WO2006098225A1

WO2006098225A1 - Method of constructing plant having nodules with high nitrogen fixation activity

Info

Publication number: WO2006098225A1
Application number: PCT/JP2006/304681
Authority: WO
Inventors: Toshiki Uchiumi; Yoshikazu Shimoda
Original assignee: Kagoshima University
Priority date: 2005-03-14
Filing date: 2006-03-03
Publication date: 2006-09-21
Also published as: JP4677568B2; JPWO2006098225A1; CA2600560A1; CN101208430A; CN101208430B; AU2006224032A1; AU2006224032B2; CA2600560C

Abstract

A method of constructing a nodulating plant capable of having nodules with an elevated nitrogen fixation activity characterized by comprising overexpressing a nonsymbiotic globin gene in a nodulating plant.

Description

Description of production method of plants that grow nodules with high nitrogen fixation activity Technical Field

The present invention relates to a method for producing a plant in which a nodule having high nitrogen fixation activity is formed. Background art

Leguminous crops such as soybean, azuki bean and kidney bean grow (form) the nodule, a symbiotic organ, by infection with rhizobia, and the rhizobia (pacteroid) symbiotic in this nodule fixes nitrogen in the air. Therefore, it can grow well even in soil with low nitrogen content. For this reason, when cultivating leguminous crops, in addition to applying normal fertilizer, pre-cultured rhizobia is applied to the seeds of crops. In order to further increase the nitrogen fixing ability of nodules in leguminous crops, development of rhizobia with enhanced nitrogen fixing ability has been carried out (Patent Document 1).

In addition to leguminous crops, leguminous trees such as acacia and nemony, and non-leguminous trees such as alder and rhododendrons, symbiotic nitrogen-fixing bacteria settle in the roots to form nodules and efficiently fix aerial nitrogen. It is known to supply nitrogen to host trees. Trees with these root nodules have high nitrogen concentration in the leaves and therefore high nitrogen concentration in the fallen leaves. For this reason, trees with root nodules are effective in increasing the amount of microorganisms in the soil and improving them into fertile soil, and are also used for greening of wasteland as so-called fertilizer trees.

Enhancing the nitrogen fixing ability of such plant nodules seems to be very useful not only for agriculture but also for environmental conservation.

By the way, it is known that the symbiotic globin gene possessed only by legumes is very strongly expressed in nodule cells of legumes. Symbiotic hemoglobin (also called leghemoglobin) composed of globin and heme, the gene product of the symbiotic globin gene, is said to account for 20-30% of the total soluble protein of nodules. There is no organization. Symbiotic hemoglobin Similar to hemoglobin in blood, it has a strong affinity with oxygen. The symbiotic hemoglobin inactivates the partial pressure of oxygen in the nodule cells, which is sufficient for the respiration of the rhizobia in the nodule, and the nitrogenase (inactivated by oxygen), which is necessary for the nitrogen fixing ability of the rhizobia. It is said to be responsible for adjusting to no level.

On the other hand, with the development of genetic analysis technology in recent years, it has become clear that plants other than legumes also have globin genes. The gubin gene found in plants other than legumes is different from the symbiotic globin genes of legumes, so it is called “non-symbiotic globin gene” (also called non-symbiotic hemoglobin gene). ). Currently, all plants are thought to have non-symbiotic globin genes. In other words, legumes have both symbiotic globin genes and non-symbiotic gubin genes, while non-legumes have only non-symbiotic globin genes. Non-symbiotic globin genes have also been reported in Miyakogusa, which is a model plant of the legume family (Non-patent Document 1).

Unlike symbiotic globin genes, nonsymbiotic globin genes are known to be expressed in all plant tissues. It has been reported that the expression level of non-symbiotic globin genes increases when plants are exposed to low temperatures (4 ° C) and low oxygen partial pressure (oxygen concentration of 5% or less). There is also a report that Arabidopsis thaliana overexpressed by introducing a non-symbiotic globin gene has enhanced resistance to hypoxic stress (Non-Patent Document 2).

Patent Document 1 Japanese Patent Laid-Open No. 2 0 0 3-3 3 1 7 4

Non-Patent Document 1 Uchiumi et al., Plant Cell Physiol. (2002) 43 (11):. 1 351-1358

Non-Patent Document 2 Hunt, P. W., et al., Proc. Natl. Acad. Sci. (2002) USA 99: p. 17197-17202 Disclosure of the Invention

An object of the present invention is to provide a method for producing a nodule-growing plant in which a nodule having an increased nitrogen-fixing activity is formed, and a nodule-growing plant exhibiting a high nitrogen-fixing activity in the nodule. As a result of intensive studies to solve the above-mentioned problems, the present inventors have introduced a Miyakogusa non-symbiotic gumbin gene and overexpressed Miyakodasa, and a Jashapsi non-symbiotic globin gene. It was found that the expressed Miyakogusa can grow root nodules with high nitrogen-fixing activity, and the present invention has been completed based on the findings.

That is, the present invention includes the following.

[1] A method for producing a nodulating plant capable of growing a nodule having increased nitrogen fixation activity, characterized by overexpressing a non-symbiotic globin gene in the nodulating plant. In this method, the non-symbiotic globin gene is more preferably a DNA comprising a DNA selected from the group consisting of the following (a) to (e):

(a) DN.A consisting of the base sequence shown in SEQ ID NO: 1 or 8

(b) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 8 and encodes a protein having non-symbiotic globin activity

(c) DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 9

(d) DNA consisting of an amino acid sequence in which 1 to 50 amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 9, and encoding a protein having a non-symbiotic globin activity

(e) DNA consisting of the base sequence shown in SEQ ID NO: 3 or 10.

In this method, preferably, a non-symbiotic globin gene is overexpressed by introducing a non-symbiotic globin gene linked to an overexpression promoter into a nodulating plant.

By this method, it is possible to produce a nodule-growing plant that can preferably grow a nodule having an increased nitrogen fixation activity at least three times that of the wild strain. The nodulating plant produced by this method is preferably capable of growing a nodule having an increased nitrogen fixing activity and exhibiting a nitrogen fixing activity of 10 to 100 nM / min / g.

In this method, the nodulating plant that overexpresses the non-symbiotic globin gene is preferably a legume. Furthermore, in this method, non-symbiotic globin inheritance It is more preferable to inoculate a nodulating plant with overexpressed offspring with a symbiotic nitrogen-fixing bacterium.

[2] A nodule-growing plant produced by the above method 1 and capable of growing nodules with increased nitrogen fixation activity. This nodule-growing plant is more preferably one having a root nodule.

[3] A vector for increasing nitrogen fixation activity of nodules, comprising a non-symbiotic globin gene linked to an overexpression promoter.

The non-symbiotic globin gene contained in this vector is preferably derived from legumes or non-legume nodulating plants. As the non-symbiotic globin gene, the DNA shown in (a) to (e) of [1] above is still more preferable.

[4] A method for increasing the nitrogen fixation efficiency in plant cultivation, characterized by cultivating the nodulating plant of [2] above. According to the method for producing a nodulating plant of the present invention, the nitrogen fixing activity of the nodule can be remarkably improved in the desired nodulating plant. When the vector for increasing the nitrogen fixation activity of the nodules of the present invention is used in this production method, the nitrogen fixation activity of the nodules can be remarkably increased. In addition, the nodulating plant obtained in the present invention can grow a nodule having high nitrogen fixation activity, and as a result, the growth of the plant is promoted and the nitrogen content is also increased. According to the present invention, the method for increasing the nitrogen fixation amount in plant cultivation by cultivating a nodule-growing plant increases the amount of nitrogen fixed from the air in a certain environment, and the nodule under the environment Not only can it increase the yield of seedlings or the amount of growth, it can also fertilize the soil by increasing the amount of nitrogen in the soil. 'The present specification includes the contents described in the specification and drawings of Japanese Patent Application No. 2 0 0 5-0 7 1 6 7 7 on which the priority of the present application is claimed. Brief Description of Drawings

Figure 1 shows the genomic structure and amino acid sequence of the non-symbiotic globin gene. It is a figure.

Fig. 2 shows the expression level of non-symbiotic globin genes in wild-type Lotus. Figure 2A shows the expression level by tissue, and Figure 2B shows the expression level under stress conditions. FIG. 3 is a diagram showing the structure of a transformation vector used for transformation of hairy roots. Fig. 4 is a photograph showing an experiment confirming non-symbiotic globin gene transfer in transformants. FIG. 4A shows the state of hairy root induction in the transformant. Figure 4B shows the GFP fluorescence observed in transformed roots. Fig. 4C shows the results of confirming the expression of the introduced gene by RT-PCR.

Fig. 5 is a photograph showing the appearance of nodulation in transformed hairy roots. Fig. 5A shows hairy roots (control roots) into which a vector not containing the LjHbl gene has been introduced. Fig. 5B shows a hairy root with the LjHbl gene introduced and overexpressed. Open arrowhead marks indicate the nodules formed on the transformed hairy roots. Shaded arrowhead marks indicate nodules formed on hairy roots that were not transformed. Only transformed nodules are shown to fluoresce.

FIG. 6 is a diagram showing data measured for the amount of ethylene by gas chromatography, which shows the nitrogen fixation activity (ARA activity) of nodules formed in transformed hairy roots.

FIG. 7 is a schematic diagram showing a vector used to express the globin protein of Lotus japonicus in E. coli.

Figure 8 shows the affinity of Miyakogusa globin protein expressed in E. coli (symbiotic [left graph, Fig. 8 A] and non-symbiotic [right graph, Fig. 8 B]) and nitric oxide. The absorbance spectrum shown. Each line shows data for each time period mixed with nitric oxide.

FIG. 9 is a photograph showing the results of confirming the expression of the Af Hb 1 gene in the Lotus japonicus transformant using RT-PCR.

FIG. 10 is a graph showing nitrogen fixation activity (ARA activity) in whole plants and formed nodules of AfHbl-introduced Lotus japonicus transformants. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. 1. Nodulation plant

In the present invention, a nodulating plant capable of growing a nodule having an increased nitrogen fixation activity is produced by overexpressing a non-symbiotic guttine gene in the nodulating plant. In the present specification, the nodulating plant means a plant species capable of growing (forming) a nodule. A nodule is a symbiotic organ in which symbiotic nitrogen-fixing bacteria settled in the root to form a granular structure. In addition to legumes (including leguminous crops and legumes), rhizobial plants include some non-leguminous plants (mainly birch family and alder family) such as birch family and alder family. Non-legume trees) are also included. The roots of legumes are symbiotic as symbiotic nitrogen-fixing bacteria and form nodules. On the other hand, in some non-leguminous plants, actinomycetes coexist in the roots as symbiotic nitrogen-fixing bacteria to form nodules. Among the nodulating plants useful in the present invention, the leguminous plants include Miyakogusa, soybean, azuki bean, yellow beans, endou, broad bean, laccasei, anolefa alfa, tanoluma palm, clover, cowpea, lentil, nise acacia, henshida, hagi, In the case of legumes such as monolecanes, there are yashapushi, alder tree, bayberry, mokumau, dokutsugi and gummy.

In the present invention, as long as the nodule-growing plant is a plant of a species that can grow (form) the nodule, at the time of introducing the non-symbiotic globin gene, at the time of regenerating the plant, or other The plant body in which the nodule has grown may be the plant body in which the nodule has not grown, or the plant body.

In the present specification, the term “nodulating plant” refers not only to a plant body (whole plant individual) but also to a plant organ (eg, leaf, petal, stem, root, seed, hypocotyl, cotyledon, etc.), plant tissue (eg, It shall include any part of the plant body such as epidermis, phloem, soft tissue, xylem, vascular bundle, palisade tissue, spongy tissue, etc.) and plant cultured cells (eg callus).

2. Non-symbiotic globin genes and their acquisition

The non-symbiotic globin gene according to the present invention encodes a globin protein that associates with heme (a complex salt of porphyrin and divalent iron) to form non-symbiotic hemoglobin. This nonsymbiotic hemoglobin, like animal hemoglobin, It is a protein with strong affinity for carbon, carbon monoxide, etc. Nonsymbiotic hemoglobin has a stronger affinity for oxygen, nitric oxide, and the like than symbiotic hemoglobin. In the present specification, the activity of globin encoded by the non-symbiotic globin gene is referred to as non-symbiotic gout bin activity.

The non-symbiotic goutin gene according to the present invention may be isolated from any plant. The non-symbiotic globin gene of the present invention is more preferably derived from a nodulating plant, and may be isolated from legumes such as Miyakodasa and soybean, or from non-legumes such as Yashapsi. It may be isolated from a nodulating plant. The non-symbiotic globin gene used in the present invention may be further isolated from plants other than nodulating plants, including monocotyledonous plants such as barley, rice, and corn. Note that known non-symbiotic globin genes that can be used in the present invention include the following: Miyakogusa (Non-Patent Document 1), Yashapushi (DD BJ / EMBL / GenBank Accession Number AB221344), Rice (DDBJ / EMBL / GenBank accession number U76030), Alphanorefa (DDBJ / EMBL / GenBank accession number AF172172; Serogelyes et al., FEBS Lett. (2000) 482, p. 125-130), Dizz (DDBJ / EMBL / GenBank Accessory 3 number U47143; Anderson, et al., Proc.

Natl. Acad. Sci. USA (1996) 93 (12), p. 5682-5687), Arabidopsis thaliana (DDBJ / EMBL / GenBank accession number U94998; Trevaskis, et al., PNAS (1997) 94 p. 12230- 12234).

The non-symbiotic globin gene used in the present invention may be cDNA, or genomic DNA containing exons and introns. As used herein, “gene” includes DNA and RNA, DNA includes at least genomic DNA, cDNA, and synthetic DNA, and RNA includes mRNA and the like. In the present specification, the “gene” may include a sequence such as an untranslated region (UTR) sequence in addition to the coding sequence.

Although it is not limited, in one preferred embodiment, the symbiotic non-symbiotic globin gene can be used as the non-symbiotic globin gene derived from the leguminous plant according to the present invention. For example, as the non-symbiotic globin gene of the present invention, DNA consisting of the nucleotide sequence of SEQ ID NO: 1 isolated from Miyakogusa, and DNA of the genomic fragment consisting of the nucleotide sequence of SEQ ID NO: 3 isolated from Miyakodasa are preferably used. Can be used The In addition, DNA encoding a non-symbiotic globin protein consisting of the amino acid sequence of SEQ ID NO: 2 can be advantageously used.

The non-symbiotic globin gene of the present invention is 1 to 50, preferably 1 to 35, more preferably 1 or several in the amino acid sequence shown in SEQ ID NO: 2 as long as it has non-symbiotic globin activity ( For example, DNA encoding a protein consisting of an amino acid sequence in which 2 to 10 amino acids have been deleted, substituted, or added may be used. The non-symbiotic globin gene used in the present invention is a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or a DNA encoding a non-symbiotic globin protein consisting of an amino acid sequence of SEQ ID NO: 2. It may be a DNA consisting of a base sequence complementary to the above base sequence and a DNA encoding a protein that is hybridized under stringent conditions and has a non-symbiotic globin activity. The non-symbiotic globin gene of the present invention is also a DNA encoding a protein having a non-symbiotic goutine activity consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 2. Anyway.

On the other hand, in another aspect, the non-symbiotic globin gene derived from the non-leguminous nodule-growing plant according to the present invention is not limited. Can do. For example, as the non-symbiotic globin gene of the present invention, DNA consisting of the nucleotide sequence of SEQ ID NO: 8 isolated from Yashabushi and DNA of the genomic fragment consisting of the nucleotide sequence of SEQ ID NO: 10 isolated from Yashapushi are preferred. Can be used appropriately. In addition, a DNA encoding a Yachabushi non-symbiotic globin protein consisting of the amino acid sequence of SEQ ID NO: 9 can be advantageously used.

As long as the non-symbiotic globin gene of the present invention has non-symbiotic globin activity, 1 to 50, preferably 1 to 35, more preferably 1 or several (for example, 2) in the amino acid sequence shown in SEQ ID NO: 9. DNA encoding a protein consisting of an amino acid sequence in which (˜10) amino acids have been deleted, substituted or appended may be used. The non-symbiotic gubin gene used in the present invention encodes a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 8 or a non-symbiotic globin protein consisting of the amino acid sequence of SEQ ID NO: 9. Hybridizes with DNA consisting of a base sequence complementary to the DNA base sequence under stringent conditions, and non-symbiotic globin activity It may be DNA encoding a protein having sex. The non-symbiotic globin gene of the present invention also encodes a protein having a non-symbiotic gubin activity comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 9. But let ’s do it.

In the present invention, “stringent conditions” refers to conditions under which a so-called specific hybrid is formed. For example, nucleic acids having high homology, that is, DNAs having a homology of 90% or more, preferably 95% or more are hybridized, and nucleic acids having lower homology are not hybridized. More specifically, the sodium salt concentration 15～750MM, preferably 50～750MM, more preferably ³ 0 ^0~750mM, temperature 25 to 70 ° C, preferably 50 ° C to 70 ° C, more preferably The conditions are 55-65 ° C, formamide concentration 0-50%, preferably 20-50%, more preferably 35-45%. Furthermore, under stringent conditions, the filter conditions after hybridization are usually such that the sodium salt concentration is 15-600 mM, preferably 50-600 mM, more preferably 300-600 mM, and the temperature 50-70. . C, preferably 55 to 70 ° (:, more preferably 60 to 65 ° C.

Although not limited, in one embodiment of the present invention, the non-symbiotic globin activity of the globin protein encoded by the non-symbiotic globin gene is the oxygen, carbon dioxide of non-symbiotic hemoglobin formed by the globin. , Or affinity for nitric oxide and the like. The non-symbiotic gustin activity of the present invention is obtained by, for example, recombinantly producing a globin protein using an expression vector containing a non-symbiotic gubin gene encoding the non-symbiotic globin. 'With respect to non-symbiotic hemoglobin reconstituted from protein and heme, the affinity for oxygen, carbon dioxide, or nitric oxide can be measured by a conventional method, and the measured value can be expressed as an index. The affinity of non-symbiotic hemoglobin to oxygen, carbon dioxide, or nitric oxide can be measured by methods known to those skilled in the art.

—As an example, the affinity of non-symbiotic hemoglobin for nitric oxide is 500 nn in the presence of nitrogen monoxide! The absorbance can be measured at a wavelength of ~ 600nm and judged from the absorbance spectrum. In general, hemoglobin absorbs light with a wavelength of 500 to 600 nm. When the curve is drawn, two characteristic peaks are observed at 540ηπι and 575nm (see Fig. 8). These two peaks are not observed in the hemogum bottle after completion of the reaction with nitric oxide. Therefore, after the hemoglobin protein is mixed with nitric oxide, the affinity spectrum of the hemoglobin protein with nitric oxide can be measured by analyzing the absorbance spectrum by a conventional method. In addition, isolated and purified hemoglobin usually exists in a state bound to oxygen (called oxyhemoglobin). Non-symbiotic hemoglobin is also isolated in a bound state with oxygen, for example when produced recombinantly. Since nonsymbiotic hemoglobin has a higher affinity for nitric oxide than oxygen, adding nitrogen monoxide to the isolated nonsymbiotic form causes two peaks to disappear in the absorbance spectrum. Transition to the spectrum after completion of the reaction with nitric oxide will be observed. For a method for measuring the affinity of hemoglobin with nitric oxide, refer to Michele Perazzoli et al., The Plant Cell (2004) 16, p. 2785-2794.

When the absorbance spectra measured as described above are compared and analyzed for non-symbiotic hemoglobin and symbiotic hemoglobin, their affinity with nitric oxide can be relatively compared. This comparison of absorbance spectra indicates that nonsymbiotic hemoglobin can bind to nitric oxide more rapidly than symbiotic hemoglobin. In the present invention, the affinity of the non-symbiotic hemoglobin to nitric oxide is used as an index, using the mixing time of the non-symbiotic hemoglobin and nitric oxide until the binding is shown in the absorbance spectrum. Judgment can be made.

In the present invention, for example, non-symbiotic globin is recombinantly produced in E. coli using a vector containing a gene encoding non-symbiotic globin (SEQ ID NO: 2 or 9), and non-symbiotic type in E. coli is produced. A non-symbiotic hemoglobin mouth reconstructed from globin and heme can be collected, and the affinity of the non-symbiotic hemoglobin for nitric oxide can be measured by the method described above. The non-symbiotic globin of the present invention is not limited, but compared with the affinity for nitric oxide of non-symbiotic hemoglobin derived from such non-symbiotic globin (SEQ ID NO: 2 or 9), It is preferable to have the same affinity for nitric oxide.

Non-symbiotic globin genes according to the present invention as described above are, for example, SEQ ID NOs: 1 to 3 And a primer designed on the basis of the sequence of 8 to 10 and performing PCR amplification using a nucleic acid derived from any plant (eg, nodulating plants; Miyakogusa, Yashabushi, etc.) It can be obtained as a fragment. In addition, the non-symbiotic globin gene of the present invention is hybridized using a nucleic acid derived from any plant (eg, nodulating plants; Miyakodasa, Yashapushi, etc.) as a cocoon and using a DNA fragment that is part of a non-symbiotic globin gene as a probe. It can be obtained as a nucleic acid fragment by performing the ditherization. The nucleic acid used as a cocoon in these methods may be, for example, genomic DNA extracted from any plant by a conventional method, or cDNA reversely synthesized from mRNA extracted by a conventional method. . The nucleic acid used as the saddle type may be purified genomic DNA, purified c 匪, one cDNA library, one genomic DNA library, or the like. Alternatively, the non-symbiotic globin gene used in the present invention may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.

Furthermore, a desired mutation may be introduced into the base sequence (and thus the encoded amino acid sequence) of the obtained non-symbiotic globin gene by site-directed mutagenesis. In order to introduce a mutation into a 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 (such as Mutan-K (TAKARA) or MutanG (TAKARA)) using site-directed mutagenesis, or TAKARA LA PCR in vitro Mutagenesis Mutation is introduced using a series kit.

Preparation of mRNA used in the present invention, preparation of cDNA, PCR, RT-PCR, preparation of library, ligation into vector, cell transformation, determination of DNA base sequence, nucleic acid chemical synthesis, protein N-terminus Experiments such as determination of the amino acid sequence on the side, mutagenesis, and protein extraction can be performed by the methods described in ordinary experiment documents. Examples of such experiments include Sambrook et al., Molecular Cloning, A laboratory manual, 2001, Eds., Sambrook, J. & Russell, DW. Cold Spring Harbor Laboratory Press.

3. Excessive occurrence of non-symbiotic globin genes in nodulating plants! ^ In the present invention, a non-symbiotic globin gene is overexpressed in a nodulating plant by a genetic engineering technique. In the present invention, “overexpressing” a gene means that the gene exceeds the amount normally expressed in the host organism by any genetic engineering technique (for example, gene transfer). It means that the gene is manipulated so that it is expressed in an amount (for example, 10% or more), or the gene is introduced into a host organism that does not have the gene and expressed. The specific means for overexpressing the non-symbiotic globin gene is not limited, and any method known to those skilled in the art can be used. Although not limited, a common method is to use a transformation vector constructed by ligating non-symbiotic globin genes so that they are expressed in the correct reading frame downstream of the overexpression promoter. A method of introducing it into a plant is mentioned. In this case, the non-symbiotic globin gene can be incorporated into the vector by, for example, excising a DNA fragment containing the non-symbiotic globin gene with an appropriate restriction enzyme and adding it to an expression vector containing an overexpression promoter. It may be inserted into an appropriate restriction enzyme site downstream of the overexpression promoter so as to be in-frame and connected. Alternatively, a DNA fragment in which a non-symbiotic globin gene is linked in advance downstream of the overexpression promoter may be incorporated into the vector. A genomic fragment of the non-symbiotic goutvin gene linked to the overexpression promoter may be incorporated into the genomic DNA of the nodulating plant by a method such as homologous recombination. As the nodulating plant that overexpresses the non-symbiotic globin gene, any of the above-mentioned nodulating plants (for example, Miyakodasa) can be preferably used. In the present invention, the “overexpression promoter” means a promoter having the ability to strongly (in large quantities) express a gene linked to the overexpression promoter in a host plant cell. In particular, the overexpression promoter of the present invention may be a promoter (nodule-specific promoter) that causes nodule-specific expression. The overexpression promoter of the present invention may be an inducible promoter or a constitutive promoter. A promoter generally refers to a DNA containing an expression control region or its modified sequence existing 5 'upstream of a structural gene. In the present invention, any promoter suitable for foreign gene expression in plant cells can be used as an overexpression promoter. Suitable examples of the overexpression promoter used in the present invention include, but are not limited to, cauliflower mosaic virus. (CaMV) 35S promoter, inactin promoter, modified 35S promoter, tobacco PRla promoter, Arabidopsis PR-1 promoter, and the like. As a transformation vector for introducing a non-symbiotic globin gene, any vector can be used as long as it is a vector for introduction into plant cells. For example, when the agrobacterium method is used, it is preferable to use a plasmid vector derived from agrobacterium (such as Ti plasmid) or a binary vector. The transformation vector used in the present invention includes a non-symbiotic globin gene and, optionally, an overexpression vector, as well as a selection marker gene, reporter gene, and binary vector system that facilitates selection of transformants. Replication origin (such as replication origin derived from Ti or Ri plasmid). Examples of the selection marker gene include drug resistance genes such as cefotax gene, hygrosine resistance gene, dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, and kanamycin resistance gene. Examples of reporter genes include green fluorescent protein gene (GFP) luciferase) gene (LUC, LU X) and the like. In the present invention, the “vector” includes a so-called expression cassette. An “expression cassette” includes a promoter DNA sequence and a DNA sequence of a gene to be expressed, and is linked to the promoter DNA sequence in an arrangement such that the DNA sequence of the gene can be expressed in plant cells. It means a DNA fragment. An expression cassette does not necessarily have autonomous replication ability.

Moreover, examples of the vector containing an overexpression promoter in advance include pBI binary vectors such as pKANNIBAL, IG121-Hm, ρΒΙ121, ρΒΙ101, ρΒΙΙΟΙ.2, ρΒΙΙΟ1.3, and pCAMBIA1301.

The present invention also provides a vector comprising such a non-symbiotic globin gene linked to an overexpression promoter. Such a transformation vector can be introduced into any nodule-growing plant in order to increase the nitrogen fixation activity of the nodule and can be used very conveniently.

The method for introducing the transformation vector into the nodulating plant is not limited. For example, the agrobatterium method, the particle gun method, the electoral pore method, the polyethylene glycol (PEG) method, the microinjection method, Plant transformation methods widely used for plants, such as protoplast fusion, can be used. These plant transformation methods are: “Isao Shimamoto, Kiyotaka Okada“ New Edition Experimental Protocol for Model Plants From Genetic Methods to Genome Analysis ”

(2001) Shujunsha ”and other general textbooks. Plant cells introduced with the transformation vector are selected by a method using a selectable marker such as kanamycin resistance, and the expression of the transgene is confirmed by detecting the reporter protein or analyzing the expression of the transgene. It is preferable to regenerate the plant body by a conventional method.

More specifically, for example, in the agrobacterium method, for example, the method of Nagel et al. Is used. First, the vector is introduced into the agrobacterium through an electoral position, and then the transformed agrobacterium is introduced. Pl nt Molecular Biology Manual (SB Gelvin et. Al., Academic Publishers), Thykaer, T. et al., Cel Biology, 2nd ed. (1998) p, 518-525, Stiller, J., et al. , J. Exp. Bot. (19 97) 48, p. 1357— 1365, Ogar, P. et al., Plant Science (1996) 116 159-168, or Hiei Y. et al., Plant J. (1994 ) The target gene may be introduced into the plant by the method described in 6, 271-282 and regenerated into the plant body.

When using the polyethylene dallicol method, first remove the cell wall by dissolving it with an enzyme, convert it to a protoplast, introduce the non-symbiotic globin gene into the cell using polyethylene glycol, and regenerate it into the plant body. Good (Datta SK: In Gene Transfer To Plants (Potrykus I and Spangenberg, Eds) pp. 66-74 (199 5)).

When using the electroporation method, first remove the cell wall by dissolving it with an enzyme, convert it to a protoplast, apply an electric pulse to introduce the non-symbiotic globin gene into the cell, and then regenerate it into the plant body. Good (Toki S, et al., Plant Physiol., 100: 1503 (1992)).

When using the particle gun method, a plant body, a plant organ, or a plant tissue itself may be used as it is, or may be used after preparing a section, or a protoplast may be prepared and used ( Chri stou P, et al., Biotechnology 9: 957 (199 1)). For the sample prepared in this way, a gene transfer device (eg PDS-1000 (BI0-R 1

AD), etc.), and the fine particles (diameter: about 1 to 2 / im) such as gold and tandasten coated with non-symbiotic globin gene are injected with high-pressure gas to bring the gene into plant cells. Introduce. 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. Once the non-symbiotic globin gene is introduced into the cell, it can be regenerated into a plant in the same manner as described above.

In the nodulating plant of the present invention transformed as described above, the non-symbiotic globin gene may be expressed in the state of being incorporated in the genomic DNA of the plant, or as extragenomic DNA. It may be expressed (eg, while retained in the vector).

For transformed plant cells or plant tissues (hairy roots, leaves, stems, nodules, etc.) overexpressing the non-symbiotic globin gene as described above, or the regenerated plants, non-symbiotic globin genes It is preferable to confirm the expression of the gene by conventional methods such as Northern plotting, Southern plotting, and reporter gene expression.

4. Measurement of nodulation and nitrogen fixation activity of nodules in plants

In the present invention, a transformed nodule-growing plant in which a non-symbiotic globin gene is overexpressed can grow a nodule by growing in an environment where symbiotic nitrogen-fixing bacteria are present. However, it is possible to artificially grow the nodule by inoculating this nodulating plant with a symbiotic nitrogen-fixing bacterium. Inoculation of symbiotic nitrogen-fixing bacteria into nodulating plants can be performed according to methods well known to those skilled in the art (for example, Higashi, S., Katahira, S. and Abe, M. Plant and Soi 81, p. 91-99 (1984)).

In the present invention, the “symbiotic nitrogen-fixing bacterium” means a microbial organism that has the ability to symbiotic to a plant to form a nodule, fix nitrogen as ammonia, and give it to a host plant (nitrogen fixing ability). Symbiotic nitrogen-fixing bacteria include Rhizobium, Bradyrishibium, Azorhizobiu, Sinorhizobium, Mesorhizobium, Mesorhizobium, Mesozobium Among actinomycetes, the genus Frankia is there. It is known that rhizobia infects leguminous plants and plants of the genus Parasponia, and Francia spp. Are known to infect mainly trees such as birch family and leguminous family. Exceptions are also known for host plant relationships. Therefore, the transformed nodulating plants may be inoculated with symbiotic nitrogen-fixing bacteria capable of infecting the plant species. For example, Miyakodasa may be inoculated with Mesorhizobiu m loti.

In the present invention, when a symbiotic nitrogen-fixing bacterium is inoculated into a nodule-growing plant in which a non-symbiotic guttine gene is overexpressed and the nodule is allowed to grow, the nitrogen-fixing activity in the nodule increases markedly. The nitrogen fixation activity in this nodule is at least 2 times, preferably at least 3 times per nodule unit weight, compared to a control of a plant of the same species that does not overexpress the non-symbiotic globin gene (wild strain). For example, it increases 3 to 6 times. The nitrogen fixation activity in the nodules may be performed using any method for measuring nitrogen fixation activity known to those skilled in the art. In the present invention, the nitrogen fixation activity in the nodules is preferably measured as an activity of reducing acetylene to ethylene (acetylene reduct ion activity; ARA activity) for the extract from the nodules. ARA activity can be expressed as the amount of ethylene generated per unit weight (eg 1 gram) of root nodules and per unit time (eg 1 hour, 1 minute). The ARA activity may be measured according to the examples described later, for example. The nodule-growing plant in which the non-symbiotic globin gene according to the present invention is overexpressed is not limited, for example, 10 to 100 nM / min / g, preferably 11 to 40 nM / min in the nodule. Shows ARA activity of / g.

The present invention also relates to a nodule-growing plant that is obtained as described above and overexpresses a non-symbiotic globin gene and has a root nodule.

5. Other embodiments

The nodule-growing plant produced by the method of the present invention grows nodules that exhibit high nitrogen-fixing activity, so that a large amount of nitrogen in the air is fixed in the cultivation environment by cultivating them. Can do. Therefore, the present invention also provides a method for increasing the nitrogen fixation efficiency in plant cultivation. “Increasing the efficiency of nitrogen fixation in plant cultivation” means that nitrogen fixation within a certain period per certain cultivation area or per individual cultivation plant. It means increasing the amount of quantification compared to the amount of nitrogen fixation by the same untransformed plant in the same environment. 'Cultivation' in the present invention means that the plant is intentionally grown in a specific place or environment. In the present invention, “cultivation” includes agricultural cultivation, but it is not always necessary to perform agricultural work (cultivation, sowing, planting, thinning, disinfection, pruning, thinning, harvesting, etc.). The cultivation in the present invention is not limited, for example, cultivation of crops and horticultural plants, landscaping, horticulture, planting for greening of degraded land and beaches, planting for fertilization of oligotrophic soil This includes planting for soil improvement such as salt and dry soils.

The nodulation plant produced by the method of the present invention increases the nitrogen fixation efficiency in plant cultivation, thereby increasing the amount of nitrogen fixed from the air under a certain environment, and the nitrogen fixation concentration in the plant tissue. And the yield or growth of the nodulating plant can be increased. Furthermore, by cultivating the nodulating plants of the present invention, it is possible to fertilize the soil by increasing the amount of nitrogen in the soil over the long term, and the yield of other plants using the soil. Can also be increased. In addition, by cultivating the nodulating plants of the present invention, it is possible to effectively greenen oligotrophic soil, salt soil, dry soil, and the like. Example

Hereinafter, the present invention will be described with reference to examples, but the technical scope of the present invention is not limited thereto.

[Example 1] Isolation and identification of non-symbiotic globin gene (LjHbl) of Lotus japonicus The non-symbiotic globin gene (LjHbl) of Lotus japonicus Res. 8: p. 311-318) was isolated by screening by PCR. The primer used for screening is based on the non-symbiotic globin gene homologue sequence (clone name: AV413959, DDBJ / EMBL / GenBank accession number: AB2 38220) present in the Miyakodasa EST library. Designed. The sequence of this primer is as follows.

LjHb lFl: 5,-TTCTCACTTCACTTCCATCGC-3 '(SEQ ID NO: 4; forward primer one) LjHblF2: 5'-TTGGTCAAGTCATGGAGCG-3, (SEQ ID NO: 5; forward primer) LjHblRl: 5'-TCACAGTGACTTTTCCAGCG-3 '(SEQ ID NO: 6; reverse primer) LjHblR2: 5'-AGACAGACATGGCATGAGGC-3' (SEQ ID NO: 7; reverse) ) GeneArap ^(R) PCR System 9700 (Applied Biosystems) was used to amplify the LjHbl gene under the following reaction conditions: 94 ° C for 30 seconds, 55 ° C for 30 seconds , 30 cycles of 30 seconds at 2 ° C.

As a result of screening of the Miyakodasa genomic library, we identified the gene LjHbl present on the TAC clone (LjTOIOl). As a result of analyzing the base sequence of LjHbl on the Lotus japonicus genome, it was found that this gene has a total length of 1012 bp from the start codon to the stop codon and encodes 161 amino acid residues. Fig. 1 shows the genomic structure and amino acid sequence of the non-symbiotic globin gene (LjHbl). The gene LjHbl has a structure containing four exons and three introns common to the plant globin gene, and was present on chromosome 3 of the six chromosomes of Miyako Dasa.

[Example 2] Expression analysis of non-symbiotic globin gene (LjHbl)

Expression analysis of LjHbl was performed in two experimental systems by RT-PCR: expression by tissue and expression under stress conditions. In the experimental system that examined the expression by tissue, four tissue samples were used: 1) leaves, 2) stems, 3) roots, and 4) nodules. In the experimental system in which the expression under stress conditions was examined, four samples were used: 1) no treatment (control), 2) sucrose addition, 3) low temperature, and 4) hypoxia. For RT-PCR, the primers LjHblFl and LjHblR2 used in Example 1 were used, and the One-step RT-PCR kit (QIAGEN) was used for reverse transcription and transcription product amplification. The expression level of the gene LjHbl was confirmed by electrophoresis. Electrophoresis photographs were imaged, and the expression level of LjHbl was expressed as a relative value based on the intensity of the band.

The results of an experiment examining the expression by tissue are shown in Fig. 2A. LjHbl was shown to be expressed in various organs of growing individuals. The expression levels (relative values) were 0, 4 for leaves, 0.4 for stems, 1.0 for roots, and 36.0 for root nodules. Thus, it was strongly expressed especially in the nodule tissue. Figure 2B shows the results of an experiment examining the expression under stress conditions. The LjHbl is strongly expressed by stress treatment such as low temperature (expression level in Fig. 2B: about 250) and hypoxia (expression level in Fig. 2B: about 450) when viewed as a relative expression level compared to no treatment. It was shown to be expressed.

[Example 3] 'Construction of vector for transformation

In order to produce transformed Lotus root plants and transgenic hairy roots that overexpress LjHbl, a gene was constructed in which LjHb1 cDNA was linked to a strong promoter. Vectors for transformation include plasmid pKANNIBAL (Wesley et al. 2001 the plant Journal 27, 581—590), pHKN29 (Kumagai and Kouchi, 2003 MPMI 16 (8), 663—668), pIG121_Hm (Ohta et al., 1990 Plant Cell Physiol., 31, 80 5-813). .

Total RNA was extracted from the root nodules of Lotus japonicus by conventional methods, cDNA was synthesized by RT-PCR, and full-length LjHbl cDNA was cloned by PCR using this cDNA as a cage. The obtained full-length LjHbl cDNA (SEQ ID NO: 1) was first ligated downstream of the 35S promoter of pKANNIBAL's california mosaic inoless. Furthermore, a 35S-LjHbl cDNA fragment was excised from this vector and ligated downstream of the GFP region of pHKN29 to obtain a plasmid vector pR35SLjHbl (Fig. 3). This pR35SLjHbl was used for the induction of transformed hairy roots as described below.

The full-length LjHbl cDNA was ligated downstream of the 35S promoter of pIG12I-Hm to obtain a plasmid vector pT35SljLbl. The _P T35S1 _jLhl, as described below, was used for production of transformants conversion Miyakodasa plants.

[Example 4] Production of transformed hairy root and introduction of gene and confirmation of expression

Using the plasmid vector prepared in Example 3, a hairy root-inducible transformation system mediated by agrobacterium lysogenes was used to produce transgenic hairy roots of Miyako-dasa introduced with LjHbl. did.

First, the vector _P R35SLjHb l constructed to overexpress LjHbl in Example 3, Aguronoku Kuteriumu rhizogenes (Agrobacterium rhizogenes) LBA1334 (Dr. Clara Diaz (Institute Molecular Plant Science, minute from Leiden University) On the other hand, it was introduced directly by electo-portion. The cell suspension of Agrobacterium lysozyme carrying this pR35SLjHbl was inoculated into Miyakogusa seedlings on the fifth day after seeding, which had been cut at the hypocotyl part. Then, this was placed on a sterilized filter paper and co-culture medium (1/10 B5, BAP 0.5 ^ g / ml, NAA 0. Οδ, α Ε / ηιΙ, MES (pH 5.2) 5 mM, acetosyringone 20 _g / ml) for 5 days. After completion of co-cultivation, put it on Gamb orgB5 medium (manufactured by Nippon Pharmaceutical Co., Ltd., sales: Wako Pure Chemical Industries, Ltd.) supplemented with the antibiotic cefotax (200 g / ml; Chugai Pharmaceutical Co., Ltd.) Induced roots. Induced hairy roots are shown in FIG. 4A. Figure 4A shows various samples with different growth stages and hairy roots.

The presence or absence of LjHbl gene introduction into hairy roots was confirmed by detecting GFP fluorescence and PCR amplification of a fusion gene of 35S promoter and L jHbl. In the hairy root into which the LjHbl gene was introduced, GFP was produced and green fluorescence was observed (Fig. 4B). The photo on the left (upper and lower) of Fig. 4B is the result of bright field observation, and the photo on the right (upper and lower) is the result of dark field observation. Roots where symbiotic globin genes are overexpressed and GFP fluorescence is observed are clearly observed.

Furthermore, total RNA was extracted from transformed hairy roots by a conventional method, and RT-PCR was performed to confirm the expression of the introduced LjHbl gene. In addition, a wild-type hairy root was used as a control. The results are shown in Fig. 4C. In Fig. 4C, the gene Lj elF-4A, which is known to be expressed at the same level in all tissues of Miyakodasa and at any time, is compared to the test sample and the control (control) sample. It was used as an index to show the same.

As shown in this result, the hairy root into which LjHbl gene was introduced had about 100 times the amount of LjHb1 gene expression induced as compared to the wild-type hairy root into which LjHbl gene was not introduced. .

.

[Example 5] Measurement of nodule formation and nodule nitrogen fixation activity in transformed hairy roots Hairy root-derived plants in which the introduction of the LjHbl gene was confirmed in Example 4 were obtained from cultured soil (Vermiculite: perlite = 4:. transplanted in 1), KN0 ₃ a Ferausu medium final concentration was added to a 1 mM (Fahraeus, (1957) J. Gen. Microbiol 16 (2) 374-38 1) and allowed to grow for 1 week. Then, it was transplanted to a new culture soil, and the hairy root was inoculated with Mesorhizobium loti AFF 303099 at a concentration of 1 × 10 ⁷ cells / ml. After inoculation with rhizobia, a medium containing no nitrogen source was given, and further grown for 4 weeks. The growth conditions of the transformed plants were 16 hours light / 8 hours dark, 25-26 ° C in the plant growth chamber. Four weeks later, nodules were formed on the hairy roots (Fig. 5).

Next, the nitrogen fixation activity of the nodules grown on the hairy roots was measured as the activity of reducing acetylene to acetylene (Acetylene reduction activity; ARA). Specifically, first, nodules collected from the hairy roots were placed in a 15 cm test tube and sealed with a rubber cap. The air in the test tube was sufficiently aspirated with an aspirator, and then filled with acetylene. After incubating the test tube at room temperature for 2 hours, the gas in the test tube was collected, and the amount of ethylene generated was measured by gas chromatography. Figure 6 shows the measurement results.

Based on the measurement results shown in Fig. 6, the ARA activity per unit weight (gram weight of root nodules) in root nodules grown on hairy roots over-expressed with LjHbl was calculated to be 11. 15 nM / min / g [1. 45 X 21. 5 -0. 5 = 30. 67 (nM / g); 30. 67 (nM) ÷ 2. 75 min = about 11.15 n M / rain / g]. On the other hand, as a control experiment, the ARA activity per unit weight (gram weight of root nodules) in root nodules grown on hairy roots without LjHbl was calculated as 2, 12 nM / min / g [1 45 X 4- 0. 5 = 5.3 (nM / g); 5.3 (nM) ÷ 2.5 minutes = 2.12 nM / min / g]. It was shown that the nodule grown on hairy roots (transformants) overexpressed with Lj Hbl increased nitrogen fixation activity by a factor of 5 or more.

[Example 6] Production of transformed plant body

1) Infection of A. tumefaciens in Miyakogusa

Hypocotyls were cut out at the boundary between the cotyledons and roots from the seedlings of Miyakogusa 5 days after sowing. On the other hand, pT35SljLbl prepared in Example 3 was directly introduced into Agrobataterum 'A. tumefaciens EHA105 (provided by Prof. Kenzo Nakamura at Nagoya University) with an electoral position. Hold the resulting pT35SljLbl Add a final concentration of 100 M of acetosyringone to the bacterial suspension of EHA105 (1.0 X 10 ⁷ cells / m 1, 0D ₆₀₀ = 0. 10 X 0. 15) The excised hypocotyl was immersed in the solution. The hypocotyl was cut into sections approximately 5 mm thick in solution. This section was directly infected with agrobacterium by immersing it in a cell suspension for 30 minutes. Place the infected sections on sterile filter paper and coculture medium (1/10 B5, BAP 0.5 μg / ml, NAA 0.05, g / ml, MES (pH 5.2) 5 mM, Toshirin Gon 20 μ β _/ πι1) in, and co-culture for 3 to 5 days at 25 ° C.

2) Callus induction

The co-cultured sections were treated with callus medium (1 X B5, 2% sucrose, BAP 0.5 μg) containing cefotax (250 1) for sterilization and hygromycin B for selection of transformants. / ml, NAA 0.05 g / ml, 10 mM NH ₄ , 0.3% phytagel) and cultured at 25 ° C. in a cycle of 14 hours light / 10 hours dark for 5 weeks. Sections were replanted every 1-2 weeks.

3) Induction of shoot from callus

Sections cultured in the above callus medium for 5 weeks were taken as shoot induction medium (1 X B5, 2% sucrose, BAP 0.5 μg / ml, NAA 0.05 / g / ml, 10 mM NH ₄ , 0.3% phytagel) and cultured at 25 ° C. in a cycle of 14 hours light (6100 lux) 10 hours darkness for 2 weeks. Thereafter, the callus sections were transplanted to a culture medium without addition of hygromycin B, and cultured for 3 weeks under the same culture conditions as described above. Callus replanting was done every 1-2 weeks.

4) Shout growth

Callus was transferred to shoot elongation medium (1 X B5, 2% sucrose, BAP 0.2 g / ml, 0.3% phy tagel) at 25 ° C for 14 hours light (6100 lux) 10 hours dark cycle And cultured for 3 weeks. Callus replanting was done every 1-2 weeks. After that, the vigor was transplanted to a shoot elongation medium containing no plant hormone, and cultured for 2 to 3 weeks under the same culture conditions as described above to promote the elongation of shout.

5) Root induction and elongation

A shoot of 5 mm or more generated from a callus placed on a shoot elongation medium was cut from the base of the stem with a force razor. Make this chute vertical and root the chute 14 hours light (6100 lux) / 10 hours dark cycle for 1 week in induction medium (1/2 B5, 1% sucrose, 0.5 ju g / ml NAA, 0.4% phytagel) The above was cultured. Thereafter, the shoots with enlarged cut ends were inserted into a root elongation medium (1/2 B5, 1% sucrose) and cultured for 2 to 3 weeks under the same culture conditions as described above to promote root elongation.

6) Cultivation of transformed plants

The plant obtained by extending the roots as described above was extracted from the medium, and the gel adhering to the roots was washed well in water. This plant was transplanted to vermiculite soaked with a commercially available B5 medium (Wako Pure Chemical Industries, Ltd.) diluted 1/10 times, and grown in a cycle of 14 hours light (6100 lux) / 10 hours darkness did. The Miyakogusa plant grown in this way was seeded and harvested. After that, the seeds were sown and cultivated with a power soil (Taleha Horticulture Soil).

The plant thus grown was confirmed by PCR amplification of the 35S promoter and LjHbl fusion gene to confirm the success of LjHbl gene transfer. The increase in LjHbl gene expression level was confirmed by RT-PCR.

[Example 7] Nodulation and nitrogen fixation activity of Miyakodasa transformed plants

The Lotus japonicus-transformed plant produced in Example 6 and confirmed to have introduced and expressed the LjHbl gene was transplanted and cultured in cultured soil (Vermiculite: perlite = 4: 1), and 1 X 10 ⁷ cells / Inoculated with Mesorhizobium loti MAFF 30309 9 at a concentration of ml. After inoculation with the rhizobia, the transformed plants were cultivated at 25-26 ° C. for 4 weeks in a plant growth chamber with a cycle of 16 hours light (6100 lux) / 8 hours dark. After 4 weeks, the grown nodules were collected from the plant body, and ARA activity was measured in the same manner as in Example 5. As a result of the measurement, the ARA activity per unit weight (gram weight of root nodules) in the root nodules that had overexpressed LjHbl was calculated to be 17. 21 nM / min / g. On the other hand, as a control experiment, the ARA activity per unit weight (gram weight of root nodules) in the root nodules that had not introduced LjHbl (normal Miyakodasa, wild type) was 5. 05 nM / min / g Calculated.

The nodules formed in the plant obtained in this example are transformed and non-transformed. The average number of all the transformants was 7. There was no particular difference in appearance such as nodule size and color.

[Example 8] Comparison of the affinity of Miyakodasa non-symbiotic globin and symbiotic globin nitric oxide

The full-length LjHbl cDNA obtained in Example 3 (the sequence from the start codon to the stop codon is shown in SEQ ID NO: 1; it encodes the amino acid sequence of SEQ ID NO: 2) is expressed in the protein expression vector PGEX4T-3 (Amersham Pharmacia Biotech) was cloned by a conventional method (FIG. 7) and introduced into E. coli to obtain a transformant. As a control sample, the symbiotic globin gene was similarly cloned into the expression vector pGEX4T-3 (Fig. 7) and introduced into E. coli to obtain a transformant. By culturing the resulting Escherichia coli transformant and inducing expression, it was possible to produce a large amount of soluble and active non-symbiotic globin in the body of Escherichia coli. Subsequently, Escherichia coli expressing the non-symbiotic globin gene was recovered by a conventional method, lysed, and then purified by protein. As a result, active non-symbiotic hemoglobin could be obtained. Since hem is simultaneously supplied in E. coli introduced with the non-symbiotic gout bin gene, the globin obtained by destroying E. coli is in a state where it has activity as hemoglobin in combination with heme derived from E. coli. It was isolated by

Subsequently, nitric oxide was mixed with the obtained symbiotic hemoglobin, and absorbance was measured over time. Fig. 8 shows absorbance spectra at wavelengths of 500 nm to 600 nm at 0 minutes, 5 minutes, 15 minutes, and 30 minutes after the start of nitric oxide mixing. As shown in Fig. 8, it is shown that two peaks at 540nm and 575nm disappeared as the mixing time of nitric oxide increased. Especially in non-symbiotic hemoglobin, the peak at 575 nm was lost earlier and almost disappeared after 15 minutes. On the other hand, in the case of symbiotic hemoglobin, the peak at 540nra did not decrease much, and even after 30 minutes, the peak at 575nm was still weak, but two peaks were observed.

[Example 9] Isolation and identification of Yashabushi non-symbiotic globin gene (AfHbl) The non-symbiotic globin gene (AfHbl) of Yashabushi (Alf firma) Sacher, F. et al., A class 1 hemoglobin gene from A lnus r irma functions in symbiotic and nonsymbiotic tissues to detoxify n itric oxide, using the DNA fragment of the non-symbiotic globin gene LjHbl as a probe. , Mol. Plant Microbe. Interact. (2006) 19 (4) during printing).

The screening probe was designed based on the sequence of Miyakogusa non-symbiotic globin gene LjHbl (clone name: AV413959, DDBJ / EMBL / GenBank accession number: AB238220), and the primer LjHblFl (5 '-TTCTCACTTCAC TTCCATCGC-3'; SEQ ID NO: 4) and LjHblRl (5'-TCACAGTGACTTTTCCAGCG-3 '; SEQ ID NO: 6) were used for PCR amplification from Miyako Sasa genomic DNA. This PCR to amplify the LjHbl fragment as a probe uses GeneAmp ⁰ PCR System 9700 (Applied Biosystems), 30 seconds at 94 ° C, 30 seconds at 55 ° C, 30 seconds at 72 ° C. The test was conducted under the condition of 30 cycles.

As a result of the screening of one of the rhododendron nodule cDNA libraries, the AfHb 1 gene was identified. As a result of nucleotide sequence analysis, it was found that the AfHbl gene has a base length of 483 bp from the start codon to the stop codon in the cDNA and encodes 160 amino acids. The nucleotide sequence from the start codon to the stop codon of cDNA of AfHbl (DDBJ / EMBL / GenBank accession number: AB221344) is shown in SEQ ID NO: 8, and the encoded amino acid sequence is shown in SEQ ID NO: 9. Furthermore, the nucleotide sequence (from the start codon to the stop codon) of the genomic DNA of Yachabushi AfHbl gene is shown in SEQ ID NO: 10.

[Example 10] Expression analysis of Yashabushi non-symbiotic globin gene (AfHbl)

The expression analysis of AfHbl was performed using RT-PCR using mRNA extracted from tissues of various organs of Yashapsi by a conventional method. The following AfHblFl and AfHblR3 primers are used for RT-PCR, One-Step RT-PCR kit (QIAGEN) is used for reverse transcription and amplification of the transcript, and the following AfHblFl primer is also used for amplification of the reverse transcript. And AfHblR3 primer. The sequences of those primers used are shown below.

AfHblFl: 5>-GCTGCTATCAAATCTGCAAT-3, (SEQ ID NO: 1 1; forward primer one)

AfHblR3: 5,-GGGGGGCTGTGATTTTAG-3, (SEQ ID NO: 12; reverse primer) The obtained amplification product was electrophoresed, and the photographed electrophoretogram was imaged to determine the AfHbl gene in each tissue from the intensity of the band. The expression level was determined.

The results of this RT-PCR expression analysis indicated that the AfHbl gene was expressed in various organs of Yashabushi growing individuals. In particular, it was strongly expressed in the nodule tissue. It was also found that the expression of the AfHbl gene was strongly induced by stress treatment such as low temperature, similar to the Miyakodasa LjHbl gene. [Example 1 1] Construction of vector for transformation

In order to elucidate the function of the AfHbl gene, we attempted to create transgenic Miyakodasa that overexpresses the AfHbl gene. The plasmids pKAN NIBAL (Wesley et al. 2001 The Plant Journal 27, 581-590) and pHKN29 (Kumagai and Kouchi. 2003 MPMI 16 (8), 663-668) were used for transformation vector construction. .

First, a full-length AfHbl cDNA was cloned by PCR using cDNA synthesized by reverse transcription from total RNA extracted from the root nodules of Yachabushi. The obtained AfH bl cDNA was ligated downstream of the 35S promoter derived from cauliflower mosaic virus of pKANNIBAL. Furthermore, this 35S-AfHbl cDNA fragment was excised and ligated downstream of the GFP region of pHKN29 to obtain the final transformation vector pAfHblS.

[Example 1 2] Production of transformed hairy roots

For the transformation of Miyakodasa using AfHbl, a hairy root-inducible transformation system via agrobacterium lysogenis was employed. This transformation method is based on the principle of co-transformation, and the induced hairy roots are transformed by agrobacterium terrizogenes. Production of transformed hairy roots in this example was performed according to the method of Example 3.

The transformation vector pAfHblS constructed in Example 11 and constructed so as to overexpress the AfHbl gene was directly introduced into Agrobacterium lysogenes LBA1334 by electroporation. Agrobatery introduced with the vector pAfHblS The cell suspension of lysogenes LBA1334 was inoculated and infected to Miyakogusa seedlings 5 days after seeding cut at the hypocotyl part. Thereafter, this was placed on sterilized filter paper and co-cultured for 5 days in a co-culture medium. After completion of co-culture, agar medium [Gambor gB5 medium supplemented with antibiotic cefotax (200 μg / ml; Chugai Pharmaceutical Co., Ltd.) I put it on top and induced a hairy root.

The presence or absence of AfHbl introduction into the hairy root was confirmed using GFP fluorescence detection and RT-PCR in the same manner as in Example 4. As a result, hairy roots that fluoresce GFP were induced in all A. thaliana individuals infected with Agrobacterium terrestrialis introduced with AfHbl. On the other hand, as a result of confirmation by RT-PCR, hairy roots (transformants) into which AfHbl had been introduced had about 100 times more AfHbl than the wild-type hairy roots into which AfHbl had not been introduced. Gene expression was induced. Figure 9 shows the results of confirmation of AfHbl gene expression using RT-PCR. In FIG. 9, LjelF-4A is a positive control. [Example 1 3] Analysis of phenotype of transformed individuals

The plant with a hairy roots transformation with AfHbl gene was confirmed in Example 1 2, potting (Bamikiyuraito: pearlite = 4: 1) to implanted, final concentration is 1 mM to KN0 ₃ Feraus medium (Fahraeus, (1957) J. Gen. Micro biol. 16 (2) 374-381) added as described above was fed and grown for 1 week. Then transferred to a new culture soil was inoculated Lotus japonicus root nodule bacteria at a concentration of IX 10 ⁷ cells / ml of (Mesorhizobium loti MAFF 3030 99) in the hairy roots. After inoculation with rhizobia, Feraus medium without nitrogen source was given and grown for another 4 weeks. The growth conditions of the transformed plants were 16 to 8 hours dark, and the temperature was 25 to 26 ° C in the plant growth chamber. Nodules were formed on the hairy roots of the grown plants. The average number of nodules was 9 per plant for both transformants and non-transformants. There was no difference in the appearance of the nodule size and color between the transformant and the non-transformant.

Next, as one of the phenotypes of the transformant, the nitrogen fixing activity of nodules grown on the hairy roots was measured. The nitrogen fixation activity was measured according to the method described in Example 5 as the activity of reducing acetylene to ethylene (Acetylene reduction activity; ARA). Set. The results are shown in FIG. In FIG. 10, the control is Miyakogusa introduced with brass pHKN29 that does not contain the AfHbl gene as a sample.

As a result of the measurement, the nodules grown on the hairy roots overexpressed with AfHbl introduced were 3 to It showed 5 times the nitrogen fixation activity. As shown in FIG. 10, nodules grown on hairy roots overexpressed with AfHbl showed an ARA activity (average value) of 7.2 nM / min / g per unit weight. Measurements were also made on the whole plant of Miyakodasa with AfHbl introduced, and an ARA activity (average value) of 13 nM / min / g per unit weight was shown. On the other hand, as a control experiment, nodules grown on wild-type hairy roots not introduced with AfHbl showed an ARA activity (average value) of 2.6 nM / min / g per unit weight. The whole wild-type plant without AfHbl introduced showed an ARA activity (average value) of 5 nM / min / g per unit weight.

From the above results, it was shown that the nitrogen fixation activity in the nodules grown on the hairy roots was also remarkably improved even in Miyakogusa introduced with the non-symbiotic globin gene AfHbl. Industrial applicability

By the method for producing a nodule-growing plant of the present invention, a nodule-growing plant in which the nitrogen-fixing activity of the nodule is significantly improved can be obtained. The method of increasing the nitrogen fixation amount in plant cultivation by cultivating the nodulating plants of the present invention is intended to increase the yield or growth amount of the nodulating plants, or to increase the amount of nitrogen in the soil. It can be used for the purpose of fertilizing soil and greening desolated land. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. Sequence table free text

The sequences of SEQ ID NOs: 4-7, 11 and 12 represent primers.

Claims

The scope of the claims

1. A method for producing a nodule-growing plant capable of growing a nodule having increased nitrogen fixation activity, characterized by overexpressing a non-symbiotic gout bin gene in the nodule-growing plant.

2. The method according to claim 1, wherein the non-symbiotic globin gene is composed of DNA selected from the group consisting of the following (a) to (e).

(a) DNA comprising the base sequence shown in SEQ ID NO: 1 or 8

(b) DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 8, and encodes a protein having non-symbiotic globin activity.

(d) DNA consisting of an amino acid sequence in which 1 to 50 amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 9, and encoding a protein having non-symbiotic globin activity

(e) DNA consisting of the base sequence shown in SEQ ID NO: 3 or 10

3. The method according to claim 1 or 2, wherein the non-symbiotic globin gene is overexpressed by introducing a non-symbiotic globin gene linked to an overexpression promoter into a nodulating plant.

4. The method according to any one of claims 1 to 3, wherein the increase in nitrogen fixation activity is at least a 3-fold increase compared to the wild type strain.

5. The method according to any one of claims 1 to 4, wherein the nodule having an increased nitrogen fixation activity is a nodule exhibiting a nitrogen fixation activity of 10 to 100 nM / min / g.

6. The method according to any one of claims 1 to 5, wherein the nodulating plant is a leguminous plant. Law

7. The method according to any one of claims 1 to 6, further comprising inoculating a nodule-growing plant overexpressing the non-symbiotic globin gene with a symbiotic nitrogen-fixing bacterium.

8. A root nodule plant produced by the method according to any one of claims 1 to 7 and capable of growing nodule having increased nitrogen fixation activity.

9. The nodule-growing plant according to claim 8, wherein the root has nodules.

10. A vector for increasing the nitrogen fixation activity of nodules, comprising a non-symbiotic gbin gene linked to an overexpression promoter.

11. The vector according to claim 10, wherein the non-symbiotic globin gene is derived from a leguminous plant or a non-legume nodulating plant.

1 2. The vector according to claim 10 or 11, wherein the non-symbiotic globin gene is composed of DNA selected from the group consisting of:

(a) DNA comprising the base sequence shown in SEQ ID NO: 1 or 8

(e) DNA consisting of the base sequence shown in SEQ ID NO: 3 or 10

1 3. A method for increasing nitrogen fixation efficiency in plant cultivation, characterized by cultivating the nodulating plant according to claim 8 or 9.