KR101660231B1 - Transgenic plants for controlling photosynthetic efficiency in the light and cold stress, and control method thereof - Google Patents

Abstract

The FBN5 gene derived from the Arabidopsis thaliana according to the present invention is composed of Plasthoquinone- 9, a photosynthetic efficiency-related substance, and Solanecyl, which is involved in the synthesis of the prenyl tail structure in the synthesis of Plastoquinol-8, an antioxidant functional substance, Regulates the production of plastoquinone-9 and plastoquinolone-8 by regulating its activity, and overexpresses it in plants using it to develop crops that maintain normal photosynthetic efficiency under low-temperature stress.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a transgenic plant having photosynthetic efficiency under light stress and low temperature stress,

The present invention relates to a transgenic plant in which photosynthetic efficiency is regulated under light stress and cold stress, and a regulating method using the transgenic plant.

Plastoquinone-9 and plastochromanol-8 are quinone compounds synthesized in chloroplasts. Plastoquinone-9 plays an important role in photosynthetic efficiency by acting as an electron transport in photosynthesis. The substance synthesized at the next metabolic step from plastoquinone-9 is Plastoquinol-8 and is known as an antioxidant. Enzyme genes involved in the synthesis of plastoquinone-9 and plastoquinolone-8 present in chloroplasts have already been discovered and known.

The present inventors have found that when Fibrillin 5 ( FBN5 ), which is presumed to function as a chloroplast lipid binding protein among proteins that are synthesized in the nuclear genome and transferred to the chloroplast, is mutated in plants, Synthesis was inhibited and the production of plastoquinone-9 and plastoquinol-8 was regulated by the regulation of FBN5 . In addition, the present inventors have developed a plant resistant to light and cold stress caused by overexpression of the FBN5 gene by studying a method for utilizing the same to improve the efficiency of photosynthesis.

Korean Patent Publication No. 10-2014-0063310

It is an object of the present invention to provide a method of controlling photosynthesis and plant growth of a plant.

It is another object of the present invention to provide a transgenic plant in which photosynthetic efficiency is regulated under light stress and cold stress.

It is another object of the present invention to provide a method for producing a transgenic plant in which photosynthetic efficiency is regulated under light stress and cold stress.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for regulating photosynthesis and plant growth of a plant, comprising the step of increasing or decreasing the expression of the Fibrillin5 ( FBN5 ) gene comprising the nucleotide sequence of SEQ ID NO: 1 in the plant (Fig. 13).

The FBN5 gene is expressed in Arabidopsis thaliana , and it is preferable to combine SPS (solanesyl-diphosphate synthase) involved in the synthesis of the prenyl tail structure during synthesis of plastoquinone-9 and plastoquinolone-8, And the synthesis of stauquinone-9 and plastoquinolone-8.

Each of the expression FBN5 increase or decrease in the gene to be due to over-expression or deletion of the gene, respectively FBN5, FBN5 But not limited to, transforming a plant by inserting T-DNA into a gene overexpressing vector or FBN5 gene.

The plant may be selected from the group consisting of Arabidopsis, rice, maize, rapeseed, tobacco, pea, kidney bean, mung bean, radish, radish, cabbage, lettuce, sweetpotato, plantain, carrot, clover, peach tree, apple tree, But it is preferable that all plants which synthesize plastoquinone-9 and plastoquinolone-8 are used, and it is more preferable that they are Arabidopsis or rice plants.

The present invention relates to a DNA sequence comprising the nucleotide sequence of SEQ ID NO: 1 (FIG. 13) The present invention provides a transgenic plant in which a plant expression vector containing a Fibrillin 5 ( FBN5 ) gene is transformed into a host plant and the photosynthetic efficiency is regulated under light stress and cold stress.

"Control of photosynthetic efficiency" refers to controlling the amount of photosynthesis of a plant. Photosynthesis of plants can vary in amount depending on light (illumination) and low temperature environmental conditions affecting plant growth. That is, the photosynthetic efficiency may vary depending on the stress environment of the plant. In particular, the regulation of the photosynthetic efficiency in the present invention means that the FBN5 gene according to the present invention can be used to sustain or promote photosynthesis even under the stress environment of plants, for example, light stress or cold stress.

The FBN 5 The genes are Arabidopsis thaliana , and it is preferable to combine SPS (solanesyl-diphosphate synthase) involved in the synthesis of the prenyl tail structure during synthesis of plastoquinone-9 and plastoquinolone-8, And the synthesis of stauquinone-9 and plastoquinolone-8.

The expression vector may include, but is not limited to, the CaMV35S promoter.

The host plants may be selected from the group consisting of Arabidopsis, rice, corn, rapeseed, tobacco, peas, kidney beans, mung beans, red beans, radishes, cabbages, lettuce, sweetpotatoes, plantains, carrots, cloves, peaches, apple trees, , But all plants which synthesize plastoquinone-9 and plastoquinolone-8 can be used, and it is more preferable that they are Arabidopsis or rice plants.

( I ) preparing a plant expression vector comprising a Fibrillin 5 ( FBN5 ) gene consisting of the nucleotide sequence of SEQ ID NO: 1; And

ii) transforming the host plant with the expression vector of step i);

Wherein the photosynthetic efficiency is regulated under light stress and low temperature stress.

The invention the FBN5 Genes, DNA, cDNA, and amino acid sequences decoded therefrom.

The variant of the above sequences is considered to be included in the scope of the present invention in addition to the gene consisting of the nucleotide sequence of SEQ ID NO: 1 (FIG. 13) or the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (FIG. 14). The mutant is a polypeptide consisting of a nucleotide sequence or amino acid sequence consisting of a nucleotide sequence having a functional characteristic similar to that of the nucleotide sequence of SEQ ID NO: 1 (FIG. 13) or the amino acid sequence of SEQ ID NO: 2 (FIG. 14) .

In one embodiment of the present invention, the FBN5 gene is mutated in the Arabidopsis plant, the amount of synthesis of plas- quinone-9 and plastoquinol-8 in the mutant is significantly reduced, inhibiting the photosynthesis ability of the plant, (Photosynthetic ability measurement: Fv / Fm = photosynthetic fluorescent indicator, wild type Fv / Fm = 0.8, FBN5 mutant Fv / Fm = 0.65). In order to confirm whether the decrease in plastoquinone-9 and plastoqualumnol-8 was due to the mutation of FBN5 , FBN5 As a result of restoring FBN5 function by transforming the gene into a mutant, the plant growth was normal, and the synthesis amount of plastoquinone-9 and plastoquinolone-8 recovered to the level of the wild-type control, so that FBN5 was synthesized And that there is a direct relationship with

In one embodiment of the present invention, binding protein was analyzed to determine whether FBN5 was involved in the synthesis of plastoquinone-9 and plastoquinol-8, and it was found that plastoquinone-9 and plastoquine- (Ss) and solanesyl-diphosphate synthase (SPS), which is involved in the synthesis of the prenyl tail structure. FBN5, in combination with SPS, modulates the synthesis of plastoquinone-9 and plastoquinarnol-8 by modulating the activity of SPS. These results suggest that the expression of FBN5 gene can be regulated to regulate the production of photosynthetic efficiency-related substance, plastoquinone-9, and the antioxidant-functional substance, plastoquinolone-8. In addition, FBN5 can be overexpressed in plants to develop crops that maintain normal photosynthetic efficiency under low temperature stress.

Meanwhile, a recombinant expression vector containing the gene according to the present invention can be prepared.

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

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

The vector of the present invention can typically be constructed as a vector for cloning or expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts. For example, when the recombinant vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter (for example, pL promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) , Ribosome binding sites for initiation of detoxification, and transcription / translation termination sequences. The vectors that can be used in the present invention include plasmids such as pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, phage such as gt4xue, Charon, z1, M13, etc.) or viruses (e.g., SV40, etc.).

The term "promoter " refers to a region of DNA upstream from a 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, the promoter may be CaMV35S, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto.

In the recombinant expression 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 and the terminator of the Octopine gene of Tumefaciens, 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.

The vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selection marker. Examples of the vector include Basta herbicide, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, There are resistance genes for geneticin, neomycin and tetracycline.

In addition, the recombinant expression vector containing the gene according to the present invention can be transformed into a plant to produce a transgenic plant whose photosynthesis efficiency is regulated under light stress and cold stress.

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 common not only for dicotyledonous plants but also for plant species including both terminal 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. Such methods are well known in the art.

The FBN5 gene can be used to regulate the production of photosynthetic efficiency-related substance, plastoquinone-9 and the antioxidant-functional substance, plastoquinolone-8, so that the plant overexpresses and develops crops that maintain normal photosynthetic efficiency under low- Can be utilized.

FIG. 1 is a cross- Analysis of gene deletion mutations; (A) Structure of FBN5 genomic gene and position of T-DNA insertion in SALK_064597 plant. (B) Genomic DNA PCR of mutant plants in which T-DNA is inserted into the FBN5 gene. (C) PCR cDNA FBN5 in fbn5 -1 plant. (D) -1 fbn5 plant in the culture medium.
2 is an analysis of the content of hydrogen peroxide _{(hydrogen Peroxide, H 2 O 2} ) in a wild-type plant and fbn5 -1; DAB: Plant filtration in DAB (3,3'-diaminobenxidine) solution, and plant filtration in Buffer: 10 mM Na ₂ HPO ₄ solution.
Figure 3 is a lethal phenotype of the plant fbn5 -1 FBN5 Analysis of changes after introduction of cDNA; (A) Expression of 35S: FBN5 in homozygous FBN5. (B) FBN5 / FBn5 DNA PCR of 35S: FBN5 transformed plants. (C) wild-type and fbn5 -1 + 35S: FBN5 transformant of the PCR DNA. (D) RT-PCR of FBN5 gene in wild-type and fbn5 -1 + 35S: FBN5 transformants in stems and leaves.
Figure 4 shows the effect of FBN5 protein on plant growth; (A) Comparison of growth of wild type and transformant. (B) Measure the size of the upper body after sowing of the wild type and the transformant. (C) Body weighing at 33 days after sowing of wild type and transformant.
Figure 5 shows the effect of FBN5 protein on the content of plastoquinone-9 and tocopherol; (A) Plastoquinone -9 (PQ-9) and plastoquinolone-8 (PC-8) content in wild type and fbn5-1 plant leaves grown on MS medium containing 1% sucrose for 3 weeks. (B) δ- tocopherol at 1% can be grown in the MS medium containing sucrose and 3 weeks wild-type plant leaves fbn5 -1 (δ-TC), γ- tocopherol (γ-TC), α- tocopherol (α-TC ) And total tocopherol (Total-TC) content. (C) 6 weeks in soil grown wild-type and transformed FBN5 Gene conversion chain in fbn5 -1 plant XVE: FBN5 (FBN5 gene expression control transformants), fbn5 -1 + 35S: FBN5 (FBN5 gene in fbn5-1 9 (PQ-9) and plastoquinolone-8 (PC-8) in WT + 35S: FBN5 (FBN5 overexpressor for wild type) leaves. (D) with a FBN5 gene in six weeks grown wild-type plant in the soil and fbn5 -1 introduced transformant chain XVE: FBN5 (FBN5 gene expression control transformants), fbn5 -1 + 35S: FBN5 (FBN5 gene in fbn5-1 Tocopherol (? -TC),? -Tocopherol (? -TC),? -Tocopherol (? -Tc) and? -Tocopherol (? -Tc) in leaves of WT + 35S: FBN5 (wild-type FBN5 overexpressor) ) And total tocopherol (Total-TC) content.
FIG. 6 is a view showing the synthesis route of tocopherol, plastoquinone-9 (PQ-9) and plastoquinolone-8 (PC-8). HPPD (4-hydroxyphenylpyruvate dioxygenase), VTE2 (homogentisate phytyltransferase), VTE3 (2-methyl-6-solanyl- 2-methyl-6-solanyl-1,4-benzoquinol methyltransferase, VTE1 (Tocopherol cyclase), VTE4 (gamma-tocopherol methyltransferase), SPS Solanesyl-diphosphate synthase), and GGPS (geranylgeranyle) pyrophosphate synthase) are enzymes involved in each pathway.
7 is a diagram showing mutual binding of FBN5 and SPS (solanesyl-diphosphate synthase).
Figure 8 is an analysis of the important domains of FBN5 in the mutual binding of SPS (solanesyl-diphosphate synthase); (A) Combination of multiple FBN5 domains combined with GAL4 DNA-binding domain and FBN5A scheme. (B) Results of analysis of FBN5 domain which is important in binding with SPS.
Figure 9 is an analysis of enzymes that are likely to interact with FBN5; (A) A schematic diagram of the synthesis route and enzymes of plastoquinone-9 (PQ-9) and tocopherol in Arabidopsis thaliana. The enzymes tested are indicated in red. (B) Mutual binding of FBN5 and enzymes As a result, only SPS1 mutually binds with FBN5. FPS1 (farnesyl-diphosphate synthase 1), SPS3 (solanesyl-diphosphate synthase 3), FPS2 (farnesyl-diphosphate synthase 2), GGPS1 1, geranylgeranyle) pyrophosphate synthase 1), GGR (geranylgeranyl reductase), SPS1 (solanesyl-diphosphate synthase 1), SPS2 (Solanecyl phosphate synthase 1) Phosphate synthase 2, solanesyl-diphosphate synthase 2), VTE2 (homogentisate phytyltransferase), HST (homogentisate solanesyltransferase), VTE3 (2-methyl- 2-methyl-6-solanyl-1,4-benzoquinol methyltransferase) refers to an enzyme gene involved in each pathway.
Fig. 10 shows the position and mutual binding of FBN5 and SPS1 (solanesyl-diphosphate synthase 1), SPS2 (solanesyl-diphosphate synthase 2) in the chloroplast; (A) Expression of SPS1-GFP / FBN5-mRFP and SPS2-GFP / FBN5-mRFP in Arabidopsis protoplasts. (B) Expression of SPS1-HA / FBN5-GFP and SPS2-HA / FBN5-GFP in Arabidopsis protoplasts and immunoprecipitating proteins separated by Western blotting using Western blotting blotting). (C) Confirmation of mutual binding of FBN5 and SPS1 or SPS2 in chloroplast. Fluorescence only in normal FBN5 and SPS1 or SPS2 protein binding.
11 shows the results of photosynthetic efficiency and reactive oxygen species measurement by short-term cold stress in wild-type and FBN5-inhibited XVE: FBN5 transformants; (A) Measurement of maximum photosynthetic efficiency of Photosynthetic System 2 under each condition. (B) Amount of absorbed light energy used through photochemical reactions ( Φ PSII), emission to controlled thermal energy ( Φ NPQ), and emission to constant thermal energy ( Φ F, D). (C) The amount of hydrogen peroxide (H ₂ O ₂ ), the active oxygen accumulated in the leaves before and after the cold stress treatment.
Fig. 12 shows the results of analysis of SPS1 (solanesyl-diphosphate synthase 1) and SPS2 (solanecyl phosphate synthetase 2, solanesyl-diphosphate synthase 1, SPS1 -diphosphate synthase 2) to synthesize a solanesyl-diphosphate (hereinafter referred to as " solanesyl-diphosphate "). HST (homogentisate solanesyltransferase), VTE3 (2-methyl-6-solanyl-1,4-benzoquinolmethyltransferase, 2-methyl-6-solanyl-1,4-benzoquinol methyltransferase ) And VTE1 (tocopherol cyclase, Tocopherol cyclase) refer to an enzyme gene involved in each pathway.
13 is the FBN5 cDNA base sequence of SEQ ID NO: 1.
14 is the amino acid sequence of the FBN5 protein of SEQ ID NO: 2.

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  1> Fibrillin 5 ( FBN5 ) Gene mutation chain fbn5 -One Isolation and photosynthetic ability assay

The process of finding mutant plants that lack the desired gene has become clearer with the advent of near-saturation T-DNA collections. The data of the precise chromosomal location of each of the inserts was analyzed by TAIL (thermal asymmetric interlaced) PCR technique (Liu et al., Genomics, Volume 25, Issue 3, 10 February 1995, Pages 674-681) Analysis, and input into the database. Users are now able to perform in silico searches on these databases for strains where T-DNA is now inserted in a specific part of the genome. More than 225,000 unique insertions have been performed at the Salk Institute to determine T-DNA insertions at sophisticated locations in about 90,000 lines (Alonso et al., Science, 1 August 2003: Vol.301 no.5633 pp. 653-657. ).

Accordingly Arabidopsis the origin Fibrillin5 (FBN5) (At5g09820) to give the T-DNA of the gene and determine its insertion position Fibrillin5 (FBN5) T-DNA inserted mutant (SALK_064597) (Fig. 1A) for gene ABRC ( Arabidopsis Biological Resource Center (http://www.arabidopsis.org)). The T-DNA insert mutant ( SALK_064597 ) on the Fibrillin5 ( FBN5 ) gene was unable to obtain homozygous mutants when seeded from the soil. When 12 plants were examined, 3 individuals were identified as wild type and 9 individuals as hybrid type mutants.

Thus, when the FBN5 gene is defective, embryo or seedling lethality may occur. In the absence or presence of 1% sucrose in the MS medium, the plants failed to develop normally and turned white and died (Fig. 1D).

To investigate whether the lethal phenotype was due to FBN5 mutation, 121 seeds obtained from FBN5 hybrid plants were planted on MS medium containing 1% sucrose. 23 individuals from 120 individuals turned white and died, and 97 individuals grew well in three weeks. (5'-GAGACGAAATCTCGAAGACCC-3 '; SEQ ID NO: 3) and RP (5'-AGAGGCATCGTATGGTGAATG-3'; SEQ ID NO: 4) on the FBN5 gene were isolated from 10 individuals, respectively, As a result of performing PCR using the primer LBa1 (5'-TGGTTCACGTAGTGGGCCATCG-3 '; SEQ ID NO: 5) (FIG. 1A) on the left edge of T-DNA, there was no 1.0 kb band corresponding to the FBN5 gene. Only the 1.2kb band was present. This lethal-type plant of the white may all be referred to as homo on mutant FBN5 -1.

PCR was performed on each of the DNAs obtained from 12 healthy plants. As a result, the 1.0 kb band of LP and RP primers was always present, and 1.2 kb of LBa1 and RP primers were present or absent. Genomic DNA was analyzed by PCR of T-DNA and selecting a mutant plant gene inserted FBN5 named fbn5 -1 (Fig. 1B). As a result, healthy plants have FBN5 It has been confirmed that the gene is wild type (WT) or heterozygous.

Performing a PCR to ';; (SEQ ID NO: 7 to remove the RNA from wild-type plants and fbn5 -1 and synthesizing cDNA with primers P1 (5'-ATGACGAGTAACCTTTTCCAG-3 5' -TTAAGGTTTCTCTATTCTTTCC-3) SEQ ID NO: 6), P2' result is not present in the transfer body FBN5 fbn5 -1, was observed only in wild-type (Fig. 1C). Thus fbn5 lethal phenotype of a plant -1 is one of the recessive mutant (120 dog 23 occurs in the object P>0.95;; χ2 = 2.17 ) in the gene can be caused by high.

fbn5 -1 plants are sucrose, growing very slowly as compared to the wild type in a medium containing MS were killed while the size is small and the color of the end of leaf pigment (Figure 1D).

In addition, plants grown in MS medium supplemented with 1% sucrose for 3 weeks were filtered with DAB (3,3'-diaminobenxidine) solution or 10 mM Na ₂ HPO ₄ solution. As a result, the leaves of fbn5-1 The maximum efficiency (Fv / Fm = 0.65) of photosynthetic system 2 was significantly lower than that of wild type (Fv / Fm = 0.8). This suggests that FBN5 is involved in the formation of photosynthesis mechanisms or photosynthesis ability. The fbn5-1 plants accumulated more hydrogen peroxide (hydrogen peroxide, H ₂ O ₂ ) than the wild type (FIG. 2).

< Example  2> FBN5  Through gene transfer fbn5 -One of Seedlings Lethal type  Recovery test

The full-length cDNA of FBN5 was amplified from the Arabidopsis leaf RNA using a combination of P1 and P2 primers and a DNA polymerase (KOD Hot Sr DNA Polymerase, Novagen), and then cloned into pENTR-Topo vector (Invitrogen) FBN5 PENTR-FBN5 in which the gene was cloned. pENTR-FBN5 and plant expression gateway (Gateway) in response to the vector of pB2GW7.0 (Karimi et al., Trends Plant Sci. 2002, Vol. 7 pp. 193-195) and LR recombinase claw (clonase) (Invitrogen) BN5 After construction of the construct 35S: FBN5 to regulate the cDNA under the CaMV35S promoter, the genotype of the plant was changed to FBN5 / fbn5 The seeds that were harvested and transformed into hybrid plants were sown in the soil. Herbicide Basta (Basta) processing the health and well grown to 32 selected from the 35S: FBN5 vector transformants dog 18 wild-type, eight FBN5 / fbn5 hybrid type, six appeared to fbn5 -1 homopolymer.

For the wild-type transformed FBN5 in PCR results using the primers P1 and P2 in the DNA of transformant 1.7kb PCR product are amplified: healthy and well-grown wild-type and mutated Homo fbn5 -1 35S to: FBN5 is introduced fbn5-1 + 35S On the other hand, fbn5 -1 + 35S: FBN5 transformants showed a 822bp PCR product amplified from the cDNA (Fig. 3C). It showed that the plants exhibit lethal fbn5 -1-type expression in the seedling stage fbn5-1 mutant lethal phenotype is restored by the introduction of what the FBN5 cDNA showing the normal growth (Fig. 3A). Wild-type and fbn5 -1 + 35S: in the stems and leaves of the transgenic FBN5 results of the RT-PCR with P1, P2 primer, as shown in FIG. 3D 35S: FBN5 transfected more than the expression of FBN5 transcripts in wild-type transformant Respectively. This proved that the lethal phenotype of the fbn5-1 plant was due to deletion of the FBN5 gene.

< Example  3> FBN5  Production of transgenic mutant transformants and growth analysis

Since fbn5 -1 plants are lethal phenotype, FBN5 the fbn5 -1 Queer basis Transformants with highly suppressed gene expression were produced. The XVE: FBN5 construct was constructed by performing the LR clonase reaction with the pENTR-FBN5 vector prepared in Example 2 with the pMDC7 vector (Curtis MD, et al., Plant Physiology 2003; 133: 462-469) . XVE: The FBN5 vector was transformed into β-estradiol by treatment with FBN5 Expression of the gene is induced. XVE: FBN5 constructs after transfection agent in the truck hybrid plant genotype FBN5 / fbn5, was selected plants with fbn5 -1 Queer background in PCR. When planted in the soil, the plant was able to grow seeds without lethal phenotype and with a small amount of FBN5 gene expression without β-estradiol treatment.

Two lines in fbn5 -1 based on Homo advanced to 4G XVE: FBN5 # 12 and XVE: FBN5 # 17 transformants, the wild type and on the respective switched fbn5 -1 overexpressing FBN5 gene based on Homo transfected chain WT + 35S: FBN5 and fbn5 -1 + 35S: it was prepared FBN5 transformants. WT + 35S: FBN5 transformants transformed the 35S: FBN5 vector into a wild-type strain, which was resistant to the herbicide Basta treatment and selected plants that were similar to wild-type plants. The size of the ground area except the root of each selected healthy growing plant was measured during the development of the plant, and the weight of the ground part of the plant was measured on the 33rd day.

As a result, XVE: FBN5 # 12 and XVE: FBN5 # 17 transformant had a small plant size in comparison to the wild type, while the growth speed is slow, WT + 35S: FBN5 and fbn5 -1 + 35S: FBN5 transformants Similar to wild type. The weights of the XVE: FBN5 # 12 and XVE: FBN5 # 17 transformants were 68% and 83%, respectively (FIG. 4).

< Example  4> FBN5  In the expression-inhibiting mutant transformants Plastoquinone -9 and Plasto Romano -8 content analysis

The MS medium containing 1% sucrose, respectively FBN5 of transformants: the wild type and the T-DNA was inserted into the gene of FBN5 fbn5 expression of the gene completely lost FBN5 -1 Homo mutant plant gene expression is very weak and FBN5 XVE , And the content of plastoquinone-9 and tochochromanol in the leaves was analyzed by HPLC (high performance liquid chromatography) (Fig. 5).

As a result, the content of the first three weeks grown in MS medium containing% sucrose fbn5 -1 plastoquinone -9 in a plant leaves was reduced 17-fold compared to wild type (Fig. 5A). The content of plastoquinolone-8 produced by VTE1 (tocopherol cyclase, Tocopherol cyclase) was not determined. In the wild type, tocopherol is composed of alpha, beta, and delta tocopherols with a ratio of about 90: 8: 2. In MS medium containing 1% sucrose 3 weeks fbn5 -1 plant grown was a total tocopherol content of 78% increase compared to the wild-type, it is mainly due to the increase of alpha-tocopherol (Fig. 5B).

The content of plastoquinone-9 in the XVE: FBN5 # 12 and XVE: FBN5 # 17 transformants of Example 3, which was grown for 6 weeks in soil, was 3-fold and 5-fold lower, respectively, And the contents were decreased 9-fold and 38-fold, respectively (Fig. 5C). The content of tocopherol after 6 weeks of soil cultivation was not statistically different from that of wild type (Fig. 5D). That is, the contents of plastoquinone-9 and plastoquinolone-8, tocopherol in WT + 35S: FBN5 and fbn5 -1 + 35S: FBN5 transformants were not significantly different from wild type.

fbn5 -1 and XVE: lack of FBN5 in FBN5-1 plants were causing a change in the content of tocopherols, plastoquinone -9 -8 playing with plastic torque Rome. Their synthetic pathways are closely interrelated (Figure 6). The homogentisate ring structure of tocopherol and plastoquinone-9 is commonly used. It is synthesized through the Shikimate pathway and the geranyl geranyl synthesized via MEP (methylerythritol phosphate) The synthesis of phytyl diphosphate and solanesyl diphosphate synthesized by the reaction of specific enzymes in geranylgeranyle diphosphate synthesizes tocopherol and plastoquinone-9. Without the expression of the FBN5 gene in this study, the synthesis of plastoquinone-9 and plastoquinol-8 was inhibited, which enhanced the synthesis of tocopherol, which is interrelated in metabolism (Fig. 6).

< Example  5> In yeast FBN5  Protein and Solanecyl phosphate  Synthetic enzyme ( Solanesyl diphosphate synthase , SPS ) &Lt; / RTI &gt;

FBN5 protein is a structural protein with a lipid-binding domain and has been shown to play an important role in the synthesis of plastoquinone-9 (PQ-9).

Thus, the possibility of this protein interacting with enzymes involved in the synthesis of plastoquinone-9 was investigated through a yeast two-hybrid system. The FBN5 protein was used as a reference for Arabidopsis cDNA prey library screening after binding to the GAL4 DNA binding domain (BD fusion) and no autotranscription activity. Twenty independent colonies in 1.24 x 10 ⁷ transformants were grown on media lacking URA3 (chromosome V of yeast) and ADE2 (yeast AIR-carboxylase gene) and showed lacZ activity.

Sequencing of cDNA fusions from these 20 colonies resulted in the gene At1g78510 expressing SPS1 (solanesyl-diphosphate synthase 1), which synthesizes the prenyl-side chain of plas-quinone-9 . This cDNA fusion consisted of DNA sequencing to synthesize a 60 amino acid-free mature peptide from the N-terminal. Premature SPSl proteins were found to be very weak or absent in FBN5 binding (Figure 7).

SPS2 (solanesyl-diphosphate synthase 2) is synthesized by At1g17050. Since the polypeptide similarity between SPS1 and SPS2 is 80%, the binding between FBN5 and SPS2 was tested. The mature SPS2 protein binds strongly to FBN5 whereas the precursor SPS2 binds weakly (Fig. 7). However, in the alternative splicing form of the FBN5 gene, the C-terminal region did not interact with any other forms of SPS1 and SPS2 than the FAN5A protein expressed by some other FBN5A transcripts than FBN5.

In order to investigate which part of FBN5 is required for mutual coupling with SPS, mutual coupling of mature SPS1 and SPS2 based on various parts of FBN5 was performed using a yeast two-hybrid system (Fields & Song, Nature 1989; 340: 245-246) (Fig. 8). FBN5 consists mainly of three domains: chloroplast migration, α-helix, and β-sheet. We have made several combinations to investigate whether the three domains are important for mutual coupling. The boundaries of the chloroplast migration domains were uncertain, and the lengths of the amino acids were varied to produce several constructs (Fig. 8A).

As a result, both α-helical and β-sheet domains were required for mutual binding with SPS, and the chloroplast migration domain (TP) was not essential. Also, since FBN5A does not mutually bind SPS1 and 2, it suggests that the completeness of the three-dimensional structure of FBN5 is important for interaction (Fig. 8B). The above results indicate that mutual binding between FBN5 and SPS is essential for the growth of Arabidopsis.

Synthesis of plastoquinone-9 and tocopherol shares a pathway to synthesize homogentisate ring, which is the head group of these, and the prenyl-side chain, while plastoquinone-9 is the solanesyl diphosphate, And tocopherol are synthesized using phytyl diphosphate, respectively (Fig. 6). The enzymes involved in the synthesis of these prenyl-side chains and the enzymes involved in the synthesis of plas-quinone-9 and tocopherol after the binding of the homogentisate ring and the prenyl-side chain are called metabolic tunneling methods , And the mutual binding of these enzymes with FBN5 was examined through a yeast two-hybrid system (Fig. 9). We also investigated the interaction of ubiquinone-9 with SPS3, which synthesizes solanesyl-diphosphate in Arabidopsis thaliana.

Figure 9A schematically shows the synthesis route of plastoquinone-9 and tochochromanol in Arabidopsis thaliana. The enzymes that were tested for mutual binding with FBN5 were red. Eight proteins and mutual binding were examined, but none of the enzymes tested except SPS1 showed mutual binding with FBN5.

< Example  6> in the chloroplast FBN5  And SPS ( Solanecyl phosphate  Synthetic enzymes, solanesyl diphosphate synthase )

To investigate the presence of FBN5 and SPS in chloroplasts, the mRFP gene (GenBank Accesion: EU262302) was ligated to the 3-terminal end of FBN5 cDNA and regulated under the CaMV35S promoter. FBN5-mRFP, SPS1-GFP, FBN5-mRFP and SPS2-GFP sets were transiently expressed in Arabidopsis protoplasts and observed under a fluorescence microscope.

As a result, it can be seen that the green fluorescence of SPS1 and SPS2 overlaps well with the red fluorescence of FBN5, indicating that these proteins are located in the same place (FIG. 10A).

In addition, co-immunoprecipitation (Co-IP) and bi-molecular fluorescence complementation (BiFC) experiments were performed to investigate whether FBN5 and SPS proteins bind to each other in plants (Min et al Plant Physiol., 2013, 161: 676-691 and Gehl et al., Mol. Plant, 2009, 2: 1051-1058). FBN5-GFP and SPS1-HA (hemagglutinin) or FBN5-GFP and SPS2-HA (hemagglutinin) proteins were transiently expressed in Arabidopsis protoplasts. Total proteins were isolated from the protoplasts and mixed with anti-GFP affinity agarose. The proteins were immunoprecipitated with an anti-GFP antibody, followed by electrophoresis. The anti-HA antibody (anti-GFP antibody) Western blotting was performed with anti-HA antibody.

As a result, in Fig. 10B, it was found that FBN5 was co-immunoprecipitated with SPS1 and SPS2, and they proved mutual binding in plants.

On the other hand, BiFC experiments confirmed the interaction of FBN5 and SPS in plants. (2) CNX6-VenusN / CNX6-VenusC, (3) FBN5A-VenusN / SPS1-SCFPC, (4) vector constructs in the epithelial cells of tobacco leaves ( Nicotiana benthamiana ) ), (5) FBN5-VenusN / SPS1-SCFPC, and (6) FBN5-VenusN / SPS2-SCFP were transiently expressed. The construct set was constructed by LR clonase reaction of pENTR-FBN5, pENTR-SPS1, pENTR-SPS2 vector and BiFC gateway vector. All of these vector constructs are regulated by the CaMV35S promoter. Green fluorescence was observed due to the mutual binding of FBN5 with SPS1 or SPS2 in the chloroplasts of transformed tobacco cells, whereas FBN5A-VenusN and SPS1-SCFPC or SPS2-SCFPC (Fig. 10C, upper, middle) because they do not bind each other in the vector combination. The vector construct set (1) FBN5-VenusN / CNX6-VenusC and (2) CNX6-VenusN / CNX6-VenusC were used as a negative control (no fluorescence) and a positive control (green fluorescence for cytoplasm) ). From this, it was confirmed that FBN5 actually binds to SPS1 and SPS2 in the chloroplast.

< Example  7> Resistance of Plants to Cold Stress

In order to investigate how the deficiency of FBN5 protein affects the photosynthetic ability of Arabidopsis, photosynthetic indicators in the wild type and two XVE: FBN5 transformants # 12 and # 17 lines with very low expression of FBN5 gene according to Example 3 Respectively. The plants grown at 22 ° C for 4 weeks were allowed to stand in a low temperature room at 5 ° C for 5 days and then transferred to normal growth conditions at 22 ° C again. Fv / Fm, which is the index of maximum photosynthetic efficiency of photosynthetic system 2, did not show much difference in the normal growth condition and the low temperature treatment in the wild type. However, the XVE: FBN5 transformants showed a significant reduction of Fv / Fm from approximately 0.8 to 0.5 after cold treatment. However, when transferred back to normal conditions, Fv / Fm recovered to 0.8 (FIG. 11A). The pathway of light energy absorbed by the photosynthesis system 2 light receptor was measured.

As a result, there was no difference in wild type and XVE: FBN5 transformants under normal conditions. However, as a result of the low-temperature stress treatment, the wild-type was not different from the normal condition, whereas the XVE: FBN5 transformant exhibited the photochemical reaction ( Φ PSII) of the photosynthetic system 2 by light energy and non-photochemical quenching The amount of light energy ( ? NPQ) emitted into the heat was reduced (FIG. 11B).

In addition, the hydrogen peroxide by the cold stress are formed in the leaf _{(hydrogen peroxide, H 2 O 2} ) the content of the result of measurement by staining with a solution-diamino benzidine (Diaminobenzidine), the wild type has, by cold stress treatment of hydrogen peroxide (hydrogen peroxide, H ₂ O ₂ ) was not significantly different, whereas XVE: FBN5 transformants were found to be very large (FIG. 11C).

< Example  8> FBN5 On by Plastoquinone -9 and Plasto Romano -8 Presentation of synthesis control model

FBN5 is an SPS (Solanesyl-Diphosphate Synthase) SPS1 (Solar), which is involved in the synthesis of the solanesyl-diphosphate (SPP; C45) chain constituting the tail structure of plastoquinone- (GGPP) (geranylgeranyle-diphosphate (C20)) and five (5-amino-5-methylphenyl) phosphite synthase 1, solanesyl-diphosphate synthase 1 and SPS2 (solanesyl-diphosphate synthase 2) IPP (isopentenyl-diphosphate) (C5) to form 45 carbon chains. FBN5 is presumed to function to efficiently release the solanesyl-diphosphate from the SPS-generated solar cell, and this function serves to maintain the optimization of the SPS activity (Fig. 12) Solanecyl has been shown to have an adverse effect on the synthesis of the solanesyl-diphosphate, thereby affecting the degradation of plastoquinone-9 and plastoquinol-8.

<110> Republic of Korea <120> Transgenic plants for controlling photosynthetic efficiency in          the light and cold stress, and the control method thereof <130> P15R12D0167 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 822 <212> DNA <213> FBN5 cDNA <400> 1 atgacgagta accttttcca gccaccatca atggcagctt cacgaggagc aatctcgaga 60 agaacaggaa acgtgaaagt attagtctct ttcaccagtt ctaatggcaa aacgctgagt 120 tttagtgaca actcatttag gctcagaccc atgttcatcg ggaaagtcac agaacagagt 180 tcttgttcct ccccaaatga acaacaacaa gatgaagaac aagaacaaga acaagaagag 240 attacagttt ctcatataaa agaagagctc tacgaagctc tcaaagggat caatagaggg 300 atattcggag ttaagtcgga taaaaagaca gagatcgagg gactggtgaa gcttttggaa 360 tgtcggaatc cgacaccgga gccgactgga gagttagaca agatcggagg ttgctggaaa 420 ctcatttaca gtacaatcac tgtattgggt tctaaacgaa ctaaattggg tcttcgagat 480 ttcgtctctc ttggtgatct tcttcaacag attgacattg ctcagggcaa aacggtccat 540 gtgttgaaat tcgatgtccg gggattgaat ctactcgacg gcgagtttag aatcgtcgct 600 tccttcaaga tctcgtccaa atcgagtgta gagattactt atgaaagctc aacgatcaaa 660 ccagaccagt tgatgaacat atttaggaag aacatggatc ttcttttggg aatcttcaac 720 cccgagggac tattcgagat ttcatactta gacgaagatt tacaagtagg gagagatggg 780 aaagggaatg tttttgtttt ggaaagaata gagaaacctt aa 822 <210> 2 <211> 273 <212> PRT <213> The FBN5 protein <400> 2 Met Thr Ser Asn Leu Phe Gln Pro Pro Ser Met Ala Ala Ser Arg Gly   1 5 10 15 Ala Ile Ser Arg Arg Thr Gly Asn Val Lys Val Leu Val Ser Phe Thr              20 25 30 Ser Ser Asn Gly Lys Thr Leu Ser Phe Ser Asp Ser Ser Phe Arg Leu          35 40 45 Arg Pro Met Phe Ile Gly Lys Val Thr Glu Gln Ser Ser Cys Ser Ser      50 55 60 Pro Asn Glu Gln Gln Gln Asp Glu Glu Gln Glu Gln Glu Gln Glu Glu  65 70 75 80 Ile Thr Val Ser Ile Lys Glu Glu Leu Tyr Glu Ala Leu Lys Gly                  85 90 95 Ile Asn Arg Gly Ile Phe Gly Val Lys Ser Asp Lys Lys Thr Glu Ile             100 105 110 Glu Gly Leu Val Lys Leu Leu Glu Cys Arg Asn Pro Thr Pro Glu Pro         115 120 125 Thr Gly Glu Leu Asp Lys Ile Gly Gly Cys Trp Lys Leu Ile Tyr Ser     130 135 140 Thr Ile Thr Val Leu Gly Ser Lys Arg Thr Lys Leu Gly Leu Arg Asp 145 150 155 160 Phe Val Ser Leu Gly Asp Leu Leu Gln Gln Ile Asp Ile Ala Gln Gly                 165 170 175 Lys Thr Val His Val Leu Lys Phe Asp Val Arg Gly Leu Asn Leu Leu             180 185 190 Asp Gly Glu Phe Arg Ile Val Ala Ser Phe Lys Ile Ser Ser Ser Ser Ser         195 200 205 Ser Val Glu Ile Thr Tyr Glu Ser Ser Thr Ile Lys Pro Asp Gln Leu     210 215 220 Met Asn Ile Phe Arg Lys Asn Met Asp Leu Leu Leu Gly Ile Phe Asn 225 230 235 240 Pro Glu Gly Leu Phe Glu Ile Ser Tyr Leu Asp Glu Asp Leu Gln Val                 245 250 255 Gly Arg Asp Gly Lys Gly Asn Val Phe Val Leu Glu Arg Ile Glu Lys             260 265 270 Pro     <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LP primer <400> 3 gagacgaaat ctcgaagacc c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RP primer <400> 4 agaggcatcg tatggtgaat g 21 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LBa1 primer <400> 5 tggttcacgt agtgggccat cg 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> P1 primer <400> 6 atgacgagta accttttcca g 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> P2 primer <400> 7 ttaaggtttc tctattcttt cc 22

Claims (12)

1. A method for regulating photosynthesis and plant growth of a plant, comprising the step of increasing or decreasing the expression of the Fibrillin 5 (FBN5) gene comprising the nucleotide sequence of SEQ ID NO: 1,
The gene binds to solanesyl-diphosphate synthase (SPS) to regulate the synthesis of plas-quinone-9 and plas- tra-rominol-8 to regulate plant photosynthesis and regulate plant growth , Photosynthesis control of plants and plant growth regulating method. delete delete The method according to claim 1, wherein the increase or decrease in expression of the FBN5 gene is caused by overexpression or deletion of the FBN5 gene, respectively. 5. The method according to claim 4, wherein the overexpression or deletion of the FBN5 gene transforms the plant by inserting the FBN5 gene overexpressing vector or the FBN5 gene into the T-DNA, respectively. The plant according to claim 1, wherein the plant is selected from the group consisting of Arabidopsis, rice, corn, rapeseed, tobacco, pea, kidney bean, mung bean, red bean, radish, Chinese cabbage, lettuce, Tomatoes, potatoes, and sweet potatoes. The method of controlling photosynthesis and controlling plant growth of plants. A plant transformed with a plant expression vector comprising a Fibrillin5 (FBN5) gene consisting of the nucleotide sequence of SEQ ID NO: 1 is transformed into a host plant and photosynthetic efficiency is regulated under light stress and cold stress.
The gene binds to solanesyl-diphosphate synthase (SPS) to regulate the synthesis of plas-quinone-9 and plas- tra-rominol-8 to regulate plant photosynthesis and regulate plant growth , Transgenic plants in which photosynthetic efficiency is regulated under light stress and cold stress. delete delete 8. The transgenic plant according to claim 7, wherein the expression vector comprises a CaMV35S promoter, wherein the photosynthetic efficiency is regulated under light stress and cold stress. The method of claim 7, wherein the host plant is selected from the group consisting of Arabidopsis, rice, corn, rapeseed, tobacco, peas, kidney beans, mung beans, red beans, radishes, cabbages, lettuce, , Tomatoes, potatoes and sweet potatoes, wherein the photosynthetic efficiency is regulated under light stress and cold stress. i) preparing a plant expression vector comprising the Fibrillin 5 (FBN5) gene consisting of the nucleotide sequence of SEQ ID NO: 1; And
ii) transforming the host plant with the expression vector of step i);
Wherein the photosynthetic efficiency is regulated under light stress and low temperature stress.

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