Improved recombinant vectors based on cucumber mosaic virus

Abstract

One aspect relates to an improved recombinant vector based on the cucumber mosaic virus. The recombinant vector according to one embodiment can significantly increase the efficiency of foreign protein expression in plants, and has a significantly higher efficiency of foreign protein expression than existing recombinant vectors, and can therefore be widely used in the production and research of useful proteins in various plant species.

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2022-0150990, filed on November 11, 2022, and Korean Patent Application No. 10-2023-0038110, filed on March 23, 2023, the entire specifications of which are incorporated herein by reference.

[0002] The present invention relates to a cucumber mosaic virus (CMV)-based recombinant vector. [Background technology]

[0003] When plant viruses infect a host plant, they cause systemic infection within a few days and use host factors to replicate their own genes. During this replication process, the virus expresses large amounts of its own proteins within the plant cells. Furthermore, because the virus genome is small, its genome structure and gene expression mechanisms can be regulated through molecular biological manipulation.

[0004] Taking advantage of these characteristics of plant viruses, active research is being conducted to develop them into gene transfer vectors by manipulating the viral genome structure to enable the insertion and expression of foreign genes. However, there is a continuing need for the development of more efficient vectors.

[0005] Against this background, the inventors conducted research efforts to develop a vector suitable for significantly increasing the expression level of a target protein, and as a result, they confirmed that a recombinant vector based on cucumber mosaic virus can significantly enhance the expression of a target protein, thereby completing the invention. Summary of the Invention [Problem to be solved by the invention]

[0006] One aspect provides a cucumber mosaic virus (CMV) recombinant vector comprising a nucleotide sequence according to SEQ ID NO:1.

[0007] Another aspect provides a transformed microorganism transformed with the recombinant vector.

[0008] Yet another aspect provides a method for producing a protein, comprising the steps of constructing a cucumber mosaic virus recombinant vector containing nucleotides according to SEQ ID NO: 1, transforming the recombinant vector into a fungus, and inoculating the transformed fungus into a plant.

[0009] However, the problems to be solved by the present invention are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0010] One embodiment provides a cucumber mosaic virus (CMV) recombinant vector comprising a nucleotide sequence according to SEQ ID NO:1.

[0011] The cucumber mosaic virus recombinant vector may further comprise a nucleotide sequence according to SEQ ID NO: 2, or may further comprise a nucleotide sequence according to SEQ ID NO: 3. Without being limited thereto, the cucumber mosaic virus recombinant vector may also comprise a nucleotide sequence according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0012] Cucumber mosaic virus (CMV) is the virus most frequently found in chili pepper cultivation in Japan, causing serious damage. It is a positive-sense single-stranded RNA virus with a wide host range, infecting 885 plant species in 65 genera. It is composed of a three-segment genome. CMV isolates can be classified into subgroups IA, IB, and II based on their nucleotide sequences. Among CMV isolates, the P0 type CMV-Fny (CMV-Fny strain) belongs to subgroup IA, while the P1 type CMV-GTN (CMV-GTN strain) belongs to subgroup IB. In this specification, the CMV-GTN refers to a highly pathogenic P1 type that was isolated purely from the chili pepper variety 'Cheongyang' grown at a chili pepper farm in Goesan County, Chungbuk Province in 2013 (Res. Plant Dis. 21(2): 99-102 (2015)).

[0013] According to one embodiment, the nucleotide sequence of SEQ ID NO: 1 also includes an operably linked multiple cloning site (MCS) that allows for insertion of a foreign gene, and further includes a 2xFLAG tag and a 6xHIS tag following the MCS for detection and extraction of the expressed foreign protein, such that the protein produced by the recombinant vector also includes the 2xFLAG tag and the 6xHIS tag at the C-terminus.

[0014] As used herein, the term "recombinant vector" refers to a vector capable of expressing a foreign gene of interest in a host cell, and containing essential regulatory elements operably linked to enable the expression of the gene insert. Suitable vectors include not only expression regulatory sequences such as a promoter, operator, initiation codon, termination codon, polyadenylation signal, and enhancer, but also a signal sequence or leader sequence for membrane targeting or secretion, and can be prepared in a variety of ways depending on the purpose.

[0015] As used herein, the term "operably linked" means that a gene whose expression is required and its regulatory sequences are joined to each other in a manner that allows gene expression.

[0016] Another aspect provides a transformed microorganism transformed with the recombinant vector.

[0017] In the transformed microorganism, the portions that overlap with the recombinant vector will not be described.

[0018] According to one embodiment, the microorganism is also of the genus Agrobacterium.

[0019] Yet another aspect provides a method for producing a protein, comprising the steps of: constructing a cucumber mosaic virus recombinant vector containing nucleotides according to SEQ ID NO: 1; transforming the recombinant vector into a fungus; and inoculating the transformed fungus into a plant.

[0020] The protein produced by the protein production method also contains a 2xFLAG tag and a 6xHIS tag at the C-terminus.

[0021] In the protein production method, the description of the parts that overlap with those of the recombinant vector and transformed microorganism will be omitted. [Effects of the Invention]

[0022] According to one embodiment, the efficiency of foreign protein expression in plants can be significantly improved through the use of a cucumber mosaic virus-based recombinant vector. The recombinant vector according to this embodiment has significantly higher foreign protein expression efficiency than existing recombinant vectors, and therefore can be widely used in the production and research of useful proteins in various plant species. [Brief explanation of the drawings]

[0023] [Figure 1A] FIG. 1 shows the structures of pCMV-GTN-R1, pCMV-GTN-R2, and pCMV-GTN-R3 according to one embodiment of the improved cucumber mosaic virus-based vector structure. [Figure 1B] FIG. 1 shows the structure of pCMV-R3V according to one embodiment of the improved cucumber mosaic virus-based vector structure. [Figure 1C] FIG. 1 shows the structure of pCMV-R2V-B2 according to one embodiment of the improved cucumber mosaic virus-based vector structure. [Figure 1D] FIG. 1 shows the structure of pCMV-R3V-GFP bound to a fluorescent molecule to confirm the effect of pCMV-R3V according to one embodiment of the present invention in the construction of an improved cucumber mosaic virus-based vector. [Figure 1E] FIG. 1 shows the structures of pCMV-Fny-R1 and pCMV-Fny-R2 according to one embodiment of the improved cucumber mosaic virus-based vector structure. [Figure 1F] FIG. 1 shows the structure of PZP-GFP according to one embodiment of the improved cucumber mosaic virus-based vector structure. [Figure 2A] This figure shows a comparative experiment with PZP-GFP to evaluate the expression efficiency of the CMV-GTN-GFP combination vector (pCMV-GTN-R1+pCMV-GTN-R2+pCMV-R3V-GFP). [Figure 2B] This figure shows a comparative experiment conducted to evaluate the expression efficiency of the CMV-GTN-GFP combination vector (pCMV-GTN-R1+pCMV-GTN-R2+pCMV-R3V-GFP) with the pCMV-Fny-R1+pCMV-Fny-R2+pCMV-R3V-GFP combination vector. [Figure 3A] This is a diagram showing the GFP expression level measured using a fluorescence measurement device to evaluate the expression efficiency of the CMV-GTN-B2-GFP combination vector (pCMV-R1+pCMV-R2V-B2+pCMV-R3V-GFP). [Figure 3B]This figure shows the results of SDS PAGE analysis of proteins extracted from the inoculation site two days after inoculation to evaluate the expression efficiency of the CMV-GTN-B2-GFP combination vector (pCMV-R1+pCMV-R2V-B2+pCMV-R3V-GFP). [Figure 3C] This figure shows GFP band quantitative analysis performed using ImageJ to evaluate the expression efficiency of the CMV-GTN-B2-GFP combination vector (pCMV-R1+pCMV-R2V-B2+pCMV-R3V-GFP). [Figure 3D] This figure shows the GFP expression level per fresh weight to evaluate the expression efficiency of the CMV-GTN-B2-GFP combination vector (pCMV-R1+pCMV-R2V-B2+pCMV-R3V-GFP). DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0025] As used herein, the singular forms "a," "an," and "the" also include the plural forms unless the context clearly dictates otherwise. Also, as used herein, "comprise," "include," and / or "comprising," "including," specify the presence of stated features, numbers, steps, operations, members, elements, and / or groups thereof, but do not exclude the presence or addition of one or more other features, numbers, operations, members, elements, and / or groups.

[0026] Example 1. Construction of improved cucumber mosaic virus-based vectors Hereinafter, a method for producing an improved vector according to an embodiment will be described with reference to Figures 1A to 1F. Figures 1A to 1F are schematic diagrams illustrating a method for producing an improved vector based on cucumber mosaic virus (CMV) according to one embodiment. Specifically, they show a schematic diagram of a cucumber mosaic virus (CMV) infectious cDNA clone, a recombinant vector, and a recombinant clone constructed for overexpression of green fluorescent protein (GFP).

[0027] The pCass-Rz vector was used as a binary vector to construct a cucumber mosaic virus (CMV)-based gene overexpression vector. The pCass-Rz vector contains, in order, the left border of the T-DNA, the CaMV double 35S promoter, multiple cloning sites (MCS) (StuI, KpnI, XbaI, BamHI), a cis-cleaving ribozyme sequence (Rz), the 35S terminator (T), and the right border of the T-DNA. It can be transformed into Agrobacterium and used to transfer genes of interest to plants via agroinfiltration. The pCass-Rz vector also contains a kanamycin resistance gene, allowing for selective cultivation of transformants when transformed into E. coli and Agrobacterium.

[0028] To construct a cucumber mosaic virus (CMV)-based recombinant protein overexpression vector, we used an infectious cDNA clone (see Virus Evolution, Volume 6, Issue 2, July 2020, veaa070) of the previously isolated CMV-GTN (Cucumber mosaic virus isolated from chili pepper) strain. The CMV genome consists of three RNA segments: RNA1, RNA2, and RNA3. RNA1 encodes the 1a gene, which is involved in RNA replication. RNA2 encodes the 2a gene, which encodes the replicase enzyme, and the 2b gene, which encodes the gene silencing suppressor. RNA3 encodes the viral movement protein (MP) and coat protein (CP) genes.

[0029] Genomic DNA corresponding to RNA1, RNA2, and RNA3 of the highly pathogenic CMV-GTN strain isolated from chili peppers in a Korean field was amplified by RT-PCR. The PCR products corresponding to each genomic RNA were cloned using the restriction enzyme sites in the MCS of the pCass-RZ vector. The resulting infectious cDNA clones corresponding to CMV-GTN RNA1, RNA2, and RNA3 were designated pCMV-GTN-R1 (SEQ ID NO: 3), pCMV-GTN-R2 (SEQ ID NO: 4), and pCMV-GTN-R3 (SEQ ID NO: 5), respectively. In addition, pCMV-R3V refers to the sequence of SEQ ID NO: 6, pCMV-R3V-GFP refers to the sequence of SEQ ID NO: 7, pCMV-Fny-R1 refers to the sequence of SEQ ID NO: 8, pCMV-Fny-R2 refers to the sequence of SEQ ID NO: 9, and PZP-GFP refers to the sequence of SEQ ID NO: 10.

[0030] Using an infectious clone of the CMV-GTN strain, we manipulated the genomic sequence of the CP ORF (open reading frame) of RNA3, which is expected to have high expression efficiency for foreign genes, and developed it as a cucumber mosaic virus (CMV)-based vector for overexpressing foreign proteins.

[0031] Specifically, the CP gene was removed from the RNA3 CP ORF, and SpeI and MluI sites were introduced as MCSs to allow for the insertion of foreign genes. A 2xFLAG tag and a 6xHIS tag were inserted after the MCS for detection and extraction of expressed foreign proteins. Therefore, when cloning is performed using the MCS, the expressed protein contains a 2xFLAG tag and a 6xHIS tag at the C-terminus. The CMV-GTN RNA3-based vector clone constructed in this manner was designated pCMV-R3V (SEQ ID NO: 1).

[0032] CMV RNA2 encodes the 2b gene, a gene silencing suppressor. In one example, a recombinant CMV RNA2 infectious clone was constructed in which the 2b gene was replaced with the B2 gene of FHV (flock house virus), which is known to be an even more potent gene silencing suppressor. The clone was named pCMV-R2V-B2 (SEQ ID NO: 2).

[0033] Specifically, the C-terminus of the 2b ORF of CMV RNA2 was removed, and the foot and mouth disease virus (FMDV) self-cleaving peptide (LLNFDLLKLAGDVESNPG / P), an MluI site, and the FHV B2 sequence were introduced into that position. The B2 gene is expressed via translation of the 2b ORF and is cleaved by the FMDV self-cleaving peptide, resulting in the inclusion of an additional proline (P) amino acid at the N-terminus.

[0034] 2. Evaluation of expression efficiency of improved vectors (1) Cloning for evaluation of expression efficiency To evaluate the efficiency of foreign gene expression in plants using the CMV-GTN-based vector constructed in section 1 above, a clone was constructed by inserting the green fluorescent protein (GFP) gene into the SpeI and MluI restriction enzyme sites of pCMV-R3V. To this end, the green fluorescent protein (GFP) gene was amplified by PCR using primers (5'-GGACTAGTATGGTGAGCAAGGGCGAGGAG-3' and 5'-AACGACGCGTGAGGATCCCCTTGTACAGCTC-3'), digested with SpeI and MluI restriction enzymes, and inserted into the SpeI and MluI restriction enzyme sites of the pCMV-R3V vector. The clone with the green fluorescent protein (GFP) inserted was designated pCMV-R3V-GFP (see Figures 1A to 1F). To generate infectious clones related to the highly pathogenic Cucumber Mosaic Virus (CMV)-Fny strain rather than the highly pathogenic CMV strain, we used the pCass-Rz vector and constructed pCMV-Fny-R1 and pCMV-Fny-R2 for CMV-Fny RNA1 and CMV-Fny RNA2, respectively, using the same cloning method as pCMV-GTN-R1 and pCMV-GTN-R2. For use as a control, we inserted green fluorescent protein (GFP) into the PZP vector to create PZP-GFP. The PZP vector contains, in order, the left border of T-DNA, the CaMV double 35S promoter, TE (translation enhance element), MCS (StuI, SpeI), the 35S terminator (T), and the right border of T-DNA. It can be transformed into Agrobacterium and transfer the target gene to plants via agroinfiltration. The PZP vector also contains a spectinomycin resistance gene, allowing for selective cultivation of transformants when transformed into Escherichia coli and Agrobacterium. The green fluorescent protein (GFP) gene was inserted into the StuI and SpeI restriction enzyme sites of the PZP vector to create a PZP-GFP clone.

[0035] Plasmid DNAs of pCMV-GTN-R1, pCMV-GTN-R2, pCMV-R3V-GFP, pCMV-R2V-B2, pCMV-Fny-R1, pCMV-Fny-R2, and PZP-GFP clones having the structures shown in Figures 1A to 1F were each transformed into the Agrobacterium strain EHA105. The transformed Agrobacterium was cultured in 5 ml of YEP liquid medium (containing 100 μg / ml kanamycin and 50 μg / ml rifampicin) at 30°C for 16 hours with shaking. Then, 1 ml of the primary culture was inoculated into 50 ml of fresh YEP medium (containing 100 μg / ml kanamycin, 50 μg / ml rifampicin, and 20 μM acetosyringone) and cultured at 30°C for 6 hours with shaking. The culture was centrifuged at 4,800×G for 10 minutes to precipitate the Agrobacterium, which was then resuspended in MMA infiltration buffer (MS salts, 10 mM MES (pH 5.6), 200 μM acetosyringone) to an OD of 0.7 at 600 nm. Thereafter, the suspension was cultured with shaking at 30°C for 4 hours.

[0036] Agrobacterium suspensions transformed with each clone, either mixed in the same ratio as presented below or singly, were pressure-infiltrated onto the hypocotyl surface of tobacco (N. benthamiana) leaves using a 1 ml syringe.

[0037] 1) CMV-GTN-GFP: Agrobacterium suspensions (OD 0.7) transformed with pCMV-GTN-R1, pCMV-GTN-R2, and pCMV-R3V-GFP were mixed in equal proportions and infiltrated.

[0038] 2) PZP-GFP: Agrobacterium suspension (OD 0.7) transformed with PZP-GFP was infiltrated alone.

[0039] 3) CMV-Fny-GFP: Agrobacterium suspensions (OD 0.7) transformed with pCMV-Fny-R1, pCMV-Fny-R2, and pCMV-R3V-GFP were mixed in equal proportions and infiltrated.

[0040] 4) CMV-GTN-B2-GFP: Agrobacterium suspensions (OD 0.7) transformed with pCMV-GTN-R1, pCMV-R2V-B2, and pCMV-R3V-GFP were mixed in equal proportions and infiltrated.

[0041] (2) Evaluation of GFP expression efficiency 1) Expression efficiency of the CMV-GTN-GFP combination vector After inoculation with each combination of Agrobacterium suspension, the time course of green fluorescent protein (GFP) expression at the inoculation site was observed using a fluorescence measurement device (FOBI system).

[0042] FIG. 2A is a diagram showing a comparative analysis of green fluorescent protein (GFP) expression efficiency between the CMV-GTN-GFP combination (pCMV-GTN-R1+pCMV-GTN-R2+pCMV-R3VGFP) and PZP-GFP.

[0043] Compared with PZP-GFP, which was constructed using a binary vector containing a 35S promoter widely used in plant transformation, the CMV-GTN-GFP combination (pCMV-GTN-R1 + pCMV-GTN-R2 + pCMV-R3V-GFP) expressed significantly higher amounts of green fluorescent protein (GFP) (Fig. 2A). The amount of GFP expressed by the CMV-GTN-GFP combination was highest 2 days postinoculation (dpi) and gradually decreased over time (Fig. 2A).

[0044] Based on the above results, it was confirmed that the vector containing pCMV-R3V represented by SEQ ID NO: 1 can significantly enhance the expression of foreign proteins in plants.

[0045] Furthermore, to confirm whether the highly pathogenic CMV-GTN strain newly constructed in the Examples has a higher foreign protein expression efficiency as a vector than other cucumber mosaic virus (CMV) strains, the CMV-GTN-GFP combination was compared with the CMV-Fny-GFP combination (pCMV-Fny-R1 + pCMV-Fny-R2 + pCMV-R3V-GFP).

[0046] Figure 2B shows a comparative analysis of green fluorescent protein (GFP) expression efficiency between the CMV-GTN-GFP combination and the CMV-Fny-GFP combination (pCMV-Fny-R1 + pCMV-Fny-R2 + pCMVR3V-GFP). After inoculation with each Agrobacterium suspension, the time course of GFP expression at the inoculation site was observed using a fluorescence measurement device (FOBI system).

[0047] As a result, it was confirmed that the CMV-GTN-GFP combination had a much higher green fluorescent protein (GFP) expression efficiency than the CMV-Fny-GFP combination (see Figure 2B). These results indicate that the efficiency of foreign gene overexpression as a vector can vary depending on the cucumber mosaic virus (CMV) strain, and that the CMV-GTN strain used in this example has excellent efficacy as a foreign gene overexpression vector.

[0048] 2) Expression efficiency of the CMV-GTN-B2-GFP combination vector Next, to confirm whether the expression efficiency could be increased by using pCMV-R2V-B2, in which the CMV2b gene was replaced with the FHV B2 gene to express a more potent gene silencing suppressor, we compared the CMV-GTN-GFP combination with the CMV-GTN-B2-GFP combination (pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V-GFP).

[0049] Figure 3A shows the time course of green fluorescent protein (GFP) expression at the inoculation site after inoculation with each combination of Agrobacterium suspension, as observed using a fluorometric device (FOBI system). Figure 3B shows SDS-PAGE analysis of total protein extracted from the inoculation site two days after inoculation. Figure 3C shows the results of a quantitative comparative analysis of the green fluorescent protein (GFP) band in Figure B using ImageJ. Figure 3D shows the results of a quantitative analysis of the green fluorescent protein (GFP) expression level of the recombinant CMVGTN-based vector relative to plant fresh weight.

[0050] Expression efficiency in inoculated tobacco plants was measured using a fluorometer (FOBI) and confirmed that the CMV-GTN-B2-GFP combination had a slightly higher expression efficiency than the CMV-GTN-GFP combination (Figure 3A). To more accurately compare the GFP expression efficiency between the two combinations, total protein was extracted from the inoculation site two days after inoculation and analyzed by SDS-PAGE. The results confirmed that the amount of GFP protein expressed using the CMV-GTN-based vector accounted for a significantly higher percentage of the total extracted protein. Furthermore, the CMV-GTN-B2-GFP combination induced slightly higher GFP expression than the CMV-GTN-GFP combination (Figure 3B). Analysis using ImageJ, a quantitative image analysis program, confirmed that the CMV-GTN-B2-GFP combination had a 30% higher GFP expression efficiency (Figure 3C).

[0051] To analyze the amount of green fluorescent protein (GFP) expressed by the CMV-GTN-based vector relative to plant fresh weight, cytoplasmic proteins were extracted from the inoculation site two days after inoculation and quantitatively analyzed for green fluorescent protein (GFP) using a GFP Quantification Kit (product number ab235672, Abcam, UK). The CMV-GTN-GFP combination showed a GFP expression efficiency of 2,846 ng / mg fresh weight, while the CMV-GTN-B2-GFP combination showed an approximately 20% increase in GFP expression efficiency of 3,444 ng / mg fresh weight (see Figure 3D).

[0052] Therefore, we cloned the green fluorescent protein (GFP) gene into each vector and expressed it in tobacco (Nicotiana benthamiana) plants. The results of a comparative analysis of expression levels showed that the highly pathogenic CMV-GTN-based vector showed significantly higher levels of GFP expression than the CMV-Fny strain, which is commonly used in research. Furthermore, we confirmed that the expression level of GFP increased by approximately 12% when using pCMV-R2V-B2 compared to the wild-type CMV RNA2 infectious clone (pCMV-GTN-R2).

[0053] Based on the above results, it can be seen that the CMV-GTN-B2-GFP combination vector (pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V-GFP) of this example shows significantly improved expression efficiency in the expression of foreign proteins in plants.

[0054] The above description of the embodiments is merely illustrative, and those skilled in the art will understand that various modifications and equivalent embodiments are possible. Therefore, the true scope of protection of the invention is defined by the appended claims, and all differences within the scope equivalent to the contents described in the claims should be construed as being included in the scope of protection defined by the claims.

Claims

1. A cucumber mosaic virus (CMV) recombinant vector comprising a nucleotide sequence according to SEQ ID NO: 1 and a nucleotide sequence according to SEQ ID NO: 2, wherein said nucleotide sequence according to SEQ ID NO: 2 is derived from FHV (flock house virus).

2. 2. The cucumber mosaic virus recombinant vector of claim 1, further comprising a nucleotide sequence according to SEQ ID NO:

3.

3. The cucumber mosaic virus recombinant vector according to claim 1 , wherein the cucumber mosaic virus is a P1 type cucumber mosaic virus.

4. The cucumber mosaic virus recombinant vector according to claim 3, wherein the P1 type cucumber mosaic virus is a CMV-GTN strain.

5. A transformed microorganism transformed with the cucumber mosaic virus recombinant vector according to any one of claims 1 to 4.

6. preparing a cucumber mosaic virus recombinant vector comprising a nucleotide sequence according to SEQ ID NO: 1 and a nucleotide sequence according to SEQ ID NO: 2, wherein the nucleotide sequence according to SEQ ID NO: 2 is derived from FHV (flock house virus); transforming the cucumber mosaic virus recombinant vector into a fungus; and inoculating the transformed fungus into a plant.

7. The method for producing a protein according to claim 6 , wherein the protein produced by the method for producing a protein contains 2×FLAG tags and 6×HIS tags at the C-terminus.

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