KR940001266B1 - Process for preparation of plant with anti-freeze protein - Google Patents

Abstract

An antifreeze protein gene bases on a gene of antifreeze protein from Arabidopsis was synthesized. The antifreeze protein gene was cloned into a plasmid pGA643 and E.coli JM109 was transformed with recombinant vector pOST1100. A strain (Agrobacterium LBA 4404) carrying the recombinant vector pOST1100 was isolated from E.coli JM109 (pOST1100), Agrobacterium LBA 4404 and E.coli MC1000 carrying pRK2013 by triparental mating method. After Agrobacterium- mediate transformation of tobacco tissue, the expression and transcription of antifreeze protein gene was analyzed by Southern and Northern hybridization and the antifreeze protein expressed in the tobacco tissue was determinated by electrophoresis.

Description

Manufacturing method of plant expressing ice protein

1 shows the nucleotide sequence of the synthesized icing protein gene and the amino acid sequence translated therefrom,

2 shows the vector pOST1100 linking the synthetic icing protein gene between the Xba I and Bg1 II restriction enzyme recognition sites of vector pGA643 to be regulated by the CaMV35S promoter,

FIG. 3 shows the results of the assay of neomycin phosphate transferase II (NPTII) from tobacco tissues into which the vector pOST1100 with the icing protein gene was introduced,

Figure 4 shows the results of Southern hybridization experiments by cutting DNA isolated from tobacco and wild-type tobacco into which the indwelling protein gene was introduced, and with restriction enzymes.

Figure 5 shows the results of Northern hybridization experiments by separating the total RNA from tobacco and wild-type tobacco introduced with the vector pOST1100,

FIG. 6 shows the results of facility staining after SDS-PAGE (SDS-polyacrylamide gel electrophoresis) by separating proteins from tobacco leaves of tobacco and wild species to which the icing protein gene was introduced.

The present invention relates to genes encoding anti-freeze proteins that are produced in the body of some species and allow the organism to survive at low temperatures. The present invention also relates to a method of introducing and expressing the gene into plant cells such as tobacco.

Most organisms are fatal because long-term exposure to low temperatures below freezing point causes the cytoplasm to freeze, dehydrate the cytoplasm by freezing, and physically destroy cell and tissue structures by the growth of ice crystals. However, a large number of cold or polar species, including fish, can survive in low temperature environments by inhibiting the formation and growth of ice crystals within the cytoplasm through the synthesis of so-called anti-freeze proteins (AFPs). (Feetnet, RE et al., Ann, Rev. Biophys. Chem., 15, 59-78 (1986)). The indwelling proteins synthesized by these species are known as at least one anti-freeze glycoprotein (AFGPs) and three types of indwelling proteins.

To date, all icing glycoproteins isolated and purified from various species, especially polar species, have been written by alanine, alanine, and threonine (Ala-ala-Thr) consisting of two alanines and a threonine with a disaccharide linked to the side chain. Glycotri peptide units are polymerized polymers that have very similar chemical structures, regardless of fish species.

In comparison, icy proteins are structurally diverse among fish, and they are classified into soluble proteins containing alanine, cysteine-containing proteins, and proteins containing neither alanine nor cysteine (see Hew, CL et al.). , 1986, Living in the Cold, Physiological and Biochemical Adaptation, (eds., Heller, HD et al.) 117-123, Elsevier, New York, USA). These ice- and ice-glycoproteins are known to be 300-500 times more effective than salt (NaCl) in terms of molecular equivalents in reducing the freezing point by monocolligative phenomena (see Yang. DSC). et al., Nature 333, 232-237 (1988)).

X-ray crystal structure analysis of the ice-rich protein containing alanine among the ice-collected proteins shows that it has a single alpha-helix structure of about 5 nm. Seven amino acid sequences of proteins have been identified. Significant freezing point degradation was also observed with an artificial α-helix synthetic polymer composed of 65 alanines and 35 aspartic acids.

Unlike fish, in the case of food, cold resistance is caused by polyhydroxy alcohol or glycolipid in the cytoplasm, but polar willow and pine white have huge icing protein molecules. It has been found to survive below ℃ (Roger, HP, 1988, Nature 333, 207-208, Levitt, J., 1980, Responses of plants to environmental stresses and high tempe rature stresses pp 10-180, Academy Press, New York., USA).

It is an object of the present invention to provide an indwelling protein gene suitably prepared for expression in a plant by applying an indwelling protein amino acid sequence of a known fish based on the above data, and also by inserting an indwelling protein gene into a suitable vector. It is intended to provide a recombinant vector suitable for transforming plant cells.

In another aspect, the present invention provides a method for producing a plant that is transformed with the recombinant vector to produce an ice-free protein in the cell to reduce the temperature constraints in cultivating plants that are restricted in growth at low temperatures, particularly in warm-tolerant plants such as tobacco It has a purpose to minimize.

Hereinafter, the present invention will be described in more detail. The endogenous protein gene is designed to have a structure and a nucleotide sequence that are easily expressed in plant cells, and then oligonucleotides are synthesized to a suitable length (about 40-100 nucleotides) using a DNA synthesizer. The synthesized oligonucleotide is denatured by treating at high temperature (about 80 to 90 ° C.), and then mixed with those having complementary nucleotide sequences, and then gradually lowered for a long time (1 hour or more) to complementarily bind. . Only the bound double-stranded DNA is separated by electrophoresis to phosphorylate the 5 'end, and then linked to other double-stranded DNA prepared in the same manner using a T ₄ DNA ligase to prepare a gene having a desired sequence.

Inland protein genes can be separated from organisms such as fish by the gene search and cloning methods commonly known in addition to the above synthetic methods, and in order to be effectively expressed in plant cells, there is no significant difference in the function of the expressed protein. Appropriate modifications may be made to the sequence within the range.

Suitable vectors with expression control devices such as promoters that can be amplified in Escherichia coli and used in plant cells can be used to introduce and express the indwelling protein genes into plant cells. Such vectors include, but are not limited to, pGA 643 (Gynheung An, et al., Plant Molecular Biology Manual A3: pp 1-19 (1988)), pGA492 (KTCC11056), pGA580 (KCTC 580), and the like. It doesn't work. In addition, warm plants such as tobacco, rice, corn, and vegetables may be preferably used as the plant for expressing the indwelling protein gene inserted into the vector, but are not limited thereto.

Agrobacterium-mediate transformation was used to introduce into the plant cells an indwelling protein gene made to be expressed in the plant, using helper plasmids in a known manner prior to plant transformation. And co-introduced plasmid genes into Agrobacterium (Stephen, GR et al., Plant Molecular Biology pp 1-12 (1988)). According to the method of Robert et al. (Ro bert, BH, et al., Plant Molecular Biology Manual A 42, 1-9 (1988)), the bacteria are used to transform genes of plant cells in MS medium and to kanamycin. (kanamycine) screening resistant shoots (shoot) are selected and passaged several times, when the leaves are formed in the root formation medium, the roots are formed in the soil to grow to complete plants.

In order to confirm the transgenic inland transgenic genes in this plant, Neo using a method of Estrella et al. (Estrella, LH, et al., Plant Molecular Biology Manual B1, pp 1-22 (1988)) Nemiccin phosphot rans-ferase II (NPT-II) enzyme assay or whole genomic DNA was isolated and digested with restriction enzymes, electrophoresed on agarose gels, and transferred to a nylon filter to provide a suitable probe. Use Southern hybridization and more.

In addition, in order to investigate whether the indwelling protein gene is transcribed in plants in which the indwelling protein gene is introduced, the whole RNA is isolated, electrophoresed in a denatured formaldehyde gel, and then adsorbed onto a nitrocellulose filter to adsorb the γ-P ³² [ATP]. Northern hybridization with terminal labeled probes. In addition, the whole protein of the plant is extracted in order to know whether the indwelling protein has been generated, and then compared with the wild species by SDS-PAGE and silver staining (silver staining) to check the protein production.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples are merely provided to illustrate the invention and are not intended to limit the scope of the invention.

Example 1

Preparation of Inland Protein Genes

Amino acid sequence of SS-8 icing protein of Shoten sculptin fish (Leutwiler, LS et al., Nucl.Acids Res. 14, 4051-4076 (1986)) and 5 'leader sequence of Arabidopsis cap I gene (see: The Iceberg protein gene was designed by modeling Pickett, MH et al., Eur. J. Biochem. 143, 35-38 (1985). Since the protein is a structure of α-heryx which is involved in the mechanism of ice-breaking, N-terminal methionine and 45 amino acids, it is assumed that the protein is produced without any problem in post-translational processing even in plant cells. .

Using this amino acid sequence, the DNA sequence was designed, and the codon usage tendency of the plants already known to be well expressed in plant cells (Murray, EE et al., Nu cl. Acids Res. 17, 477-498 ( (1989), Messing, J., Genetic Engineering, 6, 2-46, Academic Press, London), alanine almost used GCT codons, and GCC codons were used to rule out repeat and reverse repeat sequences. The 5 'leader sequence also used the Cap I gene of Arabidopsis plants to enhance transcription in plant cells. Four adhesion ends were placed in the middle for linking these genes. The restriction enzymes Xba I and Bgl II sites were placed after the leader sequence. The leader sequence was followed by a translation initiation codon (ATG) and a codon at the end of the codon (TAA).

In order to facilitate DNA synthesis, 76 bases were divided into two, and two complementary to these were designed into four strands. The oligonucleotide sequence of the icing protein gene designed in FIG. 1 and the amino acid encoded by the well are shown well. (In FIG. 1 and the present specification, nucleotides and amino acids are denoted by standard single abbreviations. In the textbook).

The designed gene was synthesized into two strands of 76 nucleotides and two strands complementary thereto. The DNA synthesizer was used by Milligen Co., Ltd. (Milligen 7500 DNA Synthe sizer, Milligen Co.) and synthesized and purified by β-cyano ethyl phosphoramidite method (see Matteucci et al., J. Am). Chem. Soc., 103: 3185 (1981)).

Take 1 μg of each purified Oligigo nucleotides A, B, C and D and mix them in different tubes with denatured buffer solution (final concentration: 0.18M NaCl, 100mM Tris, 1mM EDTA, pH 7.0) so that the final volume is 100µl. The mixture was denatured by heating at 86 ° C. for 2 hours. 0.5 μg of denatured oligonucleotides A, B, C, and D were mixed with annealing buffer solution to a final solution concentration of 0.18 M NaCl, 10 mM Tris, 1 mM EDTA, pH 7.0 and final volume of 100 μl. It was then allowed to cool slowly to 25 ° C. starting at 61 ° C. for 10 hours. After the annealing reaction was electrophoresed at 70 volts for 3 hours on a 4% low melting agarose gel, only two fragments of the oligonucleotides complementarily bound to the 0.5-fold concentration of TBE buffer were passed through the dialysis membrane. Electrolyzed in solution (45 mM Tris-borate, 45 mM boric acid, 0.1 mM EDTA) and concentrated by ethanol precipitation. It was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) to a final concentration of 0.1 μg / μl.

To phosphorylate the 5 'end of each DNA fragment, 2 μl of the DNA (200 mg), 1 μl of T ₄ polynucleotide kinase (0.5 unit), 3 μl of 10 mM ATP, 10 fold concentration kinase buffer solution (0.5M Tris, 10 mM MgCl ₂ 3 μl of 1 mM ZnCl ₂ , 10 mM spermidine and 21 μl of distilled water were mixed and reacted at 37 ° C. for 30 minutes, followed by ethanol precipitation to dissolve in TE buffer solution to a final concentration of 50ng / μl.

2 μL of the A / B DNA fragment was reacted to link the DNA fragments (A / B) to which the phosphorylated A and B oligonucleotides were bound, and the fragments (C / D) to which the C and D oligonucleotides were bound, respectively. (100 ng) and 2 μl of C / D DNA fragment (100 ng), 1 μl of T ₄ DNA ligase (2 units), 10-fold concentration ligation buffer (0.5 M Tris pH 7.4, 0.1 M MgCl ₂ , 0.1 M) 2.5 μl of DTT (dithiothreitol), 10 mM spermidine, 1 mg / ml BSA (bovine serum albumin), 2.5 μl of 10 mM ATP and 15 μl of distilled water were mixed and left to stand at 14 ° C. for 15 hours to allow the ligation reaction. Extracted with chloroform (1: 1 v / v), precipitated with 2 times the volume of ethanol, dissolved in TE buffer solution to a final concentration of 50ng / μl, and confirmed the DNA fragments connected by electrophoresis.

Example 2

Preparation of Expression Vector pOST1100 and E. Transformation of E. coli

50 ng of the 152 bp synthetic icing protein gene, the product of Example 1, was mixed with 200 ng of pGA 643 double digested with Xba I and Bgl II restriction cleavage enzymes, and 2 μl of T ₄ DNA ligase, 10-fold concentration ligation buffer After adding 2 μl of 10 mM ATP, the final volume was adjusted to 20 μl with distilled water. The mixture was reacted at 14 ° C. for 15 hours to allow a synthetic icing protein gene to be linked between the Xba I and Bgl II recognition sites of the pGA 643 vector. E. by the DNA. E. coli transformation was carried out by calcium chloride method (Maniatis et al., Molecular Cloning, A labora-tory manual, pp250-251, Cold Spring Harbor Laboratory, 1982). 13 μg / ml tetracycline was applied to an LB solid medium (10 g of bacto-trypton per liter, 5 g of yeast extract, 10 g of NaCl, 16 g of bacto-agar) and incubated at 37 ° C. for 12 to 16 hours to include a prepared vector. Colonies were selected.

The plasmid was isolated from the clones by the alkali dissolution method. Alkaline lysis method was performed according to Maniatis et al., Molecular Cloning, A laboratory manual, pp368-369, Cold Spring Har bor Laboratory, 1982, and CsCl concentration gradient ultracentrifugation method (see above) Purified by the same document pp93-94). Plasmids isolated and purified as described above were digested with Eco RI and Bgl II restriction digestases, followed by agarose gel electrophoresis to confirm that the icing protein gene fragment was inserted into the vector, and the oligonucleotide (5'-) of the promoter region of the vector was inserted. TTCGCAAGACCCTTCCTCTA-3 ') was synthesized and purified in the same manner as in Example 1, and then used as a primer to determine the sequence by Sanger's dideoxy method (Sanger, et al., Proc.Natl.Acad.Sci The nucleotide sequence of the gene synthesized by USA 74: 5463 (1977) was identified The plasmid into which the indwelling protein gene was introduced was named pOST 1100 and is shown in Fig. 2. In Fig. 2, B is the restriction enzyme Bam. HI represents the recognition site, S is Sac II, Sa is Sal I, H is Hind III, Bg is Bgl II, X is Xba I, P is Pst I, N is Nae I, St is Stu I, Hi is Hinc II , Ss is Sst I, and R is EcoR I recognition Shows were deposited with the E.coli JM109 containing the pOST 1100 by the Korea Institute of Science and Technology Genetic Engineering Research deposited in the gene bank (KCTC) No. KCTC 0015BP July 08, 1991.

Example 3

Trialal mating method of three strains

In order to introduce pOST1100 prepared in Example 2 to Agrobacterium, the following experiment was performed.

E. coli JM109 (ATCC No. 53323) containing the pOST1100 plasmid was prepared in YEP liquid medium containing 10 μg / ml of tetracycline (10 g of bacto-peptone per liter, 10 g of yeast extract, 5 g of NaCl), Agrobacterium LBA 4404 ( Clontech Laboratories Inc. # 60 27-01 USA) is an E.coli containing YEP liquid medium containing 25 μg / ml of strapomycin and 100 μg / ml of rifampicin, pRK 2013 helper plasmid (ATCC 37159). MC10 00 (ATCC 39531) was incubated in YEP liquid medium containing 50 g / ml of Panamamycin until the optical density (OD) at 650 nm was 0.4.

Each 100 μl of the culture solution was taken, mixed, and centrifuged to resuspend the precipitated cells in 1 ml YEP liquid medium, followed by centrifugation, and the precipitated cells were suspended in 100 μl of YEP liquid medium. 50 μl of the suspension was used to spot 2 or 3 spots on YEP solid medium (15 g of phytagar, Gibco Co.) in YEP liquid medium composition and incubated at 28 ° C. for 2 days. To be mated to each other.

Cultured colonies were taken, diluted with YEP liquid medium, and then reached for YEP solid medium containing strapomycin, tetracycline, and rifampicin, followed by incubation for at least 48 hours. Was isolated and then cleaved with one or more restriction enzymes to confirm the conjugation. Colonies confirmed to have been introduced into the Agrobacterium LBA 4404 by the three strains conjugated to the pOST1100 plasmid was used in the following procedure.

Example 4

Agrobacterium-mediated plant cell transformation

Sterilized in MS medium (4.3 g of MS salt mixture per liter (Gibco Co.), 30 g of sucrose, 0.1 g of inositol, phytohormones, other vitamins and nutrients, 8 g of phytaga r, Gibco Co. After seeding and incubating in sterile conditions for 3 to 4 weeks, the leaves (Nicotiana tabacco xanthileaf) were cut from the tobacco plants formed, cut in several places and placed on fresh MS medium, and then the conjugated Agrobacterium obtained in Example 3 above. LBA 4404 was cultured for 18 hours in a YEP liquid medium, and 200 µl of the culture solution was applied to the wounded area, and co-cultured for 3 days, in which the surface of the leaf was placed in MS medium with the surface facing down, and cultured for 3 days, followed by antibiotics. Washed with YEP liquid medium containing phosphorus carbenicillin (500 μg / ml) Washed leaf tissue was MS medium [MS medium containing kanamycin (300 μg / ml) and carbenicillin (500 μg / ml)] 2-3 weeks in After liver culture, shoots with kanamycin resistance formed in cultured leaf tissues were transferred back to MS medium (including 300 µg / ml of kanamycin) and cultured until they grew to 2 to 3 cm. It was transferred to rooting medium (MS rooting media, MS medium containing indole acetic acid at a concentration of 2mg / ℓ), and after 2-3 weeks, the roots were sufficiently formed, and then the plants were transferred to soil and planted as full objects.

Example 5

Identification of Naked Protein Genes Introduced into Tobacco Cells by Assay of Neomycin Phosphate Transferase II

To confirm whether the tissues were taken from the tobacco prepared in Example 4 and transformed by the icing protein gene, a test of neomycin phosphate transferase II (NPT-II) was performed, and known methods (see: Estrella, LH, et al., Plant Molecular Biology Manu al, Bl, pp 15-17 (1988), Reiss, B et al., Gene 30, 211-218 (1981)).

To 0.5 g of tobacco tissue, 0.5 ml extraction buffer [final concentration: 0.5% β-mercaptoethanol, 25 mM Tris-Hcl, pH 6.8 65 μg / ml leupeptin] was added, followed by the addition of glass beads. After crushing the tissue and centrifuged for 2 to 3 minutes in a micro centrifuge. Take the supernatant and place in another Eppendorf tube and mix 1/10 volume of 10-fold concentration buffer (50% glycerol, 0.5% SDS, 40 μg / ml bromophenol blue) to 50-60 μg in 10% polyacrylamide gel. Was added.

After gel electrophoresis at 4 ° C. (150 to 200 volts, 4 to 5 hours), the gel was removed and washed twice in cold water (4 ° C.) for 5 minutes and then at 4 ° C. reaction buffer (final concentration: 50 mM, Tris-HCl, pH 7.7., 25mM MgCl ₂ , 200mM NH ₄ Cl, 0.5mM DTT) and allowed to shake slightly for 30 minutes. A 1% agarose gel (containing 0.1 μg / ml of kanamycin and ⁴ μci / ml of ³² p-α-ATP) was poured at 45 ° C. to be evenly spread over the polyacrylamide gel and then wetted with P81 Whatman filter paper (Whatman). paper) was placed on agarose gel and placed at 37 ° C. for 30 minutes.

Paper towels were stacked on the Whatman filter paper and blotted for 2 to 3 hours or longer, and then the Whatman filter paper was removed and washed at 80 ° C. for 40 to 50 minutes with a washing solution (5 mM Na ₂ HPO ₄ ). It was. Whatman filter paper was dried and exposed to an X-ray film. When the neomycin phosphate transferase gene present in pOST1100 is expressed in plant cells, kanamycin contained in the agarose gel portion placed on the generated enzyme portion is phosphorylated by γ- ³² p-ATP, so it is adsorbed on watman filter paper to autoradiography. Through the presence of neomycin phosphate transferase can be seen.

The results are shown in FIG. 3, where column 1 represents tobacco tissue transformed with vector pGA 643, column 2 represents tobacco tissue of wild species, and columns 3 to 8 represent tobacco tissue into which pOST1100 has been introduced. Indicates. Only in cigarettes into which the vectors pGA 643 and pOST1100 were introduced, there was a positive signal, demonstrating that the neomycin phosphate transferase II gene was introduced into the cell, from which the indwelling protein gene in pOST1100 was also introduced.

Example 6

Southern hyb ridization analysis to determine the transformation of tobacco

1 g of the plant transformed with the icing protein gene prepared in Example 4, 3 ml of proteinase K reaction solution (0.2 M Tris-HCl, pH 8.0., 0.1 M EDTA (ethylenedia mine tetraacetic acid), 1% After adding sarcosyl, 100 ㎍ / ml proteinase K) and 0.5 g glass beads, liquid nitrogen was added and pulverized, and then reacted at 40 to 50 ° C. for 1 hour, followed by centrifugation at 10,00 rpm for 10 minutes. The supernatant was taken and double volume ethanol was added to precipitate DNA. The DNA precipitate was dissolved in TE buffer solution (10 mM Tris, 1 mM EDTA) and then known in a known method (e.g. Maniatis, T. et al., Molecular Cloning, A laboratory manual, Cod Spring Harbor Laboratory (1982), 281). DNA was purified by CsCl gradient ultrafast centrifugation. 10 μg of the purified DNA was digested with restriction enzymes EcoR I and Xba I, electrophoresed in 0.7% agarose gel, and then blotted to a nylon filter (ICN Biochemicals Inc., USA) having a 0.45 μm diameter. It was then crosslinked by ultraviolet irradiation. Subsequently, the filter was added to a prehybridization solution (prehybridization solution: 7% SDS, 0.5M NaPO ₄ , 1 mM EDTA, 1% BSA, pH 7.2) and reacted by shaking gently at 60 ° C. for 2 hours, followed by a hybridization solution. ( ³² P labeled probe was added to the preliminary hybridization solution) and hybridized at 60 ° C. for 14 hours.

Probes were prepared in the following manner. pOST1100 was digested with restriction enzymes Xba I, Ec oR I, electrophoresed on agarose gels, and DNA fragments of about 0.9 Kb were isolated by electroelution, followed by known methods (see Maniatis, T. et al. , Nick ranslation according to Molecu lar Cloning, A laboratory manual, Cold Spring Harbor (1982), pp 109-112), labeled with ³² P so that the specific activity is greater than 10 ⁷ cpm. Probes were prepared. The filter was washed twice at room temperature with a wash solution (2 × SSC (containing 17.53 g of NaCl, 8.82 g of sodium citrate) and 0.1% SDS) at room temperature for 5 minutes and then washed at 40 ° C. for 10 minutes with the same washing solution and dried. And exposed to the X-ray film for 24 hours (see Figure 4). In FIG. 4, columns 1 and 3 are cigarettes in which pOST1100 was introduced, column 2 was tobacco introduced only with a vector (pGA 643) without an icing protein gene, and column 4 was extracted from wild tobacco. Double cleavage with I and EcoR I, column 5 double cleavage of vector pGA643 with restriction enzymes Xba I and EcoR I, column 6 double cleavage of pOST 1100 with restriction enzymes Xba I and Bgl II, respectively Columns 7 and 8 are molecular weight indicators, column 7 is the cleavage of phagelamida DNA with HindIII, column 8 is the cleavage of lambda DNA with Bst EII, and the molecular weight is in Kb (kilobase pair) units. .

Example 7

Northern hybridization analysis of transformed tobacco

To examine the transcription level of the endogenous protein gene in tobacco in which the synthetic endogenous protein gene was introduced, total RNA from the plant was determined by Cox et al. (Cox, KHand Goldberg, RB, Plant Molecular Biology, pp1-35, IRL). Press, Oxford, 19 88), and purified, and denatured by separating total RNA from plants according to known methods (Thomas, PS, Proc. Natl. Acad. Sci. U. SA, 77, 5201 (1980)). Electrophoresis of total RNA on a formaldehyde gel followed by transfer to a nitro cellulose filter followed by Northern-labeled probes (specific activity; 10 ⁸ cpm) end-labeled B, D oligonucleotides of γ- ³² P Hybridization experiments were carried out and washed twice with a washing solution (2 × SSC, 0.1% SDS) twice for 10 minutes, further washing at 50 ° C. for 30 minutes with the same washing solution, and drying the filter.

FIG. 5 shows the result of exposing the filter to an X-ray film. The first column is RNA extracted from wild tobacco and the second row is RN A extracted from tobacco transformed with pOST1100. In addition, Figure 5 (a) is a picture of a gel stained with ethidium bromide after electrophoresis of the entire RNA and Figure 5 (b) shows an autoradiogr (autoradiogr aphy) after the northern hybridization. As a result, it can be seen that the transgenic protein is highly transcribed in tobacco transformed with pOST1100.

Example 8

Protein electrophoresis to determine whether icing protein is produced

1 g of tobacco leaves were homogenized in 3 ml of grinding buffer (100 mM Tris, pH 7.4, 5 mM DTT, 0.1% SDS), followed by centrifugation at 100,000 rpm for 20 minutes to obtain a supernatant, followed by double concentration of sample buffer ( 62.5 mM Tris, pH 6.8, 1% SDS, 15% glycerol, 15 mM DTT, 0.05% bromophenol blue) was added to the same volume and boiled for 3 minutes. 70 μl of the sample was added to a discontinuous gel made of 10% polyacrylamide and 20% polyacrylamide, each containing 10% glycerol, and electrophoresed at 60 mA for 6 hours.

The gel was silver stained with a Silver Staining Kit (PIERCE Co.) according to the manual of PIERCE. The results are shown in FIG. 6, where column 1 is protein extracted from tobacco leaves transformed with pOST1100, column 2 is protein extracted from tobacco leaves introduced only with pGA 643, and column 3 is wild tobacco. The protein extracted from the leaves, column 4, showed the standard molecular weight indicators, respectively, and the unit of the molecular weight indicator was kilodaltons. An icing protein band appeared at an arrow position only in the first column, and as a result, the icing protein was generated in tobacco cells transformed by pOST1100.

Claims (5)

Inland protein having the following amino acid sequence: MNGETPAQKAARLAAAAALAAKTAADAAAAAAAKAAAIAAAAASA DNA having a nucleotide sequence shown in FIG. 1, which coats an icing protein. Expression vector pOST 1100 comprising DNA having the nucleotide sequence shown in FIG. A method of producing a plant that produces an icy protein by transforming plant cells by culturing the Agrobacterium-mediated transformation method using an expression vector pOST1100 and then culturing them. The method of claim 4 wherein the plant is tobacco.