KR101055275B1 - How to improve the environmental resistance of plants - Google Patents

Abstract

The present invention relates to a novel environmental resistance gene (base sequence), a corresponding protein, a cloning vector comprising a nucleotide sequence, and a plant transformant using the same, and more particularly, the highest expression in ginseng and its expression under stress conditions. The present invention relates to a gene of the increasing ginseng specific abundant proteins (GSAPs), a nucleotide sequence thereof, a corresponding protein, a cloning vector comprising a nucleotide sequence, and a plant transformed by the present invention. Resistance genes and plant transformants using the same may be widely used industrially because they are excellent in resistance to external conditions such as cold, drought and salt.

Environmental resistance genes, cloning vectors, plant transformants

Description

Method for Improving Resistant to Environmental Stress of plants.

Figure 1 shows the genes of the ginseng specific high expression proteins (GSAPs) that are the highest expression among the gene group (ESTs) of the ginseng cDNA library.

Figure 2 shows the expression of the GSAP 1 gene and GSAP 2 gene of the present invention attached to the cell wall confirmed by cell fluorescence analysis.

Figure 3 shows a comparison of the response of the transformed plants and wild-type plants of the present invention to mannitol.

Figure 4 shows a comparison of the response to the salt stress environment transformed Arabidopsis expressing the GSAP gene of the present invention.

Figure 5 shows the analysis of Southern blot GSAP gene, the highest expression 5 in ginseng.

Figure 6 shows a comparison of the amino acid sequence inferred from the GSAP 1 gene and GSAP 2 gene of the present invention.                 

Figure 7 shows the expression of GSAP 1 gene and GSAP 2 gene expression of the present invention using RT-PCR.

Figure 8 shows the analysis of the GSAP 1 gene and GSAP 2 gene expression in the stress environment using RT-PCR.

Figure 9 shows the GSAP 1 gene and GSAP 2 gene of the present invention after the MeJA, SA, ABA treatment is expressed using RT-PCR analysis.

Figure 10 shows the GSAP 1 gene and GSAP 2 gene of the present invention after the abiotic stress is expressed by analyzing in real time RT-PCR method.

11 is a conceptual cleavage map of a cloning vector according to the present invention.

The present invention relates to a novel environmental resistance gene (base sequence), a corresponding protein, a cloning vector comprising a nucleotide sequence, and an environmental resistant plant using them, and more particularly, the highest expression in ginseng and under stress conditions. Ginseng specific abundant proteins whose expression is increased (hereinafter abbreviated as "GSAPs") genes, their nucleotide sequences, their corresponding proteins, cloning vectors containing nucleotide sequences, and plants transformed with them will be.

Ginseng has been shown to be effective in strengthening the lungs, strengthening the lungs, improving the spleen, and relaxing the heart. In addition, it is indicated that the new ginseng herbaceous ginseng is to promote the yang of the five intestines, namely, the liver, heart, lung, kidney and spleen, stabilize the mind, eliminate the soldiers entering the five intestines, brighten the eyes, and take longer to take longer. In addition, the herbal herb tree explains the efficacy of ginseng relatively broadly, and based on this, ginseng is used for various clinical symptoms.

As described above, ginseng is described in many Chinese medicines and its prescription examples, and it is a medicinal herb that plays a central role in all prescriptions. It is recognized as a medicine that does not harm a person.

In particular, Korean ginseng is not only different from Western ginseng in China and Jeonchisam in China, but is also recognized by the world for its efficacy because Korea has geographic conditions suitable for growing ginseng. Korean ginseng is grown cultivated mainly at 36 to 38 ° latitude and grows sufficiently for 180 days longer than the growth period of other ginseng (120 to 130 days per year). can do.

In addition to the main root, ginseng has a shape with many roots such as brain head, root, aide and root hair, and is similar to human. In particular, ginseng is the area where young shoots, which will become flowers and leaves, are stored next year. Winter in Korea is an environment where the drying and humidification is repeated due to the cold and thawing caused by cold and cold temperatures, and the expression of special genes and proteins to protect young tissues is expected in the brains of ginseng.

As a result, the present inventors continued to obtain new environmental resistance genes. As a result, the inventors examined the cDNA library obtained from ginseng to find ginseng specific genes (GSAPs), which are mainly expressed in the brain head or seeds of ginseng and whose expression is increased under stress environment. The nucleotide sequence and the amino acid sequence inferred therefrom are determined to prepare a cloning vector including the nucleotide sequence, and the Arabidopsis sp. Is transformed using the nucleotide sequence, and the plant transformant is resistant to environmental stress. The present invention was successfully completed by confirming this excellent thing.

The present invention aims to provide a new environmental resistance gene (base sequence) derived from ginseng.

It is another object of the present invention to provide an environmentally resistant protein encoded by the environmentally resistant gene.

In addition, an object of the present invention is to provide a cloning vector comprising the nucleotide sequence of the environmental resistance gene.

It is another object of the present invention to provide a plant transformed with the cloning vector.

The present invention for achieving the above object, a novel environmental resistance gene derived from ginseng, that is, environmental resistance gene GSAP 1 comprising the nucleotide sequence of SEQ ID NO: 1, environmental resistance gene GSAP comprising the nucleotide sequence of SEQ ID NO: 2 It relates to an environmental resistance gene GSAP 3 comprising the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3.

The present invention also relates to environmentally resistant proteins encoded by the environmentally resistant genes. The amino acid sequences inferred from the environmental resistance genes GSAP 1, GSAP 2 and GSAP 3 are summarized in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively (see Sequence Listing).

The present invention also relates to a cloning vector pGSAP1 (Accession No .: KCTC10735BP), pGSAP2 (Accession No .: KCTC10736BP), pGSAP3 (Accession No .: KCTC10737BP) comprising the environmental resistance genes GSAP 1, GSAP 2 and / or GSAP 3. .

The present invention also relates to a plant transformant transformed with the cloning vector.

In order to obtain the environmental resistance gene according to the present invention, from about 20,000 ESTs consisting of about 9,600 cDNA is isolated from ginseng, and among the most highly expressed genes, a new gene whose function is not known yet, namely ginseng specific Ginseng specific abundant proteins (GSAPs) genes were cloned (see FIG. 1 ).

As a result of investigating the base sequence of the cloned GSAP genes and the amino acid sequence inferred therefrom, these genes constitute a small gene family consisting of three or more in the genome of ginseng, in particular two of them. Confirmed that the base sequence and the amino acid sequence inferred therefrom are very similar to each other. The amino acid sequences deduced from the environmental resistance genes GSAP 1, GSAP 2 and GSAP 3 according to the present invention are summarized in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively (see Sequence Listing). In addition, by examining the expression pattern of the environmental resistance gene using cell fluorescence, it was confirmed that the product of the gene is mainly expressed in the cell wall (see Fig. 2 ).

The GSAP gene was inserted into an existing vector to produce a cloning vector in a conventional manner, and an environmentally resistant plant was transformed using Arabidopsis sp . The plant transformant was used to measure resistance to environmental stress, and the growth of the transformed Arabidopsis larvae was significantly increased compared to the control group under stress conditions such as salt and dehydration ( FIG. 3 and FIG. 4). Reference).

Therefore, it can be expected that the GSAP gene of the present invention can be used in various ways for plants, microorganisms, animals, etc. for the purpose of increasing resistance to environmental stress such as cold, drought and salt. In addition, the gene of the present invention may be applied to increase the resistance to microorganisms, considering that ginseng has a competitive relationship with various microorganisms in the soil for a long time.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation of Environmental Resistance Genes from the cDNA Library of Ginseng

For the purpose of studying the genes present in ginseng, cDNA banks were prepared from each tissue of ginseng and about 21,152 ESTs were generated. As a result of analyzing 17,685 ESTs, 7,037 ESTs were identified as a single gene, and the remaining 10,648 ESTs were expressed as two or more transcripts, indicating that 2,620 cDNAs were finally formed.

Genes with the largest number of transcripts among the obtained cDNAs and amino acid sequences inferred therefrom were analyzed in nr database of Genebank using blastX and blastP programs. As a result, one of the most highly expressed genes in the ginseng tissue, especially ginseng, in which young leaves and flower buds are stored during winter, was considered a new gene. This group of genes was named the gene of Ginseng Specific Abundant Protein (GSAP) (see FIG. 1 ).

The former is thought to be involved in environmental stress resistance mechanisms such as cold and water deprivation, because it is the highest expression in the head of ginseng and also highly expressed in other tissues of ginseng under stress conditions. GSAP genes were examined using the gene analysis programs cap3 and clustal W, and found to be composed of three kinds of similar genes. These genes were named GSAP1, GSAP2 and GSAP3 (see FIG. 6).

GSAP1 and GSAP2 showed about 88% similarity in the amino acid sequence, but GSAP1 and GSAP3 mutated only one amino acid. However, the gene sequence showed more difference.

Example 2 Base Sequence Determination of Environmental Resistance Gene GASP

The base sequence was determined by a conventional method using the GSAP gene isolated in Example 1 (see FIGS . 5 and 6 ).

In order to analyze the structure of GSAP in the ginseng genome, a souther blot was performed using the GSAP1 cDNA gene as a probe (see FIG. 5). To this end, genomic DNA was extracted from young leaves of ginseng and cut with restriction enzymes EcoRI (E), HindIII (H) and XbaI (X), and electrophoresed on agarose gel to separate DNA fragments by size. After electrophoresis, DNA was transferred to the membrane and hybridization was performed using GSAP1 cDNA as a probe.

As a result, it was confirmed that the GSAP gene constitutes three or more small gene families in the genome of ginseng. After separating three of these genes to determine the nucleotide sequence, each GSAP gene comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, two of which are inferred from the base sequence The amino acid sequences were found to be very similar. The amino acid sequences inferred from the GSAP gene are summarized in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 (see Sequence Listing).

Example 3 Characterization of Environmental Resistance Gene GSAP

In order to investigate the characteristics of the environmental resistance genes, the expression patterns of the three genes isolated in Example 2 were confirmed using cell fluorescence (see FIG. 6 ). As a result, it was confirmed that the protein expressed by the GSAP gene is mainly present in the cell wall (see FIG. 2 ).

In order to investigate the expression pattern of the GSAP gene, cDNA obtained by reverse transcription from mRNA was amplified by PCR and quantitatively analyzed through control experiments as well as qualitative analysis. For GSAP1 and GSAP2, which showed 88% similarity among the three similar genes, the ginseng seeds, roots, stems, leaves, and flowers of each ginseng, low temperature and dehydration treatment, and various hormone treatments over time (MeJA, ABA, SA) In the same stress environment, the tissues of ginseng plants were ground in liquid nitrogen and powdered, and total RNA was isolated using TRIzol ^TM (Invitrogen). MRNA was isolated from the extracted total RNA and 1st strand cDNA was synthesized by reverse transcription. The 1st strand cDNA synthesized by each tissue was amplified by PCR using primers specific to each gene, and the PCR product was compared by expression of electrophoresis on 1% agarose.

As a result, the gene was mainly expressed in the head or seed of ginseng (see FIG. 1 ), and it was found that the expression of the gene was increased in a stress environment such as cold environment (freezing), drought, dehydration or base ( FIG. 1 ) . 7 , 8 , 9 and 10 ). Thus, the GSAP gene of the present invention was expected to be involved in plants to overcome the abiotic environment.

Example 4 Construction of a Cloning Vector Comprising an Environmental Resistance Gene GSAP

Cloning vectors containing the GSAP gene were constructed in a conventional manner.

Specifically, DNA regions containing the nucleotide sequences of SEQ ID NOs: 1, 2, and 3 were inserted into the existing vector pBluscript KS, respectively , to produce cloning vectors pGSAP1, pGSAP2, and pGSAP3, which were assigned to the Biotechnology Research Institute, Dec. 2004 Deposited on 2 days (Accession No .: KCTC10735BP, KCTC10736BP and KCTC10737BP).

A conceptual cleavage map of each cloning vector is shown in FIG. 11 shows GSAP1, GSAP2 and GSAP3 may be interchanged.

Example 5 Preparation of Environmentally Resistant Plant Transformants

In order to prepare an environmentally resistant plant transformant using the GSAP gene, a plant transformation vector was prepared using the 35S promoter of the GSAP1 gene cloned in Example 4 above. Arabidopsis sp. Was transformed in a conventional manner using the prepared plant expression vector, and plant transformants were selected therefrom.

Transformed Arabidopsis and control seeds were sterilized and germinated and cultured in a medium containing 300mM mannitol or 100mM NaCl after 2-3 days of sterilization at 4 ° C. in a cancerous state for 2-3 days. . The growth rate of dehydration and salt stress on the 20th day of culture in a 23 ° C. growth chamber controlled by a photoperiod of 16 hours in a bright state and 8 hours in a dark state was compared with the control group. As a result, it was confirmed that the growth of plant transformants is significantly increased compared to the control group under stress and dehydration (see FIGS . 3 and 4 ).

Therefore, the GSAP gene of the present invention could prove to be a gene that increases resistance to environmental stress such as cold, drought and salt in plants. Furthermore, ginseng is expected to be involved in increasing the resistance to microorganisms, considering that it is competitive with various microorganisms in soil for a long time.

As described and demonstrated above, the environmentally resistant genes of the present invention, which are most expressed in ginseng and whose expression is increased under stress conditions, are plants that withstand environmental stress because they are excellent in resistance to external conditions such as cold, drought and salt. It can be widely used in industrial fields such as developing animals, microorganisms and the like.

<110> EUGENTECH INC. <120> Novel Genes Resistant to Environmental Stress, Nucleotide          Sequences, Proteins by the Nucleotide Sequences, Cloning Vectors          and Plant Transformants using the Vectors <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 691 <212> DNA <213> ginseng <400> 1 gaaaaaaaca cggaaaaaac atgttcattg taatcatccc agctcgttca ctgtaaaaag 60 gattgcttat aggataatct aatatacagt aaaaagtact cataactaac ggaaatggag 120 aacaagaagg ttgctctggt ggtggtagca atggtggtgg ttctgatatc taccttcgca 180 cccttggctc tggcagctga tgaggctggt aggtctgacg gtggaaatgg agatttaatt 240 gctgtggagt ccgtcagtcg ctctaacggc ggtaatggag attcagttac tgttgacacc 300 gtcagttggg taagttctaa caggaaaacg ctccggtcaa gcatctttct gccgcaggga 360 tatctcccgg acggcggcct aggctgcaaa tcttctggag cgtggtgcgg ttttgatcca 420 cacggctgct gtggcaactg tggctgtttg gttggttttt gctacggtac tggttgctga 480 gctgtggccg tggtgcttgt gcttgaggga gatggcattg tgccttttat cagtgtcttt 540 tagtaattat gtgttaatta aaacgccttc atttcattca aaccgatagt ttggatgaac 600 tatgtaaggc tcctttttgt gtgtagaaga aatttacttt gtactgcaaa gtaacataat 660 aattaaaata aatactccct ccgttaactc a 691 <210> 2 <211> 590 <212> DNA <213> ginseng <400> 2 ctcataacta acggaaatgg agaacaagaa ggttgctctg gtggtggtgg caatggtggt 60 ggttctgata tcgaccgtcg cacccttggc tatggcagtt gatgatgctg gtagctctga 120 cggtttaatt gctgtggagt ccgtcagtcg ctctaatggc ggtaatggag attcagttgc 180 tgttgacacc gtcagttggg taagttctaa caggaaaacg ctccggtcaa agatctttct 240 gccgcaggga tacctcccgg acggcggtct aggctgcaaa tctgccggaa cgtggtgcgg 300 ttttgatcca cacggctgct gcggcagctg tggttgtctg gtgggttttt gctacggtgt 360 tagttgctga gctgtggccg tggtggttgt gctcgaggga gatggcattg tgcctaattt 420 tatcagtgtc ttttagtact tatatgtgtt aattaaaacg ccttcatttc attcaaaccg 480 atagtttgga tgaactatgt aaggctcctt tctctgtgta aaagaaattt actttctacc 540 gcaaagtaac ataataatta aaataaatac tccctccgtt aattcacgct 590 <210> 3 <211> 553 <212> DNA <213> ginseng <400> 3 aaagtactca tcataactaa cggaaatgga gaacaagaag gttgctctgg tggtggtggc 60 aatggtggtg gttctaatat cgaccttcgc acccttggct ctggcagctg atgaggctgg 120 taggtctgac ggtggaaatg gagatttaat tgctgtggag tccgtcagtc gctctaacgg 180 cggtaatgga gattcagtca ctgttgacac agtcagttgg gtaagttcta acaggaaaac 240 gctccggtca agcatctttc tgccgcaggg atacctcccg gacggcggcc taggctgcaa 300 atctggcgga gcgtggtgcg gttttgatcc acacggctgc tgtggcaact gtggctgttt 360 ggttggtttt tgctacggta ctggttgctg agctgtggcc gtggtggttg tgcttgaggg 420 agatgcatga gattgtgcct tttattagtg tcttttagta cttgtatgtg ttaattaaaa 480 cgccttcatt tcattcaaac ccgatagttt ggatgaacta tgcaaggctc ctttctatgt 540 gtactgccaa gta 553 <210> 4 <211> 121 <212> PRT <213> ginseng <400> 4 Met Glu Asn Lys Lys Val Ala Leu Val Val Val Ala Met Val Val Val   1 5 10 15 Leu Ile Ser Thr Phe Ala Pro Leu Ala Leu Ala Ala Asp Glu Ala Gly              20 25 30 Arg Ser Asp Gly Gly Asn Gly Asp Leu Ile Ala Val Glu Ser Val Ser          35 40 45 Arg Ser Asn Gly Gly Asn Gly Asp Ser Val Thr Val Asp Thr Val Ser      50 55 60 Trp Val Ser Ser Asn Arg Lys Thr Leu Arg Ser Ser Ile Phe Leu Pro  65 70 75 80 Gln Gly Tyr Leu Pro Asp Gly Gly Leu Gly Cys Lys Ser Ser Gly Ala                  85 90 95 Trp Cys Gly Phe Asp Pro His Gly Cys Cys Gly Asn Cys Gly Cys Leu             100 105 110 Val Gly Phe Cys Tyr Gly Thr Gly Cys         115 120 <210> 5 <211> 117 <212> PRT <213> ginseng <400> 5 Met Glu Asn Lys Lys Val Ala Leu Val Val Val Ala Met Val Val Val   1 5 10 15 Leu Ile Ser Thr Val Ala Pro Leu Ala Met Ala Val Asp Asp Ala Gly              20 25 30 Ser Ser Asp Gly Leu Ile Ala Val Glu Ser Val Ser Arg Ser Asn Gly          35 40 45 Gly Asn Gly Asp Ser Val Ala Val Asp Thr Val Ser Trp Val Ser Ser      50 55 60 Asn Arg Lys Thr Leu Arg Ser Lys Ile Phe Leu Pro Gln Gly Tyr Leu  65 70 75 80 Pro Asp Gly Gly Leu Gly Cys Lys Ser Ala Gly Thr Trp Cys Gly Phe                  85 90 95 Asp Pro His Gly Cys Cys Gly Ser Cys Gly Cys Leu Val Gly Phe Cys             100 105 110 Tyr Gly Val Ser Cys         115 <210> 6 <211> 121 <212> PRT <213> ginseng <400> 6 Met Glu Asn Lys Lys Val Ala Leu Val Val Val Ala Met Val Val Val   1 5 10 15 Leu Ile Ser Thr Phe Ala Pro Leu Ala Leu Ala Ala Asp Glu Ala Gly              20 25 30 Arg Ser Asp Gly Gly Asn Gly Asp Leu Ile Ala Val Glu Ser Val Ser          35 40 45 Arg Ser Asn Gly Gly Asn Gly Asp Ser Val Thr Val Asp Thr Val Ser      50 55 60 Trp Val Ser Ser Asn Arg Lys Thr Leu Arg Ser Ser Ile Phe Leu Pro  65 70 75 80 Gln Gly Tyr Leu Pro Asp Gly Gly Leu Gly Cys Lys Ser Gly Gly Ala                  85 90 95 Trp Cys Gly Phe Asp Pro His Gly Cys Cys Gly Asn Cys Gly Cys Leu             100 105 110 Val Gly Phe Cys Tyr Gly Thr Gly Cys         115 120  

Claims (15)

delete delete delete delete delete delete delete delete delete delete delete delete A method of improving the environmental resistance of a plant by increasing the expression of a gene having a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The method of claim 13, By transforming with a vector comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, the expression of the gene having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is increased How to improve the environmental resistance of plants. The method of claim 14, The vector is pGSAP1 (Accession No .: KCTC 10735BP), pGSAP2 (Accession No .: KCTC 10736BP) or pGSAP3 (Accession No .: KCTC 10737BP) The method of improving the environmental resistance of the plant, characterized in that.

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