KR101370283B1 - Method for producing plant with improved growth and increased seed production transformed with gene encoding atrbp1 protein from arabidopsis thaliana - Google Patents

Abstract

The present invention relates to a method of producing a plant with an improved growth and seed production comprising a step for transforming the plant cells with a recombinant vector including a gene coding AtRBP1 protein derived from Arabidopsis thaliana and a plant produced by the method. The plant produced by the present invention shows that the growth rate and stem elongation are promoted and the seed production is increased; therefore, the present invention can be useful for producing plants with improved growth and seed production. [Reference numerals] (AA) Number of seed (piece) / plant; (BB) Weight of seed (mg) / plant

Description

Method for producing plant with improved growth and increased seed production transformed with gene encoding AtRBP1 protein from Arabidopsis thaliana}

The present invention relates to a method for producing a plant that has been transformed with a gene encoding AtRBP1 protein derived from Arabidopsis and improved seed yield, and encoding AtRBP1 (Arabidopsis thaliana RNA Binding Protein 1) protein derived from Arabidopsis edodes. A method of promoting plant growth and seed yield in comparison to a wild type plant comprising the step of transforming the plant with a recombinant vector comprising a gene, the wild type plant comprising the step of transforming the plant cells with the recombinant vector Compared to the method for producing a plant having improved growth and seed yield and a transgenic plant having improved growth and seed yield compared to the wild type plants produced by the method, the seeds thereof, and the gene encoding the AtRBP1 protein derived from the Arabidopsis herpes. Growth compared to wild-type plants containing a recombinant vector containing an active ingredient It relates to a composition for promoting and increasing seed yield.

RNA-binding proteins (RBPs) play several roles, primarily RNA metabolism, including pre-mRNA processing, transport, stabilization / disintegration and translation. As genome information is revealed, more than 200 of these RNA-binding proteins have been predicted in rice and Arabidopsis, and many RNA-binding proteins including RNA-recognition motifs (RRMs) have been identified. Arabidopsis has a gene encoding about 200 RRM-containing proteins in its genome and, along with an RNA-recognition motif (RRM), K homology (KH) domain, glycine-rich (GR) domain, zinc finger ( zinc finger; ZF) and Arginine / Aspartic acid domains, including various domains (Lorkovic and Barta., 2002, Nucleic Acids Res. 30, 623-635).

RNA-binding proteins of the invention are proteins comprising a La protein-like RNA binding protein (LARP) domain with RNA-recognition motifs (RRMs), and RNA-recognition motifs (RRMs) are conserved proteins present in RNA binding proteins. Sequences include two short conserved sequences, ribonucleic acid protein 1 (RNP1, octamer) and ribonucleic acid protein 2 (RNP2, hexamer) (Lorkovic and Barta., 2002, Nucleic Acids Res. 30, 623-635). ). In Arabidopsis, AtGRP2 and AtGRP7, which have glycine-rich domains in RNA-binding proteins, have been reported to affect seed germination, growth of nanny, and stress tolerance of Arabidopsis under various stress conditions (Kim et al., 2008, Plant J. 55, 455-466), proteomic analysis of RNA-binding protein in rice showed the presence and regulation of RNA-binding protein in dry seeds of rice. Looking at the effect of RNA binding protein on the growth and development of plants as in the present invention can contribute to understanding the function of RNA binding protein.

 On the other hand, Korean Patent No. 0671225 discloses a method for imparting low temperature or freezing resistance to plants using a nucleotide sequence encoding a glycine-rich RNA binding protein, Korean Patent No. 1149109 discloses pepper resistance-related pepper RNA Although a binding protein gene CaRBP1 and a transformed plant using the same have been disclosed, a method for producing a plant promoted growth and seed yield improved with a gene encoding an AtRBP1 protein derived from Arabidopsis as in the present invention is known. There is no bar.

The present invention is derived from the above requirements, the present invention is transformed Arabidopsis with a recombinant vector containing a gene encoding the Arabidopsis thaliana RNA Binding Protein 1 (Arabidopsis thaliana RNA Binding Protein 1) protein derived from Arabidopsis, wild type Comparing growth was confirmed and the amount of seeds increased, the present invention was completed.

In order to solve the above problems, the present invention is the growth of plants compared to wild type plants comprising the step of transforming plants with recombinant vectors comprising a gene encoding Arabidopsis thaliana RNA Binding Protein 1 (Arbidopsis thaliana RNA Binding Protein 1) protein Provides a method for promoting and increasing seed yield.

In addition, transforming plant cells with a recombinant vector comprising a gene encoding the AtRBP1 protein derived from Arabidopsis; And

It provides a method for producing a plant with improved growth and seed yield compared to wild-type plants comprising the step of regenerating the transformed plant from the transformed plant cells.

In addition, the present invention provides a transgenic plant having improved growth and seed yield compared to wild-type plants prepared by the above production method.

It also provides seeds of the plant.

The present invention also provides a composition for promoting growth and seed yield, compared to wild-type plants, containing a recombinant vector containing a gene encoding AtRBP1 protein derived from Arabidopsis as an active ingredient.

The method of producing a transgenic plant having improved seed yield and promoting growth using the AtRBP1 protein coding gene of the present invention can be very useful as a basic knowledge for improving productivity and yield of economic crops.

Figure 1 shows the expression pattern according to tissue, growth stages of RBP1 gene in Arabidopsis. Total RNA was isolated from the sample, followed by RT-PCR using 1 μg of total RNA. (A) shows the gene expression patterns of different parts of the Arabidopsis (rosette leaves, stem leaves, stems, flowers, roots). (B) shows the gene expression patterns of Arabidopsis growth stages (2, 4, 6, 8 to 8; BB before BB; BB after BB depending on the number of rosette leaves).
2 shows the growth promoting effect of RBP1 overexpressing transgenic plants. (A) shows the kidney of wild type (WT) and overexpressed transgenic plants (35S :: RBP1). (B) shows the stem thickness of wild type (WT) and overexpressing transgenic plants (35S :: RBP1). The results represent the average of three experiments.
Figure 3 shows the effect of increasing seed yield of RBP1 overexpressing transgenic plants. (A) is a photograph showing seeds harvested from wild-type (WT) and RBP1 overexpressing transgenic plants, (B) is the number of seeds per plant, (C) is a graph showing the weight of seeds per plant. The results represent the average of three experiments.
4 is a gene map of an expression vector prepared by inserting the RBP1 gene into the pBI121 vector.

In order to achieve the object of the present invention, the present invention provides a plant as compared to wild-type plants comprising the step of transforming the plant with a recombinant vector comprising a gene encoding Arabidopsis thaliana RNA Binding Protein 1 (Arbidopsis thaliana RNA Binding Protein 1) protein It provides a method of promoting the growth of seeds and increasing the seed yield.

According to one embodiment of the present invention, the plant transformed using the Arabidopsis AtRBP1 gene of the present invention is 7 ~ 13 cm in height of the plant compared to the non-transgenic wild Arabidopsis plant height, seed Yields increased by about 40%.

In the method according to an embodiment of the present invention, the AtRBP1 protein may be composed of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The gene encoding AtRBP1 protein derived from Arabidopsis of the present invention is registered in Genebank (Registration No .: At5g46250), but as for the present invention, there is no information on promoting the growth of plants and increasing seed yield.

The gene encoding the AtRBP1 protein may be preferably composed of the nucleotide sequence of SEQ ID NO: 1. 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 at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% 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 < / RTI >

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. Recombinant cells can also express genes found in natural cells, but the genes are modified and intracellularly reintroduced by artificial means.

In the present invention, the gene sequence encoding the AtRBP1 protein may be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

Expression vectors comprising the gene sequence encoding the AtRBP1 protein and appropriate transcriptional / translational control signals can be constructed by methods well known to those skilled in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a so-called T-region to 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, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance gene, aadA gene, and the like, but are not limited thereto.

In the recombinant vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS, histone promoter, Clp promoter. 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 vectors of the present invention, conventional terminators can be used, for example nopalin 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 are 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.

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

In addition, the present invention comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding the AtRBP1 protein derived from Arabidopsis; And

It provides a method for producing a plant with improved growth and seed yield compared to wild-type plants comprising the step of regenerating the transformed plant from the transformed plant cells.

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 other hybrid DNA into suitable progenitor cells in the present invention. Calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74), electroporation of protoplasts (Shillito RD et al., 195 Bio / Technol. 3, 1099-1102 ), Microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), particle bombardment methods (DNA or RNA-coated) of various plant elements (Klein TM et. al., 1987, Nature 327, 70), infection with (incomplete) virus in agrobacterium tumerfaciens mediated gene transfer by invasion of plants or transformation of mature pollen or vesicles, and the like. have. 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.

"Plant cell" used for transformation of a plant may be any plant cell. Plant cells may be in any form of cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably cultured cells.

"Plant tissue" refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, various types of cells used in culture, such as roots, stems, leaves, pollen, seeds, such as single cells, protoplasts. Contains, shoots and callus tissue. Plant tissue may be in planta or in organ culture, tissue culture or cell culture.

The plant may be a dicotyledonous plant or a monocotyledonous plant, preferably a dicotyledonous plant. Examples of dicotyledonous plants include, but are not limited to, baby pole, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, beetroot, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, mustard, There are watermelons, melons, cucumber pumpkins, gourds, strawberries, soybeans, green beans, kidney beans, and peas. The plant may be preferably Arabidopsis thaliana, but is not limited thereto.

The method of the present invention includes transforming plant cells with another recombinant vector according to the present invention, wherein the transformation can be mediated by Agrobacterium tumefaciens. In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

In addition, the present invention provides a transgenic plant and its seed having improved growth and seed yield compared to wild-type plants produced by the above method. The plant may be a dicotyledonous plant, and most preferably, a Arabidopsis plant, but is not limited thereto.

The present invention also provides a composition for promoting growth and seed yield, compared to wild-type plants, containing a recombinant vector containing a gene encoding AtRBP1 protein derived from Arabidopsis as an active ingredient. The composition of the present invention comprises a recombinant vector comprising a gene encoding the AtRBP1 protein derived from Arabidopsis as an active ingredient, the expression of the gene in the plant to improve the growth and seed yield of the plant using the gene It will be able to contribute greatly to improving the productivity of the plant and increasing the yield.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Plant material

Arabidopsis thaliana used in the present invention is Col-0 (Columbia 0). The soil was mixed with Vermiculite: Peat-moss: Perlite 2: 1: 1. After seeding, normal growth conditions (light; 100 μEm ^-2 s ^-1 , long day conditions (16 hours bright / 8 hours dark cycle, 23 ℃, watering once a week) after treatment at 4 ℃ for 3 days in dark conditions Sampling by plant tissue (roots, leaves, stems, flowers, and siliques) used in the experiment was done 4 weeks after seed germination.

2. RNA  Extraction and Reverse transcription PCR

Total RNA was extracted from frozen Arabidopsis samples using Plant RNeasy Extraction Kit (Qiagen, USA) and tested by RT-PCR method. RT-PCR was used with 0.5-1 μg of total RNA extracted and performed with gene specific primers. Primers for RBP1 used forward primers (5'-TCTTCCTCTTCGCTCCGGTGAG-3 '; SEQ ID NO: 3) and reverse primers (5'-AACTTCATGTAACGGCGATTGCG-3'; SEQ ID NO: 4). ACTIN used as a control was used as a forward primer (5'-CTCCGTGTTGCTCCTGAGGAACATC-3 '; SEQ ID NO: 5) and a reverse primer (5'-ACCTCAGGACAACGGAATCGCTC-3'; SEQ ID NO: 6).

3. Vector production and transformation of Arabidopsis

In order to prepare an overexpressing transformant, the coding region of the RBP1 cDNA was amplified by a polymerase chain reaction (PCR) and then inserted into the pGEM-T Easy vector. The pGEM-T Easy vector and the pBI121 vector having the RBP1 gene inserted therein were cleaved with Xba I and BamH I restriction enzymes and then linked to the RBP1 and pBI121 vectors. All DNA manipulations were in accordance with standard methods (Sambrook et al., 1989 "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor, NY.Press) and sequenced the RBP1 coding region, promoter region and junction region. Was confirmed by DNA sequencing. Arabidopsis transformation was followed by vacuum infiltration (Bechtold and Pelletier, 1998, Mol. Biol. 82, 259-266), using Agrobacterium turmeration GV3101. Selection of transformed plants was carried out after seed seeding in selection medium with kanamycin antibiotic (50 μg / ml) and then transformed plants were selected. After further selection of the transformed lines with a 3: 1 segregation ratio, T ₃ homozygous lines were used for phenotypic investigation.

4. Growth and seed production analysis of plants

Seeds of wild-type and transformed plants were inoculated into soil and incubated at 4 ° C. for 3 days in dark conditions, followed by long-term conditions (16 hours bright / 8 hours dark conditions) at 100 μEm ^-2 s ^-1 . ), And was grown at 23 ℃. During plant cultivation, the height of the plant and the thickness of the stems were measured, and the seed quantity was measured after seeding and the total weight of seed and seed of each plant.

Example  1: at the baby pole RBP1  Expression patterns of genes by tissue and growth stages

Tissue-specific and growth-level transcription levels of Arabidopsis RBP1 gene were analyzed by RT-PCR. Genetic transcription levels of RBP1 were similarly expressed in all tissues such as rosette leaves, stem leaves, stems, flowers, and roots in Arabidopsis (Fig. 1A). In addition, when examining the gene transcription level of RBP1 in each stage of Arabidopsis growth, the number of rosette leaves (2, 4, 6 and 8), pre- and post-flowering, and similarly expressed in all growth stages (Fig. 1B). Thus, it is shown that the RBP1 gene is expressed in all tissues throughout the growth period of Arabidopsis and affects the growth and development of plants.

Example  2: RBP1  Growth Promotion Effect of Overexpressed Transgenic Plants

To investigate the function of the RBP1 gene on plant growth and seed yield, a transgenic Arabidopsis overexpressing RBP1 under the control of Cauliflower mosaic virus 35S promoter was constructed (35S :: RBP1). Similar results were obtained by analyzing three lines of independent transformants overexpressing PBP1, and FIG. 2 and FIG. 3 show one transformant result. As a result of comparing the growth of wild type (Col-0) and RBP1 overexpressed transformants, wild type has a height of about 35 cm, whereas RBP1 overexpressed transformants have a height of about 45 cm. Increased (FIG. 2A). For stem thickness, wild type was about 1 mm thick, whereas 35S :: RBP1 transgenic plants increased stem thickness to about 1.5 mm (FIG. 2B).

Example  3: RBP1  Seed Production Enhancement Effect of Transgenic Plants Overexpressed

In order to investigate the effect of RBP1 gene on seed production, seed quantity was analyzed after wild type (Col-0) and RBP1 overexpressed transformants were grown under the same conditions (FIGS. 3A-C). RBP1 overexpressed transformants produced about 25,000 seeds per plant and wild type produced about 17,500 seeds (FIG. 3B). The seed was weighed and the transformants overexpressed RBP1 produced about 750 mg of seed per plant and the wild type produced about 550 mg of seed (FIG. 3C). This result shows that RBP1 overexpressed transformants increase about 40% in growth and seed productivity compared to wild type. RBP1 gene enhances seed production.

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Claims (11)

Promoting the growth of plants compared to wild-type plants comprising the step of transforming the plant with a recombinant vector comprising a gene encoding the Arabidopsis thaliana RNA Binding Protein 1 (Arbidopsis thaliana RNA Binding Protein 1) protein derived from Arabidopsis consisting of the amino acid sequence of SEQ ID NO: How to increase seed yield. delete Transforming a plant cell with a recombinant vector comprising a gene encoding an AtRBP1 protein derived from Arabidopsis edo consisting of the amino acid sequence of SEQ ID NO: 2; And
A method for producing a plant having improved growth and seed yield compared to a wild-type plant comprising the step of regenerating a transformed plant from the transformed plant cell. delete The method of claim 3, wherein the plant is a dicotyledonous plant. A transgenic plant having improved growth and seed yield compared to wild type plants prepared by the method of claim 3. The transformed plant according to claim 6, wherein the plant is a dicotyledonous plant. The transgenic plant of claim 7, wherein the plant is a Arabidopsis vulgaris. Seeds of plants according to claim 6. A composition for promoting growth and seed yield as compared to wild-type plants, comprising a recombinant vector comprising a gene encoding the AtRBP1 protein derived from Arabidopsis consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient. delete

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