KR101717474B1 - Composition for inducing anthocyanin biosynthesis comprising AT1G70000 and use thereof - Google Patents

Abstract

The present invention relates to a composition for inducing increased anthocyanin synthesis in plants, comprising AT1G70000 or a polynucleotide encoding the same. The present invention also relates to a recombinant vector for inducing increased anthocyanin synthesis of a plant comprising a polynucleotide encoding AT1G70000 and a plant transformed with said vector. The present invention also relates to a method for inducing an increase in anthocyanin synthesis in a plant using the vector. The AT1G70000 or the polynucleotide encoding the AT1G70000 according to the present invention effectively induces the synthesis of anthocyanin in the plant and can produce anthocyanin in a large quantity and can also be used for the production of plant as a raw material of functional food having an increased content of anthocyanin Is expected to become possible.

Description

AT1G70000 and use thereof for inducing anthocyanin synthesis in plants containing AT1G70000,

The present invention relates to a composition for inducing increased anthocyanin synthesis in plants, comprising AT1G70000 or a polynucleotide encoding the same.

The present invention also relates to a recombinant vector for inducing increased anthocyanin synthesis of a plant comprising a polynucleotide encoding AT1G70000 and a plant transformed with said vector.

The present invention also relates to a method for inducing an increase in anthocyanin synthesis in a plant using the vector.

Anthocyanin not only determines the color of plants' flowers, fruits, stems or leaves, but also plays an important role in plant growth and development. Anthocyanins attract insect and other pollen mediators from the petals and help disperse the seeds in the fruit and seed. In addition, flavonoid compounds, including anthocyanins, act as antioxidants to protect plants from damage to insects and external stresses, and in particular to prevent damage by reactive oxygen species (Gould et al., 2004, J. Biomed. Biotechnol. 5: 314-320).

Anthocyanins have been studied in animals for their efficacy. Anthocyanin is known to have excellent antioxidant activity as well as to protect somatic cells and improve immune system. The anticancer activity of anthocyanin has already been shown to be effective in several types of cancer cells. In particular, anthocyanin has been reported to have an inhibitory effect on NF-κB related to cancer growth, metastasis, and tolerance.

Anthocyanin biosynthetic pathways in plants are well known. The accumulation of anthocyanins in plants is mediated by regulatory genes that regulate structural gene families that encode key enzymes involved in the flavonoid biosynthetic pathway and regulate expression of these structural genes. CHS (chalcone isomerase), CHI (chalcone isomerase), and F3H (flavanone 3-hydroxylase) are the early biosynthetic genes (EBGs) involved in the early synthesis of flavonoid compounds in Snapdragon, Petunia and Arabidopsis All are related in common, and expression occurs at an early stage. On the other hand, DFR (dihydroflavonol 4-reductase), LDOX (leucoanthocyanidin oxygenase), ANR (anthocyanidin reductase) and UF3GT (UDP-glucose: flavonoid 3-O-glucosyltransferase) were expressed as LBGs (late biosynthetic genes) And is responsible for the second half of the anthocyanin biosynthetic pathway.

The accumulation of anthocyanin in plants is known to be regulated in response to various environmental stimuli. Light, temperature, sugars, plant hormones and inorganic salts are very important factors in the anthocyanin biosynthesis of plants. And pigment accumulation in cells. Therefore, identifying the interaction of these factors in anthocyanin biosynthesis is crucial to understanding the anthocyanin pigment accumulation and plant secondary metabolism of plants. Previous studies have shown that light is one of the most important factors in anthocyanin biosynthesis by studying the biosynthesis of anthocyanin under various light conditions. In particular, the deficiency of phosphoric acid, low temperature stress, and increased sugar are responsible for the accumulation of anthocyanins (Lea et al., 2007, Planta. 225: 1245-1253). It is also known that anthocyanin biosynthesis by the sugar is inhibited by ethylene and gibberellin in the correlation of sucrose, hormone ethylene, cytokinin, gibberellin and abscisic acid in anthocyanin biosynthesis (Das et al., 2012, Mol. Cell. : 501-507).

However, there is still a lack of diversified research on the genetic resources involved in anthocyanin synthesis in plants.

Application No. 10-2011-0078073

Accordingly, the present inventors have made intensive efforts to discover a genetic resource capable of enhancing the synthesis of anthocyanin in plants. As a result, when the AT1G70000 gene was able to induce the synthesis of anthocyanin synthesis, when the gene was overexpressed in the plant, And the present invention has been accomplished.

Accordingly, one aspect of the present invention is to provide a composition for inducing an increase in anthocyanin synthesis of a plant.

Another aspect of the present invention is to provide a recombinant vector for inducing anthocyanin synthesis increase of a plant, and a plant transformed by the vector.

Another aspect of the present invention is to provide a method for inducing increased anthocyanin synthesis in a plant.

One aspect of the present invention provides a composition for inducing increased anthocyanin synthesis in plants, comprising AT1G70000 or a polynucleotide encoding the same.

Hereinafter, the present invention will be described in detail.

In the present invention, AT1G70000 (Gene Code [ MIPS (http://pgsb.helmholtz-muenchen.de/plant/athal/reportsjsp/geneticElement.jsp?gene=AT1G70000.1)] [ TAIR (http://arabidopsis.org/ servlets / TairObject? type = locus & name = At1g70000)]) is known as myb-like transcription factor and its function is not yet known in detail. The present invention corresponds to the first identification of the relationship between AT1G70000 and anthocyanin synthesis. The AT1G70000 may comprise the amino acid sequence of SEQ ID NO: 1. The AT1G70000 may be derived from Arabidopsis thaliana.

The composition for inducing the increase of anthocyanin synthesis of a plant according to the present invention may comprise a polynucleotide encoding AT1G70000.

As used herein, the term polynucleotide is a polymer of a deoxyribonucleotide or a ribonucleotide present in single stranded or double stranded form. RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and include analogs of natural polynucleotides unless otherwise specified.

The polynucleotide may be the cDNA of SEQ ID NO: 2, but is not limited thereto.

The polynucleotide includes not only the nucleotide sequence but also a sequence complementary to the nucleotide sequence. The complementary sequence includes not only perfectly complementary sequences but also substantially complementary sequences. , Which means a sequence that can hybridize under stringent conditions known in the art, for example, to a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NOS: 1-10.

The polynucleotide may also be modified. Such modifications include addition, deletion or non-conservative substitution or conservative substitution of nucleotides. The polynucleotide is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. The above substantial identity is determined by aligning the nucleotide sequence with any other sequence as closely as possible and using at least 70% homology, preferably at least 70% sequence identity, when analyzing the aligned sequence using algorithms commonly used in the art. At least 80% homology or at least 90% homology.

"% 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 >

In addition, an aspect of the present invention provides a recombinant vector for inducing increased anthocyanin synthesis in plants, comprising a polynucleotide encoding AT1G70000.

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.

In the present invention, the polynucleotide encoding AT1G70000 may be inserted into a recombinant vector. The term "recombinant vector" means a means for expressing a gene of interest in a host cell. This includes bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. 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 polynucleotides encoding AT1G70000 can be constructed by methods well known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. 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. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the genome of a plant can be produced . 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 may 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 recombinant 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 ) Octopine gene terminator, 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 vector of the present invention may be composed of a cleaved map of Fig. 1, but is not limited thereto.

The present invention also provides a plant having an increased anthocyanin synthesis, which is transformed with the above recombinant vector.

The increase in the synthesis of anthocyanin means that the synthesis of anthocyanin is increased compared to the plant without transformation, and the concept includes the demonstration of anthocyanin synthesis function.

The plant is not limited to a variety of plants such as a monocotyledonous plant, a dicotyledonous plant, a perennial plant, a perennial plant, and the like. Examples of the plant include rice terra cotta, cabbage, cabbage, radish, mustard, oilseed rape, arugula, kale, broccoli, It may be a cruciferous plant such as Chrysanthemum japonica, Cucurbitae, Cauliflower, or Rutalin. In addition, the plant is a concept including plant cell lines, plant calluses, seeds, or some organs of plants.

In addition, an aspect of the invention provides a method of inducing increased anthocyanin synthesis in a plant, comprising transforming the plant with a vector comprising a polynucleotide encoding AT1G70000.

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 mature pollen or vesicles into 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.

The AT1G70000 or the polynucleotide encoding the AT1G70000 according to the present invention effectively induces the synthesis of anthocyanin in the plant and can produce anthocyanin in a large quantity and can also be used for the production of plant as a raw material of functional food having an increased content of anthocyanin Is expected to become possible.

Figure 1 is a cleavage map of a vector for expression of AT1G70000 in plants.
FIG. 2 shows the result of confirming overexpression of AT1G70000 in the plant transformed with the vector of FIG.
FIG. 3 shows the result of confirming that anthocyanin content was increased in plants overexpressing AT1G70000.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: AT1G70000 cDNA cloning

After 2 weeks of germination, wild-type Arabidopsis mRNA was extracted and reverse transcriptase was used to synthesize cDNA. The amplified AT1G70000 cDNA was then confirmed by electrophoresis and eluted from the gel.

The eluted cDNA fragment (786 bp) was inserted into a topo vector (pCR8 / GW / TOPO: invitrogen) and inserted into the pMDC32 vector, a vector for overexpression of the plant, with the LR reaction (LR Clonase®: invitrogen). A cleavage map of a specific vector is shown in Fig.

The thus-constructed vector was transformed into E. coli cells (TOP10 cell: invitrogen) by heat-shock transformation and cultured at 37 DEG C for 12 hours until colony formation.

The resulting colony was cultured again on LB medium and plasmid vector was extracted.

Example 2 Transformation of Plants with AT1G70000 cDNA

The vector prepared as in Example 1, that is, 35S CaMV promoter :: AT1G70000 vector was transformed into Agrobacteria by electric shock transformation method and then cultured. Transgenic Agrobacteria were transformed into wild-type Arabidopsis and rapeseed plants prepared by floral dipping transformation. The seeds were then harvested from the transgenic plants and self-crossed to the third generation to obtain homologous lines.

Subsequently, AT1G70000 overexpression was confirmed by the Rt-PCR method. The results are shown in Fig. As shown in FIG. 2, it was confirmed that AT1G70000 (MybD) increased the amount of rRNA at 1 # and 4 # lines using Northern blotting.

Example 3: Confirmation of an increase in anthocyanin content in plants

60 mg of plants germinated 7 days are used for anthocyanin extraction experiments. After rapid freezing with liquid nitrogen, the sample is crushed finely. Add 300 μL of the solution mixed with 45 mL methanol + 450 μL HCl to the ground sample and react at 16 ° C for 4 hours. Then, add 200 μL of DW and 500 μL of Chloroform, mix well and centrifuge at 3000 rpm. Measure the absorbance (OD 535-650) by transferring a certain amount of the supernatant.

The results are shown in Fig.

As shown in FIG. 3, it was confirmed that the anthocyanin content was increased in the 1 # and 4 # lines, which were confirmed to increase the expression level of AT1G70000 (MybD) in Example 2.

Claims (9)

Wherein the AT1G70000 comprises the Arabidopsis originating AT1G70000 and the AT1G70000 is contained in a vector represented by the following cleavage map:

. delete delete delete delete delete Plant having increased anthocyanin synthesis, transformed with a recombinant vector comprising Arabidopsis-derived AT1G70000. [Claim 7] The plant according to claim 7, wherein the plant is anthocyanin-synthesized plant having increased anthocyanin synthesis, wherein the plant is Arabidopsis, cabbage, cabbage, radish, mustard, oilseed, cauliflower, arugula, kale, broccoli, . A method of inducing increased anthocyanin synthesis in a plant, comprising transforming the plant with a recombinant vector comprising Arabidopsis derived AT1G70000.

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