Promoter capable of inducing gene expression in plant and use thereof

Abstract

The present invention addresses the problem of providing a promoter capable of inducing the expression of a target gene in a plant, and this problem is solved as follows.　The present invention provides DNA which has a promoter function and is selected from the group consisting of (a) to (e) below: (a) DNA consisting of a base sequence represented by SEQ ID NO: 1 or a partial sequence thereof; (b) DNA consisting of a base sequence represented by SEQ ID NO: 2 or a partial sequence thereof; (c) DNA consisting of a base sequence having at least 90% sequence identity with the base sequence of the DNA of (a) or (b); (d) DNA consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted in the base sequence of the DNA of (a) or (b); and (e) DNA which hybridizes to, under stringent conditions, DNA consisting of a base sequence complementary to the base sequence of the DNA of (a) or (b).

Description

Inducible promoter for gene expression in plants and its use

[0001] The present invention relates to promoters capable of inducing gene expression in plants.

[0002] Two important expression strategies can be applied to the production of recombinant proteins in plants: transient expression and stable expression. Transient expression in plants, typically via infiltration with Agrobacterium tumefaciens carrying a plant expression vector (the "agroinfiltration" method), is a frequently used strategy for rapid and reliable production of recombinant proteins within a few days. Among many plant species, Nicotiana benthamiana (also referred to herein as "N. benthamiana") is suitable for transient expression (Non-Patent Document 1) and is currently the primary production host. This is due to its advantageous characteristics, such as fast growth, large biomass, scalability, and susceptibility to pathogen infection (Non-Patent Document 2). For example, Medicago Inc. produced a coronavirus-like particle vaccine for the treatment of COVID-19 by transient expression in Nicotiana benthamiana (Non-Patent Document 3). On the other hand, stable expression strategies offer a more sustainable and scalable method for the large-scale production of recombinant proteins from established seed banks (Non-Patent Document 2).

[0003] A promoter is a DNA region located upstream of an encoded gene and contains binding sites for proteins involved in the initiation and regulation of transcription. Transcription begins when a preinitiation complex (PIC) is formed at the transcription start site (TSS) by general transcription factors and RNA polymerase II (Non-Patent Document 4). Promoters are composed of various initiation motifs (e.g., TATA box, Y patch, and initiator (Inr) element) that induce the formation of the TSS and PIC (Non-Patent Document 5). Promoters also contain numerous cis-regulatory elements, which allow transcription factors to bind and regulate transcription (Non-Patent Document 4). Therefore, promoters are responsible for the specificity and strength of gene expression.

[0004] However, plant biotechnology has relied heavily on a few well-established constitutive promoters, such as the cauliflower mosaic virus (CaMV) 35S promoter, to drive gene expression (Non-Patent Document 6). As a result, the strength and precise control of gene expression by endogenous plant promoters have not been thoroughly investigated.

[0005] Nosaki, S. et al., (2021). Transient protein expression systems in plants and their applications. Plant biotechnology (Tokyo, Japan), 38(3), 297-304Liu, H. et al., (2022). Improving Protein Quantity and Quality-The Next Level of Plant Molecular Farming. International journal of molecular sciences, 23(3), 1326Hager, K. J. et al., (2022). Efficacy and Safety of a Recombinant Plant-Based Adjuvanted Covid-19 Vaccine. The New England journal of medicine, 386(22), 2084-2096Haberle, V. et al., (2018). Eukaryotic core promoters and the functional basis of transcription initiation. Nature reviews. Molecular cell biology, 19(10), 621-637Zhong, V. et al., (2023). Transcriptional and post-transcriptional controls for tuning gene expression in plants. Current Opinion in Plant Biology, 71, 102315Tian, C. et al., (2022). Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research. BioDesign Research, 2022. Article ID: 9834989 DOI: 10.34133 / 2022 / 9834989

[0006] An object of the present invention is to provide a promoter capable of inducing gene expression in plants.

[0007] In order to solve the above-mentioned problems, the present invention encompasses the following inventions. [1] A DNA having promoter function, which is any one of the following DNAs selected from the group consisting of (a) to (e): (a) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a partial sequence thereof; (b) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 9 or a partial sequence thereof; (c) a DNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of the DNA of (a) or (b); (d) a DNA consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, or inserted in the nucleotide sequence of the DNA of (a) or (b); and (e) a DNA that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of the DNA of (a) or (b). [2] The DNA of [1], wherein the (a) is a DNA consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 6. [3] The DNA of [1], wherein the (b) is a DNA consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 9 to 16. [4] The DNA according to [1], wherein (a) further comprises, downstream, DNA consisting of the nucleotide sequence represented by SEQ ID NO: 17. [5] The DNA according to [1], wherein (b) further comprises, downstream, DNA consisting of the nucleotide sequence represented by SEQ ID NO: 18. [6] An expression vector comprising the DNA according to any of [1] to [5]. [7] The expression vector according to [6], further comprising a target gene for expression. [8] An Agrobacterium transformant comprising the expression vector according to [7]. [9] A plant cell or tissue transformed with the Agrobacterium transformant according to [8].

[10] A plant transformed with the Agrobacterium transformant according to [8].

[11] A method for inducing expression of a target gene in a plant, the method comprising the steps of infecting a plant or cultured plant cells with the Agrobacterium transformant according to [8], and cultivating a plant infected with the Agrobacterium transformant or culturing cultured plant cells infected with the Agrobacterium transformant.

[12] A method for inducing expression of a target gene in a plant, the method comprising the steps of infecting the transformed plant according to

[10] with Agrobacterium and cultivating the transformed plant infected with the Agrobacterium.

[13] The method according to

[12] , further comprising the step of producing a transformed plant using the Agrobacterium transformant according to [8].

[0008] According to the present invention, a promoter capable of inducing gene expression in plants can be provided. The promoter of the present invention can induce the expression of a target gene for expression operably linked to the promoter in a plant by Agrobacterium infection, such as agroinfiltration.

[0009] 1 shows sequences showing the positional relationship of CDE1 promoter candidates of each length, 5'-UTR, and start codon (continued from FIG. 2). 2 shows sequences showing the positional relationship of CDE1 promoter candidates of each length, 5'-UTR, and start codon (continued from FIG. 1). 3 shows sequences showing the positional relationship of CDE2 promoter candidates of each length, 5'-UTR, and start codon (continued from FIG. 4). 4 shows sequences showing the positional relationship of CDE2 promoter candidates of each length, 5'-UTR, and start codon (continued from FIG. 3). 5 shows a schematic diagram showing the configuration of the gene expression system in various expression vectors used in the Examples (a. pRI201-AN (vector control; VC); b. pRI201-EGFP(35SP); c. CDE1P; d. CDE2P; and e. CDE1P-AU). 6 shows fluorescence micrographs of N. benthamiana leaves infiltrated by syringe infiltration with Agrobacterium transformed with vectors containing each CDE1 promoter candidate. Figure 1 shows fluorescence micrographs of N. benthamiana leaves infiltrated by syringe infiltration with Agrobacterium transformed with vectors containing each of the CDE2 promoter candidates. Figure 2 shows Western blot results for EGFP expression in N. benthamiana leaves infiltrated by syringe infiltration with Agrobacterium transformed with vectors containing each of the CDE1 promoter candidates. Figure 3 shows Western blot results for EGFP expression in N. benthamiana leaves infiltrated by syringe infiltration with Agrobacterium transformed with vectors containing each of the CDE2 promoter candidates. Figure 4 shows graphs showing the estimated amount of EGFP expressed in N. benthamiana leaves infiltrated by vacuum infiltration with Agrobacterium transformed with vectors containing each of the CDE1 promoter candidates. 1 is a graph showing the estimated amount of EGFP expressed in N. benthamiana leaves infiltrated by vacuum infiltration with Agrobacterium transformed with vectors containing each of the CDE2 promoter candidates.These are fluorescence micrographs of N. benthamiana leaves infiltrated by syringe infiltration with Agrobacterium transformed with vectors in which the native CDE1 5'-UTR was placed downstream of each CDE1 promoter candidate, or in which the AtADH 5'-UTR was replaced. These are fluorescence micrographs of leaves of N. benthamiana transgenic plants carrying CDE2P_500-EGFP (CDE2P_500-EGFP T2 plants) and wild-type plants (WT) on days 0, 3, 5, and 7 after agroinfiltration.

[0010] [DNA with promoter function] The present inventors previously discovered two proteins (two carbohydrate-degrading enzymes, namely CDE1 and CDE2) in the apoplastic fraction of leaves of Nicotiana benthamiana plants infected with Agrobacterium tumefaciens by agroinfiltration. The present inventors further identified their signal peptides and discovered a method for producing recombinant proteins in plants using these peptides (PCT / JP2024 / 4642).

[0011] The present inventors identified promoter regions located upstream of the CDE1 and CDE2 genes from the genomic DNA of Nicotiana benthamiana, and discovered that these promoter regions function as promoters for transient and stable expression of target genes in plants, and further that expression can be induced by Agrobacterium infection, such as agroinfiltration.

[0012] Therefore, the present invention provides DNA having promoter function, which is any one of the following (a) to (e): (a) DNA consisting of the base sequence shown in SEQ ID NO: 1 or a partial sequence thereof; (b) DNA consisting of the base sequence shown in SEQ ID NO: 9 or a partial sequence thereof; (c) DNA consisting of a base sequence having 90% or more sequence identity with the base sequence of the DNA of (a) or (b); (d) DNA consisting of a base sequence in which one or more bases have been substituted, deleted, added or inserted in the base sequence of the DNA of (a) or (b); and (e) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of the DNA of (a) or (b).

[0013] A promoter is a DNA region located upstream of an encoded gene and contains binding sites for proteins involved in the initiation and control of transcription. As used herein, "promoter function" refers to the function of controlling (e.g., initiating) the transcription of a gene located downstream of the promoter into mRNA. As used herein, "DNA having promoter function" is also referred to simply as "promoter" or "promoter DNA."

[0014] As used herein, "sequence" refers to a base sequence (or nucleotide sequence) unless otherwise specified. As used herein, "upstream" in a base sequence refers to a sequence or position located on the 5' side of a reference sequence or position, and "downstream" refers to a sequence or position located on the 3' side of a reference sequence or position. Unless otherwise specified, base sequences are expressed in the 5'→3' direction.

[0015] The DNA (a) is a DNA consisting of the base sequence shown in SEQ ID NO: 1 or a partial sequence thereof, and having promoter function. As shown in standard typeface without underlines in Figures 1 and 2, SEQ ID NO: 1 is the entire sequence of the 2.5 kb (2500 base length) promoter region ("CDE1 promoter") upstream from the putative transcription initiation site of the CDE1 gene of Nicotiana benthamiana, but the origin of the "promoter DNA" of the present invention is not important.

[0016] The above (a) includes DNA consisting of any sequence selected from the nucleotide sequence represented by SEQ ID NO: 1, as long as it has promoter function. Preferably, the sequence includes the 3'-terminal nucleotide of the nucleotide sequence represented by SEQ ID NO: 1. That is, the 5'-terminal region of the nucleotide sequence represented by SEQ ID NO: 1 may be deleted as long as the promoter function is not lost. In one embodiment, the above (a) DNA is DNA consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 6. The DNAs represented by SEQ ID NOs: 2 to 6 are DNAs consisting of nucleotide sequences 2000 bases long (SEQ ID NO: 2), 1500 bases long (SEQ ID NO: 3), 1000 bases long (SEQ ID NO: 4), 500 bases long (SEQ ID NO: 5), and 250 bases long (SEQ ID NO: 6), respectively, in the upstream direction from the 3'-terminal nucleotide T of the nucleotide sequence represented by SEQ ID NO: 1, and are DNAs in which the 5'-terminal region of the nucleotide sequence represented by SEQ ID NO: 1 has been deleted. In Figures 1 and 2, the promoter region (the region shown in standard typeface without underlines) is shown in the 5' to 3' direction, and the sequences of SEQ ID NOs: 2 to 6 are regions that extend from the 3'-terminal base T of the promoter region as the starting point toward the 5'-terminal (upstream), and each corresponds to the range up to the position corresponding to the base length of the sequence (number in parentheses).

[0017] The DNA of (b) is a DNA consisting of the base sequence shown in SEQ ID NO: 9 or a partial sequence thereof, and having promoter function. As shown in standard typeface without underlining in Figures 3 and 4, SEQ ID NO: 9 is the entire sequence of the 2.5 kb (2500 base length) promoter region ("CDE2 promoter") upstream from the putative transcription initiation site of the CDE2 gene of Nicotiana benthamiana, but the origin of the "promoter DNA" of the present invention is not important.

[0018] The above (b) includes DNA consisting of any sequence selected from the nucleotide sequence represented by SEQ ID NO: 9, as long as it has promoter function. Preferably, the sequence includes the 3'-terminal nucleotide of the nucleotide sequence represented by SEQ ID NO: 9. That is, the 5'-terminal region of the nucleotide sequence represented by SEQ ID NO: 9 may be deleted as long as the promoter function is not lost. In one embodiment, the above (b) DNA is DNA consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 9 to 16. The DNAs represented by SEQ ID NOs: 10 to 16 are DNAs consisting of nucleotide sequences of 2000 bases (SEQ ID NO: 10), 1500 bases (SEQ ID NO: 11), 1000 bases (SEQ ID NO: 12), 500 bases (SEQ ID NO: 13), 250 bases (SEQ ID NO: 14), 100 bases (SEQ ID NO: 15), and 50 bases (SEQ ID NO: 16) upstream from the 3'-terminal nucleotide C of the nucleotide sequence represented by SEQ ID NO: 9, respectively, in which the 5'-terminal region of the nucleotide sequence represented by SEQ ID NO: 9 has been deleted. In Figures 3 and 4, the promoter region (the region shown in standard typeface without underlines) is shown in the 5' to 3' direction, and the sequences of SEQ ID NOs: 10 to 16 are regions extending from the 3'-terminal base C of the promoter region as the starting point toward the 5'-terminal (upstream), and each corresponds to the range up to the position corresponding to the base length of the sequence (number in parentheses).

[0019] Here, the "DNA having promoter function" of the present invention may contain a regulatory sequence such as an enhancer or repressor. Examples of regulatory sequences include, but are not limited to, the 5' untranslated regions (5'-UTRs) of the CDE1 gene and the CDE2 gene. The DNA of (a) above may further contain downstream DNA consisting of the nucleotide sequence represented by SEQ ID NO: 17. SEQ ID NO: 17 is the sequence of the 5' untranslated region (5'-UTR) of the CDE1 gene, and is the sequence represented by italics located downstream of the promoter region in Figures 1 and 2. As long as the DNA of (a) above has promoter function, it may be DNA consisting of DNA consisting of the nucleotide sequence represented by SEQ ID NO: 17 downstream of DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a partial sequence thereof. The DNA of (b) above preferably further contains downstream DNA consisting of the nucleotide sequence represented by SEQ ID NO: 18. SEQ ID NO: 18 is the sequence of the 5' untranslated region (5'-UTR) of the CDE2 gene, and is the sequence represented by italics located downstream of the promoter region in Figures 3 and 4. As long as (b) above has a promoter function, it can be a DNA consisting of a DNA consisting of the base sequence represented by SEQ ID NO: 9 or a partial sequence thereof followed downstream by a DNA consisting of the base sequence represented by SEQ ID NO: 18.

[0020] The "promoter DNA" of the present invention is not limited to the DNAs (a) and (b) above, and DNAs (c), (d), and (e) as described below can also be used in its construction, as long as they have promoter function.

[0021] The DNA (c) is defined by the sequence identity between it and the base sequence of the DNA (a) or (b) above. The sequence identity may be within the range that maintains promoter function, and may be, for example, 70% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, or 99% or more, but is not limited to these. In the present invention, the sequence identity between it and the base sequence of the DNA (a) or (b) above is, for example, 90% or more, but may also be, but is not limited to, 92% or more, 95% or more, 97% or more, or 99% or more. The DNA (c) above may be a DNA consisting of a base sequence that has 90% or more sequence identity with any base sequence selected from (a) and (b) above, within the range that maintains promoter function.

[0022] The sequence identity of the base sequences can be determined using a homology search program well known to those skilled in the art, such as NCBI's BLAST. Sequence comparison and sequence identity determination can be performed, for example, using the standard settings of NCBI's BLAST.

[0023] The DNA of (d) is defined by the substitution, deletion, addition, or insertion of one or more bases in the base sequence of the DNA of (a) or (b) above. The sequence modification by base substitution, deletion, addition, or insertion can be performed within the range that maintains promoter function. The term "multiple" refers to any number of bases as long as it maintains promoter function. The term "multiple" depends on the length of the referenced sequence, but examples include, but are not limited to, 2 to 250, 2 to 200, 2 to 150, 2 to 100, 2 to 75, 2 to 50, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, or 2 to 3 bases. A "substituted, deleted, added, or inserted base sequence" may refer to any one of substitution, deletion, addition, and insertion occurring alone, or any two or more of these. The DNA (d) above may be a DNA consisting of a base sequence in which one or more bases have been substituted, deleted, added or inserted in any base sequence selected from (a) and (b) above, within a range that has the desired promoter function.

[0024] The DNA (e) is a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence of (a) or (b) above. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. For example, two DNAs with high identity, e.g., two DNAs with identity of 90% or more, 92% or more, 95% or more, 97% or more, or 99% or more, hybridize, but two DNAs with lower identity do not hybridize. Stringent conditions include, for example, washing at 60°C to 70°C in 0.1x SSC and 0.1% SDS solution. Hybridization can be performed by methods well known to those skilled in the art, such as the method described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

[0025] The DNA of the present invention can be obtained by known DNA synthesis methods, PCR, etc. Specifically, for example, it can be chemically synthesized using a commercially available DNA synthesizer based on the sequence of the DNA of the present invention. Furthermore, primers can be designed and prepared based on the sequence of the DNA of the present invention, and the DNA can be obtained by PCR using, for example, the genomic DNA of N. benthamiana as a template. Alternatively, a probe can be prepared based on the sequence of the DNA of the present invention, and hybridization can be performed under stringent conditions as described above.

[0026] In particular, the "promoter function" of the present invention transcribes a target gene for expression located downstream of the promoter into mRNA upon Agrobacterium infection in a plant containing the gene. The "promoter function" of the DNA of the present invention can be determined by whether expression of the target gene located downstream is observed in the plant. A reporter gene is used as the target gene for this purpose. Examples of reporter genes that can be used include fluorescent protein genes such as green fluorescent protein (GFP or EGFP) gene, β-galactosidase gene (lacZ), luciferase gene, and β-lactamase gene. A target gene can be introduced into a plant by infecting the plant with the Agrobacterium transformant described below. Expression can be confirmed and the amount of expression quantified using methods well known to those skilled in the art, such as immunological techniques (e.g., Western blotting) and fluorescence intensity measurements.

[0027] The promoter DNA may be either single-stranded or double-stranded DNA. In the case of double-stranded DNA, it may be in the form of double-stranded DNA consisting of a single-stranded DNA having a sequence having promoter function and its complementary strand.

[0028] [Expression Vector and Agrobacterium Transformant] The present invention provides an expression vector comprising the promoter DNA described above. That is, the expression vector of the present invention comprises DNA (the promoter DNA of the present invention) that is any one of the above (a) to (e) and has promoter function.

[0029] Since the expression vector of the present invention is introduced into Agrobacterium, it is preferable to use a binary vector or intermediate vector containing T-DNA derived from the Ti plasmid or Ri plasmid of Agrobacterium, such as, but not limited to, a pBI-based binary vector or a pRI-based binary vector.

[0030] The expression vector of the present invention may further contain other DNA segments. The other DNA segments are not particularly limited, but examples thereof include terminators, selection markers, enhancers, etc. The transcription terminator is not particularly limited as long as it functions as a transcription termination site, and known terminators can be suitably used. Examples include, but are not limited to, terminators derived from HSP (heat shock protein) genes. Examples of selectable markers for transformants include, but are not limited to, drug resistance genes. Specific examples include, but are not limited to, drug resistance genes for hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol, bialaphos, etc.

[0031] The expression vector of the present invention may or may not contain a gene intended for expression in a plant (also referred to as a "target gene for expression"). That is, if the expression vector does not contain a target gene for expression, the target gene for expression can be inserted into the expression vector prior to use so that the gene is operably linked to the promoter DNA of the present invention. Alternatively, the expression vector may be one into which the target gene for expression has already been inserted so that the gene is operably linked to the promoter DNA of the present invention. The term "operably linked" refers to a functional link between a gene and an expression regulatory element (e.g., the promoter DNA of the present invention) so that the expression regulatory element (e.g., the promoter DNA of the present invention) controls (e.g., initiates) the transcription of the target gene for expression. Functional linkage can be achieved using recombinant DNA techniques known in the art. For example, the linkage can be achieved by, but is not limited to, typical site-specific DNA cleavage and ligation.

[0032] The target gene for expression is not particularly limited as long as it is a gene intended for expression in a host plant. Examples of target genes for expression include, but are not limited to, genes encoding proteins intended for production in a host plant. Examples of such proteins include, but are not limited to, proteins that serve as active ingredients in biopharmaceuticals and medical proteins, such as proteins of a quality suitable for use in xeno-free media for regenerative medicine. Specific examples include, but are not limited to, vaccine proteins, antibodies, antibody derivatives (e.g., single-chain antibodies), diagnostic enzymes, protease inhibitor proteins, lectins, growth factors, antibacterial proteins, proteins useful for cell culture, metabolic enzymes, cytoskeleton-constituting enzymes, and stress-responsive proteins.

[0033] The vector may also be either single-stranded or double-stranded DNA.

[0034] An Agrobacterium transformant (the Agrobacterium transformant of the present invention) can be obtained by introducing the expression vector of the present invention containing a target gene to be expressed into Agrobacterium. Introduction of the expression vector into Agrobacterium can be achieved by known methods such as, but not limited to, the calcium phosphate method, lipofection, and electroporation.

[0035] [Transformed Plant Cells, Tissues, and Plants] The transformed plant cells, tissues, and plants of the present invention can be produced using the Agrobacterium transformant of the present invention by known methods for plant transformation and transgenic plant production using Agrobacterium transformants. Transformed plant cells or tissues can be obtained, for example, by suspending the Agrobacterium transformant of the present invention in a liquid medium, contacting it with a part or tissue explant of a host plant, and co-cultivating it. Examples of plant parts or tissue explants include, but are not limited to, leaf segments, stems, hypocotyls, embryos, shoot tips, roots, and calli. Furthermore, transformed calli obtained through the co-cultivation described above can be selected, redifferentiated by tissue culture, and then rooted and acclimatized to regenerate into plants, thereby obtaining transformed plants (transgenic plants). Plant transformation and transgenic plant production using Agrobacterium transformants can be performed based on, for example, but not limited to, the method described in Yau, YY., Easterling, M., Brennan, L. (2020). Rapid Agrobacterium-mediated transformation of tobacco cotyledons using toothpicks. Climate Change, Photosynthesis and Advanced Biofuels.

[0036] [Induction of gene expression in plants] The present invention provides methods for inducing expression of a gene of interest in plants. These methods include a method using the Agrobacterium transformant of the present invention (method 1) and a method using the transformed plant of the present invention (method 2).

[0037] The first method comprises the following steps: infecting a plant or cultured plant cells with the Agrobacterium transformant of the present invention, and cultivating the plant infected with the Agrobacterium transformant or culturing the cultured plant cells infected with the Agrobacterium transformant.

[0038] According to the first method, first, a plant or cultured plant cells is infected with the Agrobacterium transformant of the present invention.

[0039] When infecting a plant with an Agrobacterium transformant, the method is not particularly limited, and any known method for infecting a plant with an Agrobacterium transformant can be appropriately selected and used. Agroinfiltration may be used as a method for infecting a plant with an Agrobacterium transformant. For agroinfiltration, an Agrobacterium transformant containing an expression vector can be suspended in a buffer solution suitable for infiltration into plant tissue. The turbidity of the Agrobacterium transformant suspension may be 0.05 to 5, 0.1 to 2, or 0.2 to 1 in terms of OD600. The Agrobacterium transformant suspension may consist of a solution containing a buffer solution, etc., the components of which have been appropriately investigated.

[0040] The plant to be used is not particularly limited as long as it can be infected with Agrobacterium, but is preferably a plant that can be efficiently subjected to agroinfiltration. Examples include, but are not limited to, Solanaceae plants such as tobacco, potato, tomato, etc., Brassicaceae plants such as arugula, komatsuna, mizuna, mustard, Arabidopsis, etc., Asteraceae plants such as chicory, endive, artichoke, etc., Leguminaceae plants such as alfalfa, mung bean, soybean, etc., Chenopodiaceae plants such as spinach, sugar beet, etc., Lamiaceae plants such as perilla, basil, etc., Umbelliferae plants such as mitsuba, etc., and Poaceae plants such as rice, wheat, barley, corn, etc.

[0041] Among them, solanaceae plants are preferred, and tobacco is more preferred. Tobacco is not particularly limited, and examples thereof include Nicotiana tabacum, Nicotiana benthamiana, Nicotiana alata, Nicotiana glauca, Nicotiana longiflora, Nicotiana persica, Nicotiana rustica, and Nicotiana sylvestris, with Nicotiana benthamiana being preferred. Glycosylation-modified Nicotiana benthamiana (Strasser R et al., Plant Biotechnol J. 2008 May; 6(4): 392-402; Limkul J et al., Plant Biotechnol J. 2016 Aug; 14(8): 1682-1694) can also be used.

[0042] Agroinfiltration is well known in the art. In the method of the present invention, it can be appropriately selected from known agroinfiltration methods. Examples of agroinfiltration include syringe infiltration using a needleless syringe and vacuum infiltration using a vacuum pump, and any of these can be used.

[0043] The cultured plant cells can be cultured cells prepared from the above plants by known methods, such as those described in "Mutation and Selection of Plant Cultured Cells (Biotechnology Series), April 1, 1985, edited by Yamada Yasuyuki, Kodansha, ISBN-10: 4061390058" and "Plant Biotechnology (Modern Chemistry, Supplementary Edition 5), April 25, 1986, edited by Yamada Yasuyuki and Okada Yoshimi, Tokyo Kagaku Dojin, ISBN-10: 4807902644."

[0044] When infecting cultured plant cells with an Agrobacterium transformant, the method is not particularly limited and can be appropriately selected from known methods. Known methods include, for example, the method of Sukenik et al. (International Journal of Molecular Sciences 2018 Apr 16;19(4):1205), the method of Poborilova et al. (Plant Cell Reports https: / / doi.org / 10.1007 / s00299-020-02544-w), and the method of Rademacher et al. (Plant Biotechnology Journal (2019) 17, pp. 1560-1566). Alternatively, a method of co-cultivating plant cultured cells with an Agrobacterium transformant and infecting them may be used.

[0045] Next, plants infected with the Agrobacterium transformant are cultivated, or plant culture cells infected with the Agrobacterium transformant are cultured.

[0046] The period for cultivating a plant infected with an Agrobacterium transformant is not particularly limited, and may be 1 day or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more. The cultivation period may also be 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, or 5 days or less.

[0047] The cultivation conditions are not particularly limited, and the plants may be cultivated under the same conditions as before infection with the Agrobacterium transformant, or under conditions different from those before infection with the Agrobacterium transformant. Plants are usually cultivated in a greenhouse where temperature, light intensity, humidity, sunshine duration, etc. are controlled. The cultivation temperature may be 15 to 28°C, or 20 to 25°C. After infection with the Agrobacterium transformant, it is preferable to apply a liquid fertilizer with a high nitrogen concentration, and it is also preferable to change the controls of temperature, sunshine duration, photon flux density, planting density, light intensity, transpiration, etc.

[0048] The period for culturing the cultured plant cells infected with the Agrobacterium transformant is not particularly limited, and may be 1 day or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more. The culture period may also be 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, or 5 days or less.

[0049] The culture conditions are not particularly limited, and the culture may be carried out under the same conditions as before infection with the Agrobacterium transformant, or under conditions different from those before infection with the Agrobacterium transformant. Cultured plant cells are usually cultured in an incubator in which the temperature, agitation state, medium components, etc. are controlled. The culture temperature may be 15 to 28°C, or 20 to 25°C. After infection with the Agrobacterium transformant, it is preferable to adjust or change the temperature, agitation state, etc. as appropriate.

[0050] The second method comprises the following steps: infecting a transformed plant of the present invention with Agrobacterium, and cultivating the transformed plant infected with the Agrobacterium. In the second method, the Agrobacterium used for infection does not need to contain an expression vector. For example, a non-transformed Agrobacterium can be used. The second method may also use an Agrobacterium transformant containing an expression vector, preferably an Agrobacterium transformant of the present invention, which may contain the same or a different gene of interest. The infection and cultivation methods are as described above for the first method.

[0051] The second method can further include the step of producing a transformed plant using the Agrobacterium transformant of the present invention. The production of a transformed plant is as described above.

[0052] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0053] The materials used in this example are listed below.

[0054] <Bacteria and host cells used> Escherichia coli DH5α was used for plasmid construction, and Agrobacterium tumefaciens LBA4404 with the plasmid introduced was used for plant transformation.

[0055] Nicotiana benthamiana (N. benthamiana) plants were used as hosts for EGFP expression / production. Plants were grown in a plant chamber at 22°C and 50% humidity, and were used 4-5 weeks after germination.

[0056] <Culture Media> LB medium (10 g / L HIPOLYPEPTON (Gibco), 5 g / L Yeast Extract (BD Bionutrients), 10 g / L NaCl) was used for E. coli culture. 2xYT medium (16 g / L HIPOLYPEPTON, 10 g / L Yeast Extract, 5 g / L NaCl) was used for Agrobacterium culture. The following antibiotics were added for transformant selection as needed: 50 mg / L kanamycin (FUJIFILM Wako Pure Chemical Corporation), 50 mg / L rifampicin (FUJIFILM Wako Pure Chemical Corporation), or 20 mg / L streptomycin (FUJIFILM Wako Pure Chemical Corporation) (final concentration). Agar (Nacalai Tesque) was added to a final concentration of 15 g / L when preparing plate media.

[0057] The soil for N. benthamiana plants was a mixture of Supermix A (Sakata) and vermiculite (Vermitec) in a ratio of 5:1, autoclaved at 121°C for 20 minutes, and then used.

[0058] The media used to generate N. benthamiana transgenic plants were as follows: MS vitamin solution: 0.1 mg / mL thiamine hydrochloride (Nacalai Tesque), 0.5 mg / mL nicotinic acid (Wako Pure Chemical Industries, Ltd.), 0.5 mg / mL pyridoxine hydrochloride (Wako Pure Chemical Industries, Ltd.), 100 mg / mL myo-inositol (Wako Pure Chemical Industries, Ltd.), 2 mg / mL glycine (Nacalai Tesque); Gamborg vitamin solution: 10 mg / mL thiamine hydrochloride (Nacalai Tesque), 1 mg / mL nicotinic acid (Wako Pure Chemical Industries, Ltd.), 1 mg / mL pyridoxine hydrochloride (Wako Pure Chemical Industries, Ltd.), 100 mg / mL myo-inositol (Wako Pure Chemical Industries, Ltd.); MS liquid medium (pH 5.8): 2.15 g / L Murashige & Skoog salt (DUCHEFA Biochemie BV), 15 g / L sucrose (Kanto Chemical Co., Ltd.), 1 mL MS vitamin solution, 0.5 g / L MES (Wako Pure Chemicals); 1 / 2 MS medium (pH 5.8): 2.15 g / L Murashige & Skoog salts (DUCHEFA Biochemie BV), 15 g / L sucrose (Kanto Chemical), 1 mL MS vitamin solution, 0.5 g / L MES (Wako Pure Chemicals), 2.5 g / L gellan gum (Kanto Chemical); MS differentiation medium (pH 5.8): 4.3 g / L Murashige & Skoog salts (DUCHEFA Biochemie BV), 30 g / L sucrose (Kanto Chemical), 1 mL Gamborg vitamin solution, 2 mg / L naphthaleneacetic acid, 0.2 mg / L 6-benzyladenine, 0.5 g / L MES (Wako Pure Chemicals), 2.5 g / L gellan gum (Kanto Chemical); MS selection medium (pH 5.8): 4.3 g / L Murashige & Skoog salts (DUCHEFA Biochemie BV), 30 g / L sucrose (Kanto Chemical), 1 mL Gamborg vitamin solution, 2 mg / L naphthaleneacetic acid, 0.2 mg / L 6-benzyladenine, 0.5 g / L MES (Wako Pure Chemicals), 2.5 g / L gellan gum (Kanto Chemical); Skoog salt (DUCHEFA Biochemie BV), 30 g / L sucrose (Kanto Chemical), 1 mL Gamborg vitamin solution, 2 mg / L naphthaleneacetic acid, 0.2 mg / L 6-benzyladenine, 250 mg / L carbenicillin (nacalai tesque), 50 mg / L kanamycin (Wako Pure Chemicals), 0.5 g / L MES (Wako Pure Chemicals), 2.5 g / L gellan gum (Kanto Chemical); 1 / 2 MS rooting medium (pH 5.8): 2.15 g / L Murashige & Skoog Salt (DUCHEFA Biochemie BV), 15 g / L Sucrose (Kanto Chemical), 1 mL MS Vitamin Solution, 100 mg / L Carbenicillin (Nacalai Tesque), 25 mg / L Kanamycin (Wako Pure Chemicals), 0.5 g / L MES (Wako Pure Chemicals), 2.5 g / L Gellan Gum (Kanto Chemical).

[0059] Example 1: Preparation of DNA fragments containing candidate CDE promoters. To isolate promoter-containing regions upstream of the coding sequences for carbohydrate degrading enzymes (CDEs) 1 and 2, polymerase chain reaction (PCR) primers were designed using genome data from the Sol Genomics Network's Nicotiana benthamiana genome database v1.0.1 (https: / / solgenomics.net / organism / Nicotiana_benthamiana / genome). Primers were synthesized for amplification of the enhanced green fluorescence protein (EGFP) gene and fragments containing candidate CDE promoters of various lengths (2500 bp, 2000 bp, 1500 bp, 1000 bp, 500 bp, 250 bp, 100 bp, or 50 bp) for subsequent ligation reactions. The lengths of the candidate CDE promoters were defined relative to the putative transcription start site, indicated as the 5' end of the native CDE 5'-untranslated region (5'-UTR). The sequences of the candidate CDE1 and CDE2 promoters are shown in SEQ ID NOS: 1-8 and 9-16, respectively: the candidate CDE1 promoters are 2500 bp (SEQ ID NOS: 1), 2000 bp (SEQ ID NOS: 2), 1500 bp (SEQ ID NOS: 3), 1000 bp (SEQ ID NOS: 4), 500 bp (SEQ ID NOS: 5), 250 bp (SEQ ID NOS: 6), 100 bp (SEQ ID NOS: 7), and 50 bp (SEQ ID NOS: 8); the candidate CDE2 promoters are 2500 bp (SEQ ID NOS: 9), 2000 bp (SEQ ID NOS: 10), 1500 bp (SEQ ID NOS: 11), 1000 bp (SEQ ID NOS: 12), 500 bp (SEQ ID NOS: 13), 250 bp (SEQ ID NOS: 14), 100 bp (SEQ ID NOS: 15), and 50 bp (SEQ ID NOS: 16). The sequences of the native 5'-untranslated regions (5'-UTRs) of CDE1 and CDE2 are shown in SEQ ID NOS: 17 and 18, respectively.

[0060] Figures 1 and 2 show the sequence (SEQ ID NO: 19) representing the relative positions of the promoter candidates, 5'-UTR, and start codon for CDE1 at various lengths. Similarly, Figures 3 and 4 show the sequence (SEQ ID NO: 20) representing the relative positions of the promoter candidates, 5'-UTR, and start codon for CDE2 at various lengths. In these figures, the sequence of the 5'-UTR region is shown in italics, the recognition sequence for the restriction enzyme NdeI is underlined, the boxed portion represents the start codon, and the remaining portion in standard type is the sequence of the candidate promoter region. In these figures, the numbers in parentheses represent the length of the sequence from the putative transcription start site (corresponding to the "number of bases," but excluding the bases at the putative transcription start site). In these figures, each sequence extending upstream from the 3'-terminal base of the candidate promoter region adjacent to the putative transcription initiation site ("T" in Figures 1 and 2, "C" in Figures 3 and 4) to the position corresponding to its base length (number in parentheses) corresponds to SEQ ID NOs: 1 to 16, respectively, as follows: SEQ ID NO: 1: Sequence up to (2500) in Figures 1 and 2 (CDE1); SEQ ID NO: 2: Sequence up to (2000) in Figures 1 and 2 (CDE1); SEQ ID NO: 3: Sequence up to (1500) in Figures 1 and 2 (CDE1); SEQ ID NO: 4: Sequence up to (1000) in Figures 1 and 2 (CDE1); SEQ ID NO: 5: Sequence up to (500) in Figures 1 and 2 (CDE1); SEQ ID NO: 6: Sequence up to (250) in Figures 1 and 2 (CDE1); SEQ ID NO: 7: Sequence up to (100) in Figures 1 and 2 (CDE1); SEQ ID NO: 8: Sequence up to (50) in Figures 1 to 2 (CDE1); SEQ ID NO: 9: Sequence up to (2500) in Figures 3 to 4 (CDE2); SEQ ID NO: 10: Sequence up to (2000) in Figures 3 to 4 (CDE2); SEQ ID NO: 11: Sequence up to (1500) in Figures 3 to 4 (CDE2); SEQ ID NO: 12: Sequence up to (1000) in Figures 3 to 4 (CDE2); SEQ ID NO: 13: Sequence up to (500) in Figures 3 to 4 (CDE2); SEQ ID NO: 14: Sequence up to (250) in Figures 3 to 4 (CDE2); SEQ ID NO: 15: Sequence up to (100) in Figures 3 to 4 (CDE2); SEQ ID NO: 16: Sequence up to (50) in Figures 3 to 4 (CDE2).

[0061] Primer synthesis was outsourced to an oligo DNA synthesis service (Thermo Fisher Scientific). The primer pairs and template combinations used are shown in Tables 1 and 2 below. Primer sequences are shown in Table 3. Templates were prepared or obtained as follows: Leaves of wild-type N. benthamiana were frozen in liquid nitrogen and crushed using a mortar and pestle. Genomic DNA was prepared from the crushed leaves according to the protocol of the DNeasy Plant Mini kit (QIAGEN). EGFP DNA was obtained from pEGFP-n1 (Clonetech).

[0062]

[0063]

[0064]

[0065] The target fragment was amplified by PCR using a Veriti 96-well thermal cycler (Applied Biosystems) with the following composition and conditions: PCR reaction solution composition: 25 μL of KOD One PCR Master Mix (TOYOBO), 1 μL of Fw primer (10 pmol / μL), 1 μL of Rv primer (10 pmol / μL), 1 μL of template DNA (1 ng / μL), and dH2O up to 50 μL. PCR reaction conditions: Step 1: 96°C for 1 minute, Step 2: (96°C for 30 seconds, (Tm -5)°C for 30 seconds, 68°C for 5 seconds / kb) x 30 cycles, Step 3: 68°C for 15 seconds / kb.

[0066] The PCR product was purified using a Gel / PCR DNA Isolation System (VIOGENE) after agarose gel electrophoresis. A solution containing equal amounts of purified PCR product and 2x DreamTaq Green Master Mix (Thermo Fisher Scientific) was incubated at 72°C for 30 minutes to add deoxyadenosine overhangs to the 3' ends of the PCR fragments. The resulting DNA solution was separated by agarose gel electrophoresis, and the DNA was extracted from the gel using a Gel / PCR DNA Isolation System (VIOGENE) as an insert DNA for the T-vector.

[0067] The ligation mixture was prepared as follows, and the prepared mixture was incubated at 16°C for 30 minutes, followed by incubation at 4°C for 30 minutes. Ligation mixture: 1 μL of pGEM®-T Easy (Promega), M μL of insert DNA (N ng / μL), and 3 μL of 2x Ligation Mix (Takara Bio) (the concentration (N) and amount (M) of insert DNA were determined using the following formula).

[0068]

[0069] (Example 2: Construction of expression vectors containing candidate CDE promoters) The insert DNA prepared as described in Example 1 was cloned using the SLiCE (Seamless Ligation Cloning Extract) method (Zhang, Y., Werling, U., & Edelmann, W. (2013). Seamless Ligation Cloning Extract (SLiCE) Cloning Method. DNA Cloning and Assembly Methods, 235-244.). The insert DNA and vector DNA (50 ng / μL) were reacted at a molar ratio of 1:1 at 37°C for 20 minutes.

[0070] EGFP expression vectors with different combinations of promoter and 5'-UTR were constructed as follows. The configuration of the gene expression system in each expression vector used in the examples is shown in Figure 5. The direction of the arrows in Figure 5 indicates the direction of transcription. Each diagram in Figure 5 shows, from left to right, the promoter, 5'-UTR, (if any) the target gene to be expressed (in Figure 5, the gene encoding "EGFP"), and terminator. "CDE1P" or "CDE2P" inside the promoter arrow represents the candidate CDE1 promoter or CDE2 promoter. "HSPT" represents the HSP terminator. The annotations at the bottom of Figure 5 indicate the names of the various expression vectors.

[0071] pRI201-AN (Takara Bio) was used as the basis for vector construction. pRI201-AN contains the full-length CaMV 35S promoter (346 bp) and the Arabidopsis alcohol dehydrogenase (AtADH) 5'-UTR (58 bp) (Fig. 5a, "pRI201-AN (vector control: VC)"). pRI201-AN also contains an HSP terminator (HSPT).

[0072] The NdeI / SacI-digested pRI201-AN was ligated with purified PCR product #1 (the purified product of PCR using the primer pair and template #1 in Table 1: EGFP fragment) to insert the EGFP gene fragment downstream of the AtADH 5'-UTR (58 bp). This resulted in the construction of an EGFP expression vector (pRI201-EGFP) driven by the constitutive CaMV 35S promoter. Insertion of the EGFP gene into the vector was confirmed by sequencing. pRI201-EGFP contains, from upstream to downstream, the full-length CaMV 35S promoter (346 bp), the AtADH 5'-UTR (58 bp), and the EGFP gene (Figure 5b, "pRI201-EGFP(35SP)").

[0073] pRI201-EGFP was digested with HindIII / NdeI to remove the full-length CaMV 35S promoter (346 bp) and the AtADH 5'-UTR (58 bp), and then ligated individually with purified PCR products #2 to #17 (the purified PCR products using each primer pair and template for #2 to #17 in Table 1). As a result, an EGFP expression vector (referred to as CDE1P_X, where X indicates the length of the promoter candidate) was constructed (Fig. 5c "CDE1P"), containing, from upstream to downstream, a CDE1 promoter candidate (CDE1P) with lengths from the putative transcription start site of 2500 bp (SEQ ID NO: 1), 2000 bp (SEQ ID NO: 2), 1500 bp (SEQ ID NO: 3), 1000 bp (SEQ ID NO: 4), 500 bp (SEQ ID NO: 5), 250 bp (SEQ ID NO: 6), 100 bp (SEQ ID NO: 7), or 50 bp (SEQ ID NO: 8). The native CDE1 5'-UTR (23 bp) (SEQ ID NO: 17) and the EGFP gene were arranged in this order from upstream to downstream (Fig. 5c "CDE1P"). Similarly, we constructed EGFP expression vectors (CDE2P_X, where X indicates the length of the promoter candidate) containing the CDE2 promoter candidate (CDE2P), which was 2500 bp (SEQ ID NO: 9), 2000 bp (SEQ ID NO: 10), 1500 bp (SEQ ID NO: 11), 1000 bp (SEQ ID NO: 12), 500 bp (SEQ ID NO: 13), 250 bp (SEQ ID NO: 14), 100 bp (SEQ ID NO: 15), or 50 bp (SEQ ID NO: 16) from the putative transcription start site (Fig. 5d). The vectors were then sequenced downstream to contain the native CDE2 5'-UTR (225 bp) (SEQ ID NO: 18) and the EGFP gene (Fig. 5d). The insertion of promoters of various lengths was confirmed by sequencing the 5' end of the insert DNA.

[0074] pRI201-EGFP was digested with HindIII / XbaI to remove the full-length CaMV 35S promoter, and purified PCR products #18–#20 (#18–#20 in Table 1) were individually ligated with the purified PCR products using each primer pair and template. This resulted in the construction of EGFP expression vectors containing either 2000-bp, 1000-bp, or 500-bp CDE1P upstream of EGFP and the AtADH 5'-UTR downstream of the native CDE1 5'-UTR (referred to as CDE1P-AU_X, where X indicates the length of the promoter candidate) (Fig. 5e, "CDE1P-AU").

[0075] Five microliters of the SLiCE reaction solution was added to Escherichia coli DH5α competent cells, pipetted, and then incubated on ice for 30 minutes. The cells were heat-shocked at 42°C for 1 minute and incubated on ice for 2 minutes. After that, 200 μL of LB medium was added and incubated at 37°C for 45 minutes. To select transformants, 100 μL was spread onto LB plates supplemented with kanamycin, incubated overnight at 37°C to obtain colonies, and then stored at 4°C. Transformants were confirmed by colony PCR using primers for the insert DNA and GoTaq® Master Mix (Promega). Transformants were cultured overnight in LB medium at 37°C, then centrifuged at 10,000 × g for 1 minute to harvest the cells. Plasmids were extracted by standard methods. Purified plasmids were obtained by extraction using the FastGene Plasmid Mini Kit (NIPPON Genetics), if necessary. The purified plasmid was subjected to sequence analysis by a DNA sequencing service (Azenta) to confirm that the desired plasmid had been constructed.

[0076] (Example 3: Transient expression of EGFP under candidate CDE promoters) Agrobacterium transformed with each of the vectors (CDE1P_X and CDE2P_X) constructed using purified PCR products #2 to #17 in Example 2 was used. The Agrobacterium transformants were infiltrated into leaves of N. benthamiana plants by agroinfiltration using a needleless syringe (syringe infiltration). For agroinfiltration, Agrobacterium transformants were used along with Agrobacterium carrying the p19 gene, a suppressor of RNA silencing (Uthailak et al., Front Plant Sci. 2021 Jun 7;12:683762).

[0077] Agrobacterium transformants were generated as follows. A frozen stock of Agrobacterium tumefaciens LBA4404 was inoculated into 5 mL of 2xYT medium and cultured overnight at 30°C with shaking. Then, 200 mL of 2xYT medium was inoculated and cultured at 30°C with shaking at 133 rpm until the OD600 reached 0.4-0.6. After cooling on ice for 10 minutes, the cells were centrifuged at 2,500 rpm and 4°C for 10 minutes and the supernatant was removed. The pellet was washed by suspending it in 20 mL of autoclaved 10% (v / v) glycerol solution, centrifuging it at 2,500 rpm and 4°C for 10 minutes, and removing the supernatant. This washing was repeated twice. The pellet was suspended in 2 mL of ice-cold 10% (v / v) glycerol solution, and 50 μL aliquots were flash-frozen in liquid nitrogen and stored at -80°C. These were used as competent cells.

[0078] Stored Agrobacterium competent cells were thawed on ice. 10 ng of purified plasmid was added to 25 μL of competent cells and suspended by pipetting. This suspension was transferred to an ice-cooled 0.2 cm cuvette (#165-2086, BIO-RAD) and subjected to an electric pulse (2.5 kV, 25 mF, 400 ohms) using a Gene Pulser (BIO-RAD). 200 μL of 2xYT medium was added to the cuvette and pipetted into a 1.5 mL tube. An additional 800 μL of 2xYT medium was added and cultured at 30°C for 2 hours. After centrifugation at 5000 × g for 3 minutes, 900 μL of the supernatant was removed and the remaining material was suspended. The suspension was then plated on 2xYT plates containing kanamycin, rifampicin, and streptomycin. After culturing at 30°C for 2 days to obtain colonies, the plates were stored at 4°C. Transformants were confirmed by colony PCR using primers for the insert DNA and Go taq® Mix (Promega).

[0079] Agroinfiltration was performed as follows. The Agrobacterium transformant and the Agrobacterium carrying the p19 gene were inoculated into 5 mL of 2xYT medium containing kanamycin, rifampicin, and streptomycin. After pre-incubation at 30°C for 24 hours, they were added to 200 mL of the same medium and cultured at 30°C until the OD600 reached 2. The culture was centrifuged at 4°C for 10 minutes at 4,000 × g to recover the Agrobacterium. The Agrobacterium transformant and the Agrobacterium carrying the p19 gene were mixed in a 1.5 mL microtube at a 1:1 ratio, and the pellets were resuspended in infiltration buffer (10 mM MES, 10 mM MgSO4, pH 5.8) to an OD600 of 0.2 (final OD600 of 0.4) to obtain an Agrobacterium suspension. The Agrobacterium suspension was placed in a needleless syringe and injected into the abaxial side of mature leaves of N. benthamiana plants grown 4–5 weeks after germination. Successful infiltration was confirmed by visual inspection of the infiltrated area, which was darkened. Infiltration was continued until the diameter of the infiltrated area was approximately the same (approximately 1.5 cm).

[0080] (Example 4: Observation of EGFP fluorescence under CDE promoter candidate) After infiltration in Example 3, the plants were cultivated in a plant room for 5 days, and the infiltrated leaves were collected. Fluorescence at each infiltration site was visually observed for the leaves 5 days post-infiltration (dpi). The entire leaf was exposed to 470 nm blue light and photographed using a Canon Kiss X5 with a 490 nm low-pass filter. The photographing conditions were a shutter speed of 0.25 seconds, F5.6, and ISO 1600.

[0081] Fluorescence micrographs of N. benthamiana leaves infiltrated by syringe infiltration with Agrobacterium transformed with vectors containing each CDE promoter candidate are shown. Figure 6 shows the results for each CDE1 promoter candidate, and Figure 7 shows the results for each CDE2 promoter candidate. The numbers in Figures 6 and 7 indicate the length of each promoter candidate from its predicted transcription start site. "35SP" represents the positive control when the vector pRI201-EGFP, which contains the full-length CaMV 35S promoter, was used, and "VC" represents the negative control when the base vector pRI201-AN, which contains the full-length CaMV 35S promoter but does not contain the EGFP coding sequence, was used. For the CDE1 promoter candidate, EGFP fluorescence was observed for candidate lengths ranging from 2500 bp to 250 bp (Figure 6). However, fluorescence was not detectable for lengths of 100 bp and 50 bp. In contrast, EGFP expression was confirmed for all CDE2 promoter candidates tested (Figure 7).

[0082] Example 5: Western Blotting Analysis of EGFP Expression Under Candidate CDE Promoters After infiltration as described in Example 3, plants were grown in a plant room for 5 days, and infiltrated leaves were harvested. At 5 days post-infiltration (dpi), leaves were excised from each infiltrated site, frozen in liquid nitrogen, and crushed. Extraction buffer (PBS; NaCl 8.0 g / L, NaHPO 1.1 g / L, KCl 0.2 g / L, KHPO 0.2 g / L) was added to 0.1 g of crushed material at a ratio of 0.2 mL per 0.1 g of crushed material. The mixture was then cooled on ice for 60 minutes with appropriate stirring using a vortex mixer. The mixture was centrifuged at 15,000 × g at 4°C for 15 minutes, and the supernatant was collected. This was then centrifuged again at 15,000 × g at 4°C for 10 minutes, and the supernatant was collected to serve as the protein extract. The samples were dissolved in sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 7% (v / v) glycerol, 5% (v / v) 2-mercaptoethanol, 0.025% bromophenol blue) and stored at -20°C.

[0083] Protein concentrations in the samples were measured using Protein Assay CBB solution (5x) (Nacalai tesque). After boiling for 5 minutes, the samples were centrifuged at 15,000 x g for 3 minutes. The supernatant was applied to an acrylamide gel and subjected to SDS-PAGE. As a positive control, 10 μg of histidine-tagged GFP (Millipore #14-392) was electrophoresed alongside the test samples. The gel was then subjected to Western blotting. Proteins were transferred to a PVDF membrane (Immobilon-P Transfer Membrane, Millipore) according to standard procedures. The primary antibody was a rabbit anti-GFP antibody (MBL598, MBL, 1:5000 dilution) and the secondary antibody was an HRP-conjugated anti-rabbit IgG antibody (Sigma-Aldrich, 1:10000 dilution). Detection was performed using Luminata Forte Western HRP substrate (Millipore) and an iBright Imaging System (Thermo Fisher).

[0084] The results of Western blot analysis are shown in Figure 8 (CDE1 promoter candidate) and Figure 9 (CDE2 promoter candidate). In Figure 8, the number attached to "1P" indicates the length of the CDE1 promoter candidate used. Similarly, in Figure 9, the number attached to "2P_" indicates the length of the CDE2 promoter candidate used. In Figures 8 and 9, "35SP" indicates the results obtained using the vector pRI201-EGFP, which contains the full-length CaMV 35S promoter; "VC" indicates the results obtained using the base vector pRI201-AN; "WT" indicates the results obtained without infiltration; and "PC" indicates the positive control (histidine-tagged GFP).

[0085] For the CDE1 promoter candidate, bands were detected at lengths of 250 bp to 2500 bp, confirming EGFP expression. However, no bands were detected at lengths of 50 bp and 100 bp. For the CDE2 promoter candidate, bands were detected at all lengths examined, confirming EGFP expression in all lengths.

[0086] (Example 6: Examination of EGFP expression levels under CDE promoter candidates) To further examine transient expression by agroinfiltration, the expression levels of EGFP were measured for CDE1 promoter candidates with lengths of 2000 bp, 1000 bp, 500 bp, and 250 bp, and for CDE2 promoter candidates with lengths of 2000 bp, 1000 bp, 500 bp, and 50 bp.

[0087] Agrobacterium transformants were produced and Agrobacterium suspensions were prepared in the same manner as in Example 3, except that vectors containing each of the above CDE promoter candidates (CDE1P_2000, CDE1P_1000, CDE1P_500, CDE1P_250, CDE2P_2000, CDE2P_1000, CDE2P_500, and CDE2P_50) were used. In this example, the Agrobacterium transformants were infiltrated into mature leaves of N. benthamiana using vacuum infiltration instead of syringe infiltration. More specifically, N. benthamiana plants grown 4 to 5 weeks after germination were immersed in an Agrobacterium suspension and infected with Agrobacterium transformants by vacuum infiltration (-0.75 hPa, 3 minutes), followed by cultivation in a plant chamber for 5 to 9 days. Protein extracts were then prepared from leaves 5, 7, and 9 days after infiltration, as described in Example 5.

[0088] 100 μL of each appropriately diluted protein extract was placed in a black microplate. For the calibration curve, recombinant GFP produced in E. coli (Sigma-Aldrich) was prepared at concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, and 50 ng / μL, and 100 μL of each was applied. Fluorescence intensity was measured using a FLUOstar Omega (BGM Labtech). The excitation wavelength was 485 nm, and the emission wavelength was 520 nm. Using a calibration curve based on the fluorescence intensity of known concentrations of EGFP, the amount of EGFP expression was calculated by back-calculation from the fluorescence intensity of the sample.

[0089] The graphs show the levels of EGFP expression in N. benthamiana leaves 5, 7, and 9 days after vacuum infiltration with Agrobacterium transformed with vectors containing each CDE promoter candidate. Figure 10 shows the results when each CDE1 promoter candidate was used, and Figure 11 shows the results when each CDE2 promoter candidate was used. In Figure 10, 1P2000, 1P1000, 1P500, and 1P250 represent the cases where vectors containing any of the CDE1 promoter candidates with lengths of 2000 bp, 1000 bp, 500 bp, and 250 bp (CDE1P_2000, CDE1P_1000, CDE1P_500, and CDE1P_250), respectively, were used. In Figure 11, 2P2000, 2P1000, 2P500, and 2P50 represent the cases where vectors containing any of the CDE2 promoter candidates with lengths of 2000 bp, 1000 bp, 500 bp, and 50 bp (CDE2P_2000, CDE2P_1000, CDE2P_500, and CDE2P_50), respectively, were used. In Figures 10 and 11, "VC" indicates the case where the base vector pRI201-AN was used, and "WT" indicates the case where no infiltration was performed. Furthermore, in both Figures 10 and 11, the horizontal axis indicates the construct and treatment used for infiltration, and the vertical axis indicates the estimated amount of EGFP expressed (μg) per gram of leaf sample. Furthermore, the horizontal axis shows the results for each construct on days 5, 7, and 9, from left to right. Individual results and average values ​​for six samples are shown for each construct and treatment.

[0090] For all promoter candidates, EGFP expression increased over time. For the CDE1 promoter candidate, expression levels varied depending on the length within the range examined, but EGFP expression increased over time in all cases (Fig. 10). For the CDE2 promoter candidate, expression levels were roughly equivalent across different lengths within the range examined (Fig. 11).

[0091] Example 7: Effect of 5'-UTR on CDE promoter function Agrobacterium transformants were prepared in the same manner as in Example 3, except that the vectors used were CDE1P_2000, CDE1P_1000, and CDE1P_500 (vectors constructed using purified PCR products #3, #5, and #6 in Example 2), CDE1P-AU_2000, CDE1P-AU_1000, and CDE1P-AU_500 (vectors constructed using purified PCR products #18 to #20 in Example 2), pRI201-AN, and pRI201-EGFP. The Agrobacterium transformants were infiltrated into mature leaves of N. benthamiana by syringe infiltration, and the fluorescence of the leaves was visually observed 5 days post-infiltration (dpi) in the same manner as in Example 4.

[0092] Figure 12 shows fluorescence micrographs of leaves infiltrated by syringe infiltration with Agrobacterium transformed with vectors containing either the native CDE1 5'-UTR downstream of each CDE1 promoter candidate or the AtADH 5'-UTR. In Figure 12, "1P" represents the native CDE1 5'-UTR downstream, "1P-AU" represents the AtADH 5'-UTR downstream, and the numbers attached to these labels indicate the length of each promoter candidate from its predicted transcription start site. In Figure 12, "35SP" represents the result using the vector pRI201-EGFP, which contains the full-length CaMV 35S promoter, and "VC" represents the result using the base vector pRI201-AN (which contains the full-length CaMV 35S promoter but does not contain the EGFP coding sequence).

[0093] Visual observation revealed that CDE1P-AU, in which the CDE1 5'-UTR was replaced with the AtADH 5'-UTR, showed similar EGFP fluorescence compared to CDE1P at all lengths tested, with some bright regions observed in CDE1P-AU, although the differences were small compared to the 35SP reference.

[0094] (Example 8: Induced Expression by Agroinfiltration in Transgenic Plants) N. benthamiana transgenic plants were produced using Agrobacterium transformants prepared in the same manner as in Example 3 using the CDE2P_500 vector. Transgenic plants were produced based on the procedure described in Yau, YY., Easterling, M., Brennan, L. (2020). Rapid Agrobacterium-mediated transformation of tobacco cotyledons using toothpicks. Climate Change, Photosynthesis, and Advanced Biofuels. More specifically, the procedure is as follows: Acetosyringone (Tokyo Chemical Industry Co., Ltd.) was added to MS liquid medium to a concentration of 40 μg / mL, and the cultured Agrobacterium transformants were scraped with a platinum loop and suspended until visually turbid (OD600 = approximately 0.5-1.0). This suspension was slowly stirred at 30°C (in the dark) for 2 hours to obtain a bacterial cell solution. Eight days after germination on 1 / 2 MS medium, Nb leaves were cut and poked with a toothpick dipped in fungal fluid. The leaves were then placed face up on MS differentiation medium. The container containing the toothpick-pierced leaf and MS differentiation medium was sealed with parafilm and left to stand at 25°C (in the dark) for 3 days. The toothpick-pierced leaf was then cut and placed face up on MS selection medium. The container containing the leaf and MS selection medium was sealed with parafilm and cultured at 25°C (16 hours light / 8 hours dark). The medium was changed every two weeks. Once shoots emerged, they were transplanted into 1 / 2 MS rooting medium. Once fully grown (approximately 8-10 cm), the medium was removed, and the roots were immersed in Menedale® for 1 hour before being transplanted into soil.

[0095] Transgenic N. benthamiana plants containing CDE2P_500-EGFP (CDE2P_500-EGFP T2 plants) or wild-type N. benthamiana plants (WT) were transplanted into soil and cultivated for approximately 5 weeks. They were immersed in a suspension of untransformed Agrobacterium (strain LBA4404) (OD600 = 0.25) and infected by vacuum infiltration (-0.75 hPa, 3 minutes). They were then cultivated in a plant chamber for 3 to 7 days. Leaves were excised from the stems and harvested on days 0, 3, 5, and 7 after infiltration. Leaf fluorescence was visually observed as described in Example 4.

[0096] Figure 13 shows fluorescence micrographs of leaves from N. benthamiana CDE2P_500-EGFP-introduced transgenic plants (CDE2P_500-EGFP T2 plants) and wild-type plants (WT) on days 0, 3, 5, and 7 after agroinfiltration. While no changes were observed in the wild-type plants after agroinfiltration, fluorescence was observed in the CDE2P_500-EGFP-introduced transgenic plants on day 7 after agroinfiltration. This indicates that EGFP expression under the CDE2P_500 promoter was induced by agroinfiltration in the transgenic plants into which CDE2P_500-EGFP had been introduced.

[0097] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. DNA having promoter function, which is any one selected from the group consisting of (a) to (e) below: (a) DNA consisting of the base sequence represented by SEQ ID NO: 1 or a partial sequence thereof; (b) DNA consisting of the base sequence represented by SEQ ID NO: 9 or a partial sequence thereof; (c) DNA consisting of a base sequence having 90% or more sequence identity with the base sequence of the DNA of (a) or (b); (d) DNA consisting of a base sequence in which one or more bases have been substituted, deleted, added or inserted in the base sequence of the DNA of (a) or (b); and (e) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of the DNA of (a) or (b).

2. The DNA according to claim 1, wherein (a) is a DNA consisting of a base sequence represented by any one of SEQ ID NOs: 1 to 6.

3. The DNA according to claim 1, wherein (b) is a DNA consisting of a base sequence represented by any one of SEQ ID NOs: 9 to 16.

4. The DNA according to claim 1, wherein (a) further comprises downstream DNA consisting of the base sequence represented by SEQ ID NO:

17.

5. The DNA of claim 1, wherein (b) further comprises downstream DNA consisting of the base sequence represented by SEQ ID NO:

18.

6. An expression vector comprising the DNA of any one of claims 1 to 5.

7. The expression vector of claim 6, further comprising a gene of interest for expression.

8. An Agrobacterium transformant containing the expression vector according to claim 7.

9. A plant cell or tissue transformed using the Agrobacterium transformant according to claim 8.

10. A plant transformed using the Agrobacterium transformant according to claim 8.

11. A method for inducing expression of a target gene in a plant, comprising the steps of infecting a plant or cultured plant cells with the Agrobacterium transformant described in claim 8, and cultivating the plant infected with the Agrobacterium transformant or cultivating the cultured plant cells infected with the Agrobacterium transformant.

12. A method for inducing expression of a target gene in a plant, comprising the steps of infecting a transformed plant body described in claim 10 with Agrobacterium, and cultivating the transformed plant infected with the Agrobacterium.

13. The method according to claim 12, further comprising the step of producing a transformed plant using the Agrobacterium transformant according to claim 8.

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