JP2003250568A - Recombinant vector for plant cell character convertion, agrobacterium introduced with the same, transformed plant cell and transformed plant body obtained therefrom - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a recombinant vector for excessively expressing a pectinmethylesterase (PME) and for preparing a transformed plant cell excellent in methanol production amount. <P>SOLUTION: A cDNA fragment encoding PME nonspecifically hydrolyzing a methyl ester contained in a pectin is isolated from cDNA library of Aspergillus niger and the isolated cDNA fragment is incorporated into a binary vector pBI121 to prepare the recombinant vector. The recombinant vector is introduced into Agrobacterium to transform the Agrobacterium and tobacco cultured cells are infected with the Agrobacterium to provide the transformed plant cell excellent in PME activity and methanol production. <P>COPYRIGHT: (C)2003,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a recombinant vector for producing a transformed plant cell having excellent methanol productivity, Agrobacterium, a transformed plant cell produced using the same, and a transformed plant body.

[0002]

2. Description of the Related Art Methanol is industrially produced from more than 90% of natural gas, and is used as a raw material for various chemical products. Furthermore, in recent years, attention has been paid to clean energy as a substitute for fossil fuels, and for example, it is being used as a supply source of hydrogen used in fuel cells. However, since most of the natural gas on the earth is located in remote areas, it has been difficult to use methanol as a fuel instead of gasoline due to transportation and liquefaction costs.

[0003] In recent years, attempts have been made to produce clean methanol from biomass, but it has not technically improved cost efficiency (Borgwardt 1998). Therefore, there is a need to develop a clean and inexpensive methanol supply line that can replace the conventional method.

[0004] Plants produce various volatile organic compounds (VOCs).
Is known to produce (Fehsenfeld, Calvert
et al. 1992; Guenther, Zimmerman et al. 1994), among which methanol is one of the major VOCs. Most of the methanol in the atmosphere is of plant origin (Fall and Ben
son 1996), is produced by the catalytic reaction of the cell wall pectin degrading enzyme pectin methylesterase (PME) (Obendorf, Koch et al. 1990) and is thought to evaporate from the stomata (Nemecek-Marshall, et al. 1995). .

[0005] This PME is present in all plants, phytopathogenic bacteria and fungi, and specifically hydrolyzes the methyl group of homogalacturonan, the main component of pectic polysaccharide, to convert methanol and galacturonic acid. Generate (Micheli 2001).
In particular, PMEs of higher plants, for example, play an important role in cell wall degradation, for example, cell growth and senescence, fruit ripening (Gaffe, Tiznado et al. 1997; Brumme
ll and Harpster 2001), interaction with pathogens (Dorokh
ov, Makinen et al. 1999).

[0006] If there is a plant that excessively expresses the PME that catalyzes the production of methanol, the supply of methanol to the atmosphere increases, and the plant itself can be used as a new energy source. Become. Also, since methanol has been reported to be involved in the growth promoting effect in C3 plants (Nonomura and Benson 1992)
Increasing methanol emissions is also useful for improving crop productivity.

[0007] For these reasons, there is a need for a plant that excessively expresses PME, and with the development of genetic engineering,
The production of transgenic plants that overexpress PME is expected.

[0008]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a recombinant vector, Agrobacterium, for producing a transformed plant cell which overexpresses PME and has excellent methanol productivity. Provided are transformed plant cells and transformed plants.

[0009]

Means for Solving the Problems In order to achieve the above object, a recombinant vector of the present invention is a recombinant vector for transforming a plant cell, wherein the vector contains methyl ester contained in pectin. A gene encoding a protein having pectin methylesterase activity that hydrolyzes nonspecifically is linked.

[0010] As a transformed plant that overexpresses PME, potatoes have already been used to investigate the effects of PME overexpression on cell elongation and organ differentiation.
Petunia in Solanum tuverosum L.
The introduction of the PME gene from flata has been reported (Pill
ing, Willmitzer et al. Planta 2000 Feb; 210 (3): 391-
9). However, in the transformed plant,
Although an increase in PME activity was observed, no accompanying change in methanol production was observed.

The present inventors have conducted intensive studies and, as a result, focused on PME derived from mold, not PME derived from plants.

The inventors focused attention on mold-derived PME instead of plant-derived PME for the following reasons. Among molds, for example, P from Aspergillus niger
ME is known to differ from plant PME not only in the optimum pH and isoelectric point but also in the mechanism of action (Khanh, Ruttkowski et al. 1991; Micheli 2001).
Specifically, plant-derived PME hydrolyzes a methyl group adjacent to a non-reducing end or a free carbosyl group, whereas PME of Aspergillus niger is a catalytic mechanism that randomly hydrolyzes a methyl ester. Because of the difference in substrate specificity between mold-derived PME and plant-derived PME, in the presence of polygalacturonase exhibiting synergistic effects with PME, highly methylesterified apple pectin (apple pectin) When a pectin degradation reaction was carried out using as a substrate, there was a report that Aspergillus niger- derived PME showed an increase in methanol production due to pectin hydrolysis as compared to orange-derived PME (Massiot, Perron et al. 1997). In other words, as described above, PM with low substrate specificity that can hydrolyze methyl esters randomly like mold-derived PME
In the case of E, an increase in the amount of methanol produced is also expected. Therefore, it was considered that the above object can be achieved by linking a gene of PME having such a catalytic function to a vector to form a recombinant vector. It is.

And, as in the above-mentioned mold-derived PEM,
P that nonspecifically hydrolyzes the methyl ester of pectin
According to the recombinant vector to which the gene encoding the protein having ME activity is linked, PME exhibiting the above-mentioned catalytic reaction can be expressed in the plant cell transformed with the recombinant vector, and methanol productivity can be improved. This has led to the present invention. As described above, the present inventors attempted to construct a recombinant vector as described above in order to provide a transformed plant cell exhibiting excellent methanol productivity as a source of methanol as clean energy. The object of the present invention has been achieved for the first time since the introduction of a gene encoding a PME showing a catalytic reaction completely different from that of plant-derived PME as described above. According to such a recombinant vector, the transformed plant cultured cells and transformed plants have excellent methanol productivity, and thus can be effectively used as a methanol supply source.

Although the present invention has been achieved by focusing on mold-derived PME in the course of research, the gene to be linked is not limited to a gene encoding mold-derived PME, Any gene may be used as long as it encodes a protein having PME activity that non-specifically hydrolyzes methyl esters. Also,
“Non-specifically hydrolyzes” refers to the plant-derived PME
Instead of specifically hydrolyzing the methyl ester adjacent to the non-reducing end or free carboxyl group contained in pectin, it is necessary to randomly hydrolyze the methyl ester contained in pectin to produce methanol. Say.

The PME activity for specifically hydrolyzing the methyl ester of pectin includes, for example, Aspergillus
It is preferable that the PME derived from the genus s belongs to the catalytic activity, and more preferably the PME derived from Aspergillus niger.
Is the catalytic activity. In addition, for example, Aspergil
PME from lus japonicus , Aspergillus foetidus, etc.
Activity is raised.

In the recombinant vector of the present invention, the gene to be ligated is preferably a gene encoding a protein shown in the following (a) or (b). (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing; (b) a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of (a), and which is contained in pectin; Proteins with pectin methylesterase activity that non-specifically hydrolyze esters

Further, in the recombinant vector of the present invention,
It is preferable that the gene to be ligated is a gene consisting of DNA shown in the following (c) or (d). (C) D consisting of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing
NA (d) consists of a base sequence in which one or more bases are deleted, substituted or added in the base sequence of (c), and has a pectin methylesterase activity of nonspecifically hydrolyzing a methyl ester contained in pectin. DNA encoding protein

A gene encoding a protein consisting of the amino acid sequence as described above,
Proteins expressed based on A can nonspecifically hydrolyze pectin methyl esters. Therefore, if a plant cell is transformed with the recombinant vector of the present invention to which such a gene is linked, a transformed plant cell or a transformed plant having excellent PME activity and methanol productivity can be obtained.

Next, the Agrobacterium of the present invention comprises:
An Agrobacterium for transforming a plant cell, wherein the recombinant vector of the present invention is introduced. The method for transforming plant cells is not particularly limited. For example, when the Agrobacterium method described below is employed, the PME activity and the Agrobacterium into which the recombinant vector of the present invention has been introduced can be used. Transformed plant cells having excellent methanol productivity can be obtained.

Next, the transformed plant cell of the present invention is a transformed cell obtained by introducing the above-mentioned recombinant vector of the present invention into a plant cell or a transformed plant cell infected with the above-mentioned Agrobacterium of the present invention. There is a feature. Such a transformed plant cell not only has excellent PEM activity as described above, but also has excellent methanol productivity, and is effective as a production source of methanol that is clean energy.

The plant cells to be transformed include:
Although not particularly limited, for example, Fragaria , Lotus , Medicag
o , Onobrychis , Trifolium , Trigonella , Vigna , Citrus , Lin
um , Geranium , Manihot , Daucus , Arabidopsis , Brassica , Ra
phanus , Sinapis , Atropa , Capsicum , Hyoscyamus , Lycopers
icon , Nicotiana , Solanum , Petunia , Digitalis , Majorana ,
Cichorium , Helianthus , Lactuca , Bromus , Asparagus , Anti
rrhinum , Hererocallis , Nemesia , Pelargonium , Panicum , P
ennisetum , Ranunculus , Senecio , Salpiglossis , Cucumis ,
Browaalia , Glycine , Lolium , Zea , Triticum , Sorghum , Datu
ra , Chrysanthemum , Dianthus , Gerbera , Euphorbia , Pelaro
nium , Ipomoea , Passiflora , Cyclamen , Malus , Prunus , Ros
a , Rubus , Populus , Santalum , Allium , Lilium , Narcissus , A
nanas , Arachis , Phaseolus , Eucomia , Heavea , Periploca
And Pisum plant cells. Specific examples include, for example, dicotyledonous plants such as tobacco, Arabidopsis, carrot, soybean, tomato, potato, eucommia, para rubber tree, periploca, barley, wheat, lily, rice, corn, asparagus, bamboo, and monocotyledonous plants such as palm. Or cells of gymnosperms such as pine, ginkgo and cycad.

Next, the transformed plant of the present invention is a plant obtained from the transformed plant cell of the present invention.
Such a plant is, for example, excellent in PME activity and increases the amount of methanol produced with the activity, and thus is useful, for example, as a source of methanol as clean energy as described above.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The recombinant vector, Agrobacterium, transformed plant cell and transformed plant of the present invention can be prepared, for example, as follows.
In addition, it is not limited to these methods.

(1) Preparation of cDNA encoding PME The PME gene of mold to be introduced into plant cells is isolated.
First, cDNA is extracted by extracting total RNA from mold by a conventionally known method and subjecting it to a reverse transcription reaction.
Is prepared.

Next, the PME gene is isolated from the obtained cDNA. The method of isolation is not particularly limited.
It may be performed by a conventionally known cloning method,
In addition, primers can be prepared based on information on the PME gene sequence of a mold that has already been known and prepared by a method such as PCR. In addition, regarding the information of such a mold PME gene sequence, not only various documents but also various databases such as “Genbank” managed by the National Institutes of Health (NIH) can be used. .

The cDNA encoding the PME preferably has, for example, at least the sequence of a structural gene, since it is sufficient to express the PME in a transformed plant cell. Because it is involved, it is preferable that it is the entire PME sequence including the nucleotide sequence encoding the signal peptide.

The cDNA prepared as described above
May be directly incorporated into a vector described later. For example, within the range showing the activity of mold-derived PME,
For NA, base deletion, substitution or addition may be performed.

Furthermore, a nucleotide sequence, such as a promoter or enhancer, for controlling the expression of the PME gene in a plant cell as a host may be added to at least one of the 3 ′ end and the 5 ′ end of the cDNA. . Such a promoter is, for example, the cauliflower mosaic virus (CaMV) 35 of the binary vector pBI121 described below.
Like the S promoter, it may be integrated into the vector itself.

(2) Preparation of Recombinant Vector for Plant Cell Transformation The obtained fungal PME gene is ligated to a vector to prepare a recombinant vector.

As the vector, for example, a plasmid vector can be used, and it is preferable that the vector has a selection marker for antibiotic resistance such as kanamycin resistance and chloramphenicol resistance. More preferably, it is a binary vector, for example, pBI121, pBI221, pBI101, pM
SH1, pMSH2, etc. can be used.

(3) Transformation of plant cells Next, the obtained recombinant vector is introduced into plant cells for transformation. As a method for transforming a plant cell, for example, a conventionally known method can be employed. For example, a method for introducing a foreign gene biologically using Agrobacterium (Agrobacterium method), or a method for directly introducing a foreign gene Method and the like. These methods can be appropriately determined depending on, for example, the type of the host plant to be transformed.

(I) Agrobacterium-introduced method When the mold-derived PME gene is introduced into plant cells by Agrobacterium, first, the recombinant vector is introduced into Agrobacterium by, for example, electroporation. . The type of Agrobacterium is not particularly limited, and can be determined, for example, by combination with the binary vector.

After the introduction of the recombinant vector, the Agrobacterium is cultured in a medium containing an antibiotic, such as kanamycin, so that a colony grown by antibiotic resistance can be selectively used as the transformed Agrobacterium. Can be obtained.

Then, when the resulting transformed Agrobacterium is infected to a plant cell, a transformed plant cell is obtained. In order for Agrobacterium to infect plants,
For example, a tissue, a callus, a cell, a protoplast, or the like may be prepared according to the type of a plant, and this may be incubated with the Agrobacterium for infection.

The plant to which the Agrobacterium method is applied is preferably a plant showing sensitivity to Agrobacterium, and examples thereof include the above-mentioned dicotyledonous plants and monocotyledonous plants.

When the Agrobacterium method is performed as described above, as the vector constituting the recombinant vector, for example, the aforementioned vector such as pBI121 can be used.

(Ii) Direct Transfection Method A method for directly introducing a foreign gene into a plant cell includes, for example, a conventionally known method such as an electroporation method, a particle gun method, and a polyethylene glycol method.

The electroporation method is preferably applied to, for example, plant cells in which protoplasts are stable and easy to cultivate and are easily regenerated. In addition, since the particle gun method is not limited to a host, it is preferably applied to, for example, plant cells that are hardly infected with Agrobacterium and plant cells that are difficult to prepare protoplasts.

When the electroporation method is performed as described above, the vectors constituting the recombinant vector include, for example, pUC18, pUC19, pBR322, pBR325, pB
luescript and the like are preferred.

(3) Preparation of Transformed Plant The transformed plant cell can be regenerated by incubating the transformed plant cell obtained as described above, for example, in a medium containing a plant differentiation inducer. As described above, the plant differentiation inducer is not particularly limited, and for example, auxins such as indoleacetic acid, indolepropionic acid, indolebutyric acid, naphthaleneacetic acid, and kinetins such as zeatin, kinetin, and benzyladenine can be used. . These inducers may be used alone or in combination of two.

The chromosomal DNAs of the obtained transformed plant cells and transformed plant bodies were prepared, and used, for example, by PCR or Southern blotting using primers and probes specific to the known mold-derived PME gene sequence. As a result, if the expression of the PME gene derived from the mold is confirmed, the transformed plant cell and the transformed plant of the present invention are obtained.

The transformed plant cells and transformed plants of the present invention obtained by the above-described methods have high PME activity and excellent methanol production because they overexpress PME derived from fungi. The PME activity and methanol production can be measured, for example, by the methods described below.

Transformation with the fungal PME gene allows the transformed plant cell of the present invention to have, for example,
A PME activity of 1.5 to 20 times is obtained, preferably in the range of 2 to 10 times, particularly preferably in the range of 2 to 5 times. In addition, the amount of methanol produced by the transformed plant cells is, for example, 1.1 to 10 times that of the non-transformed cells,
Preferably it is in the range of 1.1 to 5 times, particularly preferably 1.1 times.
The range is ~ 2 times. The same applies to the transformed plant of the present invention.

[0044]

EXAMPLES (Example 1) This example is based on Aspergillus nige
This is an example in which a r- derived PME gene (pmeA) was introduced into tobacco suspension cultured cells to prepare transformed tobacco cultured cells.

(1) Materials and Culture Conditions Black mold was subcultured on Czapek agar medium at 30 ° C. using Aspergillus niger van Tieghem KF-267 distributed by Dr. Tsuyoshi Kawaguchi, Faculty of Agriculture, Osaka Prefecture University. Nicotiana tabacum L. cv Bright Yello
w 2 (Nagata, Okada et al. 1981) is based on LS (Linsmaie
r and Skoog) KH in medium (Linsmaier and Skoog 1965)
₂ PO ₄ to 370 mg / L, thiamine HCl to 1 mg / L
L, and further cultured in a dark place at 26 ° C. in a modified LS medium containing 3% by weight of sucrose and 2,4-D at a concentration of 0.2 mg / L. Escher 's host for constructing the fusion gene is Escher
^{ichia coli DH5α (F -, φ80dlacZΔM15} , Δ (lacZYA-ar
gF) U169, deoR, recA1, endA1, hsdR17 (r _k ^- m _k ⁺ ), phoA,
^{supE44, λ -, thi-1} , gyrA96, relA1) (Hanahan 198
3) was used. Agrobacterium used for gene transfer into tobacco is Agrobacterium tumefaciens EH
Using the A105 strain (Hood, Helmer et al. 1986), Luria
Culture was performed at 28 ° C. in a Bertani medium. As a vector for constructing a fusion gene, pUC18 (Messing
1983), pBI121 (Jefferso) was used as a binary vector for producing transformed tobacco culture cells.
n, Kavanagh et al. 1987). In addition, in the culture, an antibiotic (kanamycin) was added to the medium as needed to a concentration of 100 mg / L.

[0046] (2) A. Was inoculated niger integration into PME cDNA expression for binary plasmid A. Niger van Tieghem KF-267 on the Czapek medium, 3-7 days after incubation at 30 ° C., the mycelium Sampled and crushed in the presence of liquid nitrogen. The obtained powder was supplied to RNeasy Plant Mini Kit (Qiagen, Germany), and the total RNA was purified by spin column. After treating this total RNA with RNase-free DNase (TaKaRa, Japan), reverse transcription is performed using oligo (dT) _12-18 primer (Gibco BRL) and MMLV-reverse transcriptase (Toyobo, Japan). Thus, a cDNA pool was synthesized.

The basic operation for constructing a fusion gene is carried out by the method of Sambrook et al. (Sambrook, Fritsch et al.
1989). First, PCR amplification of PME cDNA is, A. NiGe
r Known base sequence of PME from RH5344 strain (Khanh, Albrechte
tal. 1990) using a PME gene-specific primer set (PME-F and PMF-R) chemically synthesized based on 30 cycles (94 ° C, 30 seconds; 55 ° C, 30 seconds; 72 ° C, 1 minute). 30 seconds) 1
Next reaction and 20 cycles (94 ° C, 30 seconds; 55 ° C, 30 seconds; 72
(° C, 1 minute 30 seconds). PME-F of the primer set has a sequence shown in SEQ ID NO: 3 (5′-GCT
CTAGAGCATGGTTAAGTCAATTCTTGCATCCG-3 ') and PME-
R is the sequence shown in SEQ ID NO: 4 (5′-CGAGCTCGTTAGTTGATGT
AGCTAGTATCAACCC-3 '). After purifying the obtained PCR fragment, it was inserted into the SacI / XbaI site of pUC18.

The base sequence of this PCR fragment was
BI PRISM 310 Genetic Analyzer (Applied Biosystems,
USA) using a die terminator method.
As a result, the nucleotide sequence shown in SEQ ID NO: 2 was determined, which was the same amino acid sequence as the above-mentioned known nucleotide sequence (SEQ ID NO: 1).
And was found to be a PME gene.

The cloning vector pUC18
From the SacI / XbaI PME gene fragment of about 1 kb,
This was ligated to the cauliflower mosaic virus (CaMV) 35S promoter downstream of the binary vector pBI121.

(3) Transformation of Agrobacterium First, competent cells of Agrobacterium were prepared. A single colony of Agrobacterium in 5 mL LB
The medium was inoculated and cultured with shaking at 28 ° C. overnight. This culture solution was inoculated into 500 mL of LB medium, and further cultured with shaking at 28 ° C. until the turbidity at a wavelength of 600 nm became 0.5. The obtained culture solution was collected by centrifugation (5000 rpm, 10 min, 4 ° C.), and the supernatant was discarded.To wash the cells, an additional 500 mL of sterilized water was added to suspend the cells. The cells were collected again by centrifugation (5000 rpm, 10 min, 4 ° C).
After repeating this washing operation twice, 20 mL of a cold sterilized 10% by weight glycerol solution was added to the precipitated cells, suspended, and collected by centrifugation (5000 rpm, 10 min, 4 ° C). Was thrown away. Again, add 3 mL of chilled, sterile 10%
Add the glycerol solution and suspend.
Aliquots of 40 μL were dispensed into centrifuge tubes, frozen with liquid nitrogen, and stored at −80 ° C.

Next, the binary vector pBI121 incorporating the PME gene fragment was introduced into the competent cells by electroporation. First, after the frozen competent cells were thawed in ice, 2 μL of a DNA solution (the binary vector pBI121 incorporating the PME gene fragment) was added thereto, and the ice-cooled 1 m
m cuvette (trade name Gene PulserR / E. coli Pulser ^TM
Cuvette; BIO-RAD). Then, an electric pulse (1.8 KV, 25 μF, 200 W) was applied by an electroporator (trade name: Gene Pulser; BIO-RAD) to introduce the DNA into the competent cells. In addition, 1 mL SOC
The medium was added and mixed, and the mixture was transferred to a 1.5 mL centrifuge tube. The mixed solution transferred to the centrifuge tube was directly shake-cultured at 28 ° C. for 1 hour, and then spin-down to remove most of the supernatant, and the cells precipitated in the remaining medium were suspended again. Was spread on an LB agar medium containing 50 mg / L kanamycin and cultured at 28 ° C. for 2 nights. The resulting colonies were transformed Agrobacterium using kanamycin resistance as the selection marker.

(4) Transformation of Cultured Tobacco Cells The transformation of cultured tobacco cells was performed according to the method of An (An, 1985,
Plant Physiol, 79, 568-570). The transformed Agrobacterium (EHA 105 strain into which recombinant pBI121 was introduced) was transformed into LB containing kanamycin (100 mg / L).
100 μL of culture solution cultured at 28 ° C for 2 nights in medium (5 mL)
And 8 mL of the tobacco cultured cell suspension on day 4 of the culture cultured in the modified LS medium were placed in a 90 mm deep petri dish, mixed well, and allowed to stand at 25 ° C for 2 nights in a dark place. And co-cultured. After the cultivation, the culture solution in the Petri dish was transferred to a 15 mL centrifuge tube and centrifuged (10) to remove Agrobacterium.
(00 rpm, 5 min, 4 ° C.), and the supernatant was removed. further,
Add a new modified LS medium and centrifuge again (1000 rpm, 5 mi
n, 4 ° C) to wash the cultured tobacco cells. This operation
This was repeated four times to prepare cultured tobacco cells from which Agrobacterium had been removed. Transfer the cultured cells to kanamycin 100 m
modified LS containing g / L and 250 mg / L Claforan
The cells were spread on an agar medium and cultured at 25 ° C. in the dark under static conditions. The callus cells obtained after about 2 to 3 weeks of culture were transplanted to a new agar medium, and growing clones were selected.
Finally, the selected clones were transformed with a modified LS medium containing 100 mg / L kanamycin and 250 mg / L claforan.
It was transferred to 0 mL and subcultured. As a result, transformed tobacco cultured cells were obtained.

(5) Confirmation of Gene Transfer by Genomic PCR The gene transfer of the obtained transformant tobacco cultured cells was confirmed. First, the genomic DNA of the cultured tobacco cells was used under the trade name ISOPLANT DNA isolation kit (N
It was prepared using ippon Gene, Japan). Then, using the prepared genomic DNA as a template, introduction of the PME gene was confirmed by PCR using a primer set of PME-F and PME-R for specifically amplifying the full length of the PME gene described above. In the PCR, the denaturation reaction was performed at 94 ° C for 1 min, the denaturation reaction was performed at 94 ° C for 30 seconds, and the annealing reaction was performed at 55 ° C
s, extension reaction was performed at 72 ° C for 1 min 30 s, and a total of 30 cycles were repeated. Finally, extension reaction was performed at 72 ° C for 5 min. As a result, since the PCR amplification product was detected, PM
The introduction of the E gene was confirmed.

(Example 2) The PME activity and methanol production of the transformed tobacco cultured cells prepared in Example 1 were confirmed. As a control, cultured tobacco cells were prepared by infecting the Agrobacterium in the same manner except that the binary vector pBI121 having the β-glucuronidase (GUS) gene was introduced into Agrobacterium instead of pmeA. .

(1) Extraction of PME and Measurement of PME Activity The transformed tobacco cultured cells obtained in Example 1 and the control tobacco cultured cells were cultured in the modified LS medium as described above, respectively, and then cultured in the presence of liquid nitrogen. Crush and add an equal volume (w / v) of extraction buffer (20 mM sodium acetate, 2 M NaCl, 0.01 wt% Tween-20, pH 5.0)
Was added and incubated at 4 ° C. for 2 hours. After incubation, this was centrifuged (10 minutes, 1,000 rpm), and the collected supernatant was used as a crude PME extract. For the PME crude extract, the protein concentration was determined by the Bradford method (Bradford 19
76), and the PME activity was measured by the gel diffusion method (Downie, Dirk et al. 1998) shown below.
In addition, the transformed tobacco cultured cells of Example 1 and the control tobacco cultured cells were each measured for 10 samples, and the average value was determined (N = 10). These results are shown in FIG. This figure is a graph showing the average PME activity of the transformed tobacco cultured cells of Example 1 and the control tobacco cultured cells.

The activity was measured by the gel diffusion method as follows. First, 0.1% (w / v) pectin (citrus
fruit origin: about 90% esterification (degree of esterifi
cation: approx. 90%)) and 2% (w / v) agar
0.1 M citric acid / 0.2 M phosphate (dibasic) buffer (pH
After heating 5.0), the mixture was cooled to 60 ° C. or less, and 0.05% sodium azide was added to spread the mixture on the plate. After the gel had solidified, a hole was drilled with a 2 mm diameter cork borer.
To each well, a diluted solution (0.05 U, 0.1 U, 0.5 U, 1 U) of the PME crude extract and the PME preparation was added. The plate was capped and placed in a 30 ° C. incubator with a well-wetted towel for 16 hours to react. The gel after the reaction was rinsed with distilled water and stained with a 0.05% by weight aqueous ruthenium red solution for 45 minutes. After staining, the gel was rinsed with distilled water, and the staining area was measured with calipers. The PME activity of the crude extract was calculated from a standard curve created from the staining area with respect to the logarithmically converted standard PME activity value. PME samples are trade name pectinesterase from orange peel (SIGMA,
USA) was used. 1 U is defined as the amount that produces 1 microequivalent of acid per minute at pH 7.5, 30 ° C.

As a result, as shown in FIG. 1, the PME activity of the control extract was 600 U / mg _{( protein)} (10 μkatal / m
g _(protein) ), the PME activity of the extract of Example 1 was 2400 U / mg _(protein) (40 μkatal / mg _(protein) ), and the activity was improved about 4-fold.

(2) Gas chromatography-mass spec
Methanol analysis using Cutrometallic (GC-MS) Next, the transformed tobacco cultured cells and the
And control tobacco culture cells
The methanol produced was analyzed. Used to create calibration curve
Methanol and methanol-d as internal standard_Three
Was purchased from Wako Pure Chemical Industries, Ltd. GC is a product
Using Shimadzu GC-17A gas chromatograph (Japan)
The sample is a trade name with helium as carrier gas.
 CP-PoraBOND Q fused silica PLOT column (25 mx 0.25
 mm i.d., df = 3 μm film thickness; Chrompak)
did. GC conditions are column temperature 60-100 ° C (60 ° C
At a temperature of 10 ° C / min), and a vaporization chamber temperature of 270 ° C
Was. In addition, samples are injected splitlessly.
The head pressure was 60 kPa. Mass spectrometry
Is a brand name Shimadzu QP-5000 mass selective detecto
r (Japan). The detector voltage is 1.6 kV.
Was. Methanol (m / z 32) and methanol-d _Three(M / z
The spectrum of 35) is SIM (selected ion monitering)
Detected using

The calibration curve of the methanol concentration (μM) is 25 to 1500 μM when the total volume is 10 mL.
Peak ratio between methanol (m / z 32) and 100 μM methanol-d ₃ (internal standard) (m / z 35) [(m / z 35)
z 32) / (m / z 35)]. The equation of the obtained calibration curve is shown below.

Y = 0.0167x (R ² = 0.999
2) x: methanol concentration (μM) y: peak ratio [(m / z 32) / (m / z 35)]

The transformed tobacco cultured cells of Example 1 and the control tobacco cultured cells were cultured under the above-described conditions, and the obtained culture solutions were used as samples. First,
In a 40 mL vial (Supelco Inc., USA), place 10 mL of the sample and a PTFE-coated rotor,
μM methanol-d ₃ was injected. The vial was sealed with a phenolic cap with Teflon / silicon septum (Supelco Inc., USA) and
Heat treatment was performed at 15 ° C. with stirring for 15 minutes. Then, the headspace gas was changed to 75 μm Carboxen / polydimethylsiloxa
ne (PDMS) solid-phase microext with fiber
Sampling was performed using a raction (SPME) unit (Supelco Inc., USA). Specifically, the needle part of the SPME unit is pierced into a septum, and in the head space,
Volatile compounds were adsorbed by exposing the fibers at 60 ° C. for 15 minutes. After returning the fiber into the needle, the fiber was pulled out of the vial, immediately injected into the GC inlet (270 ° C.) and exposed for 30 seconds.
The fibers were conditioned by heat treatment at 270 ° C. for 10 minutes for each measurement. Then, the methanol concentration in the culture solution was determined from the obtained peak ratio [(m / z 32) / (m / z 35)] and the calibration curve. The transformed tobacco cultured cells of Example 1 and the control tobacco cultured cells were each measured for three samples. The results are shown in Table 1 below and FIG. The figure shows a graph showing the methanol concentration (μM / mg _{(dry weight)} ) in the culture solution (T1, T2, T3) of the transformed tobacco cultured cell of Example 1 and the control culture solution (C1, C2, C3). It is. In addition,
The dry weight corresponds to the dry weight of the cultured cells.

(Table 1) Methanol concentration (μM / mg) Example 1 T1 41.49 T2 60.14 T3 49.87 Control C1 34.58 C2 34.92 C3 28.40

As shown in Table 1 and FIG. 1, the culture solution of the transformed tobacco cultured cell of Example 1 had an increased amount of methanol production as compared with the control.

From the above results, the transformed plant cell of Example 1 not only improved the PME activity (about 4 times) but also increased the methanol production (about 1.2 times) as compared with the control. ~ 2 times), confirming that the transformed plant cell achieves the object of the present invention.

[0065]

As described above, according to the present invention, since a recombinant vector linked to a gene encoding PME that non-specifically hydrolyzes the methyl ester of pectin is used, the resulting transformed plant cells are Not only has excellent activity, but also has excellent methanol productivity. Therefore, if a transformed plant obtained from the transformed plant cell is used,
Methanol is increased in production, and can itself be used as a supply source of clean energy methanol.

[0066]

[Sequence list]                                SEQUENCE LISTING <110> Osaka Industrial Promotion Organization <120> Recombinant vector for plant cell transformation, Agrobacterium into which it has been introduced , Transformed plant cells and transformed plants obtained therefrom <130> R6434 <140> <141> <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 331 <212> PRT <213> Aspergillus niger <220> <221> PEPTIDE <222> (1) .. (331) <400> 1 Met Val Lys Ser Ile Leu Ala Ser Val Leu Phe Ala Ala Thr Ala Leu   1 5 10 15 Ala Ala Ser Arg Met Thr Ala Pro Ser Gly Ala Ile Val Val Ala Lys              20 25 30 Ser Gly Gly Asp Tyr Asp Thr Ile Ser Ala Ala Val Asp Ala Leu Ser          35 40 45 Thr Thr Ser Thr Glu Thr Gln Thr Ile Phe Ile Glu Glu Gly Ser Tyr      50 55 60 Asp Glu Gln Val Tyr Ile Pro Ala Leu Ser Gly Lys Leu Ile Val Tyr  65 70 75 80 Gly Gln Thr Glu Asp Thr Thr Thr Tyr Thr Ser Asn Leu Val Asn Ile                  85 90 95 Thr His Ala Ile Ala Leu Ala Asp Val Asp Asn Asp Asp Glu Thr Ala             100 105 110 Thr Leu Arg Asn Tyr Ala Glu Gly Ser Ala Ile Tyr Asn Leu Asn Ile         115 120 125 Ala Asn Thr Cys Gly Gln Ala Cys His Gln Ala Leu Ala Val Ser Ala     130 135 140 Tyr Ala Ser Glu Gln Gly Tyr Tyr Ala Cys Gln Phe Thr Gly Tyr Gln 145 150 155 160 Asp Thr Leu Leu Ala Glu Thr Gly Tyr Gln Val Tyr Ala Gly Thr Tyr                 165 170 175 Ile Glu Gly Ala Val Asp Phe Ile Phe Gly Gln His Ala Arg Ala Trp             180 185 190 Phe His Glu Cys Asp Ile Arg Val Leu Glu Gly Pro Ser Ser Ala Ser         195 200 205 Ile Thr Ala Asn Gly Arg Ser Ser Glu Ser Asp Asp Ser Tyr Tyr Val     210 215 220 Ile His Lys Ser Thr Val Ala Ala Ala Asp Gly Asn Asp Val Ser Ser 225 230 235 240 Gly Thr Tyr Tyr Leu Gly Arg Pro Trp Ser Gln Tyr Ala Arg Val Cys                 245 250 255 Phe Gln Lys Thr Ser Met Thr Asp Val Ile Asn His Leu Gly Trp Thr             260 265 270 Glu Trp Ser Thr Ser Thr Pro Asn Thr Glu Asn Val Thr Phe Val Glu         275 280 285 Tyr Gly Asn Thr Gly Thr Gly Ala Glu Gly Pro Arg Ala Asn Phe Ser     290 295 300 Ser Glu Leu Thr Glu Pro Ile Thr Ile Ser Trp Leu Leu Gly Ser Asp 305 310 315 320 Trp Glu Asp Trp Val Asp Thr Ser Tyr Ile Asn                 325 330 <210> 2 <211> 996 <212> DNA <213> Aspergillus niger van Tieghem KF-267 <220> <221> gene <222> (1) .. (996) <400> 2 atggttaagt caattcttgc atccgttctc tttgcagcga ccgcgctggc cgcgagccgc 60 atgacggctc cttccggtgc gattgttgtt gccaagtccg gaggtgacta cgacacgatc 120 agcgctgccg ttgatgctct cagcacgact tcgaccgaga cccagaccat cttcattgag 180 gagggatcct acgacgagca ggtgtatatt cccgccctca gtggaaagct gattgtctac 240 ggtcagactg aggacactac cacctacacc agcaacctgg tcaacatcac ccacgccatc 300 gctttggccg atgtcgacaa tgacgatgag actgcaaccc tccgtaacta cgctgaaggc 360 tcggccatct acaacctcaa cattgccaac acctgcggtc aggcctgcca ccaggctctc 420 gccgtgagcg cctatgccag cgagcaggga tactacgcct gccagttcac cggataccag 480 gacacccttt tggctgagac cggctaccag gtttacgccg gaacctacat cgagggtgcc 540 gttgacttca tctttggaca gcacgcccgc gcctggttcc acgagtgcga catccgcgtc 600 ctcgagggcc ccagctccgc ctccatcacc gccaacggcc gctcctccga gtcggacgac 660 tcttactacg tgatccacaa gtccaccgtc gctgctgctg atggcaacga tgtttcctcc 720 ggcacctact acctcggccg cccctggtcc cagtacgctc gcgtctgctt ccagaagacc 780 tccatgaccg atgtgatcaa ccacctcggc tggactgagt ggtcgacctc cacccccaac 840 accgagaacg tcacctttgt tgaatacggc aacaccggca ctggcgctga gggtccccgt 900 gctaacttct cttctgagct gactgagccc atcactatct cttggcttct cggatctgac 960 tgggaggact gggttgatac tagctacatc aactaa 996 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 3 gctctagagc atggttaagt caattcttgc atccg 35 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 cgagctcgtt agttgatgta gctagtatca accc 34

[Brief description of the drawings]

FIG. 1 is a graph showing PME activity of a transformed plant cell in one example of the present invention.

FIG. 2 is a graph showing the concentration of methanol in a culture solution of the transformed plant cell in the example.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Akio Kobayashi             2-1 Yamadaoka, Suita City, Osaka Prefecture Osaka University Engineering             Inside (72) Inventor Masahisa Hasunuma             2-1 Yamadaoka, Suita City, Osaka Prefecture Osaka University Engineering             Inside F term (reference) 2B030 AA02 AB03 AD20 CA06 CA17                       CA19 CB03 CD02 CD03 CD07                       CD09 CD10 CD13 CD17                 4B024 AA03 AA08 BA12 CA01 CA04                       CA11 DA01 DA05 EA01 EA04                       FA02 FA15 GA11                 4B050 CC03 DD03 LL05                 4B065 AA11X AA62Y AA88X AB01                       BA01 CA05 CA27 CA53 CA60

Claims (14)

[Claims] 1. A recombinant vector for transforming a plant cell, wherein a gene encoding a protein having a pectin methylesterase activity for non-specifically hydrolyzing a methyl ester contained in pectin is linked to the vector. Recombinant vector. 2. The recombinant vector according to claim 1, wherein the pectin methylesterase activity is a catalytic activity exhibited by mold-derived pectin methylesterase. 3. The method according to claim 1, wherein the pectin methylesterase activity is As
The recombinant vector according to claim 2, which has catalytic activity exhibited by pectin methylesterase derived from genus pergillus . 4. The method according to claim 1, wherein the pectin methylesterase activity is As
The recombinant vector according to claim 3, which has catalytic activity showing pectin methylesterase derived from pergillus niger . 5. The recombinant vector according to any one of claims 1 to 4, wherein the gene to be ligated is a gene encoding a protein shown in the following (a) or (b). (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing; (b) a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of (a), and which is contained in pectin; Proteins with pectin methylesterase activity that non-specifically hydrolyze esters 6. The gene to be ligated is a gene comprising a DNA represented by the following (c) or (d):
6. The recombinant vector according to claim 5. (C) D consisting of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing
NA (d) consists of a base sequence in which one or more bases are deleted, substituted or added in the base sequence of (c), and has a pectin methylesterase activity of nonspecifically hydrolyzing a methyl ester contained in pectin. DNA encoding a protein. 7. The recombinant vector according to claim 1, wherein the vector is a binary vector. 8. The recombinant vector according to claim 7, wherein the binary vector is pBI121. 9. An Agrobacterium for transforming a plant cell, wherein the recombinant vector according to claim 1 is introduced. 10. The method of claim 10, wherein the Agrobacterium is Agrobacteriu.
The Agrobacterium according to claim 9, which is a strain of Mtumefaciens EHA105. 11. A transformed plant cell in which the recombinant vector according to any one of claims 1 to 8 has been introduced into a plant cell. 12. A transformed plant cell obtained by infecting a plant cell with the Agrobacterium according to claim 10 or 11. 13. The method according to claim 13, wherein the plant cells are Fragaria , Lotus , Medica.
go , Onobrychis , Trifolium , Trigonella , Vigna , Citrus , Li
num , Geranium , Manihot , Daucus , Arabidopsis , Brassica , R
aphanus , Sinapis , Atropa , Capsicum , Hyoscyamus , Lycoper
sicon , Nicotiana , Solanum , Petunia , Digitalis , Majoran
a , Cichorium , Helianthus , Lactuca , Bromus , Asparagus , An
tirrhinum , Hererocallis , Nemesia , Pelargonium , Panicu
m , Pennisetum , Ranunculus , Senecio , Salpiglossis , Cucum
is , Browaalia , Glycine , Lolium , Zea , Triticum , Sorghum , D
atura , Chrysanthemum , Dianthus , Gerbera , Euphorbia , Pel
aronium , Ipomoea , Passiflora , Cyclamen , Malus , Prunus , R
osa , Rubus , Populus , Santalum , Allium , Lilium , Narcissu
s , Ananas , Arachis , Phaseolus , Eucomia , Heavea , Periploc
13. The transformed plant cell according to claim 11, which is a cell of at least one plant selected from the group consisting of a and Pisum . A plant obtained from the transformed plant cell according to any one of claims 11 to 13.