Aquaporin having carbon dioxide transport activity

Abstract

To provide an aquaporin having carbon dioxide transport activity and no water transport activity, which is different from conventional aquaporins.SOLUTION: An aquaporin has an amino acid sequence with at least one amino acid deleted, substituted, or added at positions 8 to 25 of the amino acid sequence set forth in SEQ ID NO:3.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an aquaporin having carbon dioxide transport activity. [Background technology]

[0002] Aquaporins were discovered as channels that facilitate water transport. They were first cloned in 1991 as a membrane protein (CHIP28, now AQP1) abundant in mammalian erythrocytes (Preston & Agre, 1991). Their water transport activity was measured by expressing this protein in Xenopus oocytes, revealing its function as a water channel (Preston et al., 1992). Similar proteins were subsequently discovered in plants, and numerous reviews have been published on their roles in plants (Javot & Maurel, 2002; Tyerman et al., 2002; Kaldenhoff & Fischer, 2006; Katsuhara et al., 2008; Maurel et al., 2009; Prado & Maurel, 2013; Chaumont & Tyerman, 2014; Maurel et al., 2015; Moshelion et al., 2015). Plant aquaporins form a large family containing over 30 genes and exhibit diverse substrate specificities. In recent years, it has been revealed that they transport substrates other than water, such as borate, silicate, hydrogen peroxide, arsenite, and carbon dioxide (Nakhoul et al., 1998; Takano et al., 2002; Ma et al., 2006; Bienert et al., 2007; Ma et al., 2008).

[0003] Regarding the industrial use of aquaporin water transport, Patent Document 1 describes a biomimetic aqueous membrane equipped with aquaporin water transport proteins used to generate salinity-dependent power. Aquaporins are molecules designed by nature to transport water using osmotic pressure as a driving force, and are believed to generate environmentally friendly energy. It is common technical knowledge that aquaporins have water transport properties, but no aquaporins have been known that lack water transport activity and have carbon dioxide transport activity. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2009-510301 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made to solve the above-mentioned problems, and aims to provide an aquaporin that, unlike conventional aquaporins, does not have water transport activity but has carbon dioxide transport activity. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems and have completed the present invention, which comprises the following items [1] to

[11] .

[0007] [1] Aquaporins that do not have water transport activity but have carbon dioxide transport activity; [2] The aquaporin according to [1], which has an amino acid sequence in which at least one amino acid is deleted, substituted, or added at positions 8 to 25 of SEQ ID NO: 3; [3] The aquaporin according to [1] or [2], which has an amino acid sequence in which all amino acids are deleted from positions 8 to 25 of SEQ ID NO: 3; [4] The aquaporin according to any one of [1] to [3], which is derived from a plant; [5] A gene encoding the aquaporin according to any one of [1] to [4]. [6] A carbon dioxide separation membrane comprising the aquaporin according to any one of [1] to [4]. [7] A method for improving the photosynthetic activity of a plant by expressing the aquaporin described in [4] in the mesophyll cells of the plant. [Effects of the Invention]

[0008] The present invention provides an aquaporin that does not have water transport activity but has carbon dioxide transport activity. The aquaporin of the present invention, which selectively transports carbon dioxide, can improve mesophyll conductance even under dry conditions, even when stomatal aperture is small and stomatal conductance is reduced. Therefore, by expressing the aquaporin of the present invention in the mesophyll cells of a plant, it is possible to improve the photosynthetic activity of the plant. Furthermore, the aquaporin of the present invention can be used to provide a separation membrane or the like that selectively separates carbon dioxide. [Brief explanation of the drawings]

[0009] [Figure 1] Electrophoretic images of DNA fragments (SlPIP2;1, SlPIP2;4, SlPIP2;6, SlPIP2;8, and SlPIP2;9) obtained by RT-PCR amplified. [Figure 2] This figure shows the alignment of the amino acid sequences of SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, SlPIP2;8, and SlPIP2;9, where the boxed sequences indicate diacidic motifs. [Figure 3] FIG. 1 shows the results of alignment of partial regions of the nucleotide sequences and amino acid sequences of SlPIP2;6 v1 and SlPIP2;6 v2. [Figure 4]FIG. 1 shows the results of an alignment of the amino acid sequences of SlPIP2;6 v1 and SlPIP2;6 v2 derived from Micro-Tom and SlPIP2;6 v1 and SlPIP2;6 v2 derived from Heinz 1706. [Figure 5] Electrophoretic images confirming the expression of the SlPIP2;6 variant in both leaves and fruits of Micro-Tom. [Figure 6] FIG. 1 shows an outline of subcloning into the pXβG-ev1 vector. [Figure 7] Electrophoresis images of cRNAs of SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, SlPIP2;8, and SlPIP2;9, which contain Xenopus laevis UTR portions. [Figure 8] FIG. 1 shows the dependence of the water permeability coefficient (Pf) of Xenopus oocytes on the amount of tomato aquaporin PIP2 cRNA injected. [Figure 9] FIG. 1 shows the water permeability coefficient (Pf) of the plasma membrane of Xenopus laevis oocytes injected with cRNA of tomato aquaporin SlPIP2. [Figure 10] FIG. 1 shows an example of a live recording in measuring intracellular acidification in Xenopus oocytes using a pH microelectrode method. [Figure 11] FIG. 1 shows the carbon dioxide permeability coefficient (PCO2) of the plasma membrane of Xenopus oocytes injected with cRNA for tomato aquaporin SlPIP2 and barley aquaporin HvPIP2;1. DETAILED DESCRIPTION OF THE INVENTION

[0010] The aquaporins of the present invention are characterized by not having water transport activity but having carbon dioxide transport activity. As will be described in the Examples below, the present inventors cloned cDNAs containing the full-length coding sequences of six PIP2-type aquaporins, including variants, from leaves of the dwarf tomato variety Micro-Tom. Using a heterologous expression system in Xenopus oocytes, the water permeability coefficient (P f ) and carbon dioxide permeability coefficient (P CO2 ) were measured to evaluate their water and carbon dioxide transport activities. As can be seen from Figures 9 and 11, SlPIP2;1, SlPIP2;4, SlPIP2;6v1, and SlPIP2;8 exhibit both water and carbon dioxide transport activities. On the other hand, SlPIP2;6v2 was found to have carbon dioxide transport activity but not water transport activity. SlPIP2;9 was found to have water transport activity but not carbon dioxide transport activity. These aquaporins are translocated to the plasma membrane of oocytes and are thought to have both water transport activity and / or carbon dioxide transport activity.

[0011] Among them, SlPIP2;6 v2 does not have water transport activity but does have carbon dioxide transport activity, and has characteristics different from conventional aquaporins that have water transport properties. Aquaporins that exhibit both water and carbon dioxide transport activity can transport carbon dioxide, but also transport water. Therefore, there is a risk that water in mesophyll cells will be transported into the leaf space, and that water will be further transported from the mesophyll section through the stomata. In contrast, the aquaporin of the present invention, which selectively transports carbon dioxide, can take up carbon dioxide while retaining water in mesophyll cells, and can improve the water use efficiency of plants (amount of carbon gained per unit of water lost through transpiration, mol CO2 mol -1It is possible to improve the carbon dioxide (H2O) transport rate. That is, even when stomatal aperture is small and stomatal conductance is reduced under drought conditions, mesophyll conductance can be improved. Therefore, the significance of the present invention is great because expressing the aquaporin of the present invention in plant mesophyll cells can improve the photosynthetic activity of the plant. The aquaporin of the present invention can be used to provide separation membranes and the like that selectively separate carbon dioxide. In this specification, stomatal conductance is an index of the ease of passage from stomata to the intraleaf space known as the intercellular space or stomatal cavity, and is expressed as the reciprocal of stomatal resistance, which is the resistance from stomata to the intraleaf space. Furthermore, mesophyll conductance is an index of the ease of passage from the intraleaf space to mesophyll cells, and is expressed as the reciprocal of mesophyll resistance, which is the resistance from the intraleaf space to mesophyll cells.

[0012] Furthermore, as can be seen from the sequence alignment results in Figure 3, the N-terminal sequences of SlPIP2;6 v1 and SlPIP2;6 v2 differ, and this sequence difference is thought to be significantly involved in water transport activity. From this perspective, a preferred embodiment of the present invention is an aquaporin having an amino acid sequence in which at least one amino acid is deleted, substituted, or added at positions 8 to 25 of SEQ ID NO: 3, and a gene encoding the aquaporin is also a preferred embodiment. The number of deleted, substituted, or added amino acids is preferably 2 or more, more preferably 5 or more, even more preferably 8 or more, particularly preferably 12 or more, and most preferably 15 or more. Among these, a most preferred embodiment is an aquaporin having an amino acid sequence in which all amino acids are deleted at positions 8 to 25 of SEQ ID NO: 3. That is, a most preferred embodiment is an aquaporin having the amino acid sequence shown in SEQ ID NO: 4.

[0013] In the present invention, the method for obtaining an aquaporin having an amino acid sequence in which at least one amino acid is deleted, substituted, or added is not particularly limited, and the aquaporin may be obtained by screening aquaporins derived from natural products, or by introducing a mutation into the genome of a gene encoding a protein consisting of the amino acid sequence so that an aquaporin with a partial deletion, substitution, or addition of an amino acid sequence is expressed. The method for introducing the mutation into the genome is not particularly limited, and for example, the mutation may be introduced into the genome by genome editing, or the mutation may be introduced into the genome using a mutant.

[0014] When introducing the mutation into the genome by genome editing, methods such as CRISPR / Cas9, a complex in which the part involved in binding to target DNA is RNA and the part involved in DNA cleavage is protein, and TALEN, ZFN, and the like, in which both the part involved in binding to target DNA and the part involved in DNA cleavage are protein, can be used. For example, in methods using CRISPR / Cas9, guide RNA, Cas9, etc. are introduced into target cells, while in methods using TALEN or ZFN, the mutation can be introduced into the genome by introducing into target cells a fusion protein in which a DNA binding domain and a nuclease are fused. Methods for introducing the mutation into target cells include the agrobacterial method, the RNA virus vector method, plasma treatment, particle gun (bombardment) method, PEG method, electroporation method, etc.

[0015] A method for introducing the mutation into the genome using a mutant includes, for example, selecting mutants with a mutation in the gene from natural mutants, mutants induced by UV irradiation, radiation such as gamma rays, or chemicals such as ethyl methanesulfonate (EMS) using PCR, TILLING (Targeting Induced Local Lesions in Genomes), HRM (High Resolution Melting), or the like, and obtaining mutants in which the mutation has occurred in the gene.

[0016] The aquaporin of the present invention can be derived from eukaryotes such as animals and plants, and is preferably derived from plants. Suitable plants include tomato, Arabidopsis thaliana, corn, rice, spinach, eggplant, bell pepper, potato, chili pepper, barley, wheat, sorghum, oat, soybean, broad bean, kidney bean, pea, adzuki bean, lentil, tulip, lisianthus, rose, sunflower, carnation, ranunculus, margaret, baby's breath, onion, leek, garlic, broccoli, cabbage, Chinese cabbage, rapeseed, bok choy, radish, turnip, strawberry, lettuce, kale, mizuna, shiso, celery, basil, orange, lemon, lime, grapefruit, mandarin orange, cucumber, watermelon, alfalfa, clover, and ryegrass. Among these, plasma membrane aquaporins are preferred. PIPs, which are plasma membrane aquaporins, are divided into two branches, PIP1 and PIP2, and PIP2 is a preferred embodiment of the aquaporin of the present invention. Aquaporins are known to function as tetramers, but PIP2 is thought to form a homotetramer by itself, translocate to the membrane, and exert its function. On the other hand, a model has been proposed in which PIP1, with a few exceptions, does not translocate to the plasma membrane by itself, but forms a heterotetramer with PIP2 and translocates to the membrane (Fetter et al., 2004).

[0017] As explained above, the present invention provides an aquaporin that does not have water transport activity but has carbon dioxide transport activity. The aquaporin of the present invention, which selectively transports carbon dioxide, can improve mesophyll conductance even under drought conditions, even when stomatal aperture is small and stomatal conductance is reduced. Therefore, a preferred embodiment is a method for improving the photosynthetic activity of a plant by expressing the aquaporin of the present invention in the mesophyll cells of the plant. Furthermore, the use of the aquaporin of the present invention can be used to provide suitable separation membranes that selectively separate carbon dioxide, which may contribute to solving global environmental problems. [Example]

[0018] The present invention will be explained in more detail below using examples.

[0019] 1. Cloning of plasma membrane aquaporin PIP2 cDNA from tomato leaves and characterization of the PIP2;6 variant

[0020] [Plant cultivation] Seedlings of the dwarf tomato (Solanum lycopersicum) variety "Micro-Tom" were grown in soil (Metromix). They were watered twice a week and fertilized once every two weeks with a 500-fold diluted solution of Hyponex concentrate (Hyponex Japan). They were grown in a Biotron LPH200 climate chamber (Nippon Medical Instruments) under an 8-hour / 16-hour light / dark cycle at 25°C and 60% relative humidity (Reuscher et al., 2013).

[0021] [Genomic DNA extraction] DNA was extracted from developing leaves of plants 6 weeks after sowing using the DNeasy Plant Mini Kit (QIAGEN). The procedure is as follows:

[0022] (1) Approximately 100 mg of accurately weighed expanding leaf tissue was frozen in liquid nitrogen and ground using a mortar and pestle. The powdered tissue was transferred to a 2 mL centrifuge tube. (2) 400 μL of Buffer AP1 and 4 μL of RNase A were added, and the mixture was stirred using a vortex mixer and kept at 65°C for 10 minutes. (3) 130 μL of Buffer AP2 was added, mixed, and allowed to stand on ice for 5 minutes. (4) Centrifuge at 14,000 rpm for 5 minutes at room temperature. (5) The supernatant was transferred to a QIAshredder Mini spin column and centrifuged at 14,000 rpm for 5 minutes at room temperature. (6) The flow-through that passed through the column was transferred to a new tube, and 1.5 volumes of Buffer AP3 were added and mixed with a pipette. (7) The mixed solution was transferred to a DNeasy Mini spin column and centrifuged at 8,000 rpm for 1 minute at room temperature. (8) After discarding the flow-through, 500 μL of Buffer AW was loaded onto the spin column and centrifuged at 15,000 rpm for 2 minutes at room temperature. (9) The spin column was placed in a new tube and loaded with 100 μL of Buffer AE. The column was centrifuged at 15,000 rpm for 2 minutes at room temperature to elute the DNA.

[0023] [RNA extraction] To clone the cDNA for plasma membrane aquaporins, RNA was extracted from leaves of developing plants 6 weeks after sowing using the RNeasy Plant Mini Kit (QIAGEN) according to the following procedure.

[0024] (1) Before use, 10 μL of β-ME (β-mercaptoethanol) was added per 1 mL of Buffer RLT. (2) Approximately 100 mg of accurately weighed expanding leaf tissue was frozen in liquid nitrogen and ground using a mortar and pestle. The powdered tissue was transferred to a 2 mL microcentrifuge tube. (3) After the liquid nitrogen evaporated, 450 μL of Buffer RLT was added to the powdered tissue and stirred using a vortex mixer. (4) The lysate was added to a QIAshredder spin column placed in a 2 mL collection tube and centrifuged at 15,000 rpm for 2 minutes at 4°C. The QIAshredder flow-through was transferred to a new tube, taking care not to disturb the pellet in the collection tube. (5) Half the volume of 100% ethanol was added to the clarified lysate and mixed by pipetting. (6) The sample containing the precipitate was added to an RNeasy spin column and centrifuged at 15,000 rpm for 1 minute, and the filtrate was discarded. (7) 700 μL of Buffer RW1 was added to the RNeasy spin column. The column was centrifuged at 15,000 rpm for 1 minute at room temperature, and the filtrate was discarded. (8) 500 μL of Buffer RPE was added to the RNeasy spin column, and the column membrane was washed by centrifugation at 15,000 rpm for 1 minute at room temperature. (9) After repeating the procedure in (8) above, the mixture was centrifuged at room temperature for 2 minutes to remove excess buffer solution. (10) The RNeasy spin column was placed in a new tube, and 50 μL of RNase-free water was added to the column. The column was centrifuged at 15,000 rpm for 1 minute at room temperature to elute the RNA. (11) The concentration of the recovered total RNA was measured based on the absorbance at 260 nm.

[0025] [cDNA synthesis by reverse transcription] Total RNA was reverse transcribed using the PrimeScript II 1st strand cDNA Synthesis Kit (TaKaRa) to synthesize complementary DNA (cDNA). First, DNA was removed from the RNA using a DNase-free kit (Ambion) as follows:

[0026] (1) 5 μg of RNA was mixed with 5 μL of 10X DNase I Buffer and 1 μL of rDNase I. (2) The mixture was kept at 37°C for 30 minutes. (3) 5 μL of resuspended DNase Inactivation Reagent was added and mixed. (4) The mixture was kept at room temperature for 2 minutes, mixing several times. (5) The mixture was centrifuged at 15,000 rpm for 1.5 minutes, and the supernatant was collected in a new tube.

[0027] Next, reverse transcription was performed using the PrimeScript II 1st strand cDNA Synthesis Kit (TaKaRa) with the DNA-removed total RNA as a template. The procedure is shown below.

[0028] (1) The following solutions were mixed in a tube (Table 1).

[0029] [Table 1]

[0030] (2) To denature the template RNA and increase the efficiency of reverse transcription, the mixture was incubated at 65°C for 5 minutes and then rapidly cooled on ice. (3) The following solutions were added to bring the total volume to 20 μL (Table 2).

[0031] [Table 2]

[0032] (4) After gentle stirring, the reaction was carried out under the following conditions (Table 3).

[0033] [Table 3]

[0034] (5) The reverse transcriptase was inactivated by incubating at 95°C for 5 minutes, and then cooled on ice.

[0035] [Amplification of the full-length PIP2 coding sequence by polymerase chain reaction (PCR)] Using Ex Taq polymerase (TaKaRa), amplification of a region encompassing the full-length coding sequences of SlPIP2;1, SlPIP2;4, SlPIP2;6, SlPIP2;8, and SlPIP2;9 was performed using cDNA as a template. Primers were designed using Primer 3 (a primer design tool) by referencing cDNA sequences in a database. The primers used and reaction conditions are shown in Table 5. The following solutions were mixed in a tube (Table 4).

[0036] [Table 4]

[0037] [Table 5]

[0038] Amplified DNA fragments were confirmed by electrophoresis on a 1.2% (w / v) agarose gel containing ethidium bromide. Amplification of DNA fragments of sizes roughly consistent with the expected size was confirmed for SlPIP2;1, SlPIP2;4, SlPIP2;6, SlPIP2;8, and SlPIP2;9 (Figure 1). In Figure 1, Marker is a 1 kb DNA size marker (New England Biolabs), cDNA is the RT-PCR product using cDNA from an expanding leaf of the dwarf tomato cultivar Micro-Tom as a template, and gDNA is the PCR product using genomic DNA extracted from newly expanding leaves of the dwarf tomato cultivar Micro-Tom as a template. The triangular arrows in Figure 1 indicate the sizes of the products predicted to be amplified from genomic DNA for SlPIP2;6 and SlPIP2;8. The PCR products were then extracted and purified from the agarose gel using a QIAquick Gel Extraction Kit (QIAGEN). The steps are as follows:

[0039] (1) Ethanol was added to Buffer PE before use. (2) The DNA fragment was excised from the agarose gel with a scalpel, and Buffer QG was added in an amount (μL) three times the weight of the gel fragment. (3) The mixture was kept at 50°C for 10 minutes to completely dissolve the agarose gel. (4) After confirming the pH at which DNA was efficiently adsorbed to the membrane by checking the color of the solution (yellow), an equal volume of isopropanol was added. (5) The solution from (4) was loaded onto a column set in a tube, and centrifuged at 15,000 rpm for 1 minute, after which the flow-through was discarded. (6) To completely remove the agarose, 0.5 ml of Buffer QG was loaded onto the column and centrifuged at 15,000 rpm for 1 minute. (7) For washing, 0.75 mL of Buffer PE was loaded onto the column and centrifuged at 15,000 rpm for 1 minute, after which the flow-through was discarded. (8) To remove residual ethanol, the mixture was centrifuged for another minute. (9) The column was placed in a new tube, and 30 μL of Buffer EB was loaded onto the center of the column to elute the DNA. The column was left for 4 minutes and then centrifuged at 15,000 rpm for 1 minute.

[0040] [PIP2 cloning using TA cloning vectors] Most PCR products obtained using Taq DNA polymerase have a single deoxyriboadenosine (dA) residue at their 3' ends. The PCR products containing the coding sequences of SlPIP2;1, SlPIP2;4, SlPIP2;6, SlPIP2;8, and SlPIP2;9 were cloned using the TA cloning vector pGEM-T Easy Vector (Promega), which has a single deoxyribothymidine (dT) residue at its 3' end, and were ligated using the vector, taking advantage of the complementary dA overhang of the PCR amplified products. The procedure is as follows:

[0041] (1) The following solutions were mixed, stirred, and then incubated overnight at 4° C. The volume of the PCR product (X) was calculated assuming a molar ratio of insert:vector of 3:1 (Table 6).

[0042] [Table 6]

[0043] (2) 3 μL of the ligation solution was added to 100 μL of E. coli DH5α competent cells, and transformation was carried out according to the method of Hanahan (1983). (3) The bacterial suspension was spread on an LB agar plate containing 100 μg mL-1 ampicillin and cultured overnight at 37°C. (4) The insert was checked by colony PCR using pUC / M13 forward and reverse primers.

[0044] [Plasmid DNA extraction] Plasmid DNA was extracted from E. coli using the QIAprep Spin Miniprep Kit (QIAGEN). The procedure is as follows:

[0045] (1) 3 mL of E. coli culture solution cultured overnight in LB liquid medium was centrifuged at 8,000 rpm for 3 minutes to collect the bacteria. (2) The supernatant was removed, and 250 μL of Buffer P1 was added and completely dissolved by vortexing. (3) 250 μL of Buffer P2 was added, and the tube was gently mixed by inverting it about five times. (4) 350 μL of Buffer N3 was added, and the tube was inverted and mixed gently until the blue color of LyseControl completely disappeared. (5) The lysate was clarified by centrifugation at 13,000 rpm for 10 minutes at room temperature. (6) The supernatant from (5) above was loaded onto a QIAprep spin column and centrifuged at 15,000 rpm at room temperature for 1 minute. (7) The flow-through was discarded, and 750 μL of Buffer PE was loaded onto the column, followed by centrifugation at 15,000 rpm at room temperature for 1 minute. (8) The flow-through was discarded, and the mixture was centrifuged for another minute to completely remove residual ethanol. (9) The column was placed in a new tube and eluted with 30 μL of Buffer EB.

[0046] [Sequence analysis of SlPIP2;1, SlPIP2;4, SlPIP2;6, SlPIP2;8, and SlPIP2;9] Sequencing was performed by the Sanger method using Big Dye Terminator v1.1 (Applied Biosystems). The extension reaction was carried out using the following reaction (Table 7).

[0047] [Table 7]

[0048] After the reaction was completed, the PCR product was purified by ethanol precipitation as follows:

[0049] (1) The reacted sample was mixed with 5 μL of 125 mM EDTA and 60 μL of 100% ethanol and incubated at room temperature for 15 minutes. (2) The mixture was centrifuged at 15,000 rpm for 10 minutes at room temperature, and the supernatant was discarded. (3) 70% ethanol was added, and the mixture was centrifuged at room temperature at 15,000 rpm for 3 minutes, and the supernatant was discarded. (4) After the ethanol had dried, the precipitate was dissolved in 20 μL of HiDi formamide. The prepared samples were sequenced using an in-house common equipment sequencer (ABI 3130 XL). (5) The transmembrane domain was predicted using the membrane protein structure prediction tool DeepTMHMM (https: / / dtu.biolib.com / DeepTMHMM).

[0050] [Amplification and cloning of SlPIP2;6 v1 cDNA] To efficiently isolate variant 1 (SlPIP2;6 v1), we created new primers containing SlPIP2;6 v1-specific sequences and performed PCR under the conditions listed in Table 8. The amplified DNA fragment was purified, cloned, and extracted from the plasmid as described above, and sequenced by the Sangar method.

[0051] [Table 8]

[0052] [Semi-quantitative expression analysis of SlPIP2;6 variants] We designed new primers to distinguish the expression of two variants of full-length SlPIP2;6: SlPIP2;6 v1 and SlPIP2;6 v2 (Table 9). These primers amplified short DNA fragments, 309 bp for SlPIP2;6 v1 and 255 bp for SlPIP2;6 v2, allowing the two variants to be distinguished based on their migration distance. After amplification, the DNA fragments were electrophoresed on a 2.0% (w / v) agarose gel containing ethidium bromide. The expression ratio of the two variants was analyzed semiquantitatively based on the difference in fluorescence intensity of the DNA fragments. Tomato ubiquitin was used as an internal standard (Reuscher et al., 2013).

[0053] [Table 9]

[0054] [Amplification of the full-length coding sequences of SlPIP2;1, SlPIP2;4, SlPIP2;6, SlPIP2;8, and SlPIP2;9] As described above, to clone the full-length coding sequences, total RNA was extracted from newly expanded leaves of the dwarf tomato cultivar Micro-Tom. cDNA was synthesized using this as a template, and RT-PCR was performed. Amplification was confirmed by agarose gel electrophoresis. The predicted sizes of the cDNA sequences and PCR products (including the full-length coding sequence and a portion of the untranslated region) of Micro-Tom, registered in the DDBJ / EMBL / GenBank International Nucleotide Sequence Database (hereafter referred to as the database), were 997 bp for SlPIP2;1 (AB845609), 1000 bp for SlPIP2;4 (AB845610), 865 bp for SlPIP2;6 (AB845612), 867 bp for SlPIP2;8 (AB845613), and 987 bp for SlPIP2;9 (AB845614), respectively. The accession number of the sequence is indicated in parentheses following the gene name. This was compared with the electrophoretic position of the DNA fragment amplified by RT-PCR. The amplified fragment generally matched the predicted size, suggesting that the target cDNA sequence had been amplified. Furthermore, the band size was different from that of the PCR product obtained using genomic DNA as a template, confirming that the amplified product was not derived from tomato genomic DNA contaminated with the total RNA preparation (Figure 1).

[0055] [Sequence analysis of SlPIP2;1, SlPIP2;4, SlPIP2;6, SlPIP2;8, and SlPIP2;9] As described above, the nucleotide sequence of the amplified DNA fragment inserted into the TA cloning vector (pGEM-T Easy) was determined by the Sanger method. The nucleotide sequences of the cDNAs of SlPIP2;1, SlPIP2;4, SlPIP2;8, and SlPIP2;9 were completely identical to the Micro-Tom cDNA sequence information registered in the database. The cDNA sequence of SlPIP2;6 showed polymorphism due to a deletion in the sequence. The amino acid sequence predicted from sequence analysis was also aligned. The SlPIP2;6 listed in the database is referred to as SlPIP2;6 variant 1 (SlPIP2;6 v1), and the SlPIP2;6 with the deletion site is referred to as SlPIP2;6 variant 2 (SlPIP2;6 v2). The amino acid sequences of SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, SlPIP2;8, and SlPIP2;9 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SlPIP2;9, respectively (Figure 2). As shown in Figure 2, the amino acid sequences contain the NPA box and six transmembrane domains, which are characteristic motifs of aquaporins. The NPA box, consisting of asparagine-proline-alanine, is a region conserved in almost all water-transporting aquaporin isoforms. The NPA boxes are oriented facing each other in the three-dimensional structure and are thought to contribute to the discrimination of water molecules (Maurel et al., 2015). These structural features are conserved in water-transporting aquaporins and were also conserved in the amino acid sequences predicted from the cloned cDNA.

[0056] Some Arabidopsis and maize PIP2 isoforms have a diacidic motif (a motif with two acidic amino acids at both ends), the DxE sequence, in their N-terminal region, which has been reported to aid in transport from the endoplasmic reticulum to the secretory pathway (Zelazny et al., 2009; Sorieul et al., 2011). In Figure 2, the boxed sequences are diacidic motifs. SlPIP2;6 v1 has three diacidic motifs, and SlPIP2;6 v2 has two diacidic motifs.

[0057] Figure 3 shows an alignment of the coding regions of SlPIP2;6 v1 and SlPIP2;6 v2. Figure 3a shows the region from the initiation codon to 100 bp of SlPIP2;6 v1 and SlPIP2;6 v2 (SlPIP2;6 v1 and SlPIP2;6 v2 in Figure 3a are shown by SEQ ID NO: 7 and SEQ ID NO: 8, respectively), and Figure 3b shows the N-terminal portion of the amino acid sequence deduced from the cDNA sequence (SlPIP2;6 v1 and SlPIP2;6 v2 in Figure 3b are shown by SEQ ID NO: 9 and SEQ ID NO: 10, respectively). In Figure 3, * indicates a conserved base or amino acid residue, and - indicates a deletion site. As shown in Figure 3, the nucleotide sequence of the cloned SlPIP2;6 cDNA was compared with the nucleotide sequence of Micro-Tom-derived SlPIP2;6 (AB845612) registered in the database, and it was found that 54 nucleotides (18 amino acids) were deleted at the N-terminus (Figure 3a and b).

[0058] [Cloning and sequence analysis of SlPIP2;6 v1 cDNA] The PCR product amplified with the SlPIP2;6 primers prepared as described above yielded a clone containing a variant (SlPIP2;6 v2) lacking 18 amino acids (54 bases). We attempted to clone a variant (SlPIP2;6 v1) registered in the database, suspecting that it might also be expressed. To specifically amplify SlPIP2;6 v1 by PCR, we used primers (Table 2) containing a partial sequence of the deletion site not present in SlPIP2;6 v2, and PCR was performed using expanded tomato leaf cDNA as a template. The amplified fragment was purified, cloned, and sequenced by the Sanger method.

[0059] The cDNA sequence of SlPIP2;6 v1 was completely identical to the cDNA sequence of Micro-Tom SlPIP2;6 registered in the database. Furthermore, alignment of the predicted amino acid sequences revealed the presence of an NPA box and six transmembrane domains, motifs characteristic of aquaporins (Fig. 2). Because SlPIP2;6 v1 and SlPIP2;6 v2 could be encoded by separate genome sequences or could originate from the same locus, a BLAST search was performed against the non-redundant tomato nucleotide sequence database (NCBI: National Library of Medicine, taxid:4081). The genome sequence in the database was not that of Micro-Tom but that of another cultivar (Heinz 1706). We found only one genomic sequence that shared a common sequence with SlPIP2;6 v1 (NM_001302917) and SlPIP2;6 v2 (NM_001366139). This suggests that SlPIP2;6 v1 and SlPIP2;6 v2 are transcript variants derived from the same gene. Comparing the amino acid sequences predicted from the nucleotide sequences of the two SlPIP2;6 variants from Heinz 1706 with those predicted from the nucleotide sequences of SlPIP2;6 v1 and SlPIP2;6 v2 obtained from Micro-Tom, we found that deletions were present at nearly identical positions in Micro-Tom and Heinz 1706 (Fig. 4). The amino acid sequence of SlPIP2;6 v1 derived from Heinz 1706 is shown in SEQ ID NO: 11, and the amino acid sequence of SlPIP2;6 v2 derived from Heinz 1706 is shown in SEQ ID NO: 12. These results indicate that, regardless of the variety, tomato SlPIP2;6 has two variants, one with a deletion at the N-terminus and one without.

[0060] [Semi-quantitative expression analysis of SlPIP2;6 variants] Electrophoresis of the PCR amplified product of SlPIP2;6 cDNA, including the full-length coding sequence, revealed only a single apparent band, failing to confirm the existence of two variants. Therefore, we designed new primers capable of amplifying short DNA fragments of different lengths (309 bp for SlPIP2;6 v1 and 255 bp for SlPIP2;6 v2) and compared the expression levels of the two variants based on their migration distance. RT-PCR revealed the presence of amplified DNA fragments corresponding to the sizes of SlPIP2;6 v1 and PIP2;6 v2 in both leaves and fruit (Fig. 5). In Fig. 5, v1 represents SlPIP2;6 v1, and v2 represents SlPIP2;6 v2. For the "Young leaf" template, cDNA was synthesized from RNA extracted from developing leaves 6 weeks after sowing, and for the "Fruit" template, cDNA was synthesized from RNA extracted from yellowing fruit. Marker is a 100 bp DNA size marker (New England Biolabs), and UBQ is SlUBQ (Solyc01g056940.1). In both leaf and fruit tissues, the fluorescence intensity of the SlPIP2;6 v1 band was weaker than that of SlPIP2;6 v2, suggesting that SlPIP2;6 v1 is less expressed than SlPIP2;6 v2. Furthermore, no significant differences in the fluorescence intensity of the bands were observed between leaves and fruit, suggesting that the expression ratio of the variants is similar in these tissues.

[0061] 2. Water transport activity of tomato plasma membrane aquaporin PIP2

[0062] [Subcloning into pXβG-ev1 vector] Using PCR with four primers, the full-length coding sequences of SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, SlPIP2;8, and SlPIP2;9 inserted into the pGEM-T easy vector were subcloned into the BglII site of the pXβG-ev1 plasmid in a single reaction (Figure 6). pXβG-ev1 is a vector containing the 5'-UTR and 3'-UTR of Xenopus β-globin sequences inserted into the pBluescript II plasmid, which is known to stabilize the translation of complementary RNA into protein in Xenopus oocytes (Horie et al., 2011). The primer sequences used for subcloning are listed in Table 11. KOD-FX (TOYOBO) was used for the PCR used in subcloning. The composition of the reaction mixture is shown below (Table 10).

[0063] [Table 10]

[0064] PCR was performed at 94°C for 2 min, followed by 19 cycles of 98°C for 10 s, 55°C for 5 s, and 68°C for 5 min. After the reaction, 0.2 μL of DpnI was added to the PCR reaction mixture, and the mixture was digested at 37°C for 2 hours to remove the template plasmids (pGEM-T and pXβG-ev1 containing the inserted coding sequences). Because DpnI digests methylated DNA but not unmethylated DNA, the newly synthesized circular DNA remained undigested. After DpnI treatment, Escherichia coli (DH5α strain) was transformed to obtain the desired plasmid. The coding sequence inserted into pXβG-ev1 and the surrounding DNA sequence were confirmed by Sanger sequencing in both directions using a T3 primer and a primer constructed using sequences in the 3'-UTR.

[0065] [Table 11]

[0066] [Plasmid extraction] The QIAprep Spin Miniprep Kit (QIAGEN) was used in the same manner as in "1. Cloning of plasma membrane aquaporin PIP2 cDNA from tomato leaves and analysis of PIP2;6 variants" above.

[0067] [cRNA synthesis] pXβG-ev1 containing SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, and SlPIP2;9 was linearized by digestion with BamHI, and pXβG-ev1 containing SlPIP2;8 was linearized by digestion with SpeI. Using these as templates, cRNA with a cap analog (m7G(5')ppp(5')G) was synthesized using the mMESSAGE mMACHINE T3 in vitro transcription kit (Ambion). The specific procedure is described below.

[0068] (1) The following solutions were mixed and incubated at 37°C for 2 hours (Table 12).

[0069] [Table 12]

[0070] (2) 0.5 μL of DNase was added, and the mixture was incubated at 37°C for 15 minutes. (3) 15 μL of lithium chloride and 15 μL of RNase-free water provided with the kit were added, and the mixture was left overnight at -80°C. The product was purified by ethanol precipitation. After the reaction, RNA was electrophoresed on a 1.0% (w / v) denaturing agarose gel to confirm the presence or absence of synthesis.

[0071] The sizes of the cRNAs containing the 5'-UTR and 3'-UTR of Xenopus β-globin and the full-length coding sequence were 1175 nucleotides (nt) for SlPIP2;1, 1178 nt for SlPIP2;4, 1199 nt for SlPIP2;6 v1, 1145 nt for SlPIP2;6 v2, 1193 nt for SlPIP2;8, and 1187 nt for SlPIP2;9, respectively. The predicted sizes of the synthesized cRNAs were determined by the addition of polyadenylic acid to the cRNAs. A single cRNA band was observed for SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, SlPIP2;8, and SlPIP2;9, which were approximately the predicted size. On the other hand, the cRNA for SlPIP2;1 showed multiple bands, including two distinct bands (Figure 7). In Figure 7, the marker is an ssRNA ladder (4 μg, New England Biolabs), and each cRNA was 0.5 μg. The size of the band with the lowest mobility on electrophoresis closely matched the predicted size of the cRNA. Repeated synthesis of SlPIP2;1 cRNA yielded similar results. Although the reason for the multiple bands observed is unclear, we hypothesized that SlPIP2;1 cRNA retains its higher-order structure even after denaturation, resulting in the multiple bands with apparent higher mobility. Therefore, we used this sample in subsequent experiments.

[0072] [Isolation of Xenopus oocytes] The isolated oocytes were stored in modified Barth's solution (MBS; 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 15 mM Tris-HCl (pH 7.6), 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 10 μg ml according to the method of Katsuhara et al. (2002). -1 Penicillin sodium, 10 μg ml -1 After selection under a stereomicroscope in streptomycin sulfate, the cells were incubated in MBS at 18°C ​​for 24 hours.

[0073] [cRNA injection] Glass capillaries for cRNA injection were prepared from 25 μL glass capillary micropipettes (Drummond) using a laser micropipette puller (MODEL P-2000, Sutter Instruments). 50 nL of each of the cRNA solutions for SlPIP2;1, SlPIP2;4, SlPIP2;6v1, SlPIP2;6v2, SlPIP2;8, and SlPIP2;9 was injected into selected Xenopus oocytes using an automated nanoliter injector (NanoJect II, Drummond). An equal volume of water was injected as a negative control.

[0074] [Measurement of oocyte swelling rate and water permeability coefficient (P f ) calculation] The water permeability coefficient of the oocyte plasma membrane (P f ) was measured by oocyte swelling assay after incubation in MBS at 18°C ​​for 24 hours. Xenopus oocytes were transferred from MBS to a hypotonic solution (1 / 5 concentration MBS solution). Oocytes in the hypotonic solution were observed under a microscope and photographed every 10 seconds (0 to 90 seconds). The photographed images were analyzed using image analysis software (Winroof), and the P of the oocyte plasma membrane was calculated according to the following formula (I): f was calculated (Katsuhara et al., 2002; Mahdieh et al., 2008).

[0075]

number

[0076] [Water transport activity of Xenopus oocytes injected with tomato aquaporin PIP2]

[0077] (1) Determining the amount of cRNA to be injected If the injection amount of aquaporin cRNA is excessive, oocytes may burst very rapidly when immersed in hypotonic solution. f Furthermore, if the amount of cRNA injected is too small, the amount of aquaporin expressed will be small, making it difficult to measure. f The amount of cRNA injected per cell was varied to 10 ng, 20 ng, and 40 ng, and after incubation at 18°C ​​for 24 hours, the Pf of Xenopus oocytes was measured. f The PIP2 activity was measured (Fig. 8). In Fig. 8, the error bars indicate the standard error (n = 10). In this experiment, the same batch (oocytes collected at the same time from the same Xenopus laevis individual) was used for each cRNA to minimize differences in activity due to differences in egg collection batches. Different egg collection batches were used for the six PIP2s. When 40 ng was injected, most PIP2s showed P f The increase in PIP2;4 leveled off at concentrations of 20 ng or more, suggesting that the activity was saturated due to some influence. f were compared.

[0078] (2) Semi-quantitative comparison of water transport activity among PIP2s For SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, SlPIP2;8, and SlPIP2;9, three batches of oocytes were used for each. f The P of oocytes injected with cRNA of SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, and SlPIP2;8 was measured. f are 0.74 ± 0.10 × 10, respectively.-2 cm s -1 , 0.46 ± 0.05 × 10 -2 cm s -1 , 0.55 ± 0.06 × 10 -2 cm s -1 , 0.38 ± 0.04 × 10 -2 cm s -1 , 0.75 ± 0.06 × 10 -2 cm s -1 and P in the negative control (Water control, WC) f (0.12 ± 0.00 × 10 -2 cm s -1 ) showed significantly higher values ​​compared to the control group injected with SlPIP2;6 v2 cRNA (Figure 9). In Figure 9, WC is the negative control group injected with water, and different Roman alphabet symbols indicate significant differences (α = 0.05) by Tukey-Kramer multiple comparison test. In Figure 9, WC: n = 30, SlPIP2;1: n = 29, SlPIP2;4: n = 25, SlPIP2;6 v1: n = 30, SlPIP2;6 v2: n = 29, SlPIP2;8: n = 31, SlPIP2;9: n = 33. P of oocytes injected with SlPIP2;6 v2 cRNA f is 0.20 ± 0.01 × 10 -2 cm s -1 There was no significant difference compared to WC. This suggests that SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;8, and SlPIP2;9 have water transport activity, but SlPIP2;6 v2 does not.

[0079] 3. Carbon dioxide transport activity of tomato plasma membrane aquaporin PIP2

[0080] [cRNA synthesis] cRNA synthesis for SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, SlPIP2;8, and SlPIP2;9 was performed using the same procedure as for the water transport assay. Additionally, cRNA for the barley (Hordeum vulgare) PIP2 aquaporin HvPIP2;1, which has been reported to have carbon dioxide transport activity (Mori et al., 2014), was used as a positive control. cRNA synthesis was confirmed by denaturing agarose gel electrophoresis (Figure 7). The size of the cRNA containing the 5'-UTR and 3'-UTR of Xenopus β-globin sequence and the full-length coding sequence of HvPIP2;1 was 1190 nt, and a single band was observed, almost identical to the predicted size.

[0081] [Isolation of Xenopus oocytes] This was carried out in the same manner as in "2. Water transport activity of tomato plasma membrane aquaporin PIP2" above.

[0082] [Injection of cRNA into Xenopus laevis] Ten nanograms of SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, SlPIP2;8, SlPIP2;9, and HvPIP2;1 cRNA solutions were injected in the same manner as for water transport activity. However, for carbon dioxide transport activity measurements, carbonic anhydrase [CA (derived from bovine erythrocytes, Sigma-Aldrich)] was injected simultaneously with the cRNA. CA is an enzyme that rapidly converts carbon dioxide and water to bicarbonate ions and hydrogen ions (Equation (II)). CA was used to maximize this reaction rate. Unless otherwise specified, the amount of carbonic anhydrase injected was 25 nanograms per cell.

[0083]

number

[0084] [Creating a micro pH electrode] Carbon dioxide permeability of cRNA-injected Xenopus oocytes was investigated by measuring the rate of cytoplasmic acidification in a carbon dioxide-enriched buffer solution (Nakhoul et al., 1998). Cytoplasmic pH was measured by inserting a micro pH electrode into the cells. Micro glass pipettes were prepared using a filament-type micropipette puller (MODEL P-1000, Sutter Instruments) and filament-tipped glass capillaries (TW150F, outer diameter 1.5 mm / inner diameter 1.12 mm, World Precision Instruments) using the program listed in Table 13.

[0085] [Table 13]

[0086] The fabricated glass pipettes were dried at 200°C for 2 hours, then hydrophobized with tributylchlorosilane. The tips were then backfilled with hydrogen ionophore cocktail [Hydrogen ionophore I - cocktail A (Sigma-Aldrich): 0.5% polyvinyl chloride dissolved in tetrahydrofuran: tetrahydrofuran = 1:5:4 mixture]. The pipettes were left standing for one day to allow the ionophore mixture to solidify. If necessary, the pipette tip was partially chipped under a stereomicroscope. This procedure improved the pH electrode response and insertion ability. The fabricated pipettes were backfilled with hydrogen ionophore cocktail and pH electrode solution (0.5 M KCl, 0.1 M Tris-HCl, pH 7.0) in that order.

[0087] [Measurement of intraoocyte pH and carbon dioxide permeability coefficient (P CO2 ) calculation] Xenopus oocytes were prepared as described above in "2. Water transport activity of tomato plasma membrane aquaporin PIP2." The pH in the oocytes was measured using a membrane potential microelectrode and a pH microelectrode. The membrane potential microelectrode was prepared in the same manner as the pH microelectrode, except that it was filled with an ionophore cocktail (in this case, 0.5 M KCl). The pH microelectrode was backfilled with an electrode solution containing 0.5 M KCl and 0.2 M MES / Tris (pH 6.0). The membrane potential microelectrode and pH microelectrode were attached to a headstage (HS-9A and HS-2, respectively, Axon Instruments). The reference electrode was immersed in the bath solution via an agarose gel containing 3 M KCl. The internal dimensions of the acrylic resin chamber were 3 mm (depth) × 3 mm (width) × 25 mm (path length). The amplifier used was an Axoclamp 900A (Axon Instruments), the analog-to-digital converter was a Digidata 1440A (Axon Instruments), and the electrode voltage output was acquired using a pCLAMP 10 (Axon Instruments). The recording rate was 1,000 Hz. The pH of the oocyte cytoplasm was determined from the potential difference between the two microelectrodes (Fig. 10, d). In Fig. 10, a shows a schematic diagram of the measurement. The potential difference between electrode 1 and the reference electrode (Vm_1) represents the membrane potential of the cell membrane. The potential difference between electrode 2 and the reference electrode (Vm_2) represents the sum of the membrane potential and the electromotive force due to the pH difference. The result (Math) obtained by subtracting Vm_1 from Vm_2 represents the electromotive force due to the pH difference across the cell membrane. The bath was filled with modified Barth's saline (MBS), pH 7.31, saturated with atmospheric carbon dioxide (11 μM carbon dioxide) without added sodium bicarbonate. This MBS was perfused with MBS containing 6.5 mM carbon dioxide (72 mM NaHCO3), initiating the influx of carbon dioxide into the cells. Figure 10 shows the time course of Vm_1, Vm_2, and Math. The triangle indicates the start of perfusion with MBS containing 6.5 mM carbon dioxide. The electrodes were buffered to pH 6.0, 6.5, 7.0, and 7.5 with 0.1 M MES / Tris or HEPES / Tris, and calibrated with a calibration solution containing 0.1 M KCl.

[0088] The carbon dioxide permeability coefficient (P CO2 ) was determined according to Mori et al. (2014). The final cytoplasmic pH was calculated from the measured value at the end of the recording. The time constant (τ) was calculated by curve fitting the difference trace between the two microelectrodes to an exponential equation (III) using Clampfit software (version 10.3, Axon Instruments). P CO2 was calculated from the time constant of cellular acidification (τ) using equation (IV) reported by Yang et al. (2000) according to the method of Mori et al. (2014).

[0089]

number

[0090]

number

[0091] P of Xenopus oocytes injected with cRNA for tomato aquaporin PIP2 CO2 When measuring carbon dioxide transport activity, water was injected instead of cRNA as a negative control, and barley HvPIP2;1 cRNA, which has already been reported to have carbon dioxide transport activity, was used as a positive control. The injection amount of cRNA for SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, SlPIP2;8, SlPIP2;9, and HvPIP2;1 was 10 ng, consistent with the conditions for measuring water transport activity. The P of oocytes injected with HvPIP2;1 cRNA, used as a positive control, was 10 ng.CO2 is the value of the negative control WC (0.32 ± 0.19 × 10 -5 cm s -1 ) was significantly higher (0.61 ± 0.25 × 10 -5 cm s -1 ) (Figure 11). In Figure 11, WC is the negative control group injected with water, and different Roman alphabet symbols indicate significant differences (α = 0.05) by Tukey-Kramer multiple comparison test. In Figure 11, WC: n = 31, HvPIP2;1: n = 26, SlPIP2;1: n = 13, SlPIP2;4: n = 14, SlPIP2;6 v1: n = 11, SlPIP2;6 v2: n = 15, SlPIP2;8: n = 16, SlPIP2;9: n = 15. P of oocytes injected with cRNA of SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, and SlPIP2;8 CO2 are 0.61 ± 0.77 × 10, respectively. -5 cm s -1 , 0.55 ± 0.35 × 10 -5 cm s -1 , 0.53 ± 0.19 × 10 -5 cm s -1 , 0.61 ± 0.31 × 10 -5 cm s -1 , 0.63 ± 0.77 × 10 -5 cm s -1 These P CO2 The P of oocytes injected with SlPIP2;9 cRNA was significantly higher than that of WC. CO2 is 0.40 ± 0.20 × 10 -5 cm s -1 These results suggest that SlPIP2;1, SlPIP2;4, SlPIP2;6 v1, SlPIP2;6 v2, and SlPIP2;8 have carbon dioxide transport activity, whereas SlPIP2;9 has no or very low carbon dioxide transport activity.

Claims

1. An aquaporin that does not have water transport activity but has carbon dioxide transport activity.

2. 2. The aquaporin according to claim 1, which has an amino acid sequence in which at least one amino acid is deleted, substituted or added at positions 8 to 25 of SEQ ID NO:

3.

3. 3. The aquaporin according to claim 2, which has an amino acid sequence in which all amino acids at positions 8 to 25 of SEQ ID NO: 3 are deleted.

4. The aquaporin according to claim 1, which is derived from a plant.

5. A gene encoding the aquaporin according to claim 2 or 3.

6. A carbon dioxide separation membrane comprising the aquaporin according to any one of claims 1 to 4.

7. A method for improving the photosynthetic activity of a plant by expressing the aquaporin according to claim 4 in mesophyll cells of the plant.

Images