WO2016159110A1 - Binary gene expression system - Google Patents

Abstract

The present invention addresses the problem of developing and providing a novel gene expression system with which it is possible to improve the expression efficiency of a gene of interest and to individually control the expression of multiple introduced genes of interest in a desired timeframe and/or tissue, the gene expression system also having low cytotoxicity for the host. Provided is a binary gene expression system comprising: a first expression unit that includes a promoter, a TAL region, and a transcription activity region; and a second expression unit that includes a TAL recognition sequence and a gene of interest or fragment thereof placed under the control of the TAL recognition sequence.

Description

Binary gene expression system

The present invention relates to a binary gene expression system, a transformant containing the same, and a method for producing a gene expression enhanced individual obtained by mating the transformant.

In recent years, research on production of useful proteins using genetically modified silkworms (Bombyx 、 mori), gene function analysis, and creation of medical model silkworms has progressed dramatically. For example, the silk gland of silkworm has the ability to synthesize a large amount of protein in a short period of time, and therefore, genetically modified silkworms using the properties are useful proteins such as protein for reagents, reagents for clinical testing, and proteins for cosmetics. It has already been put into practical use as a production system. In addition, the use of genetically modified silkworms modified to accept pheromones of other species for cranial nerve research has been actively used as experimental animals that combine the characteristics of silkworms with molecular biological techniques. (Non-Patent Document 1).

However, in order to further improve the expression efficiency of the target gene or to control the expression of multiple target genes individually in time or space, it is necessary to establish a more sophisticated gene expression system. Yes.

Conventionally, the GAL4 / UAS system has been mainly used to express a target gene using transgenic silkworms (Non-patent Document 2). This system consists of two systems, GAL4 and UAS. The GAL4 strain has a GAL4 gene encoding a transcription factor placed under the control of an appropriate enhancer or promoter, and the UAS strain has a UAS that is a recognition sequence for GAL4 and a target gene placed under its control. The UAS line does not express the target gene by itself, but when one individual includes these two lines by mating, etc., the transcription factor GAL4 is expressed in cells in which the enhancer or promoter of the GAL4 line is activated, The target gene is expressed by binding to UAS. By improving this GAL4 / UAS system, it is now possible to improve the expression efficiency of the target gene to nearly 10 times (Non-patent Document 3). In this system, the GAL4 strains that have been constructed so far, which are expressed in various time-specific or tissue-specific ways, and the UAS strains that express various genes are accumulated as genetic assets. Even in this case, the other system has an advantage that the existing system can be used as it is. However, in order to further improve the expression efficiency in the GAL4 / UAS system, it is necessary to introduce a new improvement completely different from the conventional method. In addition, when multiple genes are introduced into this system, since all genes are expressed only in the same tissue at the same time, the expression time and location cannot be controlled by individual genes. The strain has the problem that it is cytotoxic to the host.

The present invention improves the expression efficiency of a target gene, can control the expression of a plurality of introduced genes individually at a desired specific time and / or tissue, and is a new gene expression system with low cytotoxicity to a host The issue is to develop and provide

In order to solve the above-mentioned problems, the present inventors focused on TALEN (Transcription Activator-Like Effector Nuclease). The technology of TALEN (Cermak T., et al., 2011, Nucleic Acids Res 39: e82, patent publication 2013-513389, patent publication 2012-514976), developed as a genome editing system, can be applied to silkworms. (Daimon T., et al., 2014, Develop. Growth Differ, 56: 14-25). In recent years, TALEN has been used not only for genome editing but also for transcriptional activation and transcriptional repression of arbitrary DNA sequences using the DNA sequence binding ability of TALE (TAL Effector). For example, transcriptional activation using TALE (TALE activator) has been extensively studied using mammalian cells and plant cells (Morbitzer R., et al., 2010, PNAS, 107 (50): 21617- 21622, Zhang F., et al., 2011, Nature biotech, 29 (2): 149-153, Maeder ML, et al., 2013, Nature Methods, 10 (3): 243-245, Crocker J.et al ., 2013, Nature Methods, doi: 10.1038 / nmeth.2543), mainly used to control differentiation by controlling the expression of endogenous genes and to treat diseases. It has been reported that TALE 例 activator changes transcription activation ability depending on the position of target sequence and epigenetic state of chromosome, and transcription activation hardly occurs.

The present inventors have developed a TAL-GAL4 / UAS system that recognizes UAS using the TALE DNA binding domain TAL (Transcription Activator-Like) instead of the GAL4 DNA binding domain in the conventional GAL4 / UAS system. . However, this system has a problem that not only the transcription activity efficiency can be improved but also the transcription activity is lower than that of GAL4. Therefore, as a result of various improvements, a first expression unit in which a TAL domain having a base sequence of a specific length as a target base sequence and a transcriptional activity domain comprising a plurality of transcriptional activity units consisting of 50 to 150 amino acids, and By using a second expression unit that contains a TAL recognition sequence that includes a repetitive sequence in which multiple target base sequences are linked, the expression efficiency of the target gene is maximized in cultured cell systems compared to the conventional GAL4 / UAS system. We succeeded in increasing it by about 80 times, and up to about 6 times in transgenic silkworm strains. This invention is based on the said knowledge, Comprising: The following are provided.

(1) A binary gene expression system comprising a first expression unit and a second expression unit, wherein the first expression unit is a TAL region comprising a promoter and a base sequence encoding a TAL domain arranged under the control of the promoter. And a transcription active region consisting of a base sequence encoding a transcription active domain in a state capable of expression, wherein the TAL domain recognizes the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as a target base sequence An N-terminal site and a TAL-DNA base sequence recognition site, wherein, in the target base sequence, the N-terminal site is a 5′-terminal t, and the TAL-DNA base sequence recognition site is a main sequence following the t. Recognize each target base sequence, and the TAL-DNA base sequence recognition site is the amino acid sequence represented by SEQ ID NO: 3 for each base constituting the main target base sequence The transcriptional activity domain comprises a sequence in which 2 to 10 transcriptional activity units consisting of 50 to 150 amino acids are linked, and the second expression unit comprises a TAL recognition sequence and a gene of interest arranged under its control. Alternatively, the system comprising a fragment thereof, wherein the TAL recognition sequence consists of a base sequence containing 5 to 25 target base sequences.
(2) The binary gene expression system according to (1), wherein the main target base sequence consists of any one of the following base sequences (a) to (c):

(A) the base sequence represented by SEQ ID NO: 4,
(B) a base sequence represented by SEQ ID NO: 5; and (c) a base sequence in which 1 to 5 bases of the base sequence represented by SEQ ID NO: 6 in SEQ ID NO: 4 or 5 are substituted (3) represented by SEQ ID NO: 4 The binary gene expression system according to (2), wherein the base sequence is the base sequence represented by SEQ ID NO: 7.
(4) The binary gene expression system according to (2), wherein the base sequence represented by SEQ ID NO: 5 is the base sequence represented by SEQ ID NO: 8.
(5) The binary gene expression system according to any one of (1) to (4), wherein the transcriptional activity unit is ARII or VP16.
(6) The binary gene expression system according to any one of (3) to (5), wherein the TAL recognition sequence is composed of a repetitive sequence in which 5 to 25 nucleotide sequences represented by SEQ ID NO: 9 including the target nucleotide sequence are linked. .
(7) A transformant comprising the first expression unit of the binary gene expression system according to any one of (1) to (6).
(8) A transformant comprising the second expression unit of the binary gene expression system according to any one of (1) to (5), excluding the second expression unit of the binary gene expression system according to (6).
(9) A transformant comprising the first expression unit and the second expression unit in the binary gene expression system according to any one of (1) to (6).
(10) The transformant according to any one of (7) to (9), wherein the transformant is a Lepidoptera insect.
(11) The transformant according to (10), wherein the Lepidoptera insect is a silkworm.
(12) A step of mating the transformant having the first expression unit according to (7) and the transformant having the second expression unit according to (8), and the first and second after the mating step A method for producing an individual having enhanced expression of a target gene or a fragment thereof, comprising a step of selecting an individual having an expression unit.

This specification includes the disclosure of Japanese Patent Application No. 2015-072017, which is the basis of the priority of this application.

The binary gene expression system of the present invention can express a target gene in an amount exceeding the conventional GAL4 / UAS system.

In an individual having a plurality of two expression units of the binary gene expression system of the present invention, the expression of each target gene in a different set can be controlled in a time-specific and / or tissue-specific manner, respectively.

The first expression unit constituting the binary gene expression system of the present invention has an effect of low cytotoxicity to the host as compared with the GAL strain of the GAL4 / UAS system.

The binary gene expression system of the present invention can individually maintain and manage lines containing each of the first expression unit and the second expression unit.

The binary gene expression system of the present invention may be produced by using the GAL4 / UAS system and the accumulated GAL4 line or UAS line as it is.

It is a conceptual diagram of the 1st expression unit and the 2nd expression unit which comprise the binary gene expression system of this invention. A shows the first expression unit, and B shows the second expression unit. In the figure, the N-terminal region in the TAL region of the first expression unit is a region encoding the N-terminal site of the TAL domain, and the TAL-DNA base sequence recognition region is a region encoding the TAL-DNA base sequence recognition site of the TAL domain. The C-terminal region is a region encoding the C-terminal region of the TAL domain. The transcription active unit region in the transcription active region is a region encoding the transcription active unit of the transcription active domain. In the first expression unit of the figure, a transcription active region including four transcription active unit regions is exemplified. In the second expression unit, a TAL recognition sequence including five target base sequences is exemplified. In the binary gene expression system of the present invention used in Example 1, a part of the TAL recognition sequence (base sequence in the figure) contained in the second expression unit and the TAL domain encoded by the first expression unit are recognized. The range of the target base sequence (the range indicated by three bars in the base sequence in the figure) is shown. In Example 1, the TAL recognition sequence is composed of a sequence in which a unit containing UAS, which is a GAL4 recognition sequence, is repeated 5 times. The TAL domain encoded in the first expression unit of Example 1 recognizes UASTAL1, which recognizes 19 bases including the above unit in the TAL recognition sequence, and 20 bases from T (thymine) at the 5 ′ end most in the TAL recognition sequence. UASTAL2 and UASTAL3 that recognizes 14 bases with 6 bases shortened on the 3 ′ end of the UASTAL2 recognition sequence. The binary gene expression system of the present invention comprises a first expression unit comprising a TAL region (UASTAL) that recognizes various regions of UAS and a transcriptional active region linked downstream thereof, and UAS and a luciferase gene linked downstream thereof It is a figure which shows the result of the luciferase assay in the silkworm cultured cell which introduce | transduced the 2nd expression unit. In this figure, the expression level of luciferase in silkworm cultured cells when the first expression unit having the control GAL4 is used is 1, and the relative expression level is shown. In the binary gene expression system of this invention, it is the figure which showed the relationship between the number of the transcriptional activity unit area | regions of a 1st expression unit, and the expression level of a target gene. ARII or VP16 was used as the transcription activity unit, and the number of unit regions in the transcription activity region was 1-6. In this figure, the expression level of luciferase in silkworm cultured cells when the first expression unit having the control GAL4 is used is 1, and the relative expression level is shown. In the binary gene expression system of this invention, it is the figure which showed the relationship between the base number of the target base sequence of a 1st expression unit, the base number of a main target base sequence, and the expression level of a target gene. It is a figure which shows the specificity with respect to the target base sequence of the TAL domain encoded by the 1st expression unit in the binary gene expression system of this invention. The upper figure shows the target sequence recognized by the first expression unit, and the mutant UAS in which base substitution is introduced into the base sequence of UAS and UAS of the TAL recognition sequence in the second expression unit. In the TAL recognition sequence of the second expression unit, capital letters indicate the target base sequence of the first expression unit, and asterisks indicate the positions of the substituted bases. The lower figure shows the expression level of luciferase in cultured silkworm cells when the first and second expression units shown in the upper figure are combined. “-” Indicates that the first expression unit is not introduced. It is a figure which shows the expression efficiency of EGFP when the middle silk gland specific binary gene expression system of this invention is introduce | transduced into a silkworm. It is a figure which shows the cytotoxicity by a 1st expression unit in the transgenic silkworm which has a 1st expression unit of the binary gene expression system of this invention. A shows the layer thickness of the transgenic silkworm, B shows the yield rate, and C shows the result of the conversion rate. As a control, a first expression unit containing GAL4 in a conventional GAL4 / UAS system known to exhibit cytotoxicity was used. The numerical value shown above each bar indicates the number of samples (N). Similarly to FIG. 8, the result of SDS-PAGE which analyzed the protein derived from the middle silk gland of the transgenic silkworm having the first expression unit of the binary gene expression system of the present invention is shown. Ser1 indicates the position of sericin 1 protein, FibH indicates the position of fibroin H protein, and Ser3 indicates the position of sericin 3 protein. It is a figure which shows the silk gland of the transgenic silkworm which has a binary gene expression system specific to each of the middle silk gland and the posterior silk gland. A to C are images of the silk gland of the same field, D to F are enlarged images within the white frame of the single star in Fig. A, and G to I are within the white frame of the double star in Fig. A. An enlarged view is shown. It is the result of RT-PCR which shows the expression of EGFP and DsRed in the silk gland of the transgenic silkworm which has a binary gene expression system specific to each of the middle silk gland and the posterior silk gland. NC is the RT-PCR product from the negative control, lane 1 is the RT-PCR product from the middle silk gland, lane 2 is the RT-PCR product from the posterior silk gland, and lane 3 is from the middle and posterior silk gland. RT-PCR product is shown. It is a figure which shows the expression efficiency of EGFP when the rear part silk gland specific binary gene expression system of this invention is introduce | transduced into a silkworm. 1-4 are different strains with the same posterior silk gland specific binary gene expression system obtained independently in the production of transgenic silkworms.

1. Binary gene expression system 1-1. Overview A first aspect of the present invention is a binary gene expression system.

A “binary gene expression system” is a set of gene expression units composed of two expression units. The binary gene expression system of the present invention induces the target expression encoded by the second expression unit by expressing the transcriptional activation domain encoded by the first expression unit in a host cell having two expression units, You can also strengthen it. In addition, there is an advantage that even if the first expression unit is introduced into a cell, it is difficult to cause cytotoxicity.

1-2. Configuration The binary gene expression system of the present invention comprises two units, a first expression unit and a second expression unit. Each expression unit includes a target gene or an active domain thereof (hereinafter referred to as “target gene or the like”) in a state where it can be expressed, and includes an expression vector that can control the expression of the gene or the like. The configuration of each unit will be described below.

In this specification, the “expressible state” means that a target gene or the like is placed under the control of a promoter in a base sequence constituting a unit and can be expressed according to the activity of the promoter. “Under the control of a promoter” is in principle downstream, that is, at the 3 ′ end. Therefore, “arranged under the control of a promoter” means that the target gene or the like is linked directly downstream of the promoter or indirectly with an intervening sequence such as a spacer in between.

As used herein, “expression vector” refers to an expression unit that contains a target gene in a state where it can be expressed and can control the expression of the gene or the like. Hereinafter, the configuration of each expression unit will be described.

1-2-1. First Expression Unit As shown in FIG. 1A, the first expression unit contains a promoter, a TAL region and a transcriptional active region arranged under its control, and a mother nucleus vector as essential components. Further, if necessary, selective components such as marker genes, terminators, enhancers, 5′UTR and 3′UTR, insulators and transposon inverted terminal repeats can also be included. Hereinafter, each component will be described.

(1) TAL region and transcriptional active region The “TAL region and transcriptional active region” is a chimeric gene encoding a chimeric protein (fusion protein) comprising a TAL domain and a transcriptional active domain linked to the C-terminal side thereof.

A. TAL region In this specification, the “TAL (Transcription Activator-Like) region” is a region consisting of a base sequence encoding a TAL domain.

In this specification, “TAL domain” means DNA that lacks the transcriptional active region (effector domain) in TAL (Transcription Activator-Like) effector protein (Cermak T, et al., 2011, Nucleic Acids Res 39: e82) An amino acid site consisting only of the binding domain. The TAL domain is composed of a TAL-DNA base sequence recognition site, an N-terminal site located on the N-terminal side, and a C-terminal site located on the C-terminal side.

In this specification, the “TAL-DNA base sequence recognition site” is a site that directly recognizes a main target base sequence described later. The TAL-DNA base sequence recognition site recognizes each base of the main target base sequence in units of an amino acid sequence composed of 34 amino acids shown in SEQ ID NO: 3, respectively. The unit consists of a pair of amino acid residues at the 12th and 13th positions from the N-terminal side, and each of the four bases constituting the DNA (A: adenine, G: guanine, C: cytosine, T: thymine) Can be specifically recognized. Specifically, when the amino acid residues at positions 12 and 13 recognize A (adenine), they are N and I or N and N, respectively. When G (guanine) is recognized, N And N, when recognizing C (cytosine), they are H and D, respectively. When recognizing T (thymine), they are N and G, respectively. Therefore, the TAL-DNA base sequence recognition site repeatedly contains the unit according to the base length of the target base sequence.

In the present specification, the “target base sequence” is a base sequence recognized by the TAL domain, and a plurality of essential constituent elements are included in the second expression unit described later. The structure of the target base sequence is, as shown in SEQ ID NO: 1 or 2, t (thymine) arranged on the 5 ′ end side, followed by the main target base sequence, 14 bases in total or 15 It consists of a base sequence.

In this specification, the “main target base sequence” is a base sequence other than t (thymine) at the 5 ′ end contained in the target base sequence, and consists of an arbitrary base sequence of 13 bases or 14 bases. Therefore, an arbitrary DNA sequence can be used as a target base sequence by designing this main target base sequence into a desired base sequence. Specific examples of the main target base sequence include, for example, the base sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5. More specifically, in the GAL4-UAS system, it is a part of a GAL4 recognition sequence comprising UAS (Upstream Activating Sequence) (SEQ ID NO: 35) and consisting of a base sequence represented by SEQ ID NO: 9, An example is the base sequence represented by SEQ ID NO: 8. Furthermore, even a base sequence in which 1, 2, 3, 4, or 5 bases are substituted in the base sequence represented by SEQ ID NO: 6 included in the base sequence represented by SEQ ID NO: 4 or 5 Good.

As described above, the TAL-DNA base sequence recognition site recognizes each base constituting the main target base sequence in units of an amino acid sequence composed of 34 amino acids shown in SEQ ID NO: 3. Therefore, the TAL-DNA base sequence recognition site has a structure in which the unit consisting of the 34 amino acids is repeated 13 or 14 times. As a specific example of the base sequence encoding the TAL-DNA base sequence recognition site, for example, when the base sequence represented by SEQ ID NO: 7 is the main target base sequence, the base sequence encoding the TAL-DNA base sequence recognition site is: The nucleotide sequence is represented by SEQ ID NO: 10. When the base sequence represented by SEQ ID NO: 8 is used as the main target base sequence, the base sequence encoding the TAL-DNA base sequence recognition site is the base sequence represented by SEQ ID NO: 11.

In the present specification, the “N-terminal site” refers to a site involved in the recognition of t (thymine) located on the 5′-terminal side of the target base sequence, including a TAL domain transport signal. The N-terminal site is composed of, for example, the amino acid sequence represented by SEQ ID NO: 12, and the base sequence encoding it includes, for example, the base sequence represented by SEQ ID NO: 13.

B. Transcription active region In this specification, the “transcription active region” is a region consisting of a base sequence encoding a transcription active domain.

In the present specification, the “transcription activation domain” is a functional domain of a transcription activation factor and has a function of activating expression of a target gene. The transcriptional activity domain in the present specification includes a transcriptional activity unit consisting of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, or 2 to 5, linked repeat sequences. Become.

In this specification, the “transcriptional activity unit” is the minimum unit constituting a transcriptional activity domain and consists of 50 to 150 amino acids. As a specific example of the transcriptional activity unit, it consists of the amino acid sequence represented by SEQ ID NO: 16, and is encoded by the base sequence represented by SEQ ID NO: 17. VP16 derived from herpes simplex virus, which consists of an amino acid sequence and is encoded by the base sequence represented by SEQ ID NO: 19, can be mentioned.

(2) Promoter “Promoter” is a gene expression regulatory region capable of controlling the expression of a gene (target gene) placed under its control. The “promoter” in the first expression unit can control the expression of a chimeric gene consisting of the TAL region and the transcriptional active region, which are target genes.

Promoters can be classified into ubiquitous promoters (systemic promoters) and site-specific promoters based on the expression location of the target gene. The ubiquitous promoter is a promoter that controls the expression of a target gene in the entire host individual. A site-specific promoter is a promoter that controls the expression of a target gene in a specific cell or tissue of a host. In the binary gene expression system of the present invention, by selecting a site-specific promoter specific to a cell or tissue as the promoter in the first expression unit, the expression of a target gene or the like is induced in a specific cell or tissue in the host. Can be enhanced. Therefore, regarding the expression location, the promoter in the first expression unit is not particularly limited, and may be appropriately selected according to the use.

Further, promoters are classified into constitutively active promoters, expression-inducible promoters, or time-specific active promoters based on the expression time of the target gene. A constitutively active promoter can constitutively express the gene of interest in the host cell. An expression-inducible promoter can induce the expression of a target gene in a host cell at any time. In addition, a time-specific active promoter can induce the expression of a target gene in a host cell only at a specific stage of development. Any promoter can be interpreted as an overexpression promoter because it can cause overexpression of the gene of interest in the host cell. Regarding the expression time, the promoter in the first expression unit is not particularly limited. What is necessary is just to select suitably according to a desired expression time in the cell to introduce | transduce.

In the first expression unit, the donor species from which the promoter is derived is not particularly limited as long as it is operable in the recipient host cell into which it is introduced. The term “operable” as used herein means that it can function as a promoter and can express a target gene or the like. That is, the promoter in the first expression unit is determined by the host cell to be introduced. Preferably, it is a promoter derived from a species belonging to the same classification as the host into which the first expression unit is introduced. Promoters derived from species belonging to the same family are more preferred, and promoters derived from species belonging to the same genus are more preferred. Most preferred is a promoter from the same species as the host. For example, when the host into which the first expression unit of the present invention is introduced is a Bombyx mori, the promoter used in the first expression unit is preferably a promoter from the Lepidoptera, Bombycidae. More preferably, it is derived from a species belonging to the same genus of Bombyx such as mulberry (Bombyx mandarina). In this case, the most preferred promoter is a promoter derived from the same kind of silkworm.

When silkworm is used as a host into which the first expression unit is introduced, specific examples of the constitutively active ubiquitous promoter include actin 3 promoter derived from actin 3 gene (A3 promoter: SEQ ID NO: 20), silkworm heat shock protein 90 (hsp90 ) Gene-derived heat shock protein 90 promoter (hsp90 promoter: SEQ ID NO: 21), silkworm elongation factor 1α (Elongation Factor-1α) gene-derived elongation factor 1 promoter (EF-1 promoter: SEQ ID NO: 22), and BmNPV (Bombyx and the first gene 1 promoter (ie-1 promoter: SEQ ID NO: 23) derived from the first gene 1 (ie-1: immediate-early gene 1) of mori nuclear polyhedrosis virus). Examples of the expression-inducing ubiquitous promoter include the heat shock protein 70 promoter derived from the heat shock protein 70 (hsp70) gene (hsp70 promoter: SEQ ID NO: 24). Furthermore, specific examples of site-specific promoters include a 3xP3 gene promoter (3xP3 promoter: SEQ ID NO: 25) that expresses eye-specific expression, and a sericin 3 gene promoter (Ser3) that specifically expresses in the front part of the middle silk gland. Promoter: SEQ ID NO: 26), promoter of sericin 1 gene expressing middle silk gland-specific expression (Ser1 promoter: SEQ ID NO: 27), promoter of fibroin H gene expressing posterior silk gland-specific expression (Fib H promoter: SEQ ID NO: 28) ), Promoter of fibroin L gene (Fib L promoter: SEQ ID NO: 29), promoter of p25 gene (p25 promoter: SEQ ID NO: 30), promoter of 30K gene for fat body-specific expression (30K promoter: SEQ ID NO: 31) And the promoter of the elav like gene that is specifically expressed in the testis (e lav like promoter: SEQ ID NO: 32) and the like. The cell or tissue specificity of a site-specific promoter depends in principle on the gene that the promoter was originally regulated. For example, in the case of the Ser1 promoter described above, the Ser1 gene is specifically expressed in the middle silk gland, and thus becomes a middle silk gland-specific promoter.

In addition to the silkworm examples described above, promoters known in the art can be used depending on the host into which the first expression unit is introduced. For example, if the host is E. coli, examples of operable promoters include lac, trp or tac promoters, or phage-derived T7, T3, SP6, PR or PL promoters. When the host is yeast, examples of operable promoters include a yeast glycolytic gene promoter, an alcohol dehydrogenase gene promoter, a TPI1 promoter, and an ADH2-4c promoter.

(3) Mother Nucleus Vector The “mother nucleus vector” is a base part of the first expression unit and has a configuration necessary for the first expression unit to function as a vector. The type of vector is not particularly limited. Examples include plasmid vectors, viral vectors, cosmids, bacmids, fosmids, BACs, YACs and the like. A plasmid vector or a viral vector is preferred. The mother nucleus vector may be appropriately selected according to the host into which the first expression unit is introduced. For example, when the host is E. coli, use plasmid vectors derived from E. coli such as pBI, pPZP, pSMA, pUC, pBR, and pBluescript (Agilent technologies), and λ phage vectors such as λgt11 and λZAP. can do. When the host is yeast, plasmid vectors such as YEp13, YEp24, and YCp50 can be used. When the host is an insect cell, an insect virus vector such as baculovirus or a bacmid can be used. Furthermore, when the host is a mammal such as a human, a known vector having a mother nucleus of a viral vector such as an adenovirus, a retrovirus, a lentivirus, an adeno-associated virus, or the like can be used. In addition, shuttle vectors that can be replicated in E. coli or yeast, vectors that can be homologously or non-homologously recombined in the chromosome, or various host-specific expression vectors that are commercially available from various manufacturers may be used.

(4) Labeling gene The “labeling gene” is a gene that encodes a labeling protein that is a selective component in the first expression unit and is also called a selection marker or a reporter protein. “Labeled protein” refers to a polypeptide that can determine the presence or absence of expression of a labeled gene based on its activity. The activity may be detected directly by the activity of the labeled protein itself or indirectly by a metabolite generated by the activity of the labeled protein such as a dye. Good. Detection can be biological detection (including detection by binding of peptides or nucleic acids such as antibodies and aptamers), chemical detection (including enzymatic reaction detection), physical detection (including behavioral analysis detection), or detection It can be performed by human sensory detection (including detection by sight, touch, smell, hearing, and taste). The marker gene is used for the purpose of discriminating the host cell or transformant carrying the first expression unit.

The type of labeled protein encoded by the labeled gene is not particularly limited as long as its activity can be detected by a method known in the art. Preferably, it is a labeled protein with low invasiveness to the transformant upon detection. For example, tag peptides, drug resistant proteins, chromoproteins, fluorescent proteins, photoproteins and the like can be mentioned.

A “tag peptide” is a short peptide consisting of several tens to several tens of amino acids that can label a protein, and is used for protein detection and purification. Usually, labeling is performed by linking a base sequence encoding a tag peptide to the 5 'end side or 3' end side of a gene encoding the protein to be labeled and expressing it as a chimeric protein with the tag peptide. Although various types of tag peptides have been developed in the art, any tag peptide may be used. Specific examples of the tag peptide include FLAG, HA, His, and myc.

“Drug-resistant protein” is a protein that imparts resistance to drugs such as antibiotics added to a medium or the like to cells, and many are enzymes. For example, β-lactamase that confers resistance to ampicillin, aminoglycoside 3 ′ phosphotransferase that confers resistance to kanamycin, tetracycline efflux transporter that confers resistance to tetracycline, chloramphenicol Examples include CAT (chloramphenicol acetyltransferase) that imparts resistance.

“Chromoprotein” is a protein that is involved in pigment biosynthesis, or a protein that enables chemical detection of a transformant with a pigment by providing a substrate. The “dye” here is a low molecular compound or peptide capable of imparting a dye to a transformant, and the kind thereof is not limited. Examples thereof include β-galactosidase (LacZ), β-glucuronidase (GUS), melanin pigment synthesis protein, omochrome pigment, or pteridine pigment. Moreover, the pigment | dye which appears as an external color of an individual | organism | solid, for example, a melanin type pigment | dye (a dopamine melanin is included), an omochrome type pigment | dye, or a pteridine type pigment | dye may be sufficient.

“Fluorescent protein” refers to a protein that emits fluorescence of a specific wavelength when irradiated with excitation light of a specific wavelength. Either a natural type or a non-natural type may be used. Further, the excitation wavelength and the fluorescence wavelength are not particularly limited. Specifically, for example, CFP, RFP, DsRed (including derivatives such as 3xP3-DsRed), YFP, PE, PerCP, APC, GFP (including derivatives such as EGFP and 3xP3-EGFP), etc. It is done.

As used herein, “photoprotein” refers to a substrate protein that can emit light without the need for excitation light or an enzyme that catalyzes the light emission of the substrate protein. For example, luciferin or aequorin as a substrate protein and luciferase as an enzyme can be mentioned.

As used herein, “externally secreted protein” refers to a protein that is secreted extracellularly or externally, and includes exocrine enzymes, fiber proteins such as fibroin, and sericin. Exocrine enzymes include digestive enzymes in addition to enzymes that contribute to the degradation or inactivation of drugs such as blasticidin and impart drug resistance to the host.

The marker gene is arranged in a state that can be expressed downstream of the promoter in the first and second expression units.

In the first expression unit, the marker gene is arranged in a state where it can be expressed in a state of being linked upstream or downstream of the target gene or independently of the target gene.

(5) Terminator The “terminator” is a base sequence located at the 3 ′ end side of the chimeric gene consisting of the TAL region and the transcriptional active region, preferably downstream of the stop codon, and the terminator of the chimeric gene expressed by the promoter A sequence that can terminate transcription. The type of terminator is not particularly limited. Preferably, it is a terminator derived from the same species as the promoter. For example, in the case of E. coli, lipopolyprotein lpp 3 ′ terminator, trp operon terminator, amyB terminator, ADH1 gene terminator, and the like can be used. In the case of an insect such as a silkworm, an hsp70 terminator comprising the base sequence represented by SEQ ID NO: 33, an SV40 terminator comprising the base sequence represented by SEQ ID NO: 34, etc. can be used. A terminator paired with the promoter on the genome in the control of single gene expression is particularly preferred.

(6) 5'UTR and 3'UTR
“5′UTR (5′untranslated region)” and “3′UTR (3′untranslated region)” are polynucleotides composed of untranslated regions that do not themselves encode amino acids or functional nucleic acids. The base sequence that constitutes each UTR is not limited. In the first expression unit, 5′UTR is arranged upstream (5 ′ end side) of the start codon of the chimeric gene, and 3′UTR is arranged downstream (3 ′ end side) of the stop codon of the chimeric gene. . The 3 ′ UTR can contain a poly A signal.

(7) Enhancer An “enhancer” is a selective component in the first expression unit, and consists of a base sequence that can enhance the expression efficiency of a gene or a fragment thereof in a vector. The kind and base sequence are not particularly limited.

(8) Insulator “Insulator” is a selective component of the first expression unit, and stably controls transcription of the gene sandwiched between the sequences without being affected by the chromatin of surrounding chromosomes. It is a possible nucleotide sequence. Examples include the cHS4 sequence of chicken and the gypsy sequence of Drosophila.

(9) Inverted terminal repeat sequence of transposon “Inverted terminal repeat sequences (ITRs) of transposon” are selection structures that can be included when the first expression unit is an expression vector capable of homologous recombination. Is an element. Inverted terminal repeats are usually used in pairs. As a specific example of a transposon, piggyBac, mariner, minos, etc. can be used if the host is an insect (Shimizu, K. et al., 2000, Insect Mol. Biol., 9, 277-281; Wang W. et al., 2000, Insect Mol Biol 9 (2): 145-55).

1-2-2. Second Expression Unit As shown in FIG. 1B, the second expression unit includes a TAL recognition sequence and a target gene arranged under the control thereof, and additionally includes a mother nucleus vector as an essential component. Further, if necessary, selective components such as marker genes, terminators, enhancers, 5′UTR and 3′UTR, insulators and transposon inverted terminal repeats can also be included. Hereinafter, the TAL recognition sequence and the target gene will be described. The mother nucleus vector and the selective components included in the second expression unit are the same as the corresponding components described in the first expression unit, and thus the description thereof is omitted here.

(1) TAL recognition sequence In this specification, the "TAL recognition sequence" is a base sequence specifically recognized by the TAL domain of the chimeric protein encoded by the first expression unit. In principle, the TAL domain of the first expression unit and the TAL recognition sequence of the second expression unit are in a one-to-one relationship, and a set of binary gene expression systems is configured by this relationship. However, when a plurality of different second expression units include a TAL recognition sequence that can be recognized by the TAL domain of one first expression unit, a one-to-many relationship is established, and the first expression unit has a plurality of second expression units. A pair can be formed with the expression unit. In the binary gene expression system of the present invention, by using the one-to-one specificity of the first and second expression units and introducing different sets of binary gene expression systems into one individual, 2 Expression of the target gene contained in the expression unit can be controlled in a time-specific and / or tissue-specific manner.

The binding of the chimeric protein to the TAL recognition sequence activates the expression of the target gene or the like in which the transcriptional activation domain of the chimeric protein is placed under the control of the TAL recognition sequence. Therefore, the TAL recognition sequence in this specification can be said to be a gene expression regulatory region that can function as a promoter such as a target gene linked downstream.

The specific structure of the TAL recognition sequence is that the target base sequence of the TAL domain encoded by the first expression unit is 5 to 25, 5 to 20, 5 to 15, 5 to 10, 5 to 9, Alternatively, it consists of a repeating sequence containing 5 to 8. For example, a base sequence containing 5 to 25 target base sequences composed of a main target base sequence consisting of 5'-terminal t (thymine) followed by the base sequence represented by SEQ ID NO: 4 is applicable. A more specific example is a repeating sequence in which 5 to 25 repeating units of the GAL4 recognition sequence consisting of the base sequence represented by SEQ ID NO: 9 are linked. This sequence contains a UAS consisting of the base sequence represented by SEQ ID NO: 35, and is represented by the TAL domain encoded by the first expression unit having the base sequence represented by SEQ ID NO: 7 or 8 as the main target base sequence. Be recognized.

(2) Target gene or fragment thereof In this specification, “target gene or fragment thereof” is any gene or fragment thereof that is contained in the second expression unit and whose expression is to be enhanced by the binary gene expression system of the present invention. It is. The kind of gene is not particularly limited, and can be a nucleic acid encoding a desired protein or peptide fragment thereof, or a nucleic acid encoding a functional nucleic acid.

The “target gene” is a gene encoding a target protein, and may be any of a gene derived from a genome, a gene composed of cDNA, or a chimeric gene.

Also, the “target gene fragment” is a nucleic acid fragment composed of a part of the base sequence of the target gene. The base length of the nucleic acid fragment is not particularly limited. The target gene may be a nucleic acid fragment lacking a region encoding a signal sequence, or may be a nucleic acid fragment consisting of only one domain or one motif in the target gene.

In the present specification, the target gene encoding the target protein or the DNA encoding the active fragment is collectively referred to as “target gene etc.”. Further, in the binary gene expression system of the present invention, the above-mentioned target genes and the like include a chimeric gene comprising a TAL region and a transcriptional active region contained in the first expression unit, a target gene contained in the second expression unit, and the like. It is a superordinate concept.

The biological species derived from the target gene or the like may be different from the host biological species into which the second expression unit is introduced. For example, the target protein encoded by the second expression unit is a human-derived protein, and the host into which the second expression unit is introduced is silkworm.

In this specification, the “target protein” is a desired protein encoded by a target gene. Regardless of the type of target protein. Either a structural protein or a functional protein may be used. Examples of structural proteins include fiber proteins such as collagen, actin, myosin, fibroin, keratin, histone, and the like. Examples of functional proteins include peptide hormones (insulin, calcitonin, paratormon, growth hormone, etc.), cytokines (epidermal growth factor (EGF), fibroblast growth factor (FGF), interleukin (IL), interferon (IFN), Tumor necrosis factor α (TNF-α), transforming growth factor β (TGF-β), etc.), transcription factor (including GAL4), immunoglobulin, serum albumin, hemoglobin, enzyme and the like. The target protein may be either a wild type protein or a mutant protein. For example, a mutant protein such as a gain-of-function type may be used. Further, the target protein or peptide fragment thereof may or may not have activity. This is because even an inactive mutant protein can impart a dominant negative effect to a host into which the gene expression vector of this embodiment has been introduced. However, a protein having activity or an active peptide fragment thereof is usually preferred.

The “functional nucleic acid” refers to a specific biological function such as an enzyme function, a catalytic function, or a biological inhibition or enhancement function (for example, inhibition or enhancement of transcription, translation) in a living body or a cell. The nucleic acid molecule which has. Specific examples include RNA interference agents, nucleic acid aptamers (RNA aptamers, etc.), ribozymes, U1 adapters, transcription factor binding regions, and the like. An “RNA interference agent” is a substance that induces RNA interference (RNAi) (RNAi) in vivo and suppresses (silences) the expression of the gene through degradation of the target gene transcript. Say. For example, shRNA (short hairpin RNA), miRNA (microRNA) (including pri-miRNA and pre-miRNA) or antisense RNA can be mentioned.

In the second expression unit of this embodiment, a plurality of target genes and the like may be included. In this case, each target gene or the like may be a gene or the like encoding the same protein, or a gene or the like encoding a different protein. However, when there is only one TAL recognition sequence in the second expression unit, each target gene or the like must be located within the control region range of the TAL recognition sequence. On the other hand, a plurality of sets of expression units consisting of one TAL recognition sequence and one target gene may exist in the second expression unit.

2. Transformant 2-1. Outline | summary The 2nd aspect of this invention is a transformant. The transformant of this aspect includes in the cell the first expression unit and / or the second expression unit constituting the binary gene expression system described in the first aspect. The transformant of the present invention facilitates maintenance of a strain containing any one expression unit constituting a binary gene expression system. In addition, in an individual containing two expression units, the expression of a target gene or the like contained in the second expression unit can be enhanced. Furthermore, by introducing a plurality of sets of binary gene expression systems into an individual, the expression timing and expression location of the target gene contained in the second expression unit of each system can be individually controlled.

2-2. Configuration In the present specification, the “transformant” refers to an introducer that includes the first expression unit and / or the second expression unit constituting the binary gene expression system according to the first aspect in the cell. More specifically, the first expression unit and / or the second expression unit constituting the binary gene expression system according to the first aspect (often abbreviated as “first and / or second expression unit” in the present specification). F1 individuals comprising two expression units obtained by mating transformants containing the first generation and its second generation or later progenies or their respective generation units, The second generation and later generations fall under this category. However, when the TAL recognition sequence of the second expression unit included in the transformant is composed of a repetitive sequence in which 5 to 25 nucleotide sequences represented by SEQ ID NO: 9 are linked, the transformant is derived from the transformant of the present invention. Excluded.

In the present specification, “host (cell)” refers to a cell, tissue or individual into which the first and / or second expression unit of the binary gene expression system of the present invention is introduced. The host of the transformant is not particularly limited in principle. However, when the first and / or second expression unit introduced into the cell can be replicated and the first and second expression units coexist, the target gene contained in the second expression unit is expressed. It must be possible. As long as the above conditions are satisfied, the host may be any of bacteria, fungi, and animals (cells). Examples of bacteria include Escherichia coli and Bacillus bacteria. Examples of fungi include filamentous fungi, basidiomycetes, and yeast. Examples of animals include invertebrates (including insects and crustaceans) and vertebrates (including fish, amphibians, reptiles, birds and mammals). Insects are preferable for the host, and lepidopterous insects are particularly preferable.

“Lepidoptera” refers to insects belonging to the taxonomic Lepidoptera, butterfly or moth. The butterfly includes insects belonging to Nymphalidae, Papilionidae, Pieridae, Lycaenidae, and Hesperiidae. The moths include Saturniidae, Bombycidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Geometridae, and idae , Insects belonging to Noctuidae, Pyralidae, Sphingidae and the like. For example, in the case of moths, species belonging to the genus Bombyx, Samia, Antheraea, Saturnia, Attacus, Rhodinia, specifically silkworms, Bombyx mandarina, Shinjasan (Samia cynthia; Elysan Samia cynthia) ricini and hybrids of Shinjusan and Erisan), Antheraea yamamai, Sakusan (Antheraea pernyi), Saturnia japonica, Actias gnoma and the like. The Lepidoptera insects as the host of the transformant of the present invention are not limited to these, but silkworms with high industrial applicability are particularly preferable as the host.

In the present specification, the “progeny” is a transformant of the second generation or later obtained from the transformant of the first generation through asexual reproduction or sexual reproduction, and the binary gene expression of the first aspect An individual holding the first and / or second expression unit constituting the system. For example, in the case of a single-cell microorganism, a cell newly generated by division or budding from a transformant of the first generation or later (clone body), and a cell holding the first and / or second expression unit is applicable. To do. In addition, if it is an organism that carries out sexual reproduction, it is an individual newly generated by mating of the first generation or later gametes, an individual holding the first or second expression unit, or a trait of the first generation or later. This is an individual newly generated by joining gametes of the transformant and holding the first and second expression units.

The first and second expression units constituting the binary gene expression system of the first aspect in the transformant may exist transiently in the host cell, and are stable when introduced into the chromosome. And may exist continuously. Usually, it is preferable to exist stably and continuously.

When the first and second expression units are integrated into the host cell chromosome, the first and second expression units may be present on the same chromosome or may be present on different chromosomes. When two expression units are allowed to coexist in the same host cell by mating with a host that is a transformant, each expression unit is preferably present on a different chromosome. When present on the same chromosome, it is desirable that the two expression units be incorporated distally so as not to be linked to each other.

Moreover, it is preferable that the first and second expression units have the same type and separate hosts. By crossing a transformant of a line having only the first expression unit (preferably homozygous) with a transformant of a line having only the second expression unit (preferably homozygous), F1 A transformant having the two expression units can be easily obtained.

2-3. Host introduction method of first or second expression unit The host introduction method of each expression unit will be described.

The host into which the first or second expression unit is introduced is an individual, tissue or cell (including cell lines). Preferred hosts are individuals. When introduced into a cell or tissue, the stage of development of the collected individual is not particularly limited. When introduced into an individual, there are no particular limitations on the generation stage or sex, and any stage in the growth process may be used. Preferably, it is an embryonic time when a higher effect can be expected.

The method for introducing each expression unit may be performed according to a transformation method known in the art depending on the host to be introduced.

For example, if the host is a bacterium, a heat shock method, a calcium ion method (for example, a calcium phosphate method), an electroporation method, or the like may be used. When the host is yeast, the lithium method, electroporation method or the like may be used. These transformation methods are all known in the art, and are described in Green, M.R. and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York and so on, so you can refer to them.

In addition, when the host is an insect, particularly silkworm, each unit is a transposon inverted terminal repeat (ITRs) (Handler AM. Et al., 1998, Proc. Natl. Acad. Sci. USA 95: 7520-5) The method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84) can be applied. Briefly, the first or second expression unit diluted to an appropriate concentration may be injected into an early embryo of a silkworm egg together with a helper vector having a transposon transferase gene. For example, pHA3PIG can be used as the helper vector. When the expression unit includes a marker gene, the target transformant can be easily selected based on the expression of the gene or the like. In the transgenic silkworm obtained by this method, the expression unit is integrated into the chromosome via the transposon inverted terminal repeat sequence. If necessary, this transgenic silkworm may be sibling or inbred to obtain a homozygous nuclear expression unit inserted into the chromosome.

Furthermore, when the host is an animal cell, the lipofectin method (PNAS, 1989, Vol. 86, 6077; PNAS, 1987, Vol. 84, 7413), electroporation method, calcium phosphate method (Virology, 1973, Vol. 52). 456-467), DEAE-Dextran method and the like are preferably used, but not limited thereto.

3. 3. Method for producing individual with enhanced gene expression 3-1. Outline | summary The 3rd aspect of this invention is a production method of a gene expression enhancement individual. The production method of the present invention is a method for producing an F1 transformant containing both the first and second expression units in the binary gene expression system of the first embodiment. The obtained transformant can enhance the expression of a target gene or the like included in the second expression unit.

3-2. Production procedure The production method of the present invention includes a mating step and a selection step as essential steps. Hereinafter, each step will be described.

3-2-1. Mating Step “Mating step” is a step of mating a transformant having only the first expression unit described in the second embodiment with a transformant having only the second expression unit.

In this step, a transformant having the first expression unit and a transformant having the second expression unit may be crossed based on a conventional method. The transformant having each expression unit is preferably subjected to sibling mating or inbred mating in advance and homozygous for each expression unit.

3-2-2. Selection Step The “selection step” is a step of selecting an individual having the first and second expression units. This step can be achieved by selecting individuals having both expression units from the F1 individuals obtained after the mating step, based on the activity of the labeled protein encoded by each expression unit.

Hereinafter, embodiments of the present invention will be described with specific examples. However, the following examples are merely one concept of the present invention, and the present invention is not limited thereto.

<Example 1: Expression efficiency of target gene by first expression unit recognizing UAS (1)>
(the purpose)
In order to construct the binary gene expression system of the present invention, various first expression units encoding TAL domains that recognize UAS are constructed, and the expression efficiency of the target gene with respect to the second expression unit is verified.

(Method)
(1) Construction of the first expression unit Takasu et al. (Takasu Y., et al., 2013, PLoS ONE 8: 9, e73458. Doi: 10.1371 / journal.pone. The TALEN expression vector pBlue-TAL for silkworm constructed by (0073458) was used.

In the conventional GAL4 / UAS system, as shown in FIG. 2, a sequence consisting of 19 bases and having a GAL4 recognition sequence represented by SEQ ID NO: 9 repeated 5 times is used as a UAS construct. Therefore, TAL recognition region UASTAL1, which has this unit as the target base sequence, targets 20 bases that are shifted by 5 bases on the 3 'end side of the GAL4 recognition sequence so that the 5' end side of the target base sequence is T. UASTAL2, which is the base sequence, and 3 types of TAL, UASTAL3, which is trimmed to 13 bases by trimming the 3 'end of UASTAL2 to include 5 target base sequences on the GAL4 recognition sequence (ie repeat 5 times) Regions were created (FIG. 2). Cloning of the TAL region was performed in accordance with the method of Cermak et al. (Cermak T., et al., 2011, Nucleic Acids Res 39: e82) using the Golden gate assembly kit (Addgene). The obtained vectors were designated as “pBlue-UASTAL1”, “pBlue-UASTAL2”, and “pBlue-UASTAL3” (in this specification, these are collectively referred to as “pBlue-UASTAL (1-3)”. To do).

Next, pBlue-UASTAL (1-3) was digested with BamHI and XhoI to remove the region encoding the FokI nuclease domain. Subsequently, a BamHI-XhoI adapter obtained by annealing BamXho_ad_U consisting of the base sequence represented by SEQ ID NO: 36 and BamXho_ad_L consisting of the base sequence represented by SEQ ID NO: 37 was inserted into the BamHI / XhoI site. The obtained vector was designated as “pBlue-UASTAL (1-3) -BamHI-XhoIad”.

Subsequently, the restriction enzyme site in the N-terminal region in the TAL region was modified by PCR. Specifically, amplification was performed by PCR using TAL_N_BsmBI_U (SEQ ID NO: 38) and TAL_N_L (SEQ ID NO: 39) as primers and pBlue-TAL as a template. PCR was performed using KOD plus (TOYOBO), and the composition of the reaction solution was in accordance with the standard method described in the attached protocol. The PCR cycle conditions were 12 cycles, with 94 ° C for 30 seconds and 68 ° C for 60 seconds as one cycle. The obtained amplification product was digested with SnaBI and AgeI, and then inserted into the SnaBI / AgeI site of pBlue-UASTAL (1-3) -BamHI-XhoIad. The obtained vector was designated as “pBlue-Bsm-UASTAL (1-3) -BamHI-XhoIad”.

Furthermore, the full length of the transcriptional activation domain of GAL4 was linked to the C-terminus of the TAL2 region. BsmBI_GAL4_U30 (SEQ ID NO: 40) and BsmBI_GAL4_L30 (SEQ ID NO: 41) are used as primers, and pBac [Ser1-GAL4 / 3xP3-DsRed] (Tatematsu K, et al., 2010, Transgenic research, 19: 473-87) is used as a template. Thus, the full length of the GAL4 gene was amplified by PCR. The obtained amplification product was inserted into pZErO-2 (life technologies). The specific method was based on the method described in Tatematsu et al. (Tatematsu K, et al., 2010; mentioned above). The obtained vector was designated as “GAL4 / pZero2”.

Next, GAL4 / pZero2 was digested with ClaI and XhoI to obtain GAL4AD-ClaI-XhoI containing a region encoding the GAL4 transcriptional activation domain. After digesting pBlue-Bsm-UASTAL (1-3) -BamHI-XhoIad with ClaI and XhoI, the GAL4AD-ClaI-XhoI was inserted into the ClaI / XhoI site. The obtained vector was designated as “pBlue-Bsm-UASTAL (1-3) -GAL4AD”.

Finally, pBlue-Bsm-UASTAL (1-3) -GAL4AD was digested with SnaBI and XhoI, and the obtained SnaBI-XhoI fragment was inserted into the EcoRV / XhoI site of pIB / V5-His (life ™ technologies). pIB / V5-His has an OplE2 promoter and terminator, and the EcoRV / XhoI site is located between them. The obtained first expression unit was designated as “UASTAL (1-3) -GAL4AD / pIB”.

(2) Construction of the second expression unit The second expression unit used in this example has the same basic structure as the UAS line of the GAL4 / UAS system, and a luciferase gene is linked as a target gene downstream of the UAS repeat. It has a structure.

First, SerUAS cassette consisting of UAS repeat and 3'UTR of sericin 1 gene, serUASPCRU (SEQ ID NO: 42) and serPolyALSpe (SEQ ID NO: 43) as primers, pBac [SerUAS / 3xP3EGFP] (Tatematsu K, et al., 2010) , Transgenic research, 19: 473-87) was used as a template and amplified by PCR. PCR was performed using KOD plus (TOYOBO), and the composition of the reaction solution was in accordance with the standard method described in the attached protocol. The PCR cycle conditions were 12 cycles, with 94 ° C for 30 seconds and 68 ° C for 60 seconds as one cycle. The obtained amplification product was inserted into the EcoRV site of pBluescript SKII- (Agilent Technologies). The obtained vector was designated as “pB-SerUAS”.

Next, the firefly luciferase gene was amplified by PCR using Bln-luc U (SEQ ID NO: 44) and Bln-luc L (SEQ ID NO: 45) as primers and pGL3 (Promega) as a template. PCR conditions were in accordance with the above conditions. After digesting the amplified product with BlnI, the obtained BlnI fragment was inserted into the BlnI site of pB-SerUAS to obtain the second expression unit “pUAS-fLuc”.

(3) Luciferase assay In order to examine the transcription activation ability of the first expression unit, a luciferase assay was performed on silkworm cultured cells. The first expression unit UASTAL (1-3) -GAL4AD / pIB and the second expression unit pUAS-fLuc were transfected into silkworm cultured cells BmN4 together with pRL-TK (Promega: for internal standard). As a positive control, the transcriptional activation ability of GAL4 lines in the conventional GAL4 / UAS system was also measured. The UAS line in the positive control is the same as the second expression unit.

Three days after transfection, the cells were collected, and luciferase activity was measured by dual-luciferase assay (Promega) according to the attached protocol.

(result)
The results are shown in lanes 2 to 4 of FIG. The positive control (GAL4 / UAS system GAL4 line) is lane 1. It was clarified that the transcriptional activation ability decreased in all cases of UASTAL1 to 3 compared to the positive control only by linking the full-length coding region of GAL4AD to the TAL region. This may have been caused by the steric hindrance of UASTAL caused by the target recognition sequence being a simple repeat sequence.

<Example 2: Expression efficiency of target gene by first expression unit recognizing UAS (2)>
(the purpose)
In the first expression unit of Example 1, transcription activation ability exceeding the conventional GAL4 / UAS system could not be obtained. In order to reduce the steric hindrance of UASTAL, a new first expression unit with a shortened transcriptional active domain is constructed, and the expression efficiency of the target gene with respect to the second expression unit is verified again.

(Method)
(1) Construction of first expression unit In this example, ARII or VP16 was used in place of the transcriptional activation region in the first expression unit in place of the full length of the GAL4 transcriptional activation domain of Example 1. ARII is the minimal active unit (transcriptional activity unit) located on the C-terminal side of the GAL4 transcriptional activation domain, and VP16 is the transcriptional activation domain of herpes simplex virus VP16. For the TAL region, UASTAL3, which had the highest transcription activation ability in Example 1, and UASTAL2, which had the next highest transcription activation ability, were used (hereinafter these two were collectively referred to as “UASTAL (2-3 ) ".
・ Construction of UASTAL (2-3) -ARII (x2) First, the region encoding the ARII consisting of the amino acid sequence represented by SEQ ID NO: 16 in the GAL4 gene (ARII region; SEQ ID NO: 17) is designated as BamHI GAL4ARII U (sequence) No. 46) and Bgl GAL4ARIIL (SEQ ID NO: 47) were used as primers, and pBac [Ser1-GAL4 / 3xP3-DsRed] was used as a template for amplification by PCR. PCR was performed according to the method described in Example 1. Next, the amplified product was digested with BamHI, and the obtained BamHI fragment was inserted into the BamHI site of pBlue-Bsm-UASTAL (2-3) -BamHI-XhoIad prepared in Example 1. The obtained vector was designated as “pBlue-Bsm-UASTAL (2-3) -ARII”.

The BamHI fragment was re-inserted into the BamHI site of pBlue-Bsm-UASTAL (2-3) -ARII to make ARII a tandem. The obtained vector was designated as “pBlue-Bsm-UASTAL (2-3) -ARIIx2”.

pBlue-Bsm-UASTAL (2-3) -ARII and “pBlue-Bsm-UASTAL (2-3) -ARIIx2 were digested with SnaBI and XhoI, respectively, and the SnaBI-XhoI fragment was pIB / V5-His (life technologies) The first expression units obtained by inserting into the EcoRV / XhoI site were designated as “UASTAL (2-3) -ARII / pIB” and “UASTAL (2-3) -ARIIx2 / pIB”.
-Construction of UASTAL3-VP16 (x2) First, a region encoding VP16 consisting of the amino acid sequence represented by SEQ ID NO: 18 (VP16 region; SEQ ID NO: 19) was designated as BamHI VP16 U (SEQ ID NO: 48) and Bgl VP16operon L ( SEQ ID NO: 49) was used as a primer, and the artificially synthesized gene was used as a template to amplify by PCR. The artificially synthesized gene was commissioned to Eurofin Genomics Co., Ltd. based on the base sequence represented by SEQ ID NO: 19.

Other basic methods are based on the above UASTAL (2-3) -ARII (x2) construction method. However, only UASTAL3 was used for the TAL area. The obtained first expression units were designated as “UASTAL3-VP16 / pIB” and “UASTAL3-VP16x2 / pIB”.

(2) Luciferase assay Transfection of UASTAL (2-3) -ARII (x2) into cultured cells and luciferase assay were performed according to the method described in Example 1. As the second expression unit, pUAS-fLuc prepared in Example 1 was used.

(result)
The results are shown in lanes 5 to 10 of FIG. The positive control (GAL4 of GAL4 / UAS system) is lane 1. Lanes 5 and 6 are UASTAL2-ARII / pIB and UASTAL2-ARIIx2 / pIB, lanes 7 and 8 are UASTAL3-ARII / pIB and UASTAL3-ARIIx2 / pIB, respectively, and lanes 9 and 10 are UASTAL3-VP16 / pIB, respectively. And UASTAL3-VP16x2 / pIB.

Fig. 3 shows that UASTAL2, which recognizes the 19-base TAL region, showed only about 30% of the transcriptional activation ability of GAL4 even when two ARIIs were linked (lane 6). On the other hand, UASTAL3, which recognizes 13 bases in the TAL region, showed about 2.5 times the transcriptional activation ability of GAL4 when two ARIIs were linked (lane 8). Moreover, when the transcriptional activation domain of VP16 was used, when two were linked, the transcriptional activation ability was 7 times or more (lane 10).

From the above results, it was shown that the expression efficiency of the target gene can be improved over the conventional GAL4 by linking a plurality of ARII and VP16 under the control of UASTAL3 in the first expression unit.

<Example 3: Expression efficiency of target gene by first expression unit recognizing UAS (3)>
(the purpose)
In order to further improve the expression efficiency of the binary gene expression system of the present invention using the first expression unit constructed in Example 2, the number (repetition number) of ARII and VP16, which are transcription activity units of the first expression unit, and transcription activation Verify the relationship between Noh.

(Method)
(1) Construction of the first expression unit-Construction of UASTAL3-ARII (xX) (where "X" is an integer of 3 to 6) The basic method was in accordance with Example 2. Here, the BamHI fragment newly obtained in Example 2 was inserted into the BamHI site of pBlue-Bsm-UASTAL3-ARIIx2, and three ARIIs were ligated. In addition, the same operation was repeated for this vector, so that ARII was made into 4 linkage, 5 linkage, and 6 linkage. The obtained vectors are collectively referred to as “pBlue-Bsm-UASTAL3-ARIIx (3-6)”. Each vector was digested with SnaBI and XhoI, the SnaBI-XhoI fragment was inserted into the EcoRV / XhoI site of pIB / V5-His (life technologies), and the first expression unit was “UASTAL2-ARIIx (3-6) / pIB” It was.
Construction of UASTAL3-VP16 (xX) (where “X” is an integer of 3 to 6) The basic method was in accordance with the construction of Example 2 and UASTAL3-ARII (xX). The first expression unit obtained here was designated as “UASTAL2-VP16x (3-6) / pIB”.

(2) Luciferase assay The basic method was based on Examples 1 and 2. As controls, GAL4 / pIB constructed in Example 2 and UASTAL2-ARII / pIB, UASTAL2-ARIIx2 / pIB, UASTAL3-VP16 / pIB and UASTAL3-VP16x2 / pIB constructed in Example 2 were used.

(result)
The results are shown in lanes 2 to 13 of FIG. The positive control (GAL4 / UAS system GAL4) is lane 1. The values in the figure are relative values when the measured value of GAL4 is 1. Lanes 2 to 7 are the results using the first expression unit in which one, two, three, four, five and six ARIIs are connected, and lanes 8 to 13 each have 1 VP16. The results were obtained using the first, second, third, fourth, fifth and sixth linked first expression units.

It has been clarified that the transcriptional activation ability is improved depending on the number (repeat number) of both ARII and VP16.

<Example 4: Optimal number of bases of target base sequence>
(the purpose)
The relationship between the number of bases of the target base sequence of the second expression unit recognized by the TAL domain encoded by the first expression unit and the transcription activity efficiency is verified.

(Method)
The construction of the first expression unit basically followed the method described in the first example. However, in this example, based on UASTAL3 with 14 bases in the target base sequence, the target base sequence is extended to 20 bases on the 3 ′ side on the GAL4 recognition sequence consisting of the base sequence shown in SEQ ID NO: 9 one by one. 6 kinds of TAL regions, namely UASTAL4 (15 bases of target base sequence), UASTAL5 (16), UASTAL6 (17), UASTAL7 (18), UASTAL8 (19), and A first expression unit with UASTAL2 was made. The UASTAL2 TAL region constructed in Example 1 was diverted to the first expression unit having 20 bases in the target base sequence. The same pUAS-fLuc as in Example 1 was used as the second expression unit.

The luciferase assay was in accordance with the method described in Example 1. As a positive control, the transcriptional activation ability of GAL4 lines in the conventional GAL4 / UAS system was also measured.

(result)
The results are shown in FIG. When the number of bases in the target base sequence was 14 or 15, the transcriptional activity was higher than that of the positive control (GAL4). However, when the number was 16 or more, the transcriptional activity was significantly reduced. The low transcriptional activity when the number of bases in the target base sequence was 20 was consistent with the results of Example 1. Therefore, in the binary gene expression system of the present invention, it was revealed that the optimal base number of the target base sequence is 14 or 15 bases.

<Example 5: Sequence specificity of binary gene expression system>
(the purpose)
The target base sequence specificity of the TAL domain encoded by the first expression unit is verified.

(Method)
(1) Construction of each expression unit The first expression unit is the first expression unit containing UASTAL3-ARIIx2 constructed in Example 2, and the second expression unit is the TAL recognition sequence constructed in Example 1 as UAS. The second expression unit containing UAS-fLuc whose target gene is a gene was used as a base.

From the results of Example 4, since the number of bases in the target base sequence was 15 higher than 14, the expression level was higher on the 3 ′ side of the target base sequence consisting of 14 bases of UASTAL3. UASTAL4 with UAS4 as the target base sequence, which was made by adding 1 base of cytosine to 15 bases, was taken as one of the TAL regions of the first expression unit. In addition, a TAL recognition sequence having UAS4m2 (FIG. 6) in which two bases unnecessary for GAL4 recognition were substituted in the base sequence of UAS4 in the second expression unit and UAS4m4 (FIG. 6) in which four bases were substituted was constructed. Furthermore, UASTAL4-ARIIx2 and UASTAL4m2-ARIIx2 included in the first expression unit and capable of specifically recognizing UAS4 and UAS4m2 were constructed as TAL regions.

The cloning of the TAL region was performed in the same manner as in Example 1 using the Golden gate assembly kit (Addgene) according to the method of Cermak et al. (Cermak T, Et et al, 2011, Nucleic Acids Res 39: e82). The first expression units UASTAL4-ARII / pIB and UASTAL4m2-ARIIx2 / pIB containing the TAL region were in accordance with the method described in Example 2.

The second expression unit containing UAS4-fLuc, UAS4m2-fLuc, and UAS4m4-fLuc is represented by BsmBISer1UTR U consisting of the base sequence represented by SEQ ID NO: 19 and SEQ ID NO: 50 at the PstI-HindII site of pUAS-fLuc. After inserting a BsmSer1UTR adapter formed by annealing BsmBISer1UTR L consisting of the base sequence consisting of UAS4U consisting of the base sequence represented by SEQ ID NO: 51 and the base sequence represented by SEQ ID NO: 52 at the BsmBI site of the vector UAS4U / UAS4L formed by annealing UAS4L, UAS4Um2 composed of the base sequence represented by SEQ ID NO: 53, and UAS4m2L composed of the base sequence represented by SEQ ID NO: 54, UAS4m2U / UAS4m2L formed by annealing, or SEQ ID NO: 55 5 UAS4m4U / UAS4m4L, each of which is annealed from UAS4Um4 consisting of the base sequence and UAS4m4L consisting of the base sequence represented by SEQ ID NO: 56, are connected. Ri returned inserted.

(2) Luciferase assay The basic method was in accordance with the method described in Example 1. Here, silkworm cultured cells combining the first expression unit (GAL4 / pIB, UASTAL4-ARIIx2 / pIB, and UASTAL4m2-ARIIx2 / pIB and the second expression unit (pUAS4-fLuc, pUAS4m2-fLuc, and pUAS4m4-fLuc) BmN4 was transfected and luciferase assay was performed.

(result)
The results are shown in FIG. When the first expression unit contains GAL4, the second expression unit exhibited transcription activation ability for any of UAS4, UAS4m2, and UAS4m4. This indicates that GAL4 has low sequence specificity for the GAL4 recognition sequence. On the other hand, UASTAL4-ARIIx2 having the configuration of the binary gene expression system of the present invention showed high transcription activation ability only for UAS4 containing the target base sequence of UASTAL4. Similarly, UASTAL4m2-ARIIx2 showed high transcription activation ability only for UAS4m2 containing the target base sequence of UASTAL4m2. These results indicate that the binary gene expression system of the present invention using UASTAL has high sequence specificity for the target nucleotide sequence. Therefore, by using the binary gene expression system of the present invention, the expression of a plurality of genes can be separately controlled temporally or spatially, and compatibility with the conventional GAL4 / UAS system is maintained. Suggests that

<Example 6: Expression efficiency of binary gene expression system in transgenic silkworm (1)>
(the purpose)
In Examples 2 to 5, the expression efficiency of the binary gene expression system of the present invention was verified using cultured silkworm cells. Therefore, in this example, the expression efficiency of the binary gene expression system of the present invention when a transgenic silkworm is used is verified.

(Method)
(1) Middle silk gland-specific first expression unit In this example, the transcription activation ability of UASTAL3 in the middle silk gland of silkworms is verified.
-Construction of Ser1p-UASTAL3-ARII (xX) (where "X" is 1, 4 or 6) A first expression unit of UASTAL3-ARII linked to the sericin 1 promoter, which is a central silk gland specific promoter, was constructed.

First, PCR was performed to introduce the BsmBI site downstream of the UAS. Specifically, UASx5 was amplified by PCR using serUASPCRU (SEQ ID NO: 42) and BlnBsmSerKL (SEQ ID NO: 57) as primers and pBac [SerUAS / 3xP3EGFP] (described above) as a template. PCR conditions were the same as in Examples 2 and 3. Next, the amplified product was digested with BglII and BlnI, and the resulting BglII-BlnI fragment was inserted into the BglII-BlnI site of pB-SerUAS (described above). This vector was designated as “Bsm-SerUAS cassette”.

The pBlue-Bsm-UASTAL3-ARII, pBlue-Bsm-UASTAL3-ARIIx4, and pBlue-Bsm-UASTAL3-ARIIx6 prepared in Examples 2 and 3 were digested with BsmBI and XbaI, and the obtained BsmBI-XbaI fragments were Bsm- It was inserted into the BsmBI-BlnI site of the SerUAS cassette. Subsequently, SnaBI and XbaI were digested to obtain a SnaBI-XbaI fragment containing UASTAL3-ARII and the like. Then, pBac [Ser1-GAL4 / 3xP3-DsRed] was similarly digested with SnaBI and XbaI to replace the SnaBI-XbaI fragment containing the GAL4 gene and the SnaBI-XbaI fragment containing UASTAL3-ARII and the like. As a result, pBac [Ser1-UASTAL3-ARIIx2 / 3xP3-DsRed], pBac [Ser1-UASTAL3-ARIIx4 / 3xP3-DsRed], and pBac [Ser1-] are expressed as first expression units that specifically induce expression in the middle silk gland. UASTAL3-ARIIx6 / 3xP3-DsRed] was obtained.
Construction of Ser1p-UASTAL3-VP16 (xX) (where “X” is 3 or 6) A first expression unit of UASTAL3-VP16 linked to the sericin 1 promoter, which is a central silk gland specific promoter, was constructed. The basic method was performed according to the construction method of Ser1p-UASTAL3-ARII (xX).

(2) UAS-EGFP second expression unit A base sequence (SEQ ID NO: 59) encoding a sericin 1 gene secretion signal (SEQ ID NO: 58) downstream of UAS, and an additional base sequence for cloning added to the 3 ′ end side thereof PBacSerUAS-ser_sigEGFP / 3xP3EGFP was constructed by binding the EGFP gene (SEQ ID NO: 62) as the target water-soluble peptide DNA and linking sericin 1 3'UTR (SEQ ID NO: 63) downstream thereof. .

(3) Production of transgenic silkworm The silkworm strain used was the w1-pnd strain of white-eye, white egg, and non-dormant strain maintained by the National Institute of Agrobiological Sciences as the host strain. Breeding conditions were in a breeding room at 25-27 ° C., and all larvae were reared with artificial feed (silk mate species 1-3 years old S, Japanese agricultural industry). The artificial diet was changed every 2-3 days (Uchino K. et al., 2006, J Insect Biotechnol Sericol, 75: 89-97).

The transgenic silkworms were produced according to the method of Tamura et al. (Tamura T. et al., 2000,, Nature Biotechnology, 18, 81-84).

The middle silk gland-specific first subunit and the UAS-EGFP second subunit are respectively converted into helper plasmids pHA3PIG (Tamura T. et al., 2000,, Nature Biotechnology, 18, 81-84) expressing transposase and 1 : 1 mix and injected into silkworm eggs 2-8 hours after spawning. PBac [Ser1-GAL4 / 3xP3-DsRed] was used as a control for the first expression vector. Eggs after injection were incubated in a humidified state at 25 ° C. until hatched. The hatched larva was bred by the above method and brother-sister mating was performed. The obtained eggs were selected based on the presence or absence of eye fluorescence with the 3xP3 DsRed2 marker for the first subunit and the 3xP3EGFP marker for the second subunit, and the first and second subunits of the transgenic silkworm of the present invention were selected. Each line was obtained. A strain having both the first subunit and the second subunit is crossed, and a strain having both expression units in one individual is selected based on the presence or absence of eye fluorescence by the 3xP3EGFP marker and the 3xP3DsRed2 marker, and the middle silk gland. 4 lines of UASTAL3-ARII, 3 lines of UASTAL3-ARIIx4, 4 lines of UASTAL3-ARIIx6, 2 lines of UASTAL3-VP16x3, and 1 line of UASTAL3-VP16x6 were obtained. .

(4) Quantification of EGFP Each of the transgenic silkworms was bred, and anesthetized on ice immediately before the 5th day, 6th day of spitting. The dorsal side was incised and excised with tweezers so as not to damage the middle silk gland ( Ed. Mori, New biological experiment by silkworm, Sanseido, 1970, pp.249-255). Next, 10 mL of PBS (pH 7.2) / 1% Tween20 / 0.05% sodium azide per middle silk gland was placed in the medium and shaken at room temperature for 24 hours to extract water-soluble proteins. The obtained water-soluble protein extract was centrifuged at 2,000 × g for 10 minutes, and the supernatant was collected. The concentration of EGFP protein in the water-soluble protein contained in the supernatant was measured with Reacti-Bind Anti-GFP Coated Plates (PIERCE). Specifically, 100 μL of the supernatant was added to Reacti-Bind Anti-GFP Coated Plates and allowed to stand at room temperature for 1 hour. After washing with PBS / 0.05% Tween 20 three times, horseradish peroxidase-conjugated anti-GFP antibody (Rockland Immunochemicals) was added and allowed to stand at room temperature for 1 hour. After washing 3 times with PBS / 0.05% Tween 20, a color reaction was performed using TMB Peroxidase EIA Substrate Kit (Bio-Rad), and the reaction was stopped by adding 1N sulfuric acid. Color development was quantified with a plate reader (SpectraMax 250; Molecular Devices). A standard curve was prepared using a serial dilution (1-400 pg / μL) of recombinant GFP protein (Takara Bio; Z2373N).

(result)
The results are shown in FIG. In the UASTAL3 first expression unit in which ARII and VP16 were repeatedly fused, a very high EGFP expression was observed as compared with the control first expression unit using conventional GAL4. In particular, when UASTAL3-ARIIx4 repeated 4 times of ARII was used, an expression level of about 6 times at the maximum was obtained. From this result, similar to the result in silkworm cultured cells, the binary gene expression system of the present invention is very effective in improving the expression efficiency of the target gene etc. in the transgenic silkworm as compared with the GAL4 / UAS system. It became clear that.

<Example 7: Cytotoxicity of binary gene expression system in transgenic silkworm>
(the purpose)
In the GAL4 / UAS system, the first expression unit containing GAL4 is cytotoxic to the host. Therefore, the cytotoxicity of the first expression unit in the host in the binary gene expression system of the present invention is verified.

(Method)
Example 4: w1-pnd strain having the first expression unit pBac [Ser1-GAL4 / 3xP3-DsRed] and w1-pnd strain having pBac [Ser1-UASTAL3-ARIIx4 / 3xP3-DsRed] as transgenic silkworms Example 4 From the cocoons and cocoons that were reared according to the method described in (1) above and made by each transgenic silkworm, the cocoon layer weight, the rate of management, and the chemical yield were calculated.

The amount of protein in the middle silk gland of each transgenic silkworm was detected by SDS-PAGE. Specifically, the middle silk gland was extracted from the larvae of the 5th instar of each transgenic silkworm. The middle silk gland was placed in 5 mL of 20 mM Tris-HCl (pH 8.0) / 8 M urea / 2% SDS / 25 mM DTT, and shaken overnight at room temperature to extract the silk gland protein. 27.5 μL of H ₂ O, 12.5 μL of NuPAGE LDS Sample Buffer (life technologies) and 5 μL of NuPAGE Sample Reducing Agent (life technologies) were added to 5 μL of silk gland protein, and heated at 70 ° C. for 10 minutes to form SDS. The SDS sample was electrophoresed on a 4% SDS-PAGE gel and then stained with CBC.

(result)
FIG. 8 shows the results of cocoon layer weight, management rate, and chemical yield, and FIG. 9 shows the results of SDS-PAGE. FIG. 8A shows the cocoon layer weight, FIG. 8B shows the management rate, and FIG. 8C shows the conversion rate. In this field, it is known that when GAL4 is expressed in the middle silk gland, the cytotoxicity of GAL4 decreases the cocoon layer weight, the yield, the chemical yield, and the expression level of sericin 1 expressed in the middle silk gland. It has been.

On the other hand, (Ser1-UASTAL3-ARIIx4) having the first expression unit pBac [Ser1-UASTAL3-ARIIx4 / 3xP3-DsRed] of the present invention, compared with GAL4, has a fold layer weight, a ligation rate, a chemical yield, and It was revealed that the expression level of sericin 1 significantly recovered. This suggests that UASTAL3-ARIIx4 of the present invention is less cytotoxic to the host than GAL4.

In general, the decrease in the layer thickness and the management rate is an obstacle to recovering the target protein from the paddy. A decrease in the chemical yield makes it difficult to maintain the strain of the transgenic silkworm, and a decrease in the protein amount of sericin 1 means a decrease in the gene expression efficiency of the cells. Therefore, it is suggested that the first expression unit of the present invention in which these phenomena are recovered as compared with GAL4 is suitable for the expression of the recombinant gene in the transgenic silkworm.

<Example 8: Control according to tissue of binary gene expression system in transgenic silkworm>
(the purpose)
It is verified that the binary gene expression system of the present invention can function for each tissue in the silkworm organism.

(Method)
(1) Construction of the first expression unit specific to the middle and posterior silk glands The first expression unit specific to the middle silk gland is pBac [Ser1-UASTAL4 in which UASTAL4-ARIIx4 is linked to the sericin 1 promoter, which is the middle silk gland-specific promoter. -ARIIx4 / 3xP3-DsRed]. The specific construction method of Ser1-UASTAL4-ARIIx4 is the same as that of Ser1-UASTAL3-ARIIx4 described in “(1) Middle silk gland specific first expression unit” in Example 6, and construction of UASTAL4 was performed. According to the method described in Example 5. UASTAL4 recognizes UAS4 as shown in the result of FIG.

The first posterior silk gland-specific first expression unit was pBac [FibH-UASTAL4m2-ARIIx4 / A3-KMO] in which UASTAL4m2-ARIIx4 was linked to the fibroin H promoter, which is a posterior silk gland-specific promoter. In the construction of pBac [FibH-UASTAL4m2-ARIIx4 / A3-KMO], pBac [FibH-UASTAL4m2-ARIIx4 / 3xP3-DsRed] was first prepared. The specific construction method of FibH-UASTAL4m2-ARIIx4 is the same as that of Ser1-UASTAL3-ARIIx4 described in “(1) Central silk gland-specific first expression unit” in Example 6, and construction of UASTAL4 was performed. According to the method described in Example 5. The fibroin H promoter was constructed based on SEQ ID NO: 28. A3-KMO, a selectable marker, was obtained by PCR using the A3 promoter-KMO-SV40polyA additional sequence from pBac [A3KMO, UAS] (Kobayashi I., et al., 2007, J. Insect Biotechnol Sericol, 76: 145-48). Amplified to produce A3-KMO marker. Finally, the 3xP3-DsRed marker of pBac [FibH-UASTAL4m2-ARIIx4 / 3xP3-DsRed] was replaced with the A3-KMO marker to obtain the desired pBac [FibH-UASTAL4m2-ARIIx4 / A3-KMO]. UASTAL4m2 recognizes UAS4m2 as shown in the result of FIG.

(2) Construction of the second expression unit specific to the middle and posterior silk glands The second expression unit that forms a pair with the first expression unit specific to the middle silk gland is the EGFP gene (SEQ ID NO. 62) was ligated, and pBacUAS4-EGFP / 3xP3EYFP was obtained by ligating the 3′UTR of sericin 1 (SEQ ID NO: 63) downstream thereof. In the construction of pBacUAS4-EGFP / 3xP3EYFP, first, pBacUAS4-EGFP / 3xP3EGFP was prepared. The specific construction method of pBacUAS4-EGFP was based on the method of pBacSerUAS-ser_sigEGFP / 3xP3EGFP described in “(1) Middle silk gland-specific first expression unit” in Example 6. 3xP3EYFP was prepared according to the method described in Tada M. et al., 2015 ,. MAbs. 7 (6): 1138-1150. Thereafter, the 3xP3EGFP marker of pBacUAS4-EGFP / 3xP3EGFP was replaced with 3xP3EYFP to obtain the desired pBacUAS4-EGFP / 3xP3EYFP.

The second expression unit paired with the posterior silk gland-specific first expression unit is a pBacUAS4m2-DsRed in which the DsRed gene as the target protein gene is linked downstream of UAS4m2, and the 3'UTR of sericin 1 is linked downstream of it. / 3xP3AmCyan. In the construction of pBacUAS4m2-DsRed / 3xP3AmCyan, first, pBacUAS4m2-DsRed / 3xP3EGFP was prepared. The specific construction method of pBacUAS4m2-DsRed was based on the pBacSerUAS-ser_sigEGFP / 3xP3EGFP method described in Example 6, “(1) Middle silk gland-specific first expression unit”. The specific construction method of 3xP3-AmCyan was prepared according to the method described in Tada M. et al., 2015, MAbs. 7 (6): 1138-1150. Thereafter, the 3xP3EGFP marker of pBacUAS4m2-DsRed / 3xP3EGFP was replaced with 3xP3AmCyan to obtain the target pBacUAS4m2-DsRed / 3xP3AmCyan.

(3) Production of transgenic silkworms Two sets of binary gene expression systems, ie, pBac [Ser1-UASTAL4-ARIIx4 / 3xP3-DsRed] and second expression, which are the first expression units of the middle silk gland specific binary gene expression system PBacUAS4-EGFP / 3xP3EYFP as a unit, pBac [FibH-UASTAL4m2-ARIIx4 / A3-KMO] as the first expression unit of the posterior silk gland specific binary gene expression system, and pBacUAS4m2 as the corresponding second expression unit -A total of 4 expression units of -DsRed / 3xP3AmCyan were introduced into silkworms to produce transgenic silkworms. The production of the transgenic silkworm was in accordance with the method of “(3) Production of transgenic silkworm” described in Example 6.

(4) Fluorescence observation of silk gland GMOs having two sets of binary gene expression systems are bred by the method described in “(3) Production of transgenic silkworms” described in Example 6, and Anesthesia was performed on ice immediately before the 6th day of threading, and the dorsal side was incised and excised with tweezers so as not to damage the silk gland. This was not fixed and observed with a fluorescence microscope (Olympus SZX16, GFP HQ filter & RFP filter).

(5) Expression of the target protein gene in the silk gland The extracted silk gland is divided into a middle silk gland and a posterior silk gland, and total RNA is prepared from each by a conventional method. EGFP primer pair (SEQ ID NOs: 64 and 65) and DsRed RT-PCR was performed using a primer pair (SEQ ID NO: 66 and 67). As a negative control, a host strain (w1-pnd strain) without a binary gene expression system was used.

(Results) The results are shown in FIG.
FIG. 10-1 shows a fluorescence diagram of the silk gland of a transgenic silkworm having two sets of binary gene expression systems. The middle silk gland specific binary gene expression system encoded the EGFP gene as the target protein gene, and the posterior silk gland specific binary gene expression system encoded the DsRed gene as the target protein gene. It was confirmed that EGFP was expressed only in the middle silk gland, and DsRed was expressed only in the posterior silk gland. Furthermore, as is clear from DF and GI, the expression control of EFGP and DsRed is remarkable at the boundary between the middle and posterior silk glands, and there is a leakage of expression between the two at the fluorescence level. There wasn't.

Fig. 10-2 shows the results of RT-PCR. Only the amplified fragment of the EGFP gene was confirmed from the middle silk gland of lane 1, and only the amplified fragment of the DsRed gene was confirmed from the rear silk gland of lane 2. From these results, there was no leakage of EGFP and DsRed expression specifically expressed in the middle and posterior silk glands even at the gene expression level.

From the above results, it was proved that the binary gene expression system of the present invention can control the expression of each target gene of a different set in a silkworm organism in a tissue-specific manner.

<Example 9: Expression efficiency of binary gene expression system in transgenic silkworm (2)>
(the purpose)
In Example 6, the expression efficiency in the middle silk gland by the binary gene expression system of the present invention in the silkworm organism was verified. In this example, the expression efficiency in the posterior silk gland by the binary gene expression system of the present invention in the silkworm organism is verified.

(Method)
(1) 1st expression unit specific to the posterior silk gland The first expression unit pBac [FibH-UASTAL3-ARIIx4 / 3xP3-DsRed] was constructed by linking UASTAL3-ARII to the fibroin H promoter, a posterior silk gland-specific promoter. . The basic procedure was based on the method described in “(1) Middle silk gland-specific first expression unit” in Example 6.

(2) UAS-EGFP second expression unit pBacUAS-sigEGFP / 3xP3EYFP was constructed by binding the EGFP gene downstream of UAS4 and linking sericin 1 3 ′ UTR downstream thereof.

(3) Production of transgenic silkworm The basic procedure was in accordance with the method described in “(3) Production of transgenic silkworm” in Example 6.

(4) Quantification of EGFP The basic procedure was in accordance with the method described in “(4) Quantification of EGFP” in Example 6.

(result)
The results are shown in FIG. According to the posterior silk gland-specific binary gene expression system of the present invention, EGFP expression was observed to be 10 times higher in all lines as compared to the conventional first expression unit using GAL4. From this result, the binary gene expression system of the present invention is very effective in improving the expression efficiency of the target gene and the like in the posterior silk gland as well as the conventional GAL4 / UAS system as in the middle silk gland. It has been shown.

It should be noted that all publications, patents and patent applications cited in this specification are incorporated herein by reference as they are.

Claims (12)

1. A binary gene expression system comprising a first expression unit and a second expression unit,
   The first expression unit includes a promoter, a TAL region composed of a base sequence encoding a TAL domain, and a transcriptional active region composed of a base sequence encoding a transcriptional active domain connected downstream thereof, arranged under the control of the promoter,
   The TAL domain includes an N-terminal site for recognizing the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as a target base sequence and a TAL-DNA base sequence recognition site,
   Here, in the target base sequence, the N-terminal site recognizes t at the 5 ′ end, and the TAL-DNA base sequence recognition site recognizes the main target base sequence following the t,
   The TAL-DNA base sequence recognition site consists of an amino acid sequence represented by SEQ ID NO: 3 for each base constituting the main target base sequence,
   The transcriptional activity domain comprises a sequence in which 2 to 10 transcriptional activity units consisting of 50 to 150 amino acids are linked, and the second expression unit comprises a TAL recognition sequence and a target gene or fragment thereof arranged under its control. Including
   The TAL recognition sequence consists of a base sequence containing 5 to 25 target base sequences.
   Said system.
2. 2. The binary gene expression system according to claim 1, wherein the main target base sequence consists of any one of the following base sequences (a) to (c).
   (A) the base sequence represented by SEQ ID NO: 4,
   (B) a base sequence represented by SEQ ID NO: 5; and (c) a base sequence in which 1 to 5 bases of the base sequence represented by SEQ ID NO: 6 in SEQ ID NO: 4 or 5 are substituted.
3. The binary gene expression system according to claim 2, wherein the base sequence represented by SEQ ID NO: 4 is the base sequence represented by SEQ ID NO: 7.
4. The binary gene expression system according to claim 2, wherein the base sequence represented by SEQ ID NO: 5 is the base sequence represented by SEQ ID NO: 8.
5. The binary gene expression system according to any one of claims 1 to 4, wherein the transcriptional activity unit is ARII or VP16.
6. The binary gene expression system according to any one of claims 3 to 5, wherein the TAL recognition sequence is composed of a repetitive sequence in which 5 to 25 nucleotide sequences represented by SEQ ID NO: 9 including the target nucleotide sequence are linked.
7. A transformant comprising the first expression unit in the binary gene expression system according to any one of claims 1 to 6.
8. A transformant comprising the second expression unit of the binary gene expression system according to any one of claims 1 to 5, excluding the second expression unit of the binary gene expression system according to claim 6.
9. A transformant comprising the first expression unit and the second expression unit in the binary gene expression system according to any one of claims 1 to 6.
10. The transformant according to any one of claims 7 to 9, wherein the transformant is a Lepidoptera insect.
11. The transformant according to claim 10, wherein the Lepidoptera insect is a silkworm.
12. The step of mating the transformant having the first expression unit according to claim 7 and the transformant having the second expression unit according to claim 8, and the first and second expression units after the mating step A method for producing an individual having enhanced expression efficiency of a target gene or a fragment thereof, comprising a step of selecting an individual having the target gene.

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