WO2007148723A1 - Gène régulant la couleur de la coquille d'une huitre perlière et des perles et protéine encodée par ledit gène - Google Patents

Gène régulant la couleur de la coquille d'une huitre perlière et des perles et protéine encodée par ledit gène Download PDF

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Publication number
WO2007148723A1
WO2007148723A1 PCT/JP2007/062422 JP2007062422W WO2007148723A1 WO 2007148723 A1 WO2007148723 A1 WO 2007148723A1 JP 2007062422 W JP2007062422 W JP 2007062422W WO 2007148723 A1 WO2007148723 A1 WO 2007148723A1
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WO
WIPO (PCT)
Prior art keywords
dna
pearl
gene
shell
mantle
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PCT/JP2007/062422
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English (en)
Japanese (ja)
Inventor
Hiroshi Miyamoto
Koichi Morimoto
Masato Yano
Kouhei Nagai
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Kinki University
Japan Science And Technology Agency
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Application filed by Kinki University, Japan Science And Technology Agency filed Critical Kinki University
Publication of WO2007148723A1 publication Critical patent/WO2007148723A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a gene for a pearl oyster (Akoja), particularly a gene that is specifically expressed in the shell of a pearl oyster shell or mantle and controls the color tone, and a protein encoded by this gene. is there.
  • a core formed by processing a shell into a spherical shape and a cell piece (piece) of the mantle of a pearl shell (piece shell) are inserted into a pearl shell, which is a mother shell, and about 1 This is done by farming in the sea for years.
  • peace shellfish is determined not only by the genetic properties of peace shellfish, but also by the region of the mantle that is used. In addition to the trade secrets of traders, there is no established method for clearly identifying them.
  • Pearl colors include white, pink, cream, gold, and blue. Of these, white pearls are the most preferred and their selling price is high.
  • this aquaculture method has the following problems.
  • white pearl oysters are used as peace oysters, the probability of white pearls is higher than when ordinary pearl oysters are used. There was a problem that it was not so high.
  • white pearl oysters have a problem that it is difficult to cultivate pearl oysters because they are vulnerable to changes in water temperature.
  • Patent Document 1 Japanese Patent Laid-Open No. 05-308870
  • the present invention dramatically increases the ratio of excellent pearls by dramatically increasing information on the genes involved in the control of shell and pearl color, thereby improving the efficiency and improvement of pearl culture.
  • the challenge is to contribute.
  • the inventors have been involved in pearl and shell color efficiently by two approaches: comprehensive analysis of pearl shell mantle cDNA (EST analysis) and protein identification by TOF-MS. We searched for control factors. We identified two tyrosinase genes that are expressed specifically in the mantle and are thought to control the color of shells and pearls, and named them Pftyl and Pfty2.
  • the tyrosinase genes Pftyl and Pfty2 identified by the present invention are related to the colors of shells and pearls. Therefore, it is expected to use a probe, a primer adjusted based on this gene, and an antibody of a protein encoded by this gene to select piece shellfish and mother shellfish having a good color tone. In addition, it is possible to improve the quality of pearls in the future by manipulating the expression level of the tyrosinase gene.
  • FIG. 1 is a diagram showing the results of separating proteins isolated from a pearl ridge layer by SDS-PAGE and staining with CBB R250. The position of the molecular weight marker is shown on the left side, and the region used for TOF-MS analysis is shown in parentheses.
  • FIG. 3 is a diagram showing the correspondence between the primary structure of pearl oyster tyrosinase and the amino acid sequence obtained by MALDI-TOF / TOF MS analysis.
  • Each peptide a to h indicated by an underline in the figure corresponds to each of peaks a to h in FIG.
  • FIG. 4 shows the results of Northern plot analysis using RNA derived from adult pearl oyster tissue.
  • A A probe derived from Pftyl
  • B shows the result using a probe derived from Pfty2.
  • each lane is derived from lane 1: mantle (wild type), lane 2: gill (wild type), lane 3: midgut gland (wild type), lane 4: mantle (white-tail) total
  • lane 1 mantle (wild type)
  • lane 2 gill (wild type)
  • lane 3 midgut gland (wild type)
  • lane 4 mantle (white-tail) total
  • the results using RNA are shown.
  • the present invention is a novel tyrosinase gene that is specifically expressed in the mantle of a pearl oyster, a novel protein encoded by this DNA, a probe primer made from the DNA, and a pearl using these Shellfish sorting method.
  • Tyrosinase gene pft y l, Pfty2 is a gene specifically expressed in the mantle of the pearl, these genes nucleotide sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, or these bases It has a base sequence in which one or several bases are deleted, substituted or inserted in the sequence. From this nucleotide sequence, the protein encoded by this gene is thought to function as an enzyme in the melanin synthesis system.
  • the expression vector of the present invention can replicate autonomously in a host cell or can be integrated into a chromosome, and contains a promoter at a position suitable for transcription of the DNA. It contains the DNA in an expressible state. Moreover, genetic manipulation can be performed efficiently if the multicloning site contains an antibiotic resistance gene or the like.
  • the “state where gene function can be expressed” means that the gene product (enzyme) is expressed in a state having enzyme activity.
  • the transformant of the present invention is a transformant obtained by introducing the DNA or expression vector of the present invention into a host cell in a state where the gene function can be expressed by known means.
  • the transformant host can be used without particular limitation as long as it can be contained in a state where the gene function of the DNA can be expressed.
  • host cells include prokaryotic cells such as bacteria, microbial cells such as filamentous fungi and yeast, animal cells including molluscs such as shellfish, insect cells, plant cells, and the like. Any can be used as long as it can express a child.
  • Such a transformant can be obtained by any known method depending on the host. Specific methods include the calcium chloride method, agro-batterium method, electo-poration method, particle gun method, calcium phosphate co-precipitation method, DEAE-dextran method, electo-poration method, lipofussion method / ribosome method, etc. It is done.
  • the protein encoded by the tyrosinase gene, Pftyl, and Pfty2 is obtained by separating and purifying the protein produced by the transformant.
  • Protein separation and purification is a combination of known methods such as salting-out methods such as ammonium sulfate precipitation and column chromatography using HPLC.
  • Tyrosinase gene DNA of pft y l, Pfty2, the whole or can use some as a probe, it is possible to use a part as a primer.
  • RNA obtained by transcription of all or part of DNA of Pftyl and Pfty2 by a known transcription system can be used as a probe.
  • a part is used as a primer or a probe.
  • the DNA consists of nucleotides corresponding to at least 10, preferably 15, and more preferably about 20-30 base sequences contained in the DNA of the tyrosinase gene.
  • it may be a higher molecular weight, specifically tyrosinase gene DNA or its transcription product, mRNA itself.
  • probes and primers include, for example, DNA synthesized by a DNA synthesizer, DNA obtained by partially digesting tyrosinase gene DNA with restriction enzymes, tyrosinase gene DNA and expression vector power obtained by recombining the partial sequence thereof, Et al. RNA prepared using RNA polymerase such as T7 RNA polymerase.
  • probes and primers should be labeled with a labeling substance.
  • Tatoebahi - 32 P_dCTP fluorescent substances
  • detectable such as radioisotope
  • label engaged forming e.g. a labeling substance chemically DNA
  • the DNA can be synthesized by a known method such as by synthesizing DNA using already labeled nucleotides.
  • the transcription amount of the tyrosinase gene in the mantle is measured and used as a piece of pearl culture. It is for selecting suitable piece shells.
  • suitable piece shells include known methods such as the Northern hybridization method, RT-PCR method, in situ hybridization method using the probes and primers.
  • a template was prepared by the Rolling Cycle Amplification method, and a sequencing reaction was performed using this template by the dye terminator method.
  • T7 Promoter Primer (manufactured by Sigma) was used as a primer.
  • the base sequence of DNA was determined by MegaBACE4000 capillary sequencer (manufactured by GE Healthcare Bioscience Co., Ltd.), and the base sequence of 3214 cDNAs was determined.
  • PTA Paracel Transcript Assembler, manufactured by Paracel
  • 257 consisting of 1586 single sequences and 1628 clones were obtained. Confirmed the cluster.
  • the gel slice was digested with ⁇ trypsin at 30 ° C in 30 ⁇ l of 25 mM ammonium bicarbonate. Concentrate digestion solution with ZipTip ⁇ (Millipore) and then 2.5 mg / ml a -cyano
  • this amino acid sequence was compared with the amino acid sequence of the protein encoded by the gene contained in the mantle cDNA library.
  • genes having the amino acid sequences corresponding to the peaks a to h were found.
  • Pftyl and Pfty2 The nucleotide sequences of Pftyl and Pfty2 are shown in SEQ ID NOs: 9 and 10, respectively, and the amino acid sequences of the encoded proteins are shown in SEQ ID NOs: 11 and 12, respectively.
  • FIG. 3 shows the amino acid sequences of proteins encoded by the Pftyl and Pfty2 genes, and the underlined portions in the figure correspond to the peaks a to h in FIG.
  • RNA isolated from the pearl shell mantle, gill, midgut, and closed shell muscle was electrophoresed according to a standard method, separated into sizes, and blotted onto a membrane filter.
  • Hybond_N + (GE Healthcare Biosciences) was used as the blotting membrane.
  • A is a probe derived from Pftyl
  • B is Pfty2.
  • lane 1 is the mantle (wild type)
  • lane 2 is the gill (wild type)
  • lane 3 is the midgut (wild type)
  • lane 4 is the mantle (shirogai).
  • the result of electrophoresis of total RNA derived from is shown.

Abstract

La présente invention concerne l'amélioration de l'efficacité et du développement de la culture de perles grâce à l'accroissement spectaculaire des informations relatives à un gène associé à la couleur de la coquille et des perles ainsi que d'éléments analogues. Deux approches d'analyse exhaustive (analyse EST) de l'ADNc du manteau d'une huitre perlière et l'identification de protéines par spectrométrie de masse à temps de vol (TOF-MS) permettent d'identifier efficacement un gène associé à la régulation de la couleur des perles et de la coquille. C'est ainsi que deux gènes de tyrosinase spécifiquement exprimés dans le manteau ont été identifiés.
PCT/JP2007/062422 2006-06-23 2007-06-20 Gène régulant la couleur de la coquille d'une huitre perlière et des perles et protéine encodée par ledit gène WO2007148723A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2006-173485 2006-06-23
JP2006173485A JP2009254234A (ja) 2006-06-23 2006-06-23 真珠貝の貝殻、真珠の色調を制御する遺伝子とそのタンパク質

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2999602A1 (fr) * 2012-12-17 2014-06-20 Ifremer Signature predictive de la capacite de biomineralisation d’une huitre perliere donneuse de greffons
WO2014125054A1 (fr) * 2013-02-15 2014-08-21 Swiss Gemmological Institute Ssef Profilage d'adn de perles
CN113201543A (zh) * 2021-05-06 2021-08-03 广东海洋大学 企鹅珍珠贝Tyr基因的启动子及其应用
CN115353548A (zh) * 2022-09-20 2022-11-18 华南农业大学 一种具有美白功效的珍珠贝酪氨酸酶抑制肽及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05308870A (ja) * 1992-05-08 1993-11-22 Taiyo Shinjiyu Kk 白色系真珠の生産方法
JP2004024062A (ja) * 2002-06-24 2004-01-29 Matsushita Pearl Co Ltd 真珠の養殖方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05308870A (ja) * 1992-05-08 1993-11-22 Taiyo Shinjiyu Kk 白色系真珠の生産方法
JP2004024062A (ja) * 2002-06-24 2004-01-29 Matsushita Pearl Co Ltd 真珠の養殖方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MORIMOTO S.: "Akoyagai ni Fukumareru Tanpakushitsu ni Tsuite", EHIMEKEN KOGYOKEI KENKYU HOKOKU, no. 42, 2004, pages 121 - 124, XP003020241 *
NAGAI K. ET AL.: "Tyrosinase localization in mollusc shells", COMP. BIOCHEM. PHYSIOL. B BIOCHEM. MOL. BIOL., vol. 146, no. 2, February 2007 (2007-02-01), pages 207 - 214, XP005873165 *
WADA K. ET AL.: "The behaviour of outer mantle epithelial cells implanted into the mantle connective tissue of the freshwater muscle, hyriopsis schlegeli at an early stage of pearl-sac formation", VENUS, vol. 54, no. 2, 1995, pages 133 - 141, XP003020242 *
ZHANG C. ET AL.: "A novel putative tyrosinase involved in periostracum formation from the pearl oyster (pinctada fucata)", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 342, no. 2, April 2006 (2006-04-01), pages 632 - 639, XP005300596 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2999602A1 (fr) * 2012-12-17 2014-06-20 Ifremer Signature predictive de la capacite de biomineralisation d’une huitre perliere donneuse de greffons
WO2014096688A3 (fr) * 2012-12-17 2014-11-13 Ifremer (Institut Français De Recherche Pour L'exploitation De La Mer) Signature prédictive de la capacité de biominéralisation d'une huître perlière donneuse de greffons
WO2014125054A1 (fr) * 2013-02-15 2014-08-21 Swiss Gemmological Institute Ssef Profilage d'adn de perles
CN113201543A (zh) * 2021-05-06 2021-08-03 广东海洋大学 企鹅珍珠贝Tyr基因的启动子及其应用
CN113201543B (zh) * 2021-05-06 2022-11-08 广东海洋大学 企鹅珍珠贝Tyr基因的启动子及其应用
CN115353548A (zh) * 2022-09-20 2022-11-18 华南农业大学 一种具有美白功效的珍珠贝酪氨酸酶抑制肽及其应用
CN115353548B (zh) * 2022-09-20 2024-03-15 华南农业大学 一种具有美白功效的珍珠贝酪氨酸酶抑制肽及其应用

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