KR101322319B1 - A method increasing tiller numbers of plants using hox10 gene - Google Patents

Abstract

PURPOSE: A method for increasing plant body tiller number by using HOX10 gene is provided to supply transgenic plant, thereby realizing improvement of productivity, and smooth supply of agricultural product. CONSTITUTION: A method for increasing plant body tiller number comprises the step of increasing HOX10 protein level in cell which has sequence number 2 of amino acid sequence. To increase the level in cell, the gene coding the protein is overexpressed. The gene has base sequence represented by sequence number 1, or base sequence of 411-2930 in sequence number 1. A method of overexpressing the gene comprises the step of transforming the plant cell by the recombinant expression vector which operationally connects the gene to the promoter.

Description

A Method Increasing Tiller numbers of Plants Using HOX10 Gene}

The present invention relates to a method for selecting genes that increase the number of grains of a plant, increasing the number of grains of a plant using such genes, and a transgenic plant produced by the method.

Bundle refers to the growth of the armpits of the nodes under the stems in rice plants to form several stems. Also called abandonment. There are several stems in the base of the rice plant, and the axillary eyes come out from each stem, which is called the caudal eye, which is developed, covered with the leaf leaf of the stem, and extends outward from the end. The first bullet is called the first bullet, and the second bullet comes from the stem of the first bullet. In this way, the 34th bullet may come out. In rice barley, etc., 4-10 grains are usually produced from a single cultivation method. The grains that grow ears are called effective grains, and the grains that do not grow due to weak growth are called invalid grains. The bullet from the upper section of the base or the 23rd bullet is likely to become invalid. The number of powders varies depending on the variety, but decreases due to lack of sunshine, lack of moisture in the soil, low temperature, full meal and wheat, and the yield is reduced when there is little effective powder. Since the number of grains determines the number of ear of the most important factor related to the yield of rice plants, there is great interest in the number of grains.

Thus, the inventors of the present invention, while studying the method of increasing the number of the number of plants in the plant, confirmed that the HOX10 gene can control the number of plants in the plant and completed the present invention.

Accordingly, the problem to be solved by the present invention is to increase the number of grains of plants containing rice using the HOX10 gene, to improve the productivity of the plant and to enable the smooth supply of agricultural products.

In order to achieve the above technical problem, in one aspect, the present invention provides a method for increasing the number of minutes of plants by increasing the intracellular level of the HOX10 protein having an amino acid sequence represented by SEQ ID NO: 2.

The "intracellular level" refers to the amount present in a cell, which can be controlled by various methods known to those skilled in the art. For example, intracellular levels can be regulated through, but not limited to, regulation at the transcriptional stage or regulation at the post-transcriptional stage. Modulation at the transcriptional stage is a method for enhancing the expression of genes known to those skilled in the art, for example, linking a HOX10 gene encoding a HOX10 protein having an amino acid sequence represented by SEQ ID NO: 2 to a promoter or a homologous gene thereto A method of preparing a recombinant expression vector to enhance the expression of the gene or to enhance the expression of the gene around the HOX10 gene encoding the HOX10 protein having the amino acid sequence represented by SEQ ID NO: 2 or a homolog thereof It may be carried out by the method of inserting expression control sequences. Modulation at the post-transcriptional stage is a method for enhancing protein expression known to those of skill in the art, for example, the stability of mRNA transcribed into the HOX10 gene of the HOX10 protein having a amino acid sequence represented by SEQ ID NO: 2 or a functional equivalent thereof. It can be carried out by a method for enhancing the activity, a method for enhancing the stability of the protein or a method for enhancing the activity of the protein.

Preferably, in the present invention, increasing the intracellular level of a protein having an amino acid sequence represented by SEQ ID NO: 2 or an equivalent thereof may be performed by a method of overexpressing a gene encoding the protein. Such overexpression may use methods known to those skilled in the art, but for example, a promoter encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 or a gene encoding the equivalent thereof, preferably a nucleotide sequence represented by SEQ ID NO: 1 It is possible to enhance the expression by preparing a recombinant expression vector operably linked to the gene having a gene or its CDS (coding sequence) 411-2930 of SEQ ID NO: 1.

The protein equivalent includes a homologous protein having homology with a protein having an amino acid sequence represented by SEQ ID NO: 2.

The protein having the amino acid sequence represented by SEQ ID NO: 2 is NCBI Reference Sequence: Q6TAQ6, which is composed of 839 amino acids.

The "homologous protein" is preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% of the amino acid sequence represented by SEQ ID NO: 2. As a protein which has, it is the protein which shows the function substantially homogeneous with the HOX10 protein of this invention.

In addition, the gene equivalent includes a homology gene having homology with a gene having a nucleotide sequence represented by SEQ ID NO: 1 or a gene 411-2930 of SEQ ID NO: 1, which is a CDS thereof.

The HOX10 gene having the nucleotide sequence represented by SEQ ID NO: 1, the 411-2930 gene of SEQ ID NO: 1, or its homologous gene, which is a CDS thereof, encodes a protein. The HOX10 gene having the nucleotide sequence represented by SEQ ID NO: 1 is NCBI Reference Sequence: NM_001055242 and consists of 3252 nucleic acids. Of the 3252 nucleic acids, a region that substantially codons amino acids is a region in which 2520 bp is substantially codon of the HOX10 gene except for 410 bp and 5 322 bp downstream regions of 5 'upstream region. That is, genes 411 to 2930 of SEQ ID NO: 1 are CDS, and SEQ ID NO: 1 is a gene including UTR.

The "homologous gene" is preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, of the base sequence of SEQ ID NO: 1 or 411-2930, which is the CDS of SEQ ID NO: 1. Most preferably, a gene having a sequence homology of 99% or more refers to a gene having substantially the same function as the HOX10 gene of the present invention. Sequence homology can be analyzed by methods known in the art.

The term "substantially homogeneous function" means to be involved in increasing the number of hands. Such functional equivalents include amino acid sequence variants in which some of the amino acids of the amino acid sequence represented by SEQ ID NO: 2 are substituted, deleted or added. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows; (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids is preferably located in portions not directly involved in the activity of HOX10 of the invention. In addition, the functional equivalent range includes protein derivatives in which some chemical structures of the protein are modified while maintaining the basic skeleton of HOX10 and its physiological activity. For example, fusion proteins made by fusion with other proteins, such as GFP, while maintaining structural modifications and physiological activities to alter the stability, shelf life, volatility or solubility of the protein of the present invention.

In one embodiment, the present inventors transform a rice cell by preparing a recombinant expression vector comprising a HOX10 gene, which is a gene having a nucleotide sequence represented by SEQ ID NO: 1 or a 411-2930 gene of SEQ ID NO: 1, which is a CDS thereof. The expression level of HOX10 protein in rice cells was increased.

In addition, the transforming gene according to the present invention is a gene having a nucleotide sequence of SEQ ID NO: 1 and the 411-2930 gene of SEQ ID NO: 1 of its CDS, as well as a modification in which some bases of these genes are substituted, deleted or added As a sequence, a base sequence coding for a protein that exhibits the activity of a protein encoded from the nucleotide sequence of SEQ ID NO: 1 or 411-2930 of SEQ ID NO: This can be used.

The HOX10 gene according to the present invention can be inserted into a suitable expression vector, ie, a recombinant expression vector, to transform plant cells.

More specifically, in another aspect, the present invention also provides a gene encoding a HOX10 protein having an amino acid sequence represented by SEQ ID NO: 2, or a gene having a nucleotide sequence represented by SEQ ID NO: 1, which significantly increases the number of particles of a plant of interest. Or it provides a recombinant expression vector comprising a gene 411-2930 of SEQ ID NO: 1 of its CDS. More preferably, the vector may have a cleavage map of FIG. 3.

The expression vector refers to a plasmid, virus or other media known in the art that the gene according to the present invention can be inserted or introduced. The gene according to the present invention may be operably linked to an expression control sequence, and the operably linked gene sequence and expression control sequence may be included in one expression vector including a selection marker and a replication origin. Can be. Suitable vectors for introducing the genes of the invention into plant cells include Ti plasmids and plant viral vectors. Most preferably, pGA2897 vector may be used, but the present invention is not limited thereto, and a person skilled in the art may select a vector suitable for introducing a gene according to the present invention.

By “operably linked” is meant that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment.

The "expression control sequence" is a DNA sequence that controls the expression of genes operably linked in a particular host cell, such control sequence is a promoter for transcription, any operator sequence for controlling transcription. , Sequences encoding suitable mRNA ribosomal binding sites and sequences that control termination of transcription and translation.

The term "promoter" refers to a DNA sequence that controls the expression of a gene operably linked in a particular host cell. As a promoter, a promoter (constitutive promoter) which induces the expression of the gene of interest at all times of the day or a promoter (inducible promoter) which induces the expression of the gene of interest at a specific position and time period may be used. A promoter for inducing the expression of a gene is used, and examples thereof include, but are not limited to, a p35S promoter. In addition, the ubiquitin promoter can be used to overexpress genes in monocotyledonous or woody plants.

In one embodiment, we prepared a recombinant vector HOX10 :: pGA2897 prepared by inserting the HOX10 gene into a vector pGA2897 containing a ubiquitin promoter (FIG. 3).

Introduction of the recombinant expression vector into the plant can be used a method known in the art. For example, but not limited to, the Agrobacterium sp.-mediated method, particle gun bombardment, silicon carbide whiskers, sonication, heat shock Precipitation by heat shock, electroporation, and polyethylene glycol (PEG) can be used. In one embodiment of the present invention, a plant cell is transformed into a recombinant vector of the present invention through a method of Agrobacterium-mediated method, ie, a method of transforming plant cells using an Agrobacterium into which an expression vector according to the present invention is introduced. Although transformed, the present invention is not limited thereto.

In another aspect, the present invention provides a plant transformed with a recombinant expression vector comprising the HOX10 gene.

Plant cells into which the recombinant expression vector of the present invention is introduced are not particularly limited to any particular form as long as the cells can be regenerated into plants. These cells include, for example, cultured cell suspensions, protoplasts, leaf sections, and callus.

The target plant according to the present invention may include, but is not limited to, rice, corn, sugar cane, barley, wheat, and the like, preferably rice.

More specifically, the plant having an increased number of sesame seeds according to the present invention is transformed into a plant with a recombinant expression vector comprising a gene of SEQ ID NO: 1 or a 411-2930 gene of SEQ ID NO: 1, which is a CDS thereof, followed by callus according to a conventional method. It can be obtained through the process of induction, rooting and soil purification.

In one embodiment, the present inventors were introduced into the Agrobacterium by heat shock with a recombinant expression vector containing the HOX10 gene and transformed into rice using the introduced Agrobacterium. The HOX10 gene overexpressing transformant was obtained and analyzed for its number and phenotype. As a result, it was confirmed that the number of rice grains of the transformed rice increased compared to the wild type.

The present invention can provide a method for increasing the number of grains in a plant, and finally can provide a transformed plant having an increased number of grains. By supplying such transgenic plants, it will be possible to improve productivity and to supply agricultural products smoothly.

1 shows the results of domain analysis of the HOX10 gene.
Figure 2 shows the results of analyzing the expression pattern of the rice tissue of HOX10 gene by RT-PCR.
Figure 3 using the pGA2897 vector The recombinant expression vector (HOX10 :: pGA2897) for transformation of the HOX10 gene is shown.
Figure 4 shows plants obtained by Agrobacterium infection transformation.
Figure 5 shows the results of analyzing the expression pattern of the tissue transformant of the HOX10 gene.
Figure 6 shows the results of the analysis of the number of transformed transformants 1 month after GMO package transplantation.
FIG. 7 shows the results of the serial number analysis of the transformants 2 months after GMO package transplantation.
Figure 8 shows the results of analyzing the phenotype of the HOX10 gene overexpressed rice transformant.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example 1 Cloning and Sequence Analysis of OsHOX10 Gene by RT-PCR

< Example 1-1> First-strand cDNA Synthesis

Total RNA was extracted from the leaves of rice and first-strand cDNA was synthesized using Sprint RT Complete-Oligo (dT) (Clontech kit). PCR of the sense primer: 5'-ATGGCTGCGGCAGTGGCAATGCGA-3 '(SEQ ID NO: 3) and antisense primer: 5'-TCACACAAAGGACCAGTTGACGAAAC-3' (SEQ ID NO: 4) of the synthesized cDNA and HOX10 genes The gene amplified by electrophoresis on% agarose was gel extracted (QIAquick gel extraction kit).

<Example 1-2> Cloning to a Topo 2.1 vector (Invitrogen, TOPO TA Cloning kit)

For cloning amplified genes, white colonies obtained by plating on L-B medium added with X-gal were selected and subjected to colony PCR. As a result, the plasmid of the colonies where the band was confirmed was extracted and used for sequencing.

Example 1-3 Sequence Analysis

Sequence analysis using ABI 3700 sequencer and BLAST search of NCBI It was confirmed that the HOX10 gene (Fig. 1).

Example 2 Analysis of Expression Pattern of OsHOX10 Gene

For RNA isolation of leaves, stems, roots, medicines, fruitlets, and pears, each sample is placed in a mortar of liquid nitrogen and finely ground.Put the appropriate amount into a 2 ml tube. ㎕ was added and quickly vortexed. 400 μl of chloroform was added to the same tube, followed by simple vortex, and centrifuged at 13,000 rpm for 10 minutes using a 4 ° C. centrifuge. After repeating this process one more time, 375 μl of isopropanol was added to the final supernatant, which was mixed well, and centrifuged at 13,000 rpm for 10 minutes at 4 ° C. The supernatant was discarded and the RNA precipitate was washed with 1 ml of 70% ethanol. After centrifugation at 4 ° C. at 13,000 rpm, the supernatant was completely removed and the RNA precipitate was dried at 37 ° C. for 30 minutes. 50 μl of ddH 2 O or 1 × TE buffer was added and vortexed to completely dissolve RNA and incubated in a 60 ° C. incubator for 10 minutes. Centrifuged at 13,000 rpm at 20 ° C, the supernatant was transferred to a new 1.5 mL tube to quantify the amount of RNA by spectrophotometer and stored at -70 ° C. These RNAs were synthesized first-strand cDNA using SUPERSCRIPTII (Takara ver 3.0) mentioned above, and then RT-PCR using the same primer as the primer used in Example 1 to express the amount of HOX10 gene for each tissue. Was analyzed (FIG. 2).

Example 3 Preparation of Rice Transformation Vector Using pGA2897 Vector Production of OsHOX10 gene overexpression carrier

The HOX10 gene amplified by RT-PCR was cloned into the pCR8 / GW / TOPO vector to insert the HOX10 gene into the pGA2897 vector. In order to produce a binary vector for rice transformation, the reaction was performed by mixing the cloned HOX10 gene with the target vector pGA2897 and the pCR8 / GW / TOPO vector in an LR (Invitrogen) reaction solution according to a Gateway system. Transformation was performed. Colony PCR was performed to confirm whether the HOX10 gene was inserted into the pGA2897 vector, and a binary vector into which the HOX10 gene was inserted was completed (FIG. 3).

In order to transform the completed binary vector into Agrobacterium (LBA4404), the freeze and thaw were repeated 2-3 times, followed by a heat shock method at 37 ° C. After transformation (transformation) by YEP medium (Yeast 10g, NaCl 5g, peptone (10g), agar (Agar) 15g / 1L colonies were confirmed overnight. Colonies identified by colony PCR were transformed into rice in AB medium <AB buffer (K ₂ HPO ₄ 60g, NaH ₂ PO ₄ 20g / 1L), AB Salts (NH ₄ Cl 60g, and MgSO ₄ 7H ₂ O 6g, and cultured at 3g KCl, CaCl _{2 and 2H 2 O 0.265g, FeSO 4 .7H} 2 O 50mg / 1L), Glucose 5g / 1L>.

Example 4 Transformation of Rice Using Agrobacterium

Example 4-1 Somatic Embryonic Culture

For rice transformation by Agrobacterium, the seeds were washed in Lax and then dried appropriately and healed in 2N6 medium (Medium containing CHU (N6) vitamin from Duchefa Co.) with 2,4-D hormone. . Injured seeds were incubated at 28 ° C. for about 7 days and then embryos were extracted and used for Agrobacterium infection containing the HOX10 gene.

Example 4-2 Agrobacterium Infection and Transformant Induction (FIG. 4)

Embryogenic embryos and Agrobacterium-transformed genes were incubated in AB liquid medium for 20 minutes and then incubated for 7 days in medium containing acetosyringone in 2N6 medium. After Agrobacterium infection, the cells were cultured for 2 weeks in a medium in which cefotaxime and hygromycin were added to 2N6 medium. To induce shooting, cancer-embedded embryos are transferred to medium supplemented with MSR-cefotaxime and hygromycin, followed by culturing for 2-3 weeks, followed by rooting. In this case, the purification process was performed before moving to the greenhouse.

Example 4-3 Seed Harvest and Post Fixing

After about 5-7 days, the obtained transformant (T ₀ ) was cultivated in a greenhouse to harvest seeds, and then selected as a hygromycin antibiotic for later fixation. The seeds obtained were grown in genetically modified organism (GMO) packaging for phenotypic analysis of postfixed and overexpressed transformants, and then the seeds were used for generation progress after harvest.

< Example  5> Of Rice Transformant HOX10  Gene expression profile analysis

RT-PCR was performed to analyze the expression pattern of HOX10 gene in the rice transformant containing OsHOX10 gene, and it was confirmed that HOX10 gene was expressed in all leaves, stems, and roots (FIG. 5).

<Example 6> Analysis of Rice Flour Numbers

As a result of examining the growth state and the number of grains of the transformants for the characterization of HOX10 gene in rice transformants, it was confirmed that the number of grains was increased compared to the wild type Dongjin rice.

In addition, one month after GMO package transplantation, the growth state and the number of grains of the transformant of the HOX10 gene were examined, and the number of grains was higher than that of the wild type Dongjin rice (FIG. 6).

In addition, two months after transplantation, the growth status and number of samples of HOX10 gene transformants were examined, and the number of samples was more than that of the transformants examined one month after transplantation. It was confirmed (Fig. 7).

Example 7 Phenotypic Analysis of Rice Transformants

In order to analyze the characteristics of HOX10 gene overexpression in the transformant which confirmed the insertion of OsHOX10 gene, it was compared with the wild type Dongjin rice. It could be confirmed (Fig. 8).

Attach an electronic file to a sequence list

Claims (8)

Increasing the intracellular level of the HOX10 protein having the amino acid sequence represented by SEQ ID NO: 2, to increase the number of plants. The method of claim 1, wherein increasing the intracellular level is overexpressing a gene encoding the protein. The method of claim 2, wherein the gene has a nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence of SEQ ID NO: 411-2930. The method of claim 2, wherein the method of overexpressing the gene comprises transforming a plant cell with a recombinant expression vector operably linked to the promoter. A gene encoding a HOX10 protein having an amino acid sequence represented by SEQ ID NO: 2, which increases the number of grains of a plant; A gene having a nucleotide sequence represented by SEQ ID NO: 1; Or a recombinant expression vector comprising a gene having the nucleotides 411-2930 of SEQ ID NO: 1. The recombinant expression vector of claim 5, wherein the vector has a cleavage map of FIG. 3. Plants increased in number of minutes by transforming with the recombinant expression vector of claim 5. 8. The plant of claim 7, wherein the plant is rice, corn, sugar cane, barley, or wheat.

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