KR20060034010A - A novel asparaginase originated from soybean, a gene enconding the same, and use thereof - Google Patents

Abstract

The present invention is asparaginase involved in nitrogen metabolism derived from soybean (asparaginase), the gene encoding the asparaginase, the recombinant vector containing the gene, the microorganism transformed with the recombinant vector and transfected with the gene It relates to a low temperature resistant plant.

Asparaginase and its genes according to the present invention are useful not only for molecular genetic studies on nitrogen metabolism of plants, but also for industrially producing low temperature resistant transgenic plants.

Soybean, Asparaginase, Low Temperature, Resistant, Plants

Description

A Novel Asparaginase Originated from Soybean, a Gene Enconding the Same, and Use Thereof}             

Figure 1 compares the homology (homology) between the novel asparaginase gene according to the present invention and the gene previously known.

Figure 2 shows the results of separating the whole RNA from the soybean treated with low temperature and various stress in order to determine the expression pattern of the different asparaginase gene in the present invention and Northern blot.

Figure 3 is a photograph showing the Southern blot (Southern blot) results for the asparaginase gene.

4 is a map of a recombinant transfer vector containing an asparaginase gene according to the present invention.

5 is an electrophoresis (SDS-PAGE) photograph of a protein expressed in E. coli transformed with a recombinant vector containing an asparaginase gene according to the present invention.

6 is a quantitative determination of asparinase according to the present invention to measure the activity for each concentration.

Figure 7 shows the hourly activity of asparinase according to the present invention.

8 shows the temperature-specific activity of asparinase according to the present invention.

9 compares the activity of expressed asparaginase by cloning in the sense and antisense directions, respectively.

The present invention is asparaginase involved in nitrogen metabolism derived from soybean (asparaginase), the gene encoding the asparaginase, the recombinant vector containing the gene, the microorganism transformed with the recombinant vector and transfected with the gene It relates to a low temperature resistant plant.

Beans are known for their origin in Northeast Asia, including the Korean Peninsula, and are widely used as food sources or foods as an excellent source of vegetable protein. In addition, the value of soybeans as a healthy functional food has recently attracted great attention. Soybeans are known to contain various physiologically active substances such as isoflabone, phytic acid, saponins, protease inhibitors, asparagine, vegetable sterols and phenolic compounds. It is reported to have various physiological activities such as disease prevention.

According to the main research, the effect of low temperature in the plant is known to be water deficiency phenomenon as the intracellular water escapes out of the cell due to the imbalance of the moisture potential by freezing between the plasma and the cell membrane. This affects the mechanical breakdown of tissues due to freezing of cells and the metabolism of cells due to the imbalance of moisture, resulting in the loss of plant growth and the death of tissues. However, there are few reports of cold stress resistant proteins in soybeans.

Thus, the present inventors isolated the novel asparaginase gene from the cDNA gene library of fragments induced by cold stress of soybean through RACE PCR technique, expressing the gene in recombinant Escherichia coli, and then separating the purified recombinant protein substrate. As a result, the present invention was confirmed to have the activity of asparaginase.

It is a primary object of the present invention to provide asparaginase isolated from soybean and genes encoding the same.

Another object of the present invention is to provide a recombinant vector containing the gene and a microorganism transformed with the recombinant vector.

Still another object of the present invention is to provide a low temperature resistant plant transfected with the gene.

In order to achieve the above object, the present invention provides an asparaginase having the amino acid sequence of SEQ ID NO: 2 and a gene encoding the same ( SLTI182) . In the present invention, the gene may be characterized by having a nucleotide sequence of SEQ ID NO: 1, the gene may be characterized in that the low temperature resistance protein (asparaginase) is encoded.

The present invention also provides a recombinant vector containing the gene and a transformed microorganism obtained by introducing the recombinant vector into a host cell selected from the group consisting of bacteria, yeast and fungi.

In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, preferred host cells are prokaryotic cells. Suitable prokaryotic host cells include E. coli DH5α, E. col JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli B, E. coli B21, and the like. It includes. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the aforementioned E. coli , strains of the genus Agrobacterium, such as Agrobacterium A4, bacilli , such as Bacillus subtilis , Salmonella typhimurium or Serratia marghesen another variety of enteric bacteria and Pseudomonas (Pseudomonas) in strains such as marcescens) may be used as host cells.

Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation (Neumann et al., EMBO J., 1: 841, 1982) can also be used for transformation of these cells.

As the vector used for overexpression of the gene according to the present invention, an expression vector known in the art may be used, and it is preferable to use a pET family vector (Novagen). When the cloning is performed using the pET family vector, histidine groups are bound to the ends of the expressed protein, and thus the protein can be effectively purified. In order to separate the expressed protein from the cloned gene, a general method known in the art may be used, and specifically, it may be separated by a chromatographic method using Ni-NTA His-binding resin (Novagen). . In the present invention, the recombinant vector may be characterized in that the pET-SLTI66, the host cell may be characterized in that E. coli or Agrobacterium.

The present invention also provides a method for producing asparaginase having the amino acid sequence of SEQ ID NO: 2 characterized by culturing the transformed microorganism. More specifically, the present invention provides a method of controlling the expression of (a) a DNA sequence encoding an asparaginase having the amino acid sequence of SEQ ID NO: 2 operatively linked to this DNA sequence to regulate its expression Inserting into a vector comprising an expression control sequence; (b) transforming the host with a recombinant expression vector formed therefrom; (c) culturing the resulting transformants under medium and conditions appropriate for causing the DNA sequence to be expressed; (d) recovering the substantially pure low temperature resistant protein from the culture, the method for producing a low temperature resistant asparaginase.

The expression “expression control sequence” in the present invention means a DNA sequence essential for the expression of a coding sequence operably linked in a particular host organism. Such regulatory sequences include promoters for performing transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. For example, suitable control sequences for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells include promoters, polyadenylation signals, and enhancers. The factor that most influences the amount of gene expression in the plasmid is the promoter. As the promoter for high expression, an SRα promoter, a promoter derived from cytomegalovirus, and the like are preferably used. To express the DNA sequences of the invention, any of a wide variety of expression control sequences can be used in the vector. Useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter regions of phage lambda, fd code protein Regulatory region of, promoter for 3-phosphoglycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, promoter of yeast alpha-crossing system and gene expression of prokaryotic or eukaryotic cells or viruses Other sequences known to modulate and various combinations thereof. The T7 promoter can be usefully used to express the proteins of the invention in E. coli.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to allow gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

As used herein, the term “expression vector” generally refers to a fragment of DNA that is generally double stranded as a recombinant carrier into which fragments of heterologous DNA have been inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once expression vectors are within a host cell, they can replicate independently of the host chromosomal DNA and several copies of the vector and their inserted (heterologous) DNA can be produced.

As is well known in the art, to raise the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the host cell is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.

Host cells transformed or transfected with the expression vectors described above constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. As used herein, the term "transfection" means that the expression vector is accepted by the host cell whether or not any coding sequence is actually expressed.

Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. In selecting expression control sequences, several factors must be considered. For example, the relative strength of the sequence, the controllability, and the compatibility with the DNA sequences of the present invention should be considered, particularly with regard to possible secondary structures. Single cell hosts host a selected vector, the toxicity of the product encoded by the DNA sequence of the invention, the secretory properties, the ability to accurately fold the protein, culture and fermentation requirements, the product encoded by the DNA sequence of the invention. Should be selected in consideration of factors such as the ease of purification. Within the scope of these variables, one skilled in the art can select a variety of vector / expression control sequence / host combinations capable of expressing the DNA sequences of the invention in fermentation or large scale animal culture. As a screening method when cloning the cDNA of a protein according to the present invention by expression cloning, a binding method (binding method), a panning method, a film emulsion method and the like can be applied.

By "substantially pure" in the definition of the present invention is meant that the protein according to the invention and the DAN sequence encoding it are substantially free of other proteins derived from bacteria.

The present invention also provides a low temperature resistant plant transfected with the gene. Transfection of plants can be achieved by conventional methods using Agrobacterium, viral vectors and the like. For example, a microorganism of the genus Agrobacterium may be transformed with a recombinant vector containing a gene according to the present invention, and then the transformed Agrobacterium microorganism may be infected with tissues of a target plant to obtain a transfected plant. More specifically, (a) pre-culture the explant of the plant (preplant), and then transfected by co-culture with the transformed Agrobacterium; (b) culturing the transfected explants in a callus induction medium to obtain callus; And (c) cutting the obtained callus, and culturing it in shoot induction medium to form shoots, thereby producing a transfected plant.

In the present invention, the term "explant" refers to a slice of tissue cut out of a plant, and includes cotyledon or hypocotyl. The explants of the plant used in the method of the present invention may be cotyledons or hypocotyls, it is more preferable to use the cotyledons obtained by germinating in MS medium after disinfecting and washing the seeds of the plants.

Transfection target plants usable in the present invention include, but are not limited to, tobacco, tomatoes, peppers, beans, rice, corn, and the like. In addition, even if the plant used for transformation is a sexually propagating plant, it will be apparent to those skilled in the art that it can be repeatedly reproduced by tissue culture or the like.

The present invention cloned a new asparaginase known to be involved in nitrogen metabolism, that is, a major influence on the formation of functional proteins, and revealed its function. Asparaginase in plants is known as an enzyme to hydrolyze into aspatatic acid and ammonia with asparagine as a substrate, and acts as a regulator of asparagine and aspatate in plants. Hydrolyzed ammonia is also a useful source of nitrogen for nitrogen metabolism and has been reported to affect L-glutamine and L-glutamate formation. In the present invention, the cloned SLTI182 showed a high similarity of 84% with the asparaginase of the baby pole, and was confirmed to exist on the soybean genome through Southern blot and fluorescence in situ hybridization (FISH) analysis. Northern blot results showed that the expression of SLTI182 gene was initially induced and responded temporarily to cold stress. In order to examine the function of SLTI182 , the activity was measured after mass expression of pET- SLTI182 in E. coli , and it was confirmed that SLTI182 had the activity of L-asparaginase.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Analysis of Soybean-Derived Asparaginase Gene Induced by Low Temperature

After extracting poly (A +) mRNA from stress and non-stressed leaves in soybeans grown for more than three weeks in growth, PCR-Select cDNA Subtraction (Clontech) used for fragments expressed only by stress (cold, salt). cDNA was obtained. The obtained cDNA was cloned into pCR2.1 (Invitrogen), which is a subcloning vector, and plasmid DNA was isolated using a plasmid purification kit (purchased by NucleoGen). Sequencing was performed by requesting macrogen. Based on the sequencing results, the entire nucleotide sequence was obtained through RACE PCR.

As a result, the cDNA of the asparaginase gene induced by low temperature stress according to the present invention was composed of a total sequence of 978bp (SEQ ID NO: 1), and deduced from it was composed of 326 amino acids (SEQ ID NO: 2) there was. As a result of investigation and comparison through DNASIS and BLAST programs provided by NCBI, GenBank, EMBL and SwissProt databases, the nucleotide sequence and amino acid sequence were found to be a new gene ( STLI 182 ) induced by cold stress (FIG. 1).

Example 2: Expression of a Novel Asparaginase Gene

Soybeans grown for more than three weeks in growth were subjected to various stresses, hormones, and cold treatments over time, and total RNA was isolated from the leaves using the HOT phenol method. The total RNA isolated was electrophoresed on a 1.0% formaldehyde gel, and then the gel was transferred to a nylon membrane (Schleicher & Schuell) purchased with 20 × SSC solution (3M NaCl, 0.3M sodium citrate), and radioactive. Northern blot analysis was performed using a fragment of the asparaginase gene (500bp cDNA) labeled with isotope [α- ³² P] dCTP.

As a result, transcripts were identified at low temperature (5 ℃) 24 hours (LT 24hr), salt 6 hours (NaCl 6hr), and ABA 6 hours (ABA6 hr) among various stresses in leaves. In the most transcript was confirmed at 3 hours and 6 hours (Fig. 2).

Example 3: Asparaginase Gene Analysis on Genomics

Genomic DNA was isolated from the leaves of soybeans (Shure et al., Cell 35: 225, 1983). The separated genomic DNA was digested with EcoR I and Hind III, and then electrophoresed in 1.0% agarose gel for 5 minutes in denature solution (0.5M Tris-Cl, 1.5M NaCl, pH7.5), followed by neutralization solution. Neutralized. The neutralized gel was transferred to a nylon blotting membrane (Amasham Biosciences), fixed with UV cross-linker, and then reacted with DIG-labeled DNA probe and membrane in DIG Easy Hyb buffer (Roche, Germany) for 16 hours at 42 ° C. . After the reaction, the membrane was detected with anti-digoxigenin-AP and visualized with the chemiluminescene substrate CSPD. After washing with washing solution and exposed with X-ray flim. As a result, it was confirmed that asparaginase on the genome exists in two copies (FIG. 3). From this result, it was confirmed that the asparaginase gene according to the present invention is increased in some stress (low temperature, salt) and hormone (ABA).

Example 4 Recombinant Asparaginase Using Recombinant Escherichia Coli Expression

In this example, to confirm the function of asparaginase according to the present invention, the asparaginase gene isolated in Example 1 was inserted into pET42a (Novagen) of the E. coli expression vector. The gene cDNA and the expression vector pET42a were treated with restriction enzymes Eco Ri and Hind III, respectively, and then ligated to construct a recombinant transfer vector pET-SLTI182 (FIG. 4). The recombinant transfer vector has a characteristic of inducing expression of recombinant fusion protein expression under the control of the T7 promoter and IPTG.

To express the soybean-derived asparaginase gene in Escherichia coli, transformed Escherichia coli was prepared by inserting 5 µg of the transfection vector pET-SLTI182 into 50 µg of E. coli BL21 at a temperature shock (42 ° C). The transformed Escherichia coli was cultured in LB (Luria-Bertani) liquid medium containing 50 µg / ml of kanamycin for 1 hour, and then plated in LB-Kana solid medium and cultured at 37 ° C. for 16 hours or more. The resulting bacteria were incubated to OD 0.6 (1.0 × 10 ⁶ cells) in LB-Kana 50ml liquid medium, 0.5mM IPTG was added and incubated for 3 hours at 28 ℃.

After incubation, cells were collected by centrifugation at 3000 rpm for 5 minutes, and then 1.7 ml of 50 mM Tris-HCl (pH 8.0) buffer was added. The cells were disrupted using a cell crusher and centrifuged at 5000 rpm for 5 minutes. After centrifugation, the supernatant was separated and again centrifuged at 12,000 rpm for 10 minutes to separate soluble and non-aqueous proteins, followed by 12% SDS-protein electrophoresis (Laemmli, Nature, 227: 680, 1970) to perform recombinant fusion. Asparaginase protein expression was analyzed (FIG. 5). As a result, as shown in Figure 5, about 66kDa recombinant fusion protein bands could be confirmed. Lanes 2 and 3 show the pET42 and pET42-SLTI182 fusion proteins, respectively.

Example 5: Determination of Asparaginase Activity

L-asparaginase activity was measured by measuring the ammonia hydrolyzed from asparagine, an experimental method devised by Fawcett and Scott (1960). In this example, the purified and purified pET-SLTI182 protein in Example 4 (10 µg, 20 µg, 30 µg, 40 µg, 50 µg), hourly (0, 10 minutes, 20 minutes, 30 minutes, 40 minutes) , 50 minutes, 60 minutes, 90 minutes, 120 minutes, 140 minutes) and by reaction temperature (4 ° C., 14 ° C., 25 ° C., 37 ° C.), and the amount of the substrate and the reaction time were 10 mM and 1 hour, respectively. And measured at 450 nm using a UV spectrophotometer (Boos, J. et al., Eur. J. Cancer, 32A: 1544, 1996; Joanne, G. et al., J. Agric.Food Chem., 48: 1692- , 2000).

As a result of measuring the activity of asparaginase induced by cold stress in soybean, the concentration of the recombinant fusion protein showed the highest activity in 50 μg of the recombinant fusion protein in the concentration of 10 mM asparagine (FIG. 6). Therefore, the experiment was performed by setting the substrate (asparagine) and recombinant fusion protein concentrations to 10 mM and 50 µg, respectively, under optimal conditions. In the experiment measured by time, the reaction time increased steeply until 50 minutes elapsed and showed the highest activity at 120 minutes (FIG. 7). Experiments measured by temperature showed the highest activity at 37 ° C. (FIG. 8). As a result of the expression of asparaginase according to the present invention in Escherichia coli, it was confirmed that the enzyme had asparaginase enzymatic activity, and the optimal conditions were substrate 10mM, recombinant fusion protein 50㎍, reaction time 50min, and reaction temperature Was found to be 37 ° C.

9 compares the activity of expressed asparaginase by cloning in the sense and antisense directions, respectively. As shown in FIG. 9, it was confirmed that the activity in the case of cloning in the sense direction was high. In addition, when cloning in the antisense (antisense) direction, it was confirmed that the expression of similar asparaginase that is basically present in E. coli.

As described in detail above, the present invention provides an effect of providing a low temperature resistant asparaginase, a gene encoding the same, a recombinant vector containing the gene, a microorganism transformed with the recombinant vector, and a low temperature resistant plant transfected with the gene. There is. In addition, the low temperature resistant gene according to the present invention is useful not only for molecular genetic studies on nitrogen metabolism of plants, but also for industrially producing low temperature resistant plants.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (14)

A low temperature resistant protein having the amino acid sequence of SEQ ID NO. The protein of claim 1, having asparaginase enzyme activity. A gene encoding the amino acid sequence of SEQ ID NO. The gene according to claim 3, wherein the gene has a nucleotide sequence of SEQ ID NO: 1. The gene according to claim 3 or 4, wherein the gene encodes a low temperature resistant protein. The gene according to claim 3 or 4, characterized in that it encodes an asparaginase enzyme. Recombinant vector containing the gene of claim 3. The recombinant vector according to claim 7, wherein the recombinant vector is pET-SLTI182. A transformed microorganism obtained by introducing the recombinant vector of claim 7 into a host cell selected from the group consisting of bacteria, yeast and fungi. 10. The transformed microorganism according to claim 9, wherein the host cell is Escherichia coli or Agrobacterium. A method for producing asparaginase, comprising culturing the transformed microorganism of claim 9 or 10. 12. The method of claim 11, wherein (a) one or more expression modulators that operably linked a DNA sequence encoding an asparaginase having the amino acid sequence of SEQ ID NO: 2 to this DNA sequence Inserting into a vector comprising an expression control sequence; (b) transforming the host with a recombinant expression vector formed therefrom; (c) culturing the resulting transformants under medium and conditions appropriate for causing the DNA sequence to be expressed; (d) recovering the substantially pure cold resistant protein from the culture, the method of producing cold resistant asparaginase. A low temperature resistant plant transfected with the gene of claim 3. Low temperature resistant soybean transfected with the gene of claim 3.

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