KR101263838B1 - A preparation method of freezing―tolerant plant by a recombinant DNA technology - Google Patents

Abstract

The present invention relates to a method for producing a East Sea resistant plant by genetic engineering, more specifically NtLHS1 Transformant or NtLHS1 with Gene Since the transgenic plants have acquired resistance to East Sea stress, the method can be usefully used for the development of plants which have acquired resistance to East Sea stress.

Description

A preparation method of freezing-tolerant plant by a recombinant DNA technology

The present invention relates to a transgenic plant having a resistance to East Sea stress and a method for producing the same.

The East Sea refers to the damage caused by the formation of ice in the body, and in the case of plants, it can occur below the freezing point of water (0 ℃), but in reality, there are many differences depending on plant species, organs, and physiological conditions. The formation of ice in plants does not lead to plant damage even at temperatures below -20 ° C (Taiz, L and Zeiger, E. Plant Physiology 5th edition. Sinauer Associates, Sunderland, p. 764). However, most crops produce seas at ambient temperatures from -1 ° C to -4 ° C. In many cases, the East Sea is associated with cold seas, which occur at low temperatures above the freezing point of water. The East Sea occurs mainly in winter, but for crops, it is the case of plant cultivation, and for open field crops, the East Sea, which occurs in early spring, is mainly damaged. Except for the subtropical regions of the world, the East Sea is causing serious damage to many crops. In case of Korea, only 220 billion won of national treasury was supported for disaster recovery costs along the East Sea in winter 2009 and early spring 2010 (Ministry of Food, Agriculture, Forestry and Fisheries. It can be seen that the magnitude of the damage is great.

When plants are exposed below freezing point, freezing occurs in the body, and extracellular or intracellular freezing has been analyzed as important determinants of East Sea damage in relation to damage to plants (Taiz, L and Zeiger, E low). (2011) Plant Physiology 5th Edition, Sinauer Associates, Sunderland, p. 764). Freezing inside and outside the cells shows many differences depending on the rate of temperature drop and the physiology or developmental conditions of the plant, when considered in the same plant. Intracellular freezing results in disruption of the cell's internal structure, usually causing rapid damage to the plant, and extracellular freezing does not relatively rapidly damage the plant. The damage caused by freezing may be due to physical destruction of the cell membrane structure, causing structural changes to substances such as proteins and nucleic acids of cells, and changes in the concentration of ions. When the air temperature does not drop below freezing point, the East Sea occurs in some plants, but the cold sea occurs in other places due to the temperature maintenance period, the freezing point, and the size of the plant. Cold degeneration is accompanied by a change in protein activity due to a decrease in body temperature, and thus changes in cell function and, in many cases, changes due to apoptosis or induction of aging processes due to the genetic response of the plant due to the temperature decrease.

The present invention aims to develop a technology capable of enhancing resistance to the East Sea in plants using NtLHS1 (Korean Patent No. 10-346912), which is a molecular chaperone gene previously obtained. NtLHS1 is a molecular saffron that significantly improves the maintenance of water solubility in water-soluble proteins by inhibiting protein structural changes when protein denaturation conditions such as high temperature are applied. And this function is commonly found in molecular chaperones that share NtLHS1 and alpha crystalline domains (Eyles, S. and Gierasch, L. (2010) Nature's molecular sponges: Small heat shock proteins grow into their chaperones.Proc. Natl. Acad. Sci. USA 107: 2727-2728) Recent studies have shown that these molecular chaperones also function in the stability of cell membranes (Nakamoto, H. and Vigh, L. (2007). ) The small heat shock proteins and their clients.Cell.Mol.Life Sci. 64: 294-306).

Therefore, the present inventors have studied to develop a plant with enhanced resistance to the East Sea through genetic engineering technology, and as a result, the NtLHS1 gene is introduced into the world's crops and relatively weak lettuce in the East Sea, thereby enhancing the resistance to the East Sea stress. As a result, the present invention was completed by revealing that transgenic plants having enhanced East Sea resistance could be prepared by introducing the NtLHS1 gene.

An object of the present invention is to provide a transgenic plant having East Sea resistance, the NtLHS1 gene It is to provide a East Sea resistant transgenic plant and a method for producing the transgenic plant introduced with an expression vector.

In order to achieve the above object, the present invention is NtLHS1 having a nucleotide sequence of SEQ ID NO: Gene It provides a transformed plant having the East Sea resistance transformed with the expression vector comprising.

In addition, the present invention NtLHS1 having a nucleotide sequence of SEQ ID NO: 1 Gene Provided is a plant that is a progeny or clone of a transgenic plant having East Sea resistance transformed with an expression vector comprising.

In addition, the present invention NtLHS1 having a nucleotide sequence of SEQ ID NO: 1 Gene Provided is a seed, fruit, ear, tuber, tuber, stem, callus or protoplast of a transgenic plant having the East Sea resistance transformed with the expression vector.

In addition,

1) NtLHS1 having the nucleotide sequence set forth in SEQ ID NO: 1 Gene Preparing an expression vector comprising;

2) transforming plant cells with the expression vector prepared in step 1); And

3) It provides a method for producing a transgenic plant having East Sea resistance comprising the step of tissue culture and re-differentiation of the transformed plant cells prepared in step 2).

NtLHS1 of the present invention Genetically-transformed plants are subjected to East Sea stress and confirmed by bioanalysis (gain of function type analysis), and thus have strong resistance to East Sea stress. It can be usefully used.

1 is NtLHS1 Gene map of the NtLHS1 expression cassette of the plant expression vector pBKS1-1 / NtLHS1 used to express genes in plants. The NtLHS1 gene is transcribed by the function of P35S, RB and LB are the border sequences of T-DNA, and E. coli and Agrobacterium provided by pBKS1-1 on both sides of RB and LB. There are factors for genetic recombination in. pBKS1-1 is a circular plasmid as a whole. RB, right border sequence; LB, left border sequence; Pnos, a promoter of the nopaline synthase gene; NPTII, a neomycine phosphotransferase II gene, was introduced to use kanamycin as a selection marker; Terminator of the Tnos, nopaline synthase gene; P35S, a promoter of 35S transcript of cauliflower mosaic virus.
Figure 2 is a RNA blot analysis results confirmed that the NtLHS1 sense (sense) expression in a direction from the transformed lettuce plant is introduced into a plant expression vector pBKS1-1 / NtLHS1 of the present invention. Wild type lettuce which has not been transformed with WT, NtLHS1 gene; V, transgenic lettuce with only pBKS1-1 introduced; Individual lines of lettuce with # 2, # 4, # 5, # 6, # 7, # 8, # 9, # 11, # 12, # 13, # 14, # 15 and NtLHS1 genes transformed in the sense direction . Total RNA, In order to confirm that the amount of each sample was uniformly controlled, RNA loaded on each lane of the gel was electrophoresed and stained with ethidium bromide, followed by fluorescence generated after UV illumination.

Hereinafter, the present invention will be described in detail.

The invention NtLHS1 having the nucleotide sequence set forth in SEQ ID NO: 1 Gene It provides a transformed plant having the East Sea resistance transformed with the expression vector comprising.

NtLHS1 having a nucleotide sequence set forth in SEQ ID NO: 1 The gene is tobacco ( Nicotiana NtLHS1 isolated from tabacum ) Preferably, the gene is not limited thereto.

Variants of such genes are included within the scope of the present invention. Variant has the nucleotide sequence changed, but NtLHS1 of SEQ ID NO: 1 It is a nucleotide sequence having a functional specification similar to a gene. Specifically NtLHS1 The gene may include a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the nucleotide sequence of SEQ ID NO: 1.

The "% homology" of a sequence to a polynucleotide is determined by comparing the comparison region with two optimally arranged sequences, wherein a portion of the polynucleotide sequence in the comparison region is the reference sequence (additional or In addition to deletions (ie, gaps).

The term “vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that delivers into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” means a recombinant DNA comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.

Preferred examples of such expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumefaciens , can transfer some of their so-called T-region to plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is currently used to transfer hybrid DNA sequences to protoplasts from which plant cells or new plants can be produced which properly insert hybrid DNA into the plant genome. . A particularly preferred form of the Ti-plasmid vector is the so-called binary vector, as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the East Sea resistance genes according to the invention into plant hosts are as can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses and the like. Viral vectors such as incomplete plant viral vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The transformation is preferably a well-known method known to those skilled in the art, such as Agrobacterium-mediated method, gene gun, method using PEG, and electroporation, and the like Bacterium mediated methods are more preferred.

The plant is lettuce, rice, barley, bamboo shoots, corn, taro, asparagus, onions, garlic, leeks, leek, soothing, hemp, ginger, cedar, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, bellflower, spinach, Chard, Sweet Potato, Celery, Carrot, Buttercup, Parsley, Chinese Cabbage, Cabbage, Cocktail, Watermelon, Melon, Cucumber Pumpkin, Gourd, Strawberry, Soybean, Mung Bean, Kidney Bean, Buzzfoot Trefoil, Potato, Duckweed, Perilla, Dove Bean and Pea It is preferable that it is any one selected from the group consisting of, More preferably, it is not limited to lettuce.

In a specific embodiment of the invention, NtLHS1 For the production of transgenic plant expression vector comprising the gene, they were separated only (open reading frame) from NtLHS1 ORF, confirming then inserted into the transformed plant vector pBKS1-1 this result, the conversion is a target ORF correctly transformed The insertion into the vector was confirmed (see FIG. 1).

In a specific embodiment of the present invention, it was confirmed whether the NtLHS1 is expressed in the lettuce by confirming the production of transcripts, and confirmed a strong RNA blot results in the transgenic plants introduced in the sense direction (see Fig. 2).

In a specific embodiment of the invention, NtLHS1 is to investigate the tolerance to the East of the lettuce expressing, after the NtLHS1 the introduction of lettuce with empty vector only and the introduction of lettuce was added to the East stress, collected shoot and biomass As a result, the NtLHS1 introduced lettuce was confirmed that the biomass increased more than two times compared to the lettuce transformed with the empty vector (see Table 1).

Therefore, the transgenic lettuce overexpressed by introducing NtLHS1 has obtained resistance to the East Sea and thus can be usefully used to produce plants having resistance to East Sea stress.

In addition, the present invention NtLHS1 having a nucleotide sequence of SEQ ID NO: 1 Gene Provided is a plant that is a progeny or clone of a transgenic plant having East Sea resistance transformed with an expression vector comprising.

In addition, the present invention NtLHS1 having a nucleotide sequence of SEQ ID NO: 1 Gene Provided is a seed, fruit, ear, tuber, tuber, stem, callus or protoplast of a transgenic plant having the East Sea resistance transformed with the expression vector.

Therefore, the transgenic lettuce overexpressed by introducing NtLHS1 has acquired resistance to the East Sea, so that the seed, fruit, ear, tuber, tuber, root, callus or protoplast of the plant and progeny or clones of the transgenic plant are obtained. It can be useful for producing plants resistant to East Sea stress.

In addition,

1) NtLHS1 having the nucleotide sequence set forth in SEQ ID NO: 1 Gene Preparing an expression vector comprising;

2) transforming plant cells with the expression vector prepared in step 1); And

3) It provides a method for producing a transgenic plant having East Sea resistance comprising the step of tissue culture and re-differentiation of the transformed plant cells prepared in step 2).

NtLHS1 having a nucleotide sequence set forth in SEQ ID NO: 1 The gene is tobacco ( Nicotiana NtLHS1 isolated from tabacum ) Preferably, the gene is not limited thereto.

Variants of such genes are included within the scope of the present invention. Variant has the nucleotide sequence changed, but NtLHS1 of SEQ ID NO: 1 It is a nucleotide sequence having a functional specification similar to a gene. Specifically NtLHS1 The gene may include a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the nucleotide sequence of SEQ ID NO: 1.

The expression vector is preferably an expression vector for general plant transformation, more preferably pBKS1-1, pBI102 or pCAMBIA, and most preferably pBKS1-1, but not always limited thereto.

The plant cells used for the transformation may be any plant cells, preferably cultured cells, cultured tissues, cultured organs or whole plants, more preferably cultured cells, cultured tissues or cultured organs, most preferably cultured cells. Do.

The redifferentiation can use any method known in the art.

The plant is lettuce, rice, barley, bamboo shoots, corn, taro, asparagus, onions, garlic, leeks, leek, soothing, hemp, ginger, cedar, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, bellflower, spinach, Chard, Sweet Potato, Celery, Carrot, Buttercup, Parsley, Chinese Cabbage, Cabbage, Cocktail, Watermelon, Melon, Cucumber Pumpkin, Gourd, Strawberry, Soybean, Mung Bean, Kidney Bean, Buzzfoot Trefoil, Potato, Duckweed, Perilla, Dove Bean and Pea It is preferable that it is any one selected from the group consisting of, More preferably, it is not limited to lettuce.

Conventional plant transformation methods can be used as a method for preparing the transgenic plants (Horsch, et al., Cold Spring Harb Symp Quant Biol., 50: 433-7, 1985; Rogerset, et. Al., 1986). ), Introducing the expression vector into Agrobacterium; And co-culturing the Agrobacterium with a plant or culture cells (including callus, callus), thereby preparing a transformed plant.

In a specific embodiment of the invention, NtLHS1 For the production of transgenic plant expression vector comprising the gene, they were separated only (open reading frame) from NtLHS1 ORF, confirming then inserted into the transformed plant vector pBKS1-1 this result, the conversion is a target ORF correctly transformed The insertion into the vector was confirmed (see FIG. 1).

In a specific embodiment of the present invention, it was confirmed whether the NtLHS1 is expressed in the lettuce by confirming the production of transcripts, and confirmed a strong RNA blot results in the transgenic plants introduced in the sense direction (see Fig. 2).

In a specific embodiment of the invention, NtLHS1 is to investigate the tolerance to the East of the lettuce expressing, after the NtLHS1 the introduction of lettuce with empty vector only and the introduction of lettuce was added to the East stress, collected shoot and biomass As a result, the NtLHS1 introduced lettuce was confirmed that the biomass increased more than two times compared to the lettuce transformed with the empty vector (see Table 1).

Therefore, the transgenic lettuce overexpressed by introducing NtLHS1 has obtained resistance to the East Sea and thus can be usefully used to produce plants having resistance to East Sea stress.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited to the following examples.

< Example  1> NtLHS1 Isolation of genes

To separate the NtLHS1 gene of the present invention, first to open the tobacco plant shock treatment and to produce a cDNA library by using a from which the extraction of the RNA, and then the separated RNA fractional expression search for the separation of NtLHS1 gene (differential screening) The library was used.

Specifically, Wisconsin 38 cultivars of tobacco plants in greenhouses maintained at 25-28 ° C., light conditions of 16 hours ( Nicotiana tabacum L. cv. Wisconsin 38) was grown and subjected to heat shock treatment. Choose young tobacco plants with 5-7 leaves and apply heat shock, respectively, with relative humidity 90 ° C at 32 ° C, 36 ° C, 40 ° C, or 44 ° C for 15 minutes, 30 minutes, 1 hour, 2 hours or 4 hours. At least%, thermal shock was applied in the incubator maintained in dark conditions.

To isolate the total RNA, the leaves of the heat treated tobacco were ground with a mortar in the presence of liquid nitrogen, extracted with RNA extraction buffer containing guanidium thiocyanate, and CsCl gradient high-speed centrifugation was performed. Purification by using (Hong and Jeon, Korean Society of Plant Science, 30: 201, 1987).

Of the extracted RNA, total RNA isolated from tobacco leaves subjected to thermal shock at 39 ° C. for 1 hour was selected, and only mRNA was separated from the total RNA using oligo (dT) -cellulose column chromatography. Isolated mRNA was used as a template and cDNA was synthesized using reverse transcriptase and RNaseH (cDNA Synthesis Kit, Amersham, USA), and the synthesized cDNA was blunt inserted into the Eco RV restriction site of the pBR322 vector (Promega, USA). The recombinant vector thus prepared was transformed into E. coli strain HB101 to prepare a cDNA bank, which was then dispensed into a 96-well microtiter plate and stored at -70 ° C.

For library searching, clones of the cDNA library were incubated together in LB liquid medium (10 g / L bactotryptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing ampicillin in 10 units. E. coli was harvested from the culture by centrifugation, plasmid DNA was isolated by alkaline lysis (Sambrook et al., Molecular Cloning, Cold Spring Habor Laboratory Press, USA, 1989), and then digested with Eco RI and Hin dIII. Agarose gel electrophoresis was performed. DNA on the electrophoretic gel was transferred to a nylon membrane and library search was performed with a ³² P labeled cDNA probe. At this time, the cDNA used for the library search is the first synthetic cDNA from the mRNA extracted from tobacco grown at normal physiological temperature (25 to 28 ℃), ³² P label was added during cDNA synthesis (Sambrook et al., 1989).

As a result, the individual clones were regrown from the group containing the negative clones and the above search method was repeated to isolate the negative clones.

< Example  2> Determination of Sequence of Selected Clones

The cDNA clone isolated in <Example 1> was inserted into the Bam HI position of pBluescript II SK (+). For sequencing, unidirectional deletion sequences were prepared using a deletion kit for kilo-sequencing (Takara, Japan) using exonuclease III. Sequence determination of clones cleaved in one direction was performed by Sanger et al. (Sanger et al., Proc. Natl. Acad. Sci) using Sequenase version 2.0 (United States Biochemical Cooperation, USA). USA 74: 5463, 1977).

As a result, the cDNA clone named NtLHS1 among the nucleotide sequences determined by the above method contained a full length open reading frame (ORF), and the sequence of the ORF was as described in SEQ ID NO: 1, from which The translated 159 amino acid sequences are as described in SEQ ID NO: 2.

< Example  3> NtLHS1  Preparation of Expression Vector for Plant Transformation Comprising Genes

PBluescript SK (-) / NtLHS1 to express and insert the protein recording site of the NtLHS1 gene into the chromosome of a plant Of NtLHS1 vector ORF (open reading frame) of a forward primer (5'-ATAT GGATCC ATGTCTCTGATTCCAAGCTT-3 ' produced according to the nucleotide sequence of both the outer portion of; SEQ ID NO: 3, the underlined part of the ORF in the plant transformation vector It represents the added portion to create a Bam HI restriction enzyme to introduce the outside ORF) and the reverse primer (5'-ATAT GGATCC CTAATGGTCTCTACAGTTACCG-3 '; SEQ ID NO: 4; underlined portion is introduced into a plant transformation vector ORF To show the added portion to make the Bam HI restriction enzyme out of the ORF) to amplify the ORF of NtLHS1 . The prepared primers include restriction enzyme Bam HI position it was possible to insert a Bam HI position of the transformed plant transformation vector was cut with Bam HI and then PCR amplification. PCR amplified DNA was treated with Bam HI and electrophoresed on agarose gel, and DNA fragments corresponding to protein recording sites were purified from agarose gel using GeneClean II kit (Bio 101, USA). The pure DNA fragment was isolated from plant transformation vector pBKS1-1 (Suh MC et al. , Molecules and The expression vector pBKS1-1 / NtLHS1 for plant transformation of the NtLHS1 gene was prepared by attaching to the Bam HI position of Cells , 4, 211-219, 1994 for 8 hours at 19 ° C. using T4 DNA conjugated enzyme. 1) (Sambrook, J. Fritsch, EF and Maniatis, T., Molecular Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd Ed., 1989).

In order to confirm that the protein recording site of the NtLHS1 gene was correctly inserted between the CaMV35S promoter and the end of the nopaline synthase in the expression vector, pBKS1-1 / NtLHS1 was used as a DNA sample. The 3 'terminal portion of the CaMV35S promoter was prepared and identified using an automatic DNA sequencer (ABI 3730; Applied Bio-systems, USA) based on a didioxy chain termination method of Sanger et al. Using primers.

As a result, it was confirmed that the NtLHS1 gene was inserted in the sense direction as shown in FIG. 1 (FIG. 1).

< Example  4> pBKS1 -One/ NtLHS1  Transformation of Expression Vectors into Plants

The pBKS1-1 / NtLHS1 expression vector prepared in <Experimental Example 3> was Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) was introduced into LBA4404. Specifically, DNA was added to a transforming competent cell prepared by treating 20 mM CaCl ₂ , followed by reaction on ice for 30 minutes, and then left in liquid nitrogen for 1 minute. The solution was dissolved in a 37 ° C. water bath for 5 minutes, dissolved, and then incubated with 1 ml YEP medium for 5 hours at 28 ° C. at 200 rpm, followed by centrifugation at 12,000 rpm to precipitate Agrobacterium. Precipitated Agrobacterium was resuspended in 0.1 ml YEP medium and plated in LB solid medium containing kanamycin and incubated at 28 ° C. (An G., Methods in Enzymology , 153, 292-305, 1987). Transformation process was performed on lettuce using a transforming strain that formed colonies in kanamycin medium.

Agrobacterium tumefaciens transformed with pBKS1-1 / NtLHS1 expression vector Suspension lettuce ( Lactuca sativa L. cv. After incubation for 48 hours in the young stage of hypocotyl section of the blue stage (Suh MC, Hong CB, et) al ., Molecules and Cells , 4, 211-219. 1994), MS stem induction medium (Murashige T. and Skoog F., Physiol ) containing BAP 2.0 mg / l, NAA 0.1 mg / l, kanamycin 100 mg / l and cefotaxim 100 mg / l . Plant., in-flight and cultured for 4 to 6 weeks at 15, 473-497, 1962). When the stem is induced by the in-flight culture, MS root induction medium containing 200 mg / L of kanamycin again (Murashige T. and Skoog F., Physiolia) Plantarum ., 15, 473-497, 1962). Root-derived plants were transferred to the soil, and were grown in a greenhouse maintained at 25 ± 2 ℃ under natural light and blocked by outside and double glazing, and analyzed the development process of seeds according to growth, flowering, and self-pollination. When the seeds matured, they were harvested, dried at room temperature for 2 weeks, and then stored at 4 ° C.

< Example  5> pBKS1 -One/ NtLHS1  Identification of Expressing Transgenic Plants

Lettuce generated through the transformation process was performed by RNA blot analysis to confirm that the expression of NtLHS1 to be introduced was made as intended. Specifically, RNA extraction from lettuce was performed by Cho and Hong (Cho and Hong, J. Plant). Biol . 47: 149-159, 2004). The plants were frozen in liquid nitrogen and then ground with a mortar to prepare powder. Lysis buffer (lysis buffer: 25 mM Tris-Cl pH 8.0, 50 mM LiCl, 35 mM EDTA, 35 mM EGTA, 0.5% SDS) was added to the powder and mixed by vigorous stirring. Since the same amount of phenol (phenol) was added and then vigorously stirred. Thereafter, an additional 1/4 volume of chloroform was added and mixed with vigorous stirring, and centrifuged for 15 minutes at 6,000 g at 20 ° C. After centrifugation, the supernatant was taken, an equal amount of 4 M LiCl solution was added, and RNA was harvested as a precipitate by centrifugation at 15,000 rpm for 15 minutes at 4 ° C. with a microcentrifuge. RNA was isolated according to size by formaldehyde-agarose gel electrophoresis and blotted with a high-bond-N membrane (Sambrook, J. et al., Molecular Cloning : A laboratory manual, Cold Spring Harbor). Laboratory Press, 2nd Ed., 1989). RNA blot membranes were hybridized with NtLHS1 labeled with α- ³² P-dCTP. Hybridization methods are described by Sambrook et al. (Sambrook, J. et. al ., Molecular Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd Ed., 1989). The membranes were prehybridized in solution [0.5 M sodium phosphate, pH 7.2, 7% (w / v) SDS and 1 mM EDTA, pH 7.0), to which NtLHS1 labeled ³² P-dCTP was added Hybridization at 65 ° C. for 16-22 hours. Hybridized membranes were washed at 65 ° C. with 1 × SSPE, 0.1% SDS solution and exposed to X-ray films (Sambrook J., Fritsch EF, and Maniatis T., Molecular Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd) Ed., 1989).

As a result, as shown in Figure 2, pBKS1 -1 / NtLHS1 was introduced in the sense direction and confirmed the strong RNA blot results in 11 of 12 lines of lettuce plants analyzed in the transformed lettuce plants, wild type and vector only From the introduced plants, it was confirmed that the RNA blot result was negative. Therefore, it was confirmed that the resulting plant is a transgenic plant in which the expression of NtLHS1 is made (FIG. 2).

< Example  6> pBKS1 -One/ NtLHS1  East Sea Tolerance Analysis of Expressing Transgenic Plants

The transgenic plant confirmed the expression of NtLHS1 confirmed in <Example 5> was maintained in the greenhouse and harvested seeds by inducing self-pollination (T ₁ generation). The harvested seeds were germinated in MS-medium containing kanamycin, grown, picked and planted by turning green seedlings to obtain plants, and then subjected to bioassay experiments to determine whether the East Sea resistance was increased. Specifically, a bioassay to confirm the increase of resistance to the East Sea was performed as follows. Six to eight young lettuce plants grown in vermiculite-soil were subjected to East Sea stress for 1 or 2 hours at -4 ° C and -8 ° C in a freezer (Sanyo JP / MDF-u2086S Japan). The plants were treated for 5 days in a culture room maintained at 25 ℃, 16 hours photoperiod. Measuring the biomass of the shoot and then allowed to stand, and it was confirmed whether or not Freezing increase of transgenic plants by comparing the liver weight of the transgenic plants and transformed NtLHS1 introduced pBKS1-1 vector only and the introduction (figures in Table 1 are the Eight objects were used for one measurement, and all three measurements were taken to calculate the mean and standard error.

As a result, the NtLHS1 is introduced as shown in Table 1, the expressed transgenic plant is a biomass increase of about 20-112% compared to determine the vector only and the introduction of the plant was confirmed that the resistance East enhancement (Table 1).

Temperature Processing time Control rain
Biomass Growth Rate (%) Remarks
25 ℃
Incubation temperature
6.4 + 2.3
Comparison of shoot biomass of NtLHS1 transformant and vector transformant grown in culture chamber
4 ℃
5 days
24.1 + 3.9 NtLHS1 transformants and vector transformants grown in the culture chamber were incubated for 5 days with a 16-hour photoperiod in a low-temperature room at 4 ℃, and compared to shoot biomass.

-4 ℃
1 hours
20.5 + 15.6 Comparison of biomass measured after NtLHS1 transformant and vector transformant grown in culture chamber for 1 hour or 2 hours in -4 ℃ freezer, and transferred to the culture chamber for 5 days 2 hours 94.9 + 19.8

-8 ℃
1 hours
110.6 + 20.4 Comparison of biomass measured after NtLHS1 transformant and vector transformant grown in culture chamber for 1 hour or 2 hours in -8 ℃ freezer and transferred to the culture chamber for 5 days 2 hours 112.1 + 25.6

<110> Seoul national university R and DB foundation <120> A preparation method of freezing-tolerant plant by a recombinant          DNA technology <130> 11p-01-33 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 480 <212> DNA <213> Nicotiana tabacum <400> 1 atgtcttcga ttccaagctt ctttgatggc cgcaggagca acatcttcga tccattctcc 60 ctcaacatat gggatccttt cgagggcttc cctttctccg gcacggtcgc caacattcca 120 acctctactc gtgaaaccgc tgctttctcg agtgctcgaa ttgattggaa agagacccca 180 gaatcccatg tctttaaggt ggatcttcct ggaattaaga aagaggaggt gaaggtggag 240 gtagaagaag gaagagttct acaaattagt ggcgagagaa gcagagagca agaggagaag 300 aacgataaat ggcacagcat ggagaggagc agcggaaagt tccttaggag gtttaggttg 360 ccggagaata tcaaaatgga agagattaag gcaactatgg agaatggggt gctcactgtg 420 actgtcccaa aaatggagga aaagaaacct gaggtgaagg ccattgacat ctctggttaa 480                                                                          480 <210> 2 <211> 159 <212> PRT <213> Nicotiana tabacum <400> 2 Met Ser Leu Ile Pro Ser Phe Phe Asp Gly Arg Arg Ser Asn Ile Phe   1 5 10 15 Asp Pro Phe Ser Leu Asn Ile Trp Asp Pro Phe Glu Gly Phe Pro Phe              20 25 30 Ser Gly Thr Val Ala Asn Ile Pro Thr Ser Thr Arg Glu Thr Ala Ala          35 40 45 Phe Ser Ser Ala Arg Ile Asp Trp Lys Glu Thr Pro Glu Ser His Val      50 55 60 Phe Lys Val Asp Leu Pro Gly Ile Lys Lys Glu Glu Val Lys Val Glu  65 70 75 80 Val Glu Glu Gly Arg Val Leu Gln Ile Ser Gly Glu Arg Ser Arg Glu                  85 90 95 Gln Glu Glu Lys Asn Asp Lys Trp His Ser Met Glu Arg Ser Ser Gly             100 105 110 Lys Phe Leu Arg Arg Phe Arg Leu Pro Glu Asn Ile Lys Met Glu Glu         115 120 125 Ile Lys Ala Thr Met Glu Asn Gly Val Leu Thr Val Thr Val Pro Lys     130 135 140 Met Glu Glu Lys Lys Pro Glu Val Lys Ala Ile Asp Ile Ser Gly 145 150 155 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for ORF amplification <400> 3 atatggatcc atgtctctga ttccaagctt 30 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for ORF amplification <400> 4 atatggatcc ctaatggtct ctacagttac cg 32

Claims (10)

NtLHS1 (Nicotiana tabacum low molecular weight heat-shock protein) having the nucleotide sequence set forth in SEQ ID NO: 1 Gene Transgenic lettuce having East Sea resistance transformed with an expression vector comprising.
delete delete Lettuce which is a progeny or clone of the transgenic lettuce of claim 1.
Seed, fruit, ear, tuber, tuber, strain, callus or protoplast of claim 1 transgenic lettuce.
1) NtLHS1 (Nicotiana tabacum low molecular weight heat-shock protein) having the nucleotide sequence set forth in SEQ ID NO: 1 Gene Preparing an expression vector comprising;
2) transforming the cells of lettuce with the expression vector prepared in step 1); And
3) A method of preparing a transgenic lettuce having resistance to the East Sea comprising the step of tissue culture and re-differentiation of the transformed lettuce cells prepared in step 2).
The method of claim 6, wherein the expression vector is pBKS1-1.
7. The method according to claim 6, wherein the transformation is carried out using an Agrobacterium mediated transformation technique.









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