Patent Grant
10544425
The present invention relates to an agent for providing a plant with resistance or susceptibility to a 4-HPPD inhibitor, a transgenic plant cell capable of regenerating a plant having increased resistance or susceptibility to a 4-HPPD inhibitor, a plant regenerated from the cell, and methods for producing these. Further, the present invention relates to a method for determining whether a plant has resistance or susceptibility to a 4-HPPD inhibitor, and a method for breeding a plant having increased resistance or susceptibility to a 4-HPPD inhibitor by utilizing the determination.
Along with an increase in the demand for bioethanol fuels, the import price of feed grains from overseas has increased drastically, which has imposed heavy burden on livestock businesses in Japan. In this situation, in order to enhance domestic production and self-sufficiency rate of feeds, not paddy rice but alternate crops are cultivated by utilizing fallow fields and so forth. However, paddy fields suitable for cultivating these alternate crops are limited due to problems such as poor drainage. For this reason, use of rice as feeds and developments of exclusive feed-rice cultivars having a high productivity (high yielding cultivars) have been promoted. In order for such high yielding cultivars to demonstrate their characteristic high-yielding properties and stable growability, and also to improve the palatability and nutritional value of livestock, weed control in the cultivation paddy fields is an important cultivation management technique (NPL 1). Further, stable and economical productions of not only high yielding cultivars and rice but also crops require low-cost, energy-saving and easy weed control. Development and use of a highly selective herbicide are effective in such control (NPL 2). Hence, required are development and cultivation of crops resistant to the herbicide used.
Meanwhile, in the weed control in cultivation paddy fields, sulfonylurea (SU) herbicides are widely employed because the herbicides are effective against a wide range of weeds at a low dose and have little influence on human and also on the environment. Nevertheless, emergence of weeds such as Scirpus juncoides Roxb. having tolerance to SU herbicides has been recognized. This brings about a problem in the cultivation management for rice and so on.
Recently, as the measure against such a problem, herbicide components such as benzobicyclon (BBC), mesotrione, and tefuryltrione have been developed, which are also effective against plants tolerant to SU herbicides and have been put into practical use. All of BBC, mesotrione, and tefuryltrione are agents for inhibiting a function of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) (4-HPPD inhibitors). Inhibiting a function of this enzyme indirectly inhibits a carotenoid synthetic system and causes chlorophyll degradation to thereby whiten and wither the plant to death (see
However, susceptibility of high yielding cultivars to the 4-HPPD inhibitors was not examined sufficiently at the developmental stage or other stages. By now, it has been reported that seven high-yielding feed rice cultivars are susceptible to 4-HPPD inhibitors and may be withered to death in some cases (NPLs 1 and 3).
Developments of a method capable of surely identifying the resistance or susceptibility to a 4-HPPD inhibitor and a method capable of increasing the resistance or susceptibility to a 4-HPPD inhibitor would make it possible to utilize a 4-HPPD inhibitor for control of a germination risk (problem from fallen seeds and seedlings) of “self-sown seeds” from the previous year in crop rotation cycles with food and feed rice cultivars as shown in
Accordingly, it has been strongly desired to develop a technique for providing a plant with resistance or susceptibility to a 4-HPPD inhibitor and a technique for determining whether a plant has resistance or susceptibility to a 4-HPPD inhibitor. However, techniques which can efficiently achieve these purposes have not been developed yet.
[NPL 1] Keisuke Sekino et al., “Sensitivity of Nineteen Varieties and Line of Forage Rice to Paddy Herbicide, benzobicyclon,” Japanese Journal of Crop Science, Mar. 25, 2009, vol. 227, extra issue, pp. 120 to 121
[NPL 2] Terry R. Wright et al., Proc Natl Acad Sci USA., Nov. 23, 2010, vol. 107, no. 47, pp. 20240 to 20 245
[NPL 3] Kiyoaki Maruyama et al., “Forage rice and so forth are revealed to be susceptible to some herbicide”, [online], Mar. 26, 2010, National Agriculture and Food Research Organization Agricultural Research Center, Press Release, [retrieved on Sep. 29, 2010], the Internet <URL:http://narc.naro.affrc.go.jp/press/h22/0326/index.htm>
The present invention has been made in view of the problems of the above-described conventional techniques. An object of the present invention is to provide a technology capable of efficiently providing a plant with resistance or susceptibility to a 4-HPPD inhibitor, and to provide a technology capable of efficiently determining whether a plant has resistance or susceptibility to a 4-HPPD inhibitor.
In order to achieve the above object, the present inventors, first, attempted to identify genes associated with resistance to a 4-HPPD inhibitor in a plant. Specifically, the present inventors, first, performed a quantitative trait locus (QTL) analysis using 4-HPPD inhibitor-susceptible rice and 4-HPPD inhibitor-resistant rice. This result revealed that a gene locus for determining resistance to a 4-HPPD inhibitor was located on a short arm of chromosome 2 of rice. Then, the present inventors examined a phenotype (4-HPPD inhibitor-susceptibility) using a Nipponbare line having a retrotransposon Tos17 inserted into a hypothetical gene of an iron/ascorbate-dependent oxidoreductase gene located at the gene locus specified by the QTL analysis. It was found out that Tos17-inserted homozygous individuals showed susceptibility to a 4-HPPD inhibitor. When the iron/ascorbate-dependent oxidoreductase gene thus found out was introduced into Arabidopsis thaliana (A. thaliana) and rice, these transgenic plants showed resistance to a 4-HPPD inhibitor. This supported that this gene is a responsible gene for providing a plant with resistance to a 4-HPPD inhibitor (hereinafter also referred to as 4-hydroxyphenylpyruvate dioxygenase inhibitor sensitive gene No. 1 (HIS1)). Additionally, genes having a high homology with the HIS1 gene of rice also exist in barley, sorghum, corn, and the like.
Further, the present inventors compared the structure of the HIS1 gene between the 4-HPPD inhibitor-susceptible rice and the 4-HPPD inhibitor-resistant rice by a PCR analysis. As a result, the rice cultivars showing susceptibility to a 4-HPPD inhibitor had insertion or deletion from the fourth exon to the fifth exon of the HIS1 gene. This may suggest that suppressing a function of the HIS1 gene provide a plant with susceptibility to a 4-HPPD inhibitor.
Moreover, it was revealed that a rice gene (HSL1 gene) having the highest homology with the HIS1 gene was located on chromosome 6 of rice. Furthermore, it was also revealed that when the HSL1 gene was introduced into rice, the transgenic rice also showed resistance to a 4-HPPD inhibitor.
Based on these findings, the present inventors found out that a utilization of the HIS1 gene or a homologous gene thereof makes it possible to produce a plant having increased resistance or susceptibility to a 4-HPPD inhibitor, and that targeting the genes makes it possible to determine whether a plant has resistance or susceptibility to a 4-HPPD inhibitor. These discoveries have led to the completion of the present invention.
More specifically, the present invention is as follows.
(1) An agent for providing a plant with resistance to a 4-HPPD inhibitor, the agent comprising at least one DNA or a vector having the DNA inserted therein, the DNA encoding a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor, the DNA selected from the group consisting of the following (a) to (d):
(a) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17;
(b) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17, in which one or more amino acids are substituted, deleted, added, and/or inserted;
(c) a DNA hybridizing under stringent conditions to a DNA having a base sequence of any one of SEQ ID NOs: 1 and 16; and
(d) a DNA encoding an amino acid sequence having a homology of 60% or more with an amino acid sequence of any one of SEQ ID NOs: 2 and 17.
(2) A transgenic plant cell capable of regenerating a plant having increased resistance to a 4-HPPD inhibitor, the transgenic plant cell comprising at least one DNA or a vector having the DNA inserted therein, the DNA encoding a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor, the DNA selected from the group consisting of the following (a) to (d):
(a) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17;
(b) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17, in which one or more amino acids are substituted, deleted, added, and/or inserted;
(c) a DNA hybridizing under stringent conditions to a DNA having a base sequence of any one of SEQ ID NOs: 1 and 16; and
(d) a DNA encoding an amino acid sequence having a homology of 60% or more with an amino acid sequence of any one of SEQ ID NOs: 2 and 17.
(3) A plant having increased resistance to a 4-HPPD inhibitor, which is regenerated from the transgenic plant cell according to (2).
(4) A plant having increased resistance to a 4-HPPD inhibitor, which is any one of a progeny and a clone of the plant according to (3).
(5) A propagation material of the plant having increased resistance to a 4-HPPD inhibitor according to any one of (3) and (4).
(6) A method for producing a plant having increased resistance to a 4-HPPD inhibitor, the method comprising:
(I) a step of introducing into a plant cell at least one DNA or a vector having the DNA inserted therein, the DNA encoding a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor, the DNA selected from the group consisting of the following (a) to (d),
(a) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17,
(b) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17, in which one or more amino acids are substituted, deleted, added, and/or inserted,
(c) a DNA hybridizing under stringent conditions to a DNA having a base sequence of any one of SEQ ID NOs: 1 and 16, and
(d) a DNA encoding an amino acid sequence having a homology of 60% or more with an amino acid sequence of any one of SEQ ID NOs: 2 and 17; and
(II) a step of regenerating a plant from the transgenic plant cell in which the DNA or the vector having the DNA inserted therein has been introduced in step (I).
(7) An agent for providing a plant with susceptibility to a 4-HPPD inhibitor, the agent comprising at least one DNA or a vector having the DNA inserted therein, the DNA encoding an RNA having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor, the DNA selected from the group consisting of the following (a) to (c):
(a) a DNA encoding a double-stranded RNA complementary to a transcript of the DNA according to (1);
(b) a DNA encoding an antisense RNA complementary to a transcript of the DNA according to (1); and
(c) a DNA encoding an RNA having a ribozyme activity of specifically cleaving a transcript of the DNA according to (1).
(8) A transgenic plant cell capable of regenerating a plant having increased susceptibility to a 4-HPPD inhibitor, the transgenic plant cell comprising at least one DNA or a vector having the DNA inserted therein, the DNA encoding an RNA having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor, the DNA selected from the group consisting of the following (a) to (c):
(a) a DNA encoding a double-stranded RNA complementary to a transcript of the DNA according to (1);
(b) a DNA encoding an antisense RNA complementary to a transcript of the DNA according to (1); and
(c) a DNA encoding an RNA having a ribozyme activity of specifically cleaving a transcript of the DNA according to (1).
(9) A plant having increased susceptibility to a 4-HPPD inhibitor, which is regenerated from the transgenic plant cell according to (8).
(10) A plant having increased susceptibility to a 4-HPPD inhibitor, which is any one of a progeny and a clone of the plant according to (9).
(11) A propagation material of the plant having increased susceptibility to a 4-HPPD inhibitor according to any one of (9) and (10).
(12) A method for producing a plant having increased susceptibility to a 4-HPPD inhibitor, the method comprising:
(I) a step of introducing into a plant cell at least one DNA or a vector having the DNA inserted therein, the DNA encoding an RNA having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor, the DNA selected from the group consisting of the following (a) to (c),
(a) a DNA encoding a double-stranded RNA complementary to a transcript of the DNA according to (1),
(b) a DNA encoding an antisense RNA complementary to a transcript of the DNA according to (1), and
(c) a DNA encoding an RNA having a ribozyme activity of specifically cleaving a transcript of the DNA according to (1); and
(II) a step of regenerating a plant from the transgenic plant cell in which the DNA or the vector having the DNA inserted therein has been introduced in step (I).
(13) A method for determining whether a plant has resistance or susceptibility to a 4-HPPD inhibitor, wherein the method comprises analyzing a base sequence of at least one DNA, in a test plant, selected from the group consisting of the following (a) to (d) or a base sequence of an expression control region of the DNA:
(a) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17;
(b) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17, in which one or more amino acids are substituted, deleted, added, and/or inserted;
(c) a DNA hybridizing under stringent conditions to a DNA having a base sequence of any one of SEQ ID NOs: 1 and 16; and
(d) a DNA encoding an amino acid sequence having a homology of 60% or more with an amino acid sequence of any one of SEQ ID NOs: 2 and 17.
(14) A method for determining whether a plant has resistance or susceptibility to a 4-HPPD inhibitor, wherein the method comprises detecting an expression of at least one DNA, in a test plant, selected from the group consisting of the following (a) to (d), or a molecular weight of any one of an amplification product and an expression product of the DNA:
(a) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17;
(b) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17, in which one or more amino acids are substituted, deleted, added, and/or inserted;
(c) a DNA hybridizing under stringent conditions to a DNA having a base sequence of any one of SEQ ID NOs: 1 and 16; and
(d) a DNA encoding an amino acid sequence having a homology of 60% or more with an amino acid sequence of any one of SEQ ID NOs: 2 and 17.
(15) A method for breeding a plant having increased resistance to a 4-HPPD inhibitor, the method comprising:
(a) a step of crossing a plant cultivar resistant to a 4-HPPD inhibitor with any cultivar;
(b) a step of determining whether individuals obtained by the crossing in step (a) have resistance or susceptibility to a 4-HPPD inhibitor by the method according to any one of (13) and (14); and
(c) a step of selecting an individual determined to have resistant to the 4-HPPD inhibitor.
(16) A method for breeding a plant having increased susceptibility to a 4-HPPD inhibitor, the method comprising:
(a) a step of crossing a plant cultivar susceptible to a 4-HPPD inhibitor with any cultivar;
(b) a step of determining whether individuals obtained by the crossing in step (a) have resistance or susceptibility to a 4-HPPD inhibitor by the method according to any one of (13) and (14); and
(c) a step of selecting an individual determined to have susceptibility to the 4-HPPD inhibitor.
A utilization of genes identified in the present invention makes it possible to efficiently produce a plant having increased resistance or susceptibility to a 4-HPPD inhibitor. Moreover, targeting the genes identified in the present invention makes it possible to efficiently determine whether a plant has resistance or susceptibility to a 4-HPPD inhibitor.
<Agent for Providing Plant with Resistance to 4-HPPD Inhibitor>
An agent for providing a plant with resistance to a 4-HPPD inhibitor of the present invention is characterized in that the agent comprises a DNA or a vector having the DNA inserted therein, the DNA encoding a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor (hereinafter may be referred to as resistance DNA of the present invention).
In the present invention, a “4-HPPD inhibitor” means an agent for inhibiting a function of 4-HPPD (4-hydroxyphenylpyruvate dioxygenase) (4-HPPD inhibitor). As shown in
Note that although components of herbicides such as 4-HPPD inhibitors as described above are quite diverse compounds, it is possible to classify the herbicides into several groups based on mode of action as follows (see “From Pesticides to Agrobioregulators-disease, pest, and weed controls at present and in the future,” Japan, CMC Publishing Co., Ltd., January 2010).
(I) Acetyl-Coa Carboxylase (ACCase) Inhibiting Herbicides
This herbicide group inhibits ACCase involved at the first stage of lipid synthesis, and inhibits cell membrane synthesis, impeding the plant growth. The herbicides belonging to this group are further classified into (1) 4-aryloxyphenoxypropionate type, (2) cyclohexanedione oxime type, and (3) dione type.
(II) Acetolactate Synthase (ALS) Inhibiting Herbicides
This herbicide group, which targets ALS, inhibits ALS activity, and inhibits branched-amino acid synthesis, thereby impeding plant growth. The herbicides belonging to this group are further classified into (1) sulfonylurea type, (2) triazolinone type, (3) triazolopyrimidine type, (4) pyrimidinylsalicylate type, and (5) imidazolinone type.
(III) 4-HPPD Metabolism Inhibiting Herbicides
This herbicide group inhibits 4-HPPD metabolism in a tyrosine metabolic pathway, and indirectly inhibits a carotenoid synthetic system of a plant to whiten and wither the plant to death. The herbicides belonging to this group are further classified into (1) cyclohexanedione type, (2) pyrazole type, (3) bicyclo type, (4) isoxazole type, and (5) triketone type. Moreover, examples of (1) cyclohexanedione type include benzoylcyclohexane-1,3-dione derivatives. Examples of (2) pyrazole type include pyrazolate, benzofenap, and pyrazoxyfen. Examples of (3) bicyclo type include 3-substituted benzoyl-bicyclo[4,1,0]heptane-2,4-dione derivatives. An example of (4) isoxazole type is isoxaflutole. Examples of (5) triketone type include BBC, mesotrione, tefuryltrione, and tembotrione.
(IV) Protoporphyrinogen IX Oxidase (PPO) Inhibitor Herbicides
Herbicides of this group inhibit chlorophyll synthesis, degrade the cell membrane, and cause death by withering. The herbicides belonging to this group are further classified into (1) diphenyl ether type, (2) diallyl type, and (3) pyrazole type.
(V) Very-Long-Chain Fatty Acid Elongase (VLCFAE) Inhibiting Herbicides
Herbicides of this group inhibit biosynthetic system enzymes of very-long-chain fatty acids having C20 or longer in the plant lipid biosynthetic system, and wither the plant to death.
(VI) Phytoene Desaturase (PDS) Inhibiting Herbicides
This herbicide group inhibits a PDS enzyme in the carotenoid biosynthetic pathway, and causes plant chlorophyll degradation to whiten and wither the plant to death.
(VII) PS II Inhibiting Herbicides
Inhibitors of this group bind to plastoquinone (PQ), and as a result inhibit PQ-involving electron transfer from photosystem II (PS II) to photosystem I (PS I), so that the carbon fixation function in the plant cannot work, and the plant is withered to death.
(VIII) Synthetic Auxin Herbicides
Inhibitors of this group act like natural auxin, which is present at a low concentration in plants and regulates the plant growth, so that the plant differentiates and grows abnormally and is consequently withered to death.
(IX) EPSP Synthase (EPSPS) Inhibiting Herbicides
Inhibitors of this group bind to EPSPS in the shikimic acid pathway, and inhibit EPSP synthesis. As a result, tryptophan, phenylalanine, and tyrosine are not biosynthesized, and the plant is withered to death.
As examples of the resistance DNA of the present invention, the base sequence of a hypothetical gene (HIS1 gene; Os02g0280700) of an iron/ascorbate-dependent oxidoreductase gene derived from Nipponbare is shown in SEQ ID NO: 1, and the amino acid sequence of a protein encoded by the DNA is shown in SEQ ID NO: 2.
Moreover, as another example of the resistance DNA of the present invention, the base sequence of a gene (HIS1-LIKE (HSL) 1 gene; Os06g0176700/Os06g0178500 (AK241948) derived from Nipponbare is shown in SEQ ID NO: 16), and the amino acid sequence of a protein encoded by the DNA is shown in SEQ ID NO: 17.
One form of the resistance DNA of the present invention is a DNA encoding a protein having an amino acid sequence of SEQ ID NO: 2 (typically, a DNA comprising a coding region for the base sequence of SEQ ID NO: 1).
Moreover, another form of the resistance DNA of the present invention is a DNA encoding a protein having an amino acid sequence of SEQ ID NO: 17 (typically, a DNA comprising a coding region for the base sequence of SEQ ID NO: 16).
Once obtaining information on such DNA base sequences in the current state of the art, those skilled in the art could modify the base sequences in various ways to produce a mutation gene encoding a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor. Moreover, in nature also, a base sequence may be mutated. Thus, the resistance DNA of the present invention includes a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17, in which one or more amino acids are substituted, deleted, added, and/or inserted, as long as a protein having the above-described activity is encoded. Herein, the term “more” refers to generally 50 amino acids or less, preferably 30 amino acids or less, more preferably 10 amino acids or less, and particularly preferably several amino acids or less (for example, 5 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid) in the entire amino acid sequence of the HIS1 protein or the HSL1 protein.
Further, once obtaining a particular resistance DNA in the current state of the art, those skilled in the art could utilize information on the DNA base sequence to isolate a homologous gene, which encodes a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor, from a plant of the same or different species. As the plant from which such a homologous gene is obtained, monocots are preferable, and Poaceae plants are particularly preferable. Examples of the Poaceae plants include rice (for example, 4-HPPD inhibitor-resistant cultivars Nipponbare, Koshihikari, Kitaaoba, Akihikari, Akitakomachi, Fukuhibiki, Bekoaoba, Bekogonomi, Yumeaoba, Hokuriku 193, Leaf Star, Tachisugata, Kusanohoshi, Hoshiaoba, Nishiaoba, Tachiaoba, Makimizuho, Mogumoguaoba, Hamasari, Minamiyutaka), barley, sorghum, corn, and the like.
Examples of a method for obtaining the homologous gene include hybridization techniques (Southern, E. M., J. Mol. Biol., 98: 503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, R. K., et al. Science, 230: 1350-1354, 1985, Saiki, R. K. et al. Science, 239: 487-491, 1988). To isolate the homologous gene, generally, a hybridization reaction is carried out under stringent conditions. Examples of the stringent hybridization conditions include conditions of 6 M urea, 0.4% SDS, and 0.5×SSC; and hybridization conditions of equivalent stringency thereto. It can be expected that when higher stringency conditions are used, for example, conditions of 6 M urea, 0.4% SDS, and 0.1×SSC, a gene having a higher homology is isolated. The resistance DNA of the present invention includes a DNA hybridizing under stringent conditions to a DNA having a base sequence of any one of SEQ ID NOs: 1 and 16, as long as a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor is encoded.
The protein encoded by the homologous gene thus obtained generally has a high homology with the amino acid sequence of any one of SEQ ID NOs: 2 and 17. The high homology refers to a sequence homology of at least 60% or more, preferably 80% or more (for example, 85%, 90%, 95%, 97%, 99% or more). The homology of sequences can be determined utilizing a BLASTX (amino acid level) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). This program is based on algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993). When an amino acid sequence is analyzed with BLASTX, the parameters are set at, for example, score=50 and word length=3. Alternatively, when an amino acid sequence is analyzed using a Gapped BLAST program, the analysis can be performed as described in Altschul et al (Nucleic Acids Res. 25: 3389-3402, 1997). When BLAST and Gapped BLAST programs are used, the default parameters of each program are used. The specific procedures of these analysis methods are known. The resistance DNA of the present invention includes a DNA encoding an amino acid sequence having a homology of 60% or more with an amino acid sequence of any one of SEQ ID NOs: 2 and 17, as long as a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor is encoded. Examples of such a DNA include a barley-derived gene (HvHCP1 (AF527606)), a corn-derived gene (ZmHSL1 (BT062842), ZmHSL2 (NM_001153464)), and a sorghum-derived gene (SbHSL1 (XM_002436546)) (see
Whether or not a protein encoded by a particular gene has an activity of providing a plant with resistance to a 4-HPPD inhibitor can be determined, for example, by introducing the gene into a plant, and assaying whether or not the plant thus produced is provided with the resistance as described in Examples later (see Example 2). Specifically, in a case of using A. thaliana (ecotype Columbia) that would be whitened by an agar medium containing 0.03 μM BBC, if a transformant produced by introducing the gene into A. thaliana can grow without being whitened in the presence of BBC at the above concentration, the protein encoded by the gene can be determined to have an activity of providing a plant with resistance to a 4-HPPD inhibitor. Moreover, in a case of using a BBC susceptible rice cultivar “Kanto 239” that would be whitened by an agar medium containing 0.1 μM BBC, if a transformant produced by introducing the gene into Kanto 239 can grow without being whitened in the presence of BBC at the above concentration, the protein encoded by the gene can be determined to have an activity of providing a plant with resistance to a 4-HPPD inhibitor. Further, in a case of using a triketone type 4-HPPD inhibitor other than BBC (mesotrione, tefuryltrione, tembotrione, NTBC, or the like), if a transformant produced by introducing the gene into Kanto 239 can grow without being whitened in the presence of 1 μM mesotrione, 2.5 μM tefuryltrione, 0.5 μM tembotrione, or 1 μM NTBC, the protein encoded by the gene can be determined to have an activity of providing a plant with resistance to a 4-HPPD inhibitor.
The form of the resistance DNA of the present invention is not particularly limited, and includes, besides a cDNA, a genomic DNA and a chemically synthesized DNA. These DNAs can be prepared by utilizing conventional means for those skilled in the art. A genomic DNA can be prepared, for example, by extracting a genomic DNA from a plant, constructing a genomic library (the vector that can be utilized is a plasmid, phage, cosmid, BAC, PAC, or the like), deploying the library, followed by colony hybridization or plaque hybridization using a probe prepared based on the base sequence of the HIS1 gene (for example, DNA having SEQ ID NO: 1) or the HSL1 gene (for example, DNA having SEQ ID NO: 16). Alternatively, the genomic DNA can also be prepared by producing a primer specific to the HIS1 gene or the HSL1 gene, followed by PCR utilizing the primer. Meanwhile, the cDNA can be prepared, for example, by synthesizing a cDNA based on an mRNA extracted from a plant, inserting the cDNA into a vector such as AZAP to construct a cDNA library, deploying the library, followed by PCR or followed by colony hybridization or plaque hybridization in the same manner as above. Alternatively, using a commercially-available DNA synthesizer, a target DNA can also be prepared by synthesis.
<Agent for Providing Plant with Susceptibility to 4-HPPD Inhibitor>
Additionally, the present invention provides an agent for providing a plant with susceptibility to a 4-HPPD inhibitor. As illustrated in Examples later, suppressing a function of a protein encoded by the HIS1 gene suppresses resistance to a 4-HPPD inhibitor. Thus, the agent for providing a plant with resistance to a 4-HPPD inhibitor of the present invention is characterized in that the agent comprises a DNA or a vector having the DNA inserted therein, the DNA encoding an RNA having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor.
One form of the DNA encoding an RNA having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor, is a DNA encoding a dsRNA (double-stranded RNA) complementary to a transcript of the endogenous resistance DNA of the present invention described above. Introducing a dsRNA having a sequence the same as or similar to the target gene sequence into a cell can induce a phenomenon called RNAi (RNA interference), by which expressions of both the introduced exogenous gene and the target endogenous gene are suppressed. When approximately 40 to several hundred base pairs of dsRNA are introduced into a cell, an RNaseIII-like nuclease called Dicer, which has a helicase domain, excises approximately 21 to 23 base pairs of the dsRNA from the 3′ end at a time in the presence of ATP, forming siRNA (short interference RNA). A specific protein binds to the siRNA to form a nuclease complex (RISC: RNA-induced silencing complex). This complex recognizes and binds to a sequence identical to that of the siRNA, and cleaves a transcript (mRNA) of the target gene at a location corresponding to a central portion of the siRNA by an RNaseIII-like enzyme activity. Meanwhile, independently of this pathway, an antisense strand of the siRNA binds to mRNA and acts as a primer for an RNA-dependent RNA polymerase (RsRP), and a dsRNA is synthesized. This dsRNA again serves as a substrate of Dicer to form other siRNA, and is considered as a pathway for amplifying the action.
The DNA encoding the dsRNA of the present invention comprises: an antisense DNA encoding an antisense RNA for any region of a transcript (mRNA) of a target gene, that is, the endogenous resistance DNA of the present invention; and a sense DNA encoding a sense RNA for any region of the mRNA. The antisense RNA and the sense RNA can be expressed by the antisense DNA and the sense DNA, respectively. Moreover, the dsRNA can be prepared by these antisense RNA and sense RNA.
As the configuration to incorporate the dsRNA expression system of the present invention into a vector or the like, the antisense RNA and the sense RNA may by expressed from the same vector, or the antisense RNA and the sense RNA may be expressed from different vectors, respectively. As the configuration in which the antisense RNA and the sense RNA are expressed from the same vector, for example, an antisense RNA expression cassette and a sense RNA expression cassette are constructed, in each of which a promoter capable of expressing a short RNA, such as a pol III system, is linked upstream of the antisense DNA and the sense DNA, and these cassettes are inserted into the vector in the same direction or opposite directions.
Moreover, it is also possible to construct an expression system in which the antisense DNA and the sense DNA are arranged in opposite directions in such a manner as to face each other on the different strands. This construct includes: a single double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA-encoding strand is paired with a sense RNA-encoding strand; and promoters facing each other on both sides of the DNA so that the antisense RNA and the sense RNA can be expressed from the respective strands. In this case, in order to avoid addition of extra sequences downstream of the sense RNA and the antisense RNA, it is preferable to provide a terminator at the 3′ end of each of the strands (the antisense RNA-encoding strand, the sense RNA-encoding strand). As the terminator, a sequence of four or more consecutive A (adenine) bases, or the like can be used. In addition, in this palindromic expression system, the type of the two promoters is preferably different.
Meanwhile, as the configuration in which the antisense RNA and the sense RNA are expressed from different vectors, for example, an antisense RNA expression cassette and a sense RNA expression cassette are constructed, in each of which a promoter capable of expressing a short RNA, such as a pol III system, is linked upstream of the antisense DNA and the sense DNA, and these cassettes are incorporated into different vectors.
The dsRNA used in the present invention is preferably a siRNA. A “siRNA” means a double-stranded RNA made of short strands in such a range that no toxicity is demonstrated within a cell. The length is not particularly limited, as long as the expression of the target gene can be suppressed and no toxicity is demonstrated. The length of the dsRNA is for example 15 to 49 base pairs, preferably 15 to 35 base pairs, and further preferably 21 to 30 base pairs.
As the DNA encoding the dsRNA of the present invention, it is also possible to use such a construct including an appropriate sequence (desirably, intron sequence) inserted between inverted repeats of the target sequence as to encode a double-stranded RNA having a hairpin structure (self-complementary ‘hairpin’ RNA (hpRNA)) (Smith, N. A., et al. Nature, 407: 319, 2000, Wesley, S. V. et al. Plant J. 27: 581, 2001, Piccin, A. et al. Nucleic Acids Res. 29: E55, 2001).
The DNA encoding the dsRNA of the present invention does not necessarily have to have completely the same base sequence as that of the target gene, but the homology of the sequences is at least 70% or more, preferably 80% or more, and further preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more). The homology of the sequences can be determined with the above-described procedure (BLAST program).
In the dsRNA, a double-stranded RNA portion where RNAs are paired may include not only RNAs which are completely paired, but also an unpaired portion by a mismatch (corresponding bases are not complementary to each other), a bulge (one strand does not have a corresponding base), or the like. In the present invention, the double-stranded RNA region where RNAs of the dsRNA are paired may include both a bulge and a mismatch.
Another form of the DNA encoding an RNA having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor is a DNA (antisense DNA) encoding an antisense RNA complementary to a transcript of the endogenous resistance DNA of the present invention. Examples of an action by the antisense DNA to suppress an expression of the target gene include: inhibition of transcription initiation by triple strand formation; suppression of transcription by hybrid formation at a site where an open loop structure is locally formed by an RNA polymerase; inhibition of transcription by hybrid formation with an RNA being synthesized; suppression of splicing by hybrid formation at a boundary between an intron and an exon; suppression of splicing by hybrid formation at a site where a spliceosome is formed; suppression of translocation from the nucleus to the cytoplasm by hybrid formation with mRNA; suppression of splicing by hybrid formation at a capping site or poly (A) addition site; suppression of translation initiation by hybrid formation at a binding site for a translation initiation factor; suppression of translation by hybrid formation at a ribosome binding site near the start codon; inhibition of peptide chain elongation by hybrid formation in a translated region and at a polysome binding site of mRNA; suppression of gene expression by hybrid formation at a site where a nucleic acid interacts with a protein; and the like. These inhibit a process of transcription, splicing, or translation, and suppress an expression of the target gene (Hirajima and Inoue, “Shin Seikagaku Jikken Kouza (New Courses in Biochemistry Experiments) 2, Kakusan (nucleic acid) IV, Idenshi no Fukusei to Hatsugen (Gene Replication and Expression)”, edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, pp. 319-347, 1993). The antisense DNA used in the present invention may suppress an expression of the target gene by any of the aforementioned actions. As one mode, if an antisense sequence is designed complementary to an untranslated region near the 5′ end of an mRNA of the target gene, it will effectively inhibit translation of the gene. Nevertheless, it is also possible to use a sequence complementary to an untranslated region on the 3′ side or a coding region. As described above, the antisense DNA utilized in the present invention also includes a DNA containing an antisense sequence for sequences of not only a translated region but also an untranslated region of the gene. The antisense DNA used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3′ side.
The antisense DNA can be prepared based on the sequence information on the resistance DNA of the present invention (for example, DNA having the base sequence of SEQ ID NO: 1) by a phosphorothioate method (Stein, Nucleic Acids Res., 16: 3209-3221, 1988) or the like. The DNA thus prepared can be introduced into a plant by known methods described later. The sequence of the antisense DNA is preferably a sequence complementary to a transcript of the endogenous resistance DNA of the present invention of the plant, but does not have to be completely complementary, as long as the gene expression can be effectively inhibited. Transcribed RNA has a complementarity of preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) to a transcript of the target gene. In order to effectively inhibit an expression of the target gene, the length of the antisense DNA is at least 15 bases or longer, preferably 100 bases or longer, and further preferably 500 bases or longer. Generally, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.
Another form of the DNA encoding an RNA having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor, is a DNA encoding an RNA having a ribozyme activity of specifically cleaving a transcript of the endogenous resistance DNA of the present invention. Ribozymes include one having a size of 400 nucleotides or longer such as group I intron type and M1RNA included in RNaseP, and also one called a hammer-head or hairpin type having an active domain of approximately 40 nucleotides (Makoto Koizumi and Eiko Ohtsuka, Tanpakushitu Kakusan Koso (Protein, Nucleic Acid, Enzyme), 35: 2191, 1990).
For example, a self-cleaving domain of a hammer-head type ribozyme cleaves the 3′ side of C15 of G13U14C15; but for the activity, it is important that U14 and A at position 9 form a base pair, and the base at position 15 is shown to be cleaved if A or U is in place of C (Koizumi et al., FEBS Lett. 228: 225, 1988). A restriction enzyme-like RNA-cleaving ribozyme that recognizes a sequence such as UC, UU, or UA in the target RNA can be produced if a substrate binding site of the ribozyme is designed complementary to the RNA sequence near the target site (Koizumi et. al., FEBS Lett. 239: 285, 1988, Makoto Koizumi and Eiko Ohtsuka, Tanpakushitu Kakusan Koso (protein, nucleic acid, enzyme), 35: 2191, 1990, Koizumi et. al., Nucleic. Acids. Res. 17: 7059, 1989).
Meanwhile, a hairpin-type ribozyme is also useful for the object of the present invention. The hairpin-type ribozyme is found, for example, in the minus strand of tobacco ringspot virus satellite RNA (Buzayan, Nature 323: 349, 1986). It has been shown that this ribozyme can also be designed such that target-specific RNA cleavage occurs (Kikuchi and Sasaki, Nucleic Acids Res. 19: 6751, 1992, Yo Kikuchi, Kagaku to Seibutsu (Chemistry and Biology) 30: 112, 1992). The ribozyme designed to be capable of cleaving the target is linked to a promoter such as a cauliflower mosaic virus 35S promoter and a transcription termination sequence so that the ribozyme can be transcribed in plant cells. The effect can be increased by arranging such constituent units in tandem so that multiple sites can be cleaved in the target gene (Yuyama et al., Biochem. Biophys. Res. Commun. 186: 1271, 1992). Such a ribozyme is used to specifically cleave the target transcript of the endogenous resistance DNA of the present invention, so that an expression of the DNA can be suppressed.
<Vector having DNA According to Present Invention Inserted Therein>
The vector having the DNA of the present invention (the resistance DNA of the present invention, or the DNA encoding an RNA having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor) inserted therein is not particularly limited, as long as the inserted gene can be expressed in a plant cell. The vector according to the present invention may comprise a promoter for stably or inductively expressing the DNA of the present invention. Examples of the promoter for stable expression include a cauliflower mosaic virus 35S promoter, a rice actin promoter, a corn ubiquitin promoter, and the like. Meanwhile, examples of the promoter for inductive expression include promoters known to be expressed by external factors such as infection and invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, dryness, ultraviolet irradiation, and spraying of a certain compound; and other promoters. Examples of such promoters include a rice chitinase gene promoter and a tobacco PR protein gene promoter expressed by infection and invasion of filamentous fungi, bacteria, and virus; a rice lip19 gene promoter induced by low temperature; rice hsp80 gene and hsp72 gene promoters induced by high temperature; an Arabidopsis thaliana rab16 gene promoter induced by dryness; a parsley chalcone synthase gene promoter induced by ultraviolet irradiation; a corn alcohol dehydrogenase gene promoter induced by an anaerobic condition; and the like. In addition, the rice chitinase gene promoter and the tobacco PR protein gene promoter may also be induced by a particular compound such as salicylic acid, and rab16 may also be induced by spraying a plant hormone, abscisic acid.
The agent of the present invention may be by itself the DNA of the present invention or the vector having the DNA inserted therein, or may comprise other components mixed therewith. Such other components are not particularly limited, and examples thereof include sterile water, a saline, a vegetable oil, a surfactant, a lipid, a solubilizer, a buffer, and a preservative. Furthermore, when a transgenic plant cell of the present invention is prepared by an Agrobacterium-mediated method described below, the agent may comprise Agrobacterium having the DNA introduced therein.
<Transgenic Plant Cell of the Present Invention>
A transgenic plant cell capable of regenerating a plant having increased resistance to a 4-HPPD inhibitor of the present invention is a plant cell transformed by introducing therein the resistance DNA of the present invention or a vector having the DNA inserted therein, the DNA encoding a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor.
In addition, a transgenic plant cell capable of regenerating a plant having increased susceptibility to a 4-HPPD inhibitor of the present invention is a plant cell transformed by introducing therein the above DNA or a vector having the DNA inserted therein, the DNA encoding an RNA having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor.
A plant, from which the plant cell of the present invention is derived, is not particularly limited, and examples thereof include Poaceae plants such as rice, barley, wheat, sorghum, corn, and creeping bentgrass; Brassicaceae plants such as Arabidopsis thaliana; Solanaceae plants such as tomato; Fabaceae plants such as soybean, alfalfa, and Lotus japonicas; Malvaceae plants such as cotton plant; and Chenopodiaceae plants such as sugar beet.
Particularly, 4-HPPD inhibitor-susceptible cultivars of these plants are preferable as a target of the present invention whose resistance to a 4-HPPD inhibitor is to be increased. Examples of a 4-HPPD inhibitor-susceptible rice cultivar include Habataki, Takanari, Momiroman, Mizuhochikara, Ruriaoba, Odorokimochi, Hyogo-ushiwakamaru, Kasalath, and Kanto 239, but are not limited thereto.
Meanwhile, particularly 4-HPPD inhibitor-resistant cultivars of these plants are preferable as a target of the present invention whose susceptibility to a 4-HPPD inhibitor is to be increased. Examples of a 4-HPPD inhibitor-resistant rice cultivar include Nipponbare, Koshihikari, Kitaaoba, Akihikari, Akitakomachi, Fukuhibiki, Bekoaoba, Bekogonomi, Yumeaoba, Hokuriku 193, Leaf Star, Tachisugata, Kusanohoshi, Hoshiaoba, Nishiaoba, Tachiaoba, Makimizuho, Mogumoguaoba, Hamasari, and Minamiyutaka, but are not limited thereto.
The plant cell of the present invention includes, besides culture cells, cells in the plants. Further, the plant cell of the present invention includes plant cells in various forms, for example, suspended culture cells, protoplasts, leaf sections, calli, immature embryos, pollens, and the like.
As a method for introducing the vector having the resistance DNA of the present invention inserted therein into the plant cell, it is possible to use various methods known to those skilled in the art, such as a polyethylene glycol method, an electroporation method, an Agrobacterium-mediated method, and a particle gun method.
<Plant of Present Invention, Propagation Material thereof, and Method for Producing the Plant>
The present invention provides a plant regenerated from the above transgenic plant cell (hereinafter also referred to as transgenic plant). The plant can be regenerated from the transgenic plant cell by methods known to those skilled in the art, depending on the type of the plant cell.
For example, several techniques of the procedure for producing transgenic rice plants have been already established, such as a method in which a gene is introduced into protoplasts using polyethylene glycol and a plant is regenerated (Datta, S. K. In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.) pp 66-74, 1995); a method in which a gene is introduced into protoplasts using electric pulse and a plant is regenerated (Toki et al. Plant Physiol. 100, 1503-1507, 1992); a method in which a gene is directly introduced into cells by a particle gun method and a plant is regenerated (Christou et al. Bio/technology, 9: 957-962, 1991); and a method in which a gene is introduced using Agrobacterium and a plant is regenerated (Hiei et al. Plant J. 6: 271-282, 1994). These are widely used in the technical field of the present invention.
Moreover, examples of the procedure for producing transgenic plants of barley includes methods described in Tingay et al. (Tingay S. et al. Plant J. 11: 1369-1376, 1997), Murray et al. (Murray F et al. Plant Cell Report 22: 397-402, 2004), and Travalla et al. (Travalla S et al. Plant Cell Report 23: 780-789, 2005).
As the method for regenerating sorghum plants, preferably used are, for example, a method in which a gene is introduced into immature embryos or calli by an Agrobacterium method or a particle gun method and a plant is regenerated; and a method in which pollens having a gene introduced therein using ultrasound are used for pollination (J. A. Able et al., In Vitro Cell. Dev. Biol. 37: 341-348, 2001, A. M. Casas et al., Proc. Natl. Acad. Sci. USA 90: 11212-11216, 1993, V. Girijashankar et al., Plant Cell Rep 24: 513-522, 2005, Je. M. JEOUNG et al., Hereditas 137: 20-28, 2002, V Girijashankar et al., Plant Cell Rep 24 (9): 513-522, 2005, Zuo-yu Zhao et al., Plant Molecular Biology 44: 789-798, 2000, S. Gurel et al., Plant Cell Rep 28 (3): 429-444, 2009, Z Y Zhao, Methods Mol Biol, 343: 233-244, 2006, A K Shrawat and H Lorz, Plant Biotechnol J, 4 (6): 575-603, 2006, D Syamala and P Devi Indian J Exp Biol, 41 (12): 1482-1486, 2003, Z Gao et al., Plant Biotechnol J, 3 (6): 591-599, 2005).
Further, an example of the procedure for Arabidopsis thaliana includes a method by Akama et al. (Akama et al. Plant Cell Reports 12: 7-11, 1992). In the present invention, these methods can be preferably used.
Once a plant having the DNA of the present invention introduced in the genome is obtained, it is possible to obtain a progeny from the plant by sexual reproduction or asexual reproduction. In addition, propagation materials (for example, seeds, fruits, spikes, stubs, calli, protoplasts, and the like) are obtained from the plant or a progeny or a clone thereof, from which the plant can also be produced in mass. Thus, the present invention includes plant cells comprising the DNA of the present invention, plants comprising the cells, progenies and clones of the plants, as well as propagation materials of the plants, the progenies, and the clones.
Moreover, the present invention also provides a method for producing a plant having increased resistance to a 4-HPPD inhibitor, wherein the method comprises:
(I) a step of introducing into a plant cell the resistance DNA of the present invention or a vector having the DNA inserted therein; and
(II) a step of regenerating a plant from the transgenic plant cell in which the DNA or the vector having the DNA inserted therein has been introduced into in step (I).
Furthermore, the present invention also provides a method for producing a plant having increased susceptibility to a 4-HPPD inhibitor, wherein the method comprises:
(I) a step of introducing into a plant cell the DNA encoding an RNA having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor or a vector having the DNA inserted therein; and
(II) a step of regenerating a plant from the transgenic plant cell in which the DNA or the vector having the DNA inserted therein has been introduced in step (I).
<Method for Determining Whether Plant Has Resistance or Susceptibility to 4-HPPD Inhibitor>
A method for determining whether a plant has resistance or susceptibility to a 4-HPPD inhibitor of the present invention is characterized in that the method comprises analyzing a base sequence of the resistance DNA of the present invention or a corresponding susceptibility DNA (hereinafter referred to as detection target DNA of the present invention) in a test plant or a base sequence of an expression control region of the DNA. Note that a “susceptibility DNA” is a DNA, encoding a protein having an activity of providing a plant with susceptibility to a 4-HPPD inhibitor.
The detection target DNA of the present invention is typically at least one DNA selected from the group consisting of the following (a) to (d):
(a) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17;
(b) a DNA encoding a protein having an amino acid sequence of any one of SEQ ID NOs: 2 and 17, in which one or more amino acids are substituted, deleted, added, and/or inserted;
(c) a DNA hybridizing under stringent conditions to a DNA having a base sequence of any one of SEQ ID NOs: 1 and 16; and
(d) a DNA encoding an amino acid sequence having a homology of 60% or more with an amino acid sequence of any one of SEQ ID NOs: 2 and 17.
Note that the DNAs of (a) to (d) basically mean ones as described above, but particularly mean an endogenous DNA regarding the detection target DNA of the present invention, and mean to include both the resistance DNA and the susceptibility DNA.
As illustrated in Examples described later, in comparison with the HIS1 gene of a 4-HPPD inhibitor-resistant cultivar Nipponbare, base insertion or deletion is observed in the sequences of the corresponding genes of 4-HPPD inhibitor-susceptible cultivars Momiroman, Takanari, and Kasalath. Thus, analyzing the base sequence of the detection target DNA of the present invention makes it possible to determine whether a plant has resistance or susceptibility to a 4-HPPD inhibitor.
Moreover, as illustrated in Examples described later, the susceptibility to a 4-HPPD inhibitor is inherited in a recessive manner. Accordingly, analyzing an expression level of the detection target DNA of the present invention as well as the base sequence of a region (enhancer, promoter, silencer, insulator) controlling the expression level also makes it possible to determine whether a plant has resistance or susceptibility to a 4-HPPD inhibitor.
In analyzing the base sequence of the detection target DNA of the present invention or the expression control region, it is possible to use an amplification product of the detection target DNA of the present invention or the expression control region amplified by PCR. When the PCR is carried out, a primer used is not limited, as long as the detection target DNA of the present invention or the expression control region can be amplified specifically. The primer can be designed as appropriate based on the sequence information (for example, SEQ ID NO: 1) of the detection target DNA of the present invention or the expression control region. Examples of a preferable primer include a primer having a base sequence of SEQ ID NO: 13 and a primer having a base sequence of SEQ ID NO: 14. By combining these primers as appropriate, a particular base sequence of the detection target DNA of the present invention or the expression control region can be amplified.
Note that determining whether a test plant has resistance or susceptibility to a 4-HPPD inhibitor may comprise, for example, a comparison step with a “control base sequence.” The “control base sequence” compared with the base sequence of the detection target DNA of the present invention or the expression control region in a test plant is typically the base sequence of the detection target DNA of the present invention or the expression control region of, in a case of rice, a 4-HPPD inhibitor-resistant cultivar (for example, Nipponbare, Koshihikari, Kitaaoba, Akihikari, Akitakomachi, Fukuhibiki, Bekoaoba, Bekogonomi, Yumeaoba, Hokuriku 193, Leaf Star, Tachisugata, Kusanohoshi, Hoshiaoba, Nishiaoba, Tachiaoba, Makimizuho, Mogumoguaoba, Hamasari, Minamiyutaka) or a 4-HPPD inhibitor-susceptible cultivar (for example, Habataki, Takanari, Momiroman, Mizuhochikara, Ruriaoba, Odorokimochi, Hyogo-ushiwakamaru, Kasalath, Kanto 239).
Note that, as an example of the susceptibility DNA of the present invention, the base sequence of a hypothetical gene (mutated HIS1 gene) of an iron/ascorbate-dependent oxidoreductase gene derived from Takanari or Momiroman is shown in SEQ ID NO: 15.
By comparing the determined base sequence of the detection target DNA of the present invention or the expression control region in the test plant with the base sequence (for example, SEQ ID NO: 1, SEQ ID NO: 16) of 4-HPPD inhibitor-resistant cultivars or the base sequence (for example, SEQ ID NO: 15) of 4-HPPD inhibitor-susceptible cultivars, whether the test plant has resistance or susceptibility to a 4-HPPD inhibitor can be evaluated. For example, if the base sequence is greatly different (particularly, if the molecular weight or the amino acid sequence of the encoded protein is greatly changed due to occurrence of anew stop codon or a frame shift) in comparison with the base sequence (for example, SEQ ID NO: 1) of a 4-HPPD inhibitor-resistant cultivar, the test plant is determined to be highly likely to have susceptibility to a 4-HPPD inhibitor.
Note that, in the determination method of the present invention, a DNA can be prepared from a test plant by using ordinary methods, for example, the CTAB method. As a plant from which a DNA is prepared, it is possible to use not only a grown plant, but also a seed and a seedling. Moreover, the base sequence can be determined by ordinary methods, for example, the dideoxy method, the Maxam-Gilbert method, or the like. In determining the base sequence, commercially-available sequencing kits and sequencers can be utilized.
Whether or not the base sequence of the detection target DNA of the present invention or the expression control region in a test plant is different from the control base sequence can be indirectly analyzed by various methods, besides directly determining the base sequence as described above. Examples of such methods include the PCR-SSCP (single-strand conformation polymorphism) method, the RFLP method or PCR-RFLP method utilizing Restriction Fragment Length Polymorphism (RFLP), denaturant gradient gel electrophoresis (DGGE), Allele Specific Oligonucleotide (ASO) hybridization method, and the ribonuclease A mismatch cleavage method.
Another method for determining whether a plant has resistance or susceptibility to a 4-HPPD inhibitor of the present invention is characterized in that the method comprises detecting an expression of at least one DNA, in a test plant, selected from the group consisting of the above (a) to (d), or a molecular weight of any one of an amplification product and an expression product of the DNA.
As illustrated in Examples described later, a front half region of the fourth exon of the HIS1 gene of each 4-HPPD inhibitor-resistant cultivar of Nipponbare, Koshihikari, and Hokuriku 193 is longer than those of 4-HPPD inhibitor-susceptible cultivars Momiroman and Takanari. Thus, detecting the molecular weight of any one of the amplification product and the expression product of the detection target DNA of the present invention makes it possible to determine whether the plant has resistance or susceptibility to a 4-HPPD inhibitor.
Moreover, as illustrated in Examples described later, the susceptibility to a 4-HPPD inhibitor is inherited in a recessive manner. Accordingly, detecting an expression of the detection target DNA of the present invention makes it possible to determine whether a plant has resistance or susceptibility to a 4-HPPD inhibitor.
Herein, the phrase “detecting an expression of DNA” means to include both detecting at a transcription level and detecting at a translation level. Moreover, the phrase “detecting an expression” means to include not only detecting presence or absence of an expression, but also detecting the degree of the expression.
The detection target DNA of the present invention (for example, genomic DNA) can be amplified by the PCR (Polymerase chain reaction) method.
The DNA according to the present invention can be detected at a transcription level by ordinary methods, for example, the RT-PCR (Reverse transcribed-Polymerase chain reaction) method or the northern blotting method. A primer used when the PCR is carried out is not limited, as long as the detection target DNA of the present invention can be amplified specifically. The primer can be designed as appropriate based on the sequence information (for example, SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 15) of the resistance DNA or the susceptibility DNA of the present invention having been determined already. Examples of a preferable primer include primers having a base sequence of any of SEQ ID NOs: 3 to 14. Moreover, by combining these primers as appropriate, a particular base sequence of the detection target DNA of the present invention can be amplified.
Meanwhile, detection at a translation level can be performed by ordinary methods, for example, the western blotting method. An antibody used in western blotting may be a polyclonal antibody or a monoclonal antibody. Methods for preparing these antibodies are well-known to those skilled in the art.
In addition, an expression of the detection target DNA of the present invention can be determined by: constructing a vector having a reporter gene expressibly linked downstream of the expression control region of the detection target DNA of the present invention; introducing the vector into a plant cell; and detecting the reporter activity.
As a result of detecting a gene expression, if the expression level of the detection target DNA of the present invention of the test plant is significantly lower than the expression level of a 4-HPPD inhibitor-resistant cultivar (for example, in a case of rice, Nipponbare, Koshihikari, Kitaaoba, Akihikari, Akitakomachi, Fukuhibiki, Bekoaoba, Bekogonomi, Yumeaoba, Hokuriku 193, Leaf Star, Tachisugata, Kusanohoshi, Hoshiaoba, Nishiaoba, Tachiaoba, Makimizuho, Mogumoguaoba, Hamasari, Minamiyutaka) (for example, if the detection target DNA of the present invention is not substantially expressed), or if the molecular weight of any one of the amplification product and the expression product of the detection target DNA of the present invention is significantly different from the molecular weight in the 4-HPPD inhibitor-resistant cultivar (for example, Nipponbare, Koshihikari, Kitaaoba, Akihikari, Akitakomachi, Fukuhibiki, Bekoaoba, Bekogonomi, Yumeaoba, Hokuriku 193, Leaf Star, Tachisugata, Kusanohoshi, Hoshiaoba, Nishiaoba, Tachiaoba, Makimizuho, Mogumoguaoba, Hamasari, Minamiyutaka), the test plant is determined to be highly likely to have susceptibility to a 4-HPPD inhibitor. In fact, as illustrated in Examples described later, the molecular weight of the susceptibility DNA of the 4-HPPD inhibitor-susceptible cultivars (Momiroman, Takanari) is significantly low in comparison with the resistance DNA of the 4-HPPD inhibitor-resistant cultivars (Nipponbare, Koshihikari, Hokuriku 193).
<Method for Breeding Plant of Present Invention>
The present invention provides a method for breeding a plant having increased resistance to a 4-HPPD inhibitor. The breeding method comprises:
(a) a step of crossing a plant cultivar resistant to a 4-HPPD inhibitor with any plant cultivar;
(b) a step of determining whether individuals obtained by the crossing have resistance or susceptibility to a 4-HPPD inhibitor by the above determination method of the present invention; and
(c) a step of selecting a cultivar determined to have resistance to the 4-HPPD inhibitor.
Moreover, the present invention provides a method for breeding a plant having increased susceptibility to a 4-HPPD inhibitor. The breeding method comprises:
(a) a step of crossing a plant cultivar susceptible to a 4-HPPD inhibitor with any plant cultivar;
(b) a step of determining whether individuals obtained by the crossing have resistance or susceptibility to a 4-HPPD inhibitor by the above determination method of the present invention; and
(c) a step of selecting a cultivar determined to have susceptibility to the 4-HPPD inhibitor.
Examples of “any plant cultivar” crossed with the plant cultivar resistant to a 4-HPPD inhibitor include 4-HPPD inhibitor-susceptible cultivars, and individuals obtained by crossing 4-HPPD inhibitor-resistant cultivars with 4-HPPD inhibitor-susceptible cultivars, but are not limited thereto. Moreover, examples of “any plant cultivar” crossed with the plant cultivar susceptible to a 4-HPPD inhibitor include 4-HPPD inhibitor-resistant cultivars, and individuals obtained by crossing 4-HPPD inhibitor-resistant cultivars with 4-HPPD inhibitor-susceptible cultivars, but are not limited thereto. Since the susceptibility to a 4-HPPD inhibitor is inherited in a recessive manner, in order for individuals obtained by the crossing to show susceptibility to the 4-HPPD inhibitor, it is preferable to have the 4-HPPD inhibitor-susceptibility HIS1 gene homozygously.
A utilization of the breeding method of the present invention makes it possible to select a 4-HPPD inhibitor-resistant or -susceptible cultivar at the stage of seed or seedling, hence making it possible to breed a cultivar having the trait in a short period of time.
Hereinafter, the present invention will be described more specifically based on Examples. However, the present invention is not limited to the following Examples. Meanwhile, the experiments and analyses in the following Examples were carried out as follows.
<QTL Analysis>
A Koshihikari/Habataki chromosome segment substitution line and Tachisugata//Tachisugata/Momiroman BC1F4 were tested. Specifically, Koshihikari/Habataki chromosome segment substitution lines (KHSL) were analyzed, in which a portion of chromosome fragments of a resistant cultivar Koshihikari serving as the genetic background was substituted with a chromosome of an indica type susceptible cultivar Habataki. Note that KHSL consists of 32 lines, and enables analysis on all of the whole 12 chromosomes of Habataki (as to KHSL, see Kazumasa Murata et al., “Development and evaluation of CSSLs carrying overlapping chromosome segments of rice cultivar Habataki in a genetic background of cultivar Koshihikari, ” Breeding Research, Mar. 27, 2009, vol. 11, supplement issue 1, p. 66). In addition, 94 BC1F4 lines obtained by one-time backcrossing of a resistant cultivar Tachisugata to a susceptible cultivar Momiroman were analyzed using 80 SSR markers (as to the SSR markers used, see “Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.)”, DNA Res, 2002, vol. 9, iss. 6, pp. 199 to 207, and http://www.gramene.org/).
<Linkage Analysis Using Tos17-Inserted Lines>
A retrotransposon Tos17 discovered in rice is activated by tissue culture, and its copies are transposed into a genome. It is known that when the transposition site is inside a gene, the gene is to be disrupted, causing a mutation (see Hirochika et al., Proc. Natl. Acad. Sci. USA, 1996, vol. 93, pp. 7783 to 7788). The present Examples utilized a rice population having accumulated mutations caused by tissue culture utilizing the phenomenon (mutant panel, database name: Tos17 mutant panel database (http://tos.nias.affrc.go.jp/ to miyao/pub/tos17/)). From the Tos17 mutant panel database, two lines were selected, in each of which Tos17 was inserted in a transcription site of a hypothetical gene of an iron/ascorbate oxidoreductase gene, which was strongly suspected of being associated with BBC susceptibility located at a gene locus specified by the QTL analysis. Then, self-fertilized seeds obtained by planting 15 individuals of each of these two lines were used to examine phenotype (BBC susceptibility) and genotype (Tos17 insertion).
<Acquisition of Gene Clone>
A hypothetical mRNA (AK065581) of the iron/ascorbate-dependent oxidoreductase gene located at the gene locus specified by the QTL analysis was obtained from a rice gene bank.
<Vector Construction and Transformation>
For transformation by an Agro method, binary vectors were constructed by linking an expression cassettes a kanamycin resistance gene (NPT2) driven by a nos promoter or a hygromycin resistance gene (mHPT) driven by a CaMV35S promoter to that of AK065581 (HIS1 gene) or AK241948 (HSL1 gene), which were driven by a CaMV35S promoter (see
For transformation of A. thaliana, ecotype “Columbia” was used, and a Floral dip method was performed (see Weigel and Glazebrook, Arabidopsis, a laboratory manual, Cold Spring Harbor Laboratory Press (2002) p131-132). Specifically, first, Agrobacterium was shake-cultured in a liquid medium (LB or YEB) containing an antibiotic. Approximately 16 hours later, 2 ml of the culture solution was added to a liquid medium (LB or YEB) containing an antibiotic, and further shake-cultured. Then, the culture solution obtained approximately 16 hours thereafter was centrifuged at 8000 rpm at 4° C. for 10 minutes. The supernatant liquid was discarded, and the resulting precipitate was suspended in 500 ml of a solution containing 5% sucrose. Subsequently, immediately before the transformation, a transformation reagent silwet (registered trademark: SILWET L-77, product number: BMS-SL7755, manufactured by Bio Medical Science Inc.) was added thereto at a final concentration of 0.025%. Then, into the Agrobacterium suspension thus obtained, Arabidopsis from which already flowered and pollinated buds had been removed was dipped for 30 to 120 seconds. Thereafter, the plants were left standing for 16 hours and then grown to obtain the seeds.
For transformation of rice, a BBC susceptible rice cultivar Kanto 239 was used, and the method described in “Taniguchi et al., Plant Cell Rep., 2010, vol. 29, iss. 11, pp. 1287 to 1295” was performed with some modification. Specifically, first, sterilized mature seeds were sown on an N6D medium. After culturing at 30° C. for 7 days, the seeds were infected with Agrobacterium, and co-cultured in an acetosyringone (AS)-containing N6 medium (2N6-AS medium) under a dark condition at 25° C. for 3 days. Thereafter, the infected tissues were cultured in a carbenicillin-containing N6D medium for 4 to 6 weeks (18 hours day length) in the presence of 40 mg/L hygromycin (Hyg), and Hyg-tolerant calli were re-differentiated.
For transformation of tomatoes, a cultivar “Micro-Tom” was tested, and an Agrobacterium method was performed using vectors (35SHIS1pZK3, 35SHSL1pZK3) shown in
As a result of the tomato transformation, multiple re-differentiated plants having the HIS1 gene or the HSL1 gene introduced therein were obtained. Moreover, the genes introduced into the re-differentiated plants were confirmed by PCR
<BBC Resistance Assay on Recombinants>
A. thaliana recombinants (T2 generation) and rice recombinants (T0 generation) were produced and subjected to a BBC resistance assay using a BBC substance possessed by SDS Biotech K.K. at a concentration to be described later.
<Triketone Type 4-HPPD Inhibitor-Resistance Assay on Recombinants>
Rice recombinants (T1 and T2 generations) were produced and subjected to a resistance assay with triketone type 4-HPPD inhibitors, that is, mesotrione, tefuryltrione, tembotrione, and NTBC, using commercially-available reagents at concentrations to be described later.
<PCR>
Primers specifically amplifying five exon regions of HIS1 (AK065581) were designed and used for PCR. Note that the PCR was carried out in 35 cycles each consisting of 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 30 seconds, using the primers having base sequences shown in Table 1. Moreover, as the template DNAs, used were genomic DNAs extracted by the CTAB method from leaves of BBC susceptible rice cultivars (Momiroman, Takanari, Kasalath) and BBC resistant rice cultivars (Nipponbare, Koshihikari, Hokuriku 193).
Among japonica type rice, no cultivar is known to have susceptibility to BBC, one of 4-HPPD inhibitors. Meanwhile, a rice cultivar bred by crossing a japonica type with an indica type may have BBC susceptibility.
Accordingly, the QTL analysis was performed as described above on the Koshihikari/Habataki chromosome segment substitution lines (KHSL) using the BBC resistant rice cultivar “Koshihikari” and the susceptible rice cultivar “Habataki”. As a result, only KHSL (KHSL04) whose short arm region of chromosome 2 was substituted with that of the Habataki type showed susceptibility. This revealed that the gene locus for determining the BBC resistance was located on a short arm of chromosome 2 of Koshihikari (see Table 2). Note that Table 2 shows part of the result of the QTL analysis on the KHSL. Additionally, in Table 2, “A” indicates that the marker was derived from Koshihikari, and “B” indicates that the marker was derived from Habataki.
Further, the QTL analysis was performed as described above on Tachisugata//Tachisugata/Momiroman BC1F4 using the BBC resistant rice cultivar “Tachisugata” and the susceptible rice cultivar “Momiroman”. As a result, it was revealed that the gene locus for determining the BBC resistance was located on a short arm of chromosome 2 of Tachisugata as in the above case. It was demonstrated that the gene loci specified by the QTL analyses using different rice cultivars were located on the same region, and that 11 candidate genes existed according to the rice cultivar “Nipponbare” database information (see Table 3).
Moreover, it was also revealed that both the base sequences of the 11 candidate genes and hypothetical amino acid sequences did not have a homology with enzymes and genes thereof in the tyrosine metabolic pathway and the carotenoid biosynthetic pathway, which were influenced by BBC (see
Incidentally, according to the rice cultivar “Nipponbare” database information, a rice gene having the highest homology with the known gene encoding an HPPD enzyme whose activity is inhibited by BBC is located on the short arm of chromosome 2, but a gene locus thereof was different from that specified by the QTL analysis (see
Furthermore, in order to narrow down the region where a gene associated with the BBC resistance was located, an F2 population was produced by crossing KHSL04 with Koshihikari, and a population for analysis of the short arm of chromosome 2 was produced from the BC1F4 lines obtained by one-time backcrossing of Tachisugata to Momiroman. The analysis was attempted again using the two populations. As a result, it was revealed that the gene associated with the BBC resistance existed between the SSR markers RM12980 and RM12983. Moreover, genes existing in the region thus narrowed down were searched for by RAP-DB. The result demonstrated the existence of 10 candidate genes excluding a protein similar to glyoxalase I (741 bp).
As described above, the QTL analysis suggested that the gene for determining resistance to a 4-HPPD inhibitor be located on the short arm of chromosome 2 of rice. Accordingly, focusing on the hypothetical gene of the iron/ascorbate-dependent oxidoreductase gene located on the gene locus, the Tos17-inserted lines were tested to reveal a linkage between phenotype (high BBC susceptibility) and genotype, and also recombinants of BBC susceptible A. thaliana and rice having the gene introduced therein were produced to examine an effect of providing the BBC resistance.
Specifically, at the gene locus for determining the BBC resistance specified by the QTL analysis, the hypothetical gene of iron/ascorbate-dependent oxidoreductase (hereinafter also referred to as “target gene”) is located, similarly to the HPPD enzyme whose activity is inhibited by BBC (see Table 4).
Although the original cultivar “Nipponbare” of the Tos17-inserted lines is a BBC resistant cultivar, BBC susceptible individuals are derived from the lines having Tos17 inserted in the transcription site of the target gene. The phenotype (BBC susceptibility) and the genotype (Tos17-inserted homozygote) were examined by the linkage analysis tested on 30 individuals in total. As a result, all the progenies of six Tos17-inserted homozygous individuals showed BBC susceptibility. Meanwhile, all the progenies of 18 Tos17-inserted heterozygous individuals were segregated into BBC susceptible individuals. These results suggested that the hypothetical gene of the iron/ascorbate oxidoreductase gene be closely associated with the BBC resistance.
Accordingly, in order to verify that the target gene was a BBC resistance gene, recombinants (T2 generation) were prepared by introducing the target gene into A. thaliana (ecotype Columbia) that would be whitened by an agar medium containing 0.03 μM BBC, and the growth state of the recombinants was examined in the presence of BBC at this concentration.
Further, recombinants (T0 generation) were prepared by introducing the target gene into the BBC susceptible rice cultivar “Kanto 239” that would be whitened by an agar medium containing 0.1 μM BBC, and the growth state of the recombinants was examined in composted soil treated with BBC at 300 ga.i./ha.
Furthermore, recombinants were prepared by introducing the target gene into the BBC susceptible rice cultivar “Kanto 239” that would be whitened by an agar medium containing 0.1 μM BBC, and T1 seeds or T2 seeds were obtained. These were seeded on an agar medium containing 2 μM BBC, and the growth state of the recombinants was examined.
As apparent from the result shown in
Furthermore, as apparent from the results shown in
These results verified that the target gene was a 4-HPPD inhibitor-resistance gene (HIS1 gene), that is, a DNA encoding a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor.
Although the HIS1 gene on chromosome 2 of japonica type rice was specified, the amplification product of the HIS1 gene, amplified by PCR, was also obtained from BBC susceptible rice cultivars. In addition, among the BBC susceptible rice cultivars, it is sometimes difficult to determine whether Kasalath has susceptibility or resistance, depending on the BBC-treatment condition. For this reason, whether or not it was possible to determine a relation between the base sequence of the HIS1 gene and the degree of BBC susceptibility was examined by PCR by which a particular region of the HIS1 gene was amplified.
It should be noted that although unillustrated, all of Momiroman, Takanari, and Kasalath are BBC susceptible cultivars at different levels; it has been confirmed that Momiroman and Takanari show high BBC susceptibility in comparison with the BBC susceptibility of Kasalath.
Hence, first, BBC susceptible cultivars (Momiroman, Takanari, Kasalath) and BBC resistant cultivars (Nipponbare, Koshihikari, Hokuriku 193) were analyzed by PCR using the primers specifically amplifying the five exon regions of the HIS1 gene.
As apparent from the result shown in
Further, the genomic DNA sequences of the BBC susceptible cultivars Momiroman and Takanari were compared with that of the BBC resistant cultivar Nipponbare.
As apparent from the result shown in
Furthermore, the genomic DNA sequence of the BBC susceptible cultivar Kasalath was compared with that of the BBC resistant cultivar Nipponbare.
As apparent from the result shown in
Thus, these results revealed that an activity that a protein encoded by the HIS1 gene had so as to provide a plant with resistance to a 4-HPPD inhibitor was suppressed by the base deletion and/or insertion from the fourth exon to the fifth exon of the HIS1 gene. In addition, it seems that the difference of Momiroman and Takanari from Kasalath in susceptibility to a 4-HPPD inhibitor is attributable to whether or not a deletion is present in the front half portion of the fourth exon of the HIS1 gene, and so forth.
Next, a database was searched for a gene having a homology with the HIS1 gene. Specifically, using NCBI Blast, a tBLASTN search (default setting) was conducted using the amino acid sequence of the protein encoded by the HIS-1 gene as a query. Note that the data to be searched was nr/nt (non-redundant nucleotide collection).
As a result, it was revealed that the rice gene (HSL1 gene) having the highest homology with the HIS1 gene was located on chromosome 6, and that the homology of the hypothetical amino acid sequences was as high as approximately 86% (see
Nonetheless, it is presumed that proteins encoded by these homologous genes on chromosome 6 are also expressed in a susceptible cultivar having the HIS1 gene mutated.
Regarding this point, as shown in
Incidentally,
Moreover, it was revealed that the homologous genes of the HIS1 gene were scattered only among monocots, and were not found among dicots (see
The rice gene (HSL1 gene) having the highest homology with the HIS1 gene and located on chromosome 6 was examined whether or not the HSL1 gene was a DNA encoding a protein having an activity of providing a plant with resistance to a 4-HPPD inhibitor.
Specifically, recombinants were prepared by introducing the target gene into the BBC susceptible rice cultivar “Kanto 239” that would be whitened by an agar medium containing 0.1 μM BBC, and T1 seeds or T2 seeds were obtained. These were seeded on an agar medium containing 0.12 μM BBC, and the growth state of the recombinants was examined.
As apparent from the result shown in
When plants having increased resistance to a 4-HPPD inhibitor of the present invention are used and cultivated, the weed control can be efficiently carried out in cultivation paddy fields or cultivation fields. In addition, a method for determining whether a plant has resistance or susceptibility to a 4-HPPD inhibitor of the present invention can be utilized, for example, to reduce a germination risk of self-sown seeds from the previous year in crop rotation cycles. In this manner, the present invention can contribute greatly to stable production and yield increase of useful plants.
[Sequence Listing Free Text]
SEQ ID NOs: 3 to 14
<223> sequences of artificially synthesized primers
| Number | Date | Country | Kind |
|---|---|---|---|
| 2010-293451 | Dec 2010 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2011/080105 | 12/26/2011 | WO | 00 | 10/7/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/090950 | 7/5/2012 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6353159 | Skillings | Mar 2002 | B1 |
| 20040123343 | La Rosa | Jun 2004 | A1 |
| 20060123505 | Kikuchi | Jun 2006 | A1 |
| 20060282911 | Bull | Dec 2006 | A1 |
| 20090011936 | Hawkes | Jan 2009 | A1 |
| 20110039706 | Busch | Feb 2011 | A1 |
| 20140018241 | Sammons | Jan 2014 | A1 |
| Number | Date | Country |
|---|---|---|
| 200129080 | Feb 2001 | JP |
| 2005185101 | Jul 2005 | JP |
| 2008034648 | Mar 2008 | WO |
| 2008150473 | Dec 2008 | WO |
| Entry |
|---|
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| Number | Date | Country | |
|---|---|---|---|
| 20150047066 A1 | Feb 2015 | US |
