JP2004105136A - Gramineous plant having improved cold resistance prepared by increasing expression of active oxygen scavenging enzyme gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare a gramineous plant having increased cold resistance. <P>SOLUTION: The gramineous plant having increased cold resistance is prepared by increasing the expression of an active oxygen scavenging enzyme gene of a gramineous plant. <P>COPYRIGHT: (C)2004,JPO

Description

【図面の簡単な説明】
【図１】図１は、ベクターの構築の模式図である。図１Ａは、ｐＨＳＧ／３５ＳＩ／ｃｈｌ／ｃｌＡＰＸ／Ｎｏｓ（ｐＡＰＸ）の構築を示す。図１Ｂは、ｐＨＳＧ／３５ＳＩ／ｃｈｌ／ｃｌＡＰＸ／ｓｔｕｃｋ／Ｎｏｓ（ｐＡＰＸｓｔ）の構築を示す。
【図２】図２は、ＡＰＸ遺伝子導入イネ植物体のサザン分析の結果を示す図（写真）である。コントロールは、　非形質転換体、２７，７５，７６，７７，７８，８９は、形質転換体。
【図３】図３は、ＡＰＸ遺伝子導入イネ植物体のノザン分析の結果を示す図（写真）である。図３Ａは、ｐＡＰＸ導入個体における結果である。コントロールは非形質転換体、３，８，１３，１７は形質転換体。図３Ｂは、ｐＡＰＸｓｔ導入個体における結果である。コントロールは非形質転換体、２７，７５，７６，７７，７８，８９は形質転換体。
【図４】図４は、ＡＰＸ遺伝子導入イネ植物体のウェスタン分析の結果を示す図（写真）である。ペレットはペレット画分、上清は上清画分。コントロールは非形質転換体、７５，７６，７７，７８は形質転換体。
【図５】図５は、ＡＰＸ遺伝子導入イネの翻訳レベルの発現確認の結果を示す図（写真）である。図５Ａは活性染色とウェスタン分析の比較を示す図である。ＳＡＳは非形質転換体、ＡＰＸｓｔ８，ＡＰＸｓｔ２１，ＡＰＸｓｔ２７は形質転換体。図５Ｂは形質転換体植物の活性染色分析の結果を示す図である。コントロールは非形質転換体、７６，８９は形質転換体。
【図６】図６は、ＡＰＸ遺伝子導入イネ植物体のＡＰＸ活性測定の結果を示す図である。
【図７】図７は、ＡＰＸ遺伝子導入イネ植物体の後代（Ｔ１）のウェスタン分析の結果を示す図（写真）である。コントロールは非形質転換体、Ｍは分子量マーカー、ＡＰＸｓｔ８９　　１〜５は形質転換体。
【図８】図８は、ＡＰＸ遺伝子導入植物体のパラコート耐性（Ｆｖ／Ｆｍ値の変化）の検定結果を示す図である。図８Ａは当代（Ｔ_０）植物の比較（８９は遺伝子発現あり、１０３は遺伝子発現なし）。図８Ｂは分離後代の比較（８９−３３は遺伝子発現あり、８９−３９は遺伝子発現なし）。
【図９】図９は、ＡＰＸ遺伝子導入イネ植物体のパラコート耐性の検定結果を示す図（写真）である。分離後代（Ｔ１）葉切片を用いたパラコート（メチルビオロゲン）耐性検定。８９−３３は遺伝子発現あり、８９−３９は遺伝子発現なし。メチルビオロゲン処理は０，　５，　１０　ｍＭの３条件。
【図１０】図１０は、ＡＰＸ遺伝子導入イネ植物体の低温脱色耐性の検定結果を示す図（写真）である。１２００μｍｏｌＥの光を照射しながら、葉切片を４℃で３日間低温処理した。ＡＰＸｓｔ１０３およびＡＰＸｓｔ１０６は非形質転換体、ＡＰＸｓｔ７６およびＡＰＸｓｔ８９は形質転換体。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a grass plant with enhanced cold resistance, which is produced by enhancing the expression of an active oxygen scavenging enzyme gene. More specifically, the present invention relates to a Poaceae plant in which low-temperature decolorization resistance is enhanced by introducing a gene for ascorbate peroxidase (hereinafter sometimes abbreviated as APX) derived from Chlamydomonas. According to the present invention, active oxygen in chloroplasts, which is a cause of low-temperature decolorization, is efficiently removed, so that damage due to delayed-type cold damage during cold damage can be reduced, and direct sowing cultivation can be stabilized.
[0002]
[Prior art]
Rice (Oryza @ sativa) is a plant native to the tropics and is vulnerable to low temperatures, and rice cultivation in cold regions has long been facing the problem of cold injury. In particular, the low temperature damage encountered every few years has affected each stage of rice growth. For example, the cold damage in 1993 was due to a combination of reduced yield due to sterility due to low temperature during the ear birth season, poor ripening due to long-term exposure to low temperature from the early growth stage, and poor ripening due to blast disease. Was significantly reduced. Up until now, measures have been taken to overcome cold damage, such as breeding cold-tolerant varieties at each breeding site, but it has been clarified that these measures alone are not sufficient. In order to minimize the damage caused by the cold damage, it is required to strengthen not only the low-temperature tolerance in the panicle stage but also the low-temperature tolerance in the seedling stage.
[0003]
On the other hand, in the field of rice cultivation, the production orientation of high-quality rice has been strengthened, and the reduction of production costs has been strongly demanded. Although direct sowing has the advantages of saving facility costs and labor costs and making it easy to increase the size, there is a problem that initial growth tends to be unstable. In particular, the low temperature encountered during the seedling stage causes great damage such as leaf withering due to leaf blade wilting and yellowing and whitening of leaves. Even in direct sowing, enhancement of low temperature tolerance in the seedling stage is required.
[0004]
Low-temperature injury at the seedling stage in rice is broadly classified into two types: low-temperature withering, which is considered to be due to inhibition of water absorption at low temperatures, and low-temperature decolorization, in which leaves and stems are yellowed by low-temperature exposure under light irradiation.
Several attempts have been made to enhance cold tolerance of plants by introducing a gene related to cold tolerance. For example, an example in which proline, an osmotic pressure regulating substance, is accumulated in Arabidopsis by overexpression of a key enzyme gene or suppression of expression of a degradation system gene to enhance low temperature tolerance (for example, see Non-Patent Document 1), an environmental stress responsive gene There is also a research example in which the overexpression of the transcriptional regulatory factor DREB involved in the regulation of the expression of Arabidopsis enhances the cold stress resistance of Arabidopsis (for example, see Non-Patent Document 2). These are techniques suitable for reducing wilt damage caused by cold stress of plants.
[0005]
On the other hand, low-temperature decolorization in which leaves and stems turn yellow due to low-temperature exposure under light irradiation is thought to be due to peroxidation of biological membranes caused by active oxygen generated near photosystems I and II in chloroplasts. Have been. In chloroplasts, one of the active oxygen molecular species O₂ ^{− ・}Has been generated. O₂ ^{− ・}Spontaneously disappears in a very short period of time, but may also peroxidize biopolymers during that time. Originally, a mechanism that protects against such reactive oxygen-induced damage exists in chloroplasts of plants. The main function of this pathway is Cu / Zn-superoxide dismutase (hereinafter referred to as SOD). May be abbreviated) and elimination of active oxygen by APX. In the former, O generated near the thylakoid membrane₂ ^{− ・}Disproportionate and H₂O₂Which catalyzes the reaction₂O₂Catalyzes the reaction of decomposing water and oxygen. When low-temperature stress is applied to a plant under light irradiation, the supply of carbon dioxide gas is not sufficient because the pores are closed, and the chloroplast is in a state of excessive reducing power. In this state, it is considered that active oxygen is generated in a large amount as the capacity of the active oxygen scavenging enzyme exceeds the scavenging ability, and as a result, peroxidation of the membrane and protein molecules progresses, and chloroplasts are destroyed. Bowler, Gupta, and others have shown that overexpression of a plant-derived SOD gene can reduce cold discoloration damage (see Non-Patent Documents 3 and 4). However, the introduction of the SOD gene alone poses a problem with the generated hydrogen peroxide. There has been no example showing that the enzyme gene for eliminating hydrogen peroxide was introduced into plants to enhance the resistance of plants to low-temperature decolorization.
[0006]
Furthermore, in rice, the cold resistance at the seedling stage as well as the cold resistance at the panicle stage is practically important.²⁺-A research example of Saijo et al. (See Non-Patent Document 5) due to overexpression of a dependent protein kinase (dependent protein kinase) gene, and a research example of Sakamoto et al. (See Non-Patent Document 6) due to overexpression of a betaine synthesis gene. )). In the study of Saijo et al., The target protein of the over-expressed protein kinase gene product is not always clear, and such signal transduction is based on the fact that signals generated by receiving multiple stresses pass through the same signal transduction factor. It may not work well depending on the low-temperature conditions that rice seedlings suffer. In addition, there has been no report that there is an active oxygen scavenging enzyme gene in the chloroplast downstream of the signal of such a protein kinase gene. There is a high possibility that it will not be connected.
In addition, in the study of Sakamoto et al., In order to highly express choline oxidase, electrons are transferred to molecular oxygen, and as a result, active oxygen is produced in an equimolar amount with choline. This is an unfavorable trait especially for low-temperature light damage in the seedling stage, and it is considered that enhancement of the active oxygen scavenging system is much more effective.
However, there has been no example in which the resistance to low-temperature decolorization of rice and gramineous plants has been enhanced by introducing an active oxygen scavenging enzyme gene.
[0007]
[Non-patent document 1]
T. {Nanjo et al. 461 (3), 205-210, (1999) FEBS Lett.
[Non-patent document 2]
M. {Kasuga et al. , {17, {287-291,} (1999)} Nature {Biotechnol.
[Non-Patent Document 3]
C. {Bowler et al. , 10 (7), ２３1723-1732, (1991) EMBO Journal
[Non-patent document 4]
A. S. {Gupta et al. , {90, {1629-1633, (1993)} Proc. {Natl. {Acad. {Sci. USA
[Non-Patent Document 5]
Y. {Saijo et al. , {42 (11), {1228-1233}, {(2001)} Plant {Cell} Physiol.
[Non-Patent Document 6]
A. {Sakamoto et al. , {38 (6), {1011-1010, {(1998)} Plant {Mol. {Biol.
[0008]
[Problems to be solved by the invention]
An object of the present invention is to produce a Poaceae plant with enhanced cold resistance.
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by enhancing the expression of an active oxygen-scavenging enzyme gene in a Gramineous plant, to enhance cold resistance, By overexpressing the APX gene that eliminates hydrogen peroxide, one of the reactive oxygen species in the plant, we succeeded in enhancing the resistance to cold decolorization of grasses. APX in the chloroplasts of higher plants is an unstable enzyme, and it has been shown that APX is rapidly deactivated when a large amount of active oxygen is generated and the substrate ascorbic acid is deficient for its elimination. The present inventors have newly isolated a Chlamydomonas-derived gene that confers salt tolerance to Escherichia coli, one of which is chloroplast APX, and the stability of the enzyme against inactivation during ascorbic acid deficiency is higher than that of higher plants. (T. Takeda et al., 376 (1), 82-90, (2000) Arch. Biochem. Biophys.). Furthermore, the present inventors enhance the low-temperature photobleaching resistance of rice by introducing a stable APX gene derived from Chlamydomonas into a gramineous plant and expressing it in chloroplasts, thereby enhancing the low-temperature bleaching resistance of the rice plant. Succeeded. In addition, it has been shown that, in higher plants, a thylakoid-membrane-localized sequence is added to the stromal-type APX by alternative splicing to generate a thylakoid-membrane-type APX. It is considered that the film type APX is more effective. The present inventors have further succeeded in enhancing the low-temperature decolorization resistance of gramineous plants by adding and expressing a thylakoid-membrane localization sequence to the stable APX gene of Chlamydomonas. The present invention has been achieved in this manner.
[0009]
That is, the gist of the present invention is as follows.
(1) Poaceae plants with enhanced cold resistance produced by enhancing the expression of active oxygen-scavenging enzyme genes.
(2) The plant according to (1), wherein the Poaceae plant is rice.
(3) The plant according to (1), wherein the active oxygen-scavenging enzyme gene is an ascorbate peroxidase gene.
(4) The plant according to (3), wherein the ascorbate peroxidase gene is derived from Chlamydomonas.
(5) The plant according to (3), wherein the ascorbate peroxidase gene has any one of the following nucleotide sequences.
(A) a base sequence consisting of base numbers 32 to 991 in the base sequence described in SEQ ID NO: 1;
(B) a base sequence in which one to a plurality of bases are deleted, added or substituted in the base sequence consisting of base numbers 32 to 991 in the base sequence set forth in SEQ ID NO: 1, wherein ascorbate peroxidase is A nucleotide sequence encoding a protein having activity; or
(C) a base sequence that hybridizes under stringent conditions to a base sequence consisting of base numbers 32 to 991 or a complementary sequence thereof or a part thereof among the base sequences set forth in SEQ ID NO: 1, ascorbic acid A nucleotide sequence encoding a protein having peroxidase activity.
(6) The method according to any of (3) to (5), wherein a chloroplast translocation sequence is added to the ascorbate peroxidase gene, and the gene translation product is localized in the chloroplast. Plant.
(7) any of (3) to (6), wherein a thylakoid translocation sequence is added to the ascorbate peroxidase gene, and the gene translation product is localized near the thylakoid membrane of the chloroplast. The plant according to the present invention.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
<1> Plant of the present invention
BEST MODE FOR CARRYING OUT THE INVENTION The plant of the present invention is a Poaceae plant having enhanced cold resistance, which is produced by enhancing the expression of an active oxygen scavenging enzyme gene.
The “active enzyme elimination enzyme gene” is not particularly limited as long as it encodes an enzyme that eliminates an active enzyme species, and examples thereof include an ascorbate peroxidase gene, a catalase gene, and a superoxide dismutase gene. Acid peroxidase genes are preferred. The “active enzyme elimination enzyme gene” may be used singly or in combination of two or more. "Enhanced expression" refers to the copy number of the active oxygen-scavenging enzyme gene originally present in the target plant by introducing and expressing the active oxygen-scavenging enzyme gene derived from the same or different plant as the target plant in the target plant. Can also be carried out by introducing and expressing a more stable active oxygen scavenging enzyme gene derived from the same or different plant as the target plant into the target plant. It can also be carried out by introducing and expressing in the target plant an active oxygen scavenging enzyme gene having a higher specific activity derived from the same or different plant as the target plant. In addition, it can also be carried out by introducing and expressing in the target plant an active oxygen scavenging enzyme gene which is not originally present in the target plant. Furthermore, it can also be carried out by enhancing the expression of an active oxygen scavenging enzyme gene originally present in the target plant. In addition, "enhancement of expression" may be performed using one of these methods, or a plurality of methods may be combined.
[0011]
Whether or not the “Cold Tolerance” of the transformed grass plant is enhanced can be confirmed by assaying by the method described in detail in the following Examples. Briefly, leaf sections cut from transformants and non-transformants can be clarified by floating in Yoshida hydroponic solution, incubating at 4 ° C under high light for 3 days, and comparing the degree of decolorization of the leaves. it can. If the degree of decolorization of the transformant is suppressed as compared with the non-transformant, it can be said that the "cooling resistance" is enhanced.
The gramineous plant used in the present invention is not particularly limited as long as it can enhance the expression of the active oxygen-scavenging enzyme gene, and rice is preferable.
[0012]
A preferred embodiment of the present invention is a grass plant with enhanced cold resistance, which is produced by enhancing the expression of the APX gene. The APX gene is not particularly limited as long as its expression can be enhanced in a target plant, but a gene of an APX enzyme that is stable even under ascorbic acid deficiency is particularly preferable. Such a gene is not particularly limited as long as it is stable under ascorbic acid deficiency. For example, a gene derived from Chlamydomonas can be preferably exemplified. As the Chlamydomonas-derived APX gene, for example, a chloroplast-type gene (Genbank Accession Number: AB009084) of Chlamydomonas (Sp. W80) is particularly preferably used. This gene can be obtained, for example, by preparing appropriate primers based on the sequence and using the primers by PCR using plant cDNA as a template.
[0013]
Further, in a normal gene, naturally, mutations such as deletion, addition or substitution of one or more bases at one or more positions are present depending on differences in species, genera, individuals, and the like. Genes that can be used in the present invention also include genes containing such mutations as long as the ascorbate peroxidase activity of the encoded protein is not impaired. That is, another preferred embodiment of the present invention provides a nucleotide sequence consisting of nucleotide numbers 32 to 991 in the nucleotide sequence of SEQ ID NO: 1 as long as the ascorbate peroxidase activity of the encoded protein is not impaired. A plant having enhanced cold resistance by enhancing the expression of a gene having a nucleotide sequence in which a plurality of bases have been deleted, added or substituted from the plant. Here, the “plurality” specifically means 2 to 480, preferably 2 to 384, and more preferably 2 to 192.
[0014]
Such a deleted, added or substituted nucleotide sequence can be found in Chlamydomonas sp. Appropriate primers are prepared based on the sequence of the chloroplast APX gene of W80, and the primers can be used to obtain cDNAs of other organisms as a template by PCR or the like. Obtainable. It can also be obtained by other conventionally known mutation treatments.
[0015]
By expressing a nucleotide sequence having the above mutation in a target plant and examining the ascorbate peroxidase activity of the plant, a nucleotide sequence that can be used in the present invention can be obtained. Ascorbate peroxidase activity can be confirmed according to the method described in detail in the following examples. In addition, from a DNA having a mutation or a cell holding the same, for example, a probe having a base sequence consisting of at least base numbers 32 to 991 of the base sequence of SEQ ID NO: 1 or its complementary sequence, or a probe having a part thereof and a string The DNA used in the present invention can also be obtained by isolating a DNA that hybridizes under gentle conditions and encodes a protein having ascorbate peroxidase activity. Here, the “part” is a length effective for use as a probe. Further, “stringent conditions” refer to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify these conditions clearly, for example, DNAs having high homology, for example, at least about 50% or more, preferably at least base numbers 32 to 991 of the base sequence shown in SEQ ID NO: 1, preferably DNAs having a homology of 60% or more, more preferably 80% or more hybridize with each other, and DNAs with lower homology do not hybridize with each other, or at 65 ° C., 0.1 × SSC, 0.1% Conditions for hybridization under salt conditions corresponding to SDS are mentioned. Note that the homology is calculated by BLAST.
[0016]
Some of the genes that hybridize under the conditions described above include those in which a stop codon has been generated in the middle and those that have lost activity due to mutation of the active center, but these are introduced into the target plant, Ascorbate peroxidase activity can be easily removed by measuring in the manner detailed in the examples below.
[0017]
<2> Vector used in the present invention
Examples of a method for introducing the APX gene into a plant of the grass family include a method of constructing a vector containing the APX gene and transforming the grass with the vector.
A vector that can be used in the present invention is a vector in which a promoter and a terminator capable of expressing the APX gene in a rice plant are incorporated in a plasmid vector, or a vector in which an intron is incorporated into a plasmid vector, if necessary.
[0018]
In a preferred embodiment of the present invention, the APX gene is introduced into a gramineous plant and the chloroplast is expressed in the chloroplast, thereby enhancing the low-temperature light damage resistance of rice. It is a Poaceae plant. In addition, another preferred embodiment of the present invention provides a rice plant having enhanced cold resistance, preferably low temperature bleaching resistance, characterized in that a thylakoid membrane localization sequence is added to the APX gene and the gene is introduced and expressed in a grass plant. Family plant.
[0019]
By linking and expressing the APX gene with an appropriate chloroplast translocation sequence, the gene expression product can be expressed in the chloroplast of the target plant. Alternatively, a gene originally having a chloroplast translocation sequence may be used. Alternatively, even when a gene originally having a chloroplast translocation sequence is used, it may be used by being linked to a more efficient translocation sequence. When an APX gene derived from Chlamydomonas is used, the APX gene derived from Chlamydomonas is originally a gene having a chloroplast translocation sequence at the 5 'end, so that the gene product can be expressed as it is in a chloroplast of a grass plant. In particular, the chloroplast translocation sequence of the Chlamydomonas APX gene is replaced with a chloroplast translocation sequence of a glutamine synthase gene (Plant Molecular Biology, {13, {611} (1989)) of a Gramineae plant. Thus, the APX gene product can be localized in the chloroplast with high efficiency.
[0020]
Furthermore, the gene product can be localized in the vicinity of the chloroplast thylakoid membrane of the transformed plant by linking and expressing the thylakoid membrane localization sequence to the APX gene having the chloroplast translocation sequence. Alternatively, a gene originally having a thylakoid membrane proximal translocation sequence may be used. Alternatively, even when a gene that originally has a thylakoid-membrane translocation sequence is used, it may be used by being linked to a more efficient translocation sequence. When a stromal APX gene derived from Chlamydomonas is used, a plasmid obtained by adding a thylakoid membrane-proximal translocation sequence, which is preferably located on the c-terminal side of a thylakoid membrane-type APX gene product of tobacco or spinach, to a Chlamydomonas-derived APX gene is added to a plant belonging to the family Gramineae The gene product can be localized near the chloroplast thylakoid membrane of the transformed plant by introducing the gene into the body and expressing it.
[0021]
As a promoter to be used, for example, a promoter derived from a cauliflower mosaic virus such as CaMV35S (pBI221: EMBO. J., 6, 3901-3907） (1987)), rbcS (ribulose 1,5-bisphosphophosphate carboxylase), Cab (chlorophyllｌa /) Promoters that have been confirmed to be expressed in plants, such as b \ binding \ protein (Science, {244, {174} (1989)). Examples of the terminator include a terminator derived from a cauliflower mosaic virus, a terminator derived from a NOS (nopaline synthase) gene, and the like. In addition, a vector having an intron between the promoter and the structural gene can also be used as a high expression vector. As the intron, the first intron of castor Cat (catalase gene) (A. Tanaka et al., Nucleic Acid Research, 18, 6767). -6770, (1990)).
[0022]
<3> Gramineous plant of the present invention and method for producing the same
Hereinafter, a method for obtaining the plant of the present invention will be exemplified.
The plant of the present invention is a Poaceae plant having enhanced cold resistance, which is produced by enhancing the expression of an active oxygen scavenging enzyme gene, preferably an APX gene.
The plant of the present invention can be produced, for example, by constructing a vector containing the APX gene, transforming a grass plant with the vector, and enhancing the expression of the APX gene.
Further, in the present invention, it is preferable to use a selection marker gene such as a hygromycin phosphotransferase gene. As the selectable marker gene, one having the same vector as the APX gene may be used, or a vector having the selectable marker gene and a vector having the APX gene may be used in combination. As these vectors, vectors used for transforming gramineous plants can be used.
[0023]
As a method of transformation, for example, a protoplast derived from a gramineous plant is suspended in a liquid medium, the vector is introduced by applying an electric pulse, and then cultured in a medium containing rice cultured cells to form colonies. Plants can be regenerated from the colonies (K. Shimamoto et al., 337, 274-276, (1989) Science).
Specifically, gene transfer into rice can be performed, for example, as follows. The callus is induced by incubating Sasanishiki seeds on a medium containing 2,4-D at 2 μg / ml. Suspension cultured cells are produced from the obtained calli, and the suspension is treated with an enzyme solution containing 4% cellulase and 0.5% pectylase to obtain protoplasts. The protoplasts were suspended in a low-electrolyte buffer for electroporation containing 60 μg / ml of the vector DNA containing the hygromycin resistance gene vector and the vector DNA containing the APX gene, respectively, and placed in a Kartel cuvette (for a distance between electrodes of 4 mm). Gene transfer by electroporation is performed under the electrical conditions of cm, an electric capacity of 1000 μF, and τ = 30 msec. The protoplasts subjected to the gene transfer treatment were further incubated on ice for 30 minutes, then embedded in an R2P medium containing 0.8% of low-melting-point agarose, and agarose pieces were floated on an R2P liquid medium and cultured in an agarose bead method. Perform shaking culture in a culture room at ℃. During the cultivation, rice culture cells (Oc cells) are put into the R2P liquid medium for protection culture. On the third day after the start of the culture, the cells for protection culture are removed, and on the seventh day, 30 μg / ml of hygromycin is added. Transfer the cells together with the agar pieces to a liquid medium containing them and start selection. Seven days after the start of the selection, the agar pieces are transferred to an R2SA selection agar medium containing 30 μg / ml of hygromycin, and the culture is continued, whereby hygromycin-resistant calli can be obtained.
[0024]
The fact that a gene has been integrated into a transformed cell or a transformed plant means that DNA can be obtained therefrom using, for example, Mol. @Gen. {Genet. , 211, 27, 1988 (PCR) (Am. J. Hum. Genet., 37, 172, 1985) or the Southern method (J. Mol. Biol., 98, 505). , (1980)). The fact that the transformed cells express the Chlamydomonas APX gene integrated into the plant genome can be confirmed, for example, by the Northern method using the APX gene sequence or a part thereof as a probe (P. {Thomas et al., Proc. Natl. Acad. Sci., 77, 5201, (1980)) and a Western method using a specific antiserum against the introduced gene product (Chlamydomonas APX protein) (Towbin al., Proc. Natl. Acad. Sci., 76, 4350). , (1979)). The enzymatic activity of the APX protein, which is a translation product of the introduced gene, can be determined by the method of Zilinska et al. (R. @Mittler, \ and \ B.A. \ Zilinskas, \ Analytical \ Biochemistry, \ 212, \ 540-546), described in detail in the Examples below. )). Whether the ability to remove active oxygen in the chloroplasts of the transformed rice plants was enhanced was determined by examining leaf sections cut from the transformants and non-transformants using paraquat [methyl viologen] in Yoshida hydroponic solution. It can be revealed by measuring the change over time of photosynthetic system II activity using a chlorophyll fluorescence measuring device (Waltz, Germany) and comparing the results. If the degree of decrease in Fv / Fm of the transformant is suppressed as compared with the non-transformant, it can be said that the "active oxygen removing ability" is enhanced. As described above, whether or not the transformant rice has enhanced resistance to low-temperature decolorization is as described above. Leaf sections cut from the transformant and the non-transformant are floated in Yoshida hydroponic solution and incubated at 4 ° C for 3 days under strong light. Can be clarified by comparing the degree of decolorization of leaves. If the degree of decolorization of the transformant is suppressed as compared with the non-transformant, it can be said that the "cooling resistance" is enhanced.
[0025]
A method of calculating Fv / Fm will be described. Plant photosynthesis consists of light and dark responses. In the light reaction, a hydrogen ion concentration gradient is formed across the thylakoid membrane while electrons are transported through the two active center photosynthesis systems II and photosynthesis system I while being excited by light energy, and the energy is used to perform a dark reaction. High energy compounds such as ATP and NADPH used for the production are produced.
[0026]
The state of the bright reaction in the photosynthetic system can be known nondestructively by examining the increase / decrease of the amount of fluorescence emitted from the active center P680 of the photosynthetic system II and the infrared light absorption of the active center P700 of the photosynthetic system I. In particular, the fluorescence emitted from the photosynthetic system II greatly changes depending on the light energy intensity received by the antenna dye and the ease with which electrons flow in the electron transfer system (dark reaction state). For example, in plants adapted to dark conditions, enzymes involved in dark reactions are inactivated and electrons flow at the lowest speed. On the other hand, the photosynthetic II active center forms the complex best with the light-harvesting complex (Light @ harvesting @ complex) and is active. In addition, a xanthophyll cycle, which is an adaptation route to strong light, is also inactivated, and the emission of light energy as heat is in a minimum state. Thus, the maximum amount of fluorescence is emitted when the leaves of a plant adapted to the dark conditions are transferred to the light. When measuring the fluorescence emitted from the leaves in this state, the maximum fluorescence Fm for the excitation light is recorded. Autofluorescence is always emitted mainly from chlorophyll a from the leaves of the plant, and even when excitation light is not applied, F₀Indicates a constant value. Therefore, from the Fm value, F₀When the value obtained by subtracting the value is used as the Fv value and the index Fv / Fm value (relative value) of the photosynthetic activity is determined, the value shows a substantially constant value of 0.7 to 0.8.
[0027]
Then, when the photosynthesis system starts to move under light irradiation, a hydrogen ion concentration gradient is formed, and according to the degree, the xanthophyll cycle starts to work and light energy emission as heat starts. Also, as the chloroplast enters a reducing state, phosphorylation causes a separation between the photosynthetic II active center and the light-harvesting complex, and the number of active photosynthetic system II active centers decreases. Furthermore, since the enzymes of the dark reaction are activated and the flow of electrons becomes smooth, the amount of fluorescence Fm 'emitted from the leaves temporarily decreases. The Fv / Fm value also decreases.
[0028]
When a plant is photo-damaged and the photosynthetic system is damaged by active oxygen or the like (mostly protein degradation by oxidation), the number of active centers that can participate in photosynthesis decreases. If the stress is significant, this decrease is irreversible and does not return to the original number, even in the absence of stress. When observing the fluorescence at this time, the decrease rate of the fluorescence is faster than that in the non-stress state, and the maximum fluorescence Fm no longer comes out even when the state is adjusted to a dark state when there is no stress.
[0029]
Comparing the chlorophyll fluorescence of the non-transformed plants with the genetically engineered plant to reduce the light damage, the effect of reducing the damage is indicated by the difference in the fall of the relative fluorescence value Fv / Fm during stress, and when the addition of stress is stopped. Can be tested as a difference in the degree of recovery of the relative fluorescence value.
[0030]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.
(1) Synthesis of DNA fragment for vector construction
Based on the chloroplast-type gene sequence of Chlamydomonas (Chlamydomonas sp. W80), primer 1 APX5BAM36: GCC CAT GGC CCG ATC CAA GGC CTC CGC CGC GACT GCT GCT GCT GCT C ACT CAAACG TCG AGC CCA (SEQ ID NO: 4), primer 3 APX3STOPSAC36: G AG GTG CTT AGG ATC CCT CAA ACG TCG AGC CCA (SEQ ID NO: 5), Primer 4 STUC５GAT GAT GA GATCAGGA GA SEQ ID NO: 6), Primer 5 {STUCK3STOPSAC}: Synthesizes 5 types of primers: AAA AAA CCA CAA AGA GCT CTC A AT ATTC CCG CAA GAT ATG (SEQ ID NO: 7) (The position of each primer on the APX gene is as shown in FIG. 1) ), The Tm is set in the range of 47 to 54 ° C, and a plasmid pET / C for expression of E. coli (T. Takeda et al., 376) is obtained by using Pyrobest DNA polymerase (TAKARA) to obtain a correct APX fragment. (1), {82-90, {(2000)} Arch. {Biochem.} Biophys.) Was used as a template to perform a PCR amplification reaction for 25 to 35 cycles. An APX gene fragment of about 900 bp was obtained by combining the primers 1, 2, 1, and 3 (FIGS. 1a and 1b: fragment A and fragment B). With primers 4 and 5, the Tm was set to 50 ° C., and a reaction was performed for 25 cycles to obtain a gene fragment of about 150 bp of a thylakoid-membrane-localized sequence (hereinafter sometimes abbreviated as stack) (FIG. 1b: Fragment C).
[0031]
(2) Integration of APX gene fragment into chloroplast stroma expression cassette
Expression cassette pHSG / 35SI / chl // Nos with stromal transfer sequence (H. Hoshida et al., 43, 103-111, (2000) Plant Mol. Biol.) And fragment A cut with restriction enzymes BamHI and SacI, respectively. After that, ligation was performed using Takara ligation kit (TAKARA). Escherichia coli was transformed with the ligation reaction product to obtain an expression vector pHSG / 35SI / chl / clAPX / Nos (hereinafter abbreviated as pAPX) (FIG. 1a). After cutting pHSG / 35SI / chl // Nos and the fragment C with BamHI-SacI, ligation was performed to obtain an intermediate vector pHSG / 35SI / chl / stack / Nos. This intermediate vector and fragment B were cut with BamHI and ligated to obtain a large number of candidate clones. The selected clones were cut with HindIII and NheI (having a cleavage site inside APX) and cut correctly (1.3 Kb + 3.1 Kb), and the expression vector pHSG / 35SI / chl / clAPX / stack / Nos (hereinafter abbreviated as pAPXst) was obtained (FIG. 1b).
[0032]
(3) Introduction of the prepared vector into rice
Callus induction was carried out by incubating Sasanishiki seeds on a medium containing 2,4-D at 2 μg / ml. Suspension cultured cells were produced from the obtained calli, and treated with an enzyme solution containing 4% cellulase and 0.5% pectylase to obtain protoplasts. The protoplasts were suspended in a low-electrolyte buffer for electroporation containing 60 μg / ml of the hygromycin resistance gene vector and each vector DNA (pAPX or pAPXst), and placed in a Kartel cuvette (for a distance between electrodes of 4 mm). Gene transfer was performed by electroporation under the electrical conditions of 1 KV / cm, electric capacity of 1000 μF, and τ = 30 msec. The protoplasts subjected to the gene transfer treatment were further incubated on ice for 30 minutes, then embedded in an R2P medium containing 0.8% of low-melting-point agarose, and agarose pieces were floated on an R2P liquid medium and cultured in an agarose bead method. Shaking culture was performed in a culture room at ℃. During the culture, rice culture cells (Oc cells) were placed in the R2P liquid medium and the culture was protected. On the third day after the start of the culture, the cells for protection culture were removed, and on the seventh day, the cells were transferred together with the agar pieces to a liquid medium containing 30 μg / ml of hygromycin to start selection. Seven days after the start of the selection, the agar pieces were transferred to an R2SA agar medium containing 30 μg / ml of hygromycin to obtain a number of selected hygromycin (Hyg) -resistant calli.
[0033]
(4) Selection of transformed callus by PCR
Genomic DNA was extracted from Hyg-resistant calli, and PCR was performed with a combination of primer 6: ACACTGGCCGAGTGCCA (SEQ ID NO: 8) and primer 7: ACGTCGAGGCCCAGCTCC (SEQ ID NO: 9). A transformant was detected. Regarding the transformant into which the vector pAPXst was introduced, in particular, in order to confirm the presence of a thylakoid-membrane-localized sequence, primer 6 (described above) and primer 8: AGTTTTGTTGTAGGAGGC (SEQ ID NO: 10) were used for positive clones. When PCR was performed in combination, a band was confirmed at the same position as the plasmid. By PCR, transformants callus of 12 lines for pAPX and 38 lines for pAPXst were selected.
[0034]
(5) Confirmation of gene transfer by Southern analysis
For the transformant callus in which gene introduction was confirmed by PCR, Southern analysis was performed to confirm whether the full length of the expression cassette of the vector had been introduced. Using the full length APX gene cDNA as a probe, APXst transformant plants were subjected to Southern analysis. Genomic DNA extracted according to a conventional method was cut with a restriction enzyme Hind III-Nhe I so that the number of copies inserted into the genome could be determined. For example, as shown in FIG. 2, it was found that the APX gene was introduced into the plant transformed with the pAPXSt vector.
[0035]
(6) Confirmation of Chlamydomonas APX gene expression by Northern analysis
Northern analysis was performed to examine transcription from the introduced Chlamydomonas APX gene. Total RNA was extracted from pAPX and pAPXst transgenic plants according to a standard method, electrophoresed in 30 μg portions, and blotted on a nylon membrane. Northern analysis was performed using the APX gene fragment increased by the primers 6 and 7 as a probe. A 1.2 kb ΔAPX gene transcript was confirmed in the transformed plant into which the vector pAPX was introduced. Also, a 1.4 kb transcript was confirmed in the transformant into which pAPXst was introduced. The gene expression level had a good correlation with the copy number of the transgene (FIG. 3).
[0036]
(7) Confirmation of APX gene expression at translation level by Western analysis
Using a Chlamydomonas APX specific antibody ((T. Takeda et al., 376 (1), 82-90, (2000) Arch. Biochem. Biophys.)), The gene expression at the translation level was confirmed by Western analysis. . To 100 mg of callus, 2-3 times the amount of extraction buffer (100 mM @ Tris-HCl, @pH 7.8, @ 1% SDS, @protease inhibitor cocktail) is added on ice to grind, and centrifuged at 4 ° C., 15000 rpm for 5 to 10 minutes. Separation gave a supernatant (S) and a pellet. The pellet is added with 50 to 100 μl of a solubilization buffer (extraction buffer+0.1%@Triton X-100), suspended, and centrifuged again at 4 ° C. and 15000 rpm for 5 to 10 minutes. And Western analysis was performed using the S and P fractions of the obtained sample according to a standard method. Protein extraction from plants was performed according to a standard method, and the pAPXst transformant was used for analysis of the supernatant and the precipitate fraction. When Western analysis was performed using the anti-Chlamydomonas antibody as the primary antibody, gene expression at the translation level was confirmed in all lines in which gene expression was confirmed at the transcription level (FIG. 4). This expression level had a good correlation with the transcription level. In addition, when the gene expression levels of the supernatant and the precipitate fraction were compared, the band of the precipitate membrane fraction was deeper, indicating that the APX gene translation product was localized on the thylakoid membrane by the localization sequence near the thylakoid membrane. It was revealed.
[0037]
(8) Confirmation of translation product activity by activity staining
On ice, 100-200 mg of callus is ground with 2-3 volumes of extraction buffer (100 mM Na-P, 1 mM EDTA, 5 mM ascorbic acid, 0.1% Triton-X100, protease inhibitor cocktail), The mixture was centrifuged at 4 ° C. and 15000 rpm for 5 minutes to obtain a supernatant. A native acrylamide gel gel (containing no SDS; 5% concentrated gel, 12.5% separation gel) was prepared and subjected to 30 minutes at 4 ° C. in a running buffer (Tris @ 6 g / l, glycine @ 28 g / l) containing 2 mM ascorbic acid. Pre-run. Thereafter, a sample of 60 to 80 μg (two lanes of the same set were made) was loaded and electrophoresed for 3 to 4 hours. After completion of the electrophoresis, the gel on which the same set of samples had been electrophoresed was cut in half, and half was blotted on a membrane according to a standard method, and used for Western analysis thereafter. The other half of the gel was immersed in 50 mM Na-P buffer (pH 7.0) containing 2 mM ascorbic acid and incubated for 30 minutes, and then 2 mM H₂O₂And 4 mM ascorbic acid in 50 mM Na-P buffer (pH 7.0), incubated for 20 minutes, and then immersed in 50 mM Na-P buffer (pH 7.8) containing 28 mM TEMED and 2.45 mM NBT for 3 to 3 hours. The reaction was performed for 5 minutes. In the activity staining, in the transformants APXst8 and APXst27, a band considered to be APX activity derived from the transgene was detected in addition to a band considered to be derived from endogenous APX. This band was consistent with the position of the band reacting with the anti-Chlamydomonas APX antibody on the Western analysis membrane (transcribed from the gel run in parallel) (FIG. 5a). From this, it was revealed that active Chlamydomonas-type APX was expressed in the cells of the transformant callus. In the activity staining obtained by extracting proteins from the plant, a band derived from the introduced Chlamydomonas APX gene was detected in addition to the band considered to be derived from endogenous APX (FIG. 5b). The intensity of this band is considered to indicate the intensity of the activity, but had a good correlation with the amount of the translation product.
[0038]
(9) Regeneration of modern plants from transformed calli and production of next-generation plants
Six lines of plants were obtained from the 12 lines of pAPX callus selected by PCR. These were picked up and grown in a recombinant greenhouse to obtain T1 generation seeds for four lines. When gene expression of pAPXst callus of 38 lines obtained by PCR was examined, 18 lines were positive for Western analysis. Transformants regenerated from eight of these lines. Transformants were raised in a recombinant greenhouse and grown to obtain T1 generation seeds. The seeds were germinated to grow T1 transformed plants.
[0039]
(10) Measurement of APX activity in transgenic plants
According to the method of Zilinskas et al., A protein was extracted from the transformed plant with a 50 mM KP buffer containing 0.5 mM ascorbic acid and 1 mM PMSF, and 0.5 mM ascorbic acid was added to a 50 mM KP buffer containing 1 mM PMSF. Dialysis was performed for 3 hours or more using a dialysate and a dialysate not added. The amount of protein in the dialyzed extract was measured, and the activity was measured using 1 ml of the protein extract containing 200 μg and 400 μg (samples inactivated by boiling were also prepared). The activity was measured at a final concentration of 50 mM KP buffer containing 0.5 mM ascorbic acid, 1 mM PMSF, and 0.1 mM hydrogen peroxide. The reaction was started with the addition of hydrogen peroxide and the decrease per unit time in the Δ290 absorption of ascorbic acid was measured. Since the activity doubled when the amount of protein doubled, and the activity was lost when the sample was boiled, it was considered that APX activity could be measured correctly in this system. In the transformant, the total APX activity was increased about 5 times as compared with the control (FIG. 6).
[0040]
(11) Gene expression analysis in transgenic plant progeny
Seeds were obtained from four lines of the pAPXst recombinant plants of which the gene expression was confirmed in the current generation, and the seeds were obtained in the four lines. The seeds were sequentially germinated and the gene expression in the progeny was analyzed. Western analysis of APXst89 line progeny plants was performed according to a standard method. In the isolated progeny T1 generation of the line APXst89, gene expression at the translation level was confirmed (FIG. 7). This revealed that the introduced APX gene could be stably inherited in the next generation.
[0041]
(12) Paraquat resistance of APX transgenic plants
We succeeded in enhancing the ascorbate peroxidase activity of the transformant rice by up to 5-fold by the APX gene transfer, but we examined whether the enhanced activity could improve the ability to remove active oxygen in chloroplasts by using paraquat (methyl viologen). ) Resistance was examined. The highest developed leaves were cut out from rice grown in a recombinant greenhouse at a temperature of 26 ° C., a humidity of 55%, and a light intensity of 600 μmolE (day 14 hours, night 10 hours). A leaf section of 8 mm in length and 8 to 10 mm in width was cut out from the central portion of the cut out leaf (a 5 cm portion at the lower end was not used) and floated in Yoshida's hydroponic solution. 0, 0.1, 0.5, 1, 2, 5, 10 μM (adopt any of these concentration conditions) Yoshida's hydroponic solution (containing 0.1% Tween-20) containing methyl viologen. The dish was placed in a 24-hole dish and degassed for 10 minutes. The dish containing the leaf slices was transferred to an incubator at a temperature of 26 ° C. and a light intensity of 1200 μmolE (always bright conditions), and the Fv / Fm value was periodically measured using a PAM (Waltz, @Germany). Was examined. Also, a photograph of the leaf section was taken 2-3 days later, and the degree of bleaching was recorded. Measurement of the degree of damage by PAM: When the current plants (89 for Western + and 103 for Western-) were examined, the degree of damage was significantly lower in the lines with gene expression in the 10 μM treatment group (FIG. 8a). ). For 89 progeny with strong gene expression, it was found that they were segregated into individuals with gene expression and individuals without gene expression. Was significantly reduced (FIG. 8b). Further, three days after the paraquat treatment, the degree of decolorization of the leaves was observed, and it was revealed that the introduction of the APX gene reduced the decolorization of the leaves (collapse of chloroplasts) (FIG. 9). The above-mentioned paraquat resistance test was repeated twice or more for both the current generation and the progeny, and the effect of gene transfer could be verified with good reproducibility.
[0042]
(13) Cold resistance test of APX transgenic plants
In Western analysis, two individuals with gene expression (APXst103 and APXst106) and two individuals without expression (APXst76 and APXst89) were prepared, and the second leaf from the top was sampled. Eight leaf sections of 1.2 cm wide and 1.5 cm long were cut from the center of each leaf. Twelve leaf sections were sandwiched between 12-cm dishes (laid with Kim towels containing a hydroponic solution) and incubated on ice at 4 ° C. while irradiating with 1200 μmol E light.
In leaf sections incubated for 3 days while maintaining the low-temperature intense light irradiation conditions, individuals with strong gene expression maintained green color better (FIG. 10). From this result, it was revealed that by introducing the Chlamydomonas APX gene into rice and expressing it by localizing it near the chloroplast thylakoid membrane, the resistance of rice to low-temperature decolorization can be enhanced.
[0043]
【The invention's effect】
According to the present invention, there is provided a grass plant having enhanced cold resistance by enhancing the expression of an active oxygen-scavenging enzyme gene in the grass plant.
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a schematic diagram of the construction of a vector. FIG. 1A shows the construction of pHSG / 35SI / chl / clAPX / Nos (pAPX). FIG. 1B shows the construction of pHSG / 35SI / chl / clAPX / stuck / Nos (pAPXst).
FIG. 2 is a view (photograph) showing the results of Southern analysis of APX-transgenic rice plants. Controls are non-transformants, and 27, 75, 76, 77, 78 and 89 are transformants.
FIG. 3 is a diagram (photograph) showing the results of Northern analysis of APX-transgenic rice plants. FIG. 3A shows the results of a pAPX-introduced individual. Controls are non-transformants, and 3, 8, 13, 17 are transformants. FIG. 3B shows the results of a pAPXst-introduced individual. Controls are non-transformants, and 27, 75, 76, 77, 78, 89 are transformants.
FIG. 4 is a view (photograph) showing the results of Western analysis of APX-transgenic rice plants. The pellet is the pellet fraction, and the supernatant is the supernatant fraction. Controls are non-transformants, and 75, 76, 77 and 78 are transformants.
FIG. 5 is a diagram (photograph) showing the results of confirming the expression of the translation level of APX-transgenic rice. FIG. 5A is a diagram showing a comparison between activity staining and Western analysis. SAS is a non-transformant, and APXst8, APXst21, and APXst27 are transformants. FIG. 5B is a diagram showing the results of activity staining analysis of transformed plants. Controls are non-transformants, and 76 and 89 are transformants.
FIG. 6 is a view showing the results of APX activity measurement of APX-transgenic rice plants.
FIG. 7 is a view (photograph) showing the results of Western analysis of progeny (T1) of APX-transgenic rice plants. Control is a non-transformant, M is a molecular weight marker, APXst89 # 1-5 is a transformant.
FIG. 8 is a view showing the test results of paraquat resistance (change in Fv / Fm values) of APX-transgenic plants. FIG. 8A shows the current generation (T₀) Plant comparison (89 with gene expression, 103 without gene expression). FIG. 8B is a comparison of the progeny of the separation (89-33 has gene expression, 89-39 has no gene expression).
FIG. 9 is a diagram (photograph) showing the test results of paraquat resistance of APX-transgenic rice plants. Paraquat (methyl viologen) resistance assay using isolated progeny (T1) leaf sections. 89-33 has gene expression, 89-39 has no gene expression. Methyl viologen treatment is performed under three conditions of 0, {5, and {10} mM.
FIG. 10 is a diagram (photograph) showing the results of assaying the low-temperature decolorization resistance of APX-transgenic rice plants. The leaf sections were subjected to low-temperature treatment at 4 ° C. for 3 days while irradiating 1200 μmol E of light. APXst103 and APXst106 are non-transformants, and APXst76 and APXst89 are transformants.

Claims (7)

A grass plant with enhanced cold resistance produced by enhancing the expression of an active oxygen scavenging enzyme gene. The plant according to claim 1, wherein the gramineous plant is rice. The plant according to claim 1, wherein the active oxygen-scavenging enzyme gene is an ascorbate peroxidase gene. The plant according to claim 3, wherein the ascorbate peroxidase gene is derived from Chlamydomonas. The plant according to claim 3, wherein the ascorbate peroxidase gene has any one of the following nucleotide sequences.
(A) a base sequence consisting of base numbers 32 to 991 in the base sequence described in SEQ ID NO: 1;
(B) a base sequence in which one to a plurality of bases are deleted, added or substituted in the base sequence consisting of base numbers 32 to 991 in the base sequence described in SEQ ID NO: 1, wherein the ascorbate peroxidase is A base sequence encoding a protein having activity; or (C) a base sequence consisting of base numbers 32 to 991 or a complementary sequence thereof or a part thereof among the base sequences set forth in SEQ ID NO: 1 under stringent conditions. A nucleotide sequence that hybridizes and encodes a protein having ascorbate peroxidase activity. The plant according to any one of claims 3 to 5, wherein a chloroplast translocation sequence is added to the ascorbate peroxidase gene, and the gene translation product is localized in the chloroplast. 7. The thylakoid translocation sequence is added to the ascorbate peroxidase gene, and the gene translation product is localized near the thylakoid membrane of the chloroplast, 7. The method according to any one of claims 3 to 6, wherein Plants.

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