Tomato with increased carotene content and method for producing same

Abstract

In the present invention, it is found that the content of lycopene can be increased without decreasing the content of β-carotene in tomatoes by introducing a mutation into a nitrate transporter gene (NPF1 gene) of tomatoes to express a mutant protein in which a C-terminal region is largely deleted.

Description

Tomatoes with increased carotene content and method for producing same

[0001] The present invention relates to tomatoes having an increased content of carotenes (particularly lycopene and β-carotene) and a method for producing the same.

[0002] Carotenoids are yellow or red pigments found widely in plants and animals. They are classified as carotenes and xanthophylls and have strong antioxidant properties. Lycopene and β-carotene are well-known examples of carotenes. These carotenes inhibit and remove reactive oxygen species, and are believed to be effective in preventing arteriosclerosis, aging, and cancer. Due to these properties, carotenes have attracted attention as functional components in agricultural crops, and conventional hybridization has been used to improve carotene content. Meanwhile, research on the biosynthesis and degradation of carotenes at the genetic level has progressed, and molecular breeding techniques have been reported that regulate the expression of genes involved in carotene metabolism to enhance the accumulation of specific carotenes, such as lycopene and β-carotene (Non-Patent Documents 1-4).

[0003] Ronen, et al. 2000 Proc. Natl. Acad. Sci. U. S. A. 97, 11102-11107. Fraser, et al. 2007 Plant Cell19, 3194-3211. Sun et al. 2012 J. Exp. Bot. 63, 3097-3108. Luo et al. 2013 New Phytol. 198, 442-452.

[0004] An object of the present invention is to provide a new tomato mutant having an increased lycopene content without a decrease in β-carotene content, and a method for producing the same.

[0005] As a result of intensive research aimed at solving the above problems, the present inventors have found that by introducing a mutation into the tomato nitrate transporter gene (NPF1 gene) and expressing a mutant protein with a large deletion in the C-terminal region, it is possible to increase the lycopene content in tomato without decreasing the β-carotene content. Furthermore, they have found that a specific mutation in the NPF1 gene makes it possible to increase the contents of both lycopene and β-carotene in tomato, leading to the completion of the present invention.

[0006] The present invention relates to tomatoes having an increased carotene content due to modification of the NPF1 gene and a method for producing the same, and more specifically provides the following:

[0007] (1) A tomato having an increased lycopene content, in which a mutation has been introduced into the endogenous gene encoding the NPF1 protein so as to express an NPF1 protein that contains at least 40 amino acids from the N-terminus but does not contain at least 400 amino acids from the C-terminus.

[0008] (2) The tomato according to (1), wherein the NPF1 protein containing at least 40 amino acids from the N-terminus but not containing at least 400 amino acids from the C-terminus is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[0009] (3) The tomato according to (1), further having an increased β-carotene content.

[0010] (4) The tomato according to (3), wherein the NPF1 protein containing at least 40 amino acids from the N-terminus but not containing at least 400 amino acids from the C-terminus is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0011] (5) A method for producing tomatoes with increased lycopene content, comprising introducing a mutation into an endogenous gene encoding the NPF1 protein so as to express an NPF1 protein that contains at least 40 amino acids from the N-terminus but does not contain at least 400 amino acids from the C-terminus.

[0012] (6) The method according to (5), wherein the NPF1 protein contains at least 40 amino acids from the N-terminus but not at least 400 amino acids from the C-terminus, and is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[0013] (7) The method according to (5), wherein the tomato has an increased lycopene content and further has an increased β-carotene content.

[0014] (8) The method according to (7), wherein the NPF1 protein contains at least 40 amino acids from the N-terminus but not at least 400 amino acids from the C-terminus, and is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0015] According to the present invention, by introducing a mutation into the NPF1 gene, it is possible to increase the lycopene content in tomato without decreasing the β-carotene content or to increase the β-carotene content while also increasing it.

[0016] 1 shows the expression of NPF1 protein in wild-type (WT) tomato (cultivar: Micro-Tom) and npf1 mutants obtained by chemical (EMS) treatment or genome editing. 2 shows a graph comparing the amount of lycopene (a) and β-carotene (b) accumulated in fruit of a nitrate transporter gene mutant (npf1.9) obtained by EMS treatment of tomato (cultivar: Micro-Tom) and a wild-type (WT). 3 shows a graph comparing the amount of lycopene (a) and β-carotene (b) accumulated in fruit of a nitrate transporter gene mutant (#38A-13) obtained by genome editing in a cultivated tomato cultivar and a wild-type (WT). 4 shows a comparison of the structure of NPF1 protein in wild-type tomato (cultivar: Micro-Tom) and npf1 mutants obtained by chemical (EMS) treatment or genome editing.

[0017] The present invention provides tomatoes having an increased lycopene content, in which a mutation has been introduced into an endogenous gene encoding the NPF1 protein so that the tomato expresses an NPF1 protein that contains at least 40 amino acids from the N-terminus but does not contain at least 400 amino acids from the C-terminus.

[0018] In the present invention, the "NPF1 protein" is a protein characterized as a nitrate transporter in tomato, and is typically a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. However, the amino acid sequence of the NPF1 protein may vary depending on the variety or individual tomato. The amino acid sequence of the NPF1 protein of the present invention may also vary in this manner. Therefore, the "NPF1 protein" of the present invention includes proteins consisting of an amino acid sequence that is 90% or more (e.g., 95% or more, 96% or more, 97% or more, or 99% or more) homologous to the amino acid sequence set forth in SEQ ID NO: 2. The amino acid sequence homology can be determined by aligning amino acid sequences using the blastp algorithm with default parameters in Protein BLAST (National Library of Medicine).

[0019] In the present Examples, it was found that when a mutation was introduced into an endogenous gene encoding NPF1 protein to express a mutant protein (SEQ ID NO: 4) having a chain length of 88 amino acids, including the wild-type sequence of 40 amino acids from the N-terminus of NPF1 protein (i.e., the amino acid sequence of positions 1 to 40 in SEQ ID NO: 2) followed by a mutated sequence of 44 amino acids, the lycopene content in tomato was increased. Furthermore, when a mutation was introduced into an endogenous gene encoding NPF1 protein to express a mutant protein (SEQ ID NO: 6) having a chain length of 120 amino acids, including the wild-type sequence of 103 amino acids from the N-terminus of NPF1 protein (i.e., the amino acid sequence of positions 1 to 103 in SEQ ID NO: 2) followed by a mutated sequence of 17 amino acids, the lycopene and β-carotene contents in tomato were both increased. These results suggest that expressing a protein containing the N-terminal portion of NPF1 protein while significantly deleting the C-terminal portion is effective in increasing the lycopene content in tomato.

[0020] Thus, the tomato of the present invention is characterized in that a mutation has been introduced into an endogenous gene encoding an NPF1 protein so as to express an NPF1 protein that contains at least 40 amino acids from the N-terminus but does not contain at least 400 amino acids from the C-terminus (hereinafter referred to as the "mutant NPF1 protein of interest"). More specifically, the mutant NPF1 protein of interest is a protein that contains at least 40 amino acids (e.g., 50, 60, 70, 80, 90, or 100 amino acids) from the N-terminus but does not contain at least 400 amino acids (e.g., 410, 420, 430, 440, 450, 460, or 470 amino acids) from the N-terminus, and a non-wild-type sequence (e.g., a mutant sequence) may be added to the N-terminus or C-terminus.

[0021] One preferred embodiment of the mutant NPF1 protein of interest is, for example, an NPF1 protein that contains at least 40 amino acids from the N-terminus but does not contain at least 470 amino acids from the C-terminus. Examples of such NPF1 proteins include the protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and the protein consisting of the amino acid sequence set forth in SEQ ID NO: 6. The protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 is particularly preferred because it can increase not only the lycopene content but also the β-carotene content in tomatoes.

[0022] Furthermore, comparison of the amino acid sequence of SEQ ID NO: 4 with the amino acid sequence of SEQ ID NO: 6 suggests that an amino acid sequence effective for increasing β-carotene content is present at positions 41 to 103 of the amino acid sequence of the NPF1 protein. The minimal amino acid sequence effective for increasing β-carotene content can be determined by generating tomatoes in which mutations have been introduced into the endogenous gene encoding the NPF1 protein so that proteins of various chain lengths consisting of 40 amino acids from the N-terminus to 103 amino acids from the N-terminus are expressed, and then evaluating the β-carotene content of these proteins. The tomatoes of the present invention include tomatoes with increased contents of both lycopene and β-carotene, as identified in this manner.

[0023] In the case of tomatoes of the present invention, "the lycopene content is increased" means that the lycopene content in tomato fruit is higher compared to a control (a case in which no mutation is introduced into the endogenous gene encoding the NPF1 protein). Similarly, in the case of tomatoes of the present invention, "the β-carotene content is increased" means that the β-carotene content in tomato fruit is higher compared to a control (a case in which no mutation is introduced into the endogenous gene encoding the NPF1 protein). The lycopene and β-carotene contents in tomato fruit are preferably 10% or more, more preferably 20% or more (e.g., 30% or more, 40% or more, 50% or more) higher than the control. These contents can be evaluated using the absorbance method and calculation formula described in the Examples of the present application.

[0024] In the present invention, examples of tomatoes for which the lycopene content is increased include Solanum lycopersicum, Solanum cerasiforme, Solanum pimpinellifolium, Solanum cheesemanii, Solanum parviflorum, Solanum chmielewskii, Solanum hirsutum, Solanum pennellii, and Solanum pervianum. Examples of tomato varieties include, but are not limited to, tomato lines and varieties belonging to Solanum pennellii, Solanum chilense, Solanum lycopersicoides, and Solanum habrochaites, or derivatives thereof. In the present invention, the wild-type tomato variety Micro-Tom (Solanum lycopersicum cv. Micro-Tom) can be preferably used.

[0025] The present invention also provides a method for producing tomatoes with increased lycopene content, which comprises introducing a mutation into an endogenous gene encoding an NPF1 protein so as to express a mutant NPF1 protein of interest.

[0026] The nucleotide sequence of an endogenous gene (genomic DNA) encoding a typical NPF1 protein in tomato is shown in SEQ ID NO: 7. Various known methods can be used to introduce a mutation into an endogenous gene encoding the NPF1 protein, but it is preferable to use a genome editing system from the viewpoint of high efficiency in introducing the desired mutation. Examples of genome editing systems include class 2 CRISPR-Cas systems (e.g., type II CRISPR-Cas9 systems, type V CRISPR-Cas12 systems, etc.) and class 1 CRISPR-Cas systems (e.g., type I CRISPR-Cas3 systems, etc.). Furthermore, proteins in which a nuclease domain such as FokI is fused to a DNA binding domain such as ZF (zinc finger), TALE (transcription activator-like effector), or PPR (pentatricopeptide repeat) can also be used.

[0027] In an endogenous gene encoding the NPF1 protein, by introducing a mutation into the base near the 3' side of the base sequence encoding the region of the wild-type amino acid sequence of the target mutant NPF1 protein (the base encoding the codon adjacent to the 3' side), a frameshift and / or a stop codon can be generated on the 3' side of the base sequence encoding the wild-type amino acid sequence, thereby making it possible to express a protein in which a mutant amino acid sequence has been added to the wild-type amino acid sequence on the N-terminal side of the NPF1 protein, or a protein consisting only of the wild-type amino acid sequence on the N-terminal side of the NPF1 protein.

[0028] Furthermore, by using a donor vector containing a gene encoding the desired mutant NPF1 protein (a vector in which homology arms corresponding to the endogenous gene encoding the NPF1 protein are arranged at both ends of the gene encoding the desired mutant NPF1 protein), the endogenous gene encoding the NPF1 protein can be converted into a gene encoding the desired mutant NPF1 protein by homologous recombination.

[0029] Various known methods can be used to introduce a vector into tomato cells, including physical introduction methods such as particle gun technology, electroporation, polyethylene glycol technology, and microinjection, as well as indirect introduction methods using soil bacteria such as Agrobacterium. Tomato cells into which a target gene has been introduced can be selected, for example, by using a selection marker gene (e.g., a drug resistance gene such as a kanamycin resistance gene) carried by the vector. Tomato cells selected in this way can be regenerated into plants by culturing them in a regeneration medium containing an appropriate concentration of plant hormone.

[0030] Alternatively, the tomato of the present invention can be produced by a method in which tomatoes are subjected to a mutagen treatment (e.g., treatment with a mutagen such as ethylmethanesulfonate (EMS), ethyleneimine (MI), propane sultone, N-methyl-N-nitrosourethane (MNU), ethylnitrosourea (ENU), or sodium azide, or irradiation with radiation such as X-rays, gamma rays, neutrons, beta rays, ion beams, or ultraviolet rays), and individuals expressing the desired mutant NPF1 protein are screened.

[0031] Once a tomato plant having the desired mutation introduced therein is obtained, that plant or its progeny can be crossed with another tomato plant, and from the resulting progeny, an individual plant having the desired mutation introduced therein can be obtained. The tomato of the present invention also includes the progeny thus produced. Furthermore, the tomato of the present invention includes not only the entire plant body but also parts thereof (e.g., stems, leaves, roots, flowers, buds, fruit, seeds, and cells) as long as the desired mutation has been introduced.

[0032] 1. Materials and Methods Two lines of tomato (Solanum lycopersicum L.) having a mutation in the tomato NPF1 gene (Nitrate Transporter 1 / Peptide Transporter Family; Solyc04g005070) were used as test plants.

[0033] One of these is the npf1.9 mutant, which was isolated from the M3 generation of a large-scale Micro-Tom mutant population irradiated with EMS and γ-rays as a line with high carotenoid content. The npf1.9 mutant contains a single point mutation at the junction of the second intron and third exon. This mutation results in an aberrant splicing variant, resulting in a shorter protein than the wild-type (whereas the wild-type NPF1 protein consists of 573 amino acids, the npf1.9 mutant is identical to the wild-type up to the 103rd amino acid residue, but differs from the wild-type in the subsequent 17 amino acids; Figure 1).

[0034] The other is a cultivated tomato variety (F １ The strain #38A-13 was created by genome editing (CRISPR / Cas9) of the parent strain of the same strain, and contains a single-base insertion mutation in the second exon of the NPF1 gene. This mutation causes a frameshift, resulting in the production of a shorter protein consisting of 84 amino acids. Of these 84 amino acids, the first 40 amino acid residues are the same as those of the wild type, but the following 44 amino acids are different from those of the wild type (Figure 1).

[0035] The #38A-13 mutant was created using the genome editing technology CRISPR / Cas9 method. A gRNA (SEQ ID NO: 8) was designed in the second exon of the NPF1 gene, and a vector was constructed to express this gRNA, Cas9 protein, and a selection marker. The Agrobacterium method (Sun et al., Plant cell psychiatry, 2006; 47(3):426-431) was used for transformation, and a cultivated tomato variety (F １) The vector was introduced into the parent line of the same plant. Plants regenerated from a series of selective media containing kanamycin were subjected to primary selection as candidates for transformants. Subsequently, to confirm the transformants, genomic DNA was extracted from young tomato leaves, and primers (SEQ ID NOs: 9 and 10) that specifically bind to the CRISPR / Cas9 vector were used to confirm whether the CRISPR / Cas9 vector had been introduced. Subsequently, Sanger sequencing was performed to confirm whether a mutation had occurred near the target sequence. First, DNA fragments near the target sequence were PCR amplified using genomic DNA extracted from the tomato and primers (SEQ ID NOs: 11 and 12) designed around the target sequence. The amplified PCR fragment was used as a sequencing template and subjected to Sanger sequencing together with an NPF1 gene-specific primer (SEQ ID NO: 11). Individuals in which a mutation was confirmed near the target sequence were selfed, and a null segregant was obtained in which the mutation was homozygous in the progeny and the CRISPR / Cas9 vector had been removed (#38A-13).

[0036] The lycopene and β-carotene contents of the fruit were measured using absorbance. Mature red fruits were harvested 12 days after the start of tarnishing from wild-type, npf1.9 mutant (homozygous mutant), and #38A-13 mutant (homozygous mutant) and flash-frozen in liquid nitrogen. They were crushed into powder using a mortar and pestle while pouring liquid nitrogen over them, and 100 mg of each was used for carotenoid extraction (containing lycopene and β-carotene). Carotenoid extraction was performed by adding the powdered sample to 8 ml of a hexane-acetone-ethanol (2:1:1 v / v) solution, vortexing for 20 seconds, sonicating, stirring in the dark, and allowing to stand for 10 minutes. After 10 minutes, 1 ml of deionized water (DW) was added to the solution, vortexing, and then allowing to stand for 10 minutes in the dark without stirring to separate the organic phase. Then, 700 μl of the organic phase was transferred to a quartz cuvette and measured at 444 nm (A ４４４ ) and 503 nm (A ５０３ The volume (V) of the extract and the weight (W) of the sample were recorded, and the contents of lycopene and β-carotene were calculated using the following formula:

[0037] Lycopene: (μg / gFW) = (6.95A５０３ -1.59A ４４４ ) × 0.55 × 537 × (V / W) β-carotene: (μg / g FW) = (9.38 A ４４４ -6.710A ５０３ )×0.55×537×(V / W).

[0038] 2. Results We measured the lycopene and β-carotene contents of fruit from two tomato mutant lines (npf1.9 and #38A-13) harboring mutations in the NPF1 gene. The EMS line, npf1.9, showed increases in both lycopene and β-carotene (Figure 2). In contrast, the genome-edited line, #38A-13, showed increased lycopene content, while its β-carotene content was comparable to that of the wild type (Figure 3). The difference in β-carotene content between the npf1.9 mutant, consisting of 120 amino acids (including the 103-amino acid wild-type sequence), and the #38A-13 mutant, consisting of 84 amino acids (including the 40-amino acid wild-type sequence), suggests that the amino acid sequence between positions 41 and 103 of the NPF1 protein is important for increasing the β-carotene content of fruit (Figure 4). Furthermore, since both the npf1.9 mutant and the #38A-13 mutant showed increased lycopene content, it was suggested that a large deletion of the C-terminal end of the NPF1 protein is important for increasing the lycopene content of fruit (Figure 4).

[0039] As described above, according to the present invention, it is possible to increase the lycopene content in tomatoes without decreasing the β-carotene content or by increasing the β-carotene content. These carotenes have the effect of suppressing and removing reactive oxygen species and are effective in preventing arteriosclerosis. Therefore, the present invention will make a significant contribution, primarily to the agricultural field, as a technology for improving functional components in plants.

Claims

1. A tomato having an increased lycopene content, in which a mutation has been introduced into the endogenous gene encoding the NPF1 protein so as to express an NPF1 protein that contains at least 40 amino acids from the N-terminus but does not contain at least 400 amino acids from the C-terminus.

2. The tomato according to claim 1, wherein the NPF1 protein containing at least 40 amino acids from the N-terminus but not at least 400 amino acids from the C-terminus is a protein consisting of the amino acid sequence set forth in SEQ ID NO:

4.

3. The tomato according to claim 1, further having an increased content of β-carotene.

4. The tomato according to claim 3, wherein the NPF1 protein containing at least 40 amino acids from the N-terminus but not containing at least 400 amino acids from the C-terminus is a protein consisting of the amino acid sequence set forth in SEQ ID NO:

6.

5. A method for producing tomatoes with increased lycopene content, comprising introducing a mutation into an endogenous gene encoding an NPF1 protein so as to express an NPF1 protein that contains at least 40 amino acids from the N-terminus but does not contain at least 400 amino acids from the C-terminus.

6. The method according to claim 5, wherein the NPF1 protein containing at least 40 amino acids from the N-terminus but not containing at least 400 amino acids from the C-terminus is a protein consisting of the amino acid sequence set forth in SEQ ID NO:

4.

7. The method according to claim 5, wherein the tomato has an increased lycopene content and further has an increased β-carotene content.

8. The method according to claim 7, wherein the NPF1 protein containing at least 40 amino acids from the N-terminus but not containing at least 400 amino acids from the C-terminus is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6.

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