KR101147963B1 - Gene encoding zeaxanthine epoxidase essential to the xanthopyll cycle in soybean - Google Patents

Abstract

The present invention provides a novel zeaxanthin epoxidase gene and homologous sequence thereof isolated from soybean, and the present invention also provides a recombinant vector comprising a soybean xanthanthin epoxidase gene and a host cell transformed with the recombinant vector. .

Soybean, Zeaxanthin, Zeaxanthin Epoxidase

Description

Gene encoding zeaxanthine epoxidase essential to the xanthopyll cycle in soybean

The present invention relates to a novel zeaxanthin epoxidase gene isolated from soybeans.

Zeaxanthine is a compound belonging to xanthopyll, a group of compounds in which oxygen-containing functional groups are added to carotenoids. Zeaxanthine has anti-cancer and anti-cancer properties by scavenging singlet oxygen, reactive oxygen species, and free radicals. It has been found that it can be used for aging, immune enhancement, and the like.

This zeaxanthin is converted to antheraxanthine by zeaxanthine epoxidase, and antheranthanthin to violaxanthine by zeaxanthin epoxidase as part of the santophyll cycle in plants. Xanthine is converted to anteranthanthin by violaxanthine deepoxidase and anteranthanthin is converted back to zeaxanthin by violaxanthine diepoxidase. This santophyll cycle has been shown to catalyze the reaction to remove the photo-stresses that plants receive when too much light is applied to them (Biocimica et Biophysica Acta., Hieber et al., 1482 (2000) 84-91). Zeaxanthin accumulated by the santophyll cycle has been shown to protect plants from photooxidative stress (Plant Cell, Baroli et al., 15 (2003) 982-1008).

On the other hand, soybeans and other legumes are root nodules are formed in the roots by the root-homobacteria, thereby fixing the symbiotic nitrogen. In the early stages of symbiosis between legumes and root-homobacteria for nitrogen fixation, reactive oxygen species (ROS), which pose a threat to plants, are generated. Have

The present inventors believe that zeaxanthin, which is known to have antioxidant activity, also plays an important role in antioxidant defense mechanisms in soybean root nodules, and is expected to regulate the content of zeaxanthin in relation to antioxidant defense mechanisms in soybean root nodules. As a result of research on the epoxidase, the gene encoding the zeaxanthin epoxidase of soybean was cloned and the nucleotide sequence was confirmed.

The present invention aims to provide a novel zeaxanthin epoxidase gene isolated from soybeans.

It is also an object of the present invention to provide a recombinant vector comprising a novel zeaxanthin epoxidase gene isolated from soybeans.

It is also an object of the present invention to provide a transformant transformed with a recombinant vector comprising a novel zeaxanthin epoxidase gene isolated from soybeans.

The present invention provides a novel zeaxanthin epoxidase gene isolated from soybeans.

The present inventors conducted PCR experiments using primers based on RNA extracted from soybean leaves and the base sequence of the zeaxanthin epoxidase gene of various plant species to determine whether a gene encoding zeaxanthin epoxidase exists in soybean. Full length cDNA of the zeaxanthin epoxidase gene was isolated and sequenced.

As a result, a 2010 bp long soy zeaxanthin epoxidase gene was identified and its nucleotide sequence is the same as that of SEQ ID NO: 1 in the attached sequence list.

Accordingly, the present invention provides a soybean zeaxanthin epoxidase gene having the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a gene encoding soy zeaxanthin epoxidase and having at least 90% sequence homology with the nucleotide sequence of SEQ ID NO: 1.

In the present invention, "homology" means that the reference sequence and any other sequence have the same degree, and having 90% or more sequence homology means arranging any sequence to correspond with the reference sequence as much as possible and using a program such as ClustalX. Analysis of the sequence indicates that the two sequences are at least 90% identical.

On the other hand, the inventors obtained RNA from the leaves, stems, flowers, roots and root nodules of soybean in order to investigate the expression level of the zeaxanthin epoxidase gene according to the various tissues of soybean.

In addition, in order to investigate the expression level of the zeaxanthin epoxidase gene according to various times in the development stage of the root nodules of soybeans, RNA was extracted from the root nodule tissues by the generation periods of the root nodules.

Real-time PCR analysis was carried out using cDNA obtained using the primer set designed based on the RNA and the nucleotide sequence of the soybean zeaxanthin epoxidase gene identified in the present invention. The expression level of the zeaxanthin epoxidase gene was confirmed in the tissues according to time.

As a result, as shown in Figure 3, the expression of the zeaxanthin epoxidase gene in the stem of the various tissues of the soybean appeared the highest, the expression was extremely low in the root.

In addition, as shown in Figure 4, the expression level of the soybean root nodules at various times of occurrence is the highest in the root state, the expression of the zeaxanthin epoxidase gene gradually decreases as the root nodules develop gradually, the zeaxanthin epoxidase gene at day 27 Was found not to be expressed.

In another aspect of the invention, the invention provides a recombinant vector comprising the soy zeaxanthin epoxidase gene.

In the present invention, a "recombinant vector" is a vector comprising the soybean zeaxanthin epoxidase gene of the present invention and a regulatory element operably linked thereto, so that the protein encoding the zeaxanthin epoxidase gene or the protein encoding it in an appropriate host cell is expressed. May be an expression vector.

In the present invention, "operably linked" means that the nucleic acid expression control sequence and the target gene insert are functionally linked. For example, the promoter and the gene insert may be operably linked to affect the expression of the gene. .

Suitable vectors for use in the preparation of recombinant vectors comprising the zeaxanthin epoxidase gene of the invention include plasmids commonly used in the art (eg, pBR322, pUC8 / 9, pGEX series, pUC19, etc.), phage (eg, λ). -Charon and M13, etc.), or viruses (e.g., SV40, etc.) can be used, and expression such as appropriate promoters, initiation codons, termination codons, polyadenylation signals and enhancers depending on whether the host cell is a prokaryotic or eukaryotic cell. Control elements and the like.

The vector of the present invention may further comprise a sequence of glutathione S-transferase, maltose binding protein, FLAG or hexahistidine to facilitate purification of soy zeaxanthin epoxidase.

When the soy zeaxanthin epoxidase gene of the present invention is to be expressed in plant cells, the recombinant vector of the present invention may be RNA in the soy zeaxanthin epoxidase gene of the present invention and in promoters and plant cells which are operatively linked thereto. It is preferred to include a 3'-detoxification site that causes polyadenylation of the 3'-end of the molecule.

The preparation of recombinant vectors comprising the zeaxanthin epoxidase gene of the present invention can be accomplished using genetic recombination techniques commonly used in the art and well known, for example, as described in Sambrook et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press 201, and the like.

According to another aspect of the invention, the invention provides a host cell transformed with a recombinant vector comprising a soy zeaxanthin epoxidase gene.

In order to transform the host cell into a recombinant vector, any method known and widely used in the art may be used, and a suitable method may be selected according to the host cell. For example, electroporation, protoplast fusion, calcium phosphate precipitation, calcium chloride precipitation, Agrobacterial mediated transformation, liposome-mediated transformation, and the like can be used.

Host cells that can be used for transformation can be any prokaryotic and eukaryotic cells used for transformation in the art, for example, E. coli JM109, E. coli BL21, Bacillus subtilis , Agrobacterium, yeast, BHK, COS-7, plant cells and the like can be used.

The transformant transformed with the vector containing the zeaxanthin epoxidase gene of the present invention can be used for mass production of soy zeaxanthin epoxidase.

The soy zeaxanthin epoxidase gene of the present invention can be usefully used to elucidate the role of zeaxanthin epoxidase and zeaxanthin in the antioxidant defense mechanism of soybean root nodule, and can also be used for mass production of zeaxanthin epoxidase and zeaxanthin.

Experimental Example  One.

Big head Zeaxanthin Epoxidase  Coded Length cDNA Separation of

1) Soybean RNA Extraction and cDNA Synthesis

RNA was extracted from soybean leaves to confirm the presence of a gene ( GmZEP ) encoding zeaxanthin epoxidase in soybeans.

RNA extraction was carried out using a RNeasy Plant mini kit after grinding soybean leaf samples frozen in liquid nitrogen. The RNeasy Plant Mini Kit includes an RNeasy mini column and wash buffers to which RLT and total RNA can bind. RLT buffer is added to the ground leaf samples, mixed well to dissolve, and the insoluble material and viscosity are reduced using a QIAshredder ^™ homogenizer column. Ethanol was added to the sample passed through the QIAshredder ^™ homogenizer column, then added to the RNeasy mini column and allowed to flow, causing the total RNA to bind to the membrane of the column. After washing several times using a wash buffer, RNA was obtained by eluting with RNase-free water.

CDNA was synthesized using RNA and M-MLV reverse transcriptase (Promega, USA). For cDNA synthesis, 5X RTase buffer, RQ1 DNase, and RNase-free water were added to the extracted RNA (3-5 μg), and reacted at 37 ° C. for 15 minutes and at 65 ° C. for 10 minutes, followed by 10 mM dNTPs. , 0.5 μg / ml oligo-dT, ribonuclease inhibitor, M-MLV reverse transcriptase were mixed and reacted at 37 ° C. for 1 hour to synthesize cDNA.

2) Isolation of full-length cDNA of soybean zeaxanthin epoxidase gene

In order to amplify the soy zeaxanthin epoxidase gene, the zeaxanthin epoxidase genes of several plant species were analyzed to find the highest homology and to prepare a primer for amplifying the part. The prepared primer set is as follows.

GmZEP -F: 5'-GTAAAGTTTGATACTTTCACTCCTGCG-3 '(SEQ ID NO: 2)

GmZEP- R: 5'-GAAACAAAGTACTGTTTGTGTCCCAAG-3 '. (SEQ ID NO 3)

As a template, cDNA made from RNA extracted from the leaves of soybean was used as a template, and PCR reaction was carried out using the primer set. As a result, a PCR product of about 300bp was obtained. The PCR product was inserted into the pGEM-T easy vector by TA cloning, and then analyzed by nucleotide sequence, and showed high homology with the genes of zeaxanthin epoxidase of other plants.

In order to identify the full-length cDNA of some of the zeaxanthin epoxidase genes thus identified, CapFishing ^™ full-length cDNA premix kit (SeeGene, Korea) was used. Total RNA was extracted from the leaf tissue of soybean, and for the synthesis of first-strand full-length cDNA, SuperScript ^™ II RNase H-reverse transcriptase (Invitrogene, USA), 3 μl total RNA, 4 μl dNTP (10 mM), 2 μl dT-adaptor (10 μM) and 1.5 μl DEPC-treated water were used for reverse transcription. The mixture was incubated at 75 ° C. for 3 minutes and then stored on ice for 2 minutes, then 4 μl 5X RT buffer, 1 μl CapFishing ^™ solution, 0.5 μl RNase inhibitor (40 u / μl), 1 μl DTT (0.1 M), 2 μl BSA (1 mg / ml), 1 μl reverse transcriptase were added and incubated at 42 ° C. for 1 hour. 3 ㎕ pre-heated CapFishing ^TM After adding the adapter, 0.3 μl of additional reverse transcriptase was further incubated at 42 ° C. for 30 minutes, and then placed at 70 ° C. for 15 minutes and 94 ° C. for 5 minutes to inactivate reverse transcriptase. 180 μl of distilled water was added to the first strand cDNA thus synthesized and diluted. To obtain the sequence of the 5 'portion of the soy zeaxanthin epoxidase gene, 5' RACE PCR was first performed. The first strand cDNA was amplified using a 5 'RACE primer and a zeaxanthin epoxidase gene specific reverse primer ( GmZEP- R) and used as a template for the second PCR. The second PCR reaction was performed using a 5 'RACE primer and zeaxanthin epoxidase gene specific nested reverse primer ( GmZEP nested-R). The sequence of the primer is as follows.

5 'RACE primer: 5'-GTCTACCAGGCATTCGCTTCAT-3' (SEQ ID NO: 4)

GmZEP -R: 5'-GAAACAAAGTACTGTTTGTGTCCCAAG-3 '(SEQ ID NO: 3)

GmZEP nested-R: 5'-CTAATAACTCTTGTGACAGGAAGCCCA-3 '(SEQ ID NO: 5)

The method for identifying the sequence of the 3 'portion of the zeaxanthin epoxidase gene is similar to the 5' RACE. PCR products obtained using the 3 'RACE primer and the zeaxanthin epoxidase gene specific forward primer ( GmZEP- F) As a template, a second PCR was performed using a 3 'RACE primer and a zeaxanthin epoxidase gene specific nested forward primer ( GmZEP nested-F). The primers used are as follows.

3 'RACE primer: 5'-CTGTGAATGCTGCGACTACGAT-3' (SEQ ID NO: 6)

GmZEP -F: 5'-GTAAAGTTTGATACTTTCACTCCTGCG-3 '(SEQ ID NO: 2)

GmZEP nested-F: 5'-TGCCTGCTGACATTGAAACTGTTGGATA-3 '(SEQ ID NO: 7)

With these primers, the PCR reaction was added 5 μl first strand cDNA, 25 μl SeeAmp ^™ Taq Plus Master Mix, 18 μl distilled water. Thermal cycling conditions are 25 cycles 95 ° C. 15 min- [94 ° C. 40 sec-58 ° C. 40 sec-72 ° C. 2 min] -72 ° C. 5 min-4 ° C. Then, using the RACE technique, to clone the entire cDNA of the obtained zeaxanthin epoxidase gene, a new primer was prepared as follows.

GmZEPOX-F: 5'-CAAACTTCTAGAATGGCTACTACCTTATG-3 '(SEQ ID NO: 8)

GmZEPOX-R: 5'-GAACGGATCCTGTCACCTTAACACGGAA-3 '(SEQ ID NO: 9)

To reduce PCR errors, the zeaxanthin epoxidase gene was amplified by Top-pfu polymerase (CoreBioSystem, Korea). The PCR reaction was added 5 μl cDNA (synthesized using RNA extracted from the leaves), 10X buffer, GmZEPOX-F and GmZEPOX-R primers, 2.5 mM dNTP mixture, Top-pfu polymerase. Thermal cycling conditions are 38 cycles 95 ° C. 15 min— [94 ° C. 30 sec-55 ° C. 30 sec-68 ° C. 2 min] -68 ° C. 10 min-4 ° C. In order to confirm the error of the nucleotide sequence, the amplified 2 kb zeaxanthin epoxidase gene was inserted into the pGEM-T easy vector to confirm the nucleotide sequence, and then compared with the nucleotide sequence obtained through RACE. cDNA was cloned.

The base sequence of the soybean zeaxanthin epoxidase gene according to the present invention is as described in SEQ ID NO: 1 in the sequence listing, and based on the base sequence, the amino acid sequence of the expected soybean zeaxanthin epoxidase and the long-term zeaxanthin epoxidase The comparison result of the amino acid sequence of is shown in FIG.

Experimental Example  2.

In soybeans Zeaxanthin Epoxidase  Confirmation of gene expression pattern

Experiments were conducted to confirm the expression pattern of the zeaxanthin epoxidase gene at various times in the development of soybean tissues and root nodules.

First, RNA of various tissues of soybean and tissues obtained at various times of root nodules was extracted using RNeasy Plant Mini Kit (QIAGEN, Germany). To see the expression of the zeaxanthin epoxidase gene in various tissues, after germination, leaves and stems were obtained on the 32nd day, and roots were sampled at the 2nd to 3rd day after germination. It was obtained at the time of day 27 and used to extract RNA. To obtain samples at various times of root nodules, root nodules were inoculated at 2, 7, and 27 days, and 2-3 days old roots without root nodules were used as controls. .

The obtained plant sample was ground in liquid nitrogen, and then RNA was extracted using the RNeasy Plant Mini Kit. Extracting RNA from plant samples using the RNeasy plant mini kit is as described in Experiment 1, 1) above.

RNAs extracted from each tissue sample and root gall were synthesized as cDNA using M-MLV reverse transcriptase (Promega, USA). 5X RTase buffer, RQ1 DNase, and RNase-free water were added to the extracted RNA (3-5 ㎍) and reacted for 15 minutes at 37 ° C for 10 minutes at 65 ° C, followed by 10 mM dNTPs, 0.5 µg / ml. Oligo-dT, ribonuclease inhibitor, M-MLV reverse transcriptase was mixed and reacted for 1 hour at 37 ℃ to synthesize cDNA.

Real-time PCR reactions were then performed using Rotor-Gene 3000 (Corbett Research, Sydney, Australia). PCR reactions were performed using SYBR Premix Ex Taq. (QIAGEN, Germany) (10 μl SYBR Premix Ex Taq. And 2.0 μl cDNA solution) in a 20 μl reaction. Thermal cycling conditions of the PCR reaction are 95 ° C. 15 min— [94 ° C. 15 sec-58 ° C. 10 sec-72 ° C. 20 sec 40 cycles) -72 ° C. 10 min-4 ° C. Data collection was made during each extension phase and measurement of gene expression was performed in duplicates. Rotor-Gene 3000 has a single melting curve analysis The product was carried out using primers that result in a specific melting curve (single product-specific melting curves) , was used for soybean ubiquitin (ubiquitin) primer as a control.

The sequence of soybean ubiquitin primer is as follows.

Ubiquitin -F: 5'-GGGTTTTAAGCTCGTTGT-3 '(SEQ ID NO: 10)

Ubiquitin -R: 5'-GGACACATTGAGTTCAAC-3 '(SEQ ID NO: 11)

Primers for amplifying soy zeaxanthin epoxidase gene are as follows.

GmZEP -F: 5'-GTAAAGTTTGATACTTTCACTCCTGCG-3 '(SEQ ID NO: 2)

GmZEP -R: 5'-GAAA CAAAGTACTGTTTGTGTCCCAAG-3 '(SEQ ID NO: 3)

As a result of investigating the expression level of the zeaxanthin epoxidase gene according to the time of occurrence of various tissues and root nodules through the above experiment, as shown in FIG. It was low, and it was confirmed that the expression level of the zeaxanthin epoxidase gene decreased as the root nodules were gradually developed.

Figure 1 shows the nucleotide sequence of the zeaxanthin epoxidase gene of soybeans.

FIG. 2 shows a comparison of the expected zeaxanthin epoxidase amino acid sequence of soybean and the amino acid sequence of Zeaxanthin epoxidase of Arabidopsis.

Figure 3 shows the results of real-time PCR to confirm the expression level of the zeaxanthin epoxidase gene in various tissues of soybean.

Figure 4 shows the results of real-time PCR to confirm the expression level of the zeaxanthin epoxidase gene according to the soybean root development process.

<110> Industry-Academy Cooperation Foundation, Sookmyung Women's University <120> Gene encoding zeaxanthine epoxidase essential to the xanthopyll          cycle in soybean <160> 11 <170> Kopatentin 1.71 <210> 1 <211> 2010 <212> DNA <213> Glycine max <220> <221> gene (222) (1) .. (2010) <400> 1 atggctacta ccttatgtta caattctctt aacccttcaa caaccgtttt ctcaagaacc 60 catttctcag ttcccttgaa tacagagctt ccactggatg cttcaccttt tgttgttggc 120 tataactgtg gtgtaggatg cagaacaagg aagcaaagga agaaagtgat gcatgtgaag 180 tgtgcagtgg tggaggctcc accaggtgtt tcaccctcag caaaagatgg gaatggaacc 240 accccttcga agaagcagct tcgtatactt gtggctggtg gagggattgg agggttggtt 300 tttgctttgg ctgcaaagag aaaggggttt gaggtgatgg tgtttgagaa ggacttgagt 360 gctataagag gggagggaca gtataggggt ccaattcaga ttcagagcaa tgctttggct 420 gctttggaag ctattgattc agaggttgct gatgaagtta tgagagttgg ttgcatcact 480 ggtgatagaa tcaatggact tgtagatggg gtttctggtt cttggtacgt caagtttgat 540 agattcactc ctgcagtgga acgtgggctt cctgtcacaa gagttattag tcgaatggtt 600 ttacaagaga tccttgctcg cgcagttggg gaagatatca ttatgaatgc cagtaatgtt 660 gttaattttg tggatgatgg aaacaaggta acagtagagc tagagaatgg tcagaaatat 720 gaaggagatg tcttggttag agcggatgga atatggtcca aggtgaggaa gcagttattt 780 gggctcacag aagctgttta ctctggttat acttgttata ctggcattgc agattttgtg 840 cctgctgaca ttgaaactgt tggataccga gtattcttgg gacacaaaca atactttgta 900 tcttcagatg ttggtgcggg aaagatgcaa tggtatgcat ttcacaaaga acctcctggt 960 ggtgttgacg agcccaacgg aaaaaaggaa aggctgctta ggatatttga gggctggtgt 1020 gataatgctg tagatctgat acttgccaca gaagaagaag caattctaag acgagacata 1080 tatgacagga taccaacatt gacatgggga aagggtcgcg tgactttgct cggtgattcc 1140 gtccatgcca tgcagccaaa tatgggccaa ggagggtgca tggctattga ggacagttat 1200 caacttgcat gggagttgga gaatgcatgg gaacaaagta ttaaatcagg gagtccaatt 1260 gacattgatt cttccctaag gagctacgag agagaaagaa gactacgagt tgccattatt 1320 catggaatgg ctagaatggc ggctctcatg gcttccactt acaaggcata tctgggtgtt 1380 ggtcttggcc ctttagagtt tttgactaag tttcgtatac cacatcctgg aagagttgga 1440 ggaaggtttt ttgttgacat catgatgcct tctatgttga gctgggtctt aggtggcaat 1500 agcgacaaac ttgagggcag accactaagt tgcaggctca cagacaaagc aaatgatcag 1560 ttacgtagat ggtttgaaga tgatgaagca ctcgagcgtg ctattaatgg agaatggatt 1620 ttattaccgc atggagatgg aacaggtctt tcaaagccta tatctttaag tcgaaatgag 1680 atgaaaccct tcataatcgg gagtgcacca atgcaagata attcaggcag ttcagttaca 1740 atttcttcac cacaggtttc tccaacgcat gctcgaatta actataagga tggtgccttc 1800 ttcttgattg atttacggag tgagcatggc acctggatca ttgacaacga aggaaagcag 1860 taccgggtac ctcctaatta tcctgctcgc atccgtccat ctgatgttat tcagtttggt 1920 tctgagaagg tttcgttccg tgttaaggtg acaagctctg ttccaagagt ctcagaaaat 1980 gaaagcacac tagctttgca gggagtatga 2010 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gtaaagtttg atactttcac tcctgcg 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gaaacaaagt actgtttgtg tcccaag 27 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtctaccagg cattcgcttc at 22 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ctaataactc ttgtgacagg aagccca 27 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ctgtgaatgc tgcgactacg at 22 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tgcctgctga cattgaaact gttggata 28 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 caaacttcta gaatggctac taccttatg 29 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gaacggatcc tgtcacctta acacggaa 28 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gggttttaag ctcgttgt 18 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ggacacattg agttcaac 18  

Claims (5)

A soybean zeaxanthin epoxidase gene having the nucleotide sequence of SEQ ID NO: 1. A vector comprising the soybean zeaxanthin epoxidase gene of claim 1. The vector of claim 2, wherein the vector is a plant expression vector. A cell transformed with the vector of claim 2. The transformed cell of claim 4, wherein the cell is a plant cell.

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