KR100679715B1 - 2b THE METHOD THAT IMPROVE EXPRESSION OF THE GENE USING 2b GENE FROM CUCUMBER MOSAIC VIRUS - Google Patents

Abstract

The present invention relates to a method for enhancing the expression of a foreign gene in a plant using a Korean-derived O2 mosaic virus-derived 2b gene, and more particularly, by expressing a gene consisting of the nucleotide sequence of SEQ ID NO: 1 in a plant. Expression of a foreign gene in a plant using a Korean cucumber o mosaic virus-derived 2b gene which increases expression of other useful genes simultaneously introduced into the plant to increase expression, expression duration and stability of the protein by expression of the useful gene. It is about how to increase.

Cucumber mosaic virus 2b gene, increased expression

Description

The method for enhancing the expression of foreign genes in plants using the Korean cucumber mosaic virus-derived 2b gene {THE METHOD THAT IMPROVE EXPRESSION OF THE GENE USING 2b GENE FROM CUCUMBER MOSAIC VIRUS}

1 shows the nucleotide sequence of the Korean cucumber mosaic virus 2b gene infected with paprika of the present invention cloned by RT-PCR.

2 is a result of comparing amino acid sequence homology between Korean CMV 2b protein and other CMV 2b proteins.

3 is a phylogenetic tree of Korean CMV and other CMVs.

Figure 4 is a vector map for plant transformation that expresses the KG-CMV 2b gene.

Figure 5 shows the effect of increasing GFP expression by the expression of 2b gene in tobacco leaf tissue.

Figure 6 shows the results of analyzing the mRNA expression of GFP and 2b gene in tobacco leaf tissue.

The present invention is a novel 2b gene known to inhibit gene silencing in the process of searching for a gene required to increase and stabilize the expression level and expression period of a gene product protein by enhancing expression of a useful gene introduced into a plant. The present invention relates to a method for enhancing the expression of foreign genes in plants using a Korean cucumber omega-virus-derived 2b gene, which was developed to confirm its function and to be used for molecular farming, a key field of biotechnology.

In most eukaryotes, the homology-dependent RNA destruction system is called post-transcriptional gene silencing (PTGS), RNA interference in animals and quelling in fungi, but the same principle exists. Some plant viruses are known to encode proteins that block gene silencing. The helper component-proteinase (HC-Pro) protein of the poty virus prevents the maintenance of gene silencing and the 2b protein of the cucumber mosaic virus It prevents PTGS from occurring on the leaves. In addition, the p25 protein of potato virus X is known to block gene silencing from spreading throughout the plant (The Plant Journal (2002), 29 (5), 555-567).

Biotechnology is still rapidly developing and expanding in scope. In particular, research into the technical field of producing transgenic plants or transgenic animals by introducing foreign genes to exhibit useful properties to humans in plants or animals is active. Among them, genetic engineering of plants for animals More practical use As such, in order to obtain the effect of introducing a foreign gene in the development of new functional GM crops by introducing the foreign gene, the expression level of the introduced gene should be increased more than a certain level, and the expression of the introduced gene should be maintained stably. . However, such problems as gene silencing have arisen, and thus, a problem frequently arises that it is difficult to stably increase stable expression of useful transgenes.

In order to solve the above problems, the present invention develops a novel function of the cucumber mosaic virus-derived 2b gene that can innovatively increase the expression level and expression period of other useful genes introduced into plants and stabilize the expression proteins. Its purpose is to provide applicability to biotechnology by identifying its applicability.

The terms, techniques, and the like described in this specification are used in the meanings generally used in the technical field to which the present invention belongs unless there is a specific limitation.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, when expressing the Korean cucumber mosaic virus 2b gene in a transgenic plant to which foreign genes are introduced, not only suppresses gene silencing but also innovatively increases the expression amount and duration of the introduced gene. The problem of instability has been solved and the present invention has been found to be able to stably produce useful genes in large quantities.

According to one aspect of the present invention, there is provided a Korean cucumber mosaic virus 2b gene and its base sequence which can stably increase the expression of useful genes.

The gene of the present invention is characterized in that it comprises an open reading frame (ORF) having 110 amino acids as the nucleotide sequence described in SEQ ID NO: 1 of the sequence list having a base sequence of 333 bp. The gene of the present invention was named "2b-paprika". The homology with the 2b gene of CMV previously reported as an amino acid translated by the "2b-paprika" gene was compared as shown in FIG. Korean CMV used in the present invention was found to be systematically close to Fny, Y, O, Kor, MG8, B, and Legume.

According to another aspect of the present invention, a method of expressing the 2b gene in a plant transformation vehicle is provided. The plant transformation carrier is not particularly limited as long as it can be expressed in the plant 2b gene of the present invention and its type and method. Preferably, when the 2b gene of the present invention is inserted between the 35S promoter and the terminator, the 2b gene may be expressed in a plant to increase expression of a useful gene. As an example, in the present invention, as shown in FIG. 4, a "p2b-0229" vector having a 2S gene of the present invention inserted into a 35S promoter and a nos terminator was prepared.

According to another aspect of the present invention, a technique for transforming a plant transformation vector comprising the 2b gene into Agrobacterium and inserting the 2b gene into a plant's nuclear DNA is provided. Agrobacterium tumefaciens AGL1 was transformed with "p2b-0229". This Agrobacterium transformant was deposited with the accession number KACC 95033P on March 10, 2005, at the Korea Agricultural and Biotechnology Conservation Center, RDA, RDA.

Agrobacterium transformant production method of the present invention is characterized by comprising the following steps:

1) preparing a plant transformation vector comprising a 2b gene sequence operably linked to a DNA sequence exhibiting promoter activity and comprising SEQ ID NO: 1;

2) introducing the expression vector into Agrobacterium .

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

EXAMPLE

Stable mass production of the protein by the 2b gene of the present invention was confirmed as follows.

[Step 1: Cloning of KG-CMV 2b Gene]

Viral RNA was isolated from Korean CMV diseased leaves isolated from paprika. Reverse transcription-polymerase chain reaction (RT-PCR) using 5'-GGATCC GGG TTG AGC GTG TAA ATT CC-3 'and 5'-GAGCTC CAA TAC TGC CAA CTC AGC TCC-3' Was performed to synthesize genes. Reverse transcription was performed at 45 ° C. for 30 minutes, 94 ° C. for 15 minutes, and PCR was repeated 30 times at 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 1 minute. Thus synthesized gene was confirmed by using a sequencer (ABI 3100) 2b base and amino acid sequence of CMV, the sequence is shown in FIG.

[Step 2: Insert into plant transformation vector]

The KG-CMV 2b gene cloned in step 1 was inserted between the BamH I and Sac I sites of pBI221 to attach a 35S promoter and a nos terminator, and then the P35S-2b-TNOS cassette was transferred to the HindIII and EcoR I sites of pGreen-0229. P2b-0229 of FIG.

[Step 3: Preparation of Agrobacterium Transformant]

In order to transform the vector p2b-0229 into the host Agrobacterium tumefaciens (strain AGL1), the vector was inserted into Agrobacterium by electroporation and incubated in YEP plate medium for 2 days to transform Agrobacte Leeum colonies were identified.

[Step 4: 2b Gene Function Assay by Agro-infilteration in Tobacco]

Agrobacterium transformant AGL-1 (hereinafter referred to as 'p2b-0229 AGL-1') containing p2b-0229 was used as kanamycin 50ugmL- ¹ , ampicillin 50ugmL- ¹ , tetra After culturing LB broth containing 5ug · mL ⁻¹ of tracycline in a shaking incubator at 29 ° C., the culture solution was centrifuged (5000 g ㅧ 5 min) to precipitate Agrobacterium, and then pellets. Resuspended in 10 mM MgCl ₂ , 0.1 mM acetosyringone solution to adjust the concentration (OD600 = 0.2), and left on ice for 2 hours to prepare Agrobacterium. Agro-infiltration was performed by injecting Agrobacterium through pores using a 1 mL syringe (syringe) on the back of tobacco leaves three weeks after planting.

Experimental Example 1: Function Test of KG-CMV 2b Gene

The concentration of p2b-0229 AGL-1 prepared in steps 1 to 3 was prepared as 0.05, 0.1, 0.2, 0.4, and 0.8 in OD600.

In addition, pGFP-0229, a fluorescence protein gene (GFP, Green Fluorescence Protein) carrier, was transformed into the same Agrobacterium (strain AGL-1) and used as a marker gene for the functional test of the 2b gene.

Fix the concentration of pGFP-0229 AGL-1 to 0.2 in OD600 and infiltrate the leaf tissue of N. benthamiana with p2b-0229 AGL-1 to irradiate UV at 3, 7, 12 and 17 dpi It was confirmed and shown in FIG.

As can be seen in Figure 5, when the 2b gene according to the present invention was introduced together, GFP protein was expressed at all concentrations. In addition, when only GFP was introduced, expression decreased sharply at 7 days, but when 2b gene was introduced together, the expression period of GFP protein was extended up to 17 days, indicating a stable expression of genes. there was.

Experimental Example 2: Confirmation of Increased Expression of GFP mRNA

In the same manner as in Experimental Example 1, pGFP-0229 AGL-1 having a concentration of 0.2 and p2b-0229 AGL-1 having a concentration of 0.2 were prepared in OD600.

Example 1 (GFP + 2b / 3dpi)

Three weeks after sowing, the incubation of pGFP-0229 AGL-1 through the pores was carried out using a 1 mL syringe on the back of the leaves of the tobacco, after 4 days of infiltration of pGFP-0229 AGL-1 and p2b-0229 AGL-1. After the next three days, each day was observed for 4 days while irradiating UV, and the result is shown in FIG.

Example 2 (GFP + 2b / 10dpi)

Three weeks after sowing, the incubation of pGFP-0229 AGL-1 through the pores was carried out using a 1 mL syringe on the back of the leaves of the tobacco, after 4 days of infiltration of pGFP-0229 AGL-1 and p2b-0229 AGL-1. After the next 10 days had elapsed, each day was observed for 4 days while irradiating UV, and the results are shown in FIG.

Comparative Example 1 (Control)

The leaves of tobacco 25 days after sowing were observed every day for 4 days with UV irradiation, and the results are shown in FIG. 6.

Comparative Example 2 (GFP / 3dpi)

Three days after sowing, infiltrating pGFP-0229 AGL-1 through the pore using a 1 mL syringe on the back of the leaves of tobacco, three days after infiltration of pGFP-0229 AGL-1, , Was observed every day for 4 days while irradiating UV, the results are shown in FIG.

Comparative Example 3 (GFP / 10 dpi)

Three days after planting, 4 days after infiltration of pGFP-0229 AGL-1 through the pores using a 1 mL syringe on the back of the leaves of tobacco, 10 days after infiltration of pGFP-0229 AGL-1 , Was observed every day for 4 days while irradiating UV, the results are shown in FIG.

As shown in FIG. 6, in the case of Comparative Examples 2 and 3 in which only GFP was introduced, GFP mRNA, which was inherently expressed in tobacco transformants, was affected by the gene silencing, but when 2b gene was introduced, GFP mRNA was stable. It was expressed as and showed that the function does not occur gene silencing.

The present invention provides genes that can be usefully used in molecular farming to produce useful proteins in plants using pharmaceuticals, diagnostic reagents, functional substances, etc. in a simple and low cost by utilizing the 2b gene. Plant transformation vector production, Agrobacterium transformation and functional identification technology that can utilize the gene is provided.

Attach sequence list electronic file  

Claims (6)

delete delete delete delete A method for enhancing expression of a foreign gene in a plant by introducing a Korean-derived cucumber mosaic virus-derived 2b gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1 into a plant together with a foreign gene to be expressed in the plant. 6. The method of claim 5, wherein the 2b gene derived from the Korean cucumber mosaic virus is introduced into the plant in the form of an Agrobacterium transformant transformed with the gene. .

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