Method for producing ppGpp-enriched organisms and organisms

Abstract

To provide a ppGpp-enriched organism.SOLUTION: A method for producing a ppGpp-enriched organism employs an expression unit that includes: a ppGpp synthase gene including a region encoding a ppGpp synthesis domain but not including a region encoding a ppGpp degradation domain; an inducible promoter that controls the transfer of the ppGpp synthase gene; and a region that is adjacent to the ppGpp synthase gene and encodes a chloroplast transit peptide. The method includes growing an organism containing a chloroplast in the presence of an inducer that activates the inducible promoter.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing ppGpp-enriched organisms and organisms. [Background technology]

[0002] ppGpp (guanosine tetraphosphate), shown below, is a nucleotide that was discovered as a signaling substance in bacterial responses to nutrient starvation.

[0003] [ka]

[0004] ppGpp and pppGpp (guanosine pentaphosphate) are synthesized and decomposed as shown in the following reaction formula (1). This reaction is catalyzed by a ppGpp synthase. For example, Non-Patent Document 1 describes Rel as a ppGpp synthase found in Bacillus subtilis.

number

[0005] Plants also have ppGpp synthases. For example, Non-Patent Document 2 describes that Arabidopsis thaliana has four types of ppGpp synthases.

[0006] Furthermore, the increase or decrease in ppGpp levels in plant chloroplasts affects the plant's resistance to nutrient starvation and its overall weight. One way to increase ppGpp levels in chloroplasts is to overexpress the gene encoding the ppGpp synthase RSH (RelA-SpoT Homolog).

[0007] For example, Non-Patent Document 3 describes a method for overexpressing RSH2 or RSH3 in Arabidopsis thaliana, one of the four types of ppGpp synthases present in Arabidopsis thaliana. [Prior art documents]

Non-Patent Literature

[0008]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Summary of the Invention

[0009] An object of the present invention is to provide a ppGpp-enriched organism. [Means for solving the problem]

[0010] According to a first aspect of the present invention, there is provided a method for producing a ppGpp-enriched organism, which comprises cultivating an organism having chloroplasts, in the presence of an inducer that activates the inducible promoter, the organism comprising an expression unit including a ppGpp synthase gene that includes a region encoding a ppGpp synthesis domain but not a region encoding a ppGpp degradation domain, an inducible promoter that controls the transcription of the ppGpp synthase gene, and a region encoding a chloroplast transit peptide adjacent to the ppGpp synthase gene.

[0011] According to a second aspect of the present invention, there is provided an organism having chloroplasts, comprising an expression unit including a ppGpp synthase gene that includes a region encoding a ppGpp synthesis domain but not a region encoding a ppGpp degradation domain, an inducible promoter that controls the transcription of the ppGpp synthase gene, and a region encoding a chloroplast transit peptide adjacent to the ppGpp synthase gene. [Effects of the Invention]

[0012] According to the present invention, a ppGpp-enriched organism is provided. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram showing an example of an expression unit. [Figure 2] FIG. 2 is a schematic diagram showing the domains conserved in Arabidopsis RSH. [Figure 3] FIG. 3 is an image showing the results of Western blotting before and after estrogen induction. [Figure 4]Figure 4 is a graph showing the relationship between estrogen concentration and ppGpp concentration on the second day after estrogen induction. [Figure 5] Figure 5 is a graph showing the relationship between estrogen concentration and ppGpp concentration on the 28th day after estrogen induction.

Mode for Carrying Out the Invention

[0014] Hereinafter, embodiments of the present invention will be described. The embodiments described below are more specific forms of any of the above aspects. The matters described below can be incorporated into each of the above aspects alone or in combination.

[0015] <Method for Producing ppGpp-Enriched Organisms> Hereinafter, a method for producing ppGpp-enriched organisms according to an embodiment of the present invention will be described. First, an organism into which an expression unit is introduced is prepared. The organism into which the expression unit is introduced is an organism having an expression unit and chloroplasts. The organism into which the expression unit is introduced can be obtained, for example, by introducing an expression unit into an organism having chloroplasts. The organism containing chloroplasts is, for example, a plant cell, a plant tissue, a plant organ, a plant individual, or a combination thereof. The plant is, for example, Arabidopsis thaliana. For the introduction of the expression unit, existing gene introduction methods such as the Agrobacterium method can be used.

[0016] Figure 1 is a schematic diagram showing an example of an expression unit. Expression unit 1 is a unit for expressing ppGpp synthase. As shown in Figure 1, expression unit 1 includes a ppGpp synthase gene 10, an inducible promoter 11, a region 12 encoding a chloroplast transit peptide, and a region 13 encoding a labeled protein.

[0017] The ppGpp synthase gene 10 is a gene encoding a ppGpp synthase. The ppGpp synthase gene 10 includes a region encoding a ppGpp synthesis domain (hereinafter referred to as a first region) but does not include a region encoding a ppGpp degradation domain (hereinafter referred to as a second region). The second region is, for example, a region encoding a ppGpp hydrolysis domain.

[0018] Preferably, the ppGpp synthase gene 10 does not have a region encoding a domain that regulates the activity of the ppGpp synthesis domain. Examples of domains that regulate the activity of the ppGpp synthesis domain include a TGS (ThrRS, GTPase, SpoT) regulatory domain or an EF-hand domain (EFh). In this case, ppGpp can be more efficiently accumulated in the organism.

[0019] Preferably, the ppGpp synthase gene 10 is a yjbM gene consisting of the base sequence shown in SEQ ID NO: 1, or a yjbM-like gene consisting of a base sequence that has 90% or more sequence identity with the yjbM gene consisting of the base sequence shown in SEQ ID NO: 1, and that contains a ppGpp synthesis domain coding region but does not contain a ppGpp degradation domain coding region.

[0020] The yjbM gene is a gene that encodes the YjbM protein, a ppGpp synthase found in Bacillus subtilis. The yjbM gene has a first domain but does not have a second domain. Furthermore, the yjbM gene does not have a domain that encodes a domain that controls the activity of the ppGpp synthesis domain. Therefore, when the yjbM gene or a yjbM-like gene is used as the ppGpp synthase gene 10, ppGpp can be easily accumulated efficiently in the organism.

[0021] The nucleotide sequence of the yjbM gene is shown in the table below. In the nucleotide sequence shown below, the region from base 45 to base 164 is the first region.

[0022] [Table 1]

[0023] The inducible promoter 11 is a promoter that controls the transcription of the ppGpp synthase gene 10. The inducible promoter 11 is activated by an inducer.

[0024] The inducible promoter 11 is, for example, an estrogen-inducible promoter.

[0025] When an estrogen-inducible promoter is used as the inducible promoter 11, estrogen can be used as the inducer.

[0026] The region 12 encoding the chloroplast transit peptide (hereinafter referred to as the third region) is adjacent to the ppGpp synthase gene 10. Specifically, the third region is adjacent to the ppGpp synthase gene 10 so that the chloroplast transit peptide and the ppGpp synthase encoded by the ppGpp synthase gene 10 form a fusion protein. For example, the third region is adjacent to the ppGpp synthase gene 10 on the 5' end side of the ppGpp synthase gene 10.

[0027] The region 13 encoding the labeled protein (hereinafter referred to as the fourth region) encodes a labeled protein that labels the ppGpp synthase encoded by the ppGpp synthase gene 10. The fourth region is adjacent to the ppGpp synthase gene 10. Specifically, the fourth region is adjacent to the ppGpp synthase gene 10 so that the labeled protein and the ppGpp synthase encoded by the ppGpp synthase gene 10 form a fusion protein. The labeled protein is, for example, a FLAG (registered trademark) tag.

[0028] Next, the organism into which the expression unit has been introduced is grown in the presence of an inducer. This growth induces the expression of a first fusion protein containing a chloroplast transit peptide encoded by the third region, a ppGpp synthase encoded by the ppGpp synthase gene 10, and a marker protein encoded by the fourth region. The first fusion protein then translocates to the chloroplast. The chloroplast transit peptide contained in the first fusion protein is then cleaved, thereby producing a second fusion protein containing the ppGpp synthase and the marker protein. The second fusion protein then catalyzes the synthesis reaction of ppGpp. This reaction causes ppGpp to accumulate in the chloroplasts. In this way, a ppGpp-enriched organism is obtained.

[0029] The cultivation is carried out, for example, using a liquid medium or soil.

[0030] When a liquid medium is used, the organism into which the expression unit has been introduced can be grown in the presence of the inducer by contacting the organism with an inducer-containing liquid medium containing the inducer and the liquid medium.

[0031] The concentration of the inducer in the inducer-containing liquid medium is preferably in the range of 0.01 to 10 μM.

[0032] When soil is used, an inducer-containing liquid containing an inducer and a liquid can be supplied to the expression unit-introduced organism, allowing the expression unit-introduced organism to grow in the presence of the inducer. The liquid can be, for example, water.

[0033] The concentration of the inducer in the inducer-containing liquid is preferably in the range of 0.01 to 10 μM.

[0034] Preferably, the organism into which the expression unit has been introduced is grown in the presence of an inducer in a nutrient-poor environment.

[0035] More preferably, the organism into which the expression unit has been introduced is grown in the presence of an inducer in an environment poor in phosphate or nitrogen sources. As described in Non-Patent Document 3, Arabidopsis thaliana, which has accumulated a large amount of ppGpp by overexpressing RSH3, a ppGPp synthase found in Arabidopsis thaliana, can maintain green leaves even when cultured in an environment poor in nitrogen sources. Furthermore, Arabidopsis thaliana, which has accumulated a large amount of ppGpp, can maintain a low level of anthocyanin even when cultured in an environment poor in phosphate sources. Thus, ppGpp-enriched organisms are resistant to nutrient-poor environments. Therefore, when grown in a nutrient-poor environment, ppGpp-enriched organisms have a superior appearance compared to organisms that accumulate less ppGpp.

[0036] The above describes a method for producing a ppGpp-enriched organism.

[0037] In the above-described method, the fourth region is provided on the 3'-end side of the ppGpp synthase gene 10, but the fourth region may be provided between the third region and the ppGpp synthase gene 10. Furthermore, another region may be interposed between the fourth region and the ppGpp synthase gene 10. Furthermore, the fourth region may be omitted.

[0038] Furthermore, another region may be present between the third region and the ppGpp synthase gene 10.

[0039] Alternatively, before cultivating an expression unit-introduced organism in the presence of an inducer, the expression unit-introduced organism may be cultivated in the absence of the inducer. For example, Arabidopsis thaliana that accumulates a large amount of ppGpp exhibits lower photosynthetic ability but a larger amount of biomass than Arabidopsis thaliana that accumulates a smaller amount of ppGpp. Therefore, if an expression unit-introduced organism is cultivated in the absence of an inducer and then cultivated in the presence of an inducer, it is possible to obtain a ppGpp-enriched organism that retains a large amount of organic matter synthesized by photosynthesis and has a large amount of biomass.

[0040] <Effects> As mentioned above, Arabidopsis thaliana has four types of ppGpp synthases. Figure 2 is a schematic diagram showing the domains conserved in these ppGpp synthases, namely, RSH1, RSH2, RSH3, and CRSH4. In Figure 2, "cTP" indicates a chloroplast targeting peptide, i.e., a chloroplast transit peptide; "HD" indicates a (p)ppGpp hydrolysis domain; "SYNTH" indicates a (p)ppGpp synthesis domain; "TGS" indicates a TGS regulatory domain; "ACT(RRM)" indicates an ACT regulatory domain (RNA recognition motif domain); "EFh" indicates a calcium-binding EF-hand motif in the EF-hand domain; and "synth G 376 "S" indicates the ppGpp synthesis domain in which the 376th amino acid residue is substituted from glycine to serine, "HDc" indicates the degraded ppGpp hydrolysis domain, "TM" indicates the putative transmembrane domain, and "RPP5-ID" indicates the RPP5 interaction domain. 376 "HDc" is also thought to lack the activity of a ppGpp hydrolysis domain.

[0041] As shown in Figure 2, the ppGpp synthases RSH2 and RSH3 contain not only a ppGpp synthesis domain but also a ppGpp hydrolysis domain. Therefore, RSH2 and RSH3 catalyze not only the synthesis reaction of ppGpp but also the degradation reaction. Therefore, it is thought that the amount of ppGpp synthesis by RSH2 and RSH3 does not coincide with the amount of ppGpp accumulated.

[0042] On the other hand, the above-described expression unit-introduced organism contains the ppGpp synthase gene 10. As described above, the ppGpp synthase gene 10 contains the first region but not the second region. Therefore, according to the above-described method, the amount of ppGpp synthesis by the ppGpp synthase encoded by the ppGpp synthase gene 10 can match the amount of ppGpp accumulated. Furthermore, according to the above-described method, it is possible to efficiently accumulate ppGpp. Furthermore, according to the above-described method, it is possible to regulate the amount of ppGpp accumulated by regulating the expression level of ppGpp synthase.

[0043] Additionally, prior art includes an Arabidopsis mutant that overexpresses RSH3, a ppGpp synthase found in Arabidopsis. This Arabidopsis mutant accumulates a larger amount of ppGpp than wild-type Arabidopsis. Furthermore, this Arabidopsis mutant is more tolerant to environments lacking nitrogen sources than wild-type Arabidopsis. Furthermore, this Arabidopsis mutant has a larger biomass than wild-type Arabidopsis.

[0044] However, because RSH3 is constitutively expressed in the Arabidopsis mutants, it is not possible to express RSH3 at a desired timing, and therefore it is not possible to accumulate ppGpp at a desired timing or to regulate the amount of ppGpp accumulated.

[0045] On the other hand, in the above-mentioned method for producing a ppGpp-enriched organism, transcription of the ppGpp synthase gene is controlled by an inducible promoter. Therefore, ppGpp synthase can be expressed at any timing depending on the addition of an inducer. Therefore, it is possible to accumulate ppGpp at any timing and to adjust the amount of ppGpp accumulated.

[0046] Furthermore, ppGpp-enriched organisms have high tolerance to nutrient-poor environments and high biomass, making them useful in agriculture, e.g., crop production. [Example]

[0047] An example of the present invention will now be described.

[0048] <1. Preparation of transformants> First, a plasmid containing the expression unit shown in Figure 1 was prepared. Specifically, the yjbM gene and the region encoding the chloroplast transit peptide of the Arabidopsis thaliana recA gene (locus: At1g79050 in The Arabidopsis Information Resource (TAIR)) were amplified by PCR, and the resulting DNA fragment was cloned into the multicloning site of the binary vector pER8. The yjbM gene used was the yjbM gene described in Reference 1 below. Furthermore, pER8 used was the pER8 described in Reference 2 below. pER8 has a multicloning site downstream of the estrogen-inducible promoter.

[0049] (Reference 1) Nanamiya H., Kasai K., Nozawa A., Yun CS., Nirisawa T., Murakami K., Natori Y., Kawamura F. and Tozawa, Y. (2008) Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis. Mol. Microbiol. 67: 291-304. (Reference 2) Zuo J., Niu QW., Chua NH. (2000) An estrogen receptor-based transactivator XVE mediated highly inducible gene expression in transgenic plants. Plant J. 24: 265-273. The resulting plasmid was then used to introduce the expression unit into Arabidopsis thaliana. By the above method, organisms into which the expression unit was introduced, i.e., transformants, were obtained. Here, five transformants were obtained. The five obtained transformants are referred to as transformants #1 to #5, respectively.

[0050] <2. Western blotting> First, a portion of the plant body was collected from each of the five transformants obtained by the above-mentioned method before estrogen induction. Next, the five transformants and wild-type Arabidopsis thaliana were grown in estrogen-containing liquid medium for 2 days. Then, a portion of the plant body was collected from each of the five transformants and wild-type Arabidopsis thaliana.

[0051] Each harvested plant was homogenized with 5 μL of TE buffer (pH 7.5) per mg of plant material. 20 μL of the resulting homogenate was transferred to a new tube, and 20 μL of 2x SDS sample buffer (0.125 M Tris / HCl pH 6.8, 4% SDS, 20% glycerol, 0.01% bromphenol blue) was added. The mixture was boiled for 5 minutes to prepare the electrophoresis sample.

[0052] Next, polyacrylamide gel electrophoresis (SDS-PAGE) was performed under the following conditions: the current was set to a constant 30-50 mA, and the buffer used was a running buffer (Tris 3.03 g / L, glycine 14.31 g / L, SDS 1.0 g / L). The marker used was Precision Plus Protein (registered trademark) 2-color standard #1610374 (Bio-Rad).

[0053] Next, a PDVF membrane (manufactured by Millipore) of the same size as the gel after electrophoresis and 10 pieces of filter paper were cut out. Next, a laminate obtained by stacking 5 pieces of filter paper, the membrane, the gel, and 5 pieces of filter paper in this order was placed in a semi-dry blotting device (manufactured by ATTO), and the membrane size [cm 2 The proteins were transferred to the membrane by applying a constant current of ]x2mA for 1 hour.

[0054] Next, the membrane after transfer was washed with methanol and then immersed in blocking buffer (20 mM Tris / HCl pH 8.0, 88% NaCl, 3% skim milk) for 1 hour for blocking. After that, the membrane was immersed in a solution of blocking buffer plus 1 / 1000 volume of primary antibody anti-FLAG M2 (Sigma) and treated with the primary antibody overnight at 4°C.

[0055] The membrane was then rinsed three times with TBST buffer (20 mM Tris / HCl pH 8.0, 88% NaCl, 0.05% Tween 20). This was followed by three 5-minute washes with TBST buffer. The membrane was then immersed in a solution containing 1 / 1000 volume of secondary antibody anti-rabbit HRP (Funakoshi) in TBST buffer for 1 hour at room temperature for secondary antibody treatment. This was followed by three 5-minute washes with TBST buffer to wash away the secondary antibody. Signals were then detected using ECL Plus (GE Healthcare).

[0056] Figure 3 is an image showing the results of Western blotting before and after estrogen induction. The "WT" column in Figure 3 shows the results for wild-type Arabidopsis thaliana.

[0057] As shown in Figure 3, a band was confirmed at approximately 27 kDa. This is close to the 25.7 kDa value obtained by subtracting the molecular size of the chloroplast transit peptide from the protein size of 33.3 kDa predicted from the primary structure of the fusion protein of the chloroplast transit peptide, YjbM protein, and FLAG (registered trademark) tag. This demonstrates that the expressed YjbM protein is localized in the chloroplasts. In this way, transformants expressing the YjbM protein in response to estrogen induction were obtained.

[0058] <3. Measurement of ppGpp concentration> First, the transformant #1 was grown in an estrogen-containing liquid medium containing estrogen at concentrations of 0, 0.001, 0.01, 0.1, 1, or 10 μM. Next, a portion of the shoot of the plant was harvested 2 and 28 days after estrogen induction.

[0059] Next, the ppGpp concentration in the collected shoots was measured. The ppGpp concentration was measured using a method similar to that described in Reference 3 below. Specifically, the shoots were first crushed with liquid nitrogen. Then, a sample solution was prepared using 3 mL of formic acid and 3 mL of ammonium acetate. 1 mL of chloroform was then added to the sample solution, and solid-phase extraction was performed. The obtained sample was then subjected to column treatment and mass spectrometry using LC-MS / MS.

[0060] (Reference 3) Ihara, Y., Ohta, H. and Masuda, S. (2015) A highly sensitive quantification method for the accumulation of alarmone ppGpp in Arabidopsis thaliana using UPLC-ESI-qMS / MS. J. Plant Res. 128: 511-518. Figure 4 is a graph showing the relationship between estrogen and ppGpp concentrations on day 2 after estrogen induction. Figure 5 is a graph showing the relationship between estrogen and ppGpp concentrations on day 28 after estrogen induction.

[0061] As shown in Figures 4 and 5, estrogen treatment increased the ppGpp concentration, and as shown in Figure 5, the amount of ppGpp increased depending on the estrogen concentration.

[0062] As is clear from the results of the Western blotting and ppGpp concentration measurement, the accumulation of ppGpp in chloroplasts could be induced by expressing ppGpp synthase at any timing. [Explanation of symbols]

[0063] 1...Expression unit 10...ppGpp synthase gene 11...Inducible promoter 12...Area 13...Area

Claims

1. 1. A method for producing a ppGpp-enriched organism, comprising cultivating an organism having chloroplasts, in the presence of an inducer that activates the inducible promoter, the method comprising: growing an organism having chloroplasts, the organism comprising an expression unit including a ppGpp synthase gene that includes a region encoding a ppGpp synthesis domain but not a region encoding a ppGpp degradation domain, an inducible promoter that controls the transcription of the ppGpp synthase gene, and a region encoding a chloroplast transit peptide adjacent to the ppGpp synthase gene, The method according to claim 1, wherein the ppGpp synthase gene is a yjbM gene consisting of the base sequence represented by SEQ ID NO: 1, or a yjbM-like gene consisting of a base sequence having 90% or more sequence identity with the yjbM gene consisting of the base sequence represented by SEQ ID NO: 1, and which has the region encoding the ppGpp synthesis domain but does not have the region encoding the ppGpp degradation domain.

2. 10. The method of claim 1, further comprising growing the organism in the absence of the inducer prior to growing the organism in the presence of the inducer.

3. The method according to claim 1 , wherein the organism is grown in the presence of the inducer in a nutrient-poor environment.

4. An organism having chloroplasts, comprising an expression unit including a ppGpp synthase gene that includes a region encoding a ppGpp synthesis domain but does not include a region encoding a ppGpp degradation domain, an inducible promoter that controls the transcription of the ppGpp synthase gene, and a region encoding a chloroplast transit peptide adjacent to the ppGpp synthase gene, The ppGpp synthase gene of an organism is a yjbM gene consisting of the base sequence represented by SEQ ID NO: 1, or a yjbM-like gene consisting of a base sequence that has 90% or more sequence identity with the yjbM gene consisting of the base sequence represented by SEQ ID NO: 1, and that has the region encoding the ppGpp synthesis domain but does not have the region encoding the ppGpp degradation domain.

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