KR100747774B1 - Brassica campestris ssp. pekinensis transformed by S-adenosyl-L-methionine synthetase coding gene - Google Patents

Abstract

The invention cabbage ( Brassica) rapa ssp . pekinensis) to provide a gene encoding S-adenosyl-L-methionine synthetase isolated from and a transgenic cabbage using the same. Phosphorus is the most important step in the synthesis of ethylene and polyamine, and it is the enzyme that has the greatest influence on methylation. By controlling the growth of Chinese cabbage, you can adjust the shipping time of the Chinese cabbage farmers can be shipped at the desired time, so that the price of the Chinese cabbage can be stabilized is beneficial to both farmers and consumers.

Chinese cabbage (Brassica rapa ssp.pekinensis), esdenosyl el methionine synthase, synthetase

Description

Transformed Chinese cabbage using a gene encoding E. adenosyl el methionine synthase {Brassica campestris ssp. pekinensis transformed by S-adenosyl-L-methionine synthetase coding gene}

1 shows electrophoresis of fragments of genes encoding esdenosyl el methionine synthase amplified from cabbage by RT-PCR using BC f / mr, BC mf / mr, BC mf / r and BC f / lr primers. One picture,

FIG. 2 shows a homology comparison between the sequences of E. adenosyl el methionine synthase genes of Chinese cabbage isolated by RT-PCR and the sequences of each of Brassica juncea, Cabbage (Brassica oleracea) and Arabidopsis thaliana. Degrees,

3 is a schematic diagram for vector cloning to be transformed into Chinese cabbage,

Figure 4 shows the transformation process of Chinese cabbage by Agrobacterium, the process of coculture with Agrobacterium axle during the transformation process (A), the process of inducing transformed callus (callus) in callus induction medium (B), the process of developing a shoot in a suit differentiation medium (C), the process of creating a root from the suit (D), and the transformation of cabbage by Agrobacterium, which is being grown in a greenhouse. A diagram showing a cabbage transgenic plant (E),

Figure 5 is a diagram confirming the hygromycin resistance gene from the transformed cabbage using PCR,

Figure 6 is a diagram confirming the adenosyl el methionine synthase by RT-PCR in a transgenic plant identified a hygromycin resistance gene.

The invention cabbage ( Brassica) rapa ssp . The present invention provides a gene encoding S-adenosyl-L-methionine synthetase isolated from pekinensis and a transgenic cabbage using the same.

In general, S-adenosyl-L-methionine (S-adenosyl-L-methionine) is a compound having a methyl group (-CH ₃ ) in sulfur (sulfur-) is known to be commonly found in a variety of species, such as microorganisms, plants, animals. S-adenosyl-L-methionine acts to provide methyl groups (-CH3) in most methylation processes for the synthesis of plant secondary metabolites and proteins, carbohydrates, nucleic acids and lipids. It is believed to be a precursor of phytohormones, ethlene and polyamines, to be involved in plant cell wall formation.

S-adenosyl-L-methionine is produced by hydrolysis of ATP and L-methionine by S-adenosyl-L-methionine synthetase. At the same time, it releases pyrophosphate and inorganic orthophosphate. Esadenosyl el methionine thus produced is tissue-specific or gene-specifically regulated at various stimuli, and induces various changes in plant growth and aging. In particular, ethylene, an important hormone in plant growth and growth, is produced by ACCase from esdenosyl el methionine. Similarly, spermidine and spermine are polyamines that regulate cell differentiation, growth and aging. (spermine) is also produced from esdenosyl el methionine. In 1993, Capell et al reported that polyamines delay plant aging, and in 1981 Apelbaum et al reported that polyamines inhibit ethylene biosynthesis in plant tissues. Also, in 1984, Yang and Hoffman reported that changes in the expression of esadenosyl el methionine in plants had a significant effect on ethylene synthesis.

Various studies have been conducted to show that S-adenosyl-L-methionine synthetase plays an important role in plant growth and differentiation. Therefore, S-adenosyl-L-methionine synthetase By using the gene, it is possible to supply a stable price by controlling the growth period of the cabbage, whose price fluctuates depending on the weather, and also contribute to the study of the function of cabbage, one of the four major vegetables in Korea.

Chinese cabbage is one of the four major vegetable gowns in Korea, which is also used as a main ingredient in kimchi and soup. But Chinese cabbage rapa ssp . Transgenic cabbages using genes encoding S-adenosyl-L-methionine synthetase derived from pekinensis are not taught or disclosed anywhere above.

Accordingly, the present inventors have completed the present invention by identifying a gene encoding the S-adenosyl-L-methionine synthetase gene derived from Chinese cabbage and using this to successfully transform the Chinese cabbage. .

An object of the present invention is cabbage ( Brassica rapa ssp . The present invention provides a gene encoding S-adenosyl-L-methionine synthetase isolated from pekinensis and a transgenic cabbage using the same.

In order to achieve the above object, the present invention provides a gene encoding S-adenosyl-L-methionine synthetase described in SEQ ID NO: 1 involved in the production of S-adenosyl El-methionine.

In addition, the present invention provides a transgenic cabbage that can control the growth time of the Chinese cabbage using a gene encoding the S-adenosyl-L-methionine synthetase.

Hereinafter, the present invention will be described in detail.

Chinese cabbage ( Brassica) rapa ssp . In order to isolate a gene encoding S-adenosyl-L-methionine synthetase from pekinensis, a primer is first prepared.

In order to prepare the primers, a sequence of glutathione transferase genes is collected through NCBI GenBank, and only the coding regions are separated from the crops belonging to vegetable crops first, and then only the coding regions are aligned. alignment). Primer can be prepared by selecting the place of high homology among the alignment results.

Using the primer, the nucleotide sequence of the entire gene encoding the adenosyl el methionine synthase of Chinese cabbage was revealed from the cabbage through RT-PCR, which has the nucleotide sequence shown in SEQ ID NO: 1.

The present invention comprises the steps of preparing a primer for the cloning of the adenosyl el methionine enzyme genes of Chinese cabbage, based on the homology of the s adenosyl el methionine synthase sequences of a vegetable crop that has been published previously; Obtaining gene fragments and whole genes of esdedenosyl el methionine synthase from Chinese cabbage by RT-PCR method using the primers; And preparing a recombinant vector containing the nucleotide sequence, transforming it into Agrobacterium, and then transforming cabbage using the Agrobacterium.

Example  One: s Adenosyl  For isolating El methionine synthase gene F Writing of a reamer

In order to find a common part of the S-adenosyl-L-methionine synthetase, a previously published crop-specific S-adenosyl-L-methionine synthetase was synthesized by NCBI. National Center for Biotechnology Information). Entrez program was used to collect the entire sequence of the gene. As a search method, S-adenosyl-L-methionine synthetase was selected as a keyword.

Among the S-adenosyl-L-methionine synthetase genes collected, the nucleotide sequences of the same cabbage (Brassica napus), gat (Brassica Juncea), and Arabidopsis thaliana are Selection and a lot of homology of the nucleotide sequence was found by the BLAST program to prepare a primer around the portion. Prepared primers are shown in Table 1 below.

primer Sequence BC f (SEQ ID NO: 1)       5'-ATG GAG ACT TTT CTA TTC ACA TC-3 ' BC mf (SEQ ID NO: 2)       5'-ATG TTT GGT TAC GCC ACT GAT-3 ' BC mr (SEQ ID NO: 3)       5'-TCT TTC CCT GAG AAA GCA CCT-3 ' BC r (SEQ ID NO: 4)       5'-CAT TAC TTA AGC TTG AGG TTT GT-3 ' BC lr (SEQ ID NO: 5)       5'-TTT AAT TAT TGC TCA CAG TAG AT-3 '

Example  2: s Adenosyl  L-methionine synthase (S- adenosyl -L- methionine   synthetase) gene isolation

2-1. Isolation of RNA from Chinese Cabbage

Chinese cabbage ( Brassica) used in the present invention rapa ssp . Pekinensis) was used to isolate RNA from samples grown in the aseptic state of the aircraft.

Isolation of RNA from the cabbage was performed using the Trizol reagent provided by GibcoBRL.

Chinese cabbage rapa ssp . The leaves of pekinensis were crushed with liquid nitrogen, and then mixed well with 1 ml Trizol solution and left at room temperature for 5 minutes. After 200 μl of chloroform was added and mixed, the mixture was further centrifuged at 13,000 rpm for 10 minutes at 4 ° C., and the supernatant of the mixture was transferred to a new tube. 500 μl of isopropanol was added and precipitated by centrifugation at 13,000 rpm for 15 minutes. Thereafter, isoprophenol was removed and the precipitate was washed with 75% ethanol, dried well without ethanol, and then dissolved in DEPC-treated sterile distilled water.

2-2. s Adenosyl  Isolation of El-Methionine Synthase Gene

Isolation of the S-adenosyl-L-methionine synthetase gene was carried out using the RNA obtained in Example 2-1 as a template, using a dT primer and reverse transcriptase. cDNA was synthesized and then isolated by PCR with the prepared primers. cDNA synthesis program conditions were 1 μg of RNA was raised at 70 ℃ with dT primer to remove the RNA secondary structure, reverse transcription at 42 ℃ for 30 minutes and at 10 ℃ at 75 ℃ to inactivate the reverse transcriptase used. After that, PCR was performed using the prepared primer and tag polymerase using 1 ㎍ of the synthesized cDNA. The program allowed initial denaturation time at 96 ° C for 2 minutes, followed by 30 seconds at 96 ° C, 30 seconds at 58 ° C, 72 ° C. 40 cycles were performed at 1 ° C. for 1 minute and finally extended at 72 ° C. for 10 minutes to maintain 4 ° C. to complete the entire reaction.

The fragments amplified with the respective primers were cloned into the PCR product vector pGEM-T easy vector in order to analyze their respective sequences, and the sequences were analyzed using T7 and SP6 primers adjacent to the inserted sites.

Example  3: cabbage s Adenosyl  Identification of Whole El-Methionine Synthetase Genes

Sequencing of PCR fragments cloned into pGEM-T easy vector for sequencing was performed using GenBank BLAST program. 824bp product amplified with BC f and BC mr primers described in Table 1, about 448bp product amplified with BC mf and BC mr primers, about 830bp product amplified with BC mf and BC r primers, and BC f and BC lr primers The nucleotide sequence and amino acid sequence of each 1,265 bp amplified product was analyzed to show homology with the adenosine el methionine synthetic genes of various plants, and in particular, B. juncea s adenosyl el methionine synthase. (S-adenosyl-L-methionine synthetase) showed a high homology of 98% (see Fig. 2).

BC f / mr (FIG. 1 (1)), BC mf / mr (FIG. 1 (2)), BC mf / r (FIG. 1 (3)) and BC f / lr (FIG. 1) (4)) As a result of performing RT-PCR, it was confirmed that most of the adenosyl el methionine synthase genes of Chinese cabbage were amplified. In addition, RT-PCR using BC f / lr primers confirmed that the entire gene of S-adenosyl el methionine synthase was obtained.

After determining the start codon and the end codon of this linkage, start restriction enzyme sites at the beginning and the end respectively, attach the EcoRI cleavage sequence to the codon side, and attach the SpeI cleavage sequence to the codon side, and then prime the whole gene at once. Was prepared by PCR using pfu polymerase from cDNA of Chinese cabbage, and the entire gene was cloned into the pGEM-T easy vector for sequencing analysis to obtain and analyzed the nucleotide sequence. Final analysis The identified nucleotide sequence is determined as a gene encoding the entire S-adenosyl-L-methionine synthetase isolated from the cabbage, and the nucleotide sequence is shown in SEQ ID NO: 1.

Experimental Example   1. Cabbage of Agrobacterium s Adenosyl  Of El-Methionine Synthetase Gene Into the plant  Introduction

1-1. Preparation of the vector

S-adenosyl-L-methionine synthetase gene of Chinese cabbage, the whole S adenosyl el methionine synthetase (S) of Chinese cabbage to be inserted to create an expression recombinant vector for introduction into the plant pGEM-T easy vector containing the gene encoding -adenosyl-L-methionine synthetase) was digested with EcoRI and SpeI, and the size of the gene fragment was confirmed by electrophoresis and extracted by gel extraction. insert) was prepared.

In the same manner, pCAMBIA1390 vector was also digested with EcoRI and SpeI, and the size of the DNA fragment was confirmed and the vector (hereinafter referred to as pCSAMS) was prepared by extracting from a gel (see FIG. 3).

1-2. E. coli DH5 Transformation into α

After mixing the vector and the insert prepared in Experimental Example 1-1 to an appropriate concentration, it was bound using T4 DNA ligase (ligase) and transformed into E. coli DH5α in the following manner.

E. coli DH5α was inoculated in 50 ml of LB and incubated at 37 ° C. to OD ₅₇₀ = 0.375-0.400. The strain culture was centrifuged at 3000 rpm for 10 minutes and the precipitate obtained was released with 0.1 M CaCl ₂ solution and placed on ice for 30 minutes. Thereafter, after centrifugation at 3000 rpm for 7 minutes, the precipitate was dissolved in 0.1 M CaCl ₂ . Recombinant plasmids were mixed together and placed on ice for 30 minutes, followed by thermal shock at 42 ° C. for 90 seconds. After that, put it on ice for 10 minutes and shake the culture medium for 1 hour by adding 700 µl of LB medium, and centrifuge the culture solution for 1 hour at 12000 rpm, and remove the supernatant with only 100 µl. Melted. Ampicillin (Ampicillin) 50mg / L, X-gal (200mg / L in NN dimethylformamide), IPTG 200mg was added to the solid LB medium was added and incubated for 12 hours at 37 ℃. The transformed cells that showed resistance in solid medium and formed white colonies were selected and cultured.

Recombinant plasmid DNA was extracted from the cultured colonies by the following method.

Colonies were infected with 3 ml of LB medium to which antibiotics were added, followed by incubation at 37 ° C. for 12 hours, and the cultured cultured with colonies was centrifuged at 12,000 rpm for 10 minutes at 4 ° C. Vortex was added for several seconds by adding Solution I (200 μl / ml), carefully mixed with Solution II (200 μl / ml), and left at room temperature for 5 minutes, followed by Solution III (200 μl). / ml) was added well and left for 10 minutes on ice, then centrifuged at 12000 rpm, 4 ℃ for 10 minutes. Take the supernatant obtained after the centrifugation, add 1/10 of NaOAC, add 2.5 times 100% ethanol, precipitate for 2 hours at -20 ° C, and centrifuge to wash the precipitate with 70% ethanol. It was used after dissolving in sterile distilled water.

The DNA obtained above was digested with BglII and BbrpI, and the size and presence of the vector and the insert were confirmed by electrophoresis and confirmed to be properly bound by PCR and sequencing.

1-3. Containing the transforming carrier Acrobacterium Tumafaciens  Ready

Expression vectors in which the binding of the vector and the insert were confirmed were transformed into Acrobacterium tumafacials LBA4404 (A. tumefaciens LBA4404) by the Freeze-thaw method. The transformed Acrobacterium Tumasions LBA4404 was deposited on November 2, 2005 with the Korea Agricultural Microbiological Resource Center, and the name of the microorganism was a gene encoding S-adenosyl-L-methionine synthetase. to be.

After transformation, Agrobacterium was also cultured in colonies to confirm whether the recombinant vector was properly included by PCR. In this case, PCR is a primer that can separate the tag polymerase and the entire gene instead of putting the plasmid as a template (SAM oe 5 ': 5' gaattc atg gag act ttt cta ttc aca tc 3 ', SAM oe 3 ': 5' actagt tta agc ttg agg ttt gtc cca 3 '), a toothpick with colonies in a liquid containing 10X tag buffer and magnesium chloride solution, melt the colonies in the liquid and PCR them Among them, Agrobacterium tuumasions LBA4404 confirmed the introduction of the expression vector (vector) was used for cabbage transformation. PCR reaction conditions were treated for 2 minutes at 95 ℃, 30 cycles of 95 ℃ 20 seconds, 58 ℃ 40 seconds, 72 ℃ 2 minutes treatment and finally at 72 ℃ 5 minutes treatment.

1-4. Agrobacterium Tumapathians  Transformation of Chinese Cabbage

Chinese cabbage is transformed using Agrobacterium tumafaciens LBA4404 (KACC 95041P) as follows.

After the cabbage was germinated in aseptic condition, the embryo axis was cut into 5-10 mm, and inoculated by dipping for 10 minutes in the Agrobacterium tumafaciens suspension prepared above. Inoculated sections were co-cultured (MS base medium, 3% sucrose, 0.8% plant chicks, 4 mg / L BA, 1 mg / L NAA, 4 mg / L AgNO3, 5 mg / L acetosyringone, pH5.6) Co-culture for 3 days at. The cabbage embryos were then washed with liquid co-culture medium to wash Agrobacterium tomafaciens and then callus-induced medium (MS base medium, 3% sucrose, 0.8% plant agar, 4mg / L BA, 1mg / Chinese cabbage explants were cultured in L NAA, 4 mg / L AgNO3, 5 mg / L acetosyringone, 10 mg / L hygromycin, 200 mg / L cefotaxime, pH5.6) to induce callus in transformed tissues only. .

The induced transformed callus was cut and transferred to a chute induction medium (same as the callus induction medium) to induce the chute. The induced transformed chute was a root induction medium (1/2 MS base medium, 3% sucrose, 0.7% Plant agar, 200 mg / L cefotaxime, pH5.8) was used to induce root development. The transformants with roots were transplanted into sterile vermiculite and purified and then transplanted into pots and grown in greenhouses.

As a result, as shown in Figure 4, it can be seen that the cabbage was transformed by Agrobacterium. Agrobacterium transformed cabbage during the transformation process of coculture with Agrobacterium (see FIG. 4A), the process of inducing transformed callus (callus) in callus induction medium (see B of Figure 4) ), The process of the shoot (shoot) development in chute differentiation medium (see Fig. 4 C), the root (root) is generated from the chute (see D in Fig. 4) and the transformation process of Chinese cabbage by Agrobacterium Chinese cabbage transgenic plants being grown in the greenhouse (see E of Figure 4) was confirmed.

Experimental Example  2. Confirmation of gene insertion and stability in transformants and their subsequent

In order to confirm whether the hygromycin resistance gene and E. adenosyl el methionine synthase performed in Experimental Example 1 were successfully inserted into these transgenic individuals, genomic DNA was isolated from the cabbage transformants and confirmed by PCR.

At this time, the primer to confirm that the hygromycin gene has been inserted is a genomic DNA of the transgenic cabbage with primer pairs 3'-CCTCTACGTTATCCAGTCCGA-5 'and 3'-GGTTGTTACAGGACTGCCTGT-5' which can be amplified inside the hygromycin gene. Amplified by PCR in the template.

As a result of the experiment, as shown in FIG. 5, a band of 0.35 kb size was generated in the transformed cabbage, and it was confirmed that the hygromycin gene was stably inserted into the cabbage genome.

In addition, as shown in Figure 6, by separating the RNA from the transgenic cabbage and the part encoding the adenosyl el methionine synthase gene gene from both sides including some vectors to perform RT-PCR It was confirmed that the adenosyl el methionine synthase was stably inserted.

The invention cabbage ( Brassica) rapa ssp . The present invention provides a gene encoding S-adenosyl-L-methionine synthetase isolated from pekinensis and a transgenic cabbage using the same.

Esadenosyl el methionine synthase of the present invention is the most critical substance of the step in the synthesis of phyto hormone ethylene (poly) and polyamine (polyamine), the enzyme having the greatest effect on methylation, the adenosyl el methionine In the case of transformed cabbage using a gene encoding a synthetase, it is possible to control the growth of cabbage by controlling the growth of cabbage, so that the cabbage can be shipped at a desired time by farmers, thereby stabilizing the price of cabbage. It is a beneficial invention.

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Claims (2)

delete An expression vector pCSAMS containing a gene of SEQ ID NO: 1 encoding S adenosyl el methionine synthase was prepared from Chinese cabbage, and the vector was transformed into Acrobacterium tumafaciens LBA4404 (KACC 95042P), followed by Transgenic cabbage that can control the growth period obtained by transforming bacterium into cabbage.

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