CN111448206A - IbOr-R96H variant derived from sweet potato and use thereof - Google Patents

Abstract

The IbOr-R96H variant can remarkably improve the content of carotenoid in plants and can endow high-temperature stress resistance and high-temperature pressure resistance.

Description

IbOr-R96H variant derived from sweet potato and use thereof

technical field

The present invention relates to a variant in which the 96th amino acid of orange protein derived from sweet potato (Ipomoea batatas) is substituted by arginine to histidine and use thereof.

Background technique

Sweet potato (Ipomoea batatas L.Lam) is a representative root crop that can not only be grown on relatively thin ground, but also has an excellent yield of about 30 tons per hectare, so it is used as food and livestock feed. Purple, yellow and other colored sweet potatoes contain a variety of antioxidants. In particular, yellow sweet potatoes contain 14.7 to 20 mg/100 g of beta-carotene, and purple sweet potatoes contain about 2.28 g/100 g of anthocyanins. The antioxidant activity of reactive oxygen species that cause aging and various adult diseases is excellent. In addition, since the 1990s, sweet potato has been attempted to be transformed by Agrobacterium co-culture, and a plant redifferentiation transformation system has been developed by inducing embryogenic culture cells and somatic embryogenesis from apical and lateral bud division tissues.

Carotenoid is an essential component of the photosynthesis system, a volatile component of fruit and flower, a precursor of folic acid (ABA) as a plant hormone, and a precursor of provitamin A. Substances that are very useful to plants and animals such as humans are widely used in the medical industry such as cancer, heart disease, and ophthalmology because of their strong antioxidant effect, as well as an important substance of nutrients.

On the other hand, Korean Granted Patent Publication No. 1281071 discloses "IbOr-Ins Gene Variants Derived from Sweet Potatoes and Uses thereof", and Korean Granted Patent Publication No. 1359308 discloses "Transformation rich in carotenoids and anthocyanins" Preparation method of sweet potato plant body and plant body", but the IbOr-R96H variant derived from sweet potato of the present invention and its use have not been described.

SUMMARY OF THE INVENTION

technical problem

The present invention is obtained to meet the above requirements. The present inventors prepared a variant of IbOr-R96H in which the 96th amino acid of the orange protein derived from sweet potato was substituted by arginine to histidine, and the 96th amino acid was substituted by arginine. As a result of the transformation of sweet potato cells with the recombinant vector of the mutant gene, it was confirmed that the sweet potato transformed with the IbOr-R96H variant was compared with the untransformed sweet potato cultured cells, and the sweet potato cultured cells transformed with the wild-type IbOrange gene and the IbOr-Ins mutant gene. The total carotenoid content in the cultured cells, especially the β-carotene content, was significantly increased. Compared with the untransformed sweet potato plant body and the sweet potato plant body transformed with the wild-type IbOrange gene, it was confirmed that the IbOr-R96H variant gene was used. The transformed sweet potato plant body is more excellent in high temperature stress resistance, thereby completing the present invention.

Technical solutions

In order to solve the above problems, the present invention provides an amino acid sequence of SEQ ID NO: 1 in which the 96th amino acid of an orange protein derived from sweet potato is substituted with histidine (H) from arginine (R) The IbOr-R96H variant.

Furthermore, the present invention provides a gene encoding the above-mentioned IbOr-R96H variant.

Furthermore, the present invention provides a recombinant vector comprising the above-mentioned gene.

Also, the present invention provides a transformed plant body with increased carotenoid content and increased resistance to high temperature stress, comprising the step of using the above recombinant vector to transform plant cells to overexpress the gene encoding the IbOr-R96H variant.

Furthermore, the present invention provides a transformed plant with increased carotenoid content and enhanced high temperature stress resistance prepared by the above method, and transformed seeds thereof.

Also, the present invention provides a method for improving high temperature stress resistance and carotenoid content of a plant, comprising the step of transforming plant cells with the above recombinant vector to overexpress a gene encoding the IbOr-R96H variant.

Furthermore, the present invention provides a composition for improving the high temperature stress resistance and carotenoid content of a plant comprising the gene encoding the above-mentioned IbOr-R96H variant as an active ingredient.

effect of invention

Plants transformed using the IbOr-R96H variant of the present invention can be rich in carotenoids as functional substances, and thus can be used in various ways as materials for health food, cosmetics, feed, etc., by using the IbOr-R96H variant to develop high-quality plant bodies with increased carotenoid content. In addition, plants transformed with the IbOr-R96H variant of the present invention can also be cultivated in high-temperature regions where ordinary plants are difficult to grow.

Description of drawings

FIG. 1 is a schematic diagram of a recombinant vector containing the IbOr-R96H gene variant in which the 96th amino acid of IbOrange protein is changed from arginine (R) to histidine (H).

Fig. 2 is a photograph of confirming the phenotype of sweet potato cells transformed with untransformed sweet potato cultured cells (YM) and wild-type IbOrange gene (IbOr-wt) and IbOrange gene variants (IbOr-Ins and IbOr-R96H), respectively.

Figure 3 shows the preparation process of IbOrWT or IbOrR96H overexpressing and transforming sweet potato plants, wherein (a) part is a vector diagram, (b) part is a polymerase chain using genomic deoxyribonucleic acid (DNA) extracted from the transformed plant body Results of PCR, part (c) is the results of analyzing gene expression levels in each transformed plant using quantitative real-time PCR.

Figure 4 shows the results of high temperature stress (47°C) resistance experiments on leaf discs of sweet potato plants transformed by overexpression of IbOrWT or IbOrR96H, wherein part (a) is 3,3'-diaminobenzidine tetrahydrochloride (DAB ) stained phenotype, and part (b) is the result of measuring the ion leakage of each leaf disc.

Detailed ways

In order to achieve the object of the present invention, the present invention provides an IbOr-R96H variant consisting of the amino acid sequence of SEQ ID NO: 1, in which the 96th amino acid of the orange protein derived from sweet potato is substituted with histidine by arginine.

When the IbOr-R96H variant of the present invention is overexpressed in a transformed plant, the total carotenoid and high temperature stress resistance of the plant can be significantly increased.

In addition, the present invention also provides a gene encoding the above-mentioned IbOr-R96H variant.

The range of the gene encoding the IbOr-R96H variant of the present invention can be that the coding base of arginine is replaced by the coding base of histidine (CAT or CAC), so that the 96th amino acid of the wild type IbOr protein can be replaced by Substituted with histidine, preferably, it can be composed of the base sequence of SEQ ID NO: 2, but not limited thereto.

Furthermore, the present invention also provides a recombinant vector comprising a gene encoding the IbOr-R96H variant.

The term "recombinant" refers to a cell that replicates a heterologous nucleic acid, or expresses said nucleic acid, or a cell that expresses a protein encoded by a peptide, heterologous peptide, or heterologous nucleic acid. Recombinant cells can express genes or gene fragments not found in the natural form of the above-mentioned cells as one of a sense or antisense form. Also, recombinant cells can express genes found in the cells in their native state, but which have been modified and reintroduced into the cells by artificial means.

The term "vector" is used to refer to deoxyribonucleic acid fragment(s), nucleic acid molecules, for intracellular delivery. Vectors replicate deoxyribonucleic acid and can be independently regenerated in host cells. And the term "transporter" is often used interchangeably with "vector". The term "expression vector" refers to a recombinant deoxyribonucleic acid molecule comprising the appropriate nucleic acid sequences necessary for expression of the desired coding sequence and the operably linked coding sequence in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals that can be used in eukaryotic cells are known.

A preferable example of a plant expression vector is a Ti-plasmid vector, which can transfer a part of itself, so-called transfer deoxyribonucleic acid (T-DNA), when present in a suitable host such as Agrobacterium tumefaciens. Regions are transferred to plant cells. Currently other types of Ti-plasmid vectors (cf. EP0116718B1) are used to transfer hybrid DNA sequences into plant cells, or protoplasts of new plants can be generated by appropriate insertion of the hybrid DNA into the plant genome. A particularly preferred form of Ti-plasmid vector is the so-called binary vector as claimed in EP0120516B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the deoxyribonucleic acid of the invention into a plant host are those that can be selected from double-stranded plant viruses (eg, cauliflower mosaic virus (CaMV)), single-stranded viruses, and those that can be derived from geminiviruses and the like. Viral vectors, such as incomplete plant viruses. The use of such vectors may be advantageous especially when it is difficult to properly transform a plant host.

Preferably, the expression vector contains more than one selectable marker. The above-mentioned markers are nucleic acid sequences having properties that can be generally selected by chemical methods, and all genes capable of distinguishing transformed cells from untransformed cells correspond to this, and examples thereof include: herbicide resistance genes, such as glyphosate (glyphosate), grass phosphinothricin, glufosinate, etc.; and antibiotic endogenous genes, such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol ( Chloramphenicol), but not limited thereto.

In the plant expression vector of the present invention, the promoter may be CaMV35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto.

The term "promoter" refers to the upstream region of deoxyribonucleic acid from a structural gene, and refers to a deoxyribonucleic acid molecule that binds ribonucleic acid (RNA) polymerase in order to initiate transcription. "Plant promoter" refers to a promoter capable of initiating transcription in plant cells. A "constitutive promoter" refers to a promoter that is active under most environmental conditions and developmental states or cell differentiation. Constitutive promoters may be preferred in the present invention as selection of transformants can be carried out through various tissues at various stages. Therefore, constitutive promoters do not limit the possibilities of selection.

In the plant expression vector of the present invention, conventional terminators can be used, and examples thereof include nitric oxide (NOS), rice α-amylase RAmy1A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium tumefaciens), the Octopine gene terminator, etc., but not limited thereto. With regard to the need for terminators, it is generally believed that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly preferred in the present invention.

Furthermore, the present invention provides a method for preparing a transformed plant body with increased carotenoid content and enhanced high temperature stress resistance, comprising the steps of transforming plant cells with the above recombinant vector and overexpressing the gene encoding the IbOr-R96H variant.

In the method according to an example of the present invention, the above-mentioned IbOr-R96H variant may consist of the amino acid sequence of SEQ ID NO: 1.

The above-mentioned "gene overexpression" means that the above-mentioned gene is expressed at a level higher than the expression level in a wild-type plant. As a method of introducing the above-mentioned gene into a plant, there is a method of transforming a plant using an expression vector containing the above-mentioned gene regulated by a promoter. In the above method, the promoter is not particularly limited as long as the inserted gene can be overexpressed in a plant.

Transformation of a plant refers to any method of transferring deoxyribonucleic acid to a plant. Such transformation methods do not necessarily have regeneration and/or tissue culture periods. Transformation of plants is now common in plant species including both dicotyledonous as well as monocotyledonous plants. In principle, any transformation method can be used in the process of introducing hybrid deoxyribonucleic acids into suitable progenitor cells according to the invention. The method may be suitably selected from the calcium/polyethylene glycol method for protoplasts (Negrutiu et al., 1987, Plant Mol. Biol. 8:363-373), the electroporation method for protoplasts (Shillito et al., 1985, Bio/Technol. 3: 1099-1102), microinjection of plant factors (Crossway et al., 1986, Mol. Gen. Genet. 202: 179-185), various plant factors (deoxyribonucleic acid or ribose Nucleic acid encoded) particle impact (Klein et al., 1987, Nature 327:70), gene transfer (incomplete) virus mediated by Agrobacterium tumefaciens transformed by infiltration of plants or mature pollen or microspores Infection (EP 0301316B1) etc. A preferred method according to the invention comprises Agrobacterium-mediated delivery of DNA. In particular, preferably, so-called binary vector technology as disclosed in EPA 120516 and US Pat. No. 4,940,838 is used.

A "plant cell" used for transformation of a plant can be any plant cell. Plant cells are cultured cells, cultured tissues, cultured organs or plant whole. "Plant tissue" is a differentiated or undifferentiated plant tissue, including, for example, roots, stems, leaves, pollen, seeds, tumor tissue, and cells of various forms used in culture, i.e., single cells, protoplasts, Buds and callus, but not limited thereto. Plant tissue can be in planta or in an organ culture, tissue culture or cell culture state.

Furthermore, the present invention provides a transformed plant body and its seeds with increased carotenoid content and improved high temperature stress resistance prepared by the above method.

Compared with the untransformed plant body, the content of total carotene in the transformed plant body of the present invention is increased by about 34 times, especially, the content of beta-carotene may be increased by about 300 times, but not limited thereto.

Furthermore, it is possible that the transformed plants of the present invention are superior in high temperature stress resistance as compared with untransformed plants and plants transformed with a gene encoding wild-type IbOr. The above-mentioned high temperature may be 40-55°C, preferably, 45-49°C, more preferably, 47°C, but not limited thereto.

In addition, the above-mentioned transformed plant body with increased carotenoid content and high temperature stress resistance of the present invention may be dicotyledonous plants such as Convolvulaceae, Leguminosae, and Araliaceae, or Poaceae. (Gramineae) and other monocotyledonous plants, preferably, can be dicotyledonous plants of Convolvulaceae, more preferably, can be sweet potato, but not limited thereto.

Furthermore, the present invention provides a method for improving the carotenoid content and high temperature stress resistance of a plant, comprising transforming plant cells with a recombinant vector comprising a gene encoding the above-mentioned IbOr-R96H variant, and overexpressing the encoding IbOr-R96H variant genetic steps. The above-mentioned IbOr-R96H variant of the present invention can be composed of the amino acid sequence of SEQ ID NO: 1. If the IbOr-R96H variant is overexpressed in a plant, the total carotene content of the plant including β-carotene is increased, The high temperature stress resistance of plants is improved.

Furthermore, the present invention provides a composition for improving the carotenoid content and high temperature stress resistance of a plant comprising the gene encoding the above-mentioned IbOr-R96H variant as an active ingredient. The composition of the present invention comprises, as an active ingredient, a gene encoding an IbOr-R96H variant, which is the first orange protein derived from sweet potato, which consists of the amino acid sequence of SEQ ID NO: 1 with histidine (H) substituted for it. 96 amino acids were substituted from arginine to histidine, and the carotenoid content and high temperature stress resistance of plants can be improved by transforming plant cells with the genes encoding the above variants.

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

Example 1. Cloning and base sequence analysis of the IbOr-R96H variant gene

Based on the polymerase chain reaction-mediated site-directed mutagenesis method, the IbOr-R96H variant gene was cloned by using the QuickChange ^™ site-directed mutagenesis kit (Agilent, USA) to clone into the pDONR207 vector The IbOr wild-type (WT) complementary deoxyribonucleic acid (cDNA) was used as the template and the forward primer (5'-GAAATTCAAGACAATATTCGGAGTCACCGGAATAAAATATTTTTGCA-3', SEQ ID NO: 3) and the reverse primer (5'-TGCAAAAATATTTTATTCCGGTGACTCCGAATATTGTCTTGAATTTC-3', SEQ ID NO: 4) were used. It was confirmed by sequence analysis of the polymerase chain reaction product that arginine, which is the 96th amino acid of IbOr wild type, was substituted with histidine, and it was named as IbOr-R96H variant.

Example 2. Preparation of Recombinant Vector Containing IbOr-R96H Variant Gene and Development of Transformant Overexpressing IbOr-R96H Variant Using the Vector Above

In order to prepare transformants overexpressing the IbOr-R96H variant, the gateway reaction system of Invitrogen was used to prepare pDONR207 and pGWB5 vectors as plant expression vectors and recombinant vectors of the IbOr-R96H variant cloned by LR reaction. The recombinant vector was used to transform Yulmi culture cells of sweet potato as white (beige) pulp using Agrobacterium tumefaciens EHA105 strain. Transformed cultured cells were selected from MS (Murashige and Skoog) medium containing hygromycin. From the transformed cultured cells, genomic DNA was extracted using HiGene ^™ Genomic DNA Extraction Kit (for plants) from BIOFACT, and then the hygromycin gene as a selectable marker for transformants was analyzed by polymerase chain reaction. Expression of hygromycin phosphotransferase (HPT).

Example 3. Analysis of carotenoid content in sweet potato cultured cells transformed with IbOr-R96H recombinant vector

As a result of observing the phenotype of the sweet potato cultured cells transformed with the recombinant vector containing the IbOr-R96H variant gene, the untransformed sweet potato cultured cells were visually confirmed to be beige, whereas the sweet potato cultured cells transformed with the wild-type IbOr gene showed a beige color. Yellow, the sweet potato cultured cells transformed with the IbOr-Ins variant published in the past (Korean Granted Patent Publication No. 1281071) are dark yellow, while the sweet potato cultured cells transformed with the IbOr-R96H variant of the present invention have an almost orange color to the naked eye. color (Figure 2).

Furthermore, the carotenoid content of each transformed sweet potato cultured cell was quantified by high-performance liquid chromatography (HPLC) analysis (Table 1). As a result, the total carotenoid content of the sweet potato cultured cells transformed with the IbOr-R96H variant was approximately 24 times or more higher than that of the untransformed sweet potato cultured cells. In particular, in the case of the content of β-carotene, the sweet potato cultured cells transformed with the IbOr-R96H variant increased by about 88 times or more than the untransformed sweet potato cultured cells. In addition, it was confirmed that the total carotenoid content and the β-carotene content of the sweet potato cultured cells transformed with the IbOr-R96H variant increased by more than 13 times compared with the sweet potato cultured cells transformed with the wild-type IbOr (IbOr-WT). 39 times or more, it can be seen that the IbOr-R96H variant of the present invention has an excellent effect of increasing the content of carotenoid in plants.

Table 1

Example 4. Preparation of sweet potato plants transformed by IbOr-R96H overexpression

Transformed sweet potato plants overexpressing the wild-type IbOr gene (IbOr ^WT ) or the IbOr-R96H variant gene (IbOr ^R96H ) were prepared using the sweet potato variety Xushu 29 (one of the more cultivated varieties in northern China) body. Each of the above-mentioned IbOr ^WT or IbOr ^{R96H was} cloned into the pGWB11 vector having a 35S promoter and a FLAG tag at the C-terminus to prepare a vector (part (a) of FIG. 3 ). In the sweet potato transformation experiment, the EHA105 strain of Agrobacterium tumefaciens containing the recombinant vector was co-cultured with sweet potato embryogenic callus for 3 days, washed with MS minimal medium, cultured in selective medium, and subcultured every 3 weeks After culturing, shoots and roots were induced from the surviving embryogenic callus and redifferentiated into plantlets to prepare transformed sweet potato plants. Forward (5'-CGCACAATCCCCACTATCCTT-3) was prepared by sampling the third or fourth leaf of sweet potato plants grown for 3 weeks in a growth chamber at 25°C under the condition of 16 hours of light per day. ', SEQ ID NO: 5) and reverse (5'-TTCCCAAGCTCAGCATTCTT-3', SEQ ID NO: 6) primer sets to perform genomic DNA polymerase chain reaction. The conditions of the polymerase chain reaction are as follows. After initial denaturation at a temperature of 94°C for 5 minutes, the process of denaturation, annealing, and extension was repeated 30 times, denaturation at a temperature of 94°C for 30 seconds, and annealing at a temperature of 58°C for 30 times. seconds and extended for 60 seconds at a temperature of 72°C. The final extension was then carried out at a temperature of 72°C for 5 minutes. From the above results, it was ensured that 10 transformed plants in which IbOr ^WT or bOr ^R96H were identified in genomic deoxyribonucleic acid were confirmed (part (b) of FIG. 3 ).

Total RNA was extracted from leaves sampled in the same manner as above using Ribospin ^™ Plant kit (GeneAll, Korea). Genomic DNA contamination was removed by treatment with RNase-free DNaseI (GeneAll, Korea), using TOPscript ^™ RT DryMIX (dT18) (Enzynomics, Korea), and 1000 μg of total ribonucleic acid synthesizes complementary deoxyribonucleic acid. The polymerase chain reaction conditions were as follows, initially at a temperature of 42°C for 5 minutes, then at a temperature of 50°C for 60 minutes and at a temperature of 95°C for 5 minutes. Primers for quantitative real-time PCR for comparing gene expression were prepared as forward (5'-GCACTGGATCACTAGTCCTT-3', sequence 7) and reverse (5'-GTCAATTCGTGGGTCATGCT-3', sequence 8). Real-time quantitative polymerase chain reaction was performed by CFX real-time PCR system (Bio-Rad, USA) using a 96-well culture plate. Each reaction (20 μl final) was performed by adding 2 μl of diluted complementary DNA and 10 μl of EvaGreen PCR Master Mix (Solgent, Korea) and 1 μl of each primer. The conditions of the polymerase chain reaction are as follows, initially at a temperature of 95°C for 15 minutes, then at a temperature of 95°C for 20 seconds, at a temperature of 60°C for 40 seconds, and at a temperature of 72°C 20 seconds and repeat 40 times. As a result of the analysis, three strains with high gene expression of IbOr ^WT or IbOr ^R96H were selected (part (c) of FIG. 3 ).

Example 5. Evaluation of Environmental Stress Resistance of IbOr-R96H Overexpressed Transformed Sweet Potato Plants

In a past study, it was confirmed that IbOr-overexpressing transformed sweet potato plants (cv. Sinzami) were resistant to high temperature stress conditions compared to untransformed sweet potato plants. Based on previous research results, the present inventors conducted a comparative analysis of the high temperature stress resistance of IbOr ^R96H overexpressed and transformed sweet potato plants and IbOr ^WT overexpressed and transformed sweet potato plants. IbOr ^WT or IbOr ^R96H overexpression transformed sweet potato plants were propagated in soil, and after 4 weeks of growth in a growth chamber at 25°C and 16 hours of light per day, the (3rd and 4th from the top) leaves of the plants were cut. to prepare leaf discs. 5 leaf disks were used as one repetition, and a total of 3 repetitions were carried out. First, 3,3'-diaminobenzidine tetrahydrochloride (DAB) staining was performed and ion leakage was measured after leaf discs were exposed to high temperature (47°C) for 12 hours. In order to observe the generation degree of reactive oxygen species generated when cells undergo stress-induced apoptosis, 3,3'-diaminobenzidine tetrahydrochloride was stained, and the ion conductivity meter was used for 6 hours in units of 6 hours. The ion loss of cells due to stress was measured within 0 to 12 hours, and each assay value was calculated as 100% of the ion loss measured by treating the sample at a temperature of 80°C for 2 hours after 12 hours to completely destroy the cells . As a result, it was confirmed that the damage to the leaf discs of the IbOr overexpression-transformed plants was lighter than that of the untransformed sweet potato plants (NT), and it was found that they had resistance to high temperature. In particular, it was found that the IbOrR96H overexpression-transformed sweet potato plants were more transformed than the IbOrWT overexpression-transformed plants. The sweet potato plants had stronger high temperature resistance (Fig. 4).

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Claims (8)

1. An IbOr-R96H variant, consisting of the amino acid sequence of SEQ ID No. 1, wherein the 96 th amino acid of an orange protein from sweet potato is histidine substituted with arginine.

2. A gene encoding the IbOr-R96H variant of claim 1.

3. A recombinant vector comprising the gene of claim 2.

4. A method for producing a transformed plant having an increased carotenoid content and an improved high-temperature stress resistance, comprising the step of transforming a plant cell with the recombinant vector of claim 3 to overexpress a gene encoding the IbOr-R96H variant.

5. A transformed plant having an increased carotenoid content and improved high-temperature stress resistance, which is produced by the method for producing a transformed plant having an increased carotenoid content and improved high-temperature stress resistance according to claim 4.

6. A seed transformed with the plant body of claim 5, which has an increased carotenoid content and an increased resistance to high-temperature stress.

7. A method for increasing carotenoid content and high temperature stress resistance in a plant, comprising the step of transforming a plant cell with the recombinant vector of claim 3 to overexpress a gene encoding the IbOr-R96H variant.

8. A composition for increasing the carotenoid content and high temperature stress resistance of a plant, comprising the gene of claim 2 as an active ingredient.

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