KR101812409B1 - Mutation of LeBZR1 or LeBZR2 gene inducing biomass increase of plant and uses thereof - Google Patents

Abstract

The present invention relates to a method for increasing the biomass of a plant using the mutant gene of LeBZR1 and LeBZR2 function derived from tomato and overexpressing the transcriptional regulatory factors LeBZR1 and LeBZR2 function-acquiring mutant genes of brassinosteroid signal transduction in tomato, It has been confirmed that tomato acts positively on the development of the kidney and the stem growth of the plant. Therefore, it can be usefully used as a technique for providing a plant having increased plant production and plant biomass content.

Description

A LeBZR1 or LeBZR2 mutant gene that promotes an increase in plant biomass and a use thereof.

A LeBZR1 or LeBZR2 mutant gene that promotes plant biomass growth and uses thereof.

Recently, crop productivity improvement techniques using plant hormones have been used. Among them, brassinosteroid (BR) is a plant hormone with a steroid skeleton that promotes cell division and cell length growth and regulates various development such as vascular differentiation and optical morphogenesis. Recently, brassinosteroid hormone-induced compound intolerance (high temperature, low temperature, salt, drought, etc.) and productivity increase have been increasing. It is necessary to study the function of genes using the signal transduction system of this hormone in anticipation of the increasing technique of various beneficial effects using brassinosteroids in an agricultural field.

Brassinosteroid signaling triggers signal transduction as the brassinosteroid binds to receptors in plant cell membranes. This signal transduction ultimately activates the transcription factor BZR1 (BES1), and the activated transcription factor regulates the transcription of the various brassinosteroid response genes, resulting in various patterns of plant development.

Thus, while the brassinosteroid signaling pathway is well characterized in Arabidopsis, the function of target genes involved in the increase of yield and biomass by factors involved in these signal transduction is very rare. It is necessary to develop these target genes and to develop their functions and usage methods and to develop them in agriculture field.

Among these crops, tomatoes are produced and consumed in most countries around the world, with about 32 million tons in China and 2.4 million tons in Russia. In Korea, it has steadily increased since the 1990s and has a cultivation area of 6300 hectares (as of 2012 - National Statistical Office data) and a production scale of about 108 billion won (as of 2014 - National Statistical Office). Studies on brassinosteroids in tomatoes have been carried out a lot, but there are few studies on developmental regulation other than fruit.

As a result of overexpression of the mutation gene of LeBZR1 and LeBZR2 functioning as transcription regulators of brassinosteroid signal transduction in tomato using tomato, the present inventors have found that a large amount of plant biomass can be obtained because the primary and secondary growth is promoted in the stem of the plant The present invention has been completed.

1) Marta Ibanes et al. (2009) Brassinosteroid signaling and auxin transport are required to establish the periodic pattern of Arabidopsis shoot vascular bundles. PNAS 32, 1363013635 2) Ryu H et al. (2007) Nucleocytoplasmic shuttling of BZR1 mediated by phosphorylation is essential in Arabidopsis brassinosteroid signaling. The Plant cell 19,2749-2762.

The present invention is characterized in that a method of increasing the biomass of a plant using a gene derived from tomato is characterized.

It is therefore an object of the present invention to provide a polypeptide consisting of SEQ ID NO: 1 or SEQ ID NO: 2 which increases plant biomass.

Another object of the present invention is to provide a recombinant vector comprising the polynucleotide of SEQ ID NO: 5 or SEQ ID NO: 6.

Another object of the present invention is to provide a host cell transformed with the recombinant vector.

Another object of the present invention is to provide a method for producing a plant for increasing biomass, which comprises overexpressing a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in a plant.

Another object of the present invention is to provide transgenic plants and seeds with enhanced biomass of plants produced by the method.

In order to achieve the above object, the present invention provides a polypeptide comprising SEQ ID NO: 1 or SEQ ID NO: 2, which increases plant biomass.

Wherein the polypeptide of SEQ ID NO: 1 is encoded by the polynucleotide of SEQ ID NO: 5 and the polypeptide of SEQ ID NO: 2 is encoded by the polynucleotide of SEQ ID NO:

The present invention provides a recombinant vector comprising a polynucleotide of SEQ ID NO: 5 or SEQ ID NO: 6.

The present invention provides a host cell transformed with said recombinant vector.

In one embodiment of the present invention, the host cell may be a plant cell.

The present invention provides a method for producing a plant for increasing biomass comprising overexpressing a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in a plant.

Wherein the polypeptide of SEQ ID NO: 1 is encoded by the polynucleotide of SEQ ID NO: 5 and the polypeptide of SEQ ID NO: 2 is encoded by the polynucleotide of SEQ ID NO:

In one embodiment of the present invention, the plant with increased biomass may have increased plant length and increased volumetric growth.

The present invention provides transgenic plants and seeds with enhanced biomass of plants produced by the method.

The present invention relates to a method for increasing the biomass of a plant using a mutant gene for LeBZR1 and LeBZR2 function derived from tomato, and it has been confirmed that a mutant gene for LeBZR1 and LeBZR2 functioning acts positively on the development of the kidney and stem growth of the plant. It can be usefully used as a technique for providing plants with increased total production amount and plant biomass content.

FIG. 1 is a diagram showing a result of confirming the expression pattern of a recombinant vector containing LeBZR1 gene by transfecting tomato with GUS reporter assay.
FIG. 2 shows the results of the transfection of the transformants LeBZR1-T191A and LeBZR2-T183A (SEQ ID NO: 2) over-expressed in a tomato using a recombinant plant transformation vector containing genes in which amino acid sequences of 191-Thr and LeBZR2 183-Thr of LeBZR1 were changed to alanine Fig.
FIG. 3 is a graph showing differences in thickness and elongation of wild-type tomato, overexpressing agents LeBZR1-T191A and LeBZR2-T183A;
3A: graph showing difference in thickness of the thickest stem of wild-type tomato, overexpressing agents LeBZR1-T191A and LeBZR2-T183A
3B is a graph showing differences in height of plants between wild-type tomatoes, overexpressing plants LeBZR1-T191A and LeBZR2-T183A.
Figure 4 shows the callus induction process of tomato transgenic plants;
A: <1 week to 10 days after sowing>
B: < 5 days after callus induction >
C: <11 days after induction of callus>
D: <After 4 days of the shoot induction> The suit starts to grow
E: <3 weeks after induction of shoot> The shoot grows and grows about 2cm
F: <2 weeks after induction of rooting> A root-developed object among the induced suites.
5 is a diagram showing alignment of LeBZR1 (SEQ ID NO: 3) and LeBZR1-T191A (SEQ ID NO: 1).
6 is a diagram showing alignment of LeBZR2 (SEQ ID NO: 4) / LeBZR2-T183A (SEQ ID NO: 2).

Hereinafter, the present invention will be described in detail.

In studying plant-derived genes capable of enhancing plant biomass, the present inventors have found that transcription regulators of brassinosteroid signaling, a steroid hormone that regulates the development of tomato-derived plants, transcription regulators of brassinosteroid signaling LeBZR1 and LeBZR2 function-enhancing mutant genes have been shown to increase the production of whole plants as they increase the kidney development and stem growth of the plants.

Accordingly, the present invention provides a plant biomass derived from LeBZR1 (Brassinazole resistant1) -T191A consisting of the amino acid sequence of SEQ ID NO: 1 or LeBZR2 (Brassinazole resistant2) -T183A consisting of the amino acid sequence of SEQ ID NO: 2 and tomato (Lycopersicon esculentum) Lt; RTI ID = 0.0 &gt; mutant &lt; / RTI &gt;

The LeBZR1-T191A mutant protein provides a mutant protein in which the 191th threonine (Thr) of the LeBZR1 protein consisting of the amino acid sequence of SEQ ID NO: 3 is substituted with alanine (Alanin, Ala).

The LeBZR2-T183A mutant protein provides a mutant protein in which the 183rd threonine (Thr) of the LeBZR2 protein consisting of the amino acid sequence of SEQ ID NO: 4 is substituted with alanine (Alanin, Ala).

The range of the LeBZR1-T193A protein according to the present invention has the amino acid sequence of SEQ ID NO: 1 isolated from tomato, the range of the LeBZR2-T183A protein is the protein having the amino acid sequence of SEQ ID NO: 2 isolated from tomato, And functional equivalents of proteins. Refers to an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2 as a result of addition, substitution or deletion of an amino acid. More preferably 95% or more, and exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 1 and SEQ ID NO: 2.

The gene of the present invention includes both genomic DNAs or cDNAs encoding LeBZR1-T191A or LeBZR2-T183A mutant proteins. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the &lt; / RTI &gt;

The term "biomass" refers to the amount of biomass present in a certain space at a certain point in time, and is often referred to as biomass, biomass, or biomass. In particular, "plant biomass" is the total standing amount of organisms synthesized by plants through the process of photosynthesis using solar energy. It is a combination of leaves, branches, stem and roots.

The present invention also provides a recombinant vector comprising the polynucleotide of SEQ ID NO: 5 or SEQ ID NO: 6.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

A preferred example of a plant expression vector is a Ti-plasmid vector which is capable of transferring a so-called T-region into a plant cell when present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as Kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

In the plant expression vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens ) Terminator of the Octopine gene, and the rrnB1 / B2 terminator of E. coli, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a host cell transformed with the recombinant vector.

When the vector of the present invention is transformed into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, human cells (e.g., Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2 , 3T3, RIN and MDCK cell lines) and plant cells. The host cell is preferably a plant cell.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell.

The method of delivering the vector of the present invention into a host cell can be carried out by injecting a vector into a host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, can do.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The method is based on the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202,179-185 (Klein et al., 1987, Nature 327, 70), the infiltration of plants or the transformation of Agrobacterium tumefaciens Infection by viruses (non-integrative) in virus-mediated gene transfer (EP 0 301 316), and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

The present invention also provides a method for producing a plant for increasing biomass comprising overexpressing a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in a plant.

Wherein the polypeptide of SEQ ID NO: 1 is encoded by the polynucleotide of SEQ ID NO: 5 and the polypeptide of SEQ ID NO: 2 is encoded by the polynucleotide of SEQ ID NO:

 According to an embodiment of the present invention, the plant with increased biomass may have an increased plant length and an increased volume growth.

The present invention also provides transgenic plants and seeds with enhanced biomass of plants produced by the method.

According to an embodiment of the present invention, the range of the LeBZR1-T193A comprising the nucleotide sequence of SEQ ID NO: 5 or the LeBZR2-T183A gene comprising the nucleotide sequence of SEQ ID NO: 6 is as described above.

In the method according to one embodiment of the present invention, a method for regeneration of a transgenic plant from a transgenic plant cell can be performed by any method known in the art, and in the method according to one embodiment of the present invention, , The MS medium suitable for the growth stage of the plants was used as shown in Table 1 below. Specifically, the seeds were sterilized in the MS medium for seeding, and the cuticle was induced in callus induction MS medium to induce callus induction The callus was transferred to the MS medium for chute induction, and the chute grown on the MS medium for routing induction was transferred, followed by individual selection and plant purification.

The plants according to the present invention can be applied to plants such as Arabidopsis thaliana, eggplant, tobacco, pepper, tomato, burdock, ciliaceous lettuce, bellflower, spinach, modern, sweet potato, celery, carrot, buttercup, parsley, cabbage, It may be a dicotyledonous plant such as giblet, watermelon, melon, cucumber, pak, strawberry, soybean, mung bean, kidney bean and pea or a monocotyledon such as rice, barley, wheat, rye, maize, sorghum, oats, Is a dicotyledonous plant, more preferably a tomato (Lycopersicon esculentum), but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

< Preparation Example  1> micro-Tom transformation medium composition

< 1-1> MS medium

Types of MS medium Composition of the medium (total 500 ml) For sowing MS medium 4.4 g / L
sucrose 15g / L
MES hydrate 0.5 g / L
---- KOH pH buffering to pH 5.7 ~ 5.8 ----
Gelrite 3 g / L (plant agar 8 g / L)
DW up to 500ml Callus induction medium MS medium 4.4 g / L
sucrose 30 g / L
MES hydrate 0.5 g / L
---- KOH pH buffering to pH 5.7 ~ 5.8 ----
Gelrite 3 g / L (plant agar 8 g / L)
DW up to 500ml Suit guiding badge MS medium 4.4 g / L
sucrose 30 g / L
MES hydrate 0.5 g / L
---- KOH pH buffering to pH 5.7 ~ 5.8 ----
Gelrite 3 g / L (plant agar 8 g / L)
DW up to 500ml
zeatin 1.0 mg / L
(kanamycin 50mg / L, carbenicillin 500mg / L) Routing selection badge MS medium 4.4 g / L
sucrose 30 g / L
MES hydrate 0.5 g / L
---- KOH pH buffering to pH 5.7 ~ 5.8 ----
Gelrite 3 g / L (plant agar 8 g / L)
DW up to 500ml
(kanamycin 50mg / L, carbenicillin 500mg / L)

< Preparation Example  2> Cultivation method of transgenic plants

<2-1> Seed Disinfection and Sowing

10 to 20 seeds were sterilized in a 1.5 ml tube, rinsed with sterilized water, treated with 1 ml of 70% alcohol for 1 minute, treated with 1 ml of 50% lactase for 5 minutes, rinsed 5 times with sterilized water, 1 &lt; / RTI &gt; on the MS seeding medium for inoculation described in 1 above. Tomato seeds germinate about 1 week to 10 days later.

When seeds transfected with transgenes are sown, seeds containing the transgene are seeded in the medium inoculated with 2 ml of Agrobacterium tumefaciens antibiotic LB in the MS medium for seeding.

&Lt; 2-2 >

The germinated tomato immediately after the development of the cotyledon was prepared, and the both ends of the cotyledon were cut out. The cut cotyledon was cut perpendicularly to the leaf vein in half, and then the leaf surface was added to the callus inducing MS medium containing the composition shown in Table 1 , And the callus is induced by subculturing every 2 weeks.

During the transformation, the bacteria cultured in LB are collected and suspended in 20 ml of MS liquid medium. The cotyledonary slices were infected with Agrobacterium suspension for 10 minutes, and the slices were placed on a sterilized filter paper to absorb the suspension. The slides were dipped in a coexistent culture medium, wrapped in foil, and cultured for 3-4 days. The surface is placed upside down on the medium, and subcultured every 2 weeks to induce callus.

<2-3> Suit induction

When a chute is confirmed in a leaf section, the callus is transferred to a chute inducing MS medium containing the composition shown in Table 1 above.

<2-4> Routing induction

If the grown chute grows to 1 to 2 cm, only the chute is cut out and the chute is transferred to the MS medium for routing induction containing the composition shown in Table 1 above.

<2-5> Individual selection and plant purification

Within 2 weeks, individuals with roots are sorted into regenerated individuals and grown in a plant box for about 1 month to purify the plant.

< Example  1> LeBZR1 - T191A  And LeBZR2 - T183A  Methods for producing transformed plants containing

<1-1> Production of Transformation Vector

(SEQ ID NO: 7), LeBZR1-T191A_R: 5`-AAAGAGGTGGAGCTACAGGGGCACT-3` (SEQ ID NO: 8), LeBZR2-T183A_F: 5`-AGTGCCCCTGTAGCTCCACCTCTTT- (SEQ ID NO: 9), LeBZR2-T183A_R: 5'-AAAGAGGTGGTGCTACAGGGGCACT-3 '(SEQ ID NO: 10)), a site-specific mutagenesis PCR in a recombinant vector cloned into a Ti-plasmid vector Amplify the gene. Thereafter, the restriction enzyme Dpn1 is treated and introduced into a competent cell (E. coli) (see FIGS. 5 and 6). Substituted amino acid sequences are expressed due to the changed base sequence in protein expression in the recombinant vector.

<1-2> Production of Transgenic Plants

The vector containing the above-mentioned LeBZR1-T191A and LeBZR2-T183A was used for the recombinant vector Ti-plasmid vector (vector name: pBI121, including the kanamycin as an optional marker, CaMV 35s promoter) Agrobacterium capable of transferring into plant cells tumefaciens to make a strain of each gene. When inserted into Agrobacterium, recombinant vector is inserted using electroporation technique and cultured in LB medium at 28 ° C for 2 days. Specifically, transgenic plants were grown by the method described in <Preparation Example 2> using MS medium containing the necessary medium composition in each step described in <Preparation Example 1> by a culture method suitable for each plant growth step. Specifically, the microorganisms cultured in LB at the time of transformation are collected and suspended in 20 ml of MS liquid medium. The cotyledonary slices were infected with Agrobacterium suspension for 10 minutes, and the slices were placed on a sterilized filter paper to absorb the suspension. The slides were dipped in a coexistent culture medium, wrapped in foil, and cultured for 3-4 days. The surface is placed upside down on the medium, and subcultured every 2 weeks to induce callus.

< Example  2> LeBZR1  Analysis of gene expression site

To confirm the expression pattern of the LeBZR1 gene, the GUS reporter gene was fused to the promoter region of the LeBZR1 gene of tomato, and then the recombinant vector containing LeBZR1 gene was transformed into tomato and the expression pattern was confirmed by GUS reporter assay . Since GUS is expressed through the LeBZR1 promoter, the expression pattern of LeBZR1 can be confirmed.

As a result, it was confirmed that LeBZR1 gene was expressed in the vascular system such as stem and leaf vein of tomato (see FIG. 1).

< Example  3> LeBZR1  or LeBZR2  Gene expression analysis

The amino acid sequences of 191-Thr and LeBZR2 183-Thr of LeBZR1 were changed to alanine to produce transcription factor functional genes.

Specifically, the 191-Thr and LeBZR2 183-Thr amino acid sequences of LeBZR1 were changed to alanine, and transgenic plants transfected with recombinant plant transformation vectors containing transcription factor functional genes were transfected Expression patterns of LeBZR1-T191A and LeBZR2-T183A in the stem sections of LeBZR1-T191A and LeBZR2-T183A Respectively.

As a result, it was confirmed that the stem of LeBZR1-T191A and LeBZR2-T183A transformants overexpressing the function-acquiring transcription factor mutants was more prominent than that of the transgenic overexpressed normal BZR1 gene (control group) (See Fig. 2).

In addition, wild type tomatoes, LeBZR1-T191A, LeBZR2-T183A (See FIG. 3A), the wild-type tomatoes, LeBZR1-T191A, LeBZR2-T183A (see FIG. 3A) The length of each plant in the over-expressing transformant was 8, 22, 16 cm (see Fig. 3B).

From the above results, it was confirmed that the length growth by promoting the primary growth of tomato was increased by the tomato transcription factor genes LeBZR1 and LeBZR2 function acquiring mutant genes, and the volume growth by the secondary growth promotion was increased.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Mutation of LeBZR1 or LeBZR2 gene inducing biomass increase of          plant and uses thereof <130> PN1602-064 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 333 <212> PRT <213> LeBZR1-T191A <400> 1 Met Met Trp Glu Ala Gly Glu Ser Pro Ala Ser Ser Ser Ala Gly Ala   1 5 10 15 Gly Ala Gly Gly Gly Gly Gly Aly Gly Val Gly Leu Pro Glu Ser Gly              20 25 30 Gly Gly Gly Gly Gly Gly Arg Arg Lys Pro Ser Trp Arg Glu Arg Glu          35 40 45 Asn Asn Arg Arg Arg Glu Arg Arg Arg Arg Ala Val Ala Ala Lys Ile      50 55 60 Tyr Thr Gly Leu Arg Ala Gln Gly Asn Tyr Asn Leu Pro Lys His Cys  65 70 75 80 Asp Asn Asn Glu Val Leu Lys Ala Leu Cys Thr Glu Ala Gly Trp Ile                  85 90 95 Val Glu Pro Asp Gly Thr Thr Tyr Arg Lys Gly Cys Lys Pro Thr Pro             100 105 110 Met Glu Ile Gly Gly Thr Ser Thr Asn Ile Thr Pro Ser Ser Ser Arg         115 120 125 His Pro Ser Pro Ser Ser Tyr Phe Ala Ser Pro Ile Pro Ser Tyr     130 135 140 Gln Pro Ser Pro Thr Ser Ser Ser Phe Pro Ser Ser Ser Arg Ala Asp 145 150 155 160 Ala Asn Met Ser Ser His Pro Tyr Ser Phe Leu Gln Asn Val Val Pro                 165 170 175 Ser Ser Leu Pro Pro Leu Arg Ser Ser Asn Ser Ala Pro Val Ala Pro             180 185 190 Pro Leu Ser Ser Pro Thr Arg His Pro Lys Gln Thr Phe Asn Leu Glu         195 200 205 Thr Leu Ala Lys Glu Ser Met Phe Ala Leu Asn Ile Pro Phe Phe Ala     210 215 220 Ala Ser Ala Pro Ala Ser Pro Thr Arg Val Gln Arg Phe Thr Pro Pro 225 230 235 240 Thr Ile Pro Glu Cys Asp Glu Ser Asp Ser Ser Thr Ile Asp Ser Gly                 245 250 255 Gln Trp Ile Asn Phe Gln Lys Tyr Ala Ser Asn Val Pro Pro Ser Pro             260 265 270 Thr Phe Asn Leu Val Lys Pro Val Pro Gln Pro Leu Arg Pro Asn Asp         275 280 285 Met Ile Thr Asp Lys Gly Lys Ser Ile Asp Phe Asp Phe Glu Asn Val     290 295 300 Ser Val Lys Ala Trp Glu Gly Glu Arg Ile His Asp Val Gly Phe Asp 305 310 315 320 Asp Leu Glu Leu Thr Leu Gly Ser Gly Asn Ala Arg Ile                 325 330 <210> 2 <211> 327 <212> PRT <213> LeBZR2-T183A <400> 2 Met Trp Glu Gly Gly Gly Leu Pro Val Glu Gly Gly Gly Gly Gly Val Gly   1 5 10 15 Gly Gly Gly Gly Val Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Arg Arg              20 25 30 Lys Pro Ser Trp Arg Glu Arg Glu Asn Asn Arg Arg Arg Glu Arg Arg          35 40 45 Arg Arg Ala Ila Ala Lys Ile Tyr Ser Gly Leu Arg Ala Gln Gly      50 55 60 Asn Tyr Asn Leu Pro Lys His Cys Asp Asn Asn Glu Val Leu Lys Ala  65 70 75 80 Leu Cys Val Glu Ala Gly Trp Ile Val Glu Pro Asp Gly Thr Thr Tyr                  85 90 95 Arg Lys Gly Cys Arg Pro Thr Pro Met Glu Ile Gly Gly Thr Ser Ala             100 105 110 Asn Ile Thr Pro Ser Ser Ser Arg Asn Pro Ser Pro Pro Ser Ser Tyr         115 120 125 Phe Ala Ser Pro Ile Pro Ser Tyr Gln Val Ser Pro Thr Ser Ser Ser     130 135 140 Phe Pro Ser Pro Ser Arg Gly Asp Ala Asn Met Ser Ser His Pro Phe 145 150 155 160 Ala Phe Leu His Ser Ser Ile Pro Leu Ser Leu Pro Pro Leu Arg Ile                 165 170 175 Ser Asn Ser Ala Pro Val Ala Pro Pro Leu Ser Ser Pro Thr Arg Val             180 185 190 Pro Lys Gln Ile Phe Asn Leu Glu Thr Leu Ala Arg Glu Ser Met Ser         195 200 205 Ala Leu Asn Ile Pro Phe Phe Ala Ala Ser Ala Pro Thr Ser Pro Thr     210 215 220 Arg Gly Gln Arg Phe Thr Pro Ala Thr Ile Pro Glu Cys Asp Glu Ser 225 230 235 240 Asp Ser Ser Thr Ile Asp Ser Gly Gln Trp Met Ser Ser Phe Gln Lys Tyr                 245 250 255 Ala Ala Asn Gly Ile Pro Thr Ser Pro Thr Phe Asn Leu Ile Lys Pro             260 265 270 Val Ala Gln Arg Ile Pro Ser Asn Asp Met Ile Ile Asp Lys Gly Lys         275 280 285 Ser Ile Glu Phe Asp Phe Glu Asn Val Ser Val Lys Ala Ala Trp Glu     290 295 300 Gly Glu Lys Ile His Glu Val Gly Leu Asp Asp Leu Glu Leu Thr Leu 305 310 315 320 Gly Ser Gly Thr Ala Arg Met                 325 <210> 3 <211> 333 <212> PRT <213> LeBZR1 <400> 3 Met Met Trp Glu Ala Gly Glu Ser Pro Ala Ser Ser Ser Ala Gly Ala   1 5 10 15 Gly Ala Gly Gly Gly Gly Gly Aly Gly Val Gly Leu Pro Glu Ser Gly              20 25 30 Gly Gly Gly Gly Gly Gly Arg Arg Lys Pro Ser Trp Arg Glu Arg Glu          35 40 45 Asn Asn Arg Arg Arg Glu Arg Arg Arg Arg Ala Val Ala Ala Lys Ile      50 55 60 Tyr Thr Gly Leu Arg Ala Gln Gly Asn Tyr Asn Leu Pro Lys His Cys  65 70 75 80 Asp Asn Asn Glu Val Leu Lys Ala Leu Cys Thr Glu Ala Gly Trp Ile                  85 90 95 Val Glu Pro Asp Gly Thr Thr Tyr Arg Lys Gly Cys Lys Pro Thr Pro             100 105 110 Met Glu Ile Gly Gly Thr Ser Thr Asn Ile Thr Pro Ser Ser Ser Arg         115 120 125 His Pro Ser Pro Ser Ser Tyr Phe Ala Ser Pro Ile Pro Ser Tyr     130 135 140 Gln Pro Ser Pro Thr Ser Ser Ser Phe Pro Ser Ser Ser Arg Ala Asp 145 150 155 160 Ala Asn Met Ser Ser His Pro Tyr Ser Phe Leu Gln Asn Val Val Pro                 165 170 175 Ser Ser Leu Pro Pro Leu Arg Ser Ser Asn Ser Ala Pro Val Thr Pro             180 185 190 Pro Leu Ser Ser Pro Thr Arg His Pro Lys Gln Thr Phe Asn Leu Glu         195 200 205 Thr Leu Ala Lys Glu Ser Met Phe Ala Leu Asn Ile Pro Phe Phe Ala     210 215 220 Ala Ser Ala Pro Ala Ser Pro Thr Arg Val Gln Arg Phe Thr Pro Pro 225 230 235 240 Thr Ile Pro Glu Cys Asp Glu Ser Asp Ser Ser Thr Ile Asp Ser Gly                 245 250 255 Gln Trp Ile Asn Phe Gln Lys Tyr Ala Ser Asn Val Pro Pro Ser Pro             260 265 270 Thr Phe Asn Leu Val Lys Pro Val Pro Gln Pro Leu Arg Pro Asn Asp         275 280 285 Met Ile Thr Asp Lys Gly Lys Ser Ile Asp Phe Asp Phe Glu Asn Val     290 295 300 Ser Val Lys Ala Trp Glu Gly Glu Arg Ile His Asp Val Gly Phe Asp 305 310 315 320 Asp Leu Glu Leu Thr Leu Gly Ser Gly Asn Ala Arg Ile                 325 330 <210> 4 <211> 327 <212> PRT <213> LeBZR2 <400> 4 Met Trp Glu Gly Gly Gly Leu Pro Val Glu Gly Gly Gly Gly Gly Val Gly   1 5 10 15 Gly Gly Gly Gly Val Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Arg Arg              20 25 30 Lys Pro Ser Trp Arg Glu Arg Glu Asn Asn Arg Arg Arg Glu Arg Arg          35 40 45 Arg Arg Ala Ila Ala Lys Ile Tyr Ser Gly Leu Arg Ala Gln Gly      50 55 60 Asn Tyr Asn Leu Pro Lys His Cys Asp Asn Asn Glu Val Leu Lys Ala  65 70 75 80 Leu Cys Val Glu Ala Gly Trp Ile Val Glu Pro Asp Gly Thr Thr Tyr                  85 90 95 Arg Lys Gly Cys Arg Pro Thr Pro Met Glu Ile Gly Gly Thr Ser Ala             100 105 110 Asn Ile Thr Pro Ser Ser Ser Arg Asn Pro Ser Pro Pro Ser Ser Tyr         115 120 125 Phe Ala Ser Pro Ile Pro Ser Tyr Gln Val Ser Pro Thr Ser Ser Ser     130 135 140 Phe Pro Ser Pro Ser Arg Gly Asp Ala Asn Met Ser Ser His Pro Phe 145 150 155 160 Ala Phe Leu His Ser Ser Ile Pro Leu Ser Leu Pro Pro Leu Arg Ile                 165 170 175 Ser Asn Ser Ala Pro Val Thr Pro Pro Leu Ser Ser Pro Thr Arg Val             180 185 190 Pro Lys Gln Ile Phe Asn Leu Glu Thr Leu Ala Arg Glu Ser Met Ser         195 200 205 Ala Leu Asn Ile Pro Phe Phe Ala Ala Ser Ala Pro Thr Ser Pro Thr     210 215 220 Arg Gly Gln Arg Phe Thr Pro Ala Thr Ile Pro Glu Cys Asp Glu Ser 225 230 235 240 Asp Ser Ser Thr Ile Asp Ser Gly Gln Trp Met Ser Ser Phe Gln Lys Tyr                 245 250 255 Ala Ala Asn Gly Ile Pro Thr Ser Pro Thr Phe Asn Leu Ile Lys Pro             260 265 270 Val Ala Gln Arg Ile Pro Ser Asn Asp Met Ile Ile Asp Lys Gly Lys         275 280 285 Ser Ile Glu Phe Asp Phe Glu Asn Val Ser Val Lys Ala Ala Trp Glu     290 295 300 Gly Glu Lys Ile His Glu Val Gly Leu Asp Asp Leu Glu Leu Thr Leu 305 310 315 320 Gly Ser Gly Thr Ala Arg Met                 325 <210> 5 <211> 1002 <212> DNA <213> LeBZR1-T191A <400> 5 atgatgtggg aagctggaga atcaccagca tcttcttcgg ccggtgccgg agctggtgga 60 agtggaggtg ccggagttgg tttaccggaa agtggtggtg gtggtggtgg tgggagaagg 120 aaaccatcat ggagagaaag agagaataac aggagaagag agaggaggag gagagctgta 180 gctgctaaga tttatactgg tttaagagct caaggaaact ataatcttcc gaagcactgt 240 gataacaatg aagttcttaa agctctttgt actgaagctg gttggatcgt tgaacctgat 300 ggtaccactt atcgcaaggg atgcaagcca accccgatgg agattggagg cacttcaaca 360 aacatcacgc caagttcttc acggcatcca agtcccccat catcatactt tgctagccca 420 attccatctt atcagccaag tccaacttcc tcttctttcc ccagtccatc tcgtgctgat 480 gccaacatgt catcacatcc atattctttt ctccaaaatg tcgttccttc atcccttcct 540 ccattacgaa tatcaaacag tgcccctgta gctccacctc tttcatcacc aactaggcat 600 cctaagcaaa ctttcaattt agaaactttg gccaaagaat caatgtttgc tttaaacatc 660 cctttctttg ctgcttcagc cccagcaagc ccaactaggg ttcagcgttt tactcctcca 720 actatacccg agtgtgatga atctgactca tctaccattg attcaggcca gtggatcaac 780 tttcaaaagt atgcgtcaaa tgttccacct tctccaacat ttaatcttgt aaaacctgtg 840 cctcagccgc ttcgtcctaa tgatatgatc acagacaagg gtaagagcat agacttcgac 900 tttgaaaatg tatcagtcaa ggcatgggaa ggtgaaagga ttcacgatgt aggattcgat 960 gatctggaac tcacacttgg aagtggcaat gctcgcatat ga 1002 <210> 6 <211> 985 <212> DNA <213> LeBZR2-T183A <400> 6 atgtgggaag gtggagggtt gccggtggag ggtggtggtg gtgttggtga aggtggtggt 60 gttggtggtg gtggaggtgg tggtagtggg aggaggaagc catcatggag ggaaagggag 120 aataatagga ggagggaaag gaggagaagg gcaatagcag ctaagattta tagtggatta 180 agagcacagg ggaattataa tcttcctaaa cattgtgata acaatgaggt tttgaaggct 240 ctttgtgttg aagctggatg gattgttgag cctgatggaa ctacttatag aaagggatgc 300 aggccaactc caatggagat tggaggcact tcagccaaca ttacgccaag ttcttcacga 360 aatccaagtc ctccctcttc atactttgct agcccgattc catcttacca agttagtcca 420 acatcctcgt ctttcccaag tccatctcgt ggtgatgcta acatgtcgtc acatccattt 480 gcatttctcc atagttccat tcccttgtcg ctaccaccat tacgaatatc aaacagtgcc 540 cctgtagaca ccacctcttt catcaccaac tagagtccct aagcagatat ttaatcttga 600 gactttggct agagagtcta tgtctgctct aaatatccct ttctttgctg cttcagcccc 660 aactagccca actcgaggtc agcgattcac tcctgctaca ataccagagt gtgacgaatc 720 tgattcatcc accattgatt ctggccagtg gatgagcttt caaaagtacg cagccaatgg 780 gatccctact tctccgactt ttaatcttat taagcctgta gctcagagaa ttccttctaa 840 tgatatgatc atcgacaagg gtaagagcat tgaatttgac tttgagaatg tatcagttaa 900 ggcagcatgg gaaggtgaaa agattcatga ggttggttta gatgatctgg agctcactct 960 cggaagtggg actgctcgga tgtga 985 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LeBZR1-T191A_F <400> 7 agtgcccctg tagctccacc tcttt 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LeBZR1-T191A_R <400> 8 aaagaggtgg agctacaggg gcact 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LeBZR2-T183A_F <400> 9 agtgcccctg tagcaccacc tcttt 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LeBZR2-T183A_R <400> 10 aaagaggtgg tgctacaggg gcact 25

Claims (12)

2. A polypeptide consisting of SEQ ID NO: 2 which increases plant biomass. delete 2. The polypeptide of claim 1, wherein the polypeptide of SEQ ID NO: 2 is encoded by a polynucleotide of SEQ ID NO: A recombinant vector comprising the polynucleotide of SEQ ID NO: 6. A host cell transformed with the recombinant vector of claim 4. 6. The transformed host cell according to claim 5, wherein the host cell is a plant cell. A method for producing a plant for increasing biomass comprising overexpressing in a plant a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. delete 8. The method of claim 7, wherein the polypeptide of SEQ ID NO: 2 is encoded by a polynucleotide of SEQ ID NO: 6. 8. The method of claim 7, wherein the biomass-enhanced plant is a plant having increased length and increased volumetric growth. A transgenic plant in which the biomass of the plant produced by the method of claim 7 is enhanced. 12. Seeds of transgenic plants according to claim 11.

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