the BGIBMGA007792-93 gene or its genetic mode is clarified, it becomes possible to produce a primer for detecting a resistance gene based on the genetic information. Using such primers, it is possible to detect resistance genes and investigate the frequency of genes in adults collected from the field using pheromone traps, etc., so conventional bioassay methods (eg, inferior resistance) If, allows outdoor determine the genotype of an individual) than simply resistance gene frequencies investigated by bioassay intended for F ₁ obtained by crossing resistant strains of individuals and inbred collected from the field . As a result, when the frequency of the resistance gene is high, rapid countermeasures such as changing the type of Cry toxin to be used can be taken to prevent the development of drug resistance in pests. Therefore, the present invention is also expected to contribute to the construction of pest population management technology.

Claims (7)

Bacillus characterized by suppressing the expression or function of the endogenous DNA according to any one of the following (a) to (c) in a lepidopteran insect sensitive to an insecticidal protein produced by Bacillus thuringiensis -A method for producing lepidopteran insects with resistance to insecticidal proteins produced by Thuringiensis.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19
(C) DNA consisting of a base sequence having a homology of 90% or more with DNA consisting of the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 Including a step of recombining both alleles by introducing the DNA described in any of the following (a) to (c) into a lepidopteran insect sensitive to an insecticidal protein produced by Bacillus thuringiensis: A method for producing lepidopteran insects to which resistance to insecticidal proteins produced by Chillus thuringiensis is imparted.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 22, 24, 26, 28, 30, 32 or 34
(B) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 21, 23, 25, 27, 29, 31 or 33
(C) DNA comprising a nucleotide sequence having a homology of 90% or more with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 21, 23, 25, 27, 29, 31 or 33 Insecticide produced by Bacillus thuringiensis, comprising the step of introducing the DNA according to any of the following (i) to (iii) into a lepidopteran insect sensitive to an insecticidal protein produced by Bacillus thuringiensis A method for producing lepidopteran insects with resistance to sex proteins.
(I) DNA encoding a double-stranded RNA complementary to the transcription product of DNA described in any of (a) to (c) below
(Ii) DNA encoding an antisense RNA complementary to the transcription product of DNA described in any of (a) to (c) below
(Iii) DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcription product of the DNA according to any one of (a) to (c) below
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19
(C) DNA consisting of a base sequence having a homology of 90% or more with DNA consisting of the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 Bacillus thuringiensis sensitive to lepidopterous insects sensitive to insecticidal proteins produced by Bacillus thuringiensis , comprising the DNA according to any one of the following (a) to (c) or a vector into which the DNA is inserted A drug for imparting resistance to insecticidal proteins produced by
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 22, 24, 26, 28, 30, 32 or 34
(B) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 21, 23, 25, 27, 29, 31 or 33
(C) DNA comprising a nucleotide sequence having a homology of 90% or more with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 21, 23, 25, 27, 29, 31 or 33 Bacillus thuringiensis sensitive to lepidopteran insects sensitive to insecticidal proteins produced by Bacillus thuringiensis, comprising the DNA according to any of the following (i) to (iii) or a vector into which the DNA is inserted A drug for imparting resistance to insecticidal proteins produced by
(I) DNA encoding a double-stranded RNA complementary to the transcription product of DNA described in any of (a) to (c) below
(Ii) DNA encoding an antisense RNA complementary to the transcription product of DNA described in any of (a) to (c) below
(Iii) DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcription product of the DNA according to any one of (a) to (c) below
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19
(C) DNA consisting of a base sequence having a homology of 90% or more with DNA consisting of the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 A method for determining sensitivity or resistance to an insecticidal protein produced by Bacillus thuringiensis in a lepidopterous insect, wherein the endogenous DNA according to any one of the following (a) to (c) in a test lepidopteran insect: A method comprising analyzing the base sequence of
(A) encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 DNA
(B) DNA comprising the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33
(C) Homology with DNA comprising the nucleotide sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33 DNA consisting of more than 90% nucleotide sequence A method for breeding lepidopteran insects resistant to insecticidal proteins produced by Bacillus thuringiensis,
(A) mating a lepidopteran insect strain resistant to an insecticidal protein produced by Bacillus thuringiensis with any lepidopteran insect strain;
(B) determining the sensitivity or resistance to the insecticidal protein produced by Bacillus thuringiensis in the individual obtained by the mating in step (a) by the method according to claim 6 , and (c) Bacillus Selecting a strain determined to have resistance to an insecticidal protein produced by thuringiensis.