Translation enhancer and use of the same

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 nucleotide sequences derived from the 5'untranslated region (5'UTR) have been isolated from plants as translation enhancers that enhance the translation efficiency of genes.

[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 the 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

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0010]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0011] Even when the same translation enhancer is used, the effects exhibited vary depending on the biological species, cell type, and gene type, and in some cases, no effect is observed at all. Since this regularity has not been elucidated, it is necessary to search for and use a suitable one from many options, but currently, the types provided are few and cannot meet the demand. In many cases, due to the limited number of options, in the case of dicotyledonous plants, the 5'UTR of Arabidopsis thaliana ADH and in the case of monocotyledonous plants, the 5'UTR of rice ADH2 are blindly used. To sufficiently meet the demand, an expansion of new translation enhancers is required.

[0012] One aspect of the present invention aims to provide a novel translation enhancer for plants.

Means for Solving the Problems

[0013] As a result of intensive studies to solve the above problems, the present inventors have found a novel translation enhancer (base sequence) that can increase the protein production amount per copy number of its DNA or mRNA by linking it to the target structural gene. A plurality of them were found from the 5'UTR of the gene encoding the enzyme that catalyzes the first reaction of the fermentation pathway using pyruvic acid as a substrate in rice, and the present invention has been completed.

[0014] That is, in order to solve the above problems, the translation enhancer according to one aspect of the present invention is characterized by comprising any one of the following polynucleotides (a) to (i): (a) A polynucleotide consisting of the base sequence of SEQ ID NO: 1; (b) A polynucleotide consisting of the base sequence of SEQ ID NO: 2; (c) A polynucleotide consisting of the base sequence of SEQ ID NO: 3; (d) A polynucleotide consisting of the base sequence of SEQ ID NO: 4; (e) A polynucleotide consisting of the base sequence of SEQ ID NO: 5; (f) A polynucleotide consisting of the base sequence of SEQ ID NO: 6; (g) A polynucleotide consisting of the base sequence of SEQ ID NO: 7; (h) A polynucleotide consisting of a base sequence in which 1 to 20 bases are deleted, substituted or added in any one of the polynucleotides (a) to (g) above, and having translation enhancer activity; (i) A polynucleotide consisting of a base sequence having 90% or more sequence identity with any one of the polynucleotides (a) to (g) above, and having translation enhancer activity.

Effects of the Invention

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

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

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] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule" and is intended to mean a polymer of nucleotides. Also, "base sequence" is used interchangeably with "nucleic acid sequence" or "nucleotide sequence" and, unless otherwise specified, is intended to mean a sequence of deoxyribonucleotides or ribonucleotides. Here, the polynucleotide can exist in the form of DNA (e.g., cDNA or genomic DNA) or in the form of RNA (e.g., mRNA). When the polynucleotide is in the form of RNA, it consists of a base sequence in which T (thymine) in the DNA base sequence is replaced by U (uracil). The DNA or RNA may be double-stranded or single-stranded. The single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). As used herein, the base notation uses the one-letter notation defined by IUPAC and IUB as appropriate. Also, in this specification, the "3'-end" means the end region or position downstream of the target gene sequence, and the "5'-end" means the end region or position upstream of the target gene sequence.

[0019] [1. Translation enhancer] The translation enhancer according to one aspect of the present invention consists of any one of the following polynucleotides (a) to (i): (a) A polynucleotide consisting of the base sequence of SEQ ID NO: 1; (b) A polynucleotide consisting of the base sequence of SEQ ID NO: 2; (c) A polynucleotide consisting of the base sequence of SEQ ID NO: 3; (d) A polynucleotide consisting of the base sequence of SEQ ID NO: 4; (e) A polynucleotide consisting of the base sequence of SEQ ID NO: 5; (f) A polynucleotide consisting of the base sequence of SEQ ID NO: 6; (g) A polynucleotide consisting of the base sequence of SEQ ID NO: 7; (h) A polynucleotide comprising a nucleotide sequence in which 1 to 20 bases are deleted, substituted, or added in any one of the polynucleotides of (a) to (g) above, and having translational enhancer activity; (i) A polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with any one of the polynucleotides of (a) to (g) above, and having translational enhancer activity.

[0020] According to the translational enhancer according to one aspect of the present invention, by ligating it to a target structural gene, the protein production amount per copy number of the DNA or mRNA of the structural gene can be increased. Although techniques for producing useful proteins in plants using recombinant genes have attracted attention, by using the translational enhancer according to one aspect of the present invention in such techniques, the production efficiency of useful proteins in plants can be improved.

[0021] Furthermore, the translational enhancer according to one aspect of the present invention has a unique feature that it can exhibit a high effect not only in rice but also in potatoes, and it is rare as a translational enhancer that can be used in potatoes. Thus, it is expected that the translational enhancer according to one aspect of the present invention has a different compatibility with the type of biological species and the like from conventional translational enhancers. For example, the translational enhancer according to one aspect of the present invention can be expected to be effective in monocotyledonous and dicotyledonous plants other than rice.

[0022] In this specification, "translational enhancer" means a polynucleotide having translational enhancer activity. The "translational enhancer activity" refers to the activity of increasing the protein production amount per copy number of the DNA or mRNA of a structural gene to which a polynucleotide as a translational enhancer is ligated. In this specification, when performing a GUS activity measurement test using the transient gene expression system described in the examples, and the GUS activity measured under the condition that the target polynucleotide is ligated to the 5'-terminal side of the structural gene is higher than the GUS activity measured under the condition without 5'UTR, it can be said that the target polynucleotide has translational enhancer activity.

[0023] ((a) to (i) polynucleotides) Each of the polynucleotides of (a) to (g) is a polynucleotide isolated from the 5’UTR of the gene of an enzyme that catalyzes the first reaction of the fermentation pathway using pyruvate as a substrate in rice (scientific name: Oryza sativa, variety: Koshihikari).

[0024] More specifically, each of the polynucleotides of (a) to (e) is a polynucleotide isolated from the 5’UTR of the gene encoding pyruvate decarboxylase (PDC) in rice (scientific name: Oryza sativa, variety: Koshihikari). Also, each of the polynucleotides of (f) to (g) is a polynucleotide isolated from the 5’UTR of the gene encoding lactate dehydrogenase (LDH) in rice (scientific name: Oryza sativa, variety: Koshihikari).

[0025] In this specification, for convenience of explanation, the PDC genes of rice (scientific name: Oryza sativa, variety: Koshihikari) from which the polynucleotides of (a) to (e) were isolated are referred to as genes A to E, respectively. Also, the LDH genes of rice (scientific name: Oryza sativa, variety: Koshihikari) from which the polynucleotides of (f) to (g) were isolated are referred to as genes F to G, respectively.

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

Table 1

[0027] In plants under anaerobic conditions, in order to obtain energy without depending on oxygen, two metabolic pathways, ethanol fermentation and lactate fermentation using pyruvate as a substrate, work. Enzymes that catalyze the chemical change of pyruvate as the first reaction of each metabolic pathway are PDC (catalyzing the ethanol fermentation pathway) and LDH (catalyzing the lactate fermentation pathway).

[0028] From the results of the homology search of the amino acid sequences, it is considered that the PDC genes of rice (scientific name: Oryza sativa, variety: Koshihikari) are only genes A, B, C, D, and E, and the LDH genes of rice (scientific name: Oryza sativa, variety: Koshihikari) are only genes F and G.

[0029] Also in the KEGG ORTHOLOGY metabolic database, only genes A, B, C, D, and E are described as the PDC genes of rice, and only genes F and G are described as the LDH genes of rice [search date 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 have the common point of being genes of enzymes that catalyze the first reaction of the fermentation pathway using pyruvic acid as a substrate in rice (scientific name: Oryza sativa, variety: Koshihikari). Furthermore, each of the polynucleotides (a) to (e) has the common point of being a polynucleotide isolated from the 5'UTR of a gene of an enzyme that catalyzes the first reaction of the fermentation pathway using pyruvic acid as a substrate in rice (scientific name: Oryza sativa, variety: Koshihikari). That is, one embodiment of the present invention is based on the novel finding that a polynucleotide isolated from the 5'UTR of a gene of an enzyme that catalyzes the first reaction of the fermentation pathway using pyruvic acid as a substrate in rice (scientific name: Oryza sativa, variety: Koshihikari) has translation enhancer activity.

[0031] The polynucleotide constituting the translation enhancer according to one aspect of the present invention may be any one of the polynucleotides of (a) to (g) above. The level of the translation enhancer activity of the translation enhancer according to one aspect of the present invention is not particularly limited. However, 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'-terminal side of the structural gene is preferably 1.5 or more, more preferably 2 or more, more preferably 3 or more, more preferably 4 or more, more preferably 5 or more, more preferably 6 or more, more preferably 7 or more, more preferably 10 or more, more preferably 11 or more, more preferably 12 or more, more preferably 15 or more, and even more preferably 20 or more, in terms of the relative ratio when the GUS activity measured under the condition without 5'-UTR is set to 1.

[0032] From the viewpoint of translation enhancer activity, the polynucleotide constituting the translation enhancer according to one aspect of the present invention is preferably any one of the polynucleotides of (a), (f), or (g) above, and more preferably any one of the polynucleotides of (a) or (f). The translation enhancer composed of these polynucleotides has particularly high translation enhancer activity and is particularly excellent as a translation enhancer.

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

[0034] The polynucleotide of (h) consists of a nucleotide sequence in which 1 to 20 bases are deleted, substituted or added in any one of the polynucleotides of (a) to (g), and has translational enhancer activity. The polynucleotide of (h) is intended to be a polynucleotide such as a functionally equivalent mutant, variant, partial nucleotide, or fusion nucleotide with another nucleotide of any one of the polynucleotides of (a) to (g), and the nucleotide 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 the above functions are not lost. For example, it refers to the number that can be deleted, substituted or added by a known mutagenesis method such as the site-directed mutagenesis method. For example, it is a number that is 15% or less of the total number of bases of any one of the polynucleotides of (a) to (g), preferably 10% or less, and more preferably 5% or less (for example, 5%, 4%, 3%, 2% or 1%). Such a number of bases is preferably within 20 bases, preferably within 15 bases, more preferably within 10 bases, and most preferably within 5 bases (for example, 5, 4, 3, 2 or 1 base) in the case of the polynucleotide of (a). Similarly, for the polynucleotides of (b) to (g), the number of bases that may be deleted, substituted or added can be determined within the range of 1 to 20 bases so that the number is 15% or less, preferably 10% or less, and more preferably 5% or less of the total number of bases.

[0036] As used herein, the term "variant" mainly refers to a variant artificially introduced by a site-directed mutagenesis method or the like, but may also be a naturally occurring similar variant. As used herein, "functionally equivalent" means that a variant, a variant, a partial nucleotide, a fusion nucleotide with another nucleotide, etc. (hereinafter referred to as "variant etc.") has a biological function and a biochemical function equivalent (identical and / or similar) to any one of the polynucleotides of (a) to (g) above. More specifically, it is intended that the variant etc. has translational enhancer activity. The variant etc. only needs to have translational enhancer activity, and the level of the activity is not particularly limited. For example, the translational enhancer activity of the variant etc. may be at the same level as that of any one of the polynucleotides of (a) to (g) above, or may be at a level exceeding the activity of any one of the polynucleotides of (a) to (g) above.

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

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

[0039] The identity of a nucleotide sequence means having a sequence identity of at least 90% or more, more preferably 95% or more (for example, 95%, 96%, 97%, 98%, 99% or more) throughout the entire nucleotide sequence. The identity of a nucleotide sequence can be calculated using conventionally known software used for the purpose of calculating the identity of nucleotide sequences. For example, it can be calculated using the gene information software GENETYX Ver.9 (manufactured by Genetics Co., Ltd.).

[0040] From the viewpoint of further improving the translation enhancer activity, in one aspect of the present invention, for the translation enhancer, it is preferable that a polynucleotide consisting of the nucleotide sequence of ATGNNNNNN (N is any nucleotide) is linked to the 3'-end of any one of the polynucleotides of (a) to (i) above. By linking, in-frame, the nucleotide sequence of ATGNNNNNN linked to the 3'-end of the translation enhancer according to one aspect of the present invention upstream of the translation initiation codon (ATG) of the nucleotide sequence of the structural gene, the translation reading efficiency is improved, and as a result, the translation enhancer activity of the translation enhancer according to one aspect of the present invention can be further improved.

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

[0042] The 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, and examples include gramineous plants, among which rice (scientific name: Oryza sativa, variety: Koshihikari) can be mentioned. Also, the 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 Vector] The recombinant vector according to an embodiment of the present invention contains any of the above-described translation enhancers. Regarding the translation enhancer in the recombinant vector according to one aspect of the present invention, since it has already been described as above, the description 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 a conventionally known vector used as a vector for plant transformation can be used. Such vectors include, for example, pUC vectors, pBI vectors, pPZP vectors, and the like.

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

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

[0047] The recombinant vector according to an embodiment of the present invention may further contain, if necessary, a selection marker, a terminator (for example, NOS, etc.), a multiple cloning site, a DNA sequence encoding a protein tag, and the like. Examples of the selection marker include antibiotic resistance genes (for example, ampicillin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, hygromycin resistance gene, etc.). Examples of the protein tag include His, FLAG, HA, c-Myc, GFP, and the like.

[0048] In the recombinant vector according to one aspect of the present invention, the translation enhancer is preferably inserted between the promoter and the structural gene so as to function properly as a translation enhancer. The recombinant vector according to one embodiment of the present invention may be configured to 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 linked to the downstream (3'-terminal side) of the promoter. Also, the translation enhancer is preferably linked to the upstream (5'-terminal side) of the translation initiation codon of the structural gene. Such a nucleic acid construct is also included in the scope of the present invention.

[0049] The 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 the nucleic acid construct is amplified by the polymerase chain reaction (PCR) method using a primer with a restriction enzyme site added thereto, treated with a restriction enzyme, and ligated to an appropriate position of the restriction enzyme-treated vector, whereby the translation enhancer or the nucleic acid construct can be introduced into the vector.

[0050] [3. Transformant] The transformant into which the recombinant vector according to one aspect of the present invention is 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 as above, and thus the description will not be repeated here.

[0051] In this specification, "transformant" means a cell into which an exogenous gene (e.g., a structural gene) has been introduced and which has come to exhibit the traits of the introduced gene, and an individual organism containing such a cell. The cell may be in the state of a tissue or an organ. "Exhibiting the traits 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 "transformant" may be a progeny or clone of an organism or cell produced as a transformant as long as the protein encoded by the introduced exogenous gene is expressed. Further, the "transformant" may be a "transient transformant" obtained by transient transformation, or a "stable transformant" obtained by stable transformation.

[0052] The transformant according to one aspect of the present invention can be obtained by introducing the recombinant vector according to one aspect of the present invention into a host so that the target structural gene can be expressed. The host is not particularly limited as long as it can introduce the recombinant vector and can express the structural gene carried on the introduced recombinant vector. Examples of the species of organisms that can be used as the host include bacteria, yeasts, plants, insects, mammals, and the like. Since the translation enhancer according to one aspect of the present invention has excellent translation enhancer activity in plants, among these, it can be preferably used with plants as the host.

[0053] When the host is a plant, since the translation enhancer according to one aspect of the present invention can be expected to be effective in monocotyledonous plants and dicotyledonous plants other than rice, the type of plant used as the host is not particularly limited. The host may be, for example, either a monocotyledonous plant or a dicotyledonous plant.

[0054] Examples of monocotyledonous plants include rice, wheat, barley, foxtail millet, proso millet, Japanese millet, corn, sorghum, etc. of the grass family.

[0055] Examples of dicotyledonous plants include, for example, Arabidopsis thaliana and rapeseed of the Brassicaceae family; tobacco, tomato, potato, etc. of the Solanaceae family; melon, pumpkin, etc. of the Cucurbitaceae family; soybean of the Fabaceae family; cotton of the Malvaceae family; chrysanthemum of the Asteraceae family; tea of the Theaceae family; grape of the Vitaceae family, etc.

[0056] The transformant according to one aspect of the present invention may be any one selected from the group consisting of a plant cell, a plant tissue, a plant organ, a plant body, and a plant propagation material. The plant cell may be a cultured cell. The plant tissue is not particularly limited, and examples thereof include epidermis, phloem, parenchyma, xylem, vascular bundle, etc. The plant organ is not particularly limited, and examples thereof include leaf, petal, stem, root, rhizome, etc. Examples of the plant propagation material include seeds, cuttings, stocks, callus, protoplasts, etc. These cells, tissues, organs, and propagation materials can be obtained by known methods.

[0057] The method for introducing the recombinant vector into the host is not particularly limited, and an appropriate method according to the types of the host and the vector can be appropriately selected and used from among the conventionally known methods used for the purpose of producing a plant transformant. For example, the electroporation method, the polyethylene glycol method, the Agrobacterium binary vector method, the particle gun method, etc. can be mentioned.

[0058] Confirmation of whether the recombinant vector has been introduced into the host can be carried out by known methods such as the PCR method, the Southern hybridization method, the Northern hybridization method, etc.

[0059] [3. Method for producing recombinant protein] The method for producing a 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 a recombinant protein according to one aspect of the present invention, since the function of the above-described translation enhancer is exerted, the protein production amount per copy number of the 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 as above, and thus the description will not be repeated here.

[0060] The method for culturing the transformant is not particularly limited, and an appropriate method according to the type of the host can be appropriately selected and used from conventionally known culturing methods. Here, in the present specification, "culturing" 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, "culturing" is a concept that also includes cultivation, and the culture is also referred to as a "cultivated product".

[0061] The method for producing a recombinant protein according to one aspect of the present invention may further include a step of obtaining a transformant into which the recombinant vector according to one aspect of the present invention has been introduced before the culturing step. Further, after the culturing step, the method may further include a step of collecting the target protein from the obtained culture. As a method for collecting the target protein from the culture, conventionally known protein purification methods can be used.

[0062] A recombinant protein can also be produced by a method using a known in vitro cell-free protein synthesis system. For example, the method for producing a recombinant protein according to one aspect of the present invention may be a method including a step of synthesizing translation RNA from a nucleic acid construct in which the above-described translation enhancer is inserted between a promoter and a structural gene, and a step of bringing the translation RNA into contact with a reaction solution for in vitro cell-free protein synthesis.

[0063] The translation enhancer according to one aspect of the present invention and the nucleic acid construct containing the translation enhancer have already been described as such, and thus the description will not be repeated here. The method for synthesizing translation RNA from the nucleic acid construct and the method for synthesizing a protein using the translation RNA in the in vitro cell-free protein synthesis system are not particularly limited. For example, it can be carried out by a known method such as the method described in Patent Document 3 (Japanese Patent Application Laid-Open No. 2011-103833).

[0064] [4. Method for Producing Protein-High-Producing Transformant] The method for producing a protein-high-producing transformant according to one aspect of the present invention includes the step of obtaining a transformant into which the above-described recombinant vector has been introduced. According to the method for producing a protein-high-producing transformant according to one aspect of the present invention, since a transformant into which the above-described recombinant vector has been introduced can be obtained, it is possible to produce a protein-high-producing transformant in which the protein production amount per copy number of the DNA or mRNA of the structural gene is increased. The recombinant vector and transformant according to one aspect of the present invention have already been described as such, and thus the description will not be repeated here.

[0065] The transformant produced by the method for producing a protein-high-producing transformant 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 above-described protein production method.

[0066] [Summary] The translation enhancer according to Aspect 1 of the present invention has a configuration consisting of any one of the following polynucleotides (a) to (i): (a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1; (b) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2; (c) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3; (d) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4; (e) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5; (f) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6; (g) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7; (h) A polynucleotide consisting of a nucleotide sequence in which 1 to 20 bases are deleted, substituted or added in any one of the polynucleotides of (a) to (g) above, and having translational enhancer activity; (i) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with any one of the polynucleotides of (a) to (g) above, and having translational enhancer activity.

[0067] The translational enhancer according to Aspect 2 of the present invention is preferably composed of any one of the polynucleotides of (a), (f) or (g) in Aspect 1 above.

[0068] The vector according to Aspect 3 of the present invention has a configuration including the translational enhancer described in Aspect 1 or 2 above.

[0069] The vector according to Aspect 4 of the present invention may have a configuration including a nucleic acid construct in which the translational enhancer described in Aspect 1 or 2 above is inserted between a promoter and a 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] The transformant according to Aspect 6 of the present invention may have a configuration in which, in Aspect 5 above, the transformant is any one selected from the group consisting of plant cells, plant tissues, plant organs, plants and plant propagation materials.

[0072] The method for producing a protein according to Aspect 7 of the present invention may be a method including a step of culturing a transformant into which the recombinant vector described in Aspect 3 or 4 above is introduced.

[0073] The method for producing a protein highly productive transformant according to aspect 8 of the present invention may be a method including the step of obtaining a transformant into which the recombinant vector described in the above aspect 3 or 4 has been introduced.

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

Example

[0075] One example of the present invention will be described below, but the present invention is not limited to these examples.

[0076] [1] Construction of plasmid DNA <Experimental method> Plasmid DNA was constructed according to the following procedures (1) to (7).

[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 for amplifying from the 5'-end of cDNA to the start codon (ATG) by PCR were synthesized (for genes A, B, C, D, and E, designed so that a recognition sequence for restriction enzyme SpeI was added upstream of the 5'UTR and a recognition sequence for restriction enzyme BglII was added immediately after the start codon. Also, for genes F and G, designed so that a recognition sequence for restriction enzyme XbaI was added upstream of the 5'UTR and a recognition sequence for restriction enzyme BamHI was added 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) A plasmid DNA (containing recognition sequences of restriction enzymes BamHI, XhoI, SpeI, XbaI, SalI, and BglII in this order between CaMV35S Pro and GUS-3xFLAG) with CaMV35S Pro, GUS-3xFLAG, and Nos Ter inserted into the multiple cloning site of the pUC vector was cleaved with XbaI and BglII. (5) A DNA fragment containing the 5’UTR and start codon of genes A, B, C, D, and E was cleaved with SpeI and BglII. Also, a DNA fragment containing the 5’UTR and start codon of genes F and G was cleaved with XbaI and BamHI. The obtained DNA fragments were inserted into the plasmid DNA cleaved with XbaI and BglII by ligation reaction, respectively. (The cleavage sites of XbaI and SpeI, and BglII and BamHI are adhesive. Since there were some 5’UTRs containing SpeI or BglII recognition sequences, restriction enzymes were used in such combinations). (6) The prepared plasmid DNA was analyzed with a DNA sequencer to confirm that there were no sequence errors. (7) The prepared plasmid DNA was introduced into Escherichia coli and extracted and purified using the NucleoBond (registered trademark) Xtra Midi kit (TaKaRa).

[0078] The constructed plasmid map for transformation is shown in Figure 1. The meanings of the abbreviations in Figure 1 are as follows. 〔Explanation of Abbreviations〕 CaMV35S Pro: Cauliflower mosaic virus 35S promoter GUS-3xFLAG: β-glucuronidase gene (GUS gene) with a FLAG tag sequence added to the C-terminus Nos T: Nopaline synthase gene terminator lacZ: β-galactosidase gene containing a multiple cloning site inside AmpR: Ampicillin resistance gene pUC ori: Replication origin in Escherichia coli.

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

[0080] (1) Protoplasts were prepared from the aerial parts of young rice plants (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 amounts. (2) 10 μg of plasmid DNA was added to each tube and transfection was performed using polyethylene glycol. As a control, a vector plasmid without the 5’UTR inserted was used. (Tested in 3 tubes for each type of plasmid. n = 3). (3) After incubation at 28°C for 16 hours, the cells were collected by centrifugation. (4) An equal amount of extraction buffer [50 mM sodium phosphate (pH 7), 10 mM EDTA, 0.1% sodium N-lauroyl sarcosine, 0.1% Triton X-100, 0.1% mercaptoethanol] was added to each tube and the cells were disrupted by sonication. (5) The supernatant collected by centrifugation was mixed with an equal amount of GUS reaction solution (a solution prepared by dissolving 4-methylumbelliferyl-β-D-glucuronide (4-MUG) in the extraction buffer to a concentration of 1 mM) and incubated at 37°C for 1 hour. (6) An equal amount of 0.2 M sodium carbonate was added to each tube to stop the reaction. (7) Equal amounts were dispensed from each tube into plates for fluorescence measurement. (8) The fluorescence intensity emitted by methylumbelliferone produced from 4-MUG by the action of GUS was measured using a multiplate reader, and this value was compared as the GUS activity.

[0081] (Results) The results are shown in Fig. 2. Fig. 2 is a diagram showing the results of GUS activity measurement using a transient gene expression system. In Fig. 2, the values of the control group using a vector plasmid without inserted 5’UTR were relatively evaluated with 1 as the reference, and the standard deviation with n = 3 was shown as error bars.

[0082] As shown in Fig. 2, insertion of the 5’UTR of genes A - G increased the production amount of GUS protein per introduced DNA compared with the control group. Among them, it was found that insertion of the 5’UTR of genes A, F, and G significantly increased the production amount of GUS protein.

[0083] [3] Comparison of GUS protein by Western blotting <Experimental method> According to the following procedures (1) - (8), the amount of GUS protein was compared by Western blotting.

[0084] (1) 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.), protoplasts were prepared from the aerial parts of potato (scientific name: Solanum tuberosum, variety: Wase Shiro) and dispensed into tubes in equal amounts. (2) 10 μg of plasmid DNA was added to each tube and transfection was performed using polyethylene glycol. As control group A, a vector plasmid without inserted 5’UTR was used. (3) After incubation overnight at 28°C, the cells were collected by centrifugation. (4) An equal amount of SDS - PAGE sample buffer was added to dissolve the cells. (5) Equal amounts of the supernatant after centrifugation were electrophoresed on an SDS - polyacrylamide gel. (6) Proteins were blotted from the gel after electrophoresis onto a PVDF membrane. (7) The peroxidase-conjugated anti-FLAG tag antibody (manufactured by MBL, M185-7) was specifically bound to the FLAG-tagged GUS protein on the membrane, and a substrate solution that chemiluminesces due to peroxidase activity was added, and the signal was detected using an X-ray film. (8) After detecting the chemiluminescence signal, the membrane was stained with Coomassie Brilliant Blue (CBB) solution to confirm that there was no significant difference in the concentration of the electrophoresed protein among the samples.

[0085] (Results) The results are shown in Fig. 3. Fig. 3 is a diagram showing the analysis results by Western blotting. As shown in Fig. 3, due to the insertion of the 5'UTR of genes A to G, the production amount of GUS protein per introduced DNA increased compared to control group A. Among them, it was found that due to the insertion of the 5'UTR of genes A, F, and G, the production amount of GUS protein increased significantly compared to control group B.

[0086] In addition, it was confirmed that the 5'UTRs of genes A to G have translational enhancer activity not only in rice, which is a monocotyledonous plant, but also in potato, which is a dicotyledonous plant. Therefore, it was found that the 5'UTRs of genes A to G can be expected to have an effect as translational enhancers in plant species other than rice.

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

[0088] Using the constructed plasmid DNA, the GUS activity was measured by the same method as in [2] above.

[0089] (Result) The results are shown in Fig. 5. Fig. 5 is a diagram showing the results of measuring GUS activity using a transient gene expression system. In Fig. 5, the values of the control group using a vector plasmid without the 5’UTR inserted were relatively evaluated with 1 as the reference, and the standard deviation with n = 3 was shown as error bars.

[0090] As shown in Fig. 5, insertion of the 5’UTR of gene A or F increased the production amount of GUS protein per introduced DNA as compared with the control group. Therefore, it was found that the 5’UTRs of genes A and F can sufficiently exhibit the effect as translation enhancers even without structure α. Furthermore, structure α was considered to contribute to enhancing the activity of the 5’UTRs of genes A and F as translation enhancers.

Industrial Applicability

[0091] The present invention can be expected to be used as a translation enhancer in plants.

Claims

1. A translation enhancer comprising any one of the following polynucleotides (a) to (i): (a) a polynucleotide consisting of the base sequence of SEQ ID NO:1; (b) a polynucleotide consisting of the base sequence of SEQ ID NO: 2; (c) a polynucleotide consisting of the base sequence of SEQ ID NO: 3; (d) a polynucleotide consisting of the base sequence of SEQ ID NO: 4; (e) a polynucleotide consisting of the base sequence of SEQ ID NO:5; (f) a polynucleotide consisting of the base sequence of SEQ ID NO:6; (g) a polynucleotide consisting of the base sequence of SEQ ID NO: 7; (h) a polynucleotide consisting of a base sequence in which 1 or more and 20 or less bases are deleted, substituted or added in any one of the polynucleotides (a) to (g), and having a translation enhancer activity; (i) a polynucleotide having a base sequence having 90% or more sequence identity with any one of the polynucleotides (a) to (g) above, and having a translation enhancer activity.

2. The translation enhancer of claim 1, which consists of any one of the polynucleotides (a), (f) or (g).

3. A recombinant vector comprising the translation enhancer of claim 1 or 2.

4. A recombinant vector comprising a nucleic acid construct in which the translation enhancer according to claim 1 or 2 is inserted between a promoter and a structural gene.

5. A transformant into which the recombinant vector according to claim 4 has been introduced.

6. The transformant according to claim 5 , which is any one selected from the group consisting of a plant cell, a plant tissue, a plant organ, a plant body, and a plant propagation material.

7. A method for producing a recombinant protein, comprising the step of culturing a transformant into which the recombinant vector according to claim 4 has been introduced.

8. A method for producing a transformant having high protein productivity, comprising the step of obtaining a transformant into which the recombinant vector according to claim 4 has been introduced.

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