KR20170034771A - Transgenic plants transformed with PNGase T gene derived from tomato and methods of producing PNGase T protein by using the same - Google Patents

Abstract

The present invention relates to a transgenic plants transformed with PNGase T (peptide:N-glycanase) gene derived from tomato and a method of producing PNGase T protein by using the same. According to the present invention, in order to design a gene expression composition for producing PNGase T protein from plants, a stepwise experiment is performed and finally, a PNGase T composition directly derived by 35S promoter is produced, transformed into tobacco plants, and can be used to produce PNGase T protein which is an enzyme industrially used to remove glycosylation through molecular farming. Furthermore, when genes of recombinant protein for production are introduced into plants, in which PNGase T gene is over-expressed, and are simultaneously expressed, the utilization thereof is expected to increase because additional removal of glycosylation can be omitted.

Description

TECHNICAL FIELD The present invention relates to a plant transformed with a PNGase T gene isolated from tomato and a method for producing PNGase T protein using the same.

The present invention relates to a plant transformed with a PNGase T gene isolated from tomato and a method for producing PNGase T protein using the same. More specifically, the present invention relates to a method for producing a glycosylation enzyme PNGase T (peptide: N-glycanase), a transformed plant using the same, and a method for producing PNGase T protein using the same.

Peptide: N -glycanase (PNGase or ^{glycoamidase, peptide-N 4 - (} N-acetyl-β-D-glucosaminyl) asparagine amidase; EC 3.5.1.52) is present in the N to almost all living organisms, such as plant, animal and microorganism-binding Is an enzyme that hydrolyzes the β-aspartyl-glycosylamine linkage of glycoproteins / glycopeptides (Suzuki et al. 2002). Due to the action of PNGase, the N-linked oligosaccharide bound to the NH ₂ of the asparaginic acid residue of the glycoprotein is isolated (Wi and Park, 2014). Since PNGase T was first isolated from PNGase A in almond seeds (Takahashi, 1977; Taga et al. 1984), the bacterial Flavobacterium After PNGase F (Plummer et al. 1984) was isolated from meningosepticum , PNGase was isolated from yeast (PNGase Y) and various plants and animals including many species of bacteria.

Glycosylation is initiated by the oligosaccharide attached to the dolichol lipid at the cytoplasmic membrane of the endoplasmic reticulum and then introduced into the endoplasmic reticulum lumen to complete the oligosaccharide synthesis and the amino acid sequence motif of aspartic acid-X-serine / threonine (Song et al. 2011), which binds to proteins via an amide bond (N-glycosylation) in asparaginic acid residues in NxS / T. The process in which the oligosaccharide is transferred from the lipid to the protein takes place during the process of translocation of the protein into the endoplasmic reticulum to be secreted. Actual glycosylation occurs at about 70% of the NxS / T motif present in the secreted protein (Mellquist et al. 1998).

The glycoprotein thus formed undergoes a specific modification depending on the species in the Golgi apparatus. In the plant, a mannose is bound by mannosidase, and N- acetylglucosaminetransferase 1 cleaves N- acetylglucosamine moiety And bind to mannose. This reaction then leads to the formation of two types of oligosaccharides. One reaction involves the binding of the fucose moiety to the first N- acetylglucosamine via α-1,3 linkages or the binding of β-1,2 xylose to the end of mannose (Song et al. 2011). However, in animal glycoproteins, instead of α-1,3 fucose, α-1,6 fucose binds to N- acetylglucosamine and β-1,4 galatose is added to the oligosaccharide. Subsequently, a wide variety of oligosaccharides are added through the bone matrix to finally produce tissue-specific glycoproteins. PNGase F, an enzyme that deglycosylates animal glycoproteins, is not recognized as a glycosyltransferase in plant α-1,3 fucose-linked plants (Wi and Park, 2014).

In eukaryotic cells, glycosylation by the organozoan in the endoplasmic reticulum or Golgi affects protein folding (Helenius and Aebi 2004). Although the physiological role of the protein in animal cells has not been well studied, it is generally thought to be involved in receptor binding, cell signaling, protein folding, intracellular compartmentalization and distribution, protein stability, immune response, encapsulation, inflammatory response and pathogenicity Are reported (Song et al. 2011). However, the physiological function of glycoprotein oligosaccharides in plant cells has not been studied yet. The role of glycoprotein in plant cells is known to affect the catalytic activity of enzymes composed of glycoproteins, thermal stability, protein folding, intracellular compartmentalization and secretion. In particular, recent studies have shown that pathogenicity, It is believed that it plays an important role in interaction and is expected to contribute physiologically to tissue differentiation (Haweker et al. 2010). In particular, it is predicted that the free N-glycoside produced through the desulfurization nitrification process will play an important physiological role in growth such as differentiation and development.

Recently, therapeutic proteins and antibodies have been produced in Escherichia coli or yeast as a host for expression through gene recombination, but glycosylation pattern is different from human glycoprotein. Therefore, high-value-added pharmaceutical glycoproteins that require high protein activity and unique protein structure are produced using animal cells similar in human and oligosaccharide adhesion.

However, recent increases in the possibility of human infection with animal-derived pathogenic proteins or viruses have raised concerns about possible contamination of recombinant protein produced in animal cells. Therefore, as an alternative to these concerns, attempts have been made to utilize plant biotechnology to utilize plant-based systems that are relatively safe from animal-derived pathogenic proteins or virus contamination. In addition, plant biotechnology production of recombinant proteins has been reported to be relatively more economical than using animal cells (Wi and Park, 2014).

The recombinant proteins produced in plant cells have the same process of glycosylation in the cytoplasm, but the glycosylation process in Golgi is very different from the glycosylation type in animal cells because of the high species specificity. In particular, recombinant proteins produced by plant cell specific glycosylation are likely to cause an immune response in the human body. If necessary, the oligosaccharide of the glycoprotein may need to be removed. In particular, when abnormal glycosylation occurs in plants, it is necessary to cleave the oligosaccharide in the glycoprotein (Mamedov et al. 2012) in order to maintain the physiological characteristics of the recombinant protein and to maintain the original protein structure.

PNGase F, which is derived from bacteria ( Flavobacterium meningosepticum ), is mainly used as an enzyme that eliminates the most commonly used oligosaccharides. PNGase F can not remove α-1,3 fucose oligosaccharides from plant-produced glycoproteins Tarentino and Plummer 1981). Therefore, in order to cleave α-1,3 fucose-linked oligosaccharides from N- acetylglucosamine, plant-based recombinant proteins utilize PNGase A isolated from plant almond seeds other than microbial PNGase F (Altmann et al. 1998). PNGase A, purified and sold from almonds, is relatively expensive and, unlike PNGase F, requires a process to convert it to a peptide state as a pretreatment of the glycoprotein (Kolarich and Altmann 2000).

To overcome this limitation of PNGase A, yeast, Yarrowia (Patent No. 10-2012-0053963), and in particular, a method of simultaneously expressing a bacterial-derived PNGase F gene in a plant overexpressing a target protein for industrial production to produce an in A study confirming the elimination of glycosylation in studies that remove glycosylation in vivo has been reported (Mamedov and Yusibov, 2013).

Accordingly, the present inventors isolated a PNGase T gene that can be used for removing glycosylation from a tomato after producing an industrial animal protein or a human protein from a plant, and then producing an expression vector for expression of a plant into which a PNGase T gene has been introduced, To produce a transformed tobacco plant.

Accordingly, a technical problem to be solved by the present invention is to provide an expression vector for expression of a plant into which a gene of PNGase T isolated from tomato is introduced.

Another technical problem to be solved by the present invention is to provide a transgenic plant transformed with the expression vector.

Another object of the present invention is to provide a method for transforming a plant using the expression vector.

Another object of the present invention is to provide a method for producing recombinant PNGase T protein using the transgenic plant.

In order to accomplish the above object, the present invention provides an expression vector for plant expression comprising the gene of PNGase T having the nucleotide sequence of SEQ ID NO: 7.

The present invention also provides a plant transformed with the expression vector to achieve the above technical object.

According to another aspect of the present invention, there is provided a method for transforming a plant using the expression vector.

In order to achieve the above and other objects of the present invention, there is provided a method for producing a recombinant PNGase T protein using a plant transformed with an expression vector for plant expression comprising a PNGase T gene having the nucleotide sequence of SEQ ID NO: . ≪ / RTI >

According to the present invention, when a gene of a recombinant protein for production is introduced into a plant overexpressing the gene of PNGase T and coexpressed, additional desalting nitrification process can be omitted and its utilization can be enhanced.

In the present invention, the gene of PNGase T is a protein having the nucleotide sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8, and may be a functional equivalent to a protein encoding the PNGase T gene having the amino acid sequence of SEQ ID NO:

The "functional equivalents" have a sequence homology of at least 60%, preferably 70%, more preferably 80% or more, with the amino acid sequence of SEQ ID NO: 6 as a result of amino acid addition, substitution or deletion Quot; means a protein that exhibits substantially the same activity as the protein encoding PNGase T of the present invention.

Such functional equivalents include, for example, amino acid sequence variants in which some of the amino acids of the amino acid sequence of SEQ ID NO: 8 are substituted, deleted or added. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid is preferably located at a site that is not directly involved in the activity of the PNGase T of the present invention. The functional equivalents also include protein derivatives in which the basic structure of PNGase T and its chemical structure is modified while maintaining its physiological activity. These include, for example, fusion proteins made by fusion with other proteins such as GFP (Green Fluorescent Protein) while retaining the structural modification and physiological activity to change the stability, storage stability, volatility or solubility of the protein of the present invention .

The gene of PNGase T of the present invention is a gene having the nucleotide sequence of SEQ ID NO: 7, which is derived from tomato ( Solanum lycopersicum ).

According to a specific embodiment of the present invention, pTRAkt-PNGase T, which is a transformation vector, is prepared by inserting the above-mentioned PNGase T gene derived from tomato into a transformation vector, pTRAkt vector, and then transforming tobacco transformants And the expression of PNGase T from tomato was confirmed in transgenic tobacco plants.

As described above, the PNGase T gene has the nucleotide sequence shown in SEQ ID NO: 7, and the gene is not limited to the nucleotide sequence shown in SEQ ID NO: 7 in the attached Sequence Listing, and may be a functionally equivalent codon or a codon , Or a nucleotide sequence comprising a codon that encodes a biologically equivalent amino acid. Considering a mutation having biologically equivalent activity, the nucleotide sequence used in the present invention is interpreted to include a sequence showing substantial identity with the sequence described in the sequence listing. The above-mentioned substantial identity is determined by aligning the sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology.

According to one embodiment of the present invention, there is provided an expression vector introduced into a plant expression plasmid containing the gene of PNGase T having the nucleotide sequence of SEQ ID NO: 7. The plasmid is used for expression of a plant, and there is no restriction on its kind. In the present invention, a pTRAkt vector was used.

According to a preferred embodiment of the present invention, the expression vector for plant expression comprising the gene of PNGase T having the nucleotide sequence of SEQ ID NO: 7 is pTRAkt-PNGase T.

In the present invention, a "transgenic recombinant vector" is a gene construct containing an essential regulatory element operatively linked to the expression of a gene insert, and includes all plasmid vectors, cosmid vectors, bacteriophage vectors, .

The "recombinant vector for transformation" of the present invention is functionally linked to an expression control sequence so that the PNGase T gene can be expressed. For example, the vector may comprise a signal sequence or leader sequence for membrane targeting or secretion in addition to an expression regulatory element such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, an enhancer, . In addition, the vector may comprise a selectable marker, and the vector may be self-replicating or integrated into the host DNA. The vector of the present invention can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage are performed using enzymes generally known in the art.

In another aspect of the present invention, the present invention provides a plant transformed with said recombinant vector.

As used herein, the term "plant" is understood to include not only mature plants but also plant cells, plant tissues and plant seeds that develop into mature plants. Transformation of a plant with the vector in the present invention can be carried out by a transformation technique known to a person skilled in the art. The microorganism is preferably transformed with Agrobacterium, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome (liposome) method, mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method, and Agrobacteria spraying method can be used. And more preferably, a transformation method using Agrobacterium can be used.

In the present invention, the plant is a food crop including rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats or millet; Vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions or carrots; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut or rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots or bananas; Roses, gladioluses, gerberas, carnations, mums, lilies or tulips; And feed crops including raglass, red clover, orchardgrass, alpha-alpha, tall fescue or perennial rice, preferably ginseng, tobacco, cotton, sesame, sugarcane, beet, Perilla, peanut or rapeseed, more preferably cigarette, but not limited thereto.

According to an embodiment of the present invention, there is provided a method for transforming a plant using the expression vector.

The term "expression of a gene" in the present invention means transcription of a gene, and specifically, transcription into mRNA. Said "transcription" involves the transcription of DNA and the processing of the resulting mRNA product. In the present invention, the term " enhancement "is intended to mean at least 5% or 10%, preferably at least 20% or 40%, more preferably 50%, 60%, 70% Means 80% or more resistant to weathering, and means that the resistance to harshness is increased by introducing the gene as compared with the original resistance of the plant.

According to one embodiment of the present invention, there is provided a method for producing a PNGase T protein by transforming a plant with an expression vector for plant expression comprising the gene of PNGase T having the nucleotide sequence of SEQ ID NO: 7, followed by culturing. Any of the conventional methods for genetic engineering can be used for the transformation and culturing as long as the desired recombinant PNGase T protein can be efficiently expressed at a high yield.

Thus, according to the present invention, in order to design a gene expression construct for producing PNGase T protein in a plant, stepwise experiments were conducted to finally produce a PNGase T construct directly induced by the 35S promoter, It can be used to produce PNGase T protein, an enzyme used industrially for the elimination of glycosylation through agriculture. In addition, when the gene of the recombinant protein for production is introduced into the plant overexpressing the PNGase T gene and co-expressed, it is expected that the additional desulfurization nitrification process can be omitted and the utilization thereof can be increased.

FIG. 1 shows the result of examining the expression level of PNGase T mRNA expressed during ripening of tomato pulp from green to red.
FIG. 2 shows phylogenetic tree of amino acid sequence analysis of PNGase T gene isolated from 12 plant species.
FIG. 3 is a graph showing the results of the addition of a KDEL amino acid sequence that binds six histidines for protein cleavage to a pTRAkt vector for expression of recombinant proteins (PNGase T) derived from tomato and induces residues in the endoplasmic reticulum Is a schematic diagram of a 35S :: PNGase T consturct designed to express a recombinant gene by the 35S promoter.
FIG. 4 shows the results of genomic DNA PCR of the transformed tobacco plants selected in the antibiotic screening medium. In order to confirm the genomic DNA, 6 specific primers were constructed based on the nucleotide sequence of the recombinant PNGase T + His6 + KDEL gene construct And its nucleotide sequence was shown.
FIG. 5 shows the result of sequencing of the genomic DNA of the transgenic tobacco lines of two lines, followed by electrophoresis of the PCR product obtained by performing PCR using specific primers, .
FIG. 6 shows the phenotype of the transformed tobacco plant line PNGase T-10 after 6 weeks (left) and 12 weeks (right) in the soil.
Figure 7 shows the phenotype of plants transfected with tobacco plant line PNGase T-11 elicited 4 weeks (left) and 8 weeks (right) in soil.
FIG. 8 shows the overexpression of PNGase T-specific primers (A) and PNGase T mRNAs for quantitative RT-PCR to examine the expression level of PNGase T mRNA from transgenic tobacco plants overexpressing PNGase T B).
FIG. 9 is a graph showing changes in phenotype by measuring plant height, leaf area, number of sections and photosynthetic rate of transgenic tobacco plants overexpressing PNGase T. FIG.

Hereinafter, the present invention will be described in more detail by way of examples and the like. It is to be understood that the present invention is not limited by these examples in view of the spirit and scope of the present invention, It will be obvious to you.

Materials and methods

In this experiment, tomato ( Solanum lycopersicum L. cv MicroTom) cDNA synthesized from the total RNA isolated from the leaves was used in Chonnam National University and used for the isolation of the tomato PNGase T gene. Materials for quantitative real time RT-PCR analysis were tomatoes ( Solanum lycopersicum L. cv Styx TY) Total RNA was isolated from flesh and cDNA was synthesized.

< Example  1> Total RNA isolation and cDNA synthesis

Nitrification is the process of removing sugars from glycoproteins in plant cells. The role of N-glycosylation in the physiological processes of plant differentiation, development, and growth has hardly been studied. (Nakamura et al. 2009), PNGase T cDNA was isolated from tomato leaves with a high activity of desquamating activity, while long-chain N-glycosylation of mannose increased in the aging process of tomato pulp .

In order to investigate the expression of PNGase T in tomato, mRNA was isolated from pulp and the gene expression of PNGase T was examined by qRT-PCR according to development and aging of pulp. FIG. 1 shows the result of examining the expression level of PNGase T mRNA expressed during ripening of tomato pulp from green to red. As shown here, the expression level of the PNGase T gene was increased during the aging of the pulp.

Total RNA isolation was carried out using 1 ml of TRI Reagent. (Molecular Research Center, Inc.) for 30 seconds and then left at room temperature for 5 minutes. The supernatant obtained by centrifugation at 12,000 rpm for 10 minutes at 4 ° C was transferred to a new tube, mixed with 0.1 ml of 1-bromo-3-chloropropane (BCP), mixed for 30 seconds, and left at room temperature for 5 minutes. After centrifugation at 12,000 rpm for 15 min at 4 ° C, the supernatant was transferred to a new tube, and 0.5 ml of isopropanol was added. The RNA was precipitated at room temperature for 10 min, centrifuged at 12,000 rpm for 10 min at 4 ° C, The RNA pellet was washed with 70% ethanol and dissolved in 0.1% diethyl pyrocarbonate (DEPC) distilled water. The RNA purity and amount were determined using a spectrophotometer (UV-1601; Shimadzu, Japan) Respectively. The first strand cDNA required for gene cloning and gene expression analysis was synthesized using the PrimeScript ^™ 1st strand cDNA synthesis kit (Takara Bio Inc. Japan). As a template for cDNA synthesis, 1 ㎍ of total RNA was used and 1st strand cDNA was synthesized using oligodT primer.

< Example  2> cDNA Cloning and  Sequencing

Using the synthesized cDNA, the PNGase T gene located in the tomato genome (Locus: LOC100316898 [3,613,961 nucleotid (nt) to 3,616,373 nt, GenBank accession No. NW_004194361) was amplified by an outer primer (forward, 5'- TCTACAGTATATTTTAGTCA- 5'-AGCTTTAGCAGCAAATGAGTG-3 ') and the inner primer (forward, 5'-CCATGGCTATGGCTGCCTCCTCTTCC -3'; reverse, 5'-TCTAGATCAACCAGGAAGAGACCGC-3 ') to prepare a PCR reaction was amplified by nested PCR ^® 4-TOPO cloning kit ( Invitrogen, USA). The resulting plasmids were introduced into Escherichia coli DH5α by thermal shock method, cultured through antibiotic screening, and plasmids were extracted using plasmid midi kit (Qiagen, Germany). The extracted plasmids were separated by electrophoresis, and the nucleotide sequences were confirmed and genes were registered in NCBI (GanBank accession No. KM401550, SEQ ID NO: 1).

The ORF of the isolated PNGase T was composed of 1,767 bp and 588 amino acids in total, the expected molecular weight was 65.8 kDa and the expected pI value was 8.69.

< Example  3> Real time quantitative RT- PCR  ( qRT - PCR ) analysis

For real-time qRT-PCR analysis, the ripening process of tomato fruit was harvested by dividing the stage into 6 stages. The first stage was mature green (MG), the second stage was turning (T) (OR), Lignt-Red (LR) in step 5, and Mature Red (MR) in step 6. Total RNA was extracted from tomato fruits at each stage and used for cDNA synthesis. The specific primers of the genes to be analyzed using qRT-PCR were constructed using the manufacturer's method (Table 1). Real-time qRT-PCR analysis was performed using Chromo 4TM Continuous Fluorescence Detector (MJ Research, MA, USA) . The ^{Strip PCR tube iQTM SYBR ® Green supermix} (2 ×, Bio-Rad, USA) 10 μl, Forward primer 0.25 μl, Reverse primer 0.25 μl, into the synthesized cDNA 1 μl on the number of sterilization treatment with DEPC such that 20 μl . The PCR conditions were 95 ° C for 15 min, 45 ° C for 30 sec at 95 ° C, 30 sec at 57 ° C and 30 sec at 72 ° C, followed by 10 min at 72 ° C. And the melt curve stage was performed for 1 second. Data were analyzed using Opticon Monitor ™ software version 3.1 (MJ Research, MA, USA) based on Delta Delta CT values provided by Applied Biosystems.

The nucleotide sequence of PNGase T isolated from Cultivar Micro Tom Tomato was different from that of PNGase T gene (GenBank accession No. NW_004194361) on the chromosome 10 of heinz 1706 tomato, which provided the gene information of the primers used for the analysis, with one nucleotide base . However, in comparison with the nucleotide sequence information of PNGase T- Le (GenBank accession No. FJ804752) isolated from cultivar Momotaro tomato (Hossain et al. 2010), 3 bases of the 1,767 bp nucleotide sequence of the ORF were different, 586 out of 588 were identical, indicating 99.7% identity. At the N-terminal region of PNGase T, there was a 21-amino acid signal sequence that targets the ribosome as an endoplasmic reticulum. The amino acid sequence motif (NxS / T) of asparaginic acid-X-serine / threonine, which is the N -glycosylation site of the glycoprotein, is a total of nine amino acid sequence motifs (amino acid sequence regions 266, 386, 397, 447, 481, 489, 551, 556, 570) was present, one less than the PNGase T-Le with 10 N-glycosylation sites.

In particular, when the nucleotide sequences of the 12 PNGase T gene members reported in the past were obtained from GenBank, there were 6 regions with relatively high conserved amino acid sequence. It is determined that these sites are the active sites of PNGase T, and it was decided to use them for overexpression of this PNGase T gene.

As a result of comparing the PNGase T genes isolated from several previously reported plants, the amino acid sequence identity was about 61-62% identical to the castor bean, poplar, and grape, and the two gene members of the Arabidopsis 54% and 38%, respectively. And 50% and 43% identity with the two PNGase T gene members of rice. The other cotyledons, sorghum and maize, showed about 42% identity with PNGase T. Therefore, in the phylogenetic analysis of 12 PNGase T genes isolated from 10 plants, PNGase T showed the highest affinity with PNGase- Le , followed by PNGase T with grape Respectively.

FIG. 2 shows phylogenetic tree of amino acid sequence analysis of PNGase T gene isolated from 12 plant species.

Recently, the AtPNG1 gene, which is a PNGase T acting on ERAD, which is a process of degradation of glycoprotein due to an error of protein folding, has been isolated from Arabidopsis thaliana. Although there is no amino acid sequence identity with the previously reported PNGase T gene, Saccaromyces When the AtPNG1 gene was introduced into the S. cerevisiae mutant, the activity of N-glycanase was restored (Diepold et al. 2007). However, AtPNG1 is a protein belonging to the transglutaminase-like superfamily present in the cytoplasm (Suzuki et al. 2002), but it has been reported that ERP is involved in maintaining the accuracy of the tertiary structure of the protein through cleavage of the N- More research is needed to determine the role of N-glycanase in the ERAD process. AtPNG1 and PNGase T are thought to be isoforms that are known as peptide-N (4) - (N-acetyl-beta-glucosaminyl) asparagine amidase but do not have nucleotide sequence identity.

Although PNGase T has a physiological function of degrading protein with the error of protein folding, the production of plants overexpressing PNGase T has not yet been reported, and the study of physiological phenotypic changes due to overexpression of PNGase T Have not yet been reported. Therefore, PNGase T is produced by overexpressing genes of PNGase T F of microorganisms, which are relatively far away from each other. It is presumed that the overexpression of plant PNGase T affects the growth and development of the plant, even if it has the specificity to decompose the glycoprotein in order to maintain the accuracy of the tertiary structure of the protein of PNGase T. However, in order to use the plant as a molecular agricultural platform, it is necessary to remove the oligosaccharide attached to asparaginic acid residues, which may cause immune side effects in the human body in order to produce high value-added protein for use in plants. For this purpose, it is necessary to produce PNGase T protein from a plant PNGase T gene which can act more effectively on a plant protein in order to eliminate the glycosylation formed in the plant-produced protein.

In addition, as another use method, a specific promoter expressed at a specific time is used to transform the PNGase T If a plant which expresses PNGase T and a specific medicinal protein simultaneously is prepared by introducing a specific medical recombinant protein gene after preparing the expression inducing crop, the step of removing N-glycans after protein separation may be omitted, This will simplify the processing of the recombinant protein and produce recombinant proteins for medical use more economically.

In particular, the nucleotide sequence similarity of PNGase T gene is only about 60%. Therefore, when a recombinant protein is produced in a tobacco plant or a tomato plant as a branch plant, a tomato-derived PNGase T having relatively high protein similarity is used for cutting N- May be more effective than PNGase A derived from almonds or PNGase F derived from bacteria.

< Example  4> Production of recombinant expression vector containing PNGase T gene

In order to utilize molecular agriculture to produce PNGase T gene in actual plants, PNGase T should be stably produced in the plant. Therefore, a recombinant gene construct was prepared so that PNGase T was overexpressed in the plant. The gene was designed to introduce the full-length PNGase T gene isolated from tomato into the recombinant protein expression system (pTRAkt vector).

FIG. 3 is a graph showing the results of the addition of a KDEL amino acid sequence that binds six histidines for protein cleavage to a pTRAkt vector for expression of recombinant proteins (PNGase T) derived from tomato and induces residues in the endoplasmic reticulum Is a schematic diagram of a 35S :: PNGase T consturct designed to express a recombinant gene by the 35S promoter.

Next, in order to induce the expression of the gene introduced from the plant cell to a high state, a signal peptide to be targeted as an endoplasmic reticulum was required. The 21 amino acids at the N-terminus of PNGase T were judged to be a signal sequence. The ER retention signal, KDEL sequence, was also added to maximize the synthesis of the PNGase T protein by accumulating the resulting protein at high efficiency in the endoplasmic reticulum. In addition, the nucleotide sequence of His6-tag (CATCATCATCATCATCAT) was added to facilitate the purification of PNGase T protein.

 As a plant cell promoter for producing a high-efficiency pharmaceutical protein expressed in plants, it has been discussed with Dr. R. Fisher, a director of the Fraunhofer Institute in Germany, and a plant expression having a modified 35S promoter that further improves the expression efficiency of the 35S promoter PTRAkt vector was used. Using this vector, constructs of the previously designed recombinant PNGase T were inserted between the restriction enzymes NcoI and XbaI in a plant transformation vector and ligation was carried out to prepare plant cell expression constructs. Finally, Confirming cloning into a plant expression vector. (See FIG. 3).

< Example  5> Production of Transgenic Tobacco Plant with Tomato PNGase T

The plant expression vector into which the tomato PNGase T gene was introduced was subjected to tri-parental mating method using Agrobacterium Two colonies transformed with tumafaciens GV3101 were cultured, and plexamide DNA was isolated by a quick-screen method based on the alkali lysis method, and restriction enzyme was applied to confirm the introduction of the PNGase T gene. Subsequently, Agrobacterium with the tomato PNGase T gene introduced The leaf slices (0.5 cm ² ) of wild-type tobacco were infected in the culture medium (OD ₆₀₀ = 1.5) for 1 minute and then cultured for 2 days in the MS shooting medium. After air-conditioning, tobacco leaves were transfected with MS cultivation medium supplemented with cefotaxime (250 mg / L) and kanamycin (100 mg / L) at 2-week intervals. The transformants surviving the selection of antibiotics were transferred to roots induction medium and purified to soil. Continuous transfection experiments were repeated, and 531 tobacco leaf slices were infected. Twenty-five transformants surviving the formation of shoots from antibiotic screening The transformation efficiency was 4.7%. As a result, 15 independent transgenic tobacco plants (PNGase T- 10 , PNGase T- 11 ) were finally selected by transferring 15 surviving transformants to the root induction medium.

In order to confirm that the recombinant PNGase T transgenic plant has the PNGase T gene of tomato which is a transgene, genomic DNA is isolated and then recombinant PNGase T Six specific primers were designed based on the (+ His6 + KDEL ) gene constructs (Table 2).

The primers of SEQ ID NO: 1 and SEQ ID NO: 2 are primers in which the entire length of the tomato PNGase T gene inserted into the tobacco plant can be amplified and primers in which the sites in SEQ ID NO: 3 to SEQ ID NO: 6 pTRAkt vector are amplified.

Genomic DNA was isolated from leaf tissues of transgenic tobacco plants of line PNGase T- 10 and line PNGase T- 11 , and genomic DNA PCR was performed using each specific primer to determine whether the PNGase T gene was well inserted into the tobacco plant and whether agarose gel electrophoresis.

FIG. 5 shows the result of sequencing of the genomic DNA of the transgenic tobacco lines of two lines, followed by electrophoresis of the PCR product obtained by performing PCR using specific primers, . As shown here, when PCR was performed using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, the whole fragment of the tomato PNGase T gene was amplified and a band of 1,811 bp was amplified in the transformed tobacco plants of line PNGase T- 10 and line PNGase T -11 (See FIG. 5A). When PCR was performed using the primers of the vector sequence surrounding the PNGase T gene inserted in the pTRAkt vector, the transgenic tobacco plants transformed with line PNGase T - 10 and line PNGase T- 11 were found to contain 2,001 bp (see FIG. 5B) and 2,332 bp See Fig. 5C), respectively. When PCR was performed with the primer of the neomycin phosphotransferase II (NPT II) gene, which is an antibiotic kanamycin gene, a band of 780 bp was detected in the transformed tobacco plant of line PNGase T- 10 and line PNGase T -11 (see FIG. 5D).

Transgenic tobacco plants transfected with tomato-derived PNGase T (+ His6 + KDEL) gene were finally obtained through genomic DNA PCR analysis in Table 3 above to obtain two lines (PNGase T- 10 and PNGase T- 11 ).

These two lines of transgenic tobacco plants were subjected to purification and then grown in the greenhouse to confirm the phenotype.

FIG. 6 shows the phenotype of the transformed tobacco plant line PNGase T-10 after 6 weeks (left) and 12 weeks (right) in the soil.

Figure 7 shows the phenotype of plants transfected with tobacco plant line PNGase T-11 elicited 4 weeks (left) and 8 weeks (right) in soil.

As can be seen, the PNGase T-10 line showed a slight decrease in growth rate compared to wild-type plants and leaves, but the height of the stem was slightly larger and flowed faster than the wild type (FIG. 6) The growth rate was similar to that of wild type (Fig. 7).

< Example  6> Transformation Tobacco plants  Tomato-derived PNGase T mRNA  Expression analysis

Transgenic tobacco plant lines PNGase T- 10 and PNGase T- 11 , in which tomato-derived PNGase T (+ His6 + KEDL ) gene was introduced, were harvested and total RNA was isolated and cDNA was prepared and subjected to qRT-PCR and semiquantitative RT -PCR was performed to analyze the amount of mRNA of PNGase T from tomato in tobacco plants.

After total RNA isolation, PrimeScript ^TM First strand cDNA was synthesized by using 1st strand cDNA synthesis kit (Takara Bio Inc. Japan). 1 μg of total RNA was used as the template for cDNA synthesis and 1st strand cDNA was synthesized using oligo dT primer.

For analysis of real time quantitative RT-PCR (qRT-PCR), multiple sequence analysis of PNGase T gene of tobacco and tomatoes was performed to prevent amplification of endogenous PNGase T gene in tobacco plants. A specific primer sequence of tomato PNGase T was selected for other sites (see FIG. 9A). Forward primer: 5'- GAT GAT TCG TGC TGC TAC TCC-3 'and reverse primer: 5'-TCA ACC AGG AAG AGA CCG CTT-3' as specific primers for tomato PNGase T gene in transgenic tobacco plants Real-time qRT-PCR analysis was performed using a Chromo 4 ^TM Continuous Fluorescence Detector (MJ Research, MA, USA). A house keeping gene, actin, was used as a reference gene.

FIG. 8 shows the overexpression of PNGase T-specific primers (A) and PNGase T mRNAs for quantitative RT-PCR to examine the expression level of PNGase T mRNA from transgenic tobacco plants overexpressing PNGase T B).

Tomato results verify the transcript amount of lines PNGase T -10 PNGase T gene transgenic tobacco plants was a transcript amount of tomato PNGase T gene compared to the wild-type 2.2-fold increase in line PNGase T -11 transgenic tobacco plants is more than WT 3.3 fold (see Fig. 8B).

Although the tobacco PNGase T gene was also expressed in the wild-type tobacco plants, the expression level of the total PNGase T gene in the transgenic plants was increased in both lines because of the expression of the introduced gene (see FIG. 8C).

Table 4 and FIG. 9 show changes in phenotype by measuring plant height, leaf area, number of sections and photosynthetic rate of transgenic tobacco plants overexpressing PNGase T.

In the phenotype of transgenic tobacco plants, the phenotype of PNGase T-10 was slightly increased compared to the wild-type plant at the same growing stage, but the leaf area and number of leaves (Fig. 9A). In addition, PNGase T-11, compared to wild-type plants at the same growing stage, also significantly deteriorated growth characteristics such as plant length, leaf area, and number of sections.

However, there was no difference in the photosynthetic rates of wild-type plants of the same growth period in both transgenic tobacco plants (FIG. 9B).

Therefore, two kinds of tobacco plants (PNGase T- 10 and PNGase T- 11 ) transformed with tomato-derived PNGase T gene were able to introduce the tomato PNGase T gene ( 35S :: PNGase T- His6 - KDEL ) into the chromosome of the tobacco plant And the expression of mRNA was further increased than that of wild type. Especially, PNGase T- 11 transgenic tobacco plants showed better expression of PNGase T gene.

As a result of the above examples, it has been confirmed that PNGase T derived from tomato is expressed in leaves of tobacco plants.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Industry-academic cooperation foundation of sunchon national university <120> Transgenic plants transformed with PNGase T gene derived from          tomato and methods of producing PNGase T protein by using the          same <130> PA-15-0202 <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PNGase T-S1 primer <400> 1 ggatggctgc ctcctcttcc 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PNGase T-AS1 primer <400> 2 ggcaggaaga gaccgctttt tg 22 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> pTRAkt AS primer <400> 3 ctaagggttt cttatatgct caac 24 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P35SS-F primer <400> 4 atacatggtg gagcacgaca 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NPTII-F primer <400> 5 gaagaactcg tcaagaaggc g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NPTII-R primer <400> 6 gatggattgc acgcaggttc t 21 <210> 7 <211> 1767 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of PNGase T <400> 7 atggctgcct cctcttccat tttcttagtt tatttcttgc tacccttatt ttccacagca 60 accctccaca gtacttcttc tctttataga tcccaactca tcactcagca agaatcttca 120 cccaaaaatg ccacccccac aacatttttt gaagtcacaa aacctataaa attacccaaa 180 accaaacctt tttcacacct aattcttgaa catgattttg gttccaccta cagaaaaccc 240 ccaattcttg ctaactacac accccctttt aattgcccat ctcagaaatt ttccaaaatt 300 gtgctggaat ggagagcaac ttgtaaaggt agacaatttg atagaatttt tggtgtttgg 360 gttagtgggg ttgagatttt cagaagctgc actgctgaac caactaaaaa tgggatcttt 420 tggactgtca agaaggacat tactaggtat tcttctttgc ttatgaaaaa tcaaatcttt 480 gctgtttatt tagggaatat tgttgatagt acatatactg gtgtgtacca tgtggaaatt 540 tttgttcact tttatcctgc taaagtgaga ttgggtggat ttgattctgg ggctgatttg 600 attgttccca tttcaagaaa tatgcatttg aatgatgggt tgtggtttga gattgaaaat 660 tcagcagatg tacagtcaaa ggatttcaag attcccccaa atgtgtatag ggctgtattg 720 gaagtttatg tttcatttca tgagaatgat gagttttgga atgggaatcc acctaatgag 780 tatataagtt cgaataatct tagtatcgcg gggaatggag ctttcaggga ggtggtggtt 840 agtttggatg aaatggtagt tggtgtagtt tggcctttta ctgtgatcta tactgggggt 900 gttaatcccc tcttttggag accgattagt ggaattggat cgttcgatct tccttcttat 960 gacattgaaa ttaccccgtt gttaggaaag attttagatg gaaatagtca taagatttca 1020 tttggagtca cggatgctct gaacgtgtgg tatgttgatg caaatttgca tctttggttg 1080 gacgggaaaa gtaagaaaac ggaagggaag ttgttgagat acagcttatt gcccctttct 1140 ttatcggtgc tgaccaattt tacaggtttt gatggatcct ttatcacgaa tgctagtagg 1200 tccatcacat tgaccggaat ggtgaagtcg tcttatggaa ctatcactac taagtcgtct 1260 caaagtttaa gttatagcaa ccatatggta atgggaaatg aagggaactt gcaaatagtg 1320 gatcagataa tcgaattcaa tgatactgtt tatgccacga cgccatcttc ttatgttcac 1380 tcgcttgagt ccttcaaaaa gtatctgttg aagttgtatt ctgacaatgt agatcaagga 1440 aaccaaagct atacctcgat atcaaacttg acgttgggat tagatgacaa gagggtaaag 1500 ggttccaagt atggatcctc agtcagctct gtgaataatt tgcaaaacgc gcagggctat 1560 atgattgtaa agggccactt agtagtcaaa ggacttggaa gtacccaaca agtatataaa 1620 cataacgatg attcgtgctg ctactccagg aacataagca gctcaaatta cacaatactt 1680 tacgataagg taagcaatag ctgcagcaat agtacttggt ctcattggcc tttgaggatt 1740 gacaaaaagc ggtctcttcc tggttga 1767 <210> 8 <211> 588 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of PNGase T <400> 8 Met Ala Ala Ser Ser Ile Phe Leu Val Tyr Phe Leu Leu Pro Leu   1 5 10 15 Phe Ser Thr Ala Thr Leu His Ser Thr Ser Ser Leu Tyr Arg Ser Gln              20 25 30 Leu Ile Thr Gln Gln Glu Ser Ser Pro Lys Asn Ala Thr Pro Thr Thr          35 40 45 Phe Phe Glu Val Thr Lys Pro Ile Lys Leu Pro Lys Thr Lys Pro Phe      50 55 60 Ser His Leu Ile Leu Glu His Asp Phe Gly Ser Thr Tyr Arg Lys Pro  65 70 75 80 Pro Ile Leu Ala Asn Tyr Thr Pro Pro Phe Asn Cys Pro Ser Gln Lys                  85 90 95 Phe Ser Lys Ile Val Leu Glu Trp Arg Ala Thr Cys Lys Gly Arg Gln             100 105 110 Phe Asp Arg Ile Phe Gly Val Trp Val Ser Gly Val Glu Ile Phe Arg         115 120 125 Ser Cys Thr Ala Glu Pro Thr Lys Asn Gly Ile Phe Trp Thr Val Lys     130 135 140 Lys Asp Ile Thr Arg Tyr Ser Ser Leu Leu Met Lys Asn Gln Ile Phe 145 150 155 160 Ala Val Tyr Leu Gly Asn Ile Val Asp Ser Thr Tyr Thr Gly Val Tyr                 165 170 175 His Val Glu Ile Phe Val His Phe Tyr Pro Ala Lys Val Arg Leu Gly             180 185 190 Gly Phe Asp Ser Gly Ala Asp Leu Ile Val Pro Ile Ser Arg Asn Met         195 200 205 His Leu Asn Asp Gly Leu Trp Phe Glu Ile Glu Asn Ser Ala Asp Val     210 215 220 Gln Ser Lys Asp Phe Lys Ile Pro Pro Asn Val Tyr Arg Ala Val Leu 225 230 235 240 Glu Val Tyr Val Ser Phe His Glu Asn Asp Glu Phe Trp Asn Gly Asn                 245 250 255 Pro Pro Asn Glu Tyr Ile Ser Ser Asn Asn Leu Ser Ile Ala Gly Asn             260 265 270 Gly Ala Phe Arg Glu Val Val Val Ser Leu Asp Glu Met Val Val Gly         275 280 285 Val Val Trp Pro Phe Thr Val Ile Tyr Thr Gly Gly Val Asn Pro Leu     290 295 300 Phe Trp Arg Pro Ile Ser Gly Ile Gly Ser Phe Asp Leu Pro Ser Tyr 305 310 315 320 Asp Ile Glu Ile Thr Pro Leu Leu Gly Lys Ile Leu Asp Gly Asn Ser                 325 330 335 His Lys Ile Ser Phe Gly Val Thr Asp Ala Leu Asn Val Trp Tyr Val             340 345 350 Asp Ala Asn Leu His Leu Trp Leu Asp Gly Lys Ser Lys Lys Thr Glu         355 360 365 Gly Lys Leu Leu Arg Tyr Ser Leu Leu Pro Leu Ser Leu Ser Val Leu     370 375 380 Thr Asn Phe Thr Gly Phe Asp Gly Ser Phe Ile Thr Asn Ala Ser Arg 385 390 395 400 Ser Ile Thr Leu Thr Gly Met Val Lys Ser Ser Tyr Gly Thr Ile Thr                 405 410 415 Thr Lys Ser Ser Gln Ser Leu Ser Tyr Ser Asn His Met Met Met Gly             420 425 430 Asn Glu Gly Asn Leu Gln Ile Val Asp Gln Ile Ile Glu Phe Asn Asp         435 440 445 Thr Val Tyr Ala Thr Thr Pro Ser Ser Tyr Val His Ser Leu Glu Ser     450 455 460 Phe Lys Lys Tyr Leu Leu Lys Leu Tyr Ser Asp Asn Val Asp Gln Gly 465 470 475 480 Asn Gln Ser Tyr Thr Ser Ile Ser Asn Leu Thr Leu Gly Leu Asp Asp                 485 490 495 Lys Arg Val Lys Gly Ser Lys Tyr Gly Ser Ser Ser Val Ser Ser Val Asn             500 505 510 Asn Leu Gln Asn Ala Gln Gly Tyr Met Ile Val Lys Gly His Leu Val         515 520 525 Val Lys Gly Leu Gly Ser Thr Gln Gln Val Tyr Lys His Asn Asp Asp     530 535 540 Ser Cys Cys Tyr Ser Arg Asn Ile Ser Ser Ser Asn Tyr Thr Ile Leu 545 550 555 560 Tyr Asp Lys Val Ser Asn Ser Cys Ser Asn Ser Thr Trp Ser His Trp                 565 570 575 Pro Leu Arg Ile Asp Lys Lys Arg Ser Leu Pro Gly             580 585

Claims (7)

An expression vector for expression of a plant comprising a PNGase T gene having the nucleotide sequence of SEQ ID NO: 7. The method according to claim 1,
Wherein the PNGase T gene is derived from tomato. The method according to claim 1,
pTRAkt-PNGase T expression vector. A plant transformed with an expression vector for plant expression comprising the PNGase T gene according to claim 1. 5. The method of claim 4,
Wherein said plant is tobacco. A method for transforming a plant using an expression vector for plant expression comprising the gene of PNGase T according to claim 1. A method for producing a recombinant PNGase T protein by transforming a plant with an expression vector for plant expression comprising the gene of PNGase T according to claim 1, followed by culturing.

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