JP3004009B1 - New nacre matrix protein and its gene - Google Patents

Abstract

The present invention provides a novel nacre matrix protein and a gene encoding the same. SOLUTION: By purifying an alkali-soluble organic substrate component from a nacre of a pearl oyster shell, a 14 kD
And a novel nacre matrix protein which has a molecular weight of 17 kD and does not exist in the trabecula but only in the nacre is obtained. Using an oligonucleotide corresponding to the N-terminal amino acid sequence of this protein as a probe, a gene for a novel nacre matrix protein is cloned from a cDNA library prepared from the oyster mantle tissue.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel nacre matrix protein and its gene. More specifically, a novel nacre matrix protein that can be obtained from the nacre of the shell of the pearl oyster (Pinctada fucata), cannot be obtained from the ridge layer of the shell, and can enable artificial synthesis of pearls. The present invention relates to a gene encoding a novel nacre matrix protein.

[0002]

2. Description of the Related Art Pearls are currently mainly produced by aquaculture. Cultured pearls are transplanted into the gonads of pearl oysters with a mantle piece from the mantle that makes the nacre of pearl oysters, together with a nucleus made by shaving the shell, and the transplanted mantle cells proliferate and cover the nucleus, After one or two years of cultivation of two shellfish, the same nacre layer that forms naturally behind the shell surrounds the nucleus and forms a pearl bead. In general, shellfish shells are It has a laminar structure composed of calcite crystals on its outer surface, and a nacre layer mainly composed of aragonite crystals on its inner surface on the back side. In the nacre, if aragonite is compared to a brick, it has a layered structure in which the bricks are stacked, and an interlayer matrix called conchiolin corresponding to cement for solidifying the brick exists between the layers. Conchiolin is thought to consist of an envelope protein that surrounds the aragonite crystal layer and a sheet-like protein that supports it.

[0003] Proteins that constitute conchiolin play an important role in the formation of pearls produced from pearl oysters. It is considered open. Matsushiro and colleagues reported that one of the components of this conchiolin, a 60,000 molecular weight necrein (nac
rein) has been successfully cloned (Bioscience 1996, 48, 3
No. 126-132). The cloning of this gene was achieved from a cDNA library prepared from the pearl oyster mantle, using the N-terminal amino acid sequence of a 60,000 molecular weight necrein, which is obtained by removing the nacre from the pearl itself, as a probe. It is.

[0004]

The present inventor believes that the difference between the proteins present in the pearl layer and the trabecular layer of the pearl oyster plays a particularly important role in the formation and luster of the pearl. Were compared and examined for differences in the proteins present in the cells. As a result, they found a protein that was present in the nacre and not in the trabecula. Thus, an object of the present invention is to provide a novel nacre matrix protein which can be obtained from the pearl layer of a pearl oyster but cannot be obtained from the ridge layer. It is a further object of the present invention to provide a gene encoding the novel mother-of-pearl substrate protein that enables artificial synthesis of pearls.

[0005]

Means for Solving the Problems The present invention provides the following i) -i
ii) a novel nacre matrix protein with physicochemical properties: i) can be obtained from the nacre of the shell of the pearl oyster (Pinctada fucata) and cannot be obtained from the trabecular layer of the shell; ii) SDS The molecular weight as measured by -PAGE is 14KDa or 17K
Iii) has the following amino acid sequence as the N-terminal amino acid sequence; AFHTKKGRYSYCWIPYDIEEDRRDNGGKKKCFFCNThe present invention further relates to a novel nacre substrate protein shown in the following (a) or (b): Nacre substrate protein having the amino acid sequence shown in any one of SEQ ID NOS: 1-7; (b) In the amino acid sequence shown in any of SEQ ID NOs: 1-7 in the sequence listing, one or several amino acids are deleted or substituted Or a protein having an added amino acid sequence and having a function as a nacre matrix protein. Further, the present invention relates to a gene encoding the above protein.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION [I] Isolation and purification of novel nacre matrix protein of the present invention The novel nacre matrix protein of the present invention can be isolated and purified by the following method. Only the nacre is separated from the pearl oyster shell, crushed and powdered. The powdered nacre is then extracted with Na-azide containing EDTA and decalcified. The obtained extract is dialyzed, centrifuged, the supernatant is used as a water-soluble organic substrate, and the precipitate is used as a water-insoluble organic substrate. The water-insoluble organic substrate is extracted with diluted alkaline water, and the resulting extract is dialyzed and centrifuged. The supernatant is used as the alkali-soluble organic substrate, and the precipitate is used as the insoluble organic substrate. By subjecting the obtained alkali-soluble organic substrate to SDS-PAGE on a polyacrylamide gel, a novel nacre substrate protein of the present invention having a molecular weight of 14 KDa or 17 KDa can be separated. 14KDa
Alternatively, the protein of the present invention can be isolated from a band having a molecular weight of 17 KDa by an ordinary method. When the same operation as described above was performed using the pearl oyster ridge layer, as shown in Example 1 below, 14 KDa or from the corresponding alkali-soluble organic substrate from the ridge layer was obtained. No protein with a molecular weight of 17 KDa is obtained, nor can it be obtained from the corresponding water-soluble organic substrates. Therefore, the protein of the present invention is a protein that exists in the nacre and not in the trabecula.

[II] Physicochemical properties of the novel nacre matrix protein of the present invention The novel nacre matrix protein of the present invention has the following properties as apparent from the above-mentioned isolation and purification method. i) can be obtained from the nacre of the shell of the pearl oyster (Pinctada fucata) and cannot be obtained from the ridge layer of the shell; ii) the molecular weight measured by SDS-PAGE is 14KDa or 17KD
a. Further, the isolated and purified 14KDa of the present invention and
The N-terminal amino acid sequence of a protein having a molecular weight of 17 KDa was determined by Edman degradation, and it was found that each protein had the amino acid sequence shown below. AFHTKKGRYSYCWIPYDIEEDRRDNGGKKKCFFCN Further, as clarified in Example 2 below, the protein of the present invention has a function that can be an organic nucleus for aragonite crystal formation of pearl oyster pearls.

[III] Cloning of the gene encoding the novel nacre matrix protein of the present invention The cloning of the gene encoding the novel nacre matrix protein of the present invention comprises:
It can be performed by the method described below. (1) Synthesis of oligonucleotide probe A probe for searching for a gene encoding the protein of the present invention is prepared from a cDNA library prepared from pearl oyster tissue by the method described below.
The probe can be prepared based on the N-terminal amino acid sequence of the protein of the present invention described above. For example, the following probe can be mentioned as a nucleotide sequence encoding DRRDNGGKKK in the N-terminal amino acid sequence. 5'-GAYMGNMGNGAYAAYGGNGGNAARAARAAR-3 'The following probe can be mentioned as a base sequence encoding GGKKKCFFC in the N-terminal amino acid sequence. 5'- GGNGGNAARAARAARTGYTTYTTYTGY-3 'These probes can be used, for example, by ³² P-ATP and T4PNK.
Label the 5 'end.

(2) Preparation of cDNA Library Total from mantle piece of pearl oyster mantle
RNA is isolated and mRNA is extracted therefrom by a conventional method. m
From RNA, for example, double-stranded cDNA with EcoRI adapter
And a phage vector such as Lambda gt1
Ligation to 0 followed by in vitro packaging to prepare a cDNA library. (3) Plaque hybridization prepared cDNA library, mixed with host E. coli,
Plaques are formed by plating and incubating. Then, the phage DN in the plaque
A to a nitrocellulose filter
Alkaline phage DNA on filter is single-stranded
DNA. To this, hybridization is carried out by adding the above-mentioned probe labeled with the 5 'end. At the time of hybridization, of the two types of probes described above, first, hybridization is performed with one probe to perform primary screening to obtain a positive plaque, and then hybridization is performed again with another one probe. A second screening may be performed to obtain positive plaques.

(4) Sequencing A phage DNA, for example, Lambda gt10 phage DNA is purified from the obtained positive plaque, and a DNA insert is cut out with EcoRI to obtain a plasmid vector, for example, pGEM.
Subcloning into Then, the plasmid DNA
Is purified and subjected to sequencing. As a sequencing method, a cycle sequencing method is preferable. The cycle sequencing method is a method in which the PCR reaction and the sequencing reaction by the dideoxy method are performed simultaneously, and double-stranded DNA, which was difficult to determine the base sequence by the conventional method, is reduced to about 1 /
There is an advantage that the nucleotide sequence can be easily determined with a template amount of 4. Thus, the nucleotide sequence of the gene encoding the novel nacre matrix protein of the present invention is determined. The probe labeled at the 5′-end is the N-terminal of the protein of the present invention.
Since it is an oligonucleotide encoding the 21-30th or 26-34th amino acid sequence from the end, the resulting gene corresponds to the N-terminal part of the protein of the present invention.
It is possible that the 5 'end is missing. In such a case, the base sequence at the 5 'end can be determined by the 5' RACE method. 5'-RACE is a method for efficiently amplifying and obtaining the 5 'end of an unknown cDNA. This allows
cDNA cloning becomes possible without creating a cDNA library. Specifically, an adapter sequence is provided at the end of the prepared cDNA, and PCR is performed between the adapter sequence and the sequence of the target gene to obtain an unknown end of the target gene.

[IV] Amino acid sequence of novel nacre matrix protein of the present invention and gene encoding the same By performing the above cloning, seven types of proteins of the present invention shown in SEQ ID NOs: 1 to 7 in the sequence listing can be obtained. The encoding gene was found. Also shown in SEQ ID NOs: 1-7
From the seven types of coding genes, a protein of the present invention consisting of the seven types of amino acid sequences shown in SEQ ID NOS: 1-7 was found. In the respective amino acid sequences shown in SEQ ID NOs: 1 to 7, a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having a function as a nacre substrate protein is also within the scope of the present invention. Inside. Amino acid deletions, substitutions or additions are those which do not substantially affect the structure and function of the whole protein.For example, amino acid substitutions have the same hydrophobicity, size, charge and / or Substitution of aromatic amino acids may be mentioned. Such substitutions include, for example, substitutions of glycine for proline, tyrosine for tryptophan, and asparagine for serine. The degree of deletion, substitution, and addition of these amino acids is within the acceptable range if the homology with the original amino acid sequence is 80% or more.

In the present invention, in addition to the seven types of coding genes shown in SEQ ID NOS: 1-7, one or several amino acids are deleted or substituted in each amino acid sequence shown in SEQ ID NOs: 1-7. Alternatively, a gene encoding a protein having an added amino acid sequence and having a function as a nacre matrix protein is also within the scope of the present invention. Furthermore, a gene that hybridizes under stringent conditions with each of the encoding genes shown in SEQ ID NOs: 1 to 7 in the sequence listing and that encodes a protein having a function as a nacre substrate protein is also included in the scope of the present invention. Things. Such hybridizing DNA mutants include site-directed mutagenesis, random mutation by treatment with a mutagen, mutation of DNA fragment by restriction enzyme cleavage, deletion,
The DNA sequence may be partially changed due to ligation or the like. The degree to which these DNA mutants hybridize with the respective encoding genes shown in SEQ ID NOs: 1 to 7 is determined under stringent conditions, for example, 5 × SSPE 0.2% SDS 5% Denhart's sol 0.1 mg / ml Salmon sperm DNA 50 It hybridizes by Southern hybridization under the condition of incubation of 30% formamide at 42 ° C. overnight (12-16 hours) with the composition of% formamide.

[V] Method for producing novel nacre matrix protein of the present invention The protein of the present invention can be obtained by isolating and purifying from the nacre of pearl oyster, as described above.
Further, it can also be produced by a recombinant DNA method using the coding gene cloned according to the present invention. In the case of production by a recombinant DNA method, a recombinant vector for expression is constructed by introducing an encoding gene into an appropriate vector, a microorganism is transformed with this vector, and the resulting transformant is cultured. By recovering from culture,
The protein of the present invention can be produced. Suitable vectors used here include, for example, pUC18, pKK2
Plasmids for expression in Escherichia coli such as 23-3.
Alternatively, a normal expression vector used for expression in eukaryotic cells such as yeast and animal cells can also be used. The protein of the present invention is produced by transforming an appropriate host cell, for example, Escherichia coli, yeast, animal cell, or the like, with the expression vector and culturing the resulting transformant.

[0014]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 Isolation and purification of nacre matrix protein of the present invention (1) Isolation from pearl oyster Only nacre was separated from pearl oyster shell, and powdered nacre 100 gm was added to 10% EDTA containing 0.01% Na-azide. (pH 7.
5) Decalcified in 3 l. Decalcification was performed at room temperature under continuous stirring for 6 days. The demineralized solution contains 0.01% Na-azide.
Dialysis was performed against dH ₂ O for 4 days. The dialysate is degassed and concentrated using a rotary evaporator, and the concentrated solution is 15,000 G / 4
Centrifuged at ℃. After centrifugation, the supernatant is
(water soluble organic matrix = WSM) and precipitate in water insoluble organic matrix (WIS)
M), each was freeze-dried and stored at -20 ° C. WISM was continuously stirred overnight after polytron homogenization in dilute aqueous ammonia (pH 8.5).
The extract was centrifuged at 15,000 G / 4 ° C. After centrifugation, the supernatant is washed with an alkali soluble organic substrate (alkali soluble organic substrate).
matrix = ALSM) and precipitate insoluble organic matrix (insoluble o
rganic matrix = ISM), each was freeze-dried and stored at -20 ° C.

(2) SDS-PAGE 1 mg of each lyophilized sample of WSM and ALSM was added to 100 μl of
Dissolve in sample buffer for SDS-PAGE,
SDS-PAGE was performed on a 1-20% gradient polyacrylamide gel. CBB, Silver and Stainsall were used for staining. (3) Results of SDS-PAGE analysis The results of SDS-PAGE are shown in FIG. As is clear from FIG. 1, one band was observed at 47 kD in SDS-PAGE of WSM in nacre, and many very narrow bands were observed in the low molecular weight region. In SDS-PAGE of WSM in the ridge column,
A clear band was observed at 47 kD, and the upper 60 kD
A slightly smeared thick band was stained in the region up to. In addition, no band was observed in the low molecular weight region. 17kD and 14kD for SDS-PAGE of ALSM in nacre
However, no band was observed in the high molecular weight region. Although not shown in FIG. 1,
In the SDS-PAGE of ALSM in the trabecular layer, no band appeared and no protein component was confirmed. As a result, 14KD is present in the nacre and not in the ridge column
Proteins of the invention exhibiting a or 17 KDa molecular weight were isolated.

Example 2 In the formation of pearl oyster nacre with the protein of the present invention,
Role In the same manner as in Example 1, only the nacre is separated from the pearl oyster shell, crushed, and then decalcified with EDTA. After the demineralized solution was dialyzed, the supernatant was separated into a supernatant (water-soluble organic substrate = WSM) and a precipitate (water-insoluble organic substrate = WISM) by centrifugation. The main component of WSM is nacrein. WISM is ground with a Polytron homogenizer, stirred overnight in dilute aqueous ammonia (pH 8.5), centrifuged, and the supernatant (alkaline soluble organic substrate = ALS).
M) and a precipitate (insoluble organic substrate = ISM). The main component of ALSM is the protein of the present invention (protein having a molecular weight of 14 kD and 17 kD; hereinafter, referred to as the protein component of the present invention). AL
The protein component of the present invention is separated from the SM by centrifugal filtration,
This was used for the crystal induction experiment. The crystallization induction experiment is to determine the specific crystal form of calcium carbonate as an organic substrate component and its ability to induce a crystal form. The pearl oyster pearl layer is a hexagonal plate-shaped aragonite crystal, and the ridge column layer is made of calcite crystal, so if it is confirmed that components extracted from the pearl layer induce this form of aragonite crystal, It is confirmed that it has a guiding function. In the experiment, a mother liquor that made only aragonite inorganically and a mother liquor that made only calcite were prepared, and various organic substrate components were added to these mother liquors, and the crystal forms and crystal forms formed were observed. did. This experiment was performed in a 24-well microplate at a temperature of 18 ° C.

First, a comparison was made between the case where a dialysis tube serving as a base was attached to the bottom of the hole of the plate and the case where nothing was laid. As a result, when there was no base film, no difference was observed between the crystals formed from the mother liquor and the crystals formed by adding the organic substrate component (as shown in Fig. 2, In this case, typical needle-like aragonite crystals were formed, and from the calcite-based mother liquor, typical dice-like calcite crystals were formed.) On the other hand, when the dialysis tube was stuck, the addition of nacrein and the protein component of the present invention induced plate-like aragonite crystals in both the aragonite-based mother liquor and the calcite-based mother liquor. Particularly in the former, the crystals developed greatly and became layered. This means that the nacre is formed only when the base film component is present. Subsequently, the amount of the organic substrate component was changed, or the organic substrate component was previously adsorbed to the dialysis tube, and then another component was added to the mother liquor for further study. As a result, as shown in FIGS. 3 and 4, it was found that when the protein component of the present invention was adsorbed to the dialysis tube, the number of crystals increased, and the size of the crystals increased when nacrine was added thereto. Furthermore, instead of a dialysis membrane, an experiment was conducted based on a conchiolin membrane extracted from the nacre, and as a result, the protein component of the present invention was adsorbed to the membrane, and when a mother liquor containing nacrein was added thereto, a natural nacre was formed. Similar hexagonal plate-like aragonite crystals appeared. When a nacre is formed naturally, a nucleus centering on an organic substrate is first formed on an insoluble conchiolin film, and a crystal nucleus is formed there. The crystal grows continuously from the crystal nucleus and grows until adjacent crystals come into contact with each other to form a layer of tabular crystals unique to the nacre. For this reason, it has been pointed out that the formation of nacres has two separate components that are involved in nucleation and crystal growth. From the results of this experiment, it was estimated that the former was an N13 component and the latter was a component corresponding to conchiolin.

In summary, at the start of nacre formation, first, a membrane-like sheet of ISM (a natural base membrane corresponding to a dialysis membrane) is secreted, and then the secreted protein component of the present invention is separated from this membrane. Absorbed on the surface to become an organic nucleus for crystal formation, on which a crystal nucleus is formed. Subsequently, nacrein is secreted, and by the action of CA possessed by this component,
Araleite crystals grow. The crystal growth direction is predefined, and grows little by little in the c-axis direction (the factor controlling the crystal growth direction is unknown at this time). Eventually a layer is formed and the nacre continues to grow until it contacts the next conchiolin film. Nacrein is also the main component in the ridge column. Since the ridge column is composed of calcite, nacrein is thought to have the function of growing calcium carbonate crystals, whether aragonite or calcite. On the other hand, since the protein component of the present invention exists almost exclusively in the nacre, it is presumed to be involved in the formation of aragonite crystal nuclei in the nacre, but not to the formation of calcite crystal nuclei in the ridge column.

Example 3 Cloning of the Gene of the Protein of the Present Invention (1) Isolation and Purification of the Protein of the Present Invention In the same manner as in Example 1, (1), AL
SM was obtained and lyophilized. A 200 microgram portion of the lyophilized product was dissolved in sample buffer and subjected to SDS-PAGE with 12% acrylamide. After electrophoresis on acrylamide by electroblotting, which is a semi-dry method, proteins are blotted on a PVDF membrane. Thereafter, Ponceau S staining is performed.

Next, the PV of the band corresponding to a molecular weight of 14 kD (hereinafter referred to as N14) or 17 kD (hereinafter referred to as N17) is obtained.
Each DF membrane was cut out. After this, follow the usual method
Reduction S-alkylation (reduction carboxymethyl method) was performed on the PVDF membrane. The PVDF membrane after this treatment is applied to a Shimadzu protein sequencer PPSQ21, and the PITC method (Edman decomposition)
Was used to determine the N-terminal amino acid sequences of proteins N-14 and N17. The N-terminal amino acid sequences of N14 and N17 were as follows, and were identical amino acid sequences. AFHTKKGRYSYCWIPYDIEEDRRDNGGKKKCFFCN (2) Oligonucleotide probe sequence, its synthesis and labeling Two types of degenera based on the N-terminal amino acid sequence of N14, 17
The te oligo probes A and B were synthesized using an ABI394 DNA synthesizer. The sequence is shown below. Degenera corresponding to DRRDNGGKKK in the N-terminal amino acid sequence
te oligo Probe A: 5'-GAYMGNMGNGAYAAYGGNGGNAARAARAAR-3 ' degenera corresponding to GGKKKCFFC in N-terminal amino acid sequence
te oligo Probe B: 5'-GGNGGNAARAARAARTGYTTYTTYTGY-3 '

Next, the 5'-end of the oligonucleotide was labeled with 32P-ATP and T4PNK. That is, degenerat
Using ive oligo A and B as probe DNA, respectively
50 ng was labeled with T4 polynucleotide kinase (Promega) at 37 ° C. for 1 hour using 30-50 μCi of 32P-ATP. 0.5MEDTA 1 μl, 10 mg / ml carrier RNA2
After adding μl, the mixture was extracted with phenol-chloroform (1: 1), applied to a Sephadex G-25 column, and purified.

(3) mRNA extraction Using a SV Total RNA Isolation System (Promega), 100 mg of mantle piece of pearl oyster
Was extracted from the sample. In addition, this 100
μg of Total RNA with PolyATtract mRNA Isolation System
m (Promega) to extract 10 μg mRNA. (4) Preparation of Lambda gt10 library 5 μg of mRNA was transferred to Universal RiboClone cDNA Synthesis S
A double-stranded cDNA with an EcoRI adapter was synthesized using ystem (Promega). Specifically, the Oligo (dT) primer and
First strand synthesis is performed using AMV Reverse Transcriptase, and RNA strand removal and DNA Po
Second strand was synthesized by lymerase I.
After this, the ends are made flat using T4 DNA Polymerase, and Ec
The oRI adapter was ligated with T4 DNA Ligase. After the ligation reaction, excess adapter was removed by gel filtration using Sephacryl S-400. After gel filtration
Transfer 100 ng of double-stranded cDNA with EcoRI to Lambda gt10 EcoRI arm
s (Promega) was ligated to 1 μg. Then
In vitro packaging was performed using Packagene Lambda DNA Packaging System (Promega).

(5) Infection and plating A part of the in vitro packaged library was prepared, and a dilution series was prepared with an SM buffer. 30 μl of the dilution series was mixed with host E. coli C600hfl and incubated at 37 ° C. for 20 minutes. After clearing, 3 ml of soft agarose (0.7% LB agarose, pre-warmed to 50 ° C.) was added and spread on an LB plate. Incubated at 37 ° C for 12-16 hours. As a result, a 2 × 10 ⁶ library was completed.

(6) Primary screening After cooling the plate on which the plaque is formed at 4 ° C. for about 30 minutes,
(About 30 minutes), the nitrocellulose filter (S & S) was kept on the plate for about 3 minutes without air, and the phage DNA was transferred to the nitrocellulose filter. Then remove the filter and air dry (approximately 5
Minutes). Thereafter, the filter was placed on a filter paper impregnated with the following solution with the phage side of the filter facing up, subjected to an alkali treatment and a neutralization treatment to prepare a single-stranded DNA. 1) 0.5M NaOH / 1.5M NaCl; 30-60 seconds 2) 0.5M Tris-HCl (pH 7.5) /1.5M NaCl; 3-5 minutes 3) 2x SSC; 3-5 minutes After natural drying, 80 ° C, Heat-treated for 2 hours.

(7) Hybridization After immersing the nitrocellulose filter in 3xSSC,
Prehybridization was performed at 42 ° C. for 1-2 hours in a 1 × Denhardt solution-0.5% SDS solution. Hybridization is
Performed at 42 ° C. for about 15 hours. At this time, probe A labeled at the 5 'end with 32P-ATP was added at a standard of 200,000 cpm / ml. The composition of the hybridization solution was as follows. 1M NaCl, 1xDenhardt, 50mM Tris-HCl (pH8.0), 10mMEDT
A, 0.1% SDS, 50 μg / ml yeast RNA, 50 μg / ml denatured DNA (8) Washing and autoradiography The filter was washed three times with a 6 × SSC, 0.1% SDS solution at 50 ° C. for 30 minutes. After washing, the filters were wrapped in Saran wrap and autoradiography was performed overnight. Thirteen plaques tested positive in this primary screen.

(9) Secondary screening Thirteen plaques that became positive in the primary screening were plucked together with a soft agar around the plaque from a plate with a sterilized toothpick and suspended in 0.5 ml of an SM buffer.
C600hfl in which 10 μl of a 1/100 dilution of this solution was cultured overnight
Mix with 300 μl and add 8 ml 0.7% LB agarose to 1.5% LB
Sprinkled on agar plate (15 cm). This was followed by incubation at 37 ° C. overnight. Thereafter, in the same manner as in the primary screening, a secondary screening was performed using probe B labeled at the 5 'end with 32P-ATP. Of the 13 plaques that tested positive in the primary screen, 2 plaques became positive in the secondary screening stage.

(10) Subcloning into Plasmid Vector A 1/100 dilution of the SM buffer of the positive plaque was added to XL1 Blu
e, MRF ′ (Stratagene), and Lambdagt10 phage DNA was purified from this culture using the λQuick spin kit (BIO 101). Thereafter, the insert was cut out with EcoRI, subcloned into a plasmid vector; pGEM (Promega), and the plasmid DNA was inserted into Plasmid Midi.
It was purified using Kit (Qiagen) and used as a sample for cycle sequencing. Plasmid clones containing the positive plaque insert thus obtained were # 1 'and # 2'
And

(11) Sequencing The nucleotide sequence was determined using a cycle sequencing reaction. Here, the insert cDNA subcloned into pGEM (Promega) was used for BigDye Terminator Cycle Sequence.
T7 promoter sequence primer and SP6 promorterase using the FS FS Ready Reaction Kit (Kit used) (ABI).
The insert cDNA was sequenced from both directions using quence primer. The sequencer used was ABI PRISM 377
DNA Sequencer. The sequence primer has the following sequence. T7 promorter sequence primer; 5'-TAA TAC GAC TCA CTA TAG GG-3 'SP6 promorter sequence primer; 5'-TAT TTA GGT GAC ACT ATA G-3' Sequence of clones # 1 'and # 2' thus obtained The data were as shown in SEQ ID NOs: 8 and 9 in the sequence listing, respectively. (12) Extension to 5 'end Using Marathon cDNA Amplification Kit (Clontech) using 5'-RACE (rapid amplification of cDNA ends) method
CDNA fragments having sequences at the 5 'end of # 1' and 2 'were obtained. At this time, the first first strand CD
In the sequence of the antisense primer used for preparing NA, # 1 ′ was 5′-CATCGATGTACTCTTCCCGGAATTC-3 ′ and # 2 ′ was 5′-GAATCAAGGAAGTTTACTTGTCAT-3 ′. 1st antisense primer for nested PCR, # 1 'is 5'-CCATTTCCGTTTCCGTAACCTCCA-3', respectively
# 2 'was 5'-AGAATTCACCATTTCCGTTTCCGT-3'. Ne
2nd antisense primer for sted PCR
# 1 'is 5'-TATAAGGACTTGAGGTAATTTAAT-3' and # 2 '
Was 5'-ACCTCCATATAAAGACTTGAGGT-3 '. Following the above method, the obtained PCR products were subcloned and sequenced, respectively. MacVecto, an analysis software, analyzes the sequence of this fragment and the sequence of # 1 'and # 2'
Assembled with r6.0.1 (Teijin System Technology) and Lasergene (Teijin System Technology) and analyzed for open reading frame (ORF). As a result, the nucleotide sequences of the DNAs having the open reading frames shown in SEQ ID NOs: 1 and 2 in the sequence listing were revealed.
Further, from these nucleotide sequences, it was revealed that the protein of the present invention has the amino acid sequences shown in SEQ ID NOS: 1 and 2.

(13) Search for other DNA mutants The following RT-PCR primers were prepared so that CDS (protein coding region) could be amplified from the obtained gene sequences # 1 and # 2. 1) Sense primer TTC TGT GAG GCG GCG CCT GGA ATC 3) Antisense primer GTT CAT ACT TTA CTA TAT AAT TTT ACA From this, a sequence similar to # 1, # 2 but with some amino acid sequence mutation, This is because there is a possibility that a sequence in which amino acids have been deleted or inserted may be obtained. First, SV Total RNA I
Total RNA was isolated from 100 mg of mantle piece using manipulating the solation system (Promega), followed by PolyATtract mRNA Isolation System (Promega).
Then, 10 μg mRNA was extracted and purified. 5 μg of this mRNA
Using the Universal Ribo Clone cDNA Synthesis System (Promega), Oligo (dT) primer and AMV Reverse
First strand synthesis was performed with Transcriptase. Using this first-land cDNA as a template for PCR, amplification reaction was carried out with a thermal cycler using a 95 ° C, 30-second denature, 55 ° C, 30-second annealing, 68 ° C, 30-second PCR cycle. Purify this PCR amplification product,
E. coli was transformed by subcloning into the above pGEM vector. Five insert-positive clones were selected, the E. coli was mini-cultured, and plasmid purification was performed. This plasmid DNA was prepared using the previously described T7 promoter sequencing primer and SP6.
Promorter sequence primer Sequencing was performed using ABI377 from both directions of the insert by the Big Dye Terminator method. Analysis software Mac Vector6.0 already described.
Assembled with 1 (Teijin System Technology) and Lasergene (Teijin System Technology). Thus, the DNA sequence and amino acid sequence shown in SEQ ID NOs: 3-7 were obtained.
Geneba using NCBI's BLAST 2.0 as a search program
As a result of searching the nk database, it was shown in SEQ ID NOs: 1-7.
No gene with homology to the DNA sequence could be found. Therefore, it was found that the gene obtained in the present invention was a novel gene. Alignment was performed to examine the homology of the amino acid sequences shown in SEQ ID NOS: 1-7. The results are shown in FIG.

[0030]

Industrial Applicability According to the present invention, a novel nacre matrix protein which can be obtained from the pearl layer of the pearl oyster shell but cannot be obtained from the ridge column of the husk, and a gene encoding the same are obtained. Such a gene could enable artificial synthesis of pearls.

[0031]

[Sequence List] SEQUENCE LISTING <110> Hayashi Nakanobu and Masuo Masanori <120> New pearl layer matrix protein and gene there <130> DA-02679 <160> 9 <210> 1 <211> 697 <212> DNA <213> Pinctada fucata <400> gtcttctgtg aggcggcgcc tggaatc atg aag tgc aca ctt cgt tgg aca att 54 Met Lys Cys Thr Leu Arg Trp Thr Ile 1 5 aca gct ttg gtt ttg ctc ggc att tgc cat tta gct gac c Thrg Aca tca Leu Leu Gly Ile Cys His Leu Ala Arg Pro Ala Tyr 10 15 20 25 cat aag aag tgc gga cgt tac tca tac tgc tgg ata ccc tac gac ata 150 His Lys Lys Cys Gly Arg Tyr Ser Tyr Cys Trp Ile Pro Tyr Asp Ile 30 35 40 gag aga gat agg cgg gat aat ggg ggc aag aaa tat tgt ttc tgt aga 198 Glu Arg Asp Arg Arg Asp Asn Gly Gly Lys Lys Tyr Cys Phe Cys Arg 45 50 55 tac gct tgg tct cca tgg caa tgt aat gag gag agg tat gag tgg 246 Tyr Ala Trp Ser Pro Trp Gln Cys Asn Glu Glu Glu Arg Tyr Glu Trp 60 65 70 ctt aga tgt gga atg agg ttc tac tcc ttg tgc tgt tac acc gat gat 294 Leu Arg Cys Gly Met Arg Phe Tyr Ser Leu Cys Cys Tyr Thr Asp Asp 75 80 85 gac aac ggt aac gga aat ggc aac ggt aac ggt aac gga tta aat tac 342 Asp Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Leu Asn Tyr 90 95 100 105 ctc aag tcc tta tat gga ggt tac gga aac gga aat ggt gaa ttc cgg 390 Leu Lys Ser Leu Tyr Gly Gly Tyr Gly Asn Gly Asn Gly Glu Phe Arg 110 115 120 gaa gag tac atc gat gaa cgg tat gac aat taa aattcctgct acggattp yr Glu Glu Glu arg Tyr Asp Asn Stop 125 130 gaattttacc ctcatttaat acagtgtcag acttaacctt gacttttatg ttctgaaaat 503 aggaacgaaa cttcatgact gtatttctct atagctagaa tcttagaaaa tgtcaaagtt 563 tgaccttgct ggtgtgtaag gaaagaaaaa aatgtagtca actatgtatt acgaatatat 623 ctgaaaattc aatctcagat gtaaaattat atagtaaagt atgaacaaaa aaaaaaaaaa 683 aaaaaaaaaa aaaa 697 <210> 2 <211> 695 <212> DNA <213> Pinctada fucata <400> gtcttctgtg aggcggcgcc tggaatc atg aag tgc aca ctt cgt tgg aca att 54 Met Lys Cys Thr Leu Arg Trp Thr Ile 1 5 aca gct ttg gtt ttg ctc ggc att tgc cat tta gct cat ct gct Ala Leu Val Leu Leu Gly I le Cys His Leu Ala Arg Pro Ala Tyr 10 15 20 cat aag aag tgc gga cgt tac tca tac tgc tgg ata ccc tac gac ata 150 His Lys Lys Cys Gly Arg Tyr Ser Tyr Cys Trp Ile Pro Tyr Asp Ile 25 30 35 40 gag aga gat agg cgg gac aat ggt ggc aag aaa tgt tgc ttc tgt aga 198 Glu Arg Asp Arg Arg Asp Asn Gly Gly Lys Lys Cys Cys Phe Cys Arg 45 50 55 aac gct tgg tct cca tgg caa tgt aaa gag gac gag agg tgg 246 Asn Ala Trp Ser Pro Trp Gln Cys Lys Glu Asp Glu Arg Tyr Glu Trp 60 65 70 cta aga tgt gga cat aaa ttc tac tac atg tgt tgt tac acc gat gat 294 Leu Arg Cys Gly His Lys Phe Tyr Tyr Met Cys Cys Tyr Thr Asp Asp 75 80 85 gat aac ggt aac gga gat ggc aac ggt aac gga ttt aat tac ctc aag 342 Asp Asn Gly Asn Gly Asp Gly Asn Gly Asn Gly Phe Asn Tyr Leu Lys 90 95 100 tct tta tat gga ggt tac gga aac gga aat ggt gaa ttc tgg gaa gag 390 Ser Leu Tyr Gly Gly Tyr Gly Asn Gly Asn Gly Glu Phe Trp Glu Glu 105 110 115 120 tac atc gat gaa cgg tat gac aag taa acttccttga ttcttgaatt ttaccp Tyr Gyr L ys Stop 125 cacagtgtcc gactaaacca tgacttttat gttcttaaat aagaacgaaa ctacatgtat 504 ctctctatag cttgtatctt agaaaatatt ttttgtttgg ccttgctggt gtgtaaggaa 564 aggagagaat gtagtcaact atgtattacg aatatatctg aaaattcaat ctcagatgta 624 aaattatata gtaaagtatg aagccagatt tcaaaacaaa gcaaaaaaaa aaaaaaaaaa 684 aaaaaaaaaa a 695 <210> 3 <211> 698 <212> DNA <213> Pinctada fucata <400> gtcttctgtg aggcggcgcc tggaatc atg aag tgc aca ctt cgt tgg aca att 54 Met Lys Cys Thr Leu Arg Trp Thr Ile 1 5 aca gct ttg gtt ttg ctc ggc att tgc cat tta gct Leca Val Acacact Leu Gly Ile Cys His Leu Ala Arg Pro Ala Tyr 10 15 20 cat aag aag tgc gga cgt tac tca tac tgc tgg atc cct tat gac att 150 His Lys Lys Cys Gly Arg Tyr Ser Tyr Cys Trp Ile Pro Tyr Asp Ile 25 30 35 40 gag aga gac aga tat gat aac ggt gac aag aaa tgt tgt ttc tgt aga 198 Glu Arg Asp Arg Tyr Asp Asn Gly Asp Lys Lys Cys Cys Phe Cys Arg 45 50 55 tac gct tgg tct cca tgg caa tgt aat gag gaga gag tat gag tgg 246 Tyr Ala Trp Ser Pro Trp Gln Cys Asn Glu Glu Glu Arg Tyr Glu Trp 60 65 70 ctt aga tgt gga atg agg ttc tac tcc ttg tgc tgt tac acc gat gat 294 Leu Arg Cys Gly Met Arg Phe Tyr Ser Leu Cys Cys Tyr Thr Asp Asp 75 80 85 gac aac ggt aac gga aat ggc aac ggt aac ggt aac gga tta aat tac 342 Asp Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Leu Asn Tyr 90 95 100 ctc aag tct tta tat gga ggt tac gga aac gga aat ggt gaa ttc Leu Lys Ser Leu Tyr Gly Gly Tyr Gly Asn Gly Asn Gly Glu Phe Trp 105 110 115 120 gaa gag tac atc gat gaa cgg tat gac aat taa aattcctgct acggattctt 443 Glu Glu Tlu Ile Asp Glu Arg Tyr Asp Asn Stop cct att gac acttaacctt gacttttatg ttctgaaaat 503 aggaacgaaa cttcatgact gtatttctct atagctagaa tcttagaaaa tgtcaaagtt 563 tgaccttgct ggtgtgtaag gaaagaaaaa aatgtagtca actatgtatt acgaatatat 623 ctgaaaattc aatctcagat gtaaaattat atagtaaagt atgaacttca aaaaaaaaaa 683 aaaaaaaaaa aaaaa 698 <210> 4 <211> 678 <212> DNA <213> Pinctada fucata <400> gtcttctgtg aggcggcgcc tggaatc atg aag tgc aca c tt cgt tgg aca att 54 Met Lys Cys Thr Leu Arg Trp Thr Ile 1 5 aca gct ttg gtt ttg ctc ggc att tgc cat tta gct aga cca gct tac 102 Thr Ala Leu Val Leu Leu Gly Ile Cys His Leu Ala Arg Pro Ala Tyr 10 15 20 cat aag aag tgc gga cgt tac tca tac tgc tgg atc cct tac gac atc 150 His Lys Lys Cys Gly Arg Tyr Ser Tyr Cys Trp Ile Pro Tyr Asp Ile 25 30 35 40 gag aga gac aga tat gat aac ggt gac aag aaa tgt tgt ttc tgt aga 198 Glu Arg Asp Arg Tyr Asp Asn Gly Asp Lys Lys Cys Cys Phe Cys Arg 45 50 55 tac gct tgg tct cca tgg caa tgt aat gag gaa gag agg tat gag tgg 246 Tyr Ala Trp Ser Pro Trp Gln Cys Asn Glu Glu Glu Arg Tyr Glu Trp 60 65 70 ctt aga tgt gga atg agg ttc tac tcc ttg tgc tgt tac acc gat gat 294 Leu Arg Cys Gly Met Arg Phe Tyr Ser Leu Cys Cys Tyr Thr Asp Asp 75 80 85 gac aac ggt aac gga aat ggc aac ggt aac ggt aac gga tta aat tac 342 Asp Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Leu Asn Tyr 90 95 100 ctc aag tct tta tat gga ggt tac gga aac gga aat ggt gaa 390 Leu Lys Ser Leu Tyr Gly Gly Tyr Gly Asn Gly Asn Gly Glu Phe Trp 105 110 115 120 gaa gag tat atc gat gaa cgg tat gac aat taa aattcctgct acggattctt 443 Glu Glu Tyr Ile Asp Glu Arg Tyr Asp Asn Stop 125 130 gaattttag ctcatgca tca tca tca tca t gatt gag cttcatgact gcatttcaca cgaagaaggg gtcagacttt atcaagaatt 563 tcatgagtaa agacggtaaa gatctaacac agcatggaaa agagctactt gaccagcttg 623 ccgggaaaga taaaaatgaa ctaaaaagta acggggcaaa aaaaaaaaaa aaaaa 678 <210> 5 <211> 644 <212> DNA <213> Pinctada fucata <400> ttctgtgagg cggcgcctgg aatc atg acg tgc aca ctt cgt tgg aca att 51 Met Thr Cys Thr Leu Arg Trp Thr Ile 1 5 aca gct ttg gtt ttg ctc ggc att tgc cat tta gct aga cca gct ttc 99 Thr Ala Leu Val Leu Leu Gly Ile Cys His Leu Ala Arg Pro Ala Phe 10 15 20 25 cgt acg aag tgc gga cgt tac tca tac tgc tgg ata ccc tac gac ata 147 Arg Thr Lys Cys Gly Arg Tyr Ser Tyr Cys Trp Ile Pro Tyr Asp Ile 30 35 40 gag aga gac aga tat gac aac ggt gac aag aaa tgt tgt ttc tgt aga 195 Glu Arg Asp Arg Tyr Asp Asn Gly Asp Lys Lys Cys Cys Phe Cys Arg 45 50 55 aac gct tgg tct cca tgg caa tgt aaa gag gac gag agg tat gag tgg 243 Asn Ala Trp Ser Pro Trp Gln Cys Lys Glu Asp Glu Arg Tyr Glu Trp 60 65 70 cta aga tgt gga cat aaa ttc tac tac atg tgt tgt tac acc gat gat 291 Leu Arg Cys Gly His Lys Phe Tyr Tyr Met Cys Cys Tyr Thr Asp Asp 75 80 85 gat aac ggt aac gga aat ggc aac ggt aac gga ttt aat tac ctc aag 339 Asp Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Phe Asn Tyr Leu Lys 90 95 100 tct tta tat gga ggt tac ggg aac gga aat ggt gaa ttc tgg gaa gag 387 Ser Leu Tyr Gly Gly Tyr Gly Asn Gly Asn Glu Glu trp Glu Glu 105 110 115 120 tac atc gat gaa cgg tat gac aag taa acttccttga ttcttgaatt ttaccct 441 Tyr Ile Asp Glu Arg Tyr Asp Lys Stop 125 130 cacagtgtcc gactaaacca tgacttttat gttcttaaat aagaacgaaa ctacatgtat 501 ctctctatag cttgtatctt agaaaatatt ttttgtttga ccttgctggt gtgtaaggaa 561 aggagagaat gtagtcaact atgtattacg aatatatctg aaaattcaat ctcagatgta 621 aaattatata gtaaagtatg aac 644 <210> 6 <211> 645 <212> DNA <213> Pinctada fucata <400> tttctgtgag gcggcgcctg gaatc atg acg tgc aca ctt cgt tgg aca att 52 Met Thr Cys Thr Leu Arg Trp Thr Ile 1 5 aca gct ttg gtt ttg ctc ggc att tgc cat tta gct aga Ala Leu Val Leu Leu Gly Ile Cys His Leu Ala Arg Pro Ala Phe 10 15 20 25 cgt acg aag tgc gga cgt tac tca tac tgc tgg ata ccc tac gac ata 148 Arg Thr Lys Cys Gly Arg Tyr Ser Tyr Cys Trp Ile Pro Tyr Asp Ile 30 35 40 gag aga gac aga tat gac aac ggt gac aag aaa tgt tgt ttc tgt aga 196 Glu Arg Asp Arg Tyr Asp Asn Gly Asp Lys Lys Cys Cys Phe Cys Arg 45 50 55 aac gct tgg tct cca tgg caa tgt aaa gag gac gag agg tat gag tgg 244 Asn Ala Trp Ser Pro Trp Gln Cys Lys Glu Asp Glu Arg Tyr Glu Trp 60 65 70 cta aga tgt gga cat aaa ttc tac tac atg tgt tgt tac acc gat gat 292 Leu Arg Cys Gly His Lys Phe Tyr Tyr Met Cys Cys Tyr Thr Asp Asp 75 80 85 gat aac ggt aac gga aat ggc aac ggt aac gga ttt aat tac ctc aag 340 Asp Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Phe Asn Tyr Leu Lys 90 95 100 105 tct tta ta t gga ggt tac gga aac gga aat ggt gaa ttc tgg gaa gag 388 Ser Leu Tyr Gly Gly Tyr Gly Asn Gly Asn Gly Glu Phe Trp Glu Glu 110 115 120 tac atc gat gaa cgg tat gac aag taa acttccttga ttcttgatta ttcttgaatt Glu Arg Tyr Asp Lys Stop 125 130 cacagtgtcc gactaaacca tgacttttat gttcttaaat aagaacgaaa ctacatgtat 502 ctctctatag cttgtatctt agaaaatatt ttttgtttga ccttgctggt gtgtaaggaa 562 aggagagaat gtagtcaact atgtattacg aatatatctg aaaattcaat ctcagatgta 622 aaattatata gtaaagtatg aac 645 <210> 7 <211> 645 <212> DNA <213> Pinctada fucata <400> cttctgtgag gcggcgcctg gaatc atg acg tgc aca ctt cgt tgg aca att 52 Met Thr Cys Thr Leu Arg Trp Thr Ile 1 5 aca gct ttg gtt ttg ctc ggc att tgc cat tta gct aga cca gct Lectu Leu Val 100 Thr Gly Ile Cys His Leu Ala Arg Pro Ala Phe 10 15 20 25 cgt acg aag tgc gga cgt tac tca tac tgc tgg ata ccc tac gac ata 148 Arg Thr Lys Cys Gly Arg Tyr Ser Tyr Cys Trp Ile Pro Tyr Asp Ile 30 35 40 gag aga gac aga tat gac aac ggt gac aag aaa tgt tgt ttc tgt aga 196 Glu Arg Asp Arg Tyr Asp Asn Gly Asp Lys Lys Cys Cys Phe Cys Arg 45 50 55 aac gct tgg tct cca tgg caa tgt aaa gag gac gag agg tat gag tgg 244 Asn Ala Trp Ser Pro Trp Gln Cys Lys Glu Asp Glu Arg Tyr Glu Trp 60 65 70 cta aga tgt gga cat aaa ttc tac tac atg tgt tgt tac acc gat gat 292 Leu Arg Cys Gly His Lys Phe Tyr Tyr Met Cys Cys Tyr Thr Asp Asp 75 80 85 gat aac ggt aac gga aat ggc aac ggt aac gga ttt aat tac ctc aag 340 Asp Asn Gly Asn Gly Asn Gly Asn Gly Asn Gly Phe Asn Tyr Leu Lys 90 95 100 105 tct tta tgt gga ggt tac gga aac gga aat ggt gaa ttc tgg gaa gag 388 Ser Leu Cys Gly Tyr Gly Asn Gly Asn Gly Glu Phe Trp Glu Glu 110 115 120 tac atc gat gaa cgg tat gac aag taa acttccttga ttcttgaatt ttaccct 442 Tyr Ile Asp Glu Arg Tyr Asp Lys Stop 125 130 cacagtgtcc gactaaacca tcactatg gataaacta tcactatcat gc ccttgctggt gtgtaaggaa 562 aggagagaat gtagtcaact atgtattacg aatatatctg aaaattcaat ctcagatgta 622 aaattatata gtaaagtatg aac 645 <210> 8 <211> 550 <212> DNA <213> Pinctada fucata <400> gataggcggg ataatggggg caagaaatat tgtttctgta gatacgcttg gtctccatgg 60 caatgtaatg aggaagagag gtatgagtgg cttagatgtg gaatgaggtt ctactccttg 120 tgctgttaca ccgatgatga caacggtaac ggaaatggca acggtaacgg taacggatta 180 aattacctca agtccttata tggaggttac ggaaacggaa atggtgaatt ccgggaagag 240 tacatcgatg aacggtatga caattaaaat tcctgctacg gattcttgaa ttttaccctc 300 atttaataca gtgtcagact taaccttgac ttttatgttc tgaaaatagg aacgaaactt 360 catgactgta tttctctata gctagaatct tagaaaatgt caaagtttga ccttgctggt 420 gtgtaaggaa agaaaaaaat gtagtcaact atgtattacg aatatatctg aaaattcaat 480 ctcagatgta aaattatata gtaaagtatg aacaaaaaaa aaaaaaaaaa aaaaaaaaaa 540 atgtacaaaa 550 <210> 9 <211> 547 <212> DNA <213 > Pinctada fucata <400> gatcgtaggg acaatggtgg caagaaatgt tgcttctgta gaaacgcttg gtctccatgg 60 caatgtaaag aggacgagag gtatgagtgg ctaagatgtg gacataaatt ctactacatg 120 tgttgttaca ccgatgagcag attgaccag tattogatcaat gg ac ggaaatggtg aattctggga agagtacatc 240 gatgaacggt atgacaagta aacttccttg attcttgaat tttaccctca cagtgtccga 300 ctaaaccatg acttttatgt tcttaaataa gaacgaaact acatgtatct ctctatagct 360 tgtatcttag aaaatatttt ttgtttggcc ttgctggtgt gtaaggaaag gagagaatgt 420 agtcaactat gtattacgaa tatatctgaa aattcaatct cagatgtaaa attatatagt 480 aaagtatgaa gccagatttc aaaacaaagc aaaaaaaaaa aaaaaaaaaa aaaaaaaaat 540 gtacaaa 547

[Brief description of the drawings]

FIG. 1 is a photograph showing the results of electrophoresis. Specifically, FIG. 1 shows the results of SDS-PAGE of an alkali-soluble organic substrate from nacre and a water-soluble substrate from nacre and trabecula. It is a photograph shown.

FIG. 2 is a photograph showing a crystal structure, specifically, a photograph showing a normal inorganically formed aragonite.

FIG. 3 is a photograph showing a crystal structure, and more specifically, a photograph showing the function of the nacre matrix protein of the present invention.

FIG. 4 is a photograph showing a crystal structure, specifically, a photograph showing the function of the nacre matrix protein of the present invention.

FIG. 5 is a view showing alignment of amino acid sequences of seven kinds of proteins obtained by the present invention. FIG.
Wherein # 1.aa to # 7.aa correspond to SEQ ID NOs: 1 to 7, respectively.
Refers to the amino acid sequence of

Continued on the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/09 ZNA C07K 14/435 BIOSIS (DIALOG) WPI (DIALOG) GenBank / EMBL / DDBJ

Claims (5)

(57) [Claims] 1. A novel nacre matrix protein having the following physicochemical properties of i) -iii): i) obtainable from the nacre of the shell of the pearl oyster (Pinctada fucata), and from the trabecular layer of the shell Cannot be obtained; ii) The molecular weight as measured by SDS-PAGE is 14KDa or 17K
Da); iii) has the following amino acid sequence as the N-terminal amino acid sequence; AFHTKKGRYSYCWIPYDIEEDRRDNGGKKKCFFCN 2. A novel nacre matrix protein represented by the following (a) or (b): (a) a nacre matrix protein comprising an amino acid sequence represented by any one of SEQ ID NOs: 1-7 in the sequence listing; (b) In the amino acid sequence shown in any one of SEQ ID NOs: 1 to 7 in the sequence listing, one or several amino acids are deleted, substituted or added, including an amino acid sequence, and an organic substance for aragonite crystal formation of pearl oyster pearl. A protein that has a core function. [3] a gene encoding the protein of [2]; 4. The gene according to claim 3, which is a coding gene represented by any one of SEQ ID NOs: 1 to 7 in the sequence listing. 5. It hybridizes under stringent conditions with the coding gene shown in any one of SEQ ID NOs: 1-7 in the sequence listing, and has a function of being a core of organic matter for aragonite crystal formation of pearl oyster pearls. Gene encoding a protein.