KR101382402B1 - A gene for promoting dwarfness and branches of plants and a transgenic plant comprising the same - Google Patents

Abstract

The present invention relates to a protein derived from castor, the protein gene, a recombinant vector comprising the gene, a transgenic plant transformed from the recombinant vector and a geodetic development, the method for producing the transformed plant.

Description

D gene for promoting dwarfness and branches of plants and a transgenic plant comprising the same

The present invention relates to a method for producing a dwarf and geodetic plant by recombining a gene into a vector and overexpressing the gene in a transgenic plant.

In classical breeding, the breeding method is used in most cases breeding breeding method. However, it is not possible to introduce only one or two specific genes into the crop of interest. Therefore, to remove only the gene group that is not needed again and fix the desired gene in order to leave only the characteristic of the desired gene after mating, a long time and effort of about 5 to 20 years is usually required. In addition, these fixed varieties may exhibit susceptibility to pathogens or other weak features that were not considered in the breeding process, which often causes problems after dissemination.

Gene stacking by the sexual crossing of transformants can collect useful traits of transformants that have already been introduced for individual purposes into one plant through cross-breeding, but it takes a long time and as a result There is a disadvantage that is difficult to adjust specifically.

Since the middle of the 20th century, the genetic phenomena of plants have been understood at the molecular level, and this basic research is used for the following crop breeding.

First is the use of genetic modification technology. Previously, hybridization was the most important way to elicit genetic variation in sarcoma. Because this was done through breeding, this method could only be used between different strains within the same species. In other words, there are various natural reproductive barriers to prevent the free transfer of genes between species in the long evolutionary process, which could not be overcome by conventional artificial crosses. However, genetic modification is not a species level but a breakthrough technology that allows genes to be used across biological barriers.

Second is molecular labeling technology. Earlier breeders have been selected solely on the phenotype, an apparent trait. However, the phenotype is not accurate because it can change with environmental changes, and it takes a long time because it needs to be cultivated to check for new traits. However, this molecular labeling technology can be identified through genotype without any cultivation process, greatly improving the efficiency, accuracy and economics of selection, greatly contributing to the scientificization of crop breeding.

Recently, a technique for inducing embryos directly in somatic cells of plants rather than germ cells by expressing genes that control seed development in tissues other than seeds has been studied. To date, there have been no reports on the use of transcriptional regulatory genes that are specifically expressed in castor seeds.

Korean Unexamined Patent Publication No. 2011-0108809 Korean Unexamined Patent Publication No. 2012-0002566

Accordingly, the present invention is to provide a transgenic plant that has been transformed by overexpressing RcB3D45, a transcriptional regulatory gene that is specifically expressed only in the seed of castor, and has developed a geodetic branch.

The present invention provides a method for producing dwarf and geodetic plants by overexpressing the RcB3D45 gene.

The present invention provides a transgenic plant in which the whole plant, plant organ, seed, plant cell or progeny thereof produced by overexpressing the RcB3D45 gene is dwarfed and the geodesic is developed.

The present invention is a dwarf in which the RcB3D45 gene, which is a transcriptional regulation factor that regulates the development of a seed, an expression vector including the gene, and an RcB3D45 gene that has been artificially overexpressed and transformed into the entire tissue of a plant by the expression vector. It is possible to provide a transgenic plant capable of producing geodes and producing proteins and lipids that accumulate only in the seed in tissues other than the seed.

1 shows castor B3 domain gene, protein structure and amino acid sequence.
Figure 2 shows the caster tissue-specific expression RT-PCR analysis of the castor B3 domain gene.
Figure 3 shows castor 35S-RcB3D45 overexpression vector structure.
Figure 4 shows the morphological changes of plants by overexpression of castor RcB3D45 gene.
5 shows expression of RcB3D45 gene by RT-PCR analysis in wild-type control (WT) and castor RcB3D45 transformants.
6A shows triglyceride (TAG) production of castor RcB3D45 transformant leaves.
FIG. 6B shows fatty acid production of mono-11-eicoseno in the fatty acid composition of castor RcB3D45 transformant leaves.

The present invention provides a method for producing dwarf and geodetic plants by overexpressing the RcB3D45 gene.

More specifically, the present invention provides a method for producing a dwarf and geodetic plant, characterized in that the RcB3D45 gene consists of the nucleic acid base sequence of SEQ ID NO: 1.

In the present invention, the term dwarfness means a trait in which the size of an organism grows smaller than the standard size of the species, or a breed having such a trait. Dwarf traits are important in various aspects of agriculture and horticulture. For example, ornamental plants can be produced by reducing plant height or stem length, and crops with new commercial value such as having a bite-sized for eating by miniaturizing vegetables or fruits can be produced.

In one embodiment of the present invention, the dwarf and geodetic transformants of the present invention, the number of leaves in the basal portion is increased about three times on average compared to the wild type control, the number of branches on average about four times Increased. In addition, the basal diameter of the transformants was reduced by about 0.5 times on average compared to the wild type control, and the height of the apical branches decreased by about 0.5 times.

RcB3D45 gene according to the present invention in one embodiment, the gene having a B3 domain in the protein sequence isolated from the castor RcB3D45 ( Ricinus communis B3 domain 45 ), but RcB3D45 or similar genes from other species of interest may also be used.

The nucleic acid base sequence of SEQ ID NO: 1 may be characterized by encoding a protein having an amino acid sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the RcB3D45 gene may have substantially the same identity as the nucleic acid base sequence set forth in SEQ ID NO: 1, but is not limited thereto.

As used herein, the term "substantial identity" of a polynucleotide sequence refers to a polynucleotide having at least 60% sequence identity, typically at least 70%, more typically less than when compared to a reference sequence using the program described below using standard parameters. By at least 80% and most preferably at least 90% of sequence identity. Those skilled in the art will appreciate that these values can be appropriately adjusted to determine the corresponding identity of the protein encoded by the two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, read frame localization, and the like.

Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215: 403-410, 1990. Software for performing BLAST analyzes is available through the Web site of the National Center for Biotechnology Information.

In one embodiment of the invention, the gene expression may be under promoter control.

Promoters suitable for operatively linking and expressing plant growth related genes using the described methods of the invention can be used from the same species or from different species of the plant to be transformed. In addition, the promoter may be from the same species for the gene of the invention to be used, or may be from heterologous. Promoters for use in the methods of the invention may also include chimeric promoters which may include combinations of one or more promoters having expression profiles in common with those described below.

The term 'promoter' refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked in a specific host cell. 'Operably linked' means that one nucleic acid fragment is linked to another nucleic acid fragment. The function or expression thereof is influenced by other nucleic acid fragments. In addition, it may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation.

Methods for identifying and characterizing promoter regions in plant genomic DNA are well known to those skilled in the art, see, for example, Jordano et al, Plant Cell 1: 855-866, 1989; Bustos et al., Plant Cell 1: 839-854, 1989; Green et al., EMBO J. 7: 4035- 4044, 1988; Meier et al., Plant Cell 3: 309-316, 1991; And Zhang et al., Plant Physiol. 110: 1069-1079, 1996.

Examples of such promoter sequences include promoters for amino acid permease genes (AAP1) (eg, Arabidopsis). AAP1 promoter from thaliana (Arabidopsis thaliana ) (Hirner et al., Plant J. 14: 535-544,1998), oleate 12-hydroxylase: promoter for desaturase gene (e.g. Promoter designated LFAH12 from Lesquerella fendleri ( Lesquerella Pendleri) (Broun et al., Plant J. 13: 201-210, 1998), promoters for the 2S2 albumin gene (e.g. Arabidopsis thaliana (Arabidopsis) 2S2 promoter from Thaliana) (Guerche et al., Plant cell 2: 469-478, 1990), FAE1 promoter from fatty acid elongase gene promoter (FAEl) such as Arabidopsis thaliana ( Arabidopsis thaliana ) (See Rossak et al., Plant Mol. Biol. 46: 717-715, 2001), and the Lipi Cotileledon Gene Promoter (LEC2) (eg, Arabidopsis). LEC2 promoter from thaliana (Arabidopsis thaliana ) (Kroj et al., Development 130: 6065-6073, 2003). In addition, the CaMV (cauliflower mosaic virus) 35S promoter used in one embodiment of the present invention may also be included.

The RcB3D45 gene is characterized in that it is specifically expressed in castor seeds. In one embodiment of the present invention, the RcB3D45 gene was confirmed to be specifically expressed only in the seeds of the leaves, stems, flowers, female flowers, male flowers, seeds, seedlings, roots of the castor through RT-PCR analysis.

The present invention can also provide an expression vector for producing a dwarfed and geodetic plant comprising a nucleic acid base sequence encoding the RcB3D45 gene operably linked to one or more regulatory genes capable of promoting expression of plant transcriptional control genes. have.

The RcB3D45 gene may be a dwarf having a nucleic acid base sequence as set forth in SEQ ID NO: 1, and an expression vector for producing a plant having advanced geodes.

The term "vector" in the present invention means a DNA preparation having a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases integrate into the genome itself.

Meanwhile, in one embodiment of the present invention, the recombinant vector of the present invention was transformed into Agrobacterium GV3101, and then the gene of the present invention was introduced into plant cells by the transformed Agrobacterium.

Accordingly, the present invention can also provide Agrobacterium microorganisms transformed by the gene or recombinant vector.

The present invention also provides a transgenic plant in which the whole plant, plant organ, seed, plant cell or progeny thereof produced by overexpressing the RcB3D45 gene is dwarfed and the geodesic is developed.

The preparation of the transgenic plant can use transformation methods known in the art for introducing the vector into the plant. For example, but not by way of limitation, Agrobacterium spp . Microbial transformation, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and precipitation by PEG (Polyethylenglycol) Can be. Agrobacterium-mediated transformation can be used and the methods exemplified in the literature can be used (Horsch et al., Science 227: 1229-1231, 1985).

In one embodiment of the invention, the transgenic plant may be Arabidopsis. In one embodiment of the present invention, the gene of SEQ ID NO: 1 is subjected to LR clonase reaction with the recombinant vector pENTR-RcB3D45 comprising the gene of SEQ ID NO: 1 to pB2GW7, which is a plant expression vector having a CaMV 35S promoter. Expressed vector 35S-RcB3D45 was prepared, transformed into Agrobacterium GV3101, and transformed cells were selected and differentiated into individuals to obtain transformants.

As used herein, the term "plant" includes whole plants, plant organs (eg, leaves, stems, flowers, roots, etc.), seeds and plant cells (including tissue culture cells) and their progeny. The classes of plants that can be used in the methods of the invention are broad, such as those of higher plants applicable to transformation techniques, including both monocotyledonous and dicotyledonous plants, and certain lower plants such as algae. This includes plants of various drainage levels, including drainages, diploids, and haploids.

Transgenic plants overexpressing RcB3D45 of the present invention can be obtained, for example, by transfecting into a plant a transgenic vector (eg, plasmid, virus, etc.) encoding a promoter operably linked to the RcB3D45 gene.

The present invention therefore also provides a transgenic plant comprising the expression vector and having a dwarf and geodetic development compared to the corresponding wild type plant.

In the method for producing a transgenic plant according to the present invention, the transgenic plant may be prepared by overexpressing the RcB3D45 gene in a floristic plant, thereby providing a dwarf and geodetic ornamental plant.

The transformed plant or plant cell may be a monocotyledonous plant or a dicotyledonous plant, but is not limited thereto. The plant may be a whole plant, a plant organ, a seed, a plant cell, or a dwarf and geodetic transgenic plant. Can be.

In the present invention, the transgenic plant may produce triglyceryl (triacylglycerol, TAG) and mono-11-eicosenoic acid (11-eicosenoic acid) in the leaves. In the leaves of the transformant according to the present invention, a mono-11-eicosenoic acid having a carbon structure of only 20: 1, which is not present in the leaves of the control, which is a non-transformant, may be produced. Can be.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example >

Example 1 Isolation of the B3 Domain Protein Gene, RcB3D45, from Castors

After separating RNA from seed of castor (IT196881) distributed by RDA Genetic Resource Center, RcB3D45 gene, B3 domain gene, was isolated by RT-PCR (Reverse transcriptase-polymerase chain reaction) method, and nucleic acid base of cDNA gene. The sequence was determined. Nucleic acid base sequence of the RcB3D45 gene is composed of a total of 1,200 bp, consisting of protein information of 45 kDa molecular weight consisting of 399 amino acids encoded by the nucleic acid base sequence. The protein sequence of the RcB3D45 gene contains the B3 DNA binding domain portion from amino acids 176 to 260. The RcB3D45 gene is defined as a hypothetical protein whose function is similar to that of the 418 amino acids, which is most similar to BlastP protein homology from the National Center for Biotechnology Information (NCBI).

1 is a protein structure and amino acid sequence of the isolated castor B3 domain gene, Figure 1 (A) is a structure of castor RcB3D45 protein, amino acids 176 to 260 of the B3 domain, Figure 1 (B) is a RcB3D45 protein The amino acid sequence of and the amino acid sequence of the B3 domain are shown in red.

Example 2. Castor RcB3D45 Gene Expression Analysis

Castor tissue-specific expression analysis of the RcB3D45 gene was performed by RT-PCR method. RNA of the leaves, stems, flowers, female flowers, male flowers, seeds, seedlings, and roots of the castor was isolated and analyzed by T-PCR using RcB3D45 gene-specific primers to analyze tissue-specific expression patterns (FIG. 2). As a result, the gene was specifically expressed only in seeds. Actin gene RcACT2 gene expression analysis expressed in all tissues of castors was used as a control to demonstrate accurate expression analysis.

Figure 2 shows the caster tissue-specific expression RT-PCR analysis of castor B3 domain gene, L (leaf), St (stem), F (flower), Fe (female), M (male flower), Sd (seed), S (seedling), R (root) tissue-specific expression is shown. The RT-PCR analysis revealed that the RcB3D45 gene was specifically expressed only in castor seeds. Expression of the castor ACTIN2 (RcACT2) gene was used as a control.

Example 3 Castor RcB3D45 Gene Overexpression Plant Vector Construction

The plant expression vector pB2GW7 (10.8 kb), which contains the pENTR-RcB3D45 vector with the caster RcB3D45 gene inserted into pENTR-Topo (Invitrogen) and the CaMV 35S promoter purchased from VIB-Ghent University (Karimi et al., Trend Plant Sci) 7: 193-195, 2002) In a LR clonase reaction, a plant recombinant expression vector 35S-RcB3D45 expressing the castor RcB3D45 gene was produced under the control of the CaMV 35S promoter (FIG. 3). The produced plant expression vector was transformed into Agrobacterium GV3101 (Komcz and Schell. Mol. Gen. Genet. 204: 383-396. 1986), and then Agrobacterium was used for Arabidopsis plant transformation.

Figure 3 relates to the castor 35S-RcB3D45 overexpressing plant vector structure, RcB3D45 cDNA gene 1,200bp is coupled under the CaMV 35S promoter (LB: left border, RB: right border, bar: herbicide phosphinothricin resistance gene, T35S: CaMV 35 Terminator of gene, SpR: spectinomycin resistant gene).

Example 4. Morphological Changes of Plants due to Overexpression of Castor RcB3D45 Gene

Agrobacterium containing the RcB3D45 gene was inoculated into the flowers of Arabidopsis. Next generation seeds were harvested and germinated, and then Arabidopsis transgenic with castor RcB3D45 gene resistant to herbicide (BASTA) was selected (FIG. 4). Plants transformed with the RcB3D45 gene showed differences in normal Arabidopsis and plant growth (Fig. 4 (A), (B)). Transgenic Arabidopsis plants generally had early development of leaves, apical dominance of peduncles disappeared, and geodesic development was vigorous, altering the morphology of the plants (Fig. 4 (C)). On the 65th day after seed germination, the average number of Rosette leaf of wild type (WT) was 12, and the average number of branches branched at the bottom was 5 on average. On the other hand, the number of RcB3D45 transformants increased to 37 on the lower leaves and 21 to 21 on the branches. The size of the plant is 28 cm in height on the average apical branch of the wild type and 6.5 cm in diameter on the base of the plant, while the size of the RcB3D45 transformant is very small, 13 cm in height and 3.7 in diameter. cm (Table 1). It was confirmed by RT-PCR expression analysis using the RcB3D45 gene specific primer that this morphological change was not caused by a simple transformant but by overexpression of the RcB3D45 gene (FIG. 5). RcB3D45 gene was not expressed in the control (WT) Arabidopsis plants of Figure 5, it was confirmed that the RcB3D45 gene is strongly expressed in the transformant. This showed that morphological changes in plants were induced by overexpression of the RcB3D45 gene.

Comparison of wild type control (WT) and transformant (RcB3D45) growth (65-day plant n = 5, number ± SEM) Measurement Wild type control (WT) Transformant (RcB3D45) Number of rosette leaves 12.8 ± 0.374 37.4 ± 4.675 Number of branches 5.2 ± 0.200 21.0 ± 2.864 Diameter of plant (cm) 6.5 ± 0.356 3.7 ± 0.170 Height of apical branch (cm) 28.2 ± 0.735 13.6 ± 2.421

Example 5 Changes in Fatty Acid Production of Plant Leaves by Overexpression of Castor RcB3D45 Gene

Agrobacterium containing the RcB3D45 gene was inoculated into the flowers of Arabidopsis. Next generation seeds were harvested and germinated, and then Arabidopsis transgenic with castor RcB3D45 gene resistant to herbicide (BASTA) was selected. As a result of thin layer chromatography (TLC) analysis of the fats of the leaves of RcB3D45 gene overexpressing plants, triacylglycerol (TAG), which does not exist in the leaves, was accumulated (FIG. 6A). This triglyceride showed similar mobile phase on TLC and triglyceride isolated from seed. Gas Chromatography (GC) analysis of the leaf fatty acid showed a change in the fatty acid composition of the leaf compared to the control (Table 2, Figure 6B). Notably, 20: 1 fatty acids, which are present in Arabidopsis seeds, were produced in the overexpressed leaves of RcB3D45 (Figure 6B).

Figure 6A shows the analysis of triglycerides by separating the fat of leaves and Arabidopsis seeds of the control and RcB3D45 transformant and developed in TLC. Fatty acid bands similar to triglycerides (TAG) of seeds were detected on TLC in RcB3D45 leaves, but not on leaves of control (WT).

6B and Table 2 show the fatty acids of the control and the leaves of the RcB3D45 transformant, wherein the mono-11-eicosenoin (20: 1) fatty acids present only in Arabidopsis seeds in the leaves of the RcB3D45 transformant were total fatty acids. 2% of was present.

Fatty acid composition analysis of wild type control (WT) and transformant (RcB3D45) leaves. (n = 3) fatty acid WT (wild type) RcB3D45 (Transformer) Mol% ± SEM 16: 0 24.3 ± 2.5 21.9 ± 2.6 16: 1t 3.9 ± 0.5 2.4 ± 0.5 16: 3 8.6 ± 1.1 8.0 ± 1.6 18: 0 1.9 ± 0.1 1.7 ± 0.2 18: 1 4.6 ± 0.6 6.3 ± 0.7 18: 2 18.4 ± 0.2 15 ± 0.1 18: 3 38.5 ± 2.4 43.1 ± 2.0 20: 1 0 2.0 ± 0.8

Attach an electronic file to a sequence list

Claims (6)

(1) preparing a recombinant vector comprising the RcB3D45 gene consisting of the nucleic acid base sequence of SEQ ID NO: 1;
(2) transforming the vector into a plant using Agrobacterium; And
(3) A method for producing a dwarf and geodetic plant, comprising the step of selecting the transformant.
The method according to claim 1, wherein the RcB3D45 gene is isolated from castors.
The method of claim 1, wherein the nucleic acid base sequence of SEQ ID NO: 1 is a protein encoded by the amino acid sequence of SEQ ID NO: 2.
The method of claim 1,
The RcB3D45 gene is a method for producing a dwarf and geodetic plant is characterized in that it is specifically expressed in castor seeds. Dwarf and geodetic transgenic plant prepared by the method of claim 1.
6. The method of claim 5,
The transgenic plant is transgenic plant, characterized in that the fatty acid (11-eicosenoic acid) is produced in the triglyceride and mono-11-eicoseno.

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