Translation enhancers and their use

Abstract

To provide a novel translation enhancer for plants.SOLUTION: A translation enhancer consists of a polynucleotide which consists of a specific base sequence, a polynucleotide which consists of a base sequence in which 1 base or more and 20 bases or less are deleted, substituted or added in the polynucleotide, and has translation enhancer activity, or a polynucleotide which consists of a base sequence having 90% or more of sequence identity as the polynucleotide, and has translation enhancer activity.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a translation enhancer and its use.

Background Art

[0002] Techniques for producing useful proteins in plants using recombinant genes are known. So far, several base sequences derived from the 5'untranslated region (5'UTR) have been isolated from plants as translation enhancers that enhance gene translation efficiency.

[0003] For example, Patent Document 1 describes that a translation enhancer sequence exists in the 5'leader sequence of the ferredoxin-binding subunit gene of Nicotiana sylvestris.

[0004] Patent Document 2 describes that the full-length 5'UTR sequence of the alcohol dehydrogenase gene of Nicotiana tabacum has a function of increasing translation in dicotyledonous and monocotyledonous plants.

[0005] Patent Document 3 describes that a translation enhancer sequence exists in the 5'UTR sequence of the OsMac1 gene of Oryza sativa.

[0006] Patent Document 4 describes that translation enhancer sequences exist in the 5'UTR sequences of the OsMac2 gene and OsMac3 gene of Oryza sativa.

[0007] Patent Document 5 describes that H1-1, which is the 5'UTR of the At1g06760 gene of Arabidopsis thaliana, and COR47, which is the 5'UTR of the At1g20440 gene, improve translation efficiency in plants.

[0008] In addition, a plant transformation vector equipped with a translation enhancer has been put on the market (for example, Non-Patent Document 1). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Application Publication No. 08-256777 [Patent Document 2] Japanese Patent Publication No. 2003-079372 [Patent Document 3] Japanese Patent Publication No. 2011-103833 [Patent Document 4] International Publication No. 2015 / 174414 [Patent Document 5] International Publication No. 2016 / 175132 [Non-patent literature]

[0010] [Non-Patent Document 1] Takara Bio pRI 101 DNA Series High Expression Vector for Plant Transformation, [Accessed October 27, 2023], Internet<URL:https: / / catalog.takara-bio.co.jp / product / basic_info.php?unitid=U100006214> [Overview of the project] [Problems that the invention aims to solve]

[0011] Even when using the same translation enhancer, the effect varies depending on the species, cell type, and gene type, and in some cases, no effect is observed at all. Because the rules governing this are not yet understood, it is necessary to find and use the appropriate one from many options. However, currently, the available types are limited and do not adequately meet the demand. In many cases, due to the limited options, dicotyledonous plants are forced to blindly use the 5'UTR of Arabidopsis thaliana ADH, and monocotyledonous plants to use the 5'UTR of rice ADH2. To adequately meet the demand, an expansion of novel translation enhancers is needed.

[0012] One aspect of the present invention aims to provide a novel plant translation enhancer. [Means for solving the problem]

[0013] As a result of diligent research to solve the above problems, the inventors of this invention have discovered several novel translation enhancers (nucleotide sequences) from the 5'UTR of the gene for an enzyme that catalyzes the first reaction of the pyruvate-based fermentation pathway in rice, which can increase the protein production per copy number of DNA or mRNA when linked to the target structural gene, and have completed the present invention.

[0014] In other words, in order to solve the above problems, a translation enhancer according to one aspect of the present invention is characterized by comprising any one of the following polynucleotides (a) to (i): (a) Polynucleotides consisting of the base sequence of Sequence ID No. 1; (b) Polynucleotides consisting of the base sequence of Sequence ID No. 2; (c) A polynucleotide consisting of the base sequence of Sequence ID No. 3; (d) Polynucleotides consisting of the base sequence of Sequence ID No. 4; (e) A polynucleotide consisting of the base sequence of Sequence ID No. 5; (f) A polynucleotide consisting of the base sequence of Sequence ID No. 6; (g) A polynucleotide consisting of the base sequence of Sequence ID No. 7; (h) A polynucleotide having translational enhancer activity, comprising a base sequence in which one or more and 20 or fewer bases are deleted, substituted, or added in any one of the polynucleotides described in (a) to (g) above; (i) A polynucleotide having a base sequence that is 90% or more identical to any one of the polynucleotides described in (a) to (g) above, and which has translational enhancer activity. [Effects of the Invention]

[0015] According to one aspect of the present invention, a novel plant translation enhancer can be provided.

Brief Description of the Drawings

[0016] [Figure 1] It is a construction diagram of a transformation plasmid prepared in the example. [Figure 2] It is a diagram showing the results of the example, and is a diagram showing the results of GUS activity measurement using a transient gene expression system. [Figure 3] It is a diagram showing the results of the example, and is a diagram showing the analysis results by Western blotting. [Figure 4] It is a diagram explaining the sequence of the junction between 5'UTR and GUS in the transformation plasmid prepared in the example. [Figure 5] It is a diagram showing the results of the example, and is a diagram showing the results of GUS activity measurement using a transient gene expression system.

Modes for Carrying Out the Invention

[0017] Hereinafter, one aspect of the present invention will be described in detail. Unless otherwise specified in this specification, "A~B" representing a numerical range is intended to mean "A or more and B or less".

[0018] Where used herein, the term “polynucleotide” is interchangeable with “nucleic acid” or “nucleic acid molecule” and refers to a polymer of nucleotides. Similarly, “base sequence” is interchangeable with “nucleic acid sequence” or “nucleotide sequence” and refers to a sequence of deoxyribonucleotides or ribonucleotides unless otherwise specified. Here, polynucleotides may exist in the form of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA). When polynucleotides are in the form of RNA, they consist of a base sequence in which T (thymine) is replaced with U (uracil) in the DNA base sequence. DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). Where used herein, base notation shall be in the single-letter notation as defined by IUPAC and IUB, as appropriate. Furthermore, in this specification, "3' end" means the downstream end region or location of the gene sequence in question, and "5' end" means the upstream end region or location of the gene sequence in question.

[0019] [1. Translation Enhancer] A translation enhancer according to one aspect of the present invention comprises any one of the following polynucleotides: (a) to (i): (a) Polynucleotides consisting of the base sequence of Sequence ID No. 1; (b) Polynucleotides consisting of the base sequence of Sequence ID No. 2; (c) A polynucleotide consisting of the base sequence of Sequence ID No. 3; (d) Polynucleotides consisting of the base sequence of Sequence ID No. 4; (e) A polynucleotide consisting of the base sequence of Sequence ID No. 5; (f) A polynucleotide consisting of the base sequence of Sequence ID No. 6; (g) A polynucleotide consisting of the base sequence of Sequence ID No. 7; (h) A polynucleotide having translational enhancer activity, comprising a base sequence in which one or more and 20 or fewer bases are deleted, substituted, or added in any one of the polynucleotides described in (a) to (g) above; (i) A polynucleotide having a base sequence that is 90% or more identical to any one of the polynucleotides described in (a) to (g) above, and which has translational enhancer activity.

[0020] According to one aspect of the present invention, a translation enhancer can be linked to a target structural gene to increase the protein production per copy number of DNA or mRNA of that structural gene. While there is growing interest in technologies that use recombinant genes to produce useful proteins in plants, using a translation enhancer according to one aspect of the present invention in such technologies can improve the efficiency of useful protein production in plants.

[0021] Furthermore, the translation enhancer according to one aspect of the present invention has the unique characteristic of showing high effectiveness not only in rice but also in potatoes, making it a rare translation enhancer that can be used in potatoes. Thus, the translation enhancer according to one aspect of the present invention is expected to have different compatibility with different types of biological species compared to conventional translation enhancers. For example, the translation enhancer according to one aspect of the present invention is expected to be effective in monocots and dicots other than rice.

[0022] In this specification, "translation enhancer" means a polynucleotide having translation enhancer activity. The "translation enhancer activity" refers to the activity that increases the amount of protein produced per copy number of DNA or mRNA of a structural gene to which a polynucleotide acting as a translation enhancer is linked. In this specification, if a GUS activity measurement test is performed using the transient gene expression system described in the examples, and the GUS activity measured under conditions in which the target polynucleotide is linked to the 5' end of the structural gene is higher than the GUS activity measured under conditions without a 5'UTR, then the target polynucleotide can be said to have translation enhancer activity.

[0023] ((a)~(i) polynucleotides) The polynucleotides described in (a) to (g) above were all isolated from the 5'UTR of the gene for the enzyme that catalyzes the first reaction of the pyruvate-based fermentation pathway in rice (scientific name: Oryza sativa, variety: Koshihikari).

[0024] More specifically, the polynucleotides (a) to (e) above are all polynucleotides isolated from the 5'UTR of the gene encoding pyruvate decarboxylase (PDC) in rice (scientific name: Oryza sativa, variety: Koshihikari). Furthermore, the polynucleotides (f) to (g) above are all polynucleotides isolated from the 5'UTR of the gene encoding lactate dehydrogenase (LDH) in rice (scientific name: Oryza sativa, variety: Koshihikari).

[0025] For the sake of explanation, in this specification, the PDC genes of the rice (scientific name: Oryza sativa, variety: Koshihikari) from which the polynucleotides (a) to (e) above were isolated will be referred to as genes A to E, respectively. Similarly, the LDH genes of the rice (scientific name: Oryza sativa, variety: Koshihikari) from which the polynucleotides (f) to (g) above were isolated will be referred to as genes F to G, respectively.

[0026] Here, the information for genes A through G is shown in Table 1. [Table 1]

[0027] In plants under anaerobic conditions, two metabolic pathways are at work to obtain energy without relying on oxygen: ethanol fermentation and lactic acid fermentation, both using pyruvate as a substrate. The enzymes that catalyze the chemical change of pyruvate as the first reaction in each metabolic pathway are PDC (catalyzing the ethanol fermentation pathway) and LDH (catalyzing the lactic acid fermentation pathway).

[0028] Based on the results of the amino acid sequence homology search, it is believed that the PDC genes in rice (scientific name: Oryza sativa, variety: Koshihikari) consist only of genes A, B, C, D, and E, and the LDH genes in rice (scientific name: Oryza sativa, variety: Koshihikari) consist only of genes F and G.

[0029] In the KEGG ORTHOLOGY metabolic database, only genes A, B, C, D, and E are listed as PDC genes in rice, and only genes F and G are listed as LDH genes in rice [Accessed October 27, 2023]. <URL:https: / / www.genome.jp / entry / K01568+4.1.1.1+R00014;https: / / www.genome.jp / entry / K00016+1.1.1.27+R00703> .

[0030] From the above, genes A to G shown in Table 1 share the common characteristic of being genes for enzymes that catalyze the first reaction of the pyruvate-based fermentation pathway in rice (scientific name: Oryza sativa, variety: Koshihikari). Furthermore, all of the polynucleotides (a) to (e) share the common characteristic of being polynucleotides isolated from the 5'UTR of the genes for enzymes that catalyze the first reaction of the pyruvate-based fermentation pathway in rice (scientific name: Oryza sativa, variety: Koshihikari). In other words, one embodiment of the present invention was completed based on the novel finding that polynucleotides isolated from the 5'UTR of the genes for enzymes that catalyze the first reaction of the pyruvate-based fermentation pathway in rice (scientific name: Oryza sativa, variety: Koshihikari) possess translational enhancer activity.

[0031] The polynucleotide constituting the translational enhancer according to one aspect of the present invention may be any of the polynucleotides described in (a) to (g) above. The level of translational enhancer activity of the translational enhancer according to one aspect of the present invention is not particularly limited, but in the GUS activity measurement test using the transient gene expression system described in the examples, the GUS activity measured under the condition that the target polynucleotide is linked to the 5' end of the structural gene is preferably 1.5 or higher, more preferably 2 or higher, more preferably 3 or higher, more preferably 4 or higher, more preferably 5 or higher, more preferably 6 or higher, more preferably 7 or higher, more preferably 10 or higher, more preferably 11 or higher, more preferably 12 or higher, more preferably 15 or higher, and even more preferably 20 or higher.

[0032] From the viewpoint of translational enhancer activity, the polynucleotide constituting the translational enhancer according to one aspect of the present invention is preferably one of the polynucleotides of (a), (f), or (g), and more preferably one of the polynucleotides of (a) or (f). Translational enhancers composed of these polynucleotides have particularly high translational enhancer activity and are particularly excellent as translational enhancers.

[0033] The polynucleotide constituting the translation enhancer according to one aspect of the present invention may be the polynucleotide described in (h) or (i) above.

[0034] The polynucleotide in (h) is a polynucleotide having translational enhancer activity, consisting of a base sequence in which one or more and 20 or fewer bases are deleted, substituted, or added in any one of the polynucleotides in (a) to (g). The polynucleotide in (h) is intended to be a functionally equivalent variant, variant, partial nucleotide, or fusion nucleotide with another nucleotide of any one of the polynucleotides in (a) to (g), and its base sequence is not limited as long as it has translational enhancer activity.

[0035] Here, the number of bases that may be deleted, substituted, or added is not limited as long as it does not cause the loss of the above function. For example, it refers to a number that can be deleted, substituted, or added by known mutagenesis methods such as site-directed mutagenesis, and is, for example, 15% or less of the total number of bases of any one of the polynucleotides (a) to (g) above, preferably 10% or less, and more preferably 5% or less (e.g., 5%, 4%, 3%, 2%, or 1%). For example, in the case of the polynucleotide (a) above, the number of such bases is preferably 20 or less, preferably 15 or less, more preferably 10 or less, and most preferably 5 or less (e.g., 5, 4, 3, 2, or 1 base). Similarly for the polynucleotides (b) to (g) above, the number of bases can be determined in the range of 1 to 20 bases such that the number of bases that may be deleted, substituted, or added is 15% or less of the total number of bases, preferably 10% or less, and more preferably 5% or less.

[0036] In this specification, “mutant” primarily refers to a mutant artificially introduced by methods such as site-directed mutagenesis, but may also refer to a similar naturally occurring mutant. In this specification, “functionally equivalent” means that a mutant, variant, partial nucleotide, fusion nucleotide with another nucleotide, etc. (hereinafter referred to as “mutant, etc.”) has a biological or biochemical function equivalent to (identical and / or similar to) any one of the polynucleotides (a) to (g) above. More specifically, it is intended that the mutant, etc. has translational enhancer activity. It is sufficient that the mutant, etc. has translational enhancer activity, and the level of such activity is not particularly limited. For example, the translational enhancer activity of the mutant, etc. may be at a level equivalent to that of any one of the polynucleotides (a) to (g) above, or it may be at a level exceeding the activity of any one of the polynucleotides (a) to (g) above.

[0037] As a method for obtaining a variant of any one of the polynucleotides described above (a) to (g), commonly used polynucleotide modification methods can be used. Whether the mutated polynucleotide has the desired function can be determined by measuring the translational enhancer activity of the mutated polynucleotide.

[0038] The polynucleotide in (i) above is a polynucleotide whose base sequence has 90% or more sequence identity with any one of the polynucleotides in (a) to (g) above, and which has translational enhancer activity. Similar to the polynucleotide in (h) above, the polynucleotide in (i) above is intended to be a functionally equivalent variant or other polynucleotide of any one of the polynucleotides in (a) to (g) above, and its base sequence is not limited as long as it has translational enhancer activity.

[0039] Base sequence identity means that the entire base sequence has sequence identity of at least 90%, more preferably 95% or more (for example, 95%, 96%, 97%, 98%, 99% or more). Base sequence identity can be calculated using conventionally known software used for the purpose of calculating base sequence identity, for example, using the genetic information software GENETYX Ver.9 (manufactured by Genetics Co., Ltd.).

[0040] From the viewpoint of further improving translational enhancer activity, it is preferable that the translational enhancer according to one aspect of the present invention has a polynucleotide consisting of the base sequence ATGNNNNNN (where N is any base) ligated to the 3' end of any one of the polynucleotides described in (a) to (i). By ligating the ATGNNNNNN base sequence ligated to the 3' end of the translational enhancer according to one aspect of the present invention in-frame upstream of the translation start codon (ATG) of the base sequence of the structural gene, the translation reading efficiency is improved, and as a result, the translational enhancer activity of the translational enhancer according to one aspect of the present invention can be further improved.

[0041] In a polynucleotide consisting of the base sequence ATGNNNNNN, the base corresponding to N is not particularly limited. For example, it can be a restriction enzyme recognition sequence of 6 bases or a sequence of 6 bases produced by ligating the cleavage sites of two types of restriction enzymes, and more specifically, it may be a base sequence such as ATGAGATCT;ATGGGATCT.

[0042] A translation enhancer according to one aspect of the present invention can be obtained by isolating it from a cDNA library of any organism. The organism is not particularly limited, but examples include grasses, and among them, rice (scientific name: Oryza sativa, variety: Koshihikari). Alternatively, a translation enhancer according to one aspect of the present invention may be prepared by chemically synthesizing a polynucleotide chain according to a known polynucleotide synthesis method.

[0043] [2. Recombinant Vectors] A recombinant vector according to one embodiment of the present invention includes any of the translation enhancers described above. The translation enhancers in a recombinant vector according to one aspect of the present invention have already been described and will not be repeated here.

[0044] The recombinant vector into which the translation enhancer according to one aspect of the present invention is inserted is not particularly limited, and conventionally known vectors used as vectors for plant transformation can be used. Examples of such vectors include pUC-type vectors, pBI-type vectors, and pPZP-type vectors.

[0045] A recombinant vector according to one embodiment of the present invention may further include a promoter and a structural gene. The structural gene is not particularly limited, and any gene encoding the target protein can be appropriately selected. The structural gene can be prepared using known cloning methods.

[0046] The promoter is not particularly limited and can be appropriately selected from conventionally known promoters used for protein expression in plants, depending on the purpose. For example, as a constitutive expression promoter, the cauliflower mosaic virus 35S promoter can be used.

[0047] A recombinant vector according to one embodiment of the present invention may further include, as necessary, a selection marker, a terminator (e.g., NOS), a multicloning site, a DNA sequence encoding a protein tag, etc. Examples of selection markers include antibiotic resistance genes (e.g., ampicillin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, hygromycin resistance gene, etc.). Examples of protein tags include His, FLAG, HA, c-Myc, GFP, etc.

[0048] In a recombinant vector according to one aspect of the present invention, the translation enhancer is preferably inserted between the promoter and the structural gene so that it can function appropriately as a translation enhancer. The recombinant vector according to one embodiment of the present invention may include a nucleic acid construct in which a translation enhancer is inserted between the promoter and the structural gene. In the nucleic acid construct, the translation enhancer is preferably ligated downstream (towards the 3' end) of the promoter. Furthermore, the translation enhancer is preferably ligated upstream (towards the 5' end) of the translation start codon of the structural gene. Such nucleic acid constructs are also included in the scope of the present invention.

[0049] A recombinant vector according to one aspect of the present invention can be prepared according to known genetic engineering techniques. For example, the translation enhancer or nucleic acid construct can be introduced into a vector by amplifying it using polymerase chain reaction (PCR) with primers that have restriction enzyme sites attached, treating it with a restriction enzyme, and ligating it to an appropriate position on a restriction enzyme-treated vector.

[0050] [3. Transformed product] A transformant into which a recombinant vector according to one aspect of the present invention has been introduced is also included in the scope of the present invention. The recombinant vector according to one aspect of the present invention has already been described, so it will not be described again here.

[0051] In this specification, “transformed organism” means a cell into which an exogenous gene (e.g., a structural gene) has been introduced and which has come to exhibit the trait of the introduced gene, and an organism containing such a cell. The cell may be in the form of a tissue or organ. “Exhibiting the trait of the introduced gene” means that the protein encoded by the exogenous gene is expressed in the cell into which the exogenous gene has been introduced. A “transformed organism” may be an organism, a descendant of a cell, or a clone, produced as a transformant, as long as it expresses the protein encoded by the introduced exogenous gene. Furthermore, a “transformed organism” may be a “transient transformant” obtained by transient transformation, or a “constitutive transformant” obtained by constitutive transformation.

[0052] A transformant according to one aspect of the present invention can be obtained by introducing a recombinant vector according to one aspect of the present invention into a host such that a target structural gene can be expressed. The host is not particularly limited to any species as long as it can accept the recombinant vector and express the structural gene carried by the introduced recombinant vector. Examples of species that can be used as hosts include bacteria, yeast, plants, insects, and mammals. The translational enhancer according to one aspect of the present invention has excellent translational enhancer activity in plants, and among these, plants can be suitably used as a host.

[0053] When the host is a plant, the translation enhancer according to one aspect of the present invention is expected to be effective in monocots and dicots other than rice, so the type of plant used as the host is not particularly limited. For example, the host may be either a monocot or a dicot.

[0054] Examples of monocotyledonous plants include grasses such as rice, wheat, barley, foxtail millet, barnyard millet, corn, and sorghum.

[0055] Examples of dicotyledonous plants include Arabidopsis thaliana and rapeseed from the Brassicaceae family; tobacco, tomato, and potato from the Solanaceae family; melon and pumpkin from the Cucurbitaceae family; soybeans from the Fabaceae family; cotton from the Malvaceae family; chrysanthemums from the Asteraceae family; tea from the Theaceae family; and grapes from the Vitaceae family.

[0056] A transformant according to one aspect of the present invention may be any one selected from the group consisting of plant cells, plant tissues, plant organs, plant bodies, and plant propagation materials. Plant cells may be cultured cells. Plant tissues are not particularly limited, but examples include epidermis, phloem, parenchyma, xylem, and vascular bundles. Plant organs are not particularly limited, but examples include leaves, petals, stems, roots, and rhizomes. Plant propagation materials include, for example, seeds, scions, plants, callus, and protoplasts. These cells, tissues, organs, and propagation materials can be obtained by known methods.

[0057] The method for introducing recombinant vectors into the host is not particularly limited, and an appropriate method can be selected from conventionally known methods used for producing plant transformants, depending on the type of host and vector. Examples include electroporation, polyethylene glycol, Agrobacterium binary vector method, and particle gun method.

[0058] Confirmation of whether or not a recombinant vector has been introduced into the host can be performed by known methods such as PCR, Southern hybridization, and Northern hybridization.

[0059] [3. Methods for producing recombinant proteins] A method for producing recombinant protein according to one aspect of the present invention includes a step of culturing a transformant into which the above-described recombinant vector has been introduced (hereinafter also referred to as the "culturing step"). According to the method for producing recombinant protein according to one aspect of the present invention, the function of the above-described translational enhancer is exerted, and therefore the amount of protein produced per copy number of DNA or mRNA of the structural gene can be increased. The recombinant vector and transformant according to one aspect of the present invention have already been described, so they will not be described again here.

[0060] The method for culturing the transformant is not particularly limited, and an appropriate method can be appropriately selected from conventionally known culture methods according to the type of host. Hereinafter, "cultivation" means growing the transformant, and it is sufficient that the transformant is at least maintained. By culturing the transformant, the target protein can be obtained from the culture. When the transformant is an entire plant, plant propagation material, plant organ, or plant tissue, "cultivation" is a concept that also includes cultivation, and the culture is also called a "cultivated product."

[0061] A method for producing recombinant protein according to one aspect of the present invention may further include a step of obtaining a transformant into which a recombinant vector according to one aspect of the present invention has been introduced, before the culture step. Furthermore, a step of collecting the target protein from the obtained culture may further include a step of collecting the target protein from the culture. Conventional protein purification methods can be used as the method for collecting the target protein from the culture.

[0062] Recombinant proteins can also be produced by methods using known in vitro cell-free protein synthesis systems. For example, a method for producing a recombinant protein according to one aspect of the present invention may include the steps of synthesizing translational RNA from a nucleic acid construct in which the above-mentioned translational enhancer is inserted between a promoter and a structural gene, and contacting the translational RNA with an in vitro cell-free protein synthesis reaction solution.

[0063] A translation enhancer and a nucleic acid construct containing the translation enhancer according to one aspect of the present invention have already been described, so a repetition of that description will not be provided here. The method for synthesizing translation RNA from the nucleic acid construct, and the method for synthesizing a protein using the translation RNA with the in vitro cell-free protein synthesis system, are not particularly limited. For example, this can be done by known methods such as the method described in Patent Document 3 (Japanese Patent Application Publication No. 2011-103833).

[0064] [4. Method for producing highly protein-productive transformants] A method for producing a protein-high productivity transformant according to one aspect of the present invention includes the step of obtaining a transformant into which the above-mentioned recombinant vector has been introduced. According to the method for producing a protein-high productivity transformant according to one aspect of the present invention, a transformant into which the above-mentioned recombinant vector has been introduced can be obtained, making it possible to produce a protein-high productivity transformant with increased protein production per copy number of structural gene DNA or mRNA. The recombinant vector and transformant according to one aspect of the present invention have already been described, so they will not be described again here.

[0065] A transformant produced by a method for producing highly protein-productive transformants according to one aspect of the present invention can be suitably used as a transformant into which a recombinant vector has been introduced in the protein production method described above.

[0066] 〔summary〕 A translation enhancer according to Embodiment 1 of the present invention has a configuration consisting of one of the following polynucleotides: (a) Polynucleotides consisting of the base sequence of Sequence ID No. 1; (b) Polynucleotides consisting of the base sequence of Sequence ID No. 2; (c) A polynucleotide consisting of the base sequence of Sequence ID No. 3; (d) Polynucleotides consisting of the base sequence of Sequence ID No. 4; (e) A polynucleotide consisting of the base sequence of Sequence ID No. 5; (f) A polynucleotide consisting of the base sequence of Sequence ID No. 6; (g) A polynucleotide consisting of the base sequence of Sequence ID No. 7; (h) A polynucleotide having translational enhancer activity, comprising a base sequence in which one or more and 20 or fewer bases are deleted, substituted, or added in any one of the polynucleotides described in (a) to (g) above; (i) A polynucleotide having a base sequence that is 90% or more identical to any one of the polynucleotides described in (a) to (g) above, and which has translational enhancer activity.

[0067] In the second aspect of the present invention, the translation enhancer is preferably composed of one of the polynucleotides (a), (f), or (g) described above in the first aspect of the present invention.

[0068] The vector according to aspect 3 of the present invention has a configuration that includes the translation enhancer described in aspect 1 or 2 above.

[0069] A vector according to aspect 4 of the present invention may have a configuration that includes a nucleic acid construct in which the translation enhancer described in aspect 1 or 2 above is inserted between the promoter and the structural gene.

[0070] The transformant according to aspect 5 of the present invention may have a configuration in which the recombinant vector described in aspect 3 or 4 above is introduced.

[0071] In the embodiment 6 of the present invention, the transformant may be configured such that, in embodiment 5 above, the transformant is selected from the group consisting of plant cells, plant tissue, plant organs, plant bodies, and plant propagation materials.

[0072] A protein production method according to aspect 7 of the present invention may also include a step of culturing a transformant into which the recombinant vector described in aspect 3 or 4 has been introduced.

[0073] A method for producing a protein-high productivity transformant according to aspect 8 of the present invention may include a step of obtaining a transformant into which the recombinant vector described in aspect 3 or 4 has been introduced.

[0074] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in each embodiment are also included within the technical scope of the present invention. [Examples]

[0075] An embodiment of the present invention will be described below, but the present invention is not limited to these embodiments.

[0076] [1] Construction of plasmid DNA <Experimental Method> Plasmid DNA was constructed following the steps (1) to (7) below.

[0077] (1) cDNA clone sequences of genes A to G (see Table 1 above) were obtained from the public database (RAP-DB, https: / / rapdb.dna.affrc.go.jp / ). (2) DNA primers were synthesized for PCR amplification of the cDNA from the 5' end to the start codon (ATG). (For genes A, B, C, D, and E, the recognition sequences for restriction enzyme SpeI were added upstream of the 5'UTR, and for restriction enzyme BglII immediately after the start codon. For genes F and G, the recognition sequences for restriction enzyme XbaI were added upstream of the 5'UTR, and for restriction enzyme BamHI immediately after the start codon.) (3) Using the synthesized DNA primers, PCR was performed with rice (scientific name: Oryza sativa, variety: Koshihikari) chromosomal DNA as a template to prepare DNA fragments containing the 5'UTR and start codon of genes A to G. (4) Plasmid DNA in which CaMV35S Pro, GUS-3xFLAG, and Nos Ter were inserted into the multicloning site of a pUC vector (containing the recognition sequences of restriction enzymes BamHI, XhoI, SpeI, XbaI, SalI, and BglII in this order between CaMV35S Pro and GUS-3xFLAG) was cleaved with XbaI and BglII. (5) DNA fragments containing the 5'UTR and start codon of genes A, B, C, D, and E were cleaved with SpeI and BglII. Similarly, DNA fragments containing the 5'UTR and start codon of genes F and G were cleaved with XbaI and BamHI. The resulting DNA fragments were then inserted into plasmid DNA cleaved with XbaI and BglII, respectively, by ligation. (The cut surfaces of XbaI and SpeI, and BglII and BamHI, are adherable. These restriction enzyme combinations were used because some 5'UTRs contained SpeI or BglII recognition sequences.) (6) The prepared plasmid DNA was analyzed using a DNA sequencer to confirm that there were no errors in the sequence. (7) The prepared plasmid DNA was introduced into E. coli and extracted and purified using the NucleoBond® Xtra Midi kit (TaKaRa).

[0078] Figure 1 shows the structure diagram of the transformed plasmid. The meanings of the abbreviations in Figure 1 are as follows: [Explanation of abbreviations] CaMV35S Pro: Cauliflower Mosaic Virus 35S Promoter GUS-3xFLAG: A β-glucuronidase gene (GUS gene) with a FLAG tag sequence added to its C-terminus. Nos T: Nopalyn synthase gene terminator lacZ: A β-galactosidase gene containing multiple cloning sites. AmpR: Ampicillin resistance gene pUC ori: The origin of replication in E. coli.

[0079] [2] Measurement of GUS activity using a transient gene expression system <Experimental Method> The enzymatic activity of the GUS protein was measured according to the following procedures (1) to (8).

[0080] (1) Protoplasts were prepared from the above-ground parts of rice seedlings (scientific name: Oryza sativa, variety: Nipponbare) according to the method described in the reference (Yang Zhang et al., Plant Methods. 2011 Sep 30;7(1):30. doi: 10.1186 / 1746-4811-7-30.) and dispensed into tubes in equal volumes. (2) 10 μg of plasmid DNA was added to each tube and transfection was performed using polyethylene glycol. A vector plasmid without a 5'UTR insertion was used as a control group. (Three tubes were tested for each plasmid. n=3). (3) After incubation at 28°C for 16 hours, the cells were collected by centrifugation. (4) Equal volumes of extraction buffer [50 mM sodium phosphate (pH 7), 10 mM EDTA, 0.1% sodium N-lauroyl sarcosinate, 0.1% TritonX-100, 0.1% mercaptoethanol] were added to each tube, and the cells were disrupted by sonication. (5) The supernatant collected by centrifugation was mixed with an equal volume of GUS reaction solution (a solution of 4-methylumbelliferyl-β-D-glucuronide (4-MUG) dissolved in extraction buffer to a concentration of 1 mM) and incubated at 37°C for 1 hour. (6) Equal amounts of 0.2 M sodium carbonate were added to each tube to stop the reaction. (7) Equal amounts were dispensed from each tube into a fluorescence measurement plate. (8) The fluorescence emitted by methylumbelliferone, which is catalyzed by GUS and produced from 4-MUG, was measured using a multiplate reader, and this value was compared as GUS activity.

[0081] (result) The results are shown in Figure 2. Figure 2 shows the results of GUS activity measurement using a transient gene expression system. In Figure 2, the values ​​for the control group using a vector plasmid without a 5'UTR insertion are set to 1 for relative evaluation, and the standard deviation for n=3 is shown as error bars.

[0082] As shown in Figure 2, 5'UTR insertion of genes A-G increased the production of GUS protein per unit of introduced DNA compared to the control group. In particular, 5'UTR insertion of genes A, F, and G resulted in a significant increase in GUS protein production.

[0083] [3] Comparison of GUS proteins by Western blotting <Experimental Method> The amount of GUS protein was compared by Western blotting according to the following steps (1) to (8).

[0084] (1) Protoplasts were prepared from the above-ground parts of potato (scientific name: Solanum tuberosum, variety: Waseshiro) according to the method described in the reference (Yang Zhang et al., Plant Methods. 2011 Sep 30;7(1):30. doi: 10.1186 / 1746-4811-7-30.) and dispensed into tubes in equal volumes. (2) 10 μg of plasmid DNA was added to each tube and transfection was performed using polyethylene glycol. A vector plasmid without a 5'UTR was used as the control group A. (3) After incubation at 28°C overnight, the cells were collected by centrifugation. (4) Equal volumes of SDS-PAGE sample buffer were added to lyse the cells. (5) Equal volumes of the supernatant after centrifugation were subjected to electrophoresis on SDS-polyacrylamide gel. (6) Proteins were blotted from the gel after electrophoresis onto a PVDF membrane. (7) A peroxidase-conjugated anti-FLAG tag antibody (MBL, M185-7) was specifically bound to the FLAG-tagged GUS protein on the membrane, and a substrate solution that chemiluminescent due to peroxidase activity was added, and the signal was detected using an X-ray film. (8) After detecting the chemiluminescence signal, the membranes were stained with Coomassie Brilliant Blue (CBB) solution, and it was confirmed that there were no significant differences in the concentration of the electrophoretic proteins between the samples.

[0085] (result) The results are shown in Figure 3. Figure 3 shows the results of analysis by Western blotting. As shown in Figure 3, 5'UTR insertion of genes A-G increased the production of GUS protein per unit of introduced DNA compared to control group A. In particular, 5'UTR insertion of genes A, F, and G significantly increased the production of GUS protein compared to control group B.

[0086] Furthermore, it was confirmed that the 5'UTR of genes A-G possesses translational enhancer activity not only in rice, a monocotyledonous plant, but also in potato, a dicotyledonous plant. Therefore, it was found that the 5'UTR of genes A-G can be expected to have translational enhancer effects in plant species other than rice.

[0087] [4] Examination of the junction between the 5'UTR and GUS As shown in Figure 4, the plasmid DNA constructed in [1] had a structure called "ATGNNNNNN (where N is any base)" (hereinafter referred to as "structure α") in-frame linked between the 5'UTR of genes A-G and the translation start codon (ATG) of the structural gene. Therefore, in order to investigate the effect of this structure α on the translational enhancer activity of the 5'UTR, plasmid DNA without structure α was constructed for genes A and F.

[0088] The constructed plasmid DNA was used to measure GUS activity in the same manner as described in [2] above.

[0089] (result) The results are shown in Figure 5. Figure 5 shows the results of GUS activity measurement using a transient gene expression system. In Figure 5, the values ​​for the control group using a vector plasmid without a 5'UTR insertion are set to 1 for relative evaluation, and the standard deviation for n=3 is shown as error bars.

[0090] As shown in Figure 5, insertion of the 5'UTR of gene A or F increased the production of GUS protein per unit of introduced DNA compared to the control group. Therefore, it was found that the 5'UTRs of genes A and F exert their full effect as translational enhancers even without structure α. Furthermore, it was thought that structure α contributes to enhancing the activity of the 5'UTRs of genes A and F as translational enhancers. [Industrial applicability]

[0091] This invention is expected to be used as a translation enhancer in plants.

Claims

1. Translation enhancers consisting of the following polynucleotides: (a), (f), or (g): (a) Polynucleotides consisting of the base sequence of Sequence ID No. 1; (f) A polynucleotide consisting of the base sequence of Sequence ID No. 6; (g) A polynucleotide consisting of the base sequence of Sequence ID No.

7.

2. A nucleic acid construct comprising the translation enhancer described in claim 1, inserted between a promoter and a structural gene, If the translation enhancer is a translation enhancer consisting of the polynucleotide of (a), the structural gene is not pyruvate decarboxylase, and if the translation enhancer is a translation enhancer consisting of the polynucleotide of (f) or (g), the structural gene is not lactate dehydrogenase. Recombinant vector.

3. A transformer into which the recombinant vector described in claim 2 has been introduced.

4. The transformant according to claim 3, wherein the transformant is one selected from the group consisting of plant cells, plant tissues, plant organs, plant bodies, and plant reproductive materials.

5. A method for producing a recombinant protein, comprising the step of culturing a transformant into which the recombinant vector described in claim 2 has been introduced.

6. A method for producing a protein-high productivity transformant, comprising the step of obtaining a transformant into which the recombinant vector described in claim 2 has been introduced.

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