JP3144260B2 - Environmental stress-tolerant plant and production method thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to various environmental stresses,
For example, the present invention relates to a plant having enhanced resistance to stress due to high osmotic pressure, low temperature, high temperature, drying, and the like, and a method for producing the same.

[0002]

2. Description of the Related Art Plants existing in nature are exposed to various environmental stresses such as salt stress, drought stress, high temperature stress, and low temperature stress. In the process of evolution, plants respond to environmental stress in the area where the plants grow naturally. It has acquired a mechanism that can be developed and evolved. On the other hand, varieties have been improved by artificial means such as crossing along with the development of agriculture, and varieties with slightly increased environmental stress have been produced. However,
Deserts and salt-damaged areas have not yet responded sufficiently.

It is known that glycine betaine which is one of compatible solutes of low molecular weight organic compounds is involved in osmotic pressure regulation in plant cells. In the biosynthesis of glycine betaine, it is believed that choline is converted to betaine aldehyde with the involvement of choline dehydrogenase, and that betaine aldehyde is then converted to glycine betaine with the involvement of betaine aldehyde dehydrogenase.

[0004] In recent years, research for obtaining plants with enhanced resistance to environmental stress by genetic manipulation has been active. For example, HJBohnert et al., Science Vol. 25
9, No. 22, p508-510 (1993) reports that tobacco was introduced with mannitol synthase of Escherichia coli to impart salt tolerance. Also, The Plant J. Vol. 6, p749-758 (199
4) describes an example in which the expression of the betaine aldehyde dehydrogenase gene, which is a gene involved in the synthesis of glycine betaine, was increased to enhance the salt tolerance of plants, but the gene for choline dehydrogenase was actually introduced. It did not lead to an improvement in salt resistance.

[0005] As described above, it is known that glycine betaine is involved in the resistance of plants to environmental stress. However, what kind of gene can be introduced to increase the amount of glycine betaine in plants, It is not known whether it is possible to confer environmental stress tolerance to plants by increasing glycine betaine in plants by artificially introducing a sex gene.

[0006]

Accordingly, an object of the present invention is to provide a plant whose environmental stress tolerance has been enhanced by a genetic recombination method and a method for producing the plant.

[0007]

Means for Solving the Problems The present inventor has conducted various studies to solve the above-mentioned problems, and as a result, the present invention is one of compatible solutes of low molecular organic compounds which are considered to be involved in osmotic pressure regulation of plant cells. By introducing both choline dehydrogenase gene and betaine aldehyde dehydrogenase gene involved in the synthesis of glycine betaine into plants, glycine betaine can be synthesized or its synthesis can be enhanced, thereby conferring plant resistance to environmental stress. The inventors have found that the present invention can be performed and completed the present invention.

Accordingly, the present invention provides a plant having enhanced resistance to environmental stress by introducing a choline dehydrogenase gene and a betaine aldehyde dehydrogenase gene involved in glycine betaine synthesis. The present invention also provides a method for producing the above-mentioned plant, which comprises introducing a choline dehydrogenase gene and a betaine aldehyde dehydrogenase gene involved in glycine betaine synthesis into the plant.

[0009]

DETAILED DESCRIPTION OF THE INVENTION The origin of the choline dehydrogenase gene and the betaine aldehyde dehydrogenase gene used in the present invention is not particularly limited. For example, it can be obtained from plants and microorganisms that are naturally high in environmental stress tolerance. Bacterial genes are preferred. In particular, genes from E. coli have already been cloned (Lamark et al., Mol. Microbiol. Vol. 5, p1049-1064, 199).
1) In the present invention, the present invention will be specifically described using a gene derived from Escherichia coli as a specific example.

However, the gene of the present invention is not limited to a gene derived from Escherichia coli, since it may be expressed in a plant cell. A gene derived from Escherichia coli can be obtained as a 9 kbp BamHI fragment according to the description in the above literature, and this fragment is a choline dehydrogenase gene (betA).
And betaine aldehyde dehydrogenase gene (be
tB).

In the present invention, one kind of a freshwater cyanobacterium, Synechococcus, which is a lower plant having photosynthetic ability as a plant, is used, and glycine betaine is present from the lower plant, the cyanobacterium to the higher plants. Therefore, the present invention can be widely applied to all photosynthetic plants because it is considered to be involved in the resistance of plants to environmental stress. Such plants can be widely applied to, for example, herbaceous plants such as tobacco, corn, rice, wheat, barley, tomato, potato, soybean, cotton, and also woody plants such as eucalyptus and acacia. is there.

In order to introduce the gene into the plant, a conventional method for introducing a foreign gene into a plant cell, for example, a method such as Agrobacterium method or particle gun method may be used. Conventional tissue culture methods can also be used to regenerate a plant cell into which a foreign gene has been introduced into a plant. In a specific example of the present invention, a method was used in which a target gene was inserted into a shuttle vector capable of replicating in both Escherichia coli and cyanobacterial cells, and this vector was introduced into the cyanobacterial cells.

[0013] Many plants naturally have the ability to biosynthesize glycine betaine, and according to the present invention, to further enhance the ability to produce glycine betaine and enhance the resistance to environmental stress in those plants. Can be. It goes without saying that the present invention can be applied to a plant having substantially no glycine betaine biosynthesis ability. Many plants have the ability to synthesize choline, which is an intermediate material in glycine betaine biosynthesis, and enhance the synthesis of glycine betaine from choline by introducing the gene used in the present invention into such a plant. Can be
Furthermore, by introducing the gene used in the present invention into plant cells, an effect of increasing choline uptake can be obtained.

Since glycine betaine is present in cells and the like and is thought to contribute to the stabilization of enzymes and structural proteins of cells, the plant of the present invention can be used not only for osmotic stress due to salts and the like, but also for drought stress, low-temperature stress, It has resistance to various environmental stresses such as high temperature stress.

[0015]

Next, the present invention will be described more specifically with reference to examples. Example 1 Synechococcus sp. Remains of PCC7942
Introduction of gene (1) Cloning Cloning of a gene is Andresen et al, J. Gen. Microbiol.
Vol. 134, p1737-1764 (1988) and Lamark et al., Mol. Microbi.
ol. Vol.165, p1059-1062 (1991). By this method, 9 kb BamHI D
The NA fragment was obtained from E. coli (CSH26).

As shown in FIG. 1, the DNA fragment encodes an open reading frame (betA) encoding a choline dehydrogenase gene, an open reading frame (betB) encoding betaine aldehyde dehydrogenase, and an energy-dependent transport system of choline. Open reading frame (bet
T).

(2) Preparation of Expression Shuttle Vector The 9 kb BamHI DNA fragment was
Plasmid pBET was obtained by insertion into the BamHI site of 303-Bm. Plasmid pUC303 is commercially available as an E. coli / Synechococcus shuttle vector, and pUC303-Bm is the EcoR of pUC303.
This is a plasmid in which 12 bp containing a BamHI fragment has been introduced into the I fragment. Ba of this plasmid pUC303-Bm
At the mHI site, 9 kb Ba in the plasmid pBET was added.
The insertion of the 20 kb plasmid p
CBET was obtained. The above-described genetic recombination operation was performed using Escherichia coli DH5α as a host.

(3) Transformation of cyanobacteria Synechococcus sp. Commonly used in genetic engineering of cyanobacteria. PCC7
942 is a BG11 liquid medium supplemented with 20 mM Hepes-KOH (pH 8.0) (composition: NaNO ₃ ; 1.5 g)
_{/ L, K 2 HPO 4 ·} 7H 2 O; 40mg / l, MgSO
_{4 · 7H 2 O; 75mg /} l, CaCl 2 · 2H 2 O; 3
6 mg / l, Ciic Acid; 6 mg / l, Ferr
ic Ammonium Citrate; 6 mg, ED
TA: 1 mg / l, Na ₂ CO ₃ ; 20 mg, MnSO _4.
7H ₂ O; 2.5 mg, ZnSO ₄ .7H ₂ O; 222 μm
g / l, CuSO ₄ .5H ₂ O; 79 μg / l, H ₃ B
O ₄ ; 2.86 mg / l, NaMoO ₄ ; 21 μg / l,
Co (NO ₃ ) ₂ .6H ₂ O in 500 μg / l)
Culture was performed at 30 ° C. under continuous irradiation of fluorescent white light. Transformation was performed by mixing the cultured algae bodies with the shuttle plasmid pCBET, and selection was made based on streptomycin resistance. Cyanobacteria that grew in the presence of 10 μg / ml streptomycin were selected. The resulting colonies were screened by Southern hybridization using the ³² P-labeled 9 kb DNA fragment (FIG. 1) as a probe, and positive clones were selected.

Next, in order to confirm the expression of the target gene, H. Aiba et al., J. Biol.
m. Vol. 256, p11905-11910 (1981). Total RNA was extracted and described in Yang H. et al., Nucleic.
Acids Research vol. 21, p3337-3338 (1993), using a ³² P-labeled PstI and BglII fragment (see FIG. 1) as a probe in North.
The transcribed RNA was detected by ern blot analysis. In this test, the above-mentioned selected positive clones were cultured in a medium containing no NaCl for 2 days, further cultured in a BG11-choline medium containing 200 mM NaCl, and then detected.

As shown in lane 2 of FIG. 2, an RNA transcript of about 9 kb was detected in the cyanobacteria of the present invention.
Plasmid vector pUC without gene insertion
In the cells transformed with 303-Bm, as shown in lane 1 in FIG. 2, RNA that hybridized with the probe was not transcribed. Since the size of the transcribed RNA is about 9 kb, it is estimated that the inserted 9 kb DNA fragment was transcribed.

Example 2 Characteristics of Transformed Plants (1) Incorporation of Choline Synechococcus cells transformed with vector plasmid pUC303-Bm or plasmid pCBET were prepared with or without 200 mM NaCl.
G11 medium was grown to mid-log phase. Choline transport activity (uptake of choline) at 25 ° C.
rk et, Mol.Microbiol.Vol.5, by the method described in p1049-1064 (1991), 10μM ^[14 C] - choline (5
8.5 mCi / mmol). Endogenous choline was not detected in algal cells grown in the absence of choline. The result is shown in FIG.

As shown in FIG. 3A, the target cells (pUC
Cells transformed with 303-Bm) and cells containing the bet gene (cells transformed with pCBET)
In both cases, choline uptake progressed under non-stress conditions, but the initial rate and the intracellular choline content after 30 minutes were approximately 40% higher in pCBET-bearing cells.
This difference is due to the active choline transport system in E. coli (Lamark T.,
Vol. 5, p1049-1064 (1991)) may be the result of the functional expression of the betT gene in pCBET. In all cases, the addition of 5 μM of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) strongly inhibited choline uptake, suggesting the presence of an energy-dependent choline transport system in Synechococcus.

In FIG. 3, the hollow circle is pUC303-
The results obtained when the cells transformed with Bm were cultured in the absence of FCCP are shown. The hollow squares show the results when the cells transformed with pCBET were cultured in the absence of FCCP. The results obtained when the cells transformed with pUC303-Bm were cultured in the presence of FCCP, and the results obtained when the cells transformed with pCBET were cultured in the presence of FCCP are shown.

As shown in FIG. 3B, under salt stress conditions, choline was only taken up by Synechococcus transformed with pCBET. This is because the energy-dependent choline transport system present in the plasma membrane of Synechococcus is altered by the presence of high concentrations in the medium, while the synthesis of glycine betaine stabilizes the membrane and enables the transport of choline under high temperature conditions. Expected to have done. in this case,
By using choline in glycine betaine synthesis,
This may be because feedback suppression by choline in the transport system in the bet gene-containing cells was released.

(2) Production of glycine betaine Untransformed Synechococcus sp. PCC79
No choline dehydrogenase and betaine aldehyde dehydrogenase activities were detected in 42 and no glycine betaine accumulated under salt conditions.
By adding choline at a concentration higher than 1 mM, Synechococcus sp. Since growth of PCC7942 cells was inhibited under unstressed and stressed conditions, 1 g for production of glycine betaine in pCBET transformed cells.
00 μM choline was added.

This level of choline did not affect the growth of target cells under various conditions. Synechococcus cells grown in BG11 medium containing 100 μM choline (BG11-choline medium) for 2 days (up to mid-log phase) were transferred to BG11-choline medium with various NaCl concentrations. After three days of incubation, the quaternary ammonium compound was extracted from the cells with 1N H ₂ SO ₄ and precipitated as its periodate (Wa
ll, JS et al., Anal. Chem. Vol. 32, p870-847 (1960)). The pellet was dissolved in 600 μL of D ₂ O containing t-butanol as an internal standard.

The analysis of the quaternary ammonium compound was performed using ¹ H
The measurement was performed by a NMR spectrum method using a JEOL JMN-500 Fourier exchange NMR meter. Cell volume is Blumwa
ldE. et al., Proc. Natl. Acad. Sci. USA Vol. 80, p2599-2602.
(1983). The enriched cells are transformed into a membrane-impermeable paramagnetic eraser Na ₃ Fe (C
N) ₆ (20 mM) and Na ₂ Mn EDTA (75 mM)
Was treated with the free-permeating nitroxide spin probe THMPONE (2,2,6,6-tetramethylpiperidone-N-oxyl) in the presence of 1 mM to excite the ESR signal of the intracellular spin probe. Cell volume was also determined using ³ H ₂ O and [ ¹⁴ C] -sorbitol.
Incharoensakidi A. et al., Plant Cell Physiol. Vol. 29, p.
It was also measured by the method of 1073-1075 (1988).

FIG. 4 shows the results. In FIG. 4, A shows the ¹ H NMR spectrum of a quaternary ammonium compound from a subject (cells transformed with pUC303-Bm), and β shows that from cells transformed with pCBET. Peaks b and c indicate betaine and choline, respectively. As can be seen from FIG. 4, only cells transformed with pCBET synthesized and accumulated glycine betaine.

The level of glycine betaine in these cells varies depending on the salt concentration in the medium, and as shown in Table 1,
From about 3 mM under non-stress conditions, a NaCl concentration of 375
over 45 mM in mM. These levels of glycine betaine provide sufficient protective effects on various cellular functions (Ge
rard H. et al., Plant Cell Physiol. Vol 29, p1073-1075
(1991), and Rhodes D. et al., Annu. Rev. Plant Physiol. P.
lant Mol. Biol. Vol. 44, p357-384 (1993)).

[0030]

[Table 1]

(3) Photosynthetic activity Cells grown in BG11-choline medium for 2 days (mid-log phase) were transformed into BG11 supplemented with 200 mM NaCl.
-Transferred to choline medium and continued culture for 4 days. Since the photosynthetic activity of these cells was low, after transfer to fresh BG11-choline medium without NaCl for 1 day,
Oxygen evolution by photosynthesis and PSI (Photosystem I) and PSII (Photosystem II) were measured in a Clark-type oxygen electrode.

The reaction medium was 100 μM DCMU (3-
(3,4-dichlorophenyl) -dimethylurea), 1 mM
Sodium ascorbate, 500 μM DAD
(2,3,5,6-tetramethyl-β-phenylenediamine) and 40 for the determination of PSI electron transport activity.
For measurement of 0 μM MV (methyl viologen) or PSII electron transport activity, 1 mM PBQ (phenyl-1,
4-benzoquinone). Photosynthetic activity was also measured immediately after subjecting the cells to osmotic stress in the presence of 200 mM NaCl.

Synechococcus can grow in media containing NaCl at concentrations as high as 400 mM,
After 4 days of growth in the presence of 300 mM NaCl, the cells turned pale yellow. This effect was not observed in cells transformed with pCBET and remained green. The result is shown in FIG. 6 as a black and white photograph. In FIG. 6, the right side is a photograph of a Synechococcus culture transformed with pCBET, and the left side is a photograph of a control culture.

As expected, in cells transformed with pUC303-Bm as compared to cells transformed with pCBET, the absorption spectrum shows the phycobilisome (C-phycocyanin (λ620-630 nm)) and the plays of chlorophyll content. Significant reduction (data not shown). O by photosynthesis
_Both the development of ₂ and the activities associated with PSI and PSII were higher in cells transformed with pCBET compared to control cells transformed with pUC303-Bm. Table 2 shows the results. The values in Table 2 are the average of three measurements, and the standard error was within 5%.

[0035]

[Table 2]

Table 3 below shows the photosynthetic activity obtained after transferring cells grown under non-stress conditions to high salt conditions (200 mM NaCl). Both measurements (especially those of PSI) decreased almost instantaneously in control cells compared to the values obtained in the absence of salt stress. This decrease was not significant in cells transformed with pCBET. The values in Table 3 are the average of three measurements, and the standard error was within 5%.

[0037]

[Table 3]

According to the results of these observations,
Glycine betaine production has a stabilizing effect on phycobilisomes and PS complexes under salt stress conditions.

(4) Growth under salt stress conditions Cells transformed with pUC303-Bm (control) and cells transformed with pCBET were grown in BG11-choline medium for 2 days and then various NaCl It was grown in a concentration of BG11-choline medium. 0.3 M for both control cells and cells transformed with pCBET
It showed almost the same growth rate up to NaCl. However, at NaCl concentrations higher than 0.3 M, pCB
Cells transformed with ET had a higher growth rate than control cells (FIGS. 5B and C). In FIG. 5, A is NaCl
The results at a concentration of 0.1 M, B at 0.375 M and C at 0.4 M are shown. Overall results indicate that the bet gene present in plasmid pCBET was expressed in Synechococcus cells and produced a functional protein.
In addition, glycine betaine produced by the cells produced a generally beneficial effect on Synechococcus cells under salt stress conditions.

[Brief description of the drawings]

FIG. 1 is a diagram showing the arrangement of gene groups used in the present invention.

FIG. 2 is an electrophoretogram obtained by detecting about 9 kb mRNA transcribed from a gene introduced into Synechococcus by Northern blotting, and is a photograph substituted for a drawing.

FIG. 3 is a graph showing choline uptake under various conditions.

FIG. 4 is an NMR spectrum showing that glycine betaine (b) was produced in the transformed cells.

FIG. 5 is a graph showing the growth of Synechococcus at various NaCl concentrations.

FIG. 6 is a photograph of a culture of Synechococcus transformed with pCBET, and the left is a photograph of a control culture. Each is a drawing substitute photograph showing the form of the living thing.

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Claims (2)

(57) [Claims] 1. The large intestine involved in the synthesis of glycine betaine
Choline dehydrogenase gene from fungi and Escherichia coli
A plant having enhanced resistance to environmental stress by introducing a betaine aldehyde dehydrogenase gene of the present invention. 2. The method for producing a plant according to claim 1, wherein the Escherichia coli involved in the synthesis of glycine betaine is added to the plant.
A choline dehydrogenase gene derived from Escherichia coli and a betaine aldehyde dehydrogenase gene derived from Escherichia coli .