JP3530495B2 - Ecdysteroid 22 oxidase from Pseudomonas oryzae and molting hormone inactivation system using it - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an ecdysteroid 22-position oxidase isolated from an insect pathogenic filamentous fungus, Nomuraea rileyi, and a molting hormone inactivating system using the same.

[0002]

2. Description of the Related Art It is known that the molting of arthropods such as insects and crustaceans is induced by several ecdysteroids having molting hormone activity. At least two uses have been developed for these molting hormones.

One of the uses is to control the development of individuals such as accelerating the time of molting or metamorphosis and uniforming pupation. This makes it possible to control silkworm production in silkworms, for example.

Other uses are in cultured cell lines, transgenic animals or transgenic plants,
It is a use in a gene expression system capable of controlling the expression time of a target gene at a high level by treatment with molting hormone. This is based on the finding that molting hormone regulates the transcriptional activity of the response gene by binding to the molting hormone receptor, which is a transcription factor, and further to the molting hormone response element on the molting hormone response gene. .

For example, a target gene having a molting hormone responsive element incorporated in the molting hormone receptor and a transcriptional regulatory region has been introduced into these systems and added to a cultured cell system (Chri.
stopherson, KS et al. (1992) Proc. Natl. Acad.
Sci. USA 89, 6314-6318), injected into animals (No, D et a
l. (1996) Proc. Natl. Acad. Sci. USA 93, 3346 -335
1) Or absorbed from plant roots (Martinez, A. et
al. (1999) The Plant Journal 19, 97-106), etc., and the expression of the target gene product is induced by increasing the intracellular molting hormone concentration of each. Among these, those used for the cultured cell system have already been put to practical use as a kit.

On the other hand, a technique for enhancing molting hormone activity without using molting hormone itself has been developed. For example,
An example is the use of ecdysteroids with strong molting hormone activity and more stable and powerful molting hormone agonists that do not have an ecdysteroid skeleton in pest control. As described above, a technique for increasing the molting hormone activity has already been developed.

On the other hand, almost no method has been developed for reducing molting hormone activity, that is, for inactivating molting hormone present in the body or cells. Currently, baculovirus-derived ecdysteroid UDP-
Glucosyltransferase gene (JP-A-11-1
23079) is attracting attention as a gene for an enzyme capable of inactivating the molting hormone. However, it has a drawback that coexistence of UDP-glucose is indispensable for the enzyme to act, and thus it has not been put to practical use as a purified enzyme preparation or a recombinant protein.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a substance and system for efficiently inactivating the molting hormone.

[0009]

The present inventors have recognized that inactivation of molting hormone is extremely important for applications such as developmental control of insects and control of expression induction of target gene products. All ecdysteroids with hormonal activity have a hydroxyl group at the 22nd position, and it is noted that the molting hormone activity is significantly reduced when this site is modified. More specifically, the insect pathogenic filamentous fungus Nomuraea rileyi) from ecdysteroids 22
The inventors have found that the above problems can be solved by isolating a position oxidase and oxidizing the hydroxyl group at position 22 of ecdysteroid to a keto group by the enzyme or a modified protein thereof, and completed the present invention.

That is, the present invention is the following protein (a) or (b), that is, (a) a protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. (B) Sequence Listing The present invention relates to a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in SEQ ID NO: 2 and having ecdysteroid 22-position oxidase activity.

The present invention also provides the following DNA (a) or (b), that is, (a) a DN comprising the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.
A. (B) A gene comprising a DNA which hybridizes with the DNA comprising the nucleotide sequence of (a) under stringent conditions and which encodes a protein having an ecdysteroid 22-position oxidase activity.

The present invention further relates to a method for inactivating the molting hormone of a protein by administering the protein to an arthropod. Further, the present invention, by inactivating the molting hormone using the protein,
It relates to a method of controlling the growth of an arthropod. Furthermore, the present invention relates to a method of controlling insect growth by inactivating molting hormone using the protein. The present invention also relates to a method for producing a silkworm thread, which comprises controlling the diameter of a thread spun by a silkworm by administering the protein to the silkworm. Furthermore, the present invention relates to a method for suppressing the expression of molting hormone-inducible gene by administering the protein to a transformant.

The protein according to the present invention is an enzyme having an activity of oxidizing and inactivating the 22nd hydroxyl group of molting hormone. Therefore, if the enzyme is administered into the body of an arthropod, its growth can be controlled by inactivating the molting hormone. The enzyme according to the present invention can be used for controlling the growth of insects such as silkworms.
In particular, by administering the enzyme of the present invention to silkworms, it is possible to produce silkworm silk which has a higher commercial value because it is thinner than normal silkworm silk.

The enzyme according to the present invention can be used not only for insects but also for controlling the growth of crustaceans using ecdysteroids as molting hormones like insects. In order to obtain the enzyme, silkworms are infected with a recombinant baculovirus having the gene of the present invention, which has the nucleotide sequence shown in Sequence Listing 2, and the protein is collected in the blood by expressing the protein in a large amount. It is possible to use the system which does.

When the enzyme or gene according to the present invention is used, expression of the target gene can be optionally performed in a system in which molting hormone is added, injected or root-absorbed to increase intracellular molting hormone concentration to induce expression of the target gene product. Can be stopped. Therefore, in a transformant such as a cultured cell, a transgenic animal or a transgenic plant, it is possible to control the gene expression system using molting hormone in the negative direction, which allows gene expression using the transformant. The range of application of the system is greatly expanded. Hereinafter, the present invention will be described in detail.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Sequence Listing 1 shows the cDNA of the ecdysteroid 22-position oxidase isolated from Pseudomonas aeruginosa (Nomuraea rileyi) according to the present invention and the amino acid sequence predicted therefrom. In addition, Sequence Listing 2 shows the predicted amino acid sequence of the ecdysteroid 22-position oxidase. Regarding cloning of cDNA, first, the protein was purified from the culture solution of Pseudomonas aeruginosa using the keto-oxidation activity of the 22nd hydroxyl group of ecdysone, a type of molting hormone, as an index, and the N-terminal and internal amino acid sequences were determined. Furthermore, based on those amino acid sequences, PC
The full length cDNA was cloned by R.

FIG. 1A shows the structures of ecdysteroids found in insect bodies and their oxidation products by the enzyme according to the present invention. Moreover, as shown in FIG. 1B, these ecdysteroids can be easily detected by HPLC. That is, reverse phase column (TSKgelODS-80Ts, 4.6 mm x 150 m
(m TOSO) was attached to HPLC (LC 10-AT, Shimadzu), and the absorbance at 245 nM was measured by an ordinary ultraviolet detector. At this time, the flow rate was 0.6 ml / min, the column retention temperature was 40 ° C., and the sample was allowed to flow for 40 minutes in a gradient of acetonitrile 20-30%.

The predicted coding region of the cDNA of the present invention (A ₁₁₁ -T ₁₈₉₃ in Sequence Listing 1) was used as a protein expression system (BAC-TO-BAC Baculovirus E) using baculovirus.
Xpression Systems, Gibco BRL) was used to express in SF-9 cells, and strong ecdysteroid oxidative activity was observed in the culture medium on the 7th day from the start of culture (FIG. 2).
Western blot analysis using a purified polyclonal antibody against this enzyme also confirmed that the ecdysteroid 22-position oxidase was secreted into the culture (FIG. 3).

[0019] Generally, in the amino acid sequence encoding a protein having physiological activity, even if one or more amino acids are added, deleted or substituted,
It is obvious to those skilled in the art that the physiological activity may be maintained. The present invention also includes a DNA fragment which is modified in this way and which encodes a protein having an ecdysteroid 22-position oxidase activity.
That is, in SEQ ID NO: 2 in the Sequence Listing, a DNA which comprises an amino acid sequence in which one or several amino acids are added, deleted, substituted or inserted, and which encodes a protein having ecdysteroid 22-oxidase activity Within the scope of the present invention.

The term "one or several" as used herein means a site in the three-dimensional structure of an ecdysteroid 22-position oxidase protein of an amino acid residue to which an amino acid is added, deleted or substituted, or a lipid residue of the amino acid residue. It depends on the number, but usually 2 to 20, preferably about 2 to 15. Such modified DNA can be obtained by modifying the nucleotide sequence of the DNA of the present invention such that a specific amino acid is deleted, substituted or added by, for example, a site-directed mutagenesis method.

Further, the DNA of the present invention or cells having the same is subjected to mutation treatment, and hybridized from these DNAs or cells with, for example, the DNA having the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing under stringent sodium conditions. The modified DNA can also be obtained by selecting the DNA to be used.

The term "stringent conditions" as used herein means conditions under which a specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to clearly quantify this condition, for example, nucleic acids with high homology such as 99.5% or more hybridize with each other, but DNAs with lower homology do not hybridize with each other. Such conditions may be mentioned.

[0023]

The present invention will be described below with reference to examples.
The present invention is not limited to these. Example 1 Ecdysteroid 2 from Pseudomonas aeruginosa
Isolation of 2-position oxidase Pseudomonas aeruginosa was cultivated in a liquid medium containing the silkworm pupa extract for 9 days, and the culture broth (containing the target enzyme) was added to 0.45.
After passing through a μm filter, it was stored at 4 ° C. After collecting a sufficient amount of the culture broth, the enzyme was isolated by the following four-step extraction. 1. Precipitate with 50% ammonium sulfate (ammonium sulfate) Phenyl Hydrophobic Chromatography (phenyl-Sepharo
use se) 3. Gel filtration (using Superdex 200pg) 4. Anion chromatography (using HiTrap Q) 2-3 was performed using HPLC (Model Bio-HPLC system, Tosoh). In each process, the enzymatic reaction using ecdysone as a substrate shown in FIG. 1A was performed, and the oxidation product was monitored by HPLC to examine the fraction containing the target enzyme. Then, after the final purification, SDS PAGE was performed to confirm that the single protein was purified by silver staining.

Example 2 Sequencing of ecdysteroid 22-position oxidase gene First, N of the enzyme isolated according to the method described in Example 1 was used.
The terminal sequence was analyzed by an amino acid sequencer to determine the N-terminal amino acid sequence. Furthermore, the internal amino acid sequence of this enzyme was determined by analyzing the N-terminal sequence of the degradation product obtained by partially degrading the enzyme preparation with V8 protease using an amino acid sequencer. Based on the N-terminal sequence and the internal sequence of the enzyme thus determined, four kinds of degenerate primers, that is, E22o.6 primer as a forward primer (SEQ ID NO: 3 in the Sequence Listing; LPQGGCR (21 in amino acid sequence) ~ 27) and E22o.2 primer (SEQ ID NO: 4 in the sequence listing; CRCIPGE (26-32) of amino acid sequence)
, And Int.R1 primer as a reverse primer (SEQ ID NO: 5 in the sequence listing; QNVNNAW of amino acid sequence)
(74 to 80) in reverse direction) and Int.R2 primer (SEQ ID NO: 6 in Sequence Listing; DQGQNV of amino acid sequence)
N (71 to 77) was designed in the reverse direction). A partial cDNA of this enzyme was cloned by performing RT-PCR using these primers with mRNA extracted from the cultured P. rubrum as a template.

PCR was carried out twice by changing the primer set. That is, the first time, E22o.6 primer and Int.R1 primer were used, and the second time, E22o.2 primer and Int.R2 primer were used. Finally, the 115-nucleotide sequence from 206 to 320 in the molting hormone oxidase cDNA (full length 1963 nucleotides) was amplified and the nucleotide sequence was determined. By designing primers in this partially cloned cDNA, RT
-Use 5'RACE and 3'RACE, which are one of the modified PCR methods, as SM
By using ART RACE cDNA Amplification Kit (CLONTECH), a cDNA covering the entire length of mRNA of this enzyme was cloned. E3 in 3'RACE
209 of the entire base sequence using the 2o.RF1 primer (SEQ ID NO: 7 in the Sequence Listing; E22o corresponding to the entire base sequence of 209 to 231)
The region of -1963 was amplified and the base sequence was determined. Further, in 5'RACE, the E22o.RR1 primer (SEQ ID NO: 8 in the Sequence Listing; corresponding to the reverse strand of 268 to 290 of the E22o whole base sequence) is used to amplify the region of 1 to 290 of the whole base sequence,
The base sequence was determined. As described above, RT-PCR, 5'RACE
By overlapping the cloned regions with 3'RACE and 3'RACE, the total length of the cDNA of molting hormone oxidase of Pseudomonas aeruginosa was determined.

(Example 3) Effect of ecdysteroid 22-position oxidase on the growth of silkworm larvae An enzyme solution containing the enzyme of the present invention was injected to 4th or 5th instar larvae of silkworm at 1.6 units / 20 μl / head. The subsequent growth was examined. In addition, "1 unit" represents an enzyme activity that oxidizes 1 nM ecdysone per minute. The 4th instar larvae of silkworms usually molt into 5th instar larvae in about 5 days, and the 5th instar larvae start cocooning in about 7 days and pupate on the 11th day. However, when this enzyme was injected into the silkworm body at the beginning of the 4th instar, cocoons began to be made about 7 days after the injection, and pupation occurred after 11 days (Fig. 4A). On the other hand, when it was injected on the 5th day on the 7th day, the larva remained in a larva state for 10 days or more after the injection, and finally died without pupation (FIG. 4B).

When ecdysteroids in blood after injection of the enzyme solution were examined, the active molting hormone 20-hydroxyecdysone and its precursor, ecdysone, were hardly detected, and the ecdysone at the 22nd position was detected. A large amount of modification products in which hydroxyl groups were oxidized were accumulated (Fig. 5). Thus, by injecting the silkworm with the enzyme according to the present invention, the molting hormone in the silkworm body is inactivated, and precocious metamorphosis and extension of the spun period depending on the time of injection,
It was possible to control growth such as transformation inhibition.

(Example 4) Transcription-inducing effect of ecdysteroid whose 22-position is oxidized on molting hormone-inducible gene The molting hormone receptor (EcR) gene may be transcription-induced by molting hormone itself which is a ligand. Are known. Different concentrations of 20-hydroxyecdysone or 20-hydroxy-22-dehydroecdysone modified at position 22 by the enzyme of the present invention were added to the cultured silkworm anterior gland and EcR mRNA after several hours was added.
Was examined. As a result, when 20-hydroxyecdysone was added, the EcR mRN was dependent on the amount added.
Although the expression level of A was increased, no expression inducing effect was observed when 20-hydroxy-22-dehydroecdysone was added (Fig. 6). Thus, it was confirmed that the ecdysteroid modified at position 22 by the enzyme according to the present invention has no transcription-inducing activity for molting hormone-inducible gene.

As described above, the ecdysteroid 22-position oxidase according to the present invention has a strong ability to inactivate molting hormone, and by using this enzyme, the growth control of molluscs and molting hormone-inducible gene can be effectively performed. It was shown that the expression control of P.

[0030]

INDUSTRIAL APPLICABILITY By using the enzyme according to the present invention, it is possible to control the growth of insects by efficiently inactivating the insect molting hormone. Further, by using the enzyme, it is possible to produce a silkworm thread thinner than usual. Furthermore, by using a gene encoding the enzyme,
It also becomes possible to control the expression of the gene in a system in which the intracellular molting hormone concentration is increased to induce the expression of the target gene product.

[Brief description of drawings]

FIG. 1A is an ecdysteroid found in insects, B is a structure of its oxidation product by the enzyme according to the invention and a HPLC chart of the ecdysteroid.

FIG. 2 is a diagram showing the oxidative activity of ecdysteroid expressed by the cDNA of the present invention.

FIG. 3 is a drawing-substituting photograph showing the result of Western blot analysis using a purified polyclonal antibody against the enzyme of the present invention.

FIG. 4 A and B are of the enzyme according to the invention,
It is a drawing substitute photograph which shows the influence with respect to the 4th instar (preterm instar) and the 5th instar larva of the silkworm.

FIG. 5 Silkworm fourth-instar larvae (A) and 5 injected with the enzyme solution.
It is a figure which shows the accumulation of the modification product which the 22-position hydroxyl group was oxidized in the instar larva (B) body compared with the untreated 4th instar larva (C) and 5th instar larva (D).

FIG. 6: 2 modified at position 22 by the enzyme according to the present invention
0-hydroxy-22-dehydroecdysone is EcR
It is the figure which analyzed by the image analyzer (GS-250, Bio-Rad) which shows that it does not have mRNA expression induction effect.

[Sequence list free text]

[SEQ ID NO: 1] Ecdysteroid 22 derived from Pseudomonas aeruginosa
A nucleotide sequence of a DNA encoding a position oxidase. [SEQ ID NO: 2] Ecdysteroid 22 derived from Pseudomonas aeruginosa
Amino acid sequence of position oxidase [SEQ ID NO: 3] Description of sequence of artificial product: for RT-PCR
E22o.6 primer. Each "n" represents inosine. [SEQ ID NO: 4] Description of sequence of artificial product: for RT-PCR
E22 o.2 primer. Each "n" represents inosine. [SEQ ID NO: 5] Description of sequence of artificial product: for RT-PCR
Int.R1 primer. Each "n" represents inosine. [SEQ ID NO: 6] Description of sequence of artificial product: for RT-PCR
Int.R2 primer. Each "n" represents inosine. [SEQ ID NO: 7] Description of sequence of artificial product: modified RT-PCR
EE22o.RF1 primer for. [SEQ ID NO: 8] Description of sequence of artificial product: modified RT-PCR
EE22o.RR1 primer for.

[Sequence list]                                SEQUENCE LISTING         <110> National Institute of Sericulture and Entmological Science       KAMIMURA, Manabu <120> Ecdysteroid-22-position oxidase obtained from Nomuraea rileyi and ecdysone inactivation system using the same <130> 2007SK <140> <141> <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 1963 <212> DNA <213> Nomuraea rileyi <400> 1 atgacctcct cactcggtct cggtcatcta tcaagcctac tgcttcacag ctcccaagtt 60 tgcgaagcta cttatactac gtggcaacta gaatcgtctt atcgcccacc atgcgaagca 120 aacacatcgt ttgggcgtta tcgcttctcc ctagcacctg ggcattggct ctaccacagg 180 gcggctgtcg atgtataccc ggagaggcgt gctggccatc tgacgagact tgggatgcat 240 tcaactctac cgttgatggc aaactcatca aatccgtccc cctcgcaaag ccgtgttaca 300 cgtcaactga agggtcaggg gatcaatgcc aaaacgtcaa caatgcatgg tcgactgagc 360 gcttccaaac ggcccaggcc ctcggccgat tctatccttt caacacgacc tgccccccgg 420 ttgccaatgg acagcagcca gggacgtgca gtctgggaca gctcccagtc tatgttgtga 480 gagccactga gcattcagac gttgagaaga cgcttgggtt cgttcaagat cacaatatac 540 gtctgtctat caccaacacg ggacatgatc tgaacggccg cggcgacggg ttcggaagtc 600 tgggactctg ggttcaaaac ctccggaaag gtcttttctt ccacgaaagc tttaaatctg 660 ccacccagtg cacagaatcg ggctggaatg gcaagtcgat ccacatcgat ggcgcatatc 720 aatggggcga tgtttacgga ttcgccgaga agcataacgt tatcgttgta ggcggtggct 780 cttcaagcgt cggagccact ggaggctggt tatcaggagg cggccacgga ccggcgtcac 840 gaaactacgg actcggtgct gatcaactgc tcgaggccga ggtcatgctt gccaacggca 900 ctgtcgtcgt tgccaatcac tgccagcacg ccgatctctt ccgggccctg cgaggcggag 960 gccccggata cggagttgtc ctcggtgtca aagtcaaggc atatcccaac gtcgacaagg 1020 tgactgctca ccatctcacc atcgcccctt cgccaagtcg cctcaacacc agcgccctcg 1080 tcgatgccgt gtccatcatg atgcagtcct tcccggctct caacgagagg ggatacgcag 1140 gatacgccac ctggttccgt tacttgcctg gcccctacat cgccaacagt acatctgcct 1200 acacccatag tttctggacc atcggcatga accaggcgga cgcgagtgct gtattcgaac 1260 ctctgcgaag gaagttagcc gaccccggtc tgaatgtggt catcaacagt gacttccagg 1320 agtacaacga ctactggtca ttcttccaca acgagctgga caaggccgat atcccgggcg 1380 acactttgct cctcacctcc cgcatgctgg acaagaaggc tttgcatgat ttcgaccgcg 1440 tccgccacat ggtcgaggtt gtgagcggca gacctcaaga gtacaccatg aacttggcta 1500 tgcttgtgtc gggcggcaag gtcttcgccg atgccgccga cacctcttct ggcctcaacc 1560 ctgcctggcg aacctctcct gtggtcctcc tcaccggacg gaagatcccc aagactcaga 1620 ccctgtctct gcaagagcgt caggccattg ccgaggatat gacctcgcac aaagggcagg 1680 cgaccaagga actggccccc gatacggccg gctacatgag cgagggtgat ggcaacgatc 1740 ccgattatat caattctttc tacggccgca attatgcagc tcaccttgca gccaaggaca 1800 agtacgatcc taaacacgtg ttctactgtc ggacgtgtgt tggtgccgag cgattcatca 1860 gtcggcccga gggggcacta tgcagggctt tttagaaaga cggcccatct agatagtgta 1920 gtataagaaa gtagacgttc aattcgaaaa aaaaaaaaaa aaa 1963 <210> 2 <211> 594 <212> PRT <213> Nomuraea rileyi <400> 2 Met Arg Ser Lys His Ile Val Trp Ala Leu Ser Leu Leu Pro Ser Thr   1 5 10 15 Trp Ala Leu Ala Leu Pro Gln Gly Gly Cys Arg Cys Ile Pro Gly Glu              20 25 30 Ala Cys Trp Pro Ser Asp Glu Thr Trp Asp Ala Phe Asn Ser Thr Val          35 40 45 Asp Gly Lys Leu Ile Lys Ser Val Pro Leu Ala Lys Pro Cys Tyr Thr      50 55 60 Ser Thr Glu Gly Ser Gly Asp Gln Cys Gln Asn Val Asn Asn Ala Trp 65 70 75 80 Ser Thr Glu Arg Phe Gln Thr Ala Gln Ala Leu Gly Arg Phe Tyr Pro                  85 90 95 Phe Asn Thr Thr Cys Pro Pro Val Ala Asn Gly Gln Gln Pro Gly Thr             100 105 110 Cys Ser Leu Gly Gln Leu Pro Val Tyr Val Val Arg Ala Thr Glu His         115 120 125 Ser Asp Val Glu Lys Thr Leu Gly Phe Val Gln Asp His Asn Ile Arg     130 135 140 Leu Ser Ile Thr Asn Thr Gly His Asp Leu Asn Gly Arg Gly Asp Gly 145 150 155 160 Phe Gly Ser Leu Gly Leu Trp Val Gln Asn Leu Arg Lys Gly Leu Phe                 165 170 175 Phe His Glu Ser Phe Lys Ser Ala Thr Gln Cys Thr Glu Ser Gly Trp             180 185 190 Asn Gly Lys Ser Ile His Ile Asp Gly Ala Tyr Gln Trp Gly Asp Val         195 200 205 Tyr Gly Phe Ala Glu Lys His Asn Val Ile Val Val Gly Gly Gly Ser     210 215 220 Ser Ser Val Gly Ala Thr Gly Gly Trp Leu Ser Gly Gly Gly His Gly 225 230 235 240 Pro Ala Ser Arg Asn Tyr Gly Leu Gly Ala Asp Gln Leu Leu Glu Ala                 245 250 255 Glu Val Met Leu Ala Asn Gly Thr Val Val Val Ala Asn His Cys Gln             260 265 270 His Ala Asp Leu Phe Arg Ala Leu Arg Gly Gly Gly Pro Gly Tyr Gly         275 280 285 Val Val Leu Gly Val Lys Val Lys Ala Tyr Pro Asn Val Asp Lys Val     290 295 300 Thr Ala His His Leu Thr Ile Ala Pro Ser Pro Ser Arg Leu Asn Thr 305 310 315 320 Ser Ala Leu Val Asp Ala Val Ser Ile Met Met Gln Ser Phe Pro Ala                 325 330 335 Leu Asn Glu Arg Gly Tyr Ala Gly Tyr Ala Thr Trp Phe Arg Tyr Leu             340 345 350 Pro Gly Pro Tyr Ile Ala Asn Ser Thr Ser Ala Tyr Thr His Ser Phe         355 360 365 Trp Thr Ile Gly Met Asn Gln Ala Asp Ala Ser Ala Val Phe Glu Pro     370 375 380 Leu Arg Arg Lys Leu Ala Asp Pro Gly Leu Asn Val Val Ile Asn Ser 385 390 395 400 Asp Phe Gln Glu Tyr Asn Asp Tyr Trp Ser Phe Phe His Asn Glu Leu                 405 410 415 Asp Lys Ala Asp Ile Pro Gly Asp Thr Leu Leu Leu Thr Ser Arg Met             420 425 430 Leu Asp Lys Lys Ala Leu His Asp Phe Asp Arg Val Arg His Met Val         435 440 445 Glu Val Val Ser Gly Arg Pro Gln Glu Tyr Thr Met Asn Leu Ala Met     450 455 460 Leu Val Ser Gly Gly Lys Val Phe Ala Asp Ala Ala Asp Thr Ser Ser 465 470 475 480 Gly Leu Asn Pro Ala Trp Arg Thr Ser Pro Val Val Leu Leu Thr Gly                 485 490 495 Arg Lys Ile Pro Lys Thr Gln Thr Leu Ser Leu Gln Glu Arg Gln Ala             500 505 510 Ile Ala Glu Asp Met Thr Ser His Lys Gly Gln Ala Thr Lys Glu Leu         515 520 525 Ala Pro Asp Thr Ala Gly Tyr Met Ser Glu Gly Asp Gly Asn Asp Pro     530 535 540 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: E22o.6 primer       for RT-PCR. Each "n" represents an inosine. <400> 3 tnccncargg nggntgyag 19 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: E22o.2 primer       for RT-PCR. Each "n" represents an inosine. <400> 4 tgyagrtgya tnccnggnga 20 <210> 5 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Int.R1 primer       for RT-PCR. Each "n" represents an inosine. <400> 5 cangcntttt nacrttytg 19 <210> 6 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Int.R2 primer       for RT-PCR. Each "n" represents an inosine. <400> 6 ttnacnttnt gnccytgrtc 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: E22o.RF 1 primer       for modified RT-PCR <400> 7 gtgctggcca tctgacgaga ctt 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: E22o.RR 1 primer       for modified RT-PCR <400> 8 ctttgcgagg gggacggatt tga 23 c

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12N 15/09 ZNA C12N 15/00 ZNAA C12R 1:01) (72) Inventor Mashiko Minohara 3088 Ushiku-cho, Ushiku-shi, Ibaraki -3 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C12N 1/00-9/99 C12Q 1/68-1/70 C12P 21/00-21/08 C07K 14/00-16/46 A61K 31/00-48/00 A61P 1/00-43/00 G01N 33/00-33/98 A01K 67/033-67/04 PubMed MEDLINE (STN) BIOSIS / WPI (DIALOG ) GenBank / DDBJ / EMBL / GeneSeq SwissProt / PIR / GeneSeq

Claims (7)

(57) [Claims] 1. The following protein (a) or (b): (A) A protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. (B) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 2 in the sequence listing and having ecdysteroid 22-position oxidase activity. 2. A gene comprising the following DNA (a) or (b): (A) DN consisting of the base sequence shown in SEQ ID NO: 1 in Sequence Listing
A. (B) A DNA which hybridizes with the DNA having the nucleotide sequence of (a) under stringent conditions and which encodes a protein having ecdysteroid 22-position oxidase activity. 3. A method for inactivating the molting hormone of an arthropod by administering the protein according to claim 1 to the arthropod. 4. A method for controlling the growth of an arthropod by inactivating the molting hormone using the protein according to claim 1. 5. A method for controlling the growth of insects by inactivating molting hormone using the protein according to claim 1. 6. A method for producing a silkworm thread, which comprises controlling the diameter of the thread spun by the silkworm by administering the protein according to claim 1 to the silkworm. 7. A method for suppressing the expression of a molting hormone-inducible gene by administering the protein according to claim 1 to a transformant.