KR102319619B1 - A transgenic tobacco expressing recombination HcRNAV 34 virus like particle protein - Google Patents

Abstract

H. circularisqua-ma HU 9433-P and HA 92-1 as hosts of HcRNAV34 by extraction of total proteins from transgenic plants. As a result of treatment with non-host H. circularisquama HY 9423, HU 9436, and C. vulgaris , only HU 9433-P and HA 92-1 hosts stopped the flow of algae, changed the shape, and destroyed the cells. was observed. In addition, when observed with a confocal microscope, it is believed that GFP transformed with HcRNAV34 VLP is expressed in algae, so the morphological change and destruction of algae is thought to occur when HcRNAV34 VLP is introduced into the alga, and HcRNAV34 VLP alone is host-specific. It was confirmed that there is algae function. Therefore, if a virus that specifically infects harmful algae is selected and a part of it is mass-produced through transformation in plants to confirm its algal ability, it is expected that it can be utilized as a new biological control method for red tide.

Description

A transgenic tobacco expressing recombination HcRNAV 34 virus like particle protein

The present invention relates to a transformed tobacco expressing recombinant HcRNAV 34 virus like particle protein.

Red tide is a phenomenon in which floating organisms in seawater rapidly increase, resulting in a lack of oxygen or toxicity. Kim et al ., 2006).

Red tide organisms consume dissolved oxygen in water through respiration and carcass decomposition processes, impair the survival of other organisms, and secrete harmful substances and toxins to kill or poison fish and shellfish, causing paralysis or diarrheal poisoning when ingested. As it threatens even human health, it has become a serious socioeconomic problem (Kim, 1997; Imai et al.) al ., 2006; M, 2012).

The main marine organisms that cause red algae are about 100 species worldwide, including diatoms and flagellates. In Korea, there are about 43 species, 4 freshwater species, 13 marine diatoms, 3 rapidoalgae, and 20 flagellar algae species. have. Especially among flagellates, Gymnodinium mikimotoi , Cochlodinium polykrikoides , Gyrodinium sp. 3 species can directly kill aquatic organisms (Park, 1995; Seo et al.) al ., 2003; Ryu et al., 2004).

In Korea, the enlargement of red tides has been reported due to eutrophication of sea areas due to excessive inflow of industrial city wastewater since 1963. (Park, 1995). Since the mid-1990s, single red tides caused by toxic Cochlodinium have been continuing, and recently, the frequency of red tides has increased in coastal waters where aquaculture is concentrated, and as the causative species diversified, the toxicity is strengthened and it is characterized by long-term localization (Ryu et al., 2004; M, 2012). Due to global warming, several subtropical dinoflagellates, an indicator species of tropical waters, appeared in the waters off Jeju Island, and recently increased to 103 species, of which 32 are unrecorded species reported in Korea for the first time. In particular, due to the increase in benthic species, 2 species with high toxicity were also reported (Kim et al. al ., 2008; Kim, 2010; Kim et al ., 2011; Chung, 2011) reported the appearance of highly toxic dinoflagellates in Geomundo and Baekdo (Baek, 2012a; Baek, 2012b). Heterocapsa , a venomous dinoflagellate circularisquama ( H. circularisquama ) is the main species causing red tides in the waters of Japan recently, causing the greatest damage to bivalves (Horiguchi, 1995; Nagai et al.) al ., 1996; Matsuyama et al. al ., 1997; Yamaguchi et al. al ., 1997, Matsuyama et al. al ., 1999; Uchida et al ., 1999). Unlike other species that kill fish, protein or polypeptide hydrolyzed by trypsin attacks the heart or gills of bivalves and kills them, and the toxin kills oysters and clams, which are mainly crustaceans, causing great damage to aquaculture (Tarutani). et al ., 2001; Nagasaki et al. al ., 2003; Oh et al ., 2003; Tomaru et al ., 2004, Imai et al ., 2006; Shiraishi et al. al ., 2007; Tomaru et al ., 2007; Shiraishi et al. al ., 2008).

There are physical, chemical and biological methods to control red tides. Physical methods use ultrasonic waves to destroy cells of causative organisms or _{neutralize toxicity by injecting high-pressure ozone (O 3} ) into the generated water, but it has not reached the stage of practical use. couldn't The chemical method is _{to control the induced algae by destroying cells by spraying copper sulfate (CuSO 4} ), chlorine dioxide (ClO ₂ ), etc., but it can also affect other marine organisms and cause problems of biotoxicity and corrosion, Due to the effect, repeated use is required, and a high investment cost is required due to the relatively large amount of administration. Biological methods mainly use natural enemy relationships, but ecosystem disturbance may be caused by the proliferation of harmful algae and certain species used as natural enemies (Na et al. al ., 1996; Choi et al ., 1998; Park et al ., 1998; Kim et al ., 1999a; Matsuyama, 1999; Jeong et al ., 2000; Seo et al ., 2003; Ryu et al., 2004; Kim et al ., 2006; M, 2012). In addition, although the loess spraying method is implemented in Korea, it contains an excess of heavy metal ions, which causes poisoning by accumulation and biological concentration of the bottom layer, and has problems such as acidification of seawater and inhibition of the growth of photosynthetic organisms (Choi et al. al ., 1998; Ryu et al., 2004; Imai et al ., 2006; Kim et al ., 2006).

As part of a removal method to minimize damage to red algae without affecting the ecosystem, the algal bacterium Micrococcus sp. LG-1 (Park et al.) al ., 1998), Altermonas sp. SR-14 (Kim et al. al ., 1999a) or (Mitsutani et al.) al ., 1992; Jeong et al ., 2000; Jeoung et al ., 2012), α-mannosidase (Lee et al ., 2000), a substance with algicidal ability, or an antibacterial peptide, Mastoparan B (Seo et al.) al ., 2003) and the like are being studied (Wang et al. al ., 2005; Choi et al ., 2009). In addition, studies on biological control using marine microorganisms or viruses are in progress (Park et al ., 1998; Kim et al. al ., 1999a; Kim et al ., 1999b; Jeong et al ., 2000; Imai et al ., 2006; Jeoung et al ., 2012).

Separated from ganghwal (Notopterygii rhizoma) to the death of the toxic dinoflagellates is H. circularisquama that newly emerged in our country falcarindiol (Tamura et al ., 2010) and the use of Heterocapsa circularisquama RNA virus (HcRNAV, Tamura et al. al ., 2004) have been reported in foreign studies.

HcRNAV34 is a novel single-stranded RNA virus that specifically infects red algae-causing algae, H. circularisquama HU 9433-P and HA 92-1. This virus has an icosahedral structure, 30 nm in diameter, has no outer membrane and tail structure, and contains a single molecule of 4.4 kb single-stranded RNA. Genome consists of a complete single-stranded RNA without a cap structure at the 5' end and a poly (A) tail at the 3' end. HcRNAV has specific infectivity within a species, replicates in the cytoplasm and arranges into crystals, accumulates by targeting the nucleus, and after an incubation period of 33-48 hours, about 7,200-43,000 are released during lysis and kills the host, H. circularisquama. Make it (Tomaru et al.) al ., 2004; Nagasaki et al. al ., 2005; Mizumoto et al. al ., 2007; Tomaru et al ., 2007; Nagasaki, 2008; Wu et al ., 2012; Nakayama et al ., 2013).

Recently, virus-like particles (VLPs) related to specific infectivity of viruses are being studied in various fields, and can be produced in various cell culture systems including animal cells, yeast, and plant cells (Santi et al. al ., 2006). When using recombinant proteins made from animal cells and microorganisms as antigens, Escherichia In bacteria including coli ( E. coli ), post-translational modification does not occur, so native protein cannot be obtained. When animal cells are used, antigens more similar to those of native protein can be obtained, but the disadvantage is that the cost is high. Therefore, recently, a method for producing antigens by introducing a viral gene into an edible plant that is easy to transform and is inexpensive has been tried (Jeon, 1994; Ji, 2008).

In the past few years, the need for the development of transgenic plants through plant genetic engineering has increased for the development of virus vaccines for food or oral administration. Plants can express foreign proteins through expression, conjugation, assembly, glycosylation, etc. so that they can produce appropriate antigenic proteins through genes encoding antigenic proteins of various pathogenic viruses and bacteria (Wycoff, 2005). Due to the advantages of relatively inexpensive growth in fields or greenhouses, the ease of large-scale protein production expansion and the absence of risk of contamination by human pathogens, the use of plants as a system for expression of exogenous proteins is a traditional method for vaccine production. It has been proposed as an inexpensive alternative to the system (Walmsley et al ., 2000; Ma et al ., 2003; Howard, 2005). WHO approval for plant-based vaccine production demonstrates that the use of transgenic plants is developing as a promising tool for controlling human and livestock diseases (Lau et al. al ., 2009).

Plant-based vaccines have advantages over microbial vaccine production in that post-translational modifications that occur in eukaryotes are possible because they use plants, which are higher organisms, and can solve the problem of reduced immunity due to differences between organisms (Kong et al. al ., 2001). Plant-based vaccines and biopharmaceuticals have the advantage of no costs associated with fermentation, purification, or cold storage and sterile transport (Mitchell et al. al ., 1993). In addition, compared to other recombinant vaccines, it is expressed in plant tissues, so it is possible to reduce the possibility of damage to antigen proteins due to conditions such as pH of the digestive organs, and mass production is possible and production is possible in a certain environment (Daniell et al. al ., 2001). Through the mass production system of traditional agriculture, a large amount of antigen can be produced inexpensively, and it has the advantage that it can be applied to developing countries without additional cost investment. Currently, vegetables such as tomatoes and potatoes are used for vaccine production, and tomatoes (Lou et al. al ., 2007; Paz et al ., 2009) or carrots (Kim et al.) al ., 2011) can minimize damage to the expressed protein in that it can be consumed without a cooking process, and many studies are being conducted because potatoes are easy to ingest (Kong et al. al ., 2001; Thanvala et al. al ., 2005). Plants such as lettuce and spinach, which consume leaves, are also being studied for the development of recombinant vaccines. For this reason, research is ongoing (Yoshikazu et al ., 2008; Matsumoto et al. al ., 2009). However, the low antigen expression level in the transgenic plant is difficult to induce immunity, so the value of the vaccine is reduced (Modelska et al ., 1998), the intake requirement is increased, and the development and production period is long compared to other systems. There are disadvantages. As an improvement, it is possible to increase the yield and expression amount and shorten the cultivation period by methods such as accumulation of proteins in plants or secretion induction system through roots and leaves, use of chloroplast and virus transformation systems, and mass production through cell and tissue culture. Studies are underway to induce it (Fischer et al. al ., 2004; Edward, 2010; Kim, 2012; Lee, 2013).

Tobacco ( Nicotiana) tabacum ) can be used as a promising expression system for recombinant vaccine production because it has a high biomass yield and high water-soluble protein level compared to other crop species (GB Sunil Kimar). et al ., 2006; Kumar et al ., 2007). In addition to economic advantages, it also has the ability to produce a wide range of therapeutic proteins, including antibodies, vaccines, and the like. For this reason, so far, vaccines for foot-and-mouth disease, cholera, severe acute respiratory syndrome (SARS) virus, etc. have been developed using tobacco (Kim, 2012), but studies related to marine viruses for the control of red algae have been reported so far. none.

Republic of Korea Patent Publication No. 10-2013-0054478

Accordingly, the present inventors made it possible to mass-produce HcRNAV 34 VLPs that were specifically infected with H. circularisquama , a red algae-inducing alga, through transformation of tobacco.

Accordingly, it is an object of the present invention to provide a transformed tobacco expressing recombinant HcRNAV 34 virus like particle (VLP) protein.

In order to achieve the above object, the present invention provides a transformed tobacco expressing recombinant HcRNAV 34 virus like particle (VLP) protein.

A large amount of recombinant HcRNAV 34 virus like particle (VLP) protein can be obtained using the transformed tobacco of the present invention, which can be usefully used for controlling red tides.

1 shows a schematic diagram of a recombinant plant expression vector, pCAMBIA1304 binary vector (A), and pCAMBIA1304 binary vector (B, C) recombined with HcRNAV 34 VLP gene.
Figure 2 shows the gene base sequence of HcRNAV 34 VLP.
Figure 3 shows the results of electrophoresis by extracting the plasmid DNA transformed with the HcRNAV 34 VLP gene.
Figure 4 is the result of electrophoresis through direct PCR (A) and restriction endonuclease digestion (B) after extracting the plasmid DNA transformed with the HcRNAV 34 VLP gene.
5 is a photograph showing the growth of transformed tobacco.
6 shows the results of confirming whether the transgenic tobacco gene is introduced.
7 shows the results of RT-PCR by extracting total RNA in order to confirm the expression of the transcriptome of the transformed tobacco.
8 is a photograph showing the result of irradiating UV light to the transformed tobacco.
9 shows the results of western blot for confirming the expression of HcRNAV 34 VLP in the transformed tobacco.
10 shows the results of observing the host-specific algicidal activity of HcRNAV 34 VLP using an optical microscope.
11 is a photograph of observing the algicidal activity (H. circularisquama HU 9433-P) of the total protein containing HcRNAV 34 VLP.
12 is a photograph of observing the algicidal activity (H. circularisquama HA 92-1) of total protein containing HcRNAV 34 VLP.
Figure 13 is a photograph of observing the apoptosis (H. circularisquama HY 9423) of the total protein containing HcRNAV 34 VLP.
14 is a photograph of observing the algae activity (H. circularisquama HU 9436) of the total protein containing HcRNAV 34 VLP.
Figure 15 is a photograph of observing the apoptosis (C. vulgaris ) of the total protein containing HcRNAV 34 VLP.
Figure 16 is a photograph of observing the algal ability (H. circularisquama HU 9433-P) of the total protein containing HcRNAV 34 VLP by green fluorescence expression of GFP.
17 is a photograph of the degree of apoptosis observed using trypan blue staining after treatment with total protein extracted from tobacco transformed into H. circularisquama HU 9433-P.

material preparation

1. plant material

Tobacco (Nicotiana) for plant transformation tabacum cv. Havana) was used. For germination, the seeds are prepared by first washing the surface with 70% ethanol for 30 seconds, then washing 3 times with sterile water, sterilizing the seeds with 4% NaOCl for 30 minutes, and then washing again with sterile water 3 times. did. The sterilized tobacco seeds were sown in MS medium (Table 1, Murashige & Skoog, 1962) and aseptically germinated for 3-4 weeks at 25±1° C., light conditions for 16 hours, and dark conditions for 8 hours. After seed germination, healthy tobacco leaves with 6 or more true leaves were used for the experiment.

MS medium component Amount to add per 1 liter Sucrose 30 g Mes-monohydrate 0.5 g Vitamin 4.4 g pH 5.7

2. Birds

seawater species Heterocapsa circularisquama HU 9433-P, HA 92-1, HY 9423, HU 9436 and freshwater species Chlorella vulgaris was purchased and used by the Center for Convergence Research on Noxious Algae. Under light conditions of 12 hours and dark conditions of 12 hours, H. circularisquama was f/2 media (Tables 2 to 4, Guillard & Ryther, 1962) and C. vulgaris was BBM (Bold's Basal Medium, Tables 5 and 6). proliferated.

f/2 medium component Stock solution (200 ml) final concentration Amount to add per 1 liter NaNo ₃ 15 g 0.883 nM 1 ml NaH ₂ PO _{4 ,} 2H ₂ O 1.13 g 0.036 mM 1 ml Trace Elements Solution Table 3 1 ml Vitamin mix
Solution Table 4 0.1 ml sea water - - up to 1 L pH 8.0

Trace Elements Solution component Stock solution (200 ml) final concentration Na _{2 ,} EDTA 0.832 g 0.012 mM FeCl _{3 ,} 6H ₂ O 0.63 g 0.017 mM CuSO _{4 ,} 5H ₂ O 0.002 g 0.04 uM ZnSO _{4 ,} 7H ₂ O 0.004 g 0.076 uM CoCl _{2 ,} 6H ₂ O 0.002 g 0.042 uM MnCl _{2 ,} 4H ₂ O 0.036 g 0.91 uM Na ₂ MoO _4, 2H ₂ O 0.0012 g 0.025 uM

Vitamin Mix Solution component Stock solution (200 ml ) Cyanocobalamin (Vitamin B12) 0.001 g Thiamine HCl (Vitamine B1) 0.2 g Biotin 0.001 g

Bold's Basal medium component Stock solution (500 ml) Amount to add per 1 liter KH ₂ PO ₄ 8.75 g 10 ml CaCl _{2 ,} 2H ₂ O 1.25 g 10 ml MgSO _{4 ,} 7H ₂ O 3.75 g 10 ml NaNO ₃ 12.5 g 10 ml K ₂ HPO ₄ 3.75 g 10 ml NaCl 1.25 g 10 ml Na ₂ EDTA,2H ₂ O
KOH 5 g
3.1 g 1 ml FeSO _{4 ,} 7H ₂ O
H ₂ SO ₄ (concentrated) 2.49 g
0.5 ml 1 ml Trace Metal Solution Table 6 1 ml H ₃ BO ₃ 5.75 g 0.7 ml pH 6.8

Trace Metal Solution component Stock solution (1 L) H ₃ BO ₃ 2.86 g MnCl _{2 ,} 4H ₂ O 1.81 g ZnSO _{4 ,} 7H ₂ O 0.222 g Na ₂ MoO _{4 ,} 2H ₂ O 0.390 g CuSO _{4 ,} 5H ₂ O 0.079 g Co(NO ₃ ) _2, 6H ₂ O 0.0494 g

3. Strain and vector for plant transformation

Escherichia used in this experiment coli ( E. coli ) strain DH5α was purchased from DH5α Chemically Competent E. coli (Enzynomics, Korea) and used as a transforming host for recombinant plasmid. For proliferation, LB medium (Bacto-Trypton 10 g/L, Bacto-Yeast Extract 5 g/L, NaCl 10 g/L) was used. Agrobacterium used to transform plants tumefaciens strain GV3101 was 28± It was used after culturing for 2-3 days at 1°C and in the dark. As a recombinant plant expression vector, pCAMBIA1304 binary vector was used (FIG. 1A).

< Experimental example 1>

HcRNAV34 VLP vector construction and transformation of

<1-1> Construction of vector for plant transformation

For the VLP gene sequence of HcRNAV34, an RNA virus used in the present invention, an artificially synthesized gene clone (FIG. 2) based on Genebank (Assession No.; AB218608) was purchased from the Center for Harmful Algae Control Convergence and used. HcRNAV34 VLP plant transformation vector for gene expression in HcRNAV34 pCAMBIA1304 binary vector Two types of VLP and HcRNAV34 VLP genes were prepared by binding GFP. The primers used at this time were 34- Bgl Ⅱ -F (5'-GAA GAT CTA TGA CCC GCC C-3'), 34- Spe I -R (5'-GGA CTA GTA GCA GCC ATC AGA G-3') and 34- BstE Ⅱ- R (5'-GGG TTA CCC AGC AGC CAT CAG-3') was manufactured by Bionics (Korea) and used. PCR conditions were denatured at 95° C. for 5 minutes, followed by 35 cycles of 95° C. 30 seconds, 60° C. 45 seconds, and 72° C. 1 minute conditions, followed by reaction at 72° C. for 5 minutes, and then terminated. HcRNAV34 The amplification of the VLP gene was confirmed by performing 1% agarose gel electrophoresis. Then, the amplified HcRNAV34 VLP gene and the pCAMBIA1304 binary vector were ligated overnight at 4°C using T4 ligase (Takara, Japan).

<1-2> Escherichia coli (E. coli ) transformation

After that, the lysate and the DH5α competent cells were gently mixed on ice and reacted for 30 minutes. After heat-shock was applied at 42°C for 30 seconds, it was immediately stabilized on ice for 2 minutes. SOC (Super Optimal broth with Catabolite repression) media preheated at 37° C. was added, and then cultured with shaking at 180-200 rpm for 1 hour using a shaking incubator, and then the cell suspension was smeared and cultured at 37° C. for 16-18 hours. To select the transformed cell line, DNA was extracted from the colony generated after culture, and direct colony PCR and restriction endonuclease digestion were performed to check whether the gene was introduced.

<1-3> Agrobacterium tumefaciens transformation

The recombinant plant expression vector was introduced into A. tumefaciens GV3101 using the Freeze-Thaw method (Horsch et al ., 1985). For the culture of A. tumefaciens GV3101, in YEP medium containing rifampicin (100 mg/L) and gentamycin (50 mg/L) at 28±1°C, dark conditions, shaking for 2-3 days, the value of _{OD 600 was approximately} 0.7 was used. Remove the supernatant by centrifugation at 5000 rpm, 4°C, 5 minutes, dissolved in 0.15 M NaCl 1, centrifuged again at 5000 rpm, 4°C, 5 minutes, dissolved in _{20 mM CaCl 2 200 and recombined} 10 μl of plasmid DNA was added and reacted on ice for 30 minutes. After the reaction, it was placed in liquid nitrogen for 1 minute and then placed in a 37°C water bath for 5 minutes, then 1 ml of YEP medium was added and incubated with shaking at 28°C for 2-4 hours. After spin down at 3000 rpm for 2 minutes to remove the supernatant, resuspend in 100 μl of YEP liquid medium to contain rifampicin (100 mg/L), gentamycin (50 mg/L), and kanamycin (50 mg/L). It was spread on YEP solid medium and incubated at 28°C for 2-3 days. The transformed cell line was first selected through direct colony PCR, and the DNA of the colony selected through PCR was extracted and transformation was confirmed by sequencing (Solgent, Korea) and restriction endonuclease digestion.

< Experimental example 2>

Leaf - disk by law Agrobacterium mediated tobacco transformation

For tobacco transformation, tobacco was transformed using the leaf-disk transformation method mediated by Agrobacterium (Odel et al ., 1987). After removing the veins of healthy tobacco (N. tabacum cv. Havana) leaves grown under aseptic conditions, sections of about 0.7-1.0 cm2 were made and used for transformation. The GV3101 cell line was cultured in a medium supplemented with hygromycin (50 mg/L), rifampicin (100 mg/L) and gentamycin (50 mg/L) for 2-3 days at 28°C to adjust _{the value of OD 600} to about 0.7. which was used by centrifugation it was diluted with MS liquid medium to _^1/20. After immersing the tobacco leaf slices in MS medium for 30 minutes, gently press the leaf slices on 3MM paper to remove the medium, and then place the top surface of the leaf in TCM (Transformation Co-Cultivation media, Table 7) solid medium, which is a co-culture medium, in contact with the medium. It was cultured in the dark at 26±1°C for 2-3 days. After 2-3 days, 1% cefotaxime sodium (100 mg/ml) was added to MS broth to remove Agrobacterium remaining in the leaf sections, and the leaf sections were washed 2-3 times. The washed leaf sections are washed once again in MS liquid medium, and then lightly dried on 3MM paper, and TSIM (Transgenic Shoot Induction media) I (Table 8) solid, which is a shoot induction medium containing cefotaxime sodium (250 mg/L). After culturing in a growth chamber at 26±1° C. for 7 days on the medium, transferred to TSIMⅡ (Table 9) solid medium containing cefotaxime sodium (250 mg/L) and hygromycin (50 mg/L) and subcultured New shoots were induced. The induced shoots were planted in a TRM (Transgenic Root Induction media, Table 10) solid medium, which is a root induction medium, to induce roots, and then acclimatized and cultivated in a greenhouse.

TCM medium component Stock solution final concentration Amount to add per 1 liter MS liqiud - - 1 L Phyto agar - - 0.8% Indole Acetic Acid (IAA) 2 mM 0.5 uM 250 ul 6-Benzylaminopurine (BAP) 4 mM 2 uM 500 ul

TSIMⅠ Badge component Stock solution Final concentration Amount to add per One liter MS liqiud - - 1 L Phyto agar - - 0.8% Indole Acetic Acid (IAA) 2 mM 0.5 uM 250 ul 6-Benzylaminopurine (BAP) 4 mM 2 uM 500 ul Cefatoxime Sodium 250 mg/ml 500 mg/L 2 ml

TSIMⅡ Badge component Stock solution final concentration Amount to add per 1 liter MS liqiud - - 1 L Phyto agar - - 0.8% Indole Acetic Acid (IAA) 2 mM 0.5 uM 250 ul 6-Benzylaminopurine (BAP) 4 mM 2 uM 500 ul Cefatoxime Sodium 250 mg/ml 500 mg/L 2 ml Hygromycin 50 mg/ml 100 mg/L 2 ml

TRM medium component Stock solution Final concentration Amount to add per One liter MS liqiud - - 1 L Phyto agar - - 0.8% 1-Naphthalene acetic acid (NAA) 10 mM 0.5 uM 50 ul Cefatoxime Sodium 250 mg/ml 500 mg/L 2 ml Hygromycin 50 mg/ml 100 mg/L 2 ml

< Experimental example 3>

HcRNAV34 VLP Genetic Tobacco Transformant Analysis

<3-1> Transgenic plants genomic DNA extraction and PCR analysis

Genomic DNA from tobacco was extracted after putting 50-100 leaf tissue in a 1.7 ㎖ EP tube and pulverizing it in liquid nitrogen using a plastic pestle. Add 400 μl of DNA extraction solution (1 M Tris-HCl, pH 7.5, 5 M NaCl, 0.5 M EDTA, 20 % SDS, 10 % RNase (10 mg/ml, Takara, Japan)) and mix well, then at 60°C After 10 minutes of reaction at 4℃, centrifuged at 13,000 rpm for 15 minutes, the supernatant was transferred to a new EP tube, and Phenol/Chloroform/Isoamylalcohol (25:24:1) was added in a 1:1 ratio with the supernatant. The mixture was centrifuged at 4°C and 13,000 rpm for 10 minutes. The supernatant was transferred to a new tube and mixed with isopropanol in a 1:1 ratio. After reaction on ice for 30 minutes, centrifugation was performed at 4°C and 13,000 rpm for 20 minutes to remove the supernatant, washed with pre-cooled 70% ethanol and 95% ethanol to remove remaining moisture, and dried at room temperature. A pellet was formed. After dissolving the pellet with tertiary sterile water, 1% agarose gel electrophoresis was performed to confirm.

For genomic DNA PCR, genomic DNA extracted from transformants was used as a template, and the primer was HcRNAV34. To a specific 34-F (5'-ATG ACC CGC CCG CTG-3 '), 34-R (5'-GCT CTG ATG GCT GCT TGA-3') and GUS gene hereafter for VLP gene specifically to The prepared GUS-R (5'-CTT GTT TGC CTC CCT GCT-3') was used. PCR conditions were denatured at 95°C for 5 minutes, followed by 35 cycles of 95°C 30 seconds, 60°C 45 seconds, and 72°C 1 minute conditions, and additionally reacted at 72°C for 5 minutes and then terminated.

<3-2> Transgenic plants total RNA extraction and Reverse - Transcription polymerase Chain reaction analysis

Total RNA was extracted after 50-100 mg of tobacco leaf tissue was pulverized using liquid nitrogen, 1 ml of Tri-reagent (Molecular Research Center (MRC) Inc. USA) was added, and reacted at room temperature for 5 minutes. 0.2 ml of chloroform was added, mixed for 15 seconds, and then reacted at room temperature for 15 minutes. After centrifugation at 4°C and 13,000 rpm for 10 minutes, the supernatant was transferred to a new tube, 0.5 ml isopropanol was added, and reacted for 10 minutes, followed by centrifugation at 4°C, 13,000 rpm for 10 minutes to remove ethanol. After drying in air for 5 minutes, it was dissolved in sterile water treated with 1% DEPC (Diethyl Pyrocarbonate) and stored in a deep freezer at -80°C for use.

When performing Reverse Transcription-Polymerase Chain Reaction (RT-PCR), 500 ng of total RNA was used as a template. For first strand synthesis from total RNA (reverse transcription), the RNA was quantified in the same amount and then the First strand cDNA synthesis kit was used. (LeGene, USA) was used to reverse transcribe first strand cDNA at 65°C for 5 min, 42°C for 65 min, and 95°C for 10 min. HcRNAV34 using synthesized single-stranded cDNA as a template Secondary PCR was performed to amplify the VLP gene, and PCR conditions were denatured at 95°C for 5 minutes, followed by 27 cycles of 30 seconds at 95°C, 45 seconds at 60°C, and 1 minute at 72°C. , was further reacted at 72 °C for 5 minutes and then terminated. The PCR product was confirmed on 1% agarose gel, and the primers used were R34-F (5'-GAC GAA TGG TGG TAA CAC GAA-3') and R34-R (5'-GGG ACC GTG TAA ACA TTG G-). 3'), actin-F (5'-CCA AAG GCC AAT AGA GAG AAG-3') and actin-R (5'-CAG CTT CCA TTC CAA CAA GTG-3') were used.

<3-3> Transgenic plants GFP Expression analysis

GFP (Green Fluorescence Protein) expression of transformants was observed using a fluorescence microscope (OLYMPUS, Japan). Green fluorescence caused by the expression of the inserted GFP was confirmed by irradiating the plants into which the GFP gene was introduced by irradiating UV (360-400 nm) or blue light (440-480 nm) under a fluorescence microscope.

<3-4> transgenic plant protein extraction and western blot analysis

Protein extraction solution (RIPA buffer (Biosolution, Korea); 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1 Add 800 ul of % SDS, 1 % Triton X-100, 0.5 % Sodium deoxycholate; Protease inhibitor cocktail (Roche, Switzerland), and repeat the process 5 times with vortex and ice at 30 second intervals and 10 at 4℃, 13,000 rpm. After centrifugation for a minute, only the supernatant was recovered, the protein was quantified at 100 ug using a BCA protein quantification kit (PIERCE, Belgium), and then stored at -20°C and used for western blot analysis. 4x protein sample loading buffer (100 % Glycerol 400 ul/ml, 20 % SDS 200 ul/ml, 1 M Tris, pH 6.8, 10 % Bromo Phenol Blue 10 ul/ml, β- mercaptoethanol 100 ul/ml. dH ₂ O 90 ul/ml) and boiled for 5 minutes to prepare a sample.

For protein sample, acrylamide gel of 5 % stacking gel and 12 % separating gel was used, and MINI-PROTEIN Ⅱ (Bio-rad , USA) and electrophoresis was performed. During gel running, electrophoresis was performed with a current of 100 V in the stacking gel and 120 V in the separating gel until the protein sample loading buffer was discharged to the bottom of the gel. After electrophoresis, transfer buffer (Tris. base 3.03 g/l, Glycine 14.4 g/l, Methanol 200 ml/l) was used in Trans-Blot cell (Bio-rad, USA) at 62 V for 4 hours. After blotting on a 0.45 um PVDF membrane (PALL Life Science, USA), the membrane was stained using Ponceau S solution (Sigma, USA) to confirm transfer. After confirming the membrane was washed twice for 5 minutes with tertiary distilled water, 15 with TBS-T (TBS; 100 mM Tris-Cl, pH 7.6, 150 mM NaCl; 0.1 % Tween-20 (DaeJung, Korea)) It was washed 3 times for each minute. After washing, the membrane was blocked for 1 hour using 5% skim milk solution (skim milk/TBS-T), and then washed 3 times for 15 minutes each using TBS-T. As the primary antibody, the HcRNAV34-R2 antibody (abfrontier, Korea) prepared specifically for HcRNAV34 VLP was diluted in 5% skim milk solution at a ratio of 1:1,000 and reacted overnight at 4°C, and then the membrane was applied with TBS-T. was washed 3 times for 15 minutes each. For the secondary antibody, polyclonal anti-rabbit antibody (Abcam, USA) was diluted in 5% skim milk solution at a ratio of 1:4,000 and reacted at room temperature for 1 hour. After completion of the reaction, TBS-T for 15 minutes once, Washed 3 times for 5 minutes each. The membrane after all the reactions were completed was checked with a luminescence analyzer (LAS 3000, Fujifilm, Japan) using an Immobilon Western Chemilum ECL kit (Millipore, USA).

< Experimental example 4> HcRNAV34 VLP algae function analysis

<4-1> HcRNAV34 VLP host-specific algae function observe

H. circularisquama HU 9433-P, HA 92-1, HY 9423, HU 9436, and C. vulgaris were used for the observation of host-specific apoptosis of total proteins extracted from transformants. Total protein (2.5 ug/ul) containing HcRNAV34 VLP extracted from transgenic plants was treated with 1 ul (2.5 ug/ul) in 500 ul of algae culture medium. After treatment, host-specific algicidal activity was confirmed using an optical microscope (Nikon, Japan) and a laser scanning confocal microscope (Carl Zeiss, Germany) for each elapsed time.

<4-2> Trypan Blue using the dye algae function Verification

Total proteins extracted from transformants were treated with H. circularisquama HU 9433-P and observed through trypan blue staining. Staining with trypan blue (LPTB; 2.5 mg/ml trypan blue, 25% (w/v) lactic acid, 23% water-saturated phenol, 25% glycerol in H ₂ O) and optically measuring the degree of staining of cells according to elapsed time It was observed with a microscope (Nikon, Japan).

< Experimental example 5> HcRNAV34 VLP Plant expression vector construction and transformation

<5-1> Preparation of plant expression vector

A study on the introduction of foreign genes in plants, tobacco, carrots, peppers, including dicotyledon has mainly consisted in using the binary plant transformation vector A. One way tumafaciens-mediated transformation is a common plant in the 1980s conversion method is (Horsch et al.) al ., 1985). In this study, A. tumefaciens Strain GV3101 and pCAMBIA1304 binary vectors were used. The T-DNA of this vector has CaMV 35s promoter derived from Cauliflower Mosaic virus that is inserted into the plant genome to induce overexpression, and Nos Poly derived from nopaline synthetase of A. tumefaciens. -(A), and there is a hygromycin gene as a selective marker when inserted into a plant. In addition, a Kanamycin resistance gene is inserted into the vector and can be used as a selective marker in E. coli ( FIG. 1A ).

HcRNAV34, a single-stranded RNA virus that specifically attaches to and infects dinoflagellates, H. circularisquama , used as the material for this experiment, is a 4.4 kb genome expressed by RNA polymerase (ORF-1) and VLP (ORF-2). has (Tomoru et al. al ., 2004; Nagasaki et al. al ., 2005; Mizumoto et al. al ., 2007; Nagasaki, 2008; Wu et al ., 2012). Among them, an artificially synthesized VLP gene clone (1080 bp, FIG. 2) was sold from the Center for Harmful Algae Control Convergence Research to prepare a plant expression vector.

Recombinant vector for tobacco transformation is HcRNAV34 Vector for VLP gene expression and HcRNAV34 in plants Two vectors including the GFP gene were constructed as reporter genes to check VLP gene expression. For the first recombinant vector produced using BglⅡ and BstE Ⅱ enzyme site GFP :: GUS After the gene was removed, the HcRNAV34 VLP gene was inserted ( FIG. 1B ). In the case of the second recombinant vector , HcRNAV34 is a primer to which Bgl II and Spe I enzyme sites are attached. GFP in the pCAMBIA1304 binary vector by amplifying the VLP gene It was constructed by inserting it into the Bgl II and Spe I parts in front of the gene (FIG. 1C). The recombinant vector was a two screening the transfected cells are identified through the conversion by direct colony PCR transformed in E. coli, extracted the plasmid DNA of the selected cell lines to BglⅡ / BstE Ⅱ (Figure 3A) or Bgl Ⅱ / Spe Ⅰ (FIG. 3B), it was confirmed whether the insertion through the band of the expected size (1080 bp) (FIG. 3). These were named pCAM/34 VLP and pCAM/34 GFP, respectively.

Selected E. coli Two types of recombinant vector DNA were extracted from the cell line and transformed into A. tumefaciens GV3101 strain using the Freeze-Thaw method. The transformation efficiency of Agrobacterium through the Freeze-Thaw method was lower than that of E. coli, so it was carried out using 1 ug or more of DNA. This method reduces the possibility of homologous recombination that may occur during the transformation process and the experimental method is relatively Because it is simple, it is the most widely used transformation method to introduce foreign genes into Agrobacterium (Birnboim et al. al ., 1979).

To select the transformed cell line, colonies resistant to antibiotics rifampicin (100 mg/L), gentamycin (50 mg/L), and hygromycin (50 mg/L) were collected, followed by direct colony PCR (Fig. 4A) and restriction endonuclease. A cell line was finally selected through digestion (FIG. 4B) and used for tobacco transformation.

<5-2> Plant transformation

Plant transformation using A. tumefaciens is the most widely used method for inserting foreign genes into plants in both monocotyledons and dicotyledons. It is being used in various plant morphology, physiology, and genetic studies such as virus resistance and herbicide-resistant plant development, male infertility, and low-temperature stress resistance. It is used to develop plants.

In this study, the tobacco leaves through a leaf disk method using an A. tumefaciens GV3101 containing the produced vectors to introduce genes HcRNAV34 VLP-specific viruses that infect the enemy with the H. circularisquama saljo as part of a biological tide bangjebeop transformed. As a result of planting and culturing tobacco leaf slices transformed using the leaf disk method on TCM solid medium and TSIMⅡ solid medium, some leaves started to turn brown after one week of culture. and infertility began to form. Shoots began to differentiate after the formation of embryos, and it was confirmed from explants that the rate of shoot formation was 270 or more out of 300, or about 90% or more. After that, among the formed shoots, the healthiest ones were selected, and after 3-4 weeks, the roots were induced and redifferentiated into plantlets by placing them on a TRM solid medium to induce rooting, and then transferred to a greenhouse and grown into adults (Fig. 5). ). As a result of observing the phenotypes of transgenic tobacco and non-transgenic tobacco during adult growth, it was confirmed that they grew similarly without showing any particular difference in appearance and growth rate.

< Experimental example 6> Tobacco transgenic plant analysis and line selection

After the shoots formed after transformation were primarily selected in hygromycin antibiotic medium, HcRNAV34 VLP gene expression was confirmed in the transformant regenerated into the plant by the following experiment.

<6-1> transgenic plants genomic DNA PCR analysis

In the genomic DNA PCR performed to confirm the gene introduction of the transgenic plants, the transformants introduced with pCAM/34 VLP used 34-F and 34-R primers specifically designed for the HcRNAV34 VLP gene. For transformants into which pCAM/34 GFP was introduced, 34-F specifically for the HcRNAV34 VLP gene and GUS-R primer specifically for the GUS gene were used. In the transgenic plants into which each gene was introduced, bands of about 1.1 kb (FIG. 6A) and 3.6 kb (FIG. 6B) were confirmed.

Based on these results, it is considered that the HcRNAV34 VLP gene was inserted into the genome of tobacco in the plant in which the band appeared. When performing PCR using the genomic DNA of the transformed leaf, Agrobacterium parasitizes on tobacco leaves from the medium in the process of inducing shoots, and in some cases, it is detected during the PCR experiment. In the non-transgenic plant used as a negative control in this experiment, the introduction of the HcRNAV34 VLP gene could be confirmed only in shoots considered to be transformants due to the result that the band was not synthesized. The introduction of the HcRNAV34 VLP gene was confirmed in about 80% of the transformants primarily selected in the medium containing hygromycin antibiotics . Also, in the study result of Varsani et al (2003), the pART27 binary vector and Human papillomavirus (HPV) type 16 By recombination of the envelope protein gene, N. tabacum cv. As a result of insertion into Xanthi, the introduction of the gene was confirmed in about 80% of transformants similar to the one in this study.

<6-2> transgenic plants RT - PCR analysis

Based on the genomic DNA PCR analysis performed previously, RT-PCR was performed by extracting total RNA to confirm the transcript expression of the tobacco transgenic plants in which the introduction of the HcRNAV34 VLP gene was confirmed. Non-transgenic plant leaves were selected as a control group, and primers R34-F and R34-R specifically prepared for the HcRNAV34 VLP gene were used. The expression patterns of actin used as controls were all similar, but as a result of using the specific HcRNAV34 VLP gene primer, expression did not appear from non-transgenic plants, and only at about 522 bp in tobacco, which was expected to be a transgenic plant. The expression of the transcript was confirmed through the band (FIG. 7). During the experiment, bands appearing at positions other than the expected positions were sometimes found, and there were cases where the expression level was different even at the same position. This is thought to have occurred due to the modification of the inserted gene by long-term subculture of the selected shoots. In addition, it was confirmed that actin (480 bp) used as a positive control showed a similar expression rate, showing that the same gene was inserted into the transformant but showed a different expression rate depending on the individual. Among the 216 transformants selected through genomic DNA PCR, transformants showing the correct size band in 100 (about 50%) were selected, and the experiment was continued.

<6-3> transgenic plants GFP Expression analysis

Among the tobacco plants transformed with the pCAM/34 GFP vector, transformants that had been analyzed for genomic DNA and mRNA expression were selected, and the expression of HcRNAV34 VLP through GFP expression was observed with a fluorescence microscope (OLYMPUS, Japan). As a result of collecting shoots formed from leaf sections of transgenic and non-transgenic plants and irradiating with UV light, transgenic plants expressed bright green fluorescence by the expression of GFP (FIG. 8B), whereas non-transformed plants were not transformed. It was observed that the plants expressed rather low light ( FIG. 8A ). It is considered to be an auto-fluorescence protein or secondary metabolite that the plant basically possesses. GFP expression was confirmed in the leaf veins and stems of the transformant, and in the case of the leaf, it was confirmed that it was expressed in the whole mesophyll tissue. .

<6-4> transgenic plants western blot analysis

Western blot analysis was performed using the HcRNAV34-R2 antibody on the selected transformants to confirm the expression of HcRNAV34 VLP in the transgenic plants. HcRNAV34 The VLP gene has a total of 360 amino acids and the protein molecular weight is about 38 kDa. It was confirmed that the band of HcRNAV34 VLP was expressed at about 40 KDa, slightly higher than the expected size of the transformant, 38 KDa (FIG. 9). This is thought to be due to a factor that changes the size of a protein expressed in a plant by post-translational modification in a plant into which a foreign gene is introduced (Gomord et al ., 2004). Therefore, it is considered that the slightly increased protein expression when HcRNAV34 VLP is expressed in plants increases the size of the protein slightly during the modification process after the translation process during gene expression. For accurate comparative analysis of the protein expression level of transgenic plants, only 4, 5, 7, and 8 transgenic individuals with slightly high expression levels selected according to the results of RT-PCR and non-transgenic plants were tested. Since there is a difference in the expression amount depending on the

< Experimental example 7> HcRNAV34 VLP of algae function Verification

<7-1> HcRNAV34 VLP host-specific algae function Verification

To confirm the efficacy of HcRNAV34 VLP by extracting total protein from the selected transformants, the host H. circularisquama HU 9433-P, HA 92-1 and the non-host H. circularisquama of HcRNAV34 virus, a marine species dinoflagellate that induces red tides HY 9423, HU 9436 and freshwater species C. vulgaris were treated. Host and non-host groups were selected through a study report (Nagasaki et al ., 2005) that HcRNAV34 specifically infects only H. circularisquama HU 9433-P and HA 92-1 strains.

A total of 4 groups: untreated group, treated with total protein extracted from transgenic tobacco plants, treated with RIPA buffer (50%), a protein extraction solution, and treated with total protein extracted from non-transgenic tobacco plants was selected to observe the host-specific killing ability of the total protein including HcRNAV34 VLP. The RIPA buffer used as the protein extraction solution contains SDS, etc., which is expected to affect algae, so a control group treated with only RIPA buffer was included. was used as 1 ul (2.5 ug/ul) of the protein extracted into 500 ul (4×10 ⁵ cell/ml) of the algae culture medium was treated and the shape change, fluidity, and cell destruction degree of the algae were observed using an optical microscope at 15-minute intervals for 1 hour. and observed.

First, as a result of observing the morphological change and fluidity of algae by time period, in the case of the host alga, HU 9433-P, in the group treated with RIPA buffer and the group treated with total protein from non-transgenic tobacco plants, the algae were dented from 15 minutes after the reaction. This resulted in a shape change in which the middle part slightly swelled (Fig. 11G, L). In the algae of the group treated with RIPA buffer, it was about 15-20%, and in the algae of the group treated with the total protein of non-transgenic tobacco plants, the shape change was about 20%. The algae that caused the algae started to recover to their original form again (Fig. 11H, M), and as a result of observation 45 minutes after treatment, almost no algae with a changed form were observed, so a recovery rate of 90% or more was observed (Fig. 11I, N) . As a result of observing the fluidity of algae, the fluidity decreased by about 60-70% in both the RIPA buffer-treated group and the group treated with the total protein of non-transgenic tobacco plants 15 minutes after protein treatment (FIGS. 11G, L) As a result of observation 45 minutes after treatment, fluidity of about 95% was recovered similar to the shape change (Fig. 11I, N). As a result, the total protein of non-transgenic tobacco plants, including RIPA buffer containing SDS and various secondary metabolites such as nicotine, temporarily reduced fluidity and changed shape, but 95% after 30 minutes after treatment. It is considered that there is little effect on the destruction of host algae by showing a recovery rate of a certain degree.

As a result of observation 15 minutes after the reaction, the experimental group treated with the total protein extracted from the transgenic tobacco plants showed a change in shape in more than 80% of algae, and the fluidity was 0% (FIG. 11Q). As a result of observation 30 minutes after the reaction, morphological changes were observed in all algae, and in about 15% or more of the algae, the cell membrane was destroyed and the cell fluid was eluted (FIG. 11R). As a result of observation 45 minutes after the reaction, about 50% of the algae were destroyed (FIG. 11S), and as a result of observation 60 minutes after the reaction, it was observed that about 80-90% of the algae were destroyed (FIG. 11T).

The above results observed using an optical microscope are schematically illustrated in FIG. 10 . When the total protein concentration (10 ug/ul) extracted from the transformed tobacco was increased, more than 70% of the algae were destroyed 30 minutes after the reaction. In addition, H. circularisquama HA 92-1 showed a similar pattern to HU 9433-P (Fig. 12).

In the non-host alga, H. circularisquama HY 9423, in the group treated with RIPA buffer and in the group treated with non-transgenic tobacco plant protein, the shape change and fluidity were reduced in about 20% of the algae, as observed 15 minutes after the reaction. It exhibited a pattern similar to that of the host alga (FIGS. 13G, L), and as a result of observation 45 minutes after the reaction, it was observed that the shape change and fluidity were recovered in 90% or more of the algae (FIGS. 13I, N). In the group treated with the transgenic tobacco plant protein, as a result of observation 15 minutes after the reaction, about 20% of morphological change and about 50% of fluidity were reduced (FIG. 13Q), but as a result of observation at 30 minutes of the reaction, about 50 of the morphologically changed algae The morphology of % algae was restored, and the fluidity was also restored to about 50% (Fig. 13R). As a result of observation from 45 minutes of reaction, it was observed that the shape and fluidity were recovered in more than 90% of the algae, unlike the host algae, H. circularisquama HU 9433-P or HA 92-1 (FIG. 13S). Through this, it was found that the fluidity also increased as the shape was recovered as the reaction time passed. H. circularisquama HU 9436 (FIG. 14), which was used as another non-host alga, also showed a similar pattern to HY 9423.

C. vulgaris is a spherical single-celled microalga with a diameter of 2-10 um that exists in freshwater and is a kind of green algae. In this experiment, HcRNAV34 VLP extracted from tobacco transformants was used as a non-host to check the effect on freshwater species other than H. circularisquama. As a result of performing the experiment in the same manner as above, no morphological change or destroyed cells were found in all groups (FIG. 15). These results suggest that the HcRNAV34 VLP does not affect species other than H. circularisquama , and the total protein extracted from the transformed tobacco specifically infects the host algae, H. circularisquama HU 9433-P and HA 92-1. It was confirmed that non-host algae H. circularisquama HY 9423, HU 9436 and C. vulgaris did not cause infection.

In addition, HcRNAV34 in tobacco transformants The green fluorescence expression of GFP introduced together with the VLP gene was used to confirm the apoptosis of HcRNAV34 VLP using a confocal microscope (Fig. 16). Total protein was isolated from tobacco plants transformed with the HcRNAV34 VLP::GFP gene, treated with 1 ul (2.5 ug/ul) of H. circularisquama HU 9433-P, a host alga, and observed after 15 minutes. As a result, the protein was introduced into the algae. and (FIG. 16A), most of the introduced cells exhibited morphological changes such as swelling (FIG. 16B). Cell destruction of algae was observed 30 minutes after treatment, and strong GFP fluorescence expression was observed in the cells of the destroyed algae (FIG. 16C). Tartani et al (2001), Tomaru et al. al (2004) and Mizumoto et al. al (2007) reported that HcRNAV34 acts as a crystal in the cytoplasm of H. circularisquama , and as confirmed through the results of this paper, HcRNAV34 VLP expressed in transgenic plants also flows into algae and destroys cells. is believed to have an effect on

<7-2> Trypan Blue using the dye algae function Verification

Total proteins were extracted from the selected transformants , treated with H. circularisquama HU 9433-P, a host alga, and the degree of apoptosis was observed using trypan blue staining. Trypan blue is a reagent used as a histochemical indicator of cell membrane damage or apoptosis. Trypan blue particles pass through the cell membrane by diffusion and enter the cell. They are excluded from the cell again, but dead cells that cannot generate energy cannot perform exocytosis and are stained dark blue. When observed 15 minutes after the total protein treatment, damaged algae were observed with a change in shape (FIG. 17B), which was observed with an optical microscope after treatment with total protein. It is thought that the inside of the lining is damaged and dyed because it cannot generate ATP energy. After 30 minutes of treatment, it was observed that the cell membrane was destroyed and the inside was stained (FIG. 17C), and trypan blue particles and cell intrinsic materials released by the elution action were observed around the destroyed cells after 45 minutes (FIG. 17D).

Physicochemical and biological methods currently used as methods for removing harmful red tides that occur in the coast of Korea have many problems by affecting other marine organisms. In order to solve these problems, Research on biological control using microorganisms or viruses is being actively conducted.

Since HcRNAV34 selectively infects and destroys H. circularisquama , a red algae-induced dinoflagellate, if we confirm the induction of HcRNAV34 expression in tobacco and establish a mass production system to selectively act on the host, this method is a method for controlling harmful algae. It will be a biological control method without disturbing the ecosystem.

The HcRNAV34 VLP gene was recombined with two vectors, pCAM/34 VLP and pCAM/34 GFP, using pCAMBIA1304, a binary vector for plant transformation. Tobacco (N. tabacum cv. Havana) leaves were transformed by the leaf-disk method using the A. tumefaciens GV3101 cell line into which the recombinant pCAM/34 VLP and pCAM/34 GFP vectors were inserted, and then transformed through hygromycin antibiotic resistance. Transformants were selected. As a result of the selection, there was no particular difference in the phenotype and growth rate of transgenic tobacco and non-transgenic tobacco.

The transformants selected on the antibiotic medium was confirmed that the HcRNAV34 VLP gene in genomic DNA PCR method was confirmed that insertion into the genome of tobacco, HcRNAV34 expression VLP gene of the mRNA by RT-PCR analysis, transformed with HcRNAV34 VLP gene Transformation was confirmed through the expression of GFP. Finally, proteins were extracted from the selected transgenic tobacco plants and western blot analysis was performed using the HcRNAV34-R2 antibody. HcRNAV34 VLPs (360 amino acids) were confirmed to be expressed at the expected size of about 38 KDa. It was confirmed that it was stably expressed in plants.

H. circularisquama HU 9433-P and HA 92-1 as hosts of HcRNAV34 by extraction of total proteins from transgenic plants. As a result of treatment with non-host H. circularisquama HY 9423, HU 9436, and C. vulgaris , only HU 9433-P and HA 92-1 hosts stopped the flow of algae, changed the shape, and destroyed the cells. was observed. In addition, when observed with a confocal microscope, it is believed that GFP transformed with HcRNAV34 VLP is expressed in algae, so the morphological change and destruction of algae is thought to occur when HcRNAV34 VLP is introduced into the alga, and HcRNAV34 VLP alone is host-specific. It was confirmed that there is algae function.

Therefore, if a virus that specifically infects harmful algae is selected and a part of it is mass-produced through transformation in a plant to confirm its algal ability, it is expected that it can be utilized as a new biological control method for red tide.

<110> Industry-Academic Cooperation Foundation, Chosun University <120> A transgenic tobacco expressing recombination HcRNAV 34 virus like particle protein <130> PN1408-262 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1080 <212> DNA <213> Artificial Sequence <220> <223> HcRNAV 34 VLP <400> 1 atgacccgcc cgctggcact gacgaatggt ggtaacacga atggtggtaa taacggtggt 60 agccgccgcc gcccgccgcg ccaacgtcgt cagggccgtc gccgtaatcg ccgtcgcggc 120 ggtggcggtg gcggtccgcg caacaatgcc gcaatggttc tggcccaagg tgcaggtagt 180 gtgccgggca tgccgtttgg ttcctggccg agtcgttcca ccatgcgcgc atgggatgct 240 ttccatccgg aacatctgcc gctgccgcgt agtgtgggtc cgtattgcgt ggttcgcacc 300 agctctctga ttacgagttc cgacaaagtg atgctgtttg cgccgatggt tggcagtgcc 360 ggttgctggc tgaccgcatg tgctatgggc tcccgtacgg aaggcggtgc gatcaacggt 420 ctggataaca ccaatgttta cacggtcccg tttccgggca ttgccaccac gggttcatcg 480 atcaccgtcg tgccggcagc tctgagcgtt caggtcatga acccgaatcc gctgatgagc 540 accacgggca ttttcggcgg taccgtgtct catacgcaac tgaacctggc gggtcgtacc 600 gaaacgtgga atgattttag catggaagtg atctctttca tgcgtccgcg cctgatgtca 660 gcaggcaaac tggctctgcg cggcgttcag ggtgactcgt atccgctgaa tatgtcagca 720 ctgtcgaact tcaattgtct gtcagatgtt gctgaaggta aactgtcgtg gaccgacagc 780 tctggtcact atccggcggg tctggccccg ctggtgtttg ttaacgaagc aaaacagacc 840 atgaattacc tggtctcagt ggaatggcgt gttcgcttcg atattggcaa tccggccgtc 900 gccgcacaac gtcatcacgg tatcaccccg gaatggaaat gggatgacat gattaaaacg 960 gcgatcgccc gtggccacgg tattatggac atcgcggaac gcgttgcaaa tgctggctct 1020 tttgttgcga atgccgctat tgctgctcgt cgtgcgatgc cggctctgat ggctgcttga 1080 1080

Claims (1)

In a composition for controlling red tides, comprising the total protein (total protein) produced in the transformed tobacco into which the HcRNAV 34 virus like particle (VLP) gene represented by the nucleotide sequence of SEQ ID NO: 1 is introduced,
The total protein is a red algae control composition, characterized in that it has algae activity in H. circularisquama HU 9433-P and H. circularisquama HA 92-1, which are red algae-induced algae.

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