KR19990075102A - Transgenic Tomato Using Blood Pressure Inhibiting Peptide Gene - Google Patents

Abstract

The present invention relates to a tomato having an inhibitory effect on blood pressure increase by introduction of a functional peptide gene. In order to produce proteins, particularly short functional peptides, by introduction of foreign genes in plants, not only efficient gene expression but also stabilization of expression products, proteins or peptides is important. The present invention synthesizes a gene of a peptide having a blood pressure increase inhibitory activity derived from a casein protein of human, and also isolates the ubiquitin gene of tomato to increase its expression and stabilize the expression product in plants and Development of a functional tomato that produces a peptide for inhibiting blood pressure increase by preparing a plant transformation carrier that combines and recombines an expression enhancing regulatory site and introduces it into a tomato. This functional tomato could contribute to the development of domestic seedling industry and increase of farm income

Description

Transformed Tomato Using BP Inhibition Peptide Gene

The present invention relates to a transgenic tomato that produces an antihypertensive peptide.

More specifically, 100 amino acids derived from casein (β-casein), a human breast milk protein, synthesized in vitro and hypertension rats, have been shown to have a blood pressure increase inhibitory peptide gene that has been shown to have an effect of inhibiting blood pressure elevation, and is produced in tomato. In order to transform the tomato by producing a carrier coupled to the ubiquitin gene that acts as a signal for degradation or stabilization of the tomato gene product (protein or peptide).

Recent developments in molecular biology have enabled genetic recombination technology to isolate not only agriculturally useful traits but also genes that can give various functionalities and transform them into plants to improve genetic transformation of crops and develop new crops with new functions. Research is active. In addition, studies are being actively conducted to utilize plants as production plants, such as hepatitis vaccines and immuno-active substances, that is, plant factories. Prior art related to the present invention is the study of the blood pressure increase inhibitory peptides from food protein of Yasuo Ariyoshi (J, Food Science & Technology 4: 139-144, 1993). However, when a foreign gene is introduced into a plant, it may not be expressed well even when the foreign gene is inserted into the chromosome of the plant, or it may be rapidly decomposed even when a gene product is produced. In addition, studies to stabilize gene products are inevitable.

In the present invention, after designing and artificially synthesizing the gene of the peptide having an activity of inhibiting blood pressure as a foreign functional gene, and separating the ubiquitin gene involved in the protein expression enhancing regulatory region and tomato protein degradation or stabilization for efficient expression To produce a tomato transformant carrier conjugated with a blood pressure raising inhibitory peptide and transforming the tomato into a tomato to produce a blood pressure raising inhibitory peptide, and also a tomato having a 2-3-fold increase in production by binding an expression enhancing regulatory site and ubiquitin gene. Developed.

1 is a schematic diagram of the base sequence (A) of the synthesized blood pressure increase inhibitory peptide gene and three kinds of carriers for tomato transformation (B).

2 is a tomato transformation process of the blood pressure increase inhibitory peptide gene.

Figure 3 is a photograph showing that the transgenic tomato produces a blood pressure raising inhibitory peptide using the reaction with the blood pressure raising inhibitory peptide antibody.

Hereinafter, the present invention will be described in detail.

In the present invention, the functional peptide introduced to tomato, an anti-blood pressure raising peptide (angiotensin-converting enzyme inhibitory peptide: hereinafter referred to as "ACEI"), is the 10 amino acids corresponding to the 43-52th casein protein (β-casein) of human The sequence was SFQPQPLIYP, and the gene encoding this amino acid sequence was designed as follows.

5'-TCTTTCCAGCCACAGCCACTCATCTACCCA-3 '

S F Q P Q P L I Y P

On the other hand, the ubiquitin gene was separated by a polymerase chain reaction (PCR) with reference to the known nucleotide sequence of tomato ubiquitin gene. At this time, a primer was designed and used to directly combine the ubiquitin gene with the ACEl gene. (FIG. 1 B) The ubiquitin-binding ACEI peptide gene thus obtained was prepared in a plant transformation carrier in the same manner as in Example, and introduced into tomatoes to develop ACE1 transformed tomatoes.

The following examples are for the detailed description of the present invention and show the production of transformant carriers of the ubiquitin-binding blood pressure increase inhibitory peptide gene, tomato transformation method, and ACEl peptide generation.

Example 1 Preparation of Carrier of Blood Pressure Inhibition Peptide Gene

The ACEI gene was designed with 30 bases as described above (FIG. 1A), and for the recombination of the gene, TCTAGA, a restriction enzyme XbaI recognition sequence and ATG, a start codon at the 5 'end, and a stop codon TAA, at the 3' end. The two strands of DNA were artificially synthesized by addition of GAGCTC, a restriction enzyme SacI recognition sequence, using a DNA synthesizer. Artificially synthesized two-stranded ACEI DNA was subjected to annealing reaction and then treated with restriction enzymes XbaI and SacI to insert the ACEI gene in the site of XbaI and SacI, which removed the GUS gene of the plant vector for transgenic plant pBI121. PACE1 which was modeled in FIG. 2 was produced. ACEI Gene Transformation In order to more stably present the ACEI peptide expressed in tomato, the ubiquitin gene known to regulate protein turnover in cells was isolated and combined with the ACEI gene. Ubiquitin gene was isolated by amplification by PCR from DNA extracted from tomato. 5'-TCTAGATGCAGATCTTCGTGAAAACCCTAA-3 '(30 nt) with the XbaI recognition sequence added to the 5' end of the ubiquitin gene sequence as a forward primer for PCR amplification of the ubiquitin gene, and the reverse primer is C of the ubiquitin The 5'-GAGCTCTTATGGGTAGATGAGTGGCTGTGGCTGGAAAGAACCACCACGGAGACGGAGCACGAGATGGAG-3 '(69 nt) was designed to allow the serine residues of the N-terminus of the ACEI peptide to be directly coupled and the SacI recognition sequence was added to the ACEI C-terminus. PCR reaction was carried out using a reaction solution containing 10 mM Tris-HCI (pH8.3), 50 mM dNTP, 2.5 mM MgCl ₂ , tomato DNA l00ng, and 100 pmol of each primer and 2.5 units of Taq DNA polymerase (Takara). A total of 30 cycles were performed by denaturation for 1 minute, annealing at 58 ° C for 1 minute, and polymerization at 72 ° C for 2 minutes. PCR products were treated with restriction enzymes XbaI and SacI and pUBACE1 was prepared in the same manner as pACE1.

(Figure 1.).

To further enhance the expression of the ACEI gene, the dual 35S promoter and TEV leader were separated from the pRTL2 vector (D. Texas A & M, Dr. James Carrington) to replace pUBACE1's 35S promoter to produce pUBACE2 (FIG. 1).

Example 2. (Tomato transformation process)

Tomato transformation of the ACEI gene was performed as shown in FIG. 2 by the leaf-disc method. In other words, each of the three carriers was introduced into Agrobactrium tumefaciens LBA 4404. After germination of tomato (breed, sunshine), cotyledons are cut at both ends, and then infected with Agrobacterium tumefaciens LBA4404 with each ACEI gene carrier for 2 days of MS (Murashige and Skoog) base Co-culture to the medium. The Agrobactrium tumefaciens was then washed with sterile distilled water and re-differentiated medium (MS base medium, 3% sucrose, 0.4% gellite, 4mg / L kinetin, 0.5 Mg / L IAA, 75 mg / L kanamycin, 500 mg / L carbenicillin, pH 5.6) induced a shoot of the transformed subject. Induced shoots were induced in root-induced medium (MS base medium, 3% sucrose, 0.4% gelite, 5 mg / L IAA, 50 mg / L kanamycin, 500 mg / L carbenicillin, pH 5.6). The roots were induced and then transferred to the greenhouse for cultivation.

Example 3. (Identified fish ACEI peptide in transgenic tomato)

Figure 3 is a photograph confirming the production of ACEI peptides by immunoblot (immunoblot) analysis using the ACEI peptide antibody in crude protein extracted from the young leaves of the transgenic tomato plant.

Crude protein extraction buffer in young leaves (about 200mg) of transgenic tomato [PBST buffer: 8g / L Sodium chloride, 1.15g / L Sodium phosphate, (dibasic), 0.2g / L Potassium phosphate (monobasic), 0.2g / L Potassium chloride, 0.5g / L Tween-20, pH7.4] Add 200ul and crush it using a tissue grinder (tissue glinder (wheaton)), and then centrifuge (15000 rpm, 20 minutes) and supernatant Bradford. After quantitating proteins by the method (Anal. Biochem., 72: 248, 1976), 50 ug was used for immunoblot analysis.

Immunoblot analysis was carried out by attaching crude protein extract (50 ug) to PVDF membrane (0.2um, Bio-Rad) using a slot-blot (millipore), followed by blocking solution ( After shaking for 1 hour in TBS: 50mM Tris, 750mM NaCl, pH7.5, 5% skim milk), ACE antibody (manufactured by Takara) was added at a ratio of 1: 500 and reacted overnight at 4 ° C. Next, the solution was shaken three times for 10 minutes with a TBS solution containing 0.1% Triton X-100, and then washed with an IgG HRP rabbit antibody fused with alkaline phosphatase in a ratio of 1: 1000. After shaking for 2 hours, the solution was shaken three times for 10 minutes with a TBX solution containing 0.1% Triton X-100, and then developed using a Chemiluminescent kit (Pierce). It was developed by exposure to X-ray film for 8 seconds. It was confirmed that ACEI peptide was produced in tomato transformed with three carriers of ACEI gene, and ubiquitin was bound as compared to pACEI transformant without ubiquitin binding. At the same time, the production of ACEI peptide was increased 2-3 times in pUBACE2 transgenic tomato with dual 35S promoter and TEV leader.

Based on the above results, functional tomato producing a peptide for inhibiting blood pressure increase has been developed, and the production of such a functional peptide may be increased by ubiquitin binding, thus contributing to the development of functional new crops.

The present invention provides a gene carrier that synthesizes and binds a gene of angiotensin-converting enzyme inhibitor (ACEI) having a blood pressure increase inhibitory activity to a peptide derived from human casein (β-casein) protein in tomato ubiquitin gene. Later, the tomato was transformed using Agrobacterium tumefaciens LBA 4404 to develop a tomato that produces a blood pressure-inhibiting peptide. Using the reaction with the ACEI antibody, it was confirmed that this tomato produced a blood pressure increase inhibitory peptide, an foreign functional gene. Therefore, this tomato can contribute to the development of domestic tomato seedling industry and increase the income of farmers.

Claims (6)

In the expression of the foreign functional genes described below in plants, the structural genes of the blood pressure raising inhibitory peptide combining the blood pressure raising inhibitory gene and the ubiquitin gene Recombinant carrier for transformation comprising a structural gene combining a blood pressure raising inhibitory gene and a ubiquitin gene The method of claim 2, Recombinant carrier characterized in that the pACE1, pUBACE1 or pUBACE2 Tomato transformed by a recombinant carrier containing a structural gene combining a blood pressure raising inhibitory gene and a ubiquitin gene Plant transformed by a recombinant carrier comprising a structural gene combining a blood pressure raising inhibitory gene and a ubiquitin gene The method of claim 5, Plants are transgenic plants characterized in that they are protoplasts, plant cells, immature plants or seeds at the stage of development.