KR100805376B1 - S-adenosyl-l-methionine coding gene involved in s-adenosyl-methionine synthetase biosynthesis of chrysanthemum coronarium l. and isolation method of the same - Google Patents

Abstract

A method for isolating a gene coding S adenosyl-L-methionine synthetase of Chrysanthemum coronarium L. is provided to obtain the gene which is able to control the growth speed of a transgenic plant, thereby being very useful for a plant breeding industry and a flowering industry. A method for isolating a gene coding S adenosyl-L-methionine synthetase of Chrysanthemum coronarium L. comprises the steps of: (a) preparing a polynucleotide primer consisting of sequences described in SEQ ID : NOs. 1 to 4 cloning the S adenosyl-L-methionine synthetase to isolate the gene coding the S adenosyl-L-methionine synthetase; (b) performing an RT-PCR(reverse transcription polymerase chain reaction) using the primer and isolating an S adenosyl-L-methionine synthetase gene from the Chrysanthemum coronarium L.; (c) identifying the total sequence of the isolated S adenosyl-L-methionine synthetase gene; (d) transfecting the S adenosyl-L-methionine synthetase gene into a plant using Agrobacterium; (e) inducing shoot or root of the transgenic plant; and (f) identifying the stability of the S adenosyl-L-methionine synthetase gene. A gene sequence coding the S adenosyl-L-methionine synthetase gene is described in SEQ ID : NO. 5.

Description

S-adenosyl-L-methionine coding gene involved in S-adenosyl-methionine synthetase biosynthesis of Chrysanthemum coronarium L. and isolation method of the same}

Figure 1 shows the exact 5'-, 3'- RACE fragment of the present invention nested-PCR RT-PCR using the GeneRacer ™ primer kit, MetSyn-F (SEQ ID NO: 1) and MetSyn-R (SEQ ID NO: 2) primers This is a picture of electrophoresis of a fragment of a gene encoding S adenosyl el methionine synthase amplified from wormwood.

Figure 2a shows the amino acid sequence results of the extract of the present invention sadenosyl el methionine synthase gene of the present invention in the mugwort and reverse transcription-polymerization chain reaction.

Figure 2b is a tobacco (Nicotiana with the amino acid sequence of S-adenosyl-methionine synthetase gene of the present invention el crown daisy tabaccum), tomato (Lycopersicon esxulentum ) and mustard ( Brassica juncea ) is the result of comparison of homology between sequences.

Figure 3a shows a process of inducing transformed callus (callus) in callus induction medium during the transformation process of mugwort with Agrobacterium of the present invention, Figure 3b is a process of transformation of mugwort with Agrobacterium 3 shows a process in which a shoot is developed from a chute differentiation medium, and FIG. 3c illustrates a process of generating a root from a chute during the transformation of wormwood by Agrobacterium.

Figure 4 is a picture confirming the transgenic individual translocated wormwood S adenosyl el methionine synthase gene of the present invention by a polymerase chain reaction.

The invention garland chrysanthemum (Chrysanthemum The present invention relates to genes encoding S-adenosyl el methionine synthase, which are important in the synthesis of ethylene and polyamine, phytohormones involved in cell growth and stress reactions isolated from coronarium L.

In general, S-adenosyl-L-methionine synthetase is a compound having a methyl group (-CH ₃ ) in sulfur, which is commonly found in various species such as microorganisms, plants, and animals. It is known to be involved in the synthesis of secondary metabolites, proteins, carbohydrates, nucleic acids and lipids of plants, as well as precursors of ethlene and polyamines, and is known to be involved in plant cell wall formation.

Such S-adenosyl-L-methionine is adenosine triphosphate (ATP) and L-methionine (L-methionine) by S-adenosyl-L-methionine synthetase. methionine is produced by hydrolysis and simultaneously releases pyrophosphate and inogenic 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 (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 produced from esdedenosyl el methionine.

Mugwort is one of the crops where chrysanthemum is a nutritious crop. It has high content of vitamins and flavonoids as well as fresh and unique fragrance. However, no gene or amino acid sequence encoding S-adenosyl-L-methionine synthetase from Chrysanthemum coronarium L. has been reported.

Accordingly, the present invention has been devised in view of the above-mentioned matters, and an object of the present invention is to isolate a gene encoding es adenosyl el methionine synthase from mugwort and provide its entire sequence.

Another object of the present invention is to provide a primer that can be used to clone the gene encoding the above-mentioned adenosyl el methionine synthase and a method for separating the adenosyl el methionine synthase from wormwood using the same.

The object of the present invention was to prepare a primer for the cloning of the adenosyl el methionine synthase gene from wormwood and to isolate the entire base sequence by separating the adenosyl el methionine synthase gene from wormwood using the primer E. adenosyl el methionine synthase gene of Agrobacterium was transformed into the plant was introduced by introducing into the plant and confirmed the stability.

Hereinafter, the configuration of the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.

The present invention comprises the steps of preparing a primer that can be used to clone the adenosyl el methionine enzyme gene based on the homology of the s adenosyl el methionine synthase sequences of vegetable crops; Separating the es adenosyl el methionine synthase gene from wormwood using the prepared primer; Identifying the entire nucleotide sequence of the isolated asdenosyl el methionine synthase gene; Preparing a recombinant vector containing the nucleotide sequence and transforming it into Agrobacterium to transform the plant; Deriving the chute and root of the transformant and confirming the stability of the S. adenosyl el methionine synthase gene of wormwood introduced into the plant.

Wormwood used in the present invention ( Chrysanthemum coronarium L. ) was isolated from aseptically grown RNA.

Example  One: s Adenosyl  To isolate the El methionine synthase gene F Limer production

In order to find a common part of S-adenosyl-L-methionine synthetase (SAMS), previously published crop-specific S-adenosyl L-methionine synthetase genes were identified by the National Center for Biotechnology Information (NCBI) Genbank. Search on. 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 gene.

Among the collected S-adenosyl-L-methionine synthetase genes, Brassica Juncea ), Arabidopsis thaliana , and Eldera (Elaeagnus umbellata) were selected for sequencing, and the BLAST program was used to find the most homologous nucleotide sequences. Produced.

[Table 1] Nucleotide Sequences of Primers Prepared for Separation of S. adenosyl El-Methionine Synthase Gene

SEQ ID NO: primer Sequence SEQ ID NO: 1 Metsyn-f 5'-tgt caa cga ggg aca tcc aga c-3 ' SEQ ID NO: 2 Metsyn-r 5'-cca agt ttg gtg gcg agg ac-3 ' SEQ ID NO: 3 Nested-MetsynF 5'-aag gtc aca tgt tcg ggt atg-3 ' SEQ ID NO: 4 Nested-MetsynR 5'-tca tca gtg gcg taa cca aa-3 '

Example  2: s Adenosyl  Isolation of L-methionine Synthase Gene

CDNA was synthesized using reverse transcriptase of SAMS gene, and then isolated by polymerase chain reaction with oligo dT of Generacer ™. The conditions of the cDNA synthesis program were 1 μg of RNA and oligo dT primer at 70 ° C. to remove RNA secondary structure, reverse transcription at 42 ° C. for 30 minutes, and 10 minutes at 75 ° C. to inactivate the used reverse transcriptase. Thereafter, 2 μg of the synthesized cDNA was polymerized with the primer and tag polymerase prepared to obtain 3′-end of the SAMS gene. In the polymerization chain reaction, after the initial denaturation time at 94 ° C. for 2 minutes, the cycle was performed 34 times at 94 ° C. for 30 seconds, at 60 ° C. for 40 seconds, and at 72 ° C. for 1 minute, and finally at 72 ° C. for 7 minutes. After the final extension, the entire reaction was completed by maintaining 4 ° C. In addition, the 5'-end portion of the SAMS gene was specifically bound to the 3'-end of the Generacer ™ RNA oligo using T4 RNA ligase. Accurate 5 ′ clones were obtained using Metrac R or Nested-Metsyn R primers prepared with Generacer ™ 5 primer or Generacer ™ 5 ′ Nested primer (see FIG. 1).

Example  3: garland chrysanthemum s Adenosyl  Determination of the complete sequence of L-methionine synthase gene

The sequencing of fragments isolated by sequencing by polymerizing with pGEM-T easy vector for sequencing was performed using GenBank BLAST program. As a result, the 5 'beginning of the SAMS gene was a 417 bp product amplified by Metsyn F (SEQ ID NO: 1) and Metsyn R (SEQ ID NO: 2) primers, and a 457 bp product amplified by Generacer ™ 5' and Metsyn R (SEQ ID NO: 2) primers. , 400bp product amplified with Generacer ™ 5'Nested primer and Nested-Metsyn R (SEQ ID NO: 4) primer, approximately 1444bp product amplified with Metsyn F (SEQ ID NO: 1) and Racer-dT primers, Metsyn F (SEQ ID NO: 1) And 1400bp product amplified by Racer 3 'primer, Met400 F (SEQ ID NO: 1) and 1400bp product amplified by Racer 3' nested primer, and 1200bp amplified by Nested-Metsyn F (SEQ ID NO: 3) and Racer 3 'nested primer As a result of analyzing the base sequence and amino acid sequence of each product is shown in Figure 2a.

After determining the start codon and the end codon of S. adenosyl el methionine synthase of the present invention, XbaI is located at the beginning and end of each restriction enzyme site. End of the cleavage sequence Attach Eco RI cleavage sequence to the codon side, and prepare primers to newly clone the whole gene at once, and sequence analysis of the entire gene using polymerase chain reaction using tag polymerase from cDNA of wormwood After chromosomes were cloned into the pGEM-T easy vector for obtaining the nucleotide sequence of the gene encoding the SAMS of the present invention (SEQ ID NO: 5).

In addition, the present invention ssadenosyl el methionine synthase gene and tobacco of wormwood ( Nicotiana tabaccum), tomato (Lycopersicon Comparison of homology between esxulentum ) and E. adenosyl el methionine synthase of Brassica juncea ( L. esculentum ) It showed high homology with 93% of S-adenosyl el methionine synthase.

As a result of reverse transcription-polymerization reaction using 3'- primer and 5'- RACE primer, the entire gene of S adenosyl el methionine synthase of the present invention was secured.

Example  4: wormwood using Agrobacterium s Adenosyl  El-Methionine Synthesis Insertion of enzyme genes into plants

An expression recombinant vector was inserted to insert SAMS gene of wormwood of the present invention into a plant. The pGEM-T easy vector containing the gene encoding the whole S adenosyl el methionine synthase of the wormwood to be inserted is Eco RI And Xba I and the size of the gene fragments were confirmed by electrophoresis and then inserted into the extraction method from a gel (gel).

The pILTAB 357 vector was also digested with Eco RI and Xba I in the same manner as described above, and a vector (hereinafter referred to as pCSAMS) was prepared by checking the size of the DNA fragment and extracting it from a gel. The prepared vector and the insert were mixed at an appropriate concentration, bound using T4 DNA ligase, and transformed into DH5α ( E. coli ). Recombinant DNA was extracted by culturing the colonies obtained by transformation, and the DNA was digested with Eco RI and Xba I, and electrophoresis confirmed the size and presence of the vector and the insert, followed by polymerization and sequencing. It was confirmed.

The expression vector in which the binding of the vector and the insert was confirmed was transformed into Acrobacterium tumafasion EHA105 ( A. tumefaciens EHA105) by the Freeze-thaw method. After transformation, Agrobacterium was also cultured to determine whether the recombinant vector was properly included in the polymerase chain reaction. The polymerase chain reaction is a primer that is, to remove the whole instead of the tag (Tag) polymerase, the gene into a plasmid (plasmid) as the template (template) START: 5 'tct aga atg gag acc ttc ttg ttc aca tcc 3' , STOP: 5 ' gaa ttc tca agc ttt agg ctt gag aac ctt aac 3 'and 10X tag buffer, Magnesium chloride solution in a liquid containing a colony of toothpicks to dissolve the colony in the liquid and then the mixture was polymerized using a reaction chain expression vector Agrobacterium tuumasions EHA105 confirmed the introduction of (vector) was used for wormwood transformation.

Example  5: chute and root induction

Agrobacterium tumafaciene EHA105 containing the plant transformation vector of Example 4 was used as a transforming medium inoculated in wormwood.

When inoculated leaf fragment of chrysanthemum body by Agrobacterium-to-mapo Skien to increase the efficiency of transfection, the first carrier and transformed Agrobacterium mapo-to-Hsien (Agrobacterium which contains for tumefaciens LBA4404 / pRCV2) was plated in solid YEP medium containing kanamycin 50 mg / L and incubated for 48 hours at 28 ° C. Inoculated in 5 mL liquid YEP medium containing mg / L was incubated for 48 hours at 28 ℃. 700 μl of the cultured suspension was extracted and added to 50 mL liquid YEP medium with pH adjusted to 5.6 and suspended incubated at 28 ° C. until OD 600 became 1.0. Finally, the incubated suspension was resuspended in 25 ml liquid YEP medium by centrifugation at 4000 rpm for 15 minutes and used for inoculation of wormwood leaf sections.

The mugwort leaf explants were inoculated by germinating mugwort seeds in aseptic condition and then cutting to 5-10 mm and immersing the Agrobacterium tumafaciene suspension for 10 minutes. Inoculated sections were co-cultured in co-culture medium (MS base medium, 3% sucrose, 0.8% plant agar, 0.5 mg / L BA, 0.2 mg / L NAA, pH 5.6) for 3 days. After wormwood leaf sections were washed with liquid co-culture medium to wash Agrobacterium tumafaciene and then induction medium (MS base medium, 3% sucrose, 0.8% plant agar, 0.5 mg / L BA, 0.2 mg / L NAA, 30 mg / L kanamycin, 200 mg / L cefotaxime, pH 5.6) were incubated with the wormwood leaf sections so that the chute was induced only in the transformed tissue (see FIG. 3A).

Transgenic chute (see FIG. 3b) induced by incubation in culture with a chute induction medium was transferred to a root induction medium (1/2 MS base medium, 3% sucrose, 0.7% plant agar, 200 mg / L cefotaxime, pH 5.8). Induction occurred. Root-producing transformants were purified after incubation for 1-2 weeks in the pre-purifying phase (see FIG. 3C).

Example  6: Gene Insertion and Stability Identification in Transformants and Subsequents

In order to confirm that the hygromycin resistance gene used in the present invention was successfully inserted into the transformed individual of the SMAS gene of the present invention, the genomic DNA was isolated and confirmed by the polymerase chain reaction. At this time, the primer for confirming whether the kanamycin gene was inserted was amplified by a polymerase chain primer (primer) pair that can be amplified inside the kanamycin gene. As a result, as shown in FIG. 4, a 0.7 kb band was generated in the transformed wormwood hatch, and the kanamycin gene was stably inserted into the wormwood hatch genome.

Gene encoding the present invention S-adenosyl-L-methionine synthetase (S-adenosyl-L-methionine synthetase) is a very useful invention in the plant breeding industry and the flower industry because it can control the growth rate of plants during plant transformation.

Attach sequence list electronic file  

Claims (3)

delete a) preparing a polynucleotide primer consisting of the nucleotide sequences set forth in SEQ ID NOS: 1 to 4 to clone the es adenosyl el methionine synthase gene to isolate a gene encoding es adenosyl el methionine synthase; b) reverse transcription-polymerization chain reaction using the primer prepared in step a) and separating esdenosyl el methionine synthase gene from Chrysanthemum coronarium L .; c) identifying the entire nucleotide sequence of the adenosyl el methionine synthase gene isolated in step b); d) transforming S. adenosyl el methionine synthase gene of Wormwood ( Chrysanthemum coronarium L. ) into plants using Agrobacterium; e) inducing a chute or root of the transformant of step d) and f) confirming the stability of E. adenosyl el-methionine synthase gene of Chrysanthemum coronarium L. A method for separating genes encoding S-adenosyl-L-methionine synthetase of Chrysanthemum coronarium L. The gene sequence encoding the adenosyl el methionine synthase gene of Chrysanthemum coronarium L. of SEQ ID NO: 5.

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