JP3845661B2 - Stress resistant plant - Google Patents

Description

【００９４】
【配列表フリーテキスト】
配列番号３：合成プライマー
配列番号４：合成プライマー
配列番号５：合成プライマー
配列番号６：合成プライマー
配列番号７：合成プライマー
配列番号８：合成プライマー、第３塩基及び第１１塩基のnは、a、t、g、又はcのいずれかである。
配列番号１２：合成プライマー
配列番号１３：合成プライマー
【図面の簡単な説明】
【図１】図１は、イネ幼苗を、0、3、6、9、及び24時間にわたり42℃にて高温処理すると、６時間以上の処理区において、低温ストレス耐性が顕著に強化されることを示した図である。**は１％水準で有意であることを示す。
【図２】図２は、25℃処理した幼苗（対照）、42℃高温処理（24時間）した幼苗、42℃高温処理（24時間）後５℃で７日間低温処理した幼苗、及び25℃処理した後５℃で７日間低温処理した幼苗における、カタラーゼ(CAT)、スーパーオキシドディスムターゼ(SOD)、アスコルビン酸パーオキシダーゼ(APX)の各活性を示した図である。*は５％水準で有意、**は１％水準で有意であることを示す。
【図３】図３は、25℃処理した幼苗（対照）、42℃高温処理（1, 3, 6, 9, 12, 24時間）した幼苗、42℃高温処理（24時間）後５℃で７日間低温処理した幼苗、及び25℃処理した後５℃で７日間低温処理した幼苗における、APXa遺伝子のmRNA発現量を、ノーザンブロット解析のオートラジオグラム、及びその定量解析結果にて示した図である。オートラジオグラムについてはrRNA存在量を比較対照として示した。*は５％水準で有意であることを示す。
【図４】図４は、APXa遺伝子のプロモーター領域に、熱ショック因子(HSF)が結合するための最小限熱ショックエレメント(HSE)モチーフnGAAnnTTCnが存在することを示した図である。Aは、APXa遺伝子のプロモーター構造を示す。ATGは翻訳開始コドンを示し、APXa遺伝子のcDNAの5'端を矢印および＋１で示した。図中の数値は転写開始点からの塩基数である。Bは、イネAPXaプロモーター上のHSE（上段）とシロイヌナズナapx1プロモーター上のHSE（下段）との比較。HSEのモチーフnGAAnを大文字で示した。HSF結合のための最小限のモチーフnGAAnnTTCnを内向きの矢印で示した。その両側には１塩基置換されたモチーフが存在している。
【図５】イネに導入した組換えベクターpMLH7133-APXaの構造を示した図である。
【図６】原品種ゆきひかりと形質転換イネ（APX991217-5-2）における低温処理後の生存率の違いを示した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a transformed (transgenic) plant having enhanced resistance to active oxygen generated under environmental stress conditions.
[0002]
[Prior art]
In early cultivation and direct sowing cultivation of rice, low temperature is likely to be encountered in the early stages of growth, and significant low-temperature damage is often generated. In Asian countries, the damage caused by salt stress and drought stress is also great. In order to reduce these damages, new varieties with enhanced environmental stress resistance must be developed.
[0003]
As a method for enhancing environmental stress tolerance of rice, there is a method in which a variety / line having excellent environmental stress resistance is used as a mating parent, and an individual / line having excellent resistance is selected from the progeny of the cross. However, the breeding method using existing varieties as a material has a limit to hope for a significant improvement in environmental stress tolerance. Therefore, a new stress tolerance enhancement method is required.
[0004]
In general, many plants native to the tropics and subtropics suffer from low temperature damage when exposed to non-freezing temperatures below 12 ° C (Lafuente MT, Belver A, Guye MG, Saltveit ME. 1991. Plant Physiology 95, 443 -449). Some low temperature sensitive plants have increased resistance to low temperature stress after exposure to moderate temperatures (Moynihan MR, Ordentlich A, Raskin I. 1995. Plant Physiology 108, 995-999), Some of the cold-sensitive plants, including rice seedlings, do not show enhanced cold stress tolerance with such treatment.
[0005]
In low temperature sensitive plants, the main component of low temperature stress is oxidative stress (Hodges DM, Andrews CJ, Johnson DA, Hamilton RI. 1997.Crop Science 37, 857-863, Pinhero RG, Rao MV, Paliyath G, Murr DP, Fletcher RA. 1997 Plant Physiology 114, 695-704). Reactive oxygen species (AOS) such as hydrogen peroxide, superoxide radical, and hydroxyl radical react rapidly with DNA, lipids, and proteins to cause cell damage (Van Breusegem F, Slooten L, Stassart JM, Moens T , Botterman J, Van Montagu M, Inze D. 1999. Plant and Cell Physiology 40, 515-523). Although AOS is thought to be related to light-induced low temperature stress, molecular and biochemical evidence has shown that low temperature oxidative stress is also produced in corn seedlings that have been cold-treated under dark conditions (Prasad , TK, Anderson MD, Martin BA, Stewart CR. 1994 The Plant Cell 6, 65-74).
[0006]
Several enzymes can effectively detoxify AOS, but prolonged stress conditions can saturate such detoxification systems and cause damage (Van Breusegem F, Slooten L, Stassart JM, Moens T, Botterman J, Van Montagu M, Inze D. 1999. Plant and Cell Physiology 40, 515-523). The main hydrogen peroxide detoxification system in plant chloroplasts and cytosols is called the ascorbate-glutathione cycle, in which ascorbate peroxidase (APX) is an important enzyme (Asada K. 1992. Physiologia Plantarum 85, 235-241).
[0007]
In recent years, studies have been made to improve the environmental stress tolerance of plants by introducing such an active oxygen removal enzyme gene into plants (Plant Physiol., 111, 1177-1181, 1996, FEBS Letters, 428, 47). -51, 1998). For the production of such active oxygen-tolerant plants, a method for introducing genes of active oxygen species-removing enzymes (superoxide dismutase (SOD), peroxidase, catalase (CAT), glutathione reductase, etc.) into plants. Is adopted.
[0008]
However, it has been reported that stress tolerance is not necessarily enhanced even when the expression level of the enzyme is increased in a plant by introducing a gene for an active oxygen removal enzyme (Tepperman JM and Dunsmuir P., Plant Mol. Biol. , 14 (4): 501-511, 1990). In particular, in the case of the ascorbate peroxidase (APX) gene, which is an active oxygen scavenging enzyme, this enzyme activity decreases when the amount of ascorbic acid is insufficient. (Foyer CH, Souriau N, Perret S, Lelandais M, Kunert KJ, Pruvost C, Jouanin L., Plant Physiol., 109: 1047-1057, 1995).
[0009]
Therefore, at present, in the method for enhancing stress tolerance of plants by introducing active oxygen removal system enzymes, for example, SOD, CAT, and APX, and the activities of multiple related enzymes such as enzyme GR related to ascorbic acid supply cycle are simultaneously enhanced. Otherwise, it is said that it is difficult to effectively enhance oxidative stress tolerance.
[0010]
Ascorbate peroxidase (APX) includes a plurality of APX isozymes including cytosol type, chloroplast type and the like existing in different intracellular compartments (Asada, 1992, Physiol. Plant. 85, 235- 241; Miyake C, Asada K., 1992, Plant Cell Physiol. 33: 541-553; Yamaguchi K, Mori H, Nishimura M, 1995, Plant Cell Physiol. 36: 1157-1162), active oxygen is mainly leaf green Since it occurs in the body (Nie GY, Baker NR., Plant Physiol. 95: 184-191, 1991), introduction of a chloroplast-type APX gene has been attempted to remove active oxygen (Morita and Tanaka, Protein Nucleic acid enzyme, 44: 2232-2238, 1999).
[0011]
By the way, it has been reported that the low-temperature sensitive tissue of plants is exposed to a heat shock temperature in advance to increase the resistance to the low temperature to be exposed next (Lurie S, Klein JD. 1991. Journal of the American Society for Horticultural Science 116, 1007-1012, Saltveit ME. 1991. Physiologia Plantarum 82, 529-53). It is thought that heat shock causes oxidative stress, which induces genes involved in the oxidative stress defense system (Morgan RW, Christman MF, Jacobson FS, Storz G, Ames BN. 1986. Proceedings of the National Academy of Sciences, USA 83 , 8059-8063). In fact, the expression of the apx1 gene in pea and Arabidopsis thaliana is induced not only by oxidative stress but also by high temperature stress, and both genes have a heat shock cis element in their promoter (Mittler R, Zilinskas BA. 1992. The Journal of Biological Chemistry 267, 21802-21807, Mittler R, Zilinskas BA. 1994. The Plant Journal 5, 397-405, Storozhenko S, Pauw PD, Van Montagu M, Inze D, Kushnir S. 1998. Plant Physiology 118, 1005-1014).
[0012]
[Problems to be solved by the invention]
An object of the present invention is to provide a stress-tolerant plant and to provide a method for enhancing the tolerance of a plant to stress caused by active oxygen.
[0013]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have found that low temperature stress tolerance is remarkably increased by high-temperature treatment of rice seedlings. We isolated a gene that enhances tolerance to low-temperature stress in plants. As a result, it was found that the gene encodes a cytosolic ascorbate peroxidase, which has been considered to be less useful for enhancing stress tolerance of plants. A recombinant vector containing this gene linked to an overexpression promoter was prepared and introduced into a plant body to produce a transformed plant with enhanced stress tolerance, thereby completing the present invention.
[0014]
That is, the present invention is as follows.
(1) A stress-tolerant plant comprising a recombinant vector comprising a gene encoding the following protein (a) or (b) and an overexpression promoter.
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having ascorbate peroxidase activity
Examples of the gene encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 include those containing the following DNA (c) or (d).
(c) DNA consisting of the base sequence shown in SEQ ID NO: 1
(d) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 under a stringent condition and encodes a protein having ascorbate peroxidase activity
[0015]
Examples of stress include those caused by active oxygen (for example, environmental stress). Examples of the environmental stress include low temperature stress, high temperature stress, salt stress, drought stress, intense light stress, air pollution stress, agricultural chemical stress, disease stress, and ultraviolet light stress.
Moreover, as a plant, a plant body, a plant organ, a plant tissue, or a plant cultured cell can be used. Examples of the plant include those belonging to any family selected from the family Gramineae, Eggplant family, Brassicaceae family, Legume family, Cucurbitaceae, and Lily family. In particular, the plant belonging to the above family Gramineae may be rice.
[0016]
(2) A method for enhancing stress tolerance of a plant, comprising introducing a recombinant vector comprising a gene encoding the following protein (a) or (b) and an overexpression promoter into the plant.
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having ascorbate peroxidase activity
In the above method, examples of the gene encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 include those containing the following DNA (c) or (d).
(c) DNA consisting of the base sequence shown in SEQ ID NO: 1
(d) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 under a stringent condition and encodes a protein having ascorbate peroxidase activity
The stresses and plants targeted in this method are the same as described above.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
[0018]
In a plant, finding that the expression level of a specific gene encoding an active oxygen scavenging enzyme is increased by high-temperature treatment, resulting in enhanced resistance to low-temperature stress, and isolating the gene from the plant Can be useful for the development of plants that are highly resistant to active oxygen generated under various environmental stress conditions (high / low temperature, dryness, strong light, salt, air pollutants, pathogens, ultraviolet rays, etc.) . The present invention has been completed based on such an idea.
[0019]
In the present invention, a cytosolic ascorbate peroxidase gene was found that increases the expression level at high temperatures and enhances low temperature stress tolerance of plants in plants with enhanced low temperature stress tolerance by high temperature treatment. The present invention relates to a method for enhancing resistance to oxidative stress of a plant, which is a transformed plant into which the gene is incorporated in an overexpressed form, and is further incorporated into the plant so as to overexpress the ascorbate peroxidase gene. It is.
[0020]
The transformed plant of the present invention is one into which an ascorbate peroxidase gene encoding the amino acid sequence shown in SEQ ID NO: 2 has been incorporated. However, there may be some amino acid sequence differences between plants, depending on cultivars. Even in the same plant variety, amino acids may change due to mutation or the like. Therefore, a transformed plant containing a gene encoding an amino acid sequence in which a plurality of (1 or several, for example, 1 to 10) amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 2, It is included in the present invention.
[0021]
The transformed plant of the present invention has, for example, the ascorbate peroxidase gene shown in SEQ ID NO: 1. However, it is not limited to the said gene, All the genes which code the amino acid sequence shown to sequence number 2 are included.
The cytosolic ascorbate peroxidase gene found in the present invention and capable of enhancing low-temperature stress tolerance by increasing the expression level is considered to exist in various plant species. It is thought that can be acquired. Here, as an example, the present invention will be described using rice (Oryza sativa L.).
[0022]
1 . Enhancement of low temperature stress tolerance of rice seedlings by high temperature treatment
In the present invention, a gene to be introduced into a plant can be obtained from a rice seedling that has been found to be resistant to low temperature stress by high temperature treatment. However, the plant as the source of the gene of the present invention is not limited to rice seedlings, for example, plants grown with rice, plant organs (for example, leaves, petals, stems, roots, seeds, etc.), plant tissues (For example, epidermis, phloem, soft tissue, xylem, vascular bundle, fence-like tissue, spongy tissue, etc.) or plant cultured cells (eg, callus) may be used. Furthermore, the plant serving as the source of the gene of the present invention is a plant other than rice, such as, but not limited to, other plants belonging to the family Gramineae (such as corn), or solanaceous, cruciferous, legumes, cucumbers. Seedlings or grown plants, plant organs belonging to the family, Convolvulaceae, Lilyaceae, Lamiaceae, Asteraceae, Rosaceae, Citrusaceae, Fusariumaceae, Willowaceae, Rabbitaceae, Gentianaceae, or Gynecidae, It may be a plant tissue or a plant cultured cell. The plant is more preferably a gramineous plant, most preferably rice. As used herein, “rice seedling” means a rice plant that has been germinated and grown at 20-28 ° C., preferably 25 ° C., for 5-10 days, preferably 7 days. To do.
[0023]
Enhancement of low temperature stress tolerance of rice seedlings by high temperature treatment is achieved by treating rice seedlings at a high temperature that does not cause damage for a certain period of time and then treating them at low temperatures that would cause low temperature damage to rice seedlings. can do. “A high temperature that does not cause damage” means a temperature condition that does not cause growth failure or physiological function failure of the plant when the plant is treated at that temperature, and is appropriately set by those skilled in the art according to the experimental conditions. In the case of rice seedlings, the temperature is specifically 37 to 43 ° C, preferably 40 to 42 ° C, and most preferably 42 ° C. The “certain time” for treatment at a high temperature can be appropriately set by those skilled in the art according to the experimental conditions. In the case of rice seedlings, it is specifically 3-24 hours, preferably 6-24 hours, most preferably 24 It's time. “Low-temperature injury” means plant growth failure or death due to the influence of low temperature. “Temperature that causes low-temperature damage” is a temperature condition that causes such damage to plants, and is generally preferably a non-freezing temperature of 12 ° C. or less. Specifically, it is 0-12 degreeC, Preferably it is 3-10 degreeC, Most preferably, it is 5 degreeC. The high temperature treatment and the low temperature treatment are carried out by leaving the plant to stand under the condition of maintaining a predetermined temperature and humidity. The low temperature treatment may be performed under dark conditions or light conditions.
[0024]
It can be confirmed by observation of the appearance that the low temperature stress tolerance of the rice seedlings subjected to the above treatment has been enhanced. In order to confirm this, for example, rice seedlings are subjected to low-temperature treatment for a certain period, and then transferred to 20 to 28 ° C., preferably 25 ° C., and grown for several days. The period during which the low-temperature treatment is performed can be appropriately set by those skilled in the art, but specifically for rice seedlings, it is 5 to 10 days, preferably 7 days. The period of growth after the low-temperature treatment can be appropriately set by those skilled in the art, and specifically, it is 2 to 10 days, preferably 7 days. In the present specification, “tolerant to low temperature stress” means that a plant is subjected to a specific treatment and then left under a low temperature condition, and then a temperature suitable for growth of the plant, specifically in rice seedlings. When grown to 20 to 28 ° C., preferably 25 ° C., a temperature suitable for growth after leaving the untreated plant under low-temperature conditions in the same manner as the above plant (for example, 20 to 28 ° C. for rice seedlings, This means that the survival rate of the treated plant is significantly higher than that when the temperature is preferably returned to 25 ° C.
[0025]
The survival rate can be evaluated by observing the appearance of rice seedlings prepared by performing the treatment described above. For example, a seedling that has resumed growth is regarded as a survivor, and a seedling that has stopped growing or a dead seedling is evaluated as a non-survivor. It is desirable to repeat the above experiment at least twice.
Such evaluation of the survival rate can prove the low temperature stress tolerance enhancement of rice seedlings subjected to high temperature treatment and low temperature treatment. At the same time, rice seedlings with significantly enhanced low-temperature stress tolerance can be obtained by the above treatment.
[0026]
2 . Identification of enzymes involved in low-temperature stress tolerance enhancement by measuring the activity of active oxygen scavenging enzymes after high-temperature treatment and subsequent low-temperature treatment
By measuring the activity of an active oxygen removal system enzyme in rice seedlings after high temperature treatment and subsequent low temperature treatment, an enzyme whose activity is increased by high temperature stress and whose activity is maintained high after low temperature treatment, It can be identified as a protein involved in enhancing low temperature stress tolerance.
[0027]
In activity measurement, a combination of plants to be measured suitable for comparative analysis of activity after high temperature treatment and subsequent low temperature treatment is determined. For example, determine appropriate treatment conditions for a control rice seedling that is not subjected to high temperature treatment or low temperature treatment, a rice seedling that has been subjected to high temperature treatment, a rice seedling that has been subjected to low temperature treatment after high temperature treatment, and a rice seedling that has been subjected to low temperature treatment without being subjected to high temperature treatment, prepare.
[0028]
Next, a sample is collected from each prepared rice seedling. The sample is not particularly limited, but leaves can be used. Each sample is homogenized in buffer, the homogenate is centrifuged, and the supernatant containing the enzyme is collected. A method for obtaining an extract containing such an enzyme is not particularly limited, and any known method can be used. As the buffer solution, one having a conventional composition can be used, for example, 1% Triton X-100-containing 50 mM phosphate buffer (pH 7.0) can be used. Subsequently, the extract containing the enzyme is preferably used immediately in the next enzyme assay so as to avoid a decrease in enzyme activity.
[0029]
Examples of active enzyme removal system enzymes for activity measurement include, but are not limited to, ascorbate peroxidase (APX), superoxide dismutase (SOD), peroxidase, catalase (CAT), and glutathione reductase. It is not something. As a method for measuring these activities, a generally known method suitable for each enzyme is used. For example, APX activity can be measured by the method described in Saruyama and Tanida (1995). That is, the reaction system containing APX contains H₂O₂Is added to start the ascorbic acid oxidation reaction, measure the absorbance at 290 nm indicating the concentration of ascorbic acid, and set the decrease in the absorbance as the APX relative activity. For example, CAT activity can be measured according to the description of Tanida, 1996. In this method, the absorbance at 240 nm is measured, and the amount of decrease is taken as the relative activity of CAT. The SOD activity can be measured using, for example, an assay kit (SOD test, manufactured by Wako) based on the NBT method (Beyer and Fridovich, 1987). However, the methods for measuring these enzyme activities are not limited to those described.
[0030]
Based on the above activity measurements, it was confirmed that the APX activity level was significantly increased in rice seedlings that had been subjected to high-temperature treatment, and that high APX activity was retained even after low-temperature treatment after high-temperature treatment. it can. It is said that the main cause of low-temperature injury is peroxidation of membrane lipids due to active oxygen caused by low-temperature stress. Therefore, the increase in APX activity by high-temperature treatment contributes to the enhancement of low-temperature stress tolerance of rice seedlings. It is thought that there is.
[0031]
3 . APX Gene isolation and expression analysis
From the low temperature stress resistant rice seedlings obtained by the above treatment process, an APX gene whose activity is increased by high temperature treatment is isolated. Furthermore, by confirming the transcription level of the gene after high-temperature treatment and subsequent low-temperature treatment by Northern blot analysis, it can be confirmed that the expression of the gene is induced by high-temperature treatment.
[0032]
(1) Isolation of APX gene
Samples collected from rice seedlings after high-temperature treatment were extracted in the usual manner to extract total mRNA, and using them as templates, single-stranded cDNA was prepared using oligo dT primer and reverse transcriptase, and then the single-stranded Synthesize double-stranded cDNA from cDNA. The high-temperature treatment is performed for a time sufficient to significantly enhance the low-temperature stress tolerance of the plant to be treated according to the method described in 1. To do.
Furthermore, using this cDNA as a template, a DNA fragment containing the entire APX gene expressed after high-temperature treatment was amplified by PCR using primers designed based on the known DNA sequence of ascorbate peroxidase (APX) gene. The APX gene is isolated.
[0033]
Known APX gene sequences include, for example, the cDNA sequence of rice cytosolic APX (APXa) gene (Morita S, Kaminaka H, Yokoi H, Masumura T, Tanaka K. 1997, Plant Physiology 40, 113, 306; Accession No. D45423), but other types of APX genes or DNA sequences of other plant APX genes may be used. Preferably, a DNA fragment containing the entire APX coding sequence is amplified using primers designed to amplify the entire ORF encoding APX.
[0034]
The amplified DNA fragment thus obtained is incorporated into an appropriate cloning vector by a generally known method to prepare a recombinant vector. The base sequence of the insert APX cDNA is determined for this isolated clone. The base sequence can be determined by a known technique such as a chemical modification method of Maxam-Gilbert or a dideoxynucleotide chain termination method using M13 phage, but usually an automatic base sequencer (eg, DNA sequencer LIC-manufactured by Aloka) Sequencing is performed using 4200LS-2 etc.).
[0035]
The determined base sequence of the APX gene can be compared with a known DNA sequence of the APX gene. For comparison of base sequences, for example, the sequence and other sequences obtained from the "DDBJ / EMBL / GenBank International Base Sequence Database" (for example, DDBJ can be accessed from http://www.ddbj.nig.ac.jp) This can be done by aligning the DNA sequence of the APX gene. Alternatively, other DNA sequences having high homology with the DNA sequence of the APX gene of the present invention can be obtained from a database by a homology search against the APX cDNA sequence of the present invention and compared. From these sequence comparisons, it was found that the DNA sequence of the APX gene whose expression was induced in rice seedlings by high-temperature treatment was identical to the cDNA sequence of the rice cytosolic ascorbate peroxidase (APXa) gene. Therefore, the APX gene isolated by the method of the present invention is a cytosolic APX gene. The base sequence of the APXa gene of the present invention is shown in SEQ ID NO: 1.
[0036]
(2) APXa gene expression analysis
Using the APXa gene isolated clone obtained in (1) as a probe, expression analysis of the APXa gene in rice seedlings after high temperature treatment and subsequent low temperature treatment can be performed.
First, a combination of plants to be measured suitable for comparative analysis of APXa gene expression after high temperature treatment and subsequent low temperature treatment is determined. For example, appropriate treatment conditions are determined for a control rice seedling that is not subjected to high temperature treatment or low temperature treatment, a high temperature treated rice seedling, a rice seedling that has been subjected to low temperature treatment after high temperature treatment, and a rice seedling that has been subjected to low temperature treatment without performing high temperature treatment. These treated seedlings are prepared and then samples are taken from each. Total RNA is extracted from each of these samples by a conventional method and purified using a technique such as ethanol precipitation.
[0037]
The APXa mRNA expression level can be examined by performing Northern blot analysis on the purified total RNA using the above APXa gene probe by a generally known method. As a specific method, for example, purified total RNA is electrophoresed, blotted, and hybridized with a labeled APXa gene probe. Then, the hybridization signal in which the label is detected by an appropriate method is quantitatively read. For labeling the APX gene probe,³²P,³⁵Radioisotopes such as S, or non-radioactive labels such as digoxigenin (DIG), alkaline phosphatase, biotin, or fluorescence can be used, but are not limited thereto. As the labeling method, a random prime method is often used, but other various commonly known labeling methods can also be used. For example, an autoradiography method can be used to detect signals for these labels. Although such an autoradiogram is not limited, quantitative analysis can be performed with high accuracy by using an image analysis device such as a bioimaging analyzer.
[0038]
By analyzing the amount of APXa mRNA as described above, it is possible to confirm the increase in the expression level of the APXa gene by high temperature treatment and the maintenance of the expression level at a high level after the subsequent low temperature treatment. Thereby, it is confirmed that the expression of the APXa gene isolated by the method of the present invention is induced by high-temperature treatment.
[0039]
4 . APXa Analysis of high temperature inducible promoter of genes
Since the expression of the APXa gene is induced by high temperature treatment, it is presumed that this gene has a high temperature inducible promoter. Based on this idea, it can be proved that the gene is directly induced by high temperature by confirming that the gene has a high temperature inducible promoter by nucleotide sequencing.
[0040]
To prove this, a DNA fragment that can contain the rice APXa gene promoter can be obtained by PCR amplification of the upstream region of the APXa gene using rice genomic DNA as a template. As a PCR amplification method for obtaining this DNA fragment, the thermal asymmetric combined polymerase chain reaction (TAIL-PCR) method (Liu YG, Mitsukawa N, Oosumi T, Whittier RF. 1995, The Plant Journal 8, 457-463) is used. While other methods can be used, other suitable methods may be used. In the TAIL-PCR method, the sense direction primers to be used are designed as three kinds of gene-specific primers that are arranged stepwise inward. Furthermore, a random degenerate primer is used as the antisense direction primer, and the design of this primer should be based on the description in Liu YG, Mitsukawa N, Oosumi T, Whittier RF. 1995, The Plant Journal 8, 457-463. Can do. In this case, the three APXa gene-specific primers are used in combination with a random degenerate primer in order from the outermost one, and the three-step PCR reaction is continuously performed, thereby including the APXa promoter. The region can be amplified.
[0041]
The amplification product obtained in the final stage of this reaction is cloned into a vector by a generally known method and sequenced. Then, the DNA base sequence is compared with the promoter region of another gene containing a putative heat shock element, for example, the promoter of Arabidopsis thaliana axp1 gene. A putative heat shock element (HSE) is a sequence with a heat shock factor (HSF) binding minimal motif 5'-nGAAnnTTCn-3 'and whether the upstream region of the APXa gene has a sequence that matches this motif By confirming, it is possible to verify the possibility that it is a high temperature inducible promoter. Confirmation that the APXa gene has a high temperature-inducible promoter can support the discovery that the expression of the gene is induced by high temperature, resulting in enhanced low temperature stress tolerance of rice seedlings.
[0042]
5 . APXa Production of transgenic plants by gene transfer
The above process reveals that the expression level of the APXa gene increases at high temperatures, and as a result, enhances the low temperature stress tolerance of rice seedlings. Based on this discovery, it is conceivable that the cytosolic APX gene is introduced into plants and overexpressed to enhance the tolerance of plants to low temperature stress.
[0043]
In order to introduce a cytosolic APX gene into a plant and overexpress it, a recombinant vector comprising a cytosolic APX gene and an overexpression promoter is constructed and used to transform the plant. Is a general approach.
SEQ ID NO: 1 shows the base sequence of the cytosolic ascorbate peroxidase gene to be introduced into the transformed plant of the present invention, and SEQ ID NO: 2 shows the cytosolic ascorbic acid peroxidase encoded by the gene to be introduced into the transformed plant of the present invention. The amino acid sequence of oxidase is exemplified, but as long as the protein containing this amino acid sequence has ascorbic acid peroxidase activity, a plurality of, preferably one or several amino acids in the amino acid sequence have mutations such as deletion, substitution, addition, etc. May occur.
[0044]
For example, 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 may be deleted, and 1 to 10, preferably 1 may be deleted from the amino acid sequence represented by SEQ ID NO: 2. ˜5 amino acids may be added, or 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 may be substituted with other amino acids.
[0045]
Here, in the present invention, ascorbate peroxidase activity is
Ascorbic acid + H₂O₂→ Ascorbic acid oxide + 2H₂O
The activity of catalyzing the reaction of In addition, the activity of the protein of the present invention is as follows.₂O₂Can be confirmed by measuring the change in absorbance of the reaction solution.
[0046]
In addition, a DNA that can hybridize with the cytosolic APX gene under stringent conditions, wherein a DNA encoding a protein having ascorbate peroxidase activity is linked to an overexpression promoter. Also included in the present invention is a transformed plant comprising Stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, a nucleic acid with high homology, that is, a condition where a complementary strand of DNA having a homology of 60% or more, preferably 80% or more hybridizes, and a complementary strand of a nucleic acid with lower homology does not hybridize. . More specifically, the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C.
[0047]
Furthermore, a modified DNA encoding cytosolic APX can be synthesized by site-directed mutagenesis or the like, and a recombinant vector comprising it and an overexpression promoter can be constructed, thereby transforming the plant. In order to introduce a mutation into the APXa gene, a known method such as the Kunkel method or the Gapped duplex method, or a method equivalent thereto can be employed. For example, using a mutagenesis kit (for example, Mutan-K (TAKARA) or Mutan-G (TAKARA)) using site-directed mutagenesis, or TAKARA LA PCR in vitro Mutagenesis Mutation is introduced using a series kit.
[0048]
The cytosolic APX DNA fragment to be incorporated into the vector is not only obtained from the cytosolic APX gene cloned according to the above method, but once the base sequence has been determined, it can be obtained by chemical synthesis or by the cytosolic APX gene. It can also be obtained by PCR amplification using the clone as a template.
[0049]
(1) Construction of overexpression vector incorporating cytosolic APX gene
A recombinant vector containing a cytosolic APX gene for introduction into a plant, such as an APXa gene, is obtained by overexpressing a cytosolic APX DNA fragment obtained according to the above method, such as an APXa cDNA amplified fragment, in an appropriate vector. It can be constructed by incorporating it so as to be linked. The vector into which the cytosolic APX gene is inserted is not particularly limited as long as it contains an overexpression promoter and can be replicated in the target plant host, and examples thereof include plasmid DNA and phage DNA. Alternatively, the cytosolic APX gene may be inserted into an appropriate vector in advance linked to an overexpression promoter.
[0050]
An example of a particularly suitable recombinant vector containing an overexpression promoter is the GUS gene in pMLH7133 (Mitsuhara et al., Plant Cell Rhysiology 37: 49-59, 1996), which can effectively overexpress the inserted gene. Are substituted with the cytosolic APX gene of the present invention.
[0051]
In order to incorporate the cytosolic APX gene into the vector, a method is used in which the purified DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate restriction enzyme site downstream of the vector DNA overexpression promoter, and linked to the vector. It is done.
The cytosolic APX gene needs to be incorporated into a vector so that the function of the gene is exerted. Therefore, in addition to an overexpression promoter, cytosolic APX gene, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence), etc. may be linked to the vector. it can. Examples of the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.
[0052]
(2) Production of transformants
A transgenic plant can be obtained by introducing the recombinant vector constructed as described above into a plant.
Plants to be transformed in the present invention include whole plants, plant organs (e.g. leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g. epidermis, phloem, soft tissue, xylem, vascular bundle, It means any of a palisade tissue, a spongy tissue, etc.) or a plant cultured cell (for example, callus). Examples of the plant used for transformation include, but are not limited to, the plants described below, preferably a plant in which enhanced low-temperature stress tolerance is found by high-temperature treatment. Plants to which it belongs are preferred, and rice is more preferred.
[0053]
Eggplant family: Eggplant (Solanum melongena L.), tomato (Lycopersicon esculentum Mill), pepper (Capsicum annuum L. var. Angulosum Mill.), Red pepper (Capsicum annuum L.), tobacco (Nicotiana tabacum L.)
Brassicaceae: Arabidopsis thaliana, Brassica (Brassica campestris L.), Chinese cabbage (Brassica pekinensis Rupr.), Cabbage (Brassica oleracea L. var. Capitata L.), Japanese radish (Raphanus sativus L.), Rapeseed (Brassica camp) L., B. napus L.)
Gramineae: corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.)
Legumes: soybean (Glycine max), azuki bean (Vigna angularis Willd.), Green beans (Phaseolus vulgaris L.), broad bean (Vicia faba L.)
Cucurbitaceae: Cucumber (Cucumis sativus L.), Melon (Cucumis melo L.), Watermelon (Citrullus vulgaris Schrad.), Pumpkin (C. moschata Duch., C. maxima Duch.)
Convolvulaceae: Sweet potato (Ipomoea batatas)
Liliaceae: Allium fistulosum L., onion (Allium cepa L.), leek (Allium tuberosum Rottl.), Garlic (Allium sativum L.), asparagus (Asparagus officinalis L.)
Lamiaceae: Perilla frutescens Britt. Var. Crispa
Asteraceae: Chrysanthemum morifolium, chrysanthemum coronarium L., lettuce (Lactuca sativa L. var. Capitata L.)
Rosaceae: Rose (Rose hybrida Hort.), Strawberry (Fragaria x ananassa Duch.)
Citrus: Citrus unshiu, Salamander (Zanthoxylum piperitum DC.)
Myrtaceae: Eucalyptus globulus Labill
Willow family: Poplar (Populas nigra L. var. Italica Koehne)
Rabbitaceae: Spinach (Spinacia oleracea L.), sugar beet (Beta vulgaris L.)
Gentianaceae: Gentian (Gentiana scabra Bunge var. Buergeri Maxim.)
Nadesico: Carnation (Dianthus caryophyllus L.)
[0054]
The recombinant vector can be introduced into plants by a conventional transformation method such as the Agrobacterium method, particle gun method, PEG method, electroporation method and the like. For example, when using the Agrobacterium method, the constructed plant expression vector is introduced into an appropriate Agrobacterium, such as Agrobacterium tumefaciens, and this strain is inoculated into rice callus. It is possible to infect and obtain transformed plants.
[0055]
When the particle gun method is used, a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. The sample thus prepared can be processed using a gene transfer apparatus (for example, PDS-1000 (BIO-RAD) or the like). Treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.
[0056]
When plant cultured cells are used as a host, transformation is performed by introducing a recombinant vector into the cultured cells by the particle gun method, electroporation method or the like.
Tumor tissue, shoots, hairy roots, and the like obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture, and can be used at an appropriate concentration using a conventionally known plant tissue culture method. Of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).
[0057]
Whether or not a gene has been incorporated into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing DNA-specific primers. PCR can be performed under the same conditions as those used to amplify the cDNA fragment inserted into the recombinant vector. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, It can be confirmed that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, it is possible to employ a method in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzyme reaction.
[0058]
6 . Evaluation of stress tolerance of transformed plants
When an overexpression recombinant vector containing the cytosolic APX gene is introduced into a plant, the resulting transformed plant has enhanced resistance to stress. Therefore, the transformed plant can be used as a stress resistant plant. In conventional methods for enhancing stress tolerance of plants by introducing genes for reactive oxygen scavenging enzymes, APX is not only less effective when introduced alone, but cytosolic APX is more effective than chloroplast APX. Therefore, in the present invention, it is an unexpected effect that the low temperature stress tolerance of plants is enhanced by introducing cytosolic APX alone.
[0059]
Here, the stress is based on active oxygen. The stress based on active oxygen means stress based on environmental stress that generates active oxygen in the plant body, and refers to stress that causes a growth disorder or death if a wild-type plant is left in this environment. For example, there is a stress state when a rice seedling is left at a low temperature of 5 ° C. and a relative humidity of 80%. The stress-tolerant plant of the present invention can grow even under the above-mentioned active oxygen conditions.
[0060]
Active oxygen is generated when exposed to various environmental stresses. Environmental stress includes high temperature stress, low temperature stress, drought stress, strong light stress, salt stress, air pollution stress, pesticide stress, disease stress, UV stress, etc. These stresses can be used alone or in combination. Good.
[0061]
“High temperature stress” means stress when a temperature of 35 ° C. or higher is continuously or temporarily applied for several minutes or longer.
“Low-temperature stress” means stress when a condition of 10 ° C. or lower is continuously or temporarily applied for several minutes or longer.
“Dry stress” means stress when a state of depletion of water is sustained or temporarily loaded.
`` Strong light stress '' means stress when a plant is exposed to strong light that exceeds the photosynthetic capacity. For example, 2,000 μmol / s / m<sup>2</sup>The case where the above light is irradiated continuously or temporarily corresponds.
“Salt stress” means stress when damaging the physiological function of a plant body, such as the water potential of the soil being lowered by salts accumulated in the soil and the plant body being unable to absorb moisture.
“Air pollution stress” means stress when an air pollutant (such as ozone, PAN: peroxyacetyl nitrate, sulfur dioxide, hydrogen fluoride and ethylene) is continuously or temporarily loaded.
“Agricultural chemical stress” means stress when a plant body is in continuous or temporary contact with agricultural chemicals.
“Disease stress” means stress when suffered by fungi and the like, and examples of the harm include soybean purpura.
“Ultraviolet light stress” corresponds to a case where ultraviolet light having a wavelength of 100 to 400 nm is continuously or temporarily irradiated.
[0062]
A stress-tolerant plant is produced by breeding the transformed plant (transgenic plant) of the present invention in which a cytosolic APX gene is incorporated in an overexpressed form to such an extent that it can be used as the stress-tolerant plant. Can do. In this case, it is only necessary to select a plant exhibiting tolerance without damaging physiological functions under conditions where the above-described various stresses occur, and without causing growth inhibition or death.
The start of use as a stress-tolerant plant may be any time after selecting a plant exhibiting resistance.
The stress tolerance of the transformed plant can be relatively quantified by evaluating the survival rate after leaving it under stress conditions according to the above description.
[0063]
For example, the plant breeded from the transformant introduced with the cytosolic APX gene of the present invention is subjected to low-temperature treatment, and then returned to 25 ° C. to evaluate the survival rate of the plant grown. At the same time, by evaluating the survival rate of the wild-type plant when the same low-temperature treatment is applied, and comparing it with the survival rate of the transformed plant, it will demonstrate the enhanced stress tolerance of the transformed plant. Can do.
[0064]
【Example】
EXAMPLES Hereinafter, although this invention is demonstrated further more concretely using an Example, the technical scope of this invention is not limited to these Examples.
[0065]
[Example 1]
Enhancement of low temperature stress tolerance by high temperature treatment of rice seedlings
Rice (Oryza sativa L., variety: Kirara 397) seeds are soaked in water at 27 ° C for 3 days, soaked in a vermiculite moistened in a petri dish with a diameter of 9 cm, 25 ° C and 80% relative humidity And germinated under dark conditions. Seven days later, the seedlings were exposed to 100% relative humidity, 42 ° C. for 1, 3, 9, and 24 hours, and then exposed to a low temperature of 5 ° C. for 7 days under dark conditions of 80% relative humidity. Control seedlings were exposed to 25 ° C. After low temperature treatment, rice seedlings were transferred to a growth chamber at 25 ° C. and grown for 7 days. The survival rate of the seedlings was evaluated based on appearance observation. At that time, sufficiently growing seedlings were regarded as survivors, and non-growing seedlings and dead seedlings were regarded as non-surviving bodies. All experiments were repeated at least twice.
[0066]
Control rice seedlings that had undergone only 7 days of low temperature treatment (5 ° C.) showed low temperature injury. Rice seedlings that had been subjected to high temperature treatment (42 ° C.) for 24 hours before moving to 5 ° C. did not cause such low-temperature damage. When the high temperature treated rice seedlings were placed at 25 ° C. for 4 days before being transferred to low temperatures, the seedlings no longer showed tolerance to low temperature stress.
[0067]
The effect of high temperature treatment for various times on low temperature stress tolerance of rice seedlings is shown in FIG. After high temperature treatment at 42 ° C. for 6 hours and low temperature treatment at 5 ° C. for 7 days, more than 50% of the seedlings survived. The low temperature stress tolerance was enhanced in proportion to the high temperature treatment time. Low temperature stress tolerance was remarkably enhanced by high temperature treatment at 42 ° C. for 6 hours or more. Particularly, high temperature treatment at 42 ° C. for 24 hours dramatically increased the survival rate after low temperature treatment.
[0068]
[Example 2]
For high temperature stress and low temperature stress APX , CAT , SOD Activity measurement
By measuring the activity of an active oxygen removing enzyme for high temperature treatment and subsequent low temperature treatment, an enzyme involved in enhancing low temperature stress tolerance was identified.
[0069]
Rice seedlings treated at 25 ° C for 24 hours (control), rice seedlings treated at a high temperature of 42 ° C (24 hours), and rice treated at a low temperature of 5 ° C for 7 days after a high temperature treatment of 42 ° C for 24 hours The seedlings and the leaves of rice seedlings treated at 25 ° C. for 24 hours and then subjected to low temperature treatment at 5 ° C. for 7 days were homogenized together with 50 mM phosphate buffer (pH 7.0) containing 1% Triton X-100. Each homogenate was centrifuged at 12,000 g for 20 minutes at 4 ° C., and the supernatant was immediately used for enzyme assay.
[0070]
First, ascorbate peroxidase (APX) activity was measured by the method described in Saruyama and Tanida (1995). The reaction mixture (1.0 ml) is NaN<sub>Three</sub>, 0.5 mM ascorbic acid, 1.54 mM H<sub>2</sub>O<sub>2</sub>And 50 mM potassium phosphate (pH 7.0) containing the above enzyme fraction. H<sub>2</sub>O<sub>2</sub>Ascorbic acid oxidation reaction was started by addition of and the absorbance at 290 nm indicating the ascorbic acid concentration was measured. As a result, a decrease in absorbance was measured. This decrease was measured as the relative activity of APX. Catalase (CAT) activity was measured according to the description of Tanida, 1996, the absorbance at 240 nm was measured, and the decrease was measured as the relative activity of CAT. Superoxide dismutase (SOD) activity was measured using an assay kit (SOD test, manufactured by Wako) based on the NBT method (Beyer and Fridovich, 1987).
[0071]
As a result of the above measurement, the seedlings that had been treated at a high temperature of 42 ° C. had significantly improved APX activity levels, and retained high activity even after being subjected to a low temperature treatment at 5 ° C. for 7 days after the high temperature treatment. (FIG. 2). The CAT activity of the seedlings treated with high temperature decreased to a lower level, and further decreased dramatically due to low temperature stress. (FIG. 2). There was no significant difference in the SOD activity between the high temperature treatment group and the low temperature treatment group (FIG. 2). Similarly, glutathione reductase activity and reduced ascorbic acid content were also examined, but no effect of high-temperature treatment was observed. Therefore, it was considered that the increase in APX activity contributed to the enhancement of low temperature stress tolerance of seedlings after high temperature treatment.
[0072]
[Example 3]
APX Gene isolation and expression analysis
The APX gene whose expression was induced by high-temperature treatment was isolated, and Northern blot analysis was performed on the transcription level after high-temperature treatment and subsequent low-temperature treatment of the gene.
(1) Isolation of APX gene
According to the method described in Example 1, the leaves of rice seedlings subjected to high temperature treatment at 42 ° C. for 24 hours were collected and ground in liquid nitrogen in a mortar. Thus, the powdered leaves were treated by a conventional method to extract total RNA, and cDNA was prepared using reverse transcriptase as a template. For cDNA production, cDNA Synthesis Kit (TaKaRa) was used.
[0073]
Using this cDNA as a template, a DNA fragment using the APX gene probe as a cDNA sequence of a rice APXa gene (Morita S, Kaminaka H, Yokoi H, Masumura T, Tanaka K. 1997, Plant Physiology 40, 113, 306 Amplification was performed by PCR using the following primers designed to amplify the entire ORF encoding APXa based on the accession number D45423).
Pri-1: 5'-ACCCGCAGCCATGGCTAAGAACTAC-3 '(SEQ ID NO: 3)
Pri-2: 5'-ACTAGAAACCTCTTAAGCATCAGCG-3 '(SEQ ID NO: 4)
The composition of the PCR reaction solution used is as follows.
cDNA 100ng
Pri-1 0.5μM
Pri-2 0.5μM
dNTPs 0.2mM
Taq DNA polymerase (GIBCO BRL) 1U
Taq DNA polymerase is MgCl at a final concentration of 1.5 mM.<sub>2</sub>What was dissolved in Taq DNA polymerase reaction buffer containing was used. The PCR reaction was performed for 30 cycles, with a cycle of 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 1 minute.
[0074]
The PCR amplified fragment thus obtained was electrophoresed on a low melting point agarose gel, and the amplified band was cut out from the gel and purified. The purified PCR amplification product was cloned into the pCR2.1 vector using TA cloning (Invitrogen). Each clone cloned into the pCR2.1 vector was transformed into Escherichia coli, and the plasmid was recovered by a conventional method, treated with restriction enzymes (BamHI and EcoRV), and screened for the size and orientation of the insert fragment.
[0075]
The resulting clone containing the approximately 770 bp cDNA fragment was subjected to a sequencing reaction using Thermo Sequenase Cycle Sequencing Kit (Amersham), and the nucleotide sequence was determined using a DNA sequencer (LIC-4200LS-2, Aloka). Made a decision. As a result, this cDNA sequence (SEQ ID NO: 1) is the DNA sequence of the rice APXa gene used for probe design (Morita S, Kaminaka H, Yokoi H, Masumura T, Tanaka K. 1997, Plant Physiology 40, 113, 306; It was found that the isolated APX gene encoded rice cytosol type ascorbate peroxidase APXa.
[0076]
(2) Confirmation of expression induction by high-temperature treatment by expression analysis of APXa gene
Using the clone containing the APXa gene obtained in (1) as a probe, Northern blot analysis was performed on APXa mRNA extracted from rice seedlings after high temperature treatment and subsequent low temperature treatment.
[0077]
25 ° C control rice seedlings prepared according to the method described in Example 1, 42 ° C high temperature treatment (1, 3, 6, 9, 12, 24 hours), 42 ° C high temperature treatment (24 ° C Shoots were collected from rice seedlings that had been transferred to 5 ° C. after 7 hours and subjected to low-temperature treatment for 7 days and rice seedlings that had been subjected to low-temperature treatment for 24 hours after being placed at 25 ° C. for 24 hours. Total RNA was extracted from 1 g of this shoot by the phenol / SDS method, and purified by ethanol precipitation and LiCl precipitation.
[0078]
Purified total RNA (20μg) was electrophoresed on 1.2% agarose gel, transferred to nylon membrane,<sup>32</sup>Hybridization with the above-mentioned APXa gene probe labeled with P was performed. The hybridization signal was visualized by image analysis of the autoradiogram with a bioimaging analyzer (manufactured by FUJIX, BAS 1000).
[0079]
The result is shown in FIG. As shown in the figure, it was shown that the APXa mRNA level of rice seedlings increased about 1.8 times by high temperature treatment for 1 hour. Similarly, APXa mRNA levels after high temperature treatment for 3, 6, 9, 12, 24 hours were also significantly higher compared to untreated controls. APXa mRNA levels after low-temperature treatment are lower in rice seedlings treated with high-temperature treatment than in rice seedlings not treated with high-temperature treatment, but still show higher values. It was.
Therefore, it was confirmed that the cloned APXa gene was induced by high temperature treatment.
[0080]
[Example 4]
APXa Analysis of high temperature inducible promoter of genes
Using rice genomic DNA as a template, the upstream region of the APXa gene was analyzed by the thermal asymmetric combined polymerase chain reaction (TAIL-PCR) method (Liu YG, Mitsukawa N, Oosumi T, Whittier RF. 1995, The Plant Journal 8, 457-463). Amplified. APXa-specific primers TR1 (5'-GTAGGAGATGGTGGGTATCT-3 ': SEQ ID NO: 5), TR2 (5'-TATCTCCTCCTTGATGGGGCT-3': SEQ ID NO: 6) and TR3 (5'-TCACGACGGGGTAGTTCTTA-3 ': SEQ ID NO: 7) Was designed based on the known cDNA sequence of rice APXa (Morita S, Kaminaka H, Yokoi H, Masumura T, Tanaka K. 1997, Plant Physiology 40, 113, 306). Furthermore, AD (GTNCGASWCANAWGTT: SEQ ID NO: 8) as a random degenerate primer was synthesized according to the method described in Liu Y-G, Mitsukawa N, Oosumi T, Whittier RF. 1995, The Plant Journal 8, 457-463. Using the three APXa-specific primers (TR1, TR2, TR3) as the sense direction primer and the AD primer as the antisense direction primer, three-step PCR reaction is performed continuously, and the region containing the APXa gene promoter is detected. Amplified. The composition of the first PCR reaction solution (20 μl) is as follows.
Rice genomic DNA 20ng
TR1 primer 4pmol
AD primer 100pmol
dNTPs 200μM
Taq DNA polymerase (GIBCO BRL) 0.8U
[0081]
Taq DNA polymerase is MgCl at a final concentration of 1.5 mM.<sub>2</sub>What was dissolved in Taq DNA polymerase reaction buffer containing was used. The PCR reaction was performed continuously under the following temperature conditions (1) to (5).
(1) 94 ° C for 1 minute, then 95 ° C for 1 minute
(2) 94 cycles for 1 minute, 65 ° C for 1 minute, 72 ° C for 3 minutes, 5 cycles
(3) After 94 ° C for 1 minute, cool at 25-30 ° C for 3 minutes, increase to 72 ° C over 3 minutes, then 72 ° C for 3 minutes, 1 cycle
(4) 94 ° C 30 seconds, 68 ° C 1 minute, 72 ° C 3 minutes, 94 ° C 30 seconds, 68 ° C 1 minute, 72 ° C 3 minutes, 94 ° C 30 seconds, 44 ° C 1 minute, 72 ° C 3 minutes As 15 cycles
(5) 72 ° C 5 minutes
[0082]
1 μl of the amplification product obtained by the first PCR was diluted with 50 μl of sterilized water, and 1 μl thereof was added to the following reaction solution (19 μl).
TR2 primer 4pmol
AD primer 80pmol
dNTPs 200μM
Taq DNA polymerase (GIBCO BRL) 0.6U
[0083]
Taq DNA polymerase is MgCl at a final concentration of 1.5 mM.<sub>2</sub>What was dissolved in Taq DNA polymerase reaction buffer containing was used. The PCR reaction was performed continuously under the following temperature conditions (6) and (7).
(6) 94 ° C 30 seconds, 64 ° C 1 minute, 72 ° C 3 minutes, 94 ° C 30 seconds, 64 ° C 1 minute, 72 ° C 3 minutes, 94 ° C 30 seconds, 44 ° C 1 minute, 72 ° C 3 minutes As 12 cycles
(7) 72 ℃ 5 minutes
[0084]
1 μl of the amplification product obtained by the second PCR was diluted in 10 μl of sterilized water, and 1 μl thereof was added to the following reaction solution (99 μl).
TR3 primer 30pmol
AD primer 400pmol
dNTPs 200μM
Taq DNA polymerase (GIBCO BRL) 3U
[0085]
Taq DNA polymerase is MgCl at a final concentration of 1.5 mM.<sub>2</sub>What was dissolved in Taq DNA polymerase reaction buffer containing was used. The PCR reaction was performed continuously under the following temperature conditions (8) and (9).
(8) 20 cycles with 94 ° C for 1 minute, 44 ° C for 1 minute, 72 ° C for 3 minutes as one cycle
(9) 72 ° C 5 minutes
The amplification product obtained by the third step reaction using TR3 and AD was electrophoresed on a 1.2% agarose gel, a 400 bp fragment was extracted from the gel, and this was subjected to TA cloning (manufactured by Invitrogen). And cloned into the pCRII vector. The insert DNA was sequenced using a DNA sequencer (LIC-4200LS-2, manufactured by Aloka). The sequence of the promoter region of this rice APXa gene is shown in SEQ ID NO: 9. Furthermore, the DNA base sequence was compared with the promoter region of Arabidopsis thaliana axp1 gene.
[0086]
The promoter region of the APXa gene of the present invention had very low homology with the Arabidopsis apx1 gene except for one region located 81 bp upstream of the TATA box. One region contains putative heat shock elements (HSE) nGAAn and nTTCn (FIG. 4A). HSE has a heat shock factor (HSF) binding minimal motif 5′-nGAAnnTTCn-3 ′, which is identical to the HSE of the Arabidopsis apx1 promoter (FIG. 4B). A CCAAT motif was also found at position -215.
Therefore, it was found that the APXa gene of the present invention has a putative high temperature inducible promoter. This is a result of supporting that the APXa gene is induced at high temperature to enhance low temperature stress tolerance of rice seedlings.
[0087]
[Example 5]
APXa Production of transgenic rice by gene transfer
(1) Construction of overexpression vector incorporating APXa gene
Using the APXa gene DNA cloned in Experimental Example 3 as a template, primer Pri-3 (5'-ACCCGGATCCATGGCTAAGAACTAC-3 ': SEQ ID NO: 12) in which a restriction enzyme BamHI recognition sequence was inserted immediately before the translation start point and the translation end point Immediately after, DNA amplification was performed by PCR using the primer Pri-4 (5′-ACTAGAGAGCTCTTAAGCATCAGCG-3 ′: SEQ ID NO: 13) in which a recognition sequence for the restriction enzyme SacI was inserted, and restriction enzymes BamHI and SacI were recognized at both ends of the coding region. The APXa DNA fragment to which the sequence was assigned was obtained. The composition of the PCR reaction solution (20 μl) used is as follows.
DNA 100ng
Pri-3 0.5μM
Pri-4 0.5μM
dNTPs 0.2mM
Taq DNA polymerase (GIBCO BRL) 1U
Taq DNA polymerase is MgCl at a final concentration of 1.5 mM.<sub>2</sub>What was dissolved in Taq DNA polymerase reaction buffer containing was used. The PCR reaction was performed for 30 cycles, with one cycle consisting of 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 1 minute.
[0088]
The PCR amplified fragment thus obtained was cleaved with BamHI and SacI, and electrophoresed on a 1% agarose gel, and the DNA fragment of about 760 pb was extracted from the gel and purified. The vector to be incorporated was the pMLH7133 vector (Mitsuhara et al., Plant Cell Rhysiology 37: 49-59, 1996) developed by the National Institute of Agrobiological Sciences (currently the National Institute for Agrobiological Sciences), Ministry of Agriculture, Forestry and Fisheries. This vector was digested with restriction enzymes BamHI and SacI, and a DNA fragment containing the APXa gene prepared as described above was inserted to construct a recombinant vector in which the GUS of the insert DNA of pMLH7133 was replaced with APXa. This recombinant vector was called pMLH7133-APXa, and its structure is shown in FIG.
[0089]
This recombinant vector pMLH7133-APXa was introduced into the rice cultivar “Yukihikari” by the Agrobacterium method. This “Yukihikari” is a rice cultivar in Hokkaido that excels in low-temperature stress tolerance. Specifically, pMLH7133-APXa was introduced into Agrobacterium (Agrobacterium tumefaciens LBA4404) by electroporation, and infected with scutellum-derived callus of rice `` Yukihikari '' by co-cultivation, thereby pMLH7133-APXa Was introduced. Subsequently, the callus obtained by this transformation was regenerated into a plant body by culturing in a regeneration medium containing hygromycin (50 mg / l).
[0090]
[Example 6]
APXa Evaluation of tolerance to low temperature stress in transgenic rice
The T2 generation obtained by self-propagating the transformed plant obtained in Example 5 twice is subjected to low-temperature treatment at 5 ° C. for 10 days and then returned to 25 ° C. as described in Example 1. The survival rate of rice was examined. For comparison, the original cultivar “Yukihikari” was also subjected to low-temperature treatment under the same conditions.
[0091]
As a result, the survival rate of the original cultivar “Yukihikari” was 0%, whereas rice APX991217-5-2, which has a transgene homozygously, showed a survival rate of 48% (FIG. 6). That is, the rice introduced with the cytosolic ascorbate peroxidase APXa gene of the present invention is prominent that 48% can survive even under severe low temperature conditions such that 100% of "Yukihikari" with excellent resistance to low temperature stress dies. Enhanced cold stress tolerance was shown.
[0092]
【The invention's effect】
INDUSTRIAL APPLICABILITY The present invention provides a transgenic plant with enhanced resistance to active oxygen generated under various environmental stress conditions (high and low temperature, dryness, strong light, salt, air pollutant gas, pathogenic bacteria, ultraviolet rays, etc.). . The method of introducing a cytosolic ascorbate peroxidase gene according to the present invention into a plant has an activity generated under various environmental stress conditions (high temperature, dryness, strong light, salt, air pollutant gas, pathogen, ultraviolet ray, etc.). It is useful for the development of plants that are highly resistant to oxygen.
[0093]
[Sequence Listing]

[0094]
[Sequence Listing Free Text]
Sequence number 3: Synthetic primer
SEQ ID NO: 4: Synthetic primer
SEQ ID NO: 5: synthetic primer
SEQ ID NO: 6: synthetic primer
SEQ ID NO: 7: synthetic primer
Sequence number 8: n of a synthetic primer, the 3rd base, and the 11th base is either a, t, g, or c.
SEQ ID NO: 12: synthetic primer
SEQ ID NO: 13: synthetic primer
[Brief description of the drawings]
FIG. 1 shows that when rice seedlings are treated at 42 ° C. for 0, 3, 6, 9, and 24 hours at a high temperature, the resistance to low temperature stress is remarkably enhanced in the treatment section of 6 hours or more. FIG. ** indicates significance at the 1% level.
FIG. 2 shows seedlings treated at 25 ° C. (control), seedlings treated at a high temperature of 42 ° C. (24 hours), seedlings treated at a low temperature of 5 ° C. for 7 days after a high temperature treatment of 42 ° C. (24 hours), and 25 ° C. It is the figure which showed each activity of catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX) in the seedling which was low-temperature-treated at 5 ° C. for 7 days after the treatment. * Means significant at the 5% level, and ** means significant at the 1% level.
[Fig. 3] Fig. 3 shows seedlings treated at 25 ° C (control), seedlings treated at 42 ° C high temperature (1, 3, 6, 9, 12, 24 hours), and at 5 ° C after high temperature treatment at 42 ° C (24 hours). The figure shows the expression level of APXa gene mRNA in seedlings treated for 7 days at low temperature and seedlings treated at 25 ° C for 7 days at 5 ° C, using the autoradiogram of Northern blot analysis and the results of quantitative analysis. It is. For the autoradiogram, the amount of rRNA present was shown as a comparative control. * Indicates significance at 5% level.
FIG. 4 is a view showing that a minimal heat shock element (HSE) motif nGAAnnTTCn for binding heat shock factor (HSF) is present in the promoter region of the APXa gene. A shows the promoter structure of the APXa gene. ATG indicates a translation initiation codon, and the 5 ′ end of the APXa gene cDNA is indicated by an arrow and +1. The numerical value in the figure is the number of bases from the transcription start point. B, Comparison of HSE on rice APXa promoter (upper) and HSE on Arabidopsis apx1 promoter (lower). The HSE motif nGAAn is shown in capital letters. The minimal motif nGAAnnTTCn for HSF binding is indicated by an inward arrow. There are motifs with one base substitution on both sides.
FIG. 5 shows the structure of a recombinant vector pMLH7133-APXa introduced into rice.
FIG. 6 is a graph showing the difference in survival rate after low-temperature treatment between the original cultivar Yukihikari and transformed rice (APX991217-5-2).

Claims (8)

A plant resistant to low temperature stress in Gramineae, wherein a recombinant vector comprising a gene encoding the following protein (a) or (b) and an overexpression promoter is introduced into the cytoplasm .
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having ascorbate peroxidase activity A cold stress-resistant plant of the family Gramineae, into which a recombinant vector comprising a gene comprising the following DNA (c) or (d) and an overexpression promoter is introduced into the cytoplasm .
(c) DNA consisting of the base sequence shown in SEQ ID NO: 1
(d) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 under a stringent condition and encodes a protein having ascorbate peroxidase activity Plants, plant, plant organ, plant tissue, or low temperature stress tolerant plants according to claim 1 or 2 is cultured plant cells. Gramineous plant is rice, cold stress tolerant plants according to any one of claims 1 to 3. A method for enhancing resistance to low-temperature stress in gramineous plants, which comprises introducing a recombinant vector comprising a gene encoding the protein (a) or (b) below and an overexpression promoter into the cytoplasm of the gramineous plant .
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having ascorbate peroxidase activity A method for enhancing low temperature stress tolerance of gramineous plants, which comprises introducing a recombinant vector comprising the following gene containing DNA of (c) or (d) and an overexpression promoter into the cytoplasm of gramineous plants.
(c) DNA consisting of the base sequence shown in SEQ ID NO: 1
(d) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 under a stringent condition and encodes a protein having ascorbate peroxidase activity The method according to claim 5 or 6 , wherein the plant is a plant body, a plant organ, a plant tissue, or a plant cultured cell. The method according to any one of claims 5 to 7 , wherein the gramineous plant is rice.

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