JP2009219487A - Growth-related gene of germ line of pinctada fucata, and method for inhibiting growth of germ cell line - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new gene related to the growth of a germ cell line of Pinctada fucata. <P>SOLUTION: The new gene related to the growth of the germ cell line of the Pinctada fucata includes a base sequence represented by one of sequence numbers 1-3. The emergence of the germ cell line in the Pinctada fucata can be inhibited by inhibiting the expression of the gene. The method for inhibiting the expression of an mRNA by using, for example, an siRNA, an siRNA precursor, an miRNA, an miRNA precursor, an antisense RNA or a ribozyme is cited as the method for inhibiting the expression. The Pinctada fucata in which the expression of a new pov gene is inhibited by such the method does not require the treatment such as the removal of an egg, for example when being used as a mother shell for pearl culture, because the growth of the germ cell line is inhibited in the Pinctada fucata in which the expression of the new pov gene is inhibited by such the method. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel gene involved in germline development of pearl oysters and a method for inhibiting germline development.

In Japan, pearl oysters ( Pincta fucata ) are widely used as mother shells for pearl farming. Pearl culture using pearl oysters is generally performed as follows. First, eggs are collected from female shells of pearl oysters, sperm are collected from male shells, artificially mated, and hatched. After hatching the hatched larvae in a water tank, they are placed in an offshore cage and grown in the sea. The larvae are transferred to the cultivation cage as the larvae grow, and the mother shellfish are grown. Then, after pre-processing the mother shell, a pearl is inserted into the core (intubation) to grow the surgical shell and form a pearl.

  In this series of operations, pretreatment before nucleation greatly affects the growth of pearl oysters, which are mother shellfish, and the quality of pearls to be cultivated. In the intubation process, since the nucleus is inserted into the gonad gonad, there is a problem that insertion becomes difficult if the gonad is filled with a large amount of eggs. In addition, egg formation and maturation can lead to a decrease in pearl quality and cause enucleation. For this reason, in order to suppress the maturation of the eggs, the “suppression treatment” for growing the mother shellfish in a cramped state and the “egg removal treatment” for stimulating the mother shellfish to lay eggs are performed as pretreatment. Yes. However, since the suppression process extremely weakens the pearl oyster, there is a problem that the mortality rate increases. Moreover, since the egg removal process requires a period of about 2 months, there is a problem that the period during which the subsequent nucleation process can be performed is very short, about 3 months. Therefore, it is desired to enable nucleation processing without performing suppression processing or egg removal processing.

Continued and scientific pearl farming -Pearl Culture Q & A (2)-, Hiroshi Wada, Pearl Newspaper Company Pearl Story-Living Jewels-Akira Machii, Hanafusa

  For this reason, establishment of the pearl oyster in which the generation | occurrence | production and development of the reproductive cell which enabled the nucleation process without performing a suppression process and an egg removal process was suppressed is calculated | required. Therefore, an object of the present invention is to provide a novel gene involved in germline development in the pearl oyster. Furthermore, it aims at providing the method of suppressing the development of a germ line by suppressing the expression of such a gene.

  In order to achieve the above object, the novel gene of the present invention is a gene related to the development of the pearl oyster germline, and the nucleotide sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 It is characterized by comprising. Hereinafter, the novel gene of the present invention is also referred to as “development-related gene”.

  The developmental suppression method of the present invention is a method for suppressing the development of germline of pearl oysters, characterized by suppressing the development of germline by suppressing the expression of RNA from the development-related gene of the present invention or its partial sequence. And

The development inhibitor of the present invention is a development inhibitor for use in the method for suppressing the development of the pearl oyster germ line of the present invention, and is at least one selected from the group consisting of the following (x1) to (x3): It contains a nucleic acid.
(X1) Decomposing RNA transcribed from the germline development-related gene according to claim 1 or a partial sequence thereof, degrading with siRNA or ribonuclease, and degrading with siRNA precursor, miRNA or ribonuclease that liberates siRNA. At least one nucleic acid (x2) selected from the group consisting of a miRNA precursor, an antisense and a ribozyme that liberates miRNA by transcription of RNA from the germline development-related gene or partial sequence thereof according to claim 1 Inhibiting, selected from the group consisting of siRNA, a siRNA precursor that liberates siRNA by being degraded by ribonuclease, a miRNA, a miRNA precursor that liberates miRNA by being degraded by ribonuclease, an antisense, and a ribozyme An expression vector that expresses at least one of the at least one nucleic acid of the nucleic acid (x3) wherein (x1) and (x2) was

The method for producing pearl oysters of the present invention is a method for producing pearl oysters in which germline generation is suppressed, and includes the following steps (A1) and (A2) or the following steps (B1) and (B2). And
(A1) Step of introducing the development inhibitor of the present invention into at least one germ cell of pearl oyster egg and sperm (A2) Step of crossing said egg and sperm to obtain a fertilized egg (B1) Egg of pearl oyster A step of mating a sperm and obtaining a fertilized egg (B2) a step of introducing the development inhibitor of the present invention into the fertilized egg

  The method for producing pearls of the present invention is a method for producing pearls by introducing nuclei into pearl oysters, comprising the step of producing pearl oysters by the pearl oyster manufacturing method of the present invention prior to introduction of the nuclei. To do.

  The sterilization method of the present invention is a method for sterilizing pearl oysters, and includes the step of suppressing the development of the germline of pearl oysters by the method for inhibiting development of the present invention.

  The sterilizing agent of the present invention is a sterilizing agent for use in the method for sterilizing pearl oysters of the present invention, and is characterized by including the development inhibitor of the present invention.

  As a result of intensive studies, the present inventors have identified three types of genes involved in germline development from the pearl oyster genomic DNA. These development-related genes of the present invention were first discovered by the present inventors. Since the development-related gene of the present invention is involved in germline development as described above, germline development can be suppressed by suppressing the expression of this gene. Moreover, according to the suppression method of the present invention, for example, pearl oysters (sterile pearl oysters) in which the number of germ cells is reduced or maturation of germ cells is suppressed can be obtained. If such a pearl oyster is used, for example, as a mother shell in pearl culture, the suppression process and the egg removal process in the conventional method are not required, so that the pearl culture can be performed more easily.

FIG. 1 is a photograph showing the results of in situ hybridization showing the expression sites of the pov I gene, pov II gene and pov IV gene in germ cells in Example 1 of the present invention. FIG. 2 is a photograph of a tissue section of a gonad in Example 2 of the present invention, the right figure is a photograph of an example in which RNAi treatment was performed, and the left figure is a photograph of a control not treated with RNAi. is there. FIG. 3 is a graph showing individual distributions belonging to each GSI in Example 2 of the present invention. FIG. 4 is a photograph of a tissue section of a gonad in Example 3 of the present invention, the right figure is a photograph of an example in which RNAi treatment was performed, and the left figure is a photograph of a control not treated with RNAi. is there. FIG. 5 is a graph showing individual distributions belonging to each GSI in Example 3 of the present invention. FIG. 6 is an electrophoretogram showing the results of RT-PCR in Example 3 of the present invention.

<Development-related genes>
As described above, the development-related gene of the present invention is involved in the development of the pearl oyster germline, characterized by comprising the base sequence represented by any of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. It is a novel gene.

  Hereinafter, the gene consisting of the base sequence represented by SEQ ID NO: 1 is referred to as “pov I or pov I gene”, and the protein consisting of the amino acid sequence encoded by the gene is referred to as “POV I”. An example of the amino acid sequence of POV I is shown in SEQ ID NO: 2. The gene consisting of the base sequence represented by SEQ ID NO: 3 is hereinafter referred to as “pov II or pov II gene”, and the protein consisting of the amino acid sequence encoded by the gene is referred to as “POV II”. An example of the amino acid sequence of POV II is shown in SEQ ID NO: 4. Hereinafter, the gene consisting of the nucleotide sequence represented by SEQ ID NO: 5 is referred to as “pov IV or pov IV gene”, and the protein consisting of the amino acid sequence encoded by the gene is referred to as “POV IV”. An example of the amino acid sequence of POV IV is shown in SEQ ID NO: 6. In the present invention, the pov I gene, the pov II gene, and the pov IV gene are also collectively referred to as “pov gene”. The base sequence represented by each SEQ ID NO is the sequence of the sense strand of each pov gene. The development-related gene of the present invention may be a gene containing the aforementioned base sequence.

  The pov gene of the present invention is, for example, a gene that is specifically expressed in the gonad and is involved in germline development and development. Therefore, by suppressing the expression of the pov gene of the present invention, for example, germline development can be suppressed. Therefore, the pov gene of the present invention can be a marker of germline developmental or developmental ability, for example.

<Methods for inhibiting germline development and development inhibitors>
As described above, the present invention is a method for suppressing the generation of pearl oyster germline, wherein the development of germline is suppressed by suppressing the expression of RNA from the pov gene of the present invention or a partial sequence thereof. And

  As described above, the pov gene of the present invention is a gene involved in germline development. For this reason, germline development can be suppressed by suppressing RNA expression (for example, mRNA expression) from the pov gene of the present invention. And by suppressing the development of germ cells in this way, for example, sterility of pearl oysters as described later becomes possible. The present invention is characterized in that a novel gene involved in germline development has been identified, and means for suppressing the expression of RNA from the novel gene is not limited at all.

  In the present invention, “suppression of germline development” means that the formation of mature germ cells is consequently suppressed. Inhibition of the formation of mature germ cells can be achieved, for example, in the development of germ line, in the division of oocytes and spermatogonia, growth and differentiation into primary germ cells, the first meiotic division of primary germ cells, It may be realized by suppressing any process such as meiotic second division from secondary germ cells. In addition, according to the method of the present invention described later, in particular, suppression of formation of germ cells themselves can be confirmed, but is not limited thereto.

  In the present invention, RNA that is subject to expression suppression is also referred to as “target RNA”. The target RNA is not particularly limited, and may be, for example, RNA (mRNA) for the entire sequence of the pov gene or RNA (mRNA) for a partial sequence of the pov gene.

  In the present invention, RNA expression suppression may be, for example, (1) suppression by decomposing (cutting) RNA transcribed from the pov gene or a partial sequence thereof. In this case, for example, even if the target RNA is transcribed, the transcript is decomposed (cut), and as a result, the expression of the target RNA is suppressed. On the other hand, RNA expression suppression may be, for example, (2) inhibition (suppression) of transcription from the pov gene or a partial sequence thereof. In this case, the expression of the target RNA is suppressed by suppressing the generation (transcription) of the target RNA itself. In the above (2), “transfer suppression” includes, for example, not only complete transfer stop but also transfer decrease. In addition, “transcriptional repression” includes, for example, RNA having no original function by replacing, deleting, inserting or adding one or several bases in the pov gene or a partial sequence thereof. It also includes transcription, or transcription of RNA with reduced function. In the present invention, the “function” is, for example, a function of developing a germ line, and as a result, a function of forming a mature germ cell. Specifically, for example, division of oocytes and spermatogonia, growth and differentiation into primary germ cells, reduction of the primary germ cells, first division, secondary reduction from secondary germ cells, etc. The function to perform is given.

  The method for suppressing the expression of RNA is not particularly limited, and for example, a conventionally known method used for suppressing the expression of RNA can be employed. As specific examples, for example, at least one of the pearl oyster germ cell and fertilized egg, siRNA precursor that releases siRNA by being degraded by siRNA or ribonuclease, miRNA that releases miRNA by being degraded by miRNA or ribonuclease Examples thereof include a method of introducing at least one nucleic acid selected from the group consisting of a precursor, an antisense, a ribozyme, and an expression vector expressing them. By using such a nucleic acid, for example, as shown in (1) above, RNA transcribed from the pov gene or a partial sequence thereof can be degraded, or as shown in (2) above, the pov gene or its It is also possible to inhibit transcription of RNA from a partial sequence. In the present invention, the nucleic acid may be any one kind or a combination of two or more kinds, for example.

  Examples of the siRNA precursor include double-stranded RNA (dsRNA). For example, dsRNA suppresses RNA expression by RNA interference (RNAi) having the following mechanism. That is, when the dsRNA is introduced into a cell, it is first degraded by ribonuclease to produce siRNA. The siRNA becomes a single strand by forming a RISC complex. The RISC complex recognizes the target RNA, the single strand binds to the mRNA, and cleaves or decomposes the target RNA. Examples of the ribonuclease include Dicer and Drosha (hereinafter the same).

Although it does not restrict | limit especially as said dsRNA, For example, it is preferable that the antisense strand is a polynucleotide which consists of a base sequence of the following (a1)-(a6). In the base sequence of the dsRNA, thymine may be uracil.
(A1) In the base sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, a base sequence complementary to a base sequence consisting of consecutive 1,080 to 1,260 bases (a2) SEQ ID NO: 7, SEQ ID NO: Nucleotide sequence complementary to the nucleotide sequence represented by any of 8 and SEQ ID NO: 9 (a3) 360 to 420 amino acids continuous in the amino acid sequence represented by any of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 Complementary to the base sequence encoding the amino acid sequence consisting of residues (a4) Complementary to the base sequence encoding the amino acid sequence consisting of the first to 364th amino acids in the amino acid sequence represented by SEQ ID NO: 2 Base sequence

  For example, the sequence of the antisense strand may be completely the same as or partially the same as the sequence of the pov gene (antisense strand) of the present invention. The sequence of the antisense strand has, for example, homology to the complementary sequence of the target RNA (complementarity to the sequence of the target RNA), for example, 70% or more, preferably 80% or more, more preferably 90% or more, Particularly preferred is 95% or more, and most preferred is 100%.

  The length of the dsRNA is not particularly limited, but is, for example, 500 to 3000 bases, preferably 500 to 700 bases, and more preferably 500 to 700 bases. The dsRNA is preferably used, for example, when introduced into the cell using the uptake ability of at least one of a germ cell and a fertilized egg, as described later.

  The siRNA precursor may be, for example, a single-stranded RNA. Examples of the single-stranded RNA include a foldback RNA precursor in which a plurality of hairpin structures are linked by self-annealing, a hairpin-type miRNA-like RNA precursor, a hairpin-type siRNA precursor, and the like. These single-stranded RNAs can also generate siRNA by, for example, ribonuclease.

  The siRNA usually comprises an antisense strand complementary to RNA such as mRNA and a sense strand complementary thereto. The length of each strand of siRNA is not particularly limited, but is usually an oligonucleotide having 19 to 25 bases, and preferably 19 bases. The siRNA may have, for example, an overhang at the 3 ′ end of each strand of oligonucleotide, and in this case, each 3 ′ end has a protruding shape. The length of the overhang is not particularly limited, but is usually 2 bases. The sequence of the overhang is not particularly limited, and may be, for example, a sequence complementary to the RNA to be bound in the antisense strand, or a sequence complementary to the antisense strand in the sense strand, that is, It may be the same sequence as the RNA to which the antisense strand binds. Moreover, other arrangement | sequences may be sufficient, for example, arrangement | sequences, such as uu, au, ag, gc, cc, are mention | raise | lifted. The sense strand corresponding to the antisense strand may be, for example, a completely complementary sequence to the antisense or not a completely complementary sequence. In the latter case, for example, the internal sequence may be a sequence shorter by about 2 bases than the antisense strand, or a sequence having a mismatched base with respect to the antisense.

  In the siRNA, the sequence of the antisense strand may be completely the same as the complementary sequence of the RNA to be bound, or may not be the completely identical sequence. The sequence of the antisense strand has, for example, homology to the complementary sequence of the target RNA (complementarity to the sequence of the target RNA), for example, 70% or more, preferably 80% or more, more preferably 90% or more, Particularly preferred is 95% or more, and most preferred is 100% (perfect match). Further, the sequence excluding the overhang in the antisense strand may be completely the same as the complementary sequence of the RNA to be bound, or may not be the completely same sequence. The sequence excluding the overhang has, for example, homology to the complementary sequence of the target RNA (complementarity to the sequence of the target RNA), for example, 70% or more, preferably 80% or more, more preferably 90% or more, Particularly preferred is 95% or more, and most preferred is 100% (perfect match).

  In the target RNA of the pov gene, the region to which the siRNA binds is not particularly limited. Usually, RNA is cleaved by siRNA first in a region where siRNA is bound, but it is also known that cleaving occurs in other regions, and finally the target RNA is cleaved completely. Yes. Therefore, siRNA may be designed, for example, so as to bind to a region to be cleaved, or designed so as to bind to a region other than the region. In the latter case, cleavage within the binding region of siRNA is triggered, and as a result, the target region (target RNA) is also degraded.

  miRNA usually has a sequence complementary to RNA such as mRNA. Although it does not restrict | limit especially as length of miRNA, For example, 18-25 bases are mention | raise | lifted.

  Examples of the antisense include antisense consisting of a sequence complementary to the target RNA. The antisense may be, for example, DNA or RNA. Examples of the mechanism for suppressing the expression by antisense RNA include, for example, inhibition of transcription initiation by triplex formation, transcription inhibition by hybridization with a site where an open loop structure is locally formed by RNA polymerase, and synthesis are in progress. Examples include transcription inhibition by hybridization with a certain RNA (Hirashima and Inoue; Laboratory for Neonatal Chemistry 2 Nucleic acid IV gene replication and expression (Tokyo Biochemical Society, pp. 319-347, 1993)). The length of the antisense RNA is not particularly limited, but is, for example, 15 bases or more, preferably 100 bases or more, and more preferably 500 bases or more. The length of general antisense RNA is, for example, 5 kb or less, and preferably 2.5 kb or less.

  The ribozyme generally means an RNA molecule having catalytic activity. Examples of the ribozyme in the present invention include ribozymes that cleave (cleave) the target RNA in a site-specific manner. The ribozyme sequence is not particularly limited, and can be designed to have a sequence complementary to the sequence to be cleaved, for example. The type of ribozyme is not particularly limited, and examples thereof include hammerhead ribozymes and hairpin ribozymes.

  The siRNA, siRNA precursor, miRNA, miRNA precursor, antisense, and ribozyme technologies have been established as, for example, means for degrading RNA transcribed from genes and means for inhibiting transcription of RNA from genes. . Therefore, those skilled in the art can design siRNA, siRNA precursor, miRNA, miRNA precursor, antisense, ribozyme and the like for the purpose of target RNA degradation and target RNA transcription inhibition in the practice of the present invention. Can be done based on common sense. In addition, the constituent materials such as siRNA, siRNA precursor, antisense and ribozyme are not particularly limited, and may be, for example, a naturally-derived nucleic acid or an artificial nucleic acid such as PNA, and is not limited at all.

  As described above, the nucleic acid to be introduced into the germ cell or fertilized egg may be an expression vector that expresses various nucleic acids such as the siRNA precursor. That is, in the present invention, for example, siRNA, siRNA precursor, miRNA, miRNA precursor, antisense and ribozyme may be introduced into the germ cells or fertilized eggs, and these nucleic acids are expressed. An expression vector may be introduced into the germ cell or fertilized egg. Thus, by introducing the expression vector into the germ cell or fertilized egg, the siRNA precursor or the like can be expressed in the germ cell or fertilized egg. The expression vector is not particularly limited as long as it can express the nucleic acid, and examples thereof include viral vectors and non-viral vectors.

  In the present invention, the germ cell into which the nucleic acid is introduced may be, for example, either an egg or a sperm, or both. Examples of the target to which the nucleic acid is introduced include fertilized eggs in addition to the germ cells. The fertilized egg may be, for example, a fertilized egg (eg, an embryo) that has been divided into eggs, and is particularly preferably a fertilized egg at an early stage of egg division.

  In the present invention, the method for introducing the nucleic acid into the germ cell or fertilized egg is not particularly limited. For example, injection into the germ cell or fertilized egg, immersion of the germ cell or fertilized egg in the nucleic acid-containing solution, etc. Can be given. A specific method will be described in a method for manufacturing pearl oysters described later.

<Development inhibitor>
Next, as described above, the development inhibitor of the present invention is a development inhibitor for use in the development suppression method of the present invention, and is at least selected from the group consisting of the following (x1) to (x3): It contains one nucleic acid.
(X1) An siRNA precursor, miRNA, or ribonuclease that decomposes RNA transcribed from the germline development-related gene (pov gene) of the present invention or a partial sequence thereof, and releases siRNA by being degraded by siRNA or ribonuclease. At least one nucleic acid (x2) selected from the group consisting of miRNA precursor, antisense and ribozyme that liberates miRNA upon being degraded (x2) from the germline development related gene (pov gene) of the present invention or a partial sequence thereof Inhibits transcription of RNA, siRNA, siRNA precursor that releases siRNA by being degraded by ribonuclease, miRNA, miRNA precursor that releases miRNA by being degraded by ribonuclease, antisense and ribozyme Expression vector expressing at least one nucleic acid of at least one nucleic acid (x3) the selected from Ranaru group (x1) and (x2)

  Each nucleic acid shown in the above (x1) to (x3) is as described above. Each of (x1), (x2) or (x3) may be any one kind of nucleic acid, or two or more kinds may be used in combination. In addition, any one of the nucleic acids (x1) to (x3) may be used, or two or more of them may be used in combination.

The form of the development inhibitor of the present invention is not particularly limited, but is preferably a liquid containing the nucleic acid, for example, because it is excellent in handleability. The liquid solvent is not particularly limited, and examples thereof include seawater, water containing NaCl, and a buffer solution. The concentration of NaCl is not particularly limited, but is, for example, about 30 to 35 mmol / L, and preferably about 30 mmol / L. The buffer solution may contain, for example, KCl, CaCl ₂ , CaCl ₂ .2H ₂ O, Hepes, and the like. The type of the buffer solution is not particularly limited. The solvent preferably contains no Mg ions, for example. The pH of the solvent is not particularly limited, but is, for example, pH 7.5 to 8, preferably pH 8.

  In addition, the detail of the usage method of a development inhibitor is demonstrated in the manufacturing method of the pearl oyster mentioned later.

<Manufacturing method of pearl oyster>
The method for producing pearl oysters of the present invention is a method for producing pearl oysters in which germline development is suppressed. In the present invention, the pearl oysters whose germline development is suppressed may be, for example, fertilized eggs or pearl oysters (including juveniles) grown from fertilized eggs.

The production method of the present invention includes a first production method including the following steps (A1) and (A2), wherein target fertilized germ cells are subjected to target RNA expression suppression treatment; And a second production method including the following steps (B1) and (B2).
(A1) Step of introducing the development inhibitor of the present invention into at least one germ cell of pearl oyster egg and sperm (A2) Step of crossing said egg and sperm to obtain a fertilized egg (B1) Egg of pearl oyster A step of mating a sperm and obtaining a fertilized egg (B2) a step of introducing the development inhibitor of the present invention into the fertilized egg

  The treated fertilized eggs obtained by such first and second production methods, for example, suppress the expression of the RNA of the pov gene of the present invention. For this reason, pearl oysters (including juveniles) whose germline development is suppressed can be obtained by growing the fertilized eggs. Thus, the pearl oysters whose germline development is suppressed are extremely useful as mother shells for pearl manufacture as described later, for example, because the formation and maturation of egg cells are suppressed. Hereinafter, the step of introducing the expression inhibitor of the present invention is also referred to as “expression suppression treatment”.

  First, the first manufacturing method will be described. In the first production method, the type of germ cell to be subjected to the expression suppression treatment is not particularly limited and may be either an egg or a sperm. For example, an egg is preferable because it is easy to handle. Moreover, you may perform an expression suppression process to both an egg and a sperm. The eggs and sperm are preferably mature cells because they are fertilized, for example.

  The method for introducing the development inhibitor into the germ cells is not particularly limited. For example, the development inhibitor is brought into contact with a liquid containing the development inhibitor in contact with the germ cells, and the germ cell uptake ability allows the development inhibitor to be introduced. Examples thereof include a method for introducing it into the germ cells. Examples of the contact method include immersion of the gonad or germ cell in the liquid, injection of the liquid into the gonad, and the like. For example, the gonad is preferably immersed in the liquid as it is. In addition, the development inhibitor can be directly injected into the germ cells by microinjection or the like. Among them, since it is not necessary to take out germ cells from the gonad and the operation is simple, immersion of the gonad in the liquid, injection of the liquid into the gonad, and the like are preferable.

  The content of the development inhibitor in the liquid is not particularly limited. For example, the concentration of the development inhibitor is preferably 100 μg / ml to 1000 μg / ml, and more preferably 400 μg / ml.

  When gonads or germ cells are immersed in the liquid containing the development inhibitor, the immersion time is not particularly limited, but is preferably, for example, 30 minutes to 12 hours, and more preferably 3 to 6 hours. Moreover, although immersion temperature is not restrict | limited in particular, For example, it is 20-27 degreeC, and is 23-25 degreeC normally.

  In the case of injecting a liquid containing the development inhibitor, for example, the injection ratio to the gonad is preferably 200 to 400 μl, particularly preferably 200 μl per 10 to 15 g of body weight. For example, the pearl oyster after injection is preferably allowed to stand for 30 minutes to 12 hours, more preferably 3 to 6 hours in a state in which the shellfish are not dried. The condition of not drying is, for example, a condition in which a wet wipe or the like is laid on a bat and a shell is placed on it. The temperature is the same as described above, for example.

  In addition, examples of the method for introducing the development inhibitor include calcium phosphate method, polyethylene glycol method, lipofection method, electroporation method, ultrasonic nucleic acid introduction method, gene gun introduction method, DEAE-dextran method, Examples thereof include a direct injection method using a glass tube or the like, a hydrodynamic method, a cationic liposome method, a method using atelocollagen, and the like. In addition, as an introduction aid, for example, liposomes such as lipofectamine and cationic liposome, atelocollagen, and the like can be used.

  The mating of germ cells after the developmental inhibition treatment can be performed by, for example, a conventionally known method. When the development inhibitor is introduced without taking out germ cells from the pearl oyster, for example, egg collection and semen collection may be performed from a female shellfish and a male shellfish, and artificial mating may be performed. By such a method, a fertilized egg to be a pearl oyster (including juvenile shellfish) whose germline development is suppressed can be obtained.

  Next, the second manufacturing method will be described. In the second production method, the treatment can be performed in the same manner as the first production method described above, unless otherwise indicated, except that the expression suppression treatment is performed on the fertilized egg.

  First, a pearl oyster egg and sperm are fertilized by a conventionally known method to obtain a fertilized egg. And the said development inhibitor is introduce | transduced with respect to the obtained fertilized egg. The introduction method to the fertilized egg is not particularly limited, but, for example, the liquid containing the development inhibitor is brought into contact with the fertilized egg, and the development inhibitor is introduced into the germ cell by the uptake ability of the fertilized egg. There are methods. Examples of the contact method include immersion of the fertilized egg in the liquid, or injection of the liquid into the fertilized egg. By such a method, a fertilized egg to be a pearl oyster (including juvenile shellfish) whose germline development is suppressed can be obtained.

  The first and second production methods of the present invention preferably further include, as the step (C), a step of growing the fertilized egg after the step (A2) or the step (B2). By growing fertilized eggs in this manner, it is possible to obtain pearl oysters (including juveniles) with suppressed germline development and a small number of germ cells or low maturity.

<Pearl production method>
The pearl production method of the present invention is a pearl production method for producing a pearl by introducing a nucleus into a pearl oyster, which includes the step of producing pearl oyster by the production method of the present invention prior to the introduction of the nucleus. And

  The pearl oyster obtained by the production method of the present invention has a low germ cell number and maturity because the development of germ cells is suppressed. For this reason, it is not necessary to nurture the mother shellfish in a cramped state to suppress the maturation of the eggs or to remove the eggs as in the prior art. Therefore, according to the manufacturing method of the present invention, it is possible to manufacture pearls that are superior in quality in a shorter period of time. In addition, this invention is characterized by preparing an pearl oyster by the manufacturing method of the pearl oyster of this invention, and other processes and conditions are not restrict | limited at all.

<Sterilization method and sterilization agent>
The sterilization method of the present invention is a method for sterilizing pearl oysters, and includes the step of suppressing the development of the germline of pearl oysters by the method for inhibiting development of the present invention. In the present invention, “sterilization” means, for example, suppression of germline development-related gene expression of pearl oysters and suppression of germ cell maturation.

  As described above, according to the method for suppressing development of the present invention, germline development can be suppressed, so that sterility can be achieved. The present invention is characterized in that the method for inhibiting development of the present invention is performed, and other processes and conditions are not limited at all. Specifically, for example, sterilized pearl oysters can be obtained by the above-described method for producing pearl oysters of the present invention.

  The sterilizing agent of the present invention is a sterilizing agent for use in the sterilization method of the present invention, and includes the development inhibitor of the present invention. The sterilizing agent of the present invention is characterized in that it contains the development inhibitor of the present invention, the other components are not limited at all, and can be used under the same conditions as those of the aforementioned development inhibitor.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercially available reagents were used based on their protocols unless otherwise indicated.

[Example 1]
The expression sites in the pearl oyster were confirmed for the novel genes pov I, pov II and pov IV identified by the present inventors.

Based on the base sequences of the pov I gene, the pov II gene and the pov IV gene, the following probes complementary to these were prepared. Each of these probes was labeled at the 3 ′ end with digoxigenin (DIG).
probe for pov I antisense SEQ ID NO: 10
Sense SEQ ID NO: 11
probe for pov II antisense SEQ ID NO: 12
Sense SEQ ID NO: 13
Probe for pov IV Antisense SEQ ID NO: 14
Sense SEQ ID NO: 15

  The gonad extracted from the pearl oyster in the first year of growth was fixed with Davidson's solution to prepare a tissue section having a thickness of 8 μm. After the deparaffinized section was treated with Proteinase K, the aforementioned labeled probe was reacted at 50 ° C. for 40 hours. After the reaction, unreacted labeled probe was removed by washing, and an alkaline phosphatase labeled anti-DIG antibody was further added. As a result, a hybrid of the labeled probe and the labeled anti-DIG antibody was formed, and a color development reaction was performed using NBT and BCIP as the color development substrates. The section subjected to the color reaction was observed with an optical microscope. These results are shown in FIG. The figure is a photograph showing the results of in situ hybridization showing the expression sites of the pov I gene, the pov II gene and the pov IV gene in germ cells. In the figure, each left figure shows the result of using a sense strand probe, and each right figure shows the result of using an antisense strand probe. As a control, a photograph showing the result of staining the section with hematoxylin-eosin (HE) is also shown in FIG. In the control, the right figure is an enlarged view of the left figure.

  As shown in the figure, it was confirmed that the pov I gene, the pov II gene and the pov IV gene were specifically expressed in germ cells.

[Example 2]
(1) Inhibition of increase in germ cells By suppressing RNA expression of the pov I gene by RNA interference, increase in germ cells was suppressed.

Using the pov I gene as a template, dsRNA was prepared using MEGAscript (trademark) kit (Ambion). The dsRNA is a polynucleotide whose sense strand is composed of a base sequence represented by SEQ ID NO: 7, and its antisense strand is a polynucleotide composed of a complementary sequence of the sense strand. The dsRNA was dispersed in a soaking buffer at 400 μg / ml. This was designated as an RNAi reagent. The composition of the soaking buffer (pH 8) was Mg ²⁺ free, 530.2 mmol / L NaCl, 10.0 mmol / L KCl, 9.2 mmol / L CaCl ₂ .H ₂ O, 10.0 mmol / L Hepes.

  The RNAi reagent (200 μL) was injected into a plurality of mature female pearl oyster gonads, and 3 hours later, an RNAi-treated female pearl oyster and an RNAi-untreated male pearl oyster were artificially mated. Subsequently, the obtained juvenile shellfish (333 pieces in total) were reared for one year (seawater, natural water temperature). The grown pearl oyster is referred to as RNAi-treated group (Example). A gonad was taken out from the grown pearl oyster, and a tissue section was prepared and HE-stained in the same manner as in Example 1 to confirm germ cells in the gonad. As a control, females not treated with RNAi and male pearl oysters not treated with RNAi were mated and reared in the same manner. The grown pearl oyster is called a control group (control). Then, as in Example 1, tissue sections were prepared, and germ cells were confirmed by HE staining. An example of the embodiment and an example of the control are shown in FIG. The figure is a photograph of a tissue section of a male pearl oyster gonad, the right is a photograph of an example in which RNAi treatment was performed (RNAi treatment group), and the left is a control of RNAi untreated control (control group). It is a photograph.

  In the control group not subjected to RNAi treatment, an increase in germ cells was confirmed as shown on the left of FIG. On the other hand, in the RNAi treatment group of the example, as shown on the right side of FIG. 2, there were individuals having very few germ cells and individuals in which no germ cells could be confirmed.

Furthermore, for a total of 182 pearl oysters bred in the same manner as described above, the gonad index (GSI) represented by the following formula was calculated in May, which is the first spawning season from the start of breeding. It can be determined that the larger the value of GSI, the more germ cells, and the smaller the value, the fewer germ cells. The individual distribution belonging to each GSI is shown in the graph of FIG. In the figure, A shows the result of Control 2-1, and B shows the result of Example 2. “˜0.16” is less than 0.16, “0.18” is from 0.16 to less than 0.18, “0.2” is from 0.2 to less than 0.22, and “0.22” is 0.22 or more and less than 0.24, “0.24” is 0.24 or more and less than 0.26, “0.26” is 0.26 or more and less than 0.28, and “0.28” is 0.28 or more. Is the result of
GSI = 100 × (gonad weight / body weight)

  As shown in FIG. 3, in the control, it was found that nearly 94% showed GSI of 0.26 or more, and the majority was a population with a large number of germ cells. On the other hand, in the Examples, the number of individuals distributed in a low GSI increased compared to the control, and nearly 89% were individuals showing GSI less than 0.26. From this result, it was found that the number of germ cells in the gonad can be reduced by performing RNAi treatment.

(2) Suppression of Maturation In the same manner as in (1) above, mature female pearl oysters were treated with RNAi and artificially mated with RNAi-untreated male pearl oysters, and the resulting nymphs were bred. The gonads of pearl oysters bred for 1 year were observed for tissue, and the maturity was evaluated according to the following evaluation criteria from the number of germ cells and the presence or absence of sperm (Example). And the ratio (%) of the individual which belongs to each evaluation level in the whole pearl oyster was calculated | required. In addition, the same evaluation was performed on the pearl oysters that were artificially mated with female and male pearl oysters that were not treated with RNAi. These results are shown in Table 1 below.
(Evaluation criteria)
1: Individuals whose germ cells cannot be confirmed 2: Individuals whose germ cells can be confirmed but few in number and whose sperm cannot be confirmed 3: Individuals whose germ cells can be confirmed and large in number but whose sperm cannot be confirmed 4: Those whose germ cells can be confirmed Individuals who can confirm sperm

  As shown in the above table, it was confirmed that the maturity of germ cells was suppressed in the Examples as compared with the control.

[Example 3]
(1) Suppression of increase in germ cells Suppression of germ cells was suppressed by suppressing mRNA expression of pov I gene, pov II gene and pov IV gene by RNA interference.

  An RNAi reagent was prepared in the same manner as in Example 2 except that the pov I gene, the pov II gene, and the pov IV gene were used as templates. The dsRNA for the pov II gene was a polynucleotide whose sense strand was represented by SEQ ID NO: 8, and its antisense strand was a polynucleotide that was a complementary sequence to the sense strand. The dsRNA required for the pov IV gene was a polynucleotide whose sense strand was composed of the base sequence represented by SEQ ID NO: 9, and whose antisense strand was a polynucleotide composed of a complementary sequence of the sense strand.

  A plurality of mature female pearl oysters were injected with 200 μL of the RNAi reagent, and 4 hours later, the female pearl oysters were artificially crossed with RNAi-untreated male pearl oysters. The obtained juveniles were reared for one year in the same manner as in Example 2, and germ cell confirmation and GSI calculation were performed by HE staining. In addition, 104 juveniles obtained by mating with female pearl oysters treated with RNAi reagent for pov I gene, 185 juveniles obtained by mating female pearl oysters treated with RNAi reagent for pov II gene 109 larvae obtained by mating female pearl oysters treated with the RNAi reagent for the pov IV gene, and 80 larvae obtained by mating female pearl oysters not treated with RNAi.

  The results of HE staining are shown in FIG. (A)-(C) are photographs of tissue sections of gonad gonads treated with RNAi reagent for pov I gene, RNAi reagent for pov II gene, and RNAi reagent for pov IV gene, respectively. . In each (A)-(C), the upper panel shows the results for male pearl oysters and the lower panel shows the results for female pearl oysters. In each of (A) to (C), the left panel shows the results of the RNAi-untreated control, and the middle and right panels show the results of the RNAi-treated examples. Moreover, the bar in each figure shows 10 micrometers in length.

  As shown in the left panel of the panels (A) to (C), in the RNAi-untreated control, an increase in male pearl oyster spermatogonia and an increase in female pearl oyster ovary cells were observed. On the other hand, as shown in the middle and right panels of FIGS. (A) to (C), in the examples treated with RNAi, male pearl oyster spermatogonia and female pearl oyster ovary cells were very different. There were few individuals or individuals that could not be confirmed at all.

  The result of GSI is shown in FIG. (A) to (D) show the results of the gonad index of the pearl oyster treated with the control, the RNAi reagent for the pov I gene, the RNAi reagent for the pov II gene, and the RNAi reagent for the pov IV gene, respectively. It is a graph to show. It is a photograph of the tissue section of the gonad. In the figure, “0.001” is less than 0.001, “0.003” is 0.003 or more and less than 0.005, “0.005” is 0.005 or more and less than 0.007, “0.007”. Is 0.007 or more and less than 0.009, “0.009” is 0.009 or more and less than 0.01, “0.01” is 0.01 or more and less than 0.03, and “0.03” is 0.03 or more. Less than 0.05, “0.05” is 0.05 or more and less than 0.07, “0.07” is 0.07 or more and less than 0.09, “0.09” is 0.09 or more and less than 0.1, “0.1” is a result of 0.1 or more and less than 0.13, and “0.13” is a result of 0.13 or more.

  As shown in FIG. 6A, more than half of the RNAi-untreated controls showed GSI of 0.07 or more. On the other hand, as shown in FIGS. 7B to 7D, in the RNAi-treated examples, the number of individuals distributed in a lower GSI increased compared to the control, and more than half of the individuals were GSI 0. It was an individual showing less than .07. From this result, it was found that the number of germ cells in the gonad can be reduced by performing RNAi treatment.

  Furthermore, total RNA was prepared from pearl oysters raised for one year, and the pov I gene, the pov II gene or the pov IV gene was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) method. These results are shown in FIG. The figure is an electrophoretogram obtained by analyzing the RT-PCR amplification product, and (A), (B), and (C) show the amplification results of the pov I gene, the pov II gene, and the pov IV gene, respectively. The left lane shows the results of the RNAi-untreated control, and the other lanes show the results of the examples treated with RNAi.

  As shown in FIGS. 9A to 9C, amplification of the pov I gene, the pov II gene, and the pov IV gene was confirmed in the RNAi untreated control. On the other hand, in the examples treated with RNAi, amplification of the pov I gene, the pov II gene and the pov IV gene could not be confirmed. From these results, it was found that the expression of the pov I gene, the pov II gene and the pov IV gene was effectively suppressed by the RNAi treatment.

  As described above, since the novel pov gene of the present invention is involved in germline development, it is possible to suppress germline development by suppressing the expression of this pov gene. Moreover, according to such a method, for example, the pearl oyster (sterile pearl oyster) in which the number of germ cells is reduced or the germ cell maturation is suppressed can be obtained. If such a pearl oyster is used as a mother shell in pearl culture, for example, the suppression process and the egg removal process in the conventional method are not required, so that the pearl culture can be performed more easily.

Claims (21)

A germline-related developmental gene of the pearl oyster, characterized by comprising the base sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. A method for suppressing the germline development of pearl oysters,
A developmental suppression method comprising suppressing germline development by suppressing the expression of RNA from the germline development-related gene or partial sequence thereof according to claim 1. Suppression of RNA expression from the germline development-related gene or a partial sequence thereof,
The method of suppressing development according to claim 2, which is degradation of RNA transcribed from the germline development-related gene or a partial sequence thereof, or inhibition of transcription of RNA from the germline development-related gene or a partial sequence thereof. . At least one of the pearl oyster germ cell and fertilized egg is siRNA, a siRNA precursor that releases siRNA by being degraded by ribonuclease, a miRNA, a miRNA precursor that releases miRNA by being degraded by ribonuclease, an antisense, Introducing at least one nucleic acid selected from the group consisting of ribozymes and expression vectors expressing them, to degrade RNA transcribed from the germline development-related gene or a partial sequence thereof, or the germline The method for suppressing development according to claim 3, wherein transcription of RNA from a development-related gene or a partial sequence thereof is inhibited. The siRNA precursor is a double-stranded RNA;
The method for inhibiting development according to claim 4, wherein the antisense strand of the double-stranded RNA is a polynucleotide comprising the following base sequence (a1) or (a2).
(A1) In the base sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, a base sequence complementary to a base sequence consisting of consecutive 1,080 to 1,260 bases (a2) SEQ ID NO: 7, SEQ ID NO: Base sequence complementary to the base sequence represented by any of 8 and SEQ ID NO: 9 Bringing the liquid containing the nucleic acid into contact with at least one of the germ cells and the fertilized egg, and introducing the nucleic acid into at least one of the germ cells and the fertilized egg by the uptake ability of at least one of the germ cells and the fertilized egg. The method for inhibiting development according to claim 4 or 5. The developmental suppression according to claim 6, wherein the method of contacting at least one of the germ cell and the fertilized egg with the liquid is immersion of the gonad or germ cell in the liquid, or injection of the liquid into the gonad. Method. A developmental inhibitor for use in the method of inhibiting germline development of pearl oysters according to any one of claims 2 to 7,
A development inhibitor comprising at least one nucleic acid selected from the group consisting of the following (x1) to (x3).
(X1) Decomposing RNA transcribed from the germline development-related gene according to claim 1 or a partial sequence thereof, degrading with siRNA or ribonuclease, and degrading with siRNA precursor, miRNA or ribonuclease that liberates siRNA. At least one nucleic acid (x2) selected from the group consisting of a miRNA precursor, an antisense and a ribozyme that liberates miRNA by transcription of RNA from the germline development-related gene or partial sequence thereof according to claim 1 Inhibiting, selected from the group consisting of siRNA, a siRNA precursor that liberates siRNA by being degraded by ribonuclease, a miRNA, a miRNA precursor that liberates miRNA by being degraded by ribonuclease, an antisense, and a ribozyme An expression vector that expresses at least one of the at least one nucleic acid of the nucleic acid (x3) wherein (x1) and (x2) was The siRNA precursor is a double-stranded RNA;
The development inhibitor of Claim 8 whose antisense strand of the said double stranded RNA is a polynucleotide which consists of a base sequence of the following (a1) or (a2).
(A1) In the base sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, a base sequence complementary to a base sequence consisting of consecutive 1,080 to 1,260 bases (a2) SEQ ID NO: 7, SEQ ID NO: Base sequence complementary to the base sequence represented by any of 8 and SEQ ID NO: 9 A method for producing pearl oysters in which germline development is suppressed,
The manufacturing method characterized by including the following (A1) and (A2) process.
(A1) A step of introducing the development inhibitor of claim 8 or 9 into at least one germ cell of a pearl oyster egg or sperm (A2) a step of mating the egg and sperm to obtain a fertilized egg The manufacturing method according to claim 10, wherein in the step (A1), the germ cell is brought into contact with a liquid containing the development inhibitor, and the development inhibitor is introduced into the germ cell by the ability of the germ cell to be taken up. . The manufacturing method according to claim 11, wherein the method of contacting the germ cell and the liquid is immersion of the gonad or germ cell in the liquid or injection of the liquid into the gonad. The production method according to claim 11, wherein in the step (A1), the development inhibitor is injected into the germ cells. The manufacturing method of Claim 10 which replaces with the said (A1) and (A2) process, and has the following (B1) and (B2) process.
(B1) a step of mating a pearl oyster egg and a sperm to obtain a fertilized egg (B2) a step of introducing the development inhibitor of claim 7 or 8 into the fertilized egg The manufacturing method according to claim 14, wherein, in the step (B2), the fertilized egg is contacted with a liquid containing the development inhibitor, and the development inhibitor is introduced into the fertilized egg by the uptake ability of the fertilized egg. . The manufacturing method according to claim 15, wherein the method of contacting the fertilized egg with the liquid is immersion of the fertilized egg in the liquid. The production method according to claim 14, wherein in the step (B2), the development inhibitor is injected into the fertilized egg. Furthermore, the manufacturing method as described in any one of Claims 10-17 which has a (C) process.
(C) After the step (A2) or the step (B2), a step of growing the fertilized egg A pearl manufacturing method for manufacturing a pearl by introducing a nucleus into a pearl oyster,
A method for producing pearls comprising a step of producing pearl oysters by the production method according to any one of claims 10 to 18 prior to introduction of the nucleus. A method for sterilizing pearl oysters,
A method for sterilization comprising the step of suppressing the development of the germline of the pearl oyster by the method for inhibiting development according to any one of claims 2 to 7. A sterilizing agent for use in the method for sterilizing pearl oysters according to claim 20,
A sterilizing agent comprising the development inhibitor according to claim 8 or 9.