JP5229603B2 - Calmodulin-binding protein involved in the regulation of brassinosteroid biosynthesis - Google Patents

Description

  The present invention relates to a novel regulatory factor that activates or suppresses transcription of a plurality of enzyme genes that catalyze brassinosteroid biosynthesis reaction. Furthermore, it is related with obtaining efficiently the useful plant which increased the biosynthesis ability of brassinosteroid, and the plant by which the morphogenesis was controlled by controlling the expression level of this regulator.

  Brassinosteroids are plant hormones essential for plant growth and development. Its physiological effects are diverse, such as cell elongation, vascular differentiation, and stress tolerance, and it is extremely useful for agriculture. However, due to the high production cost of synthetic brassinosteroids, they are not used in actual agricultural fields.

In recent years, genes involved in brassinosteroid biosynthesis, metabolism, and signal transduction have been isolated one after another, and the biosynthesis pathway of brassinosteroid has been elucidated (for example, see Non-Patent Documents 1 to 3). The brassinosteroid biosynthesis pathway of Arabidopsis thaliana is shown in FIG.
Furthermore, a novel gene (CYP90D1) encoding brassinosteroid synthase protein has been found in Arabidopsis thaliana (see, for example, Patent Document 1).

  Regarding the regulation of brassinosteroid biosynthesis, the expression of the brassinosteroid biosynthesis gene is subjected to negative feedback control by treatment with brassinolide, which is an active brassinosteroid (see, for example, Non-Patent Document 4). It has also been confirmed that the expression of the CYP90A1 (CPD) gene changes daily (for example, see Non-Patent Document 5). In addition, it has been shown that calcium ion-dependent calmodulin is essential for the enzyme function of DWF1 that catalyzes the early stage reaction of brassinosteroid biosynthesis (see, for example, Non-Patent Document 6).

In addition, regarding the enzyme genes involved in biosynthesis, it has been confirmed that useful plants with enhanced brassinosteroid biosynthesis ability can be obtained by overexpressing them.
For example, in Arabidopsis overexpression of the CYP90B1 (DWF4) gene that catalyzes an early stage reaction in biosynthesis, the flower stem lengthened and seed production increased, but this overexpression increased brassinolide production. It has been confirmed that they do not (see, for example, Non-Patent Document 7). The overexpressed CYP85A2 gene, which catalyzes the final stage of brassinolide synthesis, showed the same phenotype as the overexpressed DWF4 gene, but it was confirmed that the overexpressed body increased the production of brassinolide. (See, for example, Non-Patent Document 8). In addition, it has been confirmed that an overexpressing body of CYP90C1 (ROT3) gene increases only the size (vertical direction) of flowers and leaves (see, for example, Non-Patent Document 9).

In this way, because the type and amount of brassinosteroid that accumulates phenotypes and accumulate depending on the overexpressed biosynthetic enzyme gene, it is possible to efficiently obtain useful plants with enhanced brassinosteroid biosynthesis ability. was difficult. In order to efficiently produce such useful plants, while considering the types of active brassinosteroids in organs that impart plant species and useful traits, activate transcription of multiple biosynthetic enzyme genes, Or it may be necessary to suppress.
Fujioka, S. and Yokota, T. (2003) Biosynthesis and metabolism of brassinosteroids. Annu. Rev. Plant Biol. 54: 137-164. Li, J. (2005) Brassinosteroid signaling: from receptor kinases to transcription factors. Curr. Opin. Plant Biol. 8: 526-531. Choe, S. (2006) Brassinosteroid biosynthesis and inactivation. Physiol. Plant. 126: 539-548. Tanaka, K. Asami, T. Yoshida, S. Nakamura, Y. Matsuo, T. and Okamoto, S. (2005) Brassinosteroid homeostasis in Arabidopsis is ensured by feedback expressions of multiple genes involved in its metabolism.Plant Physiol.138: 1117-1125 Bancos, S. Szatmari, AM Castle, J. Kozma-Bognar, L. Shibata, K. Yokota, T. Bishop, GJ Nagy, F. and Szekeres, M. (2006) Diurnal regulation of the brassinosteroid-biosynthetic CPD gene in Arabidopsis. Plant Physiol. 141: 299-309. Du, L. and Poovaiah, BW (2005) Ca2 + / calmodulin is critical for brassinosteroid biosynthesis and plant growth.Nature. 437: 741-745. Choe, S. Fujioka, S. Noguchi, T. Takatsuto, S. Yoshida, S. and Feldmann, KA (2001) Overexpression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis.Plant J. 26 : 573-582. Kim, TW Hwang, JY Kim, YS Joo, SH Chang, SC Lee, JS Takatsuto, S. and Kim, SK (2005) Arabidopsis CYP85A2, a cytochrome P450, mediates the Baeyer-Villiger oxidation of castasterone to brassinolide in brassinosteroid biosynthesis. Plant Cell. 17: 2397-2412. Kim, GT Tsukaya, H. Saito, Y. and Uchimiya, H. (1999) Changes in the shapes of leaves and flowers upon overexpression of cytochrome P450 in Arabidopsis.Proc. Natl. Acad. Sci. USA. 96: 9433-9437 . JP2003-334089

  An object of the present invention is to obtain a novel regulatory factor that activates or suppresses the transcription of a plurality of enzyme genes that catalyze the brassinosteroid biosynthesis reaction. Furthermore, it is an object of the present invention to efficiently obtain useful plants having enhanced brassinosteroid biosynthesis ability and plants with regulated morphogenesis by controlling the expression level of the regulator.

  As a result of intensive studies to solve the above problems, the present inventors have found a regulatory factor responsible for transcriptional control of a plurality of brassinosteroid biosynthetic enzyme genes universal to plants. It was confirmed that this regulator is expressed in all organs of plants and binds to calmodulin. Moreover, by controlling the expression of this regulatory factor, it has been found that the morphogenesis of leaf blades and petiole changes, and the present invention has been completed.

That is, this invention relates to the polynucleotide, plant, etc. as described in following (1)-(10).
(1) A polynucleotide having the base sequence of SEQ ID NO: 1 in the sequence listing.
(2) The polynucleotide according to (1) above, wherein the polynucleotide is a gene encoding a regulator of brassinosteroid biosynthesis reaction.
(3) The polynucleotide according to (1) or (2) above, wherein the regulatory factor is a regulatory factor that activates or suppresses transcription of an enzyme gene that catalyzes the brassinosteroid biosynthesis reaction.
(4) The polynucleotide according to (3) above, wherein the enzyme genes are CYP85A1, CYP90C1 and CYP90D1.
(5) A plasmid containing the polynucleotide according to any one of (1) to (4) above.
(6) A plant transformed with the polynucleotide according to any one of (1) to (4) above.
(7) The plant according to (6), wherein one or more forms of leaf blade and petiole are changed by transformation.
(8) The plant according to the above (6) or (7), which is a leguminous or cruciferous plant.
(9) Transforming a plant with the polynucleotide according to any one of (1) to (4) above, and activating transcription of an enzyme gene that catalyzes brassinosteroid biosynthesis reaction by expression of the polynucleotide or A method of changing the form of the plant by inhibiting.
(10) A protein comprising the amino acid sequence of (a) or (b).
(A) The amino acid sequence of SEQ ID NO: 2 in the sequence listing.
(B) an amino acid sequence of a protein that regulates brassinosteroid biosynthesis reaction, wherein one or several amino acid sequences are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 in Sequence Listing.

By controlling the expression of the regulatory factor of the present invention, it becomes possible to efficiently obtain useful plants having enhanced brassinosteroid biosynthesis ability. Moreover, the plant which changed the shape and the magnitude | size of a leaf can be obtained for the objectives, such as ornamental use.
Since the regulatory factor obtained by the present invention may exist universally in plants other than Arabidopsis thaliana, there is a high possibility that it can be used to control the morphogenesis of various plants.

  The polynucleotide of the present invention may be a natural polynucleotide extracted and isolated from a plant, or may be synthesized by a known method such as PCR according to its base sequence.

The regulator of brassinosteroid biosynthesis reaction of the present invention refers to a factor that regulates the transcription activity of the enzyme gene that catalyzes the reaction in the brassinosteroid biosynthesis reaction pathway.
For example, in the Arabidopsis brassinosteroid biosynthesis reaction pathway, the regulator of the present invention is preferably a factor that regulates the expression of genes encoding the enzyme genes CYP85A1, CYP90C1, and CYP90D1. Since the gene whose expression is regulated by the regulatory factor of the present invention varies depending on the plant, it is not limited thereto.

Plasmid A of the present invention, in order to amplify the gene incorporated into a plasmid, to express the plasmid transformed into E. coli, a gene incorporated, any plasmid to be transformed into a plant via Agrobacterium (Agrobacterium) Is also included. A known vector can be used depending on the subject to be transformed.

The method for changing the morphology of the plant of the present invention is obtained by introducing the plasmid prepared above into Rhizobium radiobactor (former name Agrobacterium tumefaciens ) EHA101 strain by a known method such as electroporation method or particle gun method. The obtained transformant of R. radiobactor can be incorporated into a plant by a known method such as an inflorescence dipping method.

  The plant in which the form of the present invention has changed is an enzyme that catalyzes the brassinosteroid biosynthesis reaction by the expression of the regulator of the brassinosteroid biosynthesis reaction of the present invention by a plant transformed by the method as described above. A plant whose shape has been changed by activating or repressing gene transcription.

The protein of the present invention may be a natural protein extracted and isolated from a plant, or may be synthesized by a known method according to its amino acid sequence or base sequence encoding it. .
Hereinafter, the details of the present invention will be described with reference to examples and the like, but the present invention is not limited thereto.

Cloning of Arabidopsis BPR1 cDNA Total RNA was extracted from rosette leaves of Arabidopsis ecotype Columbia grown in culture using the SV Total RNA Isolation System (Promega) and Superscript first-strand synthesis system for RT-PCR (Invitrogen) Was used to obtain cDNA. Using this cDNA as a template, primers of At2g33990ox_U1 (SEQ ID NO: 3) and At2g33990ox_L1, (SEQ ID NO: 4) and KOD-plus (manufactured by Toyobo) were used at 94 ° C. for 2 minutes, then at 94 ° C. for 15 seconds. A PCR reaction consisting of 10 cycles was carried out with 60 ° C. for 30 seconds and 68 ° C. for 1 minute as one cycle.

  Furthermore, for cloning using Gateway technology (manufactured by Invitrogen), primers of attB1 (SEQ ID NO: 5) and attB2 (SEQ ID NO: 6) and KOD-plus were used with the obtained reaction product as a template. The PCR reaction was performed at 94 ° C. for 2 minutes, followed by 20 cycles of incubation at 94 ° C. for 15 seconds, then 55 ° C. for 30 seconds and then 68 ° C. for 1 minute, and an attB adapter sequence was added.

  The PCR product to which the attB adapter sequence was added was cloned into a donor vector pDONR221 (Invitrogen) compatible with Gateway technology using BP clonase (Invitrogen). DNA sequencing was performed on the insert part of the obtained plasmid (referred to as pDONR221-AtBPR1) using ABI PRISM 3100 Genetic Analyzer (manufactured by Applied Biosystems). The resulting sequence is shown in SEQ ID NO: 1 in the sequence listing. Moreover, the amino acid sequence which seems to correspond from this base sequence was shown to sequence number 2.

Construction of E. coli expression vector of AtBPR1 Using the Arabidopsis cDNA prepared from Example 1 as a template, primers of At2g33990pET21a_U1 (SEQ ID NO: 7) and At2g33990pET21a_L1 (SEQ ID NO: 8) and TaKaRa Ex Taq (manufactured by Takara Bio) Then, after 94 ° C. for 3 minutes, a PCR reaction comprising 25 cycles was carried out, with one cycle of 94 ° C. for 30 seconds, then 60 ° C. for 30 seconds, and 72 ° C. for 1 minute.
The reaction product was electrophoresed, an about 0.9 kb amplified fragment was excised, and purified using GENECLEAN II Kit (manufactured by Kew Biogene). The cDNA fragment was cloned into pGEM-T Easy Vector (Promega) using DNA Ligation Kit <Mighty Mix> (Takara Bio). The insert part of the obtained plasmid (hereinafter referred to as pGEM-AtBPR1) was subjected to DNA sequencing using ABI PRISM 3100 Genetic Analyzer, and the sequence was confirmed to be the base sequence of SEQ ID NO: 1 in the sequence listing.

  pGEM-AtBPR1 was digested with the restriction enzyme EcoRI, and a fragment of about 0.9 kb was excised and purified. On the other hand, E. coli expression vector pET21a (Novagen) was also digested with restriction enzyme EcoRI. These were ligated using DNA Ligation Kit <Mighty Mix> (manufactured by Takara Bio) to obtain an AtBPR1 E. coli expression vector (hereinafter referred to as pET21-AtBPR1). The insert part of pET21-AtBPR1 was subjected to DNA sequencing using ABI PRISM 3100 Genetic Analyzer, and it was confirmed again that the sequence was the base sequence of SEQ ID NO: 1 in the sequence listing. E. coli Rosetta (DE3) strain (Novagen) was transformed with pET21-AtBPR1.

Calmodulin binding assay
E. coli Rosetta (DE3) strain supplemented with 1 mM IPTG was cultured at 28 ° C. for 4 hours to induce expression of AtBPR1 T7 fusion protein. After completion of the culture, the cells were collected by centrifugation, washed with ice-cold phosphate buffer (pH 7.3), and suspended in 10 ml of the same buffer. The cells were disrupted by sonication on ice, centrifuged, and the supernatant was collected and filtered through a 0.45 μm membrane filter (Advantech). 100 μl of calmodulin agarose beads (manufactured by Sigma) equilibrated with phosphate buffer and 500 μl of crude protein extract supplemented with 1 mM CaCl ₂ or 5 mM EGTA were mixed and incubated overnight at 4 ° C.

  After collecting the calmodulin agarose beads by centrifugation, the beads were washed 4 times with 500 μl phosphate buffer and finally with 100 μl. The washed calmodulin agarose beads were suspended in 50 μl of 4 × SDS sample treatment solution (Novagen) and heated at 100 ° C. for 3 minutes to elute the bound protein. Next, according to the Laemmli method, the crude protein extract, the calmodulin agarose bead unbound fraction, the final washed fraction, and the bound fraction were separated by SDS-PAGE using a 10% polyacrylamide gel.

  After electrophoresis, the protein from the polyacrylamide gel is transferred to a buffer solution (15.6 mM Tris, 120 mM glycine, 20% methanol, 0.1% SDS) using a semi-dry type transfer device (manufactured by Bio-Rad) at 30 V at 20 V. Transferred to a PVDF membrane (manufactured by Bio-Rad) under the condition of minutes. The transferred PVDF membrane was immersed in a blotting solution and shaken overnight at room temperature. Thereafter, the sample was immersed in a solution of alkaline phosphatase-labeled T7 tag antibody (T7 • Tag AP Western Blot Kit, manufactured by Novagen) diluted 10,000 times with TBST buffer and gently shaken at room temperature for 30 minutes. Next, the transfer membrane was washed 5 times with TBST solution for 2 minutes and then treated with CDP-Star, which is a substrate for alkaline phosphatase, to detect chemiluminescence. As a result, as shown in FIG. 2, it was found that AtBPR1 expressed in E. coli as a T7 tag fusion protein preferentially binds to calmodulin in the presence of calcium ions in vitro.

Construction of plant expression vector of AtBPR1
Using LR clonase (Invitrogen), the AtBPR1 cDNA site of pDONR221-AtBPR1 prepared in Example 1 was recombined with the destination vector pGWB2. The obtained AtBPR1 plant expression vector was designated as pGWB2-AtBPR1.

Transformation of Arabidopsis thaliana The pGWB2-AtBPR1 prepared in Example 4 was introduced into Rhizobium radiobactor (former name Agrobacterium tumefaciens) EHA101 strain by electroporation. The transformed R. radiobactor EHA101 strain was cultured at 28 ° C. for 2 days in an LB agar medium containing hygromycin (100 mg / l) and kanamycin (50 mg / l). The resulting colonies were precultured overnight at 28 ° C. in an LB liquid medium containing hygromycin (100 mg / l) and kanamycin (50 mg / l), followed by main culture at 28 ° C. overnight. Transformation of Arabidopsis thaliana was performed by the inflorescence dipping method (see Reference 1). The obtained T1 seeds were surface sterilized and sown in an MS medium containing hygromycin (40 mg / l) and carbenicillin (400 mg / l). T2 seeds were collected and homozygous individuals were selected.
[Reference 1]
Clough, SJ and Bent, AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J. 16: 735-743.

Isolation of AtBPR1 gene disruption mutant
From the database of The Salk Institute Genome Analysis Laboratory (SIGnAL) (http://signal.salk.edu/tabout.html), an individual in which T-DNA was inserted into the promoter region of AtBPR1 gene was found. The SALK_041348 line was ordered from The Arabidopsis Biological Resource Center (ABRC), and the expression of AtBPR1 gene was suppressed by RT-PCR using At2g33990RT_U3 (SEQ ID NO: 9) and At2g33990RT_L3 (SEQ ID NO: 10) primers. It was confirmed.

Phenotypic analysis of AtBPR1 overexpression and disruption mutants
The wild type, GUS gene-expressing plant, the AtBPR1 overexpression mutant prepared in Example 5, and the AtBPR1 disruption mutant isolated in Example 6 were seeded on MS medium. Compared with the seedlings 22 days after germination, as shown in Fig. 3, the overexpressing mutant of AtBPR1 has an extended petiole and a longitudinally extending leaf blade, and in contrast, a mutant mutant of AtBPR1. The petioles were shortened and the blades were shortened vertically. This was a phenotype similar to the CYP90C1 (ROT3) mutant shown in Non-Patent Document 9.

Complementation of AtBPR1 disruption mutation by brassinolide treatment Wild type and AtBPR1 disruption mutant isolated in Example 6 were seeded in MS medium supplemented with 100 nM brassinolide (manufactured by Wako Pure Chemical Industries) and ethanol as a control. . As shown in FIG. 4, the addition of brassinolide eliminated the germination delay seen in the AtBPR1 disruption mutant. Moreover, as shown in FIG. 5, the shortening of leaf blades and petiole seen in the AtBPR1 disruption mutant was restored by the addition of brassinolide. Therefore, it was suggested that AtBPR1 is involved in the biosynthesis of brassinosteroids.

Expression analysis of brassinosteroid biosynthetic enzyme gene Example 22 from wild-type, GUS gene-expressing plant 22 days after germination, AtBPR1 overexpression mutant prepared in Example 5, AtBPR1 disruption mutant isolated in Example 6 CDNA was prepared in the same manner as above. RT-PCR uses DET2, CYP90B1 (DWF4), CYP90A1 (CPD), CYP72B1 (BAS1), CYP90C1 (ROT3), CYP90D1, CYP85A1, and CYP85A2 brassinosteroid biosynthetic enzyme genes from the sequence numbers shown in the following sequence listing. Specific primers were used.
DET2 primers: DET2_U1 (SEQ ID NO: 11) and DET2_L1 (SEQ ID NO: 12)
CYP90B1 (DWF4) primers: CYP90B1_U1 (SEQ ID NO: 13) and CYP90B1_L1 (SEQ ID NO: 14)
CYP90A1 (CPD) primers: CYP90A1_U1 (SEQ ID NO: 15) and CYP90A1_L1 (SEQ ID NO: 16)
CYP90C1 (ROT3) primers: CYP90C1_U1 (SEQ ID NO: 17) and CYP90C1_L1 (SEQ ID NO: 18)
CYP90D1 primers: CYP90D1_U1 (SEQ ID NO: 19) and CYP90D1_L1 (SEQ ID NO: 20)
CYP85A1 primers: CYP85A1_U1 (SEQ ID NO: 21) and CYP85A1_L1 (SEQ ID NO: 22)
CYP85A2 primers: CYP85A2_U1 (SEQ ID NO: 23) and CYP85A2_L1 (SEQ ID NO: 24)
CYP72B1 (BAS1) primers: CYP72B1_U1 (SEQ ID NO: 25) and CYP72B1_L1 (SEQ ID NO: 26)

  Using TaKaRa Ex Taq, PCR reaction was performed with 94 ° C. for 3 minutes, 94 ° C. for 30 seconds, then 55-62 ° C. for 30 seconds and further 72 ° C. for 1 minute for one cycle. The PCR cycle was optimized for each gene. As internal standards, control primers AtUBQ10_U1 (SEQ ID NO: 27) and AtUBQ10_L1 (SEQ ID NO: 28) were used. As a result, as shown in FIG. 6, AtBPR1 regulates the transcription of CYP90C1 (ROT3), an enzyme involved in the late reaction of brassinosteroid biosynthesis, and CYP90D1, which functions in cooperation with CYP85A1 and CYP90C1. It became clear that

Expression analysis of AtBPR1 by organ cDNA was prepared from Arabidopsis ecotype Columbia flowers, long-horned fruit, stem, secondary leaf, rosette leaf, and root in the same manner as in Example 1. Using this cDNA as a template, primers of At2g33990RT_U1 (SEQ ID NO: 29) and At2g33990RT_L1 (SEQ ID NO: 30) and TaKaRa Ex Taq were used at 94 ° C. for 3 minutes, then at 94 ° C. for 30 seconds and then at 63 ° C. for 30 seconds. A PCR reaction consisting of 28 cycles was carried out with 1 minute incubation at 72 ° C as one cycle. As a result, as shown in FIG. 7, AtBPR1 was expressed in all organs examined.

These results suggest that BPR1 is a regulator of brassinosteroid biosynthesis that specifically regulates gene expression of enzymes (CYP90C1, CYP85A1) that catalyze late reactions of brassinosteroid biosynthesis. By using this, in the morphogenesis of plants, for example, the shape of leaves can be changed.
The present invention suggested that calmodulin-binding proteins other than BPR1 may be involved in the regulation of brassinosteroid biosynthesis.

  By controlling the expression of the regulatory factor of the present invention, it becomes possible to efficiently obtain useful plants having enhanced brassinosteroid biosynthesis ability. Moreover, the plant which changed the shape and the magnitude | size of a leaf can be obtained for the objectives, such as ornamental use.

It is the figure which showed the brassinosteroid biosynthesis pathway of Arabidopsis thaliana. It is the figure which showed the result of the calmodulin binding assay (Example 3). FIG. 6 shows the results of phenotypic analysis of AtBPR1 overexpression and disruption mutants (Example 4). It is the figure which showed the result of complementation of the AtBPR1 destruction | mutation mutation by a brassinolide process (Example 8). It is the figure which showed the result of complementation of the AtBPR1 destruction | mutation mutation by a brassinolide process (Example 8). It is the figure which showed the result of the expression analysis of a brassinosteroid biosynthesis enzyme gene (Example 9). It is the figure which showed the result of the expression analysis according to organ of AtBPR1 (Example 10).

Claims (5)

Arabidopsis transformed with a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. The Arabidopsis thaliana according to claim 1, transformed by a plasmid containing a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. The Arabidopsis thaliana according to claim 1 or 2, wherein one or more forms of leaf blade and petiole are changed by transformation. By transforming Arabidopsis thaliana with a polynucleotide having the base sequence of SEQ ID NO: 1 and activating or repressing transcription of an enzyme gene that catalyzes the brassinosteroid biosynthesis reaction by expression of the polynucleotide, A method of changing form. The method according to claim 4, wherein Arabidopsis thaliana is transformed with a plasmid containing a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing.