KR20090043754A - Ibtip1 gene derived from a root of ipomoea batatas and a promoter thereof - Google Patents

Abstract

The present invention relates to a IbTIP1 gene and its promoter from potato roots and stems, more specifically from potato is strongly expressed, and the expression is induced by various stresses in the sweet potato root (Ipomoea batatas L. Lam) root and IbTIP1 gene and its promoter. The IbTIP1 gene and its promoter of the present invention can be usefully used to develop environmentally friendly alternative energy crops for the production of transformed sweet potatoes or bioethanol, which produce useful materials such as environmental stress resistant plants, starch, functional proteins, etc. Can be.

IbTIP1 gene, promoter, stress, RT-PCR

Description

{IbTIP1 gene derived from a root of Ipomoea batatas and a promoter}

The present invention relates to a IbTIP1 gene and its promoter from potato roots and stems, more specifically from potato is strongly expressed, and the expression is induced by various stresses in the sweet potato root (Ipomoea batatas ) roots derived from the IbTIP1 gene and its promoter.

Sweet potato ( Ipomoea batatas L. Lam) can be grown on relatively poor lands and has a yield of about 22 tons per hectare, which is a representative root crop used for food and livestock feed. In particular, about 70% of the buildings are made of starch, which has been used as a raw material for alcoholic beverages for a long time, and has recently been re-examined as an environmentally friendly alternative energy crop for bioethanol production.

Recently, due to the rapid industrialization and population increase, environmental and food problems on the global scale have been raised, and the development of environmentally resistant crops that grow well even in areas where desertification, high seas and cold regions are disadvantageous has been actively conducted. In particular, in order to develop industrial sweet potatoes suitable for the disadvantaged area, research on environmentally resistant transformed sweet potatoes by molecular breeding has been attempted (Lim et al. Mol Breeding 19, 227-239, 2007).

In order to develop environmentally resistant sweet potatoes to improve the early growth of seedlings in poor soil such as dry and cold, the development of genes that are highly expressed in the sweet potato roots (fibrous root) is urgently needed. Under dry stress conditions due to lack of moisture, the development of thread roots is thought to be important for tuber growth. In addition, since the roots develop and grow into tubers, it is thought that the development of genes that are highly expressed in the roots is important when producing useful materials from the tubers. However, the genes that are specifically expressed in young roots in sweet potatoes have not been reported. However, genes encoding the sporamin protein, which is highly expressed in tubers of sweet potatoes, have long been isolated and studied (Hattori et al. Plant Mol Biol 14, 595-604, 1990).

In this regard, the isolation of genes that are strongly expressed in sweet potato roots may be useful for developing transgenic sweet potatoes producing environmentally resistant plants and various useful materials. In other words, metabolic engineering using advanced biotechnologies, especially when growing sweet potato varieties such as dry land, reclaimed land, and high sea areas, producing industrially useful materials such as starch, functional protein, and so on. It is expected to be able to produce eco-friendly bioenergy without affecting.

TIP (tonoplast intrinsic protein) of the present invention is a gene that is classified as aquaporin gene group that is a water channel protein (water channel protein) closely related to dry resistance (Chrispeels et al. Trends Biochem Sci 19, 421 -425, 1994). TIP genes isolated from the dry-resistant Craterostigma plantagineum have been reported to increase expression by drying and treatment of the plant stress hormone abscisic acid (ABA) (Mariaux et al. Plant Mol Biol 38, 1089- 1099, 1998). TIP genes isolated from olive trees (Olea europaea) have also been reported to be induced by water stress (Secchi et al. Genbank accession number ABB76813, 2005). However, no TIP gene has been reported in sweet potatoes.

The present inventors confirmed that the IbTIP1 gene isolated from the dried sweet potato roots was strongly expressed in the roots of sweet potatoes, especially in the root root tissues, as well as determining the sequencing of the promoter of the gene to determine stress-related major cis-activation factor ( The present invention was completed by confirming the presence of a cis-acting element.

It is an object of the present invention to provide an IbTIP1 gene derived from sweet potato root and its promoter.

In order to achieve the above object, the present invention provides IbTIP1 protein and its amino acid sequence derived from sweet potato root.

The present invention also provides a polynucleotide encoding the IbTIP1 protein.

The present invention also provides a vector comprising the polynucleotide.

In addition, the present invention provides a transformant transformed with the vector comprising the polynucleotide.

The present invention also provides a polynucleotide exhibiting promoter activity that induces thread-root specific gene expression.

The present invention also provides a vector comprising all or part of the polynucleotide.

The present invention also provides a transformant transformed with a vector comprising all or part of the polynucleotide.

The present invention also provides a method for producing a transformant capable of inducing the production of a foreign protein under stress.

The present invention also provides a method for the high expression of foreign proteins in the roots.

Hereinafter, the present invention will be described in detail.

The present invention provides IbTIP1 protein and its amino acid sequence derived from sweet potato root.

The IbTIP1 protein of the present invention is characterized by having an amino acid sequence set forth in SEQ ID NO: 2 .

The IbTIP1 protein of the present invention is composed of 251 amino acids, and the NPA (Asn-Pro-Ala, Asparagine-Proline-Alanine) motifs present on loops B and E in the aquaporin protein structure have an IbTIP1 protein amino acid sequence. Was well preserved in (see FIG. 1). Further, when the coding portion of IbTIP1 of the present invention was examined by blast (BlastX), TIP1; 2 of Minosa pudica , gamma-TIPs group of Arabidopsis thaliana [At4g01470 (AtTIP1; 3), At3g26520 (AtTIP1) 2), At2g36830 (AtTIP1; 1)] and high homology (74% -78% amino acids) (see FIGS. 2A and 2B).

The present invention also provides a polynucleotide encoding the IbTIP1 protein.

The polynucleotide encodes a protein having an amino acid sequence set forth in SEQ ID NO: 2, and preferably has a nucleotide sequence set forth in SEQ ID NO: 1 .

The IbTIP1 cDNA gene of the present invention has a total length of 1065 bp and is composed of 82 bp 5'-untranslated region (UTR), 197 bp 3'-untranslated region, and 753 bp coding portion (consisting of 251 amino acids). (See FIG. 1).

The gene encoding the sweet potato-derived IbTIP1 protein of the present invention exists in a large number of families (see FIG. 5) on the sweet potato genome, strongly expressed in fibrous root, thick pigmented root, and storage root. It was also expressed in tuberous root tissues. However, leaf and stem tissues were very weakly expressed (see FIG. 3). Therefore, the IbTIP1 gene of the present invention isolated from the dried sweet potato root tissue was found to be a gene that is highly expressed in the root, including the roots.

In addition, the IbTIP1 gene of the present invention gradually increased from 1 hour after drying treatment in the leaf tissues showed an expression peak at 4-8 hours and then decreased (see FIG. 4A). On the other hand, no increase or decrease in the expression level of IbTIP1 was observed in dry root tissues (see FIG. 4A). This is presumably due to the high expression level of IbTIP1 gene in the steady state.

In addition, the dry stress-related plant hormone, Apis acid (ABA) treatment also showed an expression pattern similar to the drying treatment (see Fig. 4b).

The present invention also provides a vector comprising the polynucleotide.

The gene included in the vector of the present invention includes a polynucleotide encoding IbTIP1 of the present invention, and preferably includes a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 1 . As the vector used to insert the gene, it is preferable to use any one of pKBS1-1 vector, pBI101 vector, and pCAMBIA vector, and any expression vector for general plant transformation may be used.

In addition, the present invention provides a transformant transformed with the vector comprising the polynucleotide.

The transformants include transformed microorganisms, animal cells, plant cells, transformed animals or plants, and cultured cells derived therefrom.

The present invention also provides a polynucleotide exhibiting promoter activity that induces thread-root specific gene expression.

The polynucleotide of the present invention is a promoter of the IbTIP1 gene, and is characterized by having a nucleotide sequence set forth in SEQ ID NO: 3 . That is, the promoter of the IbTIP1 gene is composed of the nucleotide sequence corresponding to the region up to -1,597 bp from the top of the translation start point of IbTIP1p_1.6 (see Fig. 7). In the IbTIP1p_1.6 promoter of the present invention, various cis-acting elements responsive to absic acid and dry stress were present, and a number of factors related to plant disease resistance were present. In addition, the presence of factors related to root expression may be useful for the study of root-specific expression promoters. In particular, since it contains a lot of dry stress response factors, it can be very useful for developing plants having resistance to dry stress.

Specifically, as a result of nucleotide sequence analysis of the promoter, it was confirmed that the IbTIP1p_1.6 promoter has regulatory factor sites of various eukaryotic promoters as follows. The TATA-box for transcription initiation was in the -115--106 position and the CAAT-box was in the -155--152 position. ACGTG sequence, the consensus sequence of ABRE, known as an important factor responding to Absic acid (ABA) as a binding site of transcriptional regulator protein, is repeatedly present in two positions -208 ~ -204 and -196 ~ -191 By -196 ~ -191 It was also in place. MYB-recognition site common sequence (CNGTTR or C / TAACG / TG) to which MYB protein responds to signal transduction process and dry stress of absic acid (ABA) is -1577 ~ -1572, -1406 ~ -1401 (reverse direction) , -1398 ~ -1393, -470 ~ -465, -468 ~ -462 (reverse) position. In addition, the MYC-identical consensus sequence (CANNTG) to which the MYC protein reacted with drying was found at the positions -926 ~ -921 and -149 ~ -144. A common base sequence (G / ACCGAC) of the dehydration-responsive element / C-repeat (DRE / CRT) factor, which is a site to which the DREB1 / CBF protein responds to dry stress and low temperature, was found at -332 ~ -327 positions. The nucleotide sequence of the W-box (TTGAC) to which WRKY protein, which plays an important role in disease resistance, is located at -1236 ~ -1232, -738 ~ -734 (reverse), -672 ~ -668, -648 ~ -644 Many existed. GARE-factor common base sequence (TAACAGA), regulated by gibberellin (GA), a signaling agent in plants, was found at the positions -431--425 and -371--365. In addition, the GCCACGTGGC sequence, which is an ACGT-motif associated with root expression, was also found at the positions -198 and -189. In addition, a number of GT1-boxes (GGTTAA), which are known as factors that react to light, have also been found (see FIG. 7).

As described above, the promoter of the IbTIP1 gene of the present invention can effectively induce the expression of the gene by stress. To this end, the promoter of the present invention includes factors for recognizing stress caused by absic acid, drying, plant disease, light or low temperature. The promoter of the IbTIP1 gene consists of all or part of the nucleotide sequence set forth in SEQ ID NO: 3 utilizing these properties, and consists of a DNA sequence exhibiting promoter activity and a structural gene operably linked to the DNA sequence. It can be used for manufacture. The fusion gene construct useful for producing a transformant for useful substance production, because expression of a useful substance by the control of the promoter of IbTIP1 gene under various stress by connecting the structural gene associated with the production of a useful substance to the promoter of IbTIP1 gene Can be used.

In addition, when a gene representing resistance to various environmental stresses to the fusion gene construct is used as a structural gene, it may be used to prepare a transformant having resistance against external stress.

The present invention also provides a vector comprising the polynucleotide.

The polynucleotide included in the vector of the present invention is IbTIP1 of the present invention. It is all or part of the promoter of a gene, Preferably it contains all or part of the base sequence described by SEQ ID NO. As the vector used to insert the gene, it is preferable to use any one of pKBS1-1 vector, pBI101 vector, and pCAMBIA vector, and any expression vector for general plant transformation may be used.

In addition, the present invention provides a transformant transformed with the vector comprising the polynucleotide.

The transformants include transformed microorganisms, animal cells, plant cells, transformed animals or plants, and cultured cells derived therefrom.

In addition, the present invention provides a method for producing a transformant capable of inducing the production of a foreign protein under various stresses using the captive motor of the IbTIP1 gene.

The method for producing the transformant

1) an expression vector comprising all or a portion of the nucleotide sequence set forth in SEQ ID NO: 3 and comprising a gene construct consisting of a polynucleotide showing a promoter activity and a polynucleotide encoding a foreign protein operably linked with the polynucleotide; Preparing a;

2) introducing the expression vector into a host cell; And

3) selecting a transformant into which the expression vector is introduced.

The "transformer" refers to a cell or plant transformed by an expression vector comprising a gene construct consisting of a poromotor of an IbTIP1 gene and a DNA sequence encoding a foreign protein operably linked thereto. it means. In the present invention, transformants include transformed microorganisms, animal cells, plant cells, transformed animals or plants, and cultured cells derived therefrom.

The foreign protein in the method of the present invention includes a protein that confers stress to the protein or transformant exerting a pharmacological effect. Therefore, it is possible to prepare a transformant capable of producing a useful substance and a transformant resistant to various environmental stresses by the method of the present invention.

The various environmental stresses are caused by abscisic acid, drying, plant diseases, light or low temperature, and factors that recognize them are included in the promoter.

In addition, the present invention

1) consists of all or part of the nucleotide sequence set forth in SEQ ID NO: 3 , Preparing an expression vector comprising a gene construct comprising a polynucleotide exhibiting promoter activity and a polynucleotide encoding a foreign protein operably linked with the polynucleotide;

2) introducing the expression vector into a host plant cell;

3) selecting a transgenic plant cell into which the expression vector is introduced;

4) preparing a callus by inducing germination of the transgenic plant cells;

5) preparing a transgenic plant by inducing differentiation of the callus; And

6) provides a method for high expression of the foreign protein in the root comprising the step of culturing the transgenic plant.

Preferably, the plant cell is preferably a monocotyledonous plant or a dicotyledonous plant, but is not limited thereto.

The monocotyledonous plants include Alismataceae, Hydrocharitaceae, Juncaginaceae, Schuchzeriaceae, Pomomotontonaceae, Najadaceae, Zosteraceae, Liliaceae, Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, Pontederiaceae, Iridaceae, Burmanniaceae, Juncaceae, Commelinaceae , Eriocaulaceae, Panaxaceae (Gramine, Gramineae, Poaceae), Araceae, Lemnaceae, Spaganiaceae, Typhaceae, Forageaceae (Cyperaceae), Pachoaceae (Musaceae) ), Ginger (Zingiberaceae), Redaceae (Cannaceae), Orchidaceae (Orchidaceae) is preferably, but not limited to.

The dicotyledonous plants include Asteraceae (Asteraceae, Diapensiaceae), Asteraceae (Clethraceae), Pyrolaceae, Ericaceae, Myrsinaceae, Primaceae, Primaceae (Plumbaginaceae), Persimmonaceae (Ebenaceae) , Styracaceae, Stink bug, Symplocaceae, Ash (Oleaceae), Loganiaceae, Gentianaceae, Menyanthaceae, Oleaceae, Apocynaceae , Asclepiadaceae, Rubisaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Labiatae, Solanaceae, Scrophulariaceae , Bignoniaceae, Acanthaceae, Sesame (Pedaliaceae), Fructose (Orobanchaceae). Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, (Perox Adoxaceae), Valerianaceae, Dipsacaceae, Campanaceae ( Campanulaceae, Compositae, Myraceae, Juglandaceae, Salicaceae, Birchaceae, Beechaceae, Fagaceae, Elmaceae, Moraceae, Nettle Urticaceae, Santalaceae, Mistletoe, Lothanthaceae, Polygonaceae, Apoaceae, Phytolaccaceae, Nyctaginaceae, Aizoaceae, Portulacaceae ), Caryophyllaceae, Chenopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cecidiphyllaceae, Minaria (Ranunculac eae), Berberidaceae, Lardizabalaceae, Bird sandaceae (Amanispermaceae), Nymphaeaceae, Ceratophyllaceae, Cabombaceae, Saururaceae , Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Camellia, Theaceae, Guttaiferae, Droseraceae, Poppyaceae (Papaveraceae), Capparidaceae, Cruciferaceae (Cruciferae), Plantanaceae (Platanuaceae, Platanaceae), Periaceae (Gelaceae, Hamamelidaceae), Pheasant's Asteraceae, Crassulaceae, Saxifragaceae Eucommiaceae, Pittosporaceae, Rosaceae, Leguminosae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae Euphorbiaceae, star Callitrichaceae, Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Mapleaceae, Aceraceae, Sapindaceae, Mapleaceae (Hippocastanaceae), Sabiaceae, Balsam (Bamsamaceae), Aquifoliaceae, Novaxaceae, Celastraceae, Staphyleaceae, Buxaceae, Empetraceae , Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Adenaceae, Thymelaeaceae, Ellaeagnaceae Flacourtiaceae, Violaceae, Passifloraceae, Tamaricaceae, Elatinaceae, Begoniaceae, Cucurbitaceae, Lymphaceae, Lythraceae, Pomegranate ( Punicaceae, Onnagraceae, Haloragaceae, Bataceae (Alangiaceae), Dogwood (Hornaceae, Cornaceae), Arboraceae (Araliaceae), Umbelliferae (Apiaceae) Is preferably, but is not limited thereto.

Most preferably the plant cells are selected from plants belonging to the Cruciferaceae, Rosaceae and Rosaceae.

The step of introducing the expression vector of step 2) into the host plant cell may be introduced into the plant by a known plant transformation method. That is, a well-known method known to those skilled in the art, such as Agrobacterium mediated method, gene gun, method using PEG, and electroporation, can be used. Preferably, the dicot plants use the Agrobacterium mediated method, and the monocots use the method using a gene gun, but are not limited thereto.

After transformation, plant cells should be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known for many different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989 Momillan, N.Y.). Thus, once transformation is realized, it is within the knowledge of the art to regenerate mature plants from transformed plant cells.

The foreign protein in the method of the present invention includes a protein that confers stress to the protein or transformant exerting a pharmacological effect.

Since the IbTIP1 gene of the present invention is strongly expressed in sweet potato roots and its expression is induced by various stresses, the gene of the present invention and its promoters are useful materials such as environmental stress resistant plants, starches, functional proteins, etc., which are suitable for adverse conditions. It can be useful for developing environmentally friendly alternative energy crops for the production of transgenic sweet potatoes or bioethanol.

Hereinafter, the present invention will be described in detail by way of examples.

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

Example 1 Sweet Potato IbTIP1  Cloning of genes

The fibrous root tissue of sweet potato ( Ipomoea batatas (L.) Lam. Cv.White star) was dried for 6 hours, followed by CTAB method (Kim and Hamada, Biotechnol Lett 27, 1841-1845, 2005). RNA was isolated. MRNA was isolated using the Poly (A) Tract mRNA separation system (Promega) and sweet potato dried root cDNA library was prepared using SMART cDNA Library Construction Kit (Clontech). The primary titer of the library showed a value of about 1.5 × 10 ⁶ pfu / ml. 983 EST clones were obtained through sequencing through random extraction.

T3 primers were used to determine the base sequence of the 5 'portion of the cDNA, and relevant genetic information and data were obtained through analysis of the Blast database of NCBI (www.ncbi.nlm.nih.gov). Among them, TIP (tonoplast intrinsic protein) gene (Chrispeels et al. Trends Biochem Sci) belonging to the aquaporin family, which is a water channel protein, is a candidate that is expected to show specific expression in dry and root through data analysis. 19, 421-425, 1994) were cloned and the total sequence of cDNA was determined. This cDNA gene was named ' IbTIP1 ' cDNA. The total length of the IbTIP1 cDNA gene was 1065 bp, which was composed of 82 bp 5'-untranslated region (UTR), 197 bp 3'-untranslated region, and 753 bp coding region (consisting of 251 amino acids). 1 (SEQ ID NO: 1). NPA (Asn-Pro-Ala, Asparagine-Proline-Alanine) motifs present on loops B and E in the aquaporin protein structure were well conserved in the amino acid sequence of IbTIP1 protein (FIG. 1, SEQ ID NO: 2) .

The coding portion of sweet potato IbTIP1 showed the highest homology (84% amino acid) with TIP1; 2 of Minosa pudica and the gamma-TIPs group of Arabidopsis thaliana [At4g01470 (BlastX). AtTIP1; 3), At3g26520 (AtTIP1; 2), At2g36830 (AtTIP1; 1)] and showed high homology (74% -78% amino acids) (FIGS. 2A and 2B). The soft relationship between the amino acid sequence of the putative protein of sweet potato IbTIP1 and the membrane endogenous protein (MIP) of Arabidopsis is investigated by using the ClustalW program (). ).

<Example 2> by sweet potato tissue IbTIP1  Gene expression analysis

RT-PCR was performed to analyze the tissue-specific expression patterns of the sweet potato water transfer pathway protein gene IbTIP1 isolated from the dried sweet potato root. Extraction of total RNA from sweet potato tissues (leaf, stem, root, coarse root, storage root) by CTAB method (Kim and Hamada 2005), and ImPro-H Reverse Transcription System cDNA synthesis kit (Promega) CDNA was synthesized. The expression pattern of IbTIP1 gene was investigated using Ex-Taq DNA polymerase (TaKaRa) using IbTIP1 gene specific primer. Specific primers of IbTIP1 include the nucleotide sequence (Forward: 5'-GTCACGTAAACCCTGCTGTCAC-3 ': SEQ ID NO: 4). And (Reverse: 5'-GCTAGAATGTTGGCACCCACTA-3 ': SEQ ID NO: 5).

As a result, IbTIP1 gene was strongly expressed in fibrous root and also in thick pigmented root and tuberous root tissue. However, leaf and stem tissues were very weakly expressed (FIG. 3).

Therefore, the IbTIP1 gene of the present invention isolated from the dried sweet potato root tissues was found to be a gene that is specifically expressed in root-specific, in particular, root-root.

Example 3 Drying and Hormone Treatment IbTIP1  Gene expression analysis

In order to analyze the degree to which the IbTIP1 gene of the present invention reacts by drying treatment, the leaves and thread root tissues of sweet potato were dried by time zones (0, 1, 2, 4, 8, 16, 24 hours), and the above example. RNA was isolated by the method performed in step 2, and cDNA was synthesized, respectively. RT-PCR was performed with the IbTIP1 gene specific primer used in Example 2 using the Accel Taq Premix kit (Genedocs).

As a result, the IbTIP1 gene gradually increased from 1 hour after drying in the leaf tissues, and showed an expression peak at 4-8 hours, and thereafter decreased (Fig. 4a). On the other hand, no increase or decrease in the expression level of IbTIP1 was observed in dry root tissues. This is presumably due to the high expression level of IbTIP1 gene in the steady state.

In general, under dry stress, the amount of plant hormone abscis acid (ABA) increases, and it is known that abscis acid controls the pore opening and closing of plants in response to drying. In order to observe whether the IbTIP1 gene in response to drying treatment increased the expression level even by abscitic acid treatment, the leaf and silt tissues of sweet potato were treated with abscisic acid (100 μM) for each hour (0, 12, 24, 36, 48 hours). RNA was extracted in the same manner as above, cDNA was synthesized, and RT-PCR was performed.

As a result, after peak treatment in the leaf tissues increased from 12 hours to 36 hours, the expression peaked, and then decreased. On the other hand, there was no change in the expression level in the roots tissue (Fig. 4b). This is also considered to be due to the high expression level of IbTIP1 gene in steady state.

<Example 4> IbTIP1  Southern blot analysis of genes

Southern blot analysis was performed to observe the presence of the IbTIP1 gene in the sweet potato genome and other homologous genes of the present invention. Genomic DNA was isolated and purified from sweet potato leaves by CTAB method, and then digested with EcoRI, HindII, HindIII, and XbaI. After electrophoresis of the cleaved genomic DNA, Southern blots were performed using a probe labeled C-terminal HindII fragment (463 bp) of IbTIP1 cDNA with ³² P radioisotopes.

As a result Since two or more DNA bands appear to bind to the probe of IbTIP1 in each fragment, it can be seen that the IbTIP1 gene of the present invention exists in a large number of families on the sweet potato genome (FIG. 5).

<Example 5> IbTIP1  Isolation and Sequencing of Top 5 'Regulatory Promoter Regions of Genes

Using a GenomeWalker kit (Clontech), the promoter region of the IbTIP1 gene belonging to the aquaporin family, which is a sweet potato water channel gene (water channel) gene, was isolated according to the manufacturer's instructions. 2.5 μg of sweet potato genomic DNA isolated by a conventional method was digested with restriction enzymes EcoRV, DraI, PvuII, StuI, and purified by digestion with phenol / chloroform and ethanol. The genomic library for genome working was prepared by binding the adapter provided in the kit to the purified genomic DNA using a DNA ligase.

Using these libraries, IbTIP1 genomic DNA was obtained by genomic PCR. The GSP1 primer (gTIP1) described in SEQ ID NO: 6 prepared based on the 5'-terminal sequence information of the isolated water transfer pathway protein gene IbTIP1 cDNA. -r1: 5'-CGAGGTAGCCTCACCCACGCTTCCAAT-3 ') and adapter primer AP1 (5'-GTAATACGACTCACTATAGGGC-3') provided in the kit were used, and genomic PCR was performed using the genomic library DNA as a template. . The PCR reaction was performed 7 times for 25 seconds at 94 ° C. and 3 minutes at 72 ° C., and was repeated 32 times for 25 seconds at 94 ° C. and 3 minutes at 67 ° C. And finally amplified for 7 minutes at 67 ℃. After the PCR reaction, a part of the reaction was confirmed by electrophoresis (FIG. 6). After diluting the reaction of the primary PCR by 50-fold, the 5'-terminal sequence information of the sweet potato water transfer pathway protein gene IbTIP1 cDNA was prepared. PCR was repeated using one GSP2 primer of SEQ ID NO. It was. PCR conditions at this time are as follows. The reaction was performed 5 times for 25 minutes at 94 ° C. and 3 minutes at 72 ° C., 20 times for 25 seconds at 94 ° C. and 3 minutes at 67 ° C., and finally amplified at 67 ° C. for 7 minutes. A portion of the reaction was electrophoresed to confirm the reaction product (FIG. 6). Two different PCR products from the DraI and StuI genome libraries were cloned into PCR2.1-TOPO vector (Invitrogen) and pGEM-T Easy vector (Promega) and sequenced.

Thus, the nucleotide sequence was determined to obtain the 5'-upstream nucleotide sequence of the IbTIP1 gene, and the genomic gene sequence was named ' IbTIP1p_3.4 ' promoter and ' IbTIP1p_1.0 ' promoter, respectively. The IbTIP1 promoters isolated from the sweet potato genomic library were 3,400 bp and 914 bp in length, respectively, from the translation start site to the top. Through sequencing, these two promoter sequences are considered to be of the same kind. These promoter sequences were found to completely match the nucleotide sequence of the 5'-untranslated region (UTR) of IbTIP1 cDNA. Among them, IbTIP1p_3.4 clone corresponding to 3,400 bp in total length was subcloned with SpeI-BglII restriction enzyme and named as IbTIP1p_1.6 (1,597 bp size) (FIG. 7).

<Example 6> IbTIP1  Analysis of cis-acting elements in promoters of genes

The IbTIP1 gene promoter of the present invention consists of a nucleotide sequence corresponding to a region from the top of the start of translation of IbTIP1p_1.6 up to -1,597 bp (FIG. 7). The nucleotide sequence of the IbTIP1p_1.6 promoter was analyzed using the PLACE database (www.dna.affrc.go.jp/PLACE/signalup.html), a cis-activator analysis program.

As a result of sequencing of the promoter, it was confirmed that the IbTIP1p_1.6 promoter has regulatory region of various eukaryotic promoters. The TATA-box for transcription initiation was in the -115--106 position and the CAAT-box was in the -155--152 position. ACGTG sequence, which is a consensus sequence of ABRE, known as an important factor responding to Absic acid (ABA) as a binding site of transcriptional regulator protein, is repeatedly present in two positions -208 ~ -204 and -196 ~ -191. It was also in the -196--191 position in the reverse direction. MYB-recognition site common sequence (CNGTTR or C / TAACG / TG) to which MYB protein responds to signal transduction process and dry stress of absic acid (ABA) is -1577 ~ -1572, -1406 ~ -1401 (reverse direction) , -1398 ~ -1393, -470 ~ -465, -468 ~ -462 (reverse direction). In addition, the MYC-identical consensus sequence (CANNTG) to which the MYC protein reacted with drying was found at the positions -926 ~ -921 and -149 ~ -144. A common sequence of dehydration-responsive element / C-repeat (DRE / CRT) -factor (G / ACCGAC), the site of binding of DREB1 / CBF protein in response to dry stress and low temperature, was found at -332 ~ -327 position . In addition, the nucleotide sequence of the W-box (TTGAC) to which WRKY protein plays an important role in disease resistance is -1236 ~ -1232, -738 ~ -734 (reverse direction), -672 ~ -668, -648 ~ -644 There were many at the location. GARE-factor common base sequence (TAACAGA), regulated by gibberellin (GA), a signaling agent in plants, was found at the positions -431--425 and -371--365. In addition, the GCCACGTGGC sequence, which is an ACGT-motif associated with root expression, was also found at -198-189. In addition, many GT1-boxes (GGTTAA), known as factors that react to light, were found (FIG. 7).

As described above, in the IbTIP1p_1.6 promoter of the present invention, there are many various cis-activators in response to absic acid and dry stress, and also many factors related to plant disease resistance. In addition, the presence of factors related to root expression may be useful for the study of root-specific expression promoters. In particular, since it contains a lot of dry stress response factors, it can be very useful for developing plants having resistance to dry stress.

1 is a diagram showing the nucleotide sequence of the sweet potato root-derived IbTIP1 gene of the present invention and the amino acid sequence deduced therefrom. NPA is an asparagine-proline-alanine motif present in aquaporin protein.

Figure 2a is a view showing a flexible relationship between the amino acid sequence of the inferred protein of the IbTIP1 gene of the present invention and the membrane-mediated protein (MIP) of Arabidopsis. PIP is a plasma membrane intrinsic protein, SIP is a small and basic intrinsic protein, and NIP is a nodulin-26 like intrinsic protein.

Figure 2b is a view comparing the amino acid sequence of the inferred protein of the IbTIP1 gene of the present invention and the TIP gene amino acid sequence of Arabidopsis plants.

Figure 3 is a picture of the root of the sweet potato and RT-PCR electrophoresis of the expression of the IbTIP1 gene of the present invention in various tissues of sweet potato. L, leaf; S, stem; Fr, fibrous root (root); Tp, thick pigmented root; Tb, tuberous root (storage root).

Figure 4 is a photograph of RT-PCR electrophoresis of the expression of the IbTIP1 gene of the present invention after treatment with sweet potato leaves and roots dried and plant hormone ABA.

5 is a Sheridan blot photograph confirming that the IbTIP1 gene of the present invention exists on the genome of sweet potatoes.

Figure 6 is an electrophoresis picture of genomic walking for isolation of IbTIP1 gene promoter derived from sweet potato root of the present invention. Secondary PCR yielded two different PCR products, 1.0 kb and 3.4 kb, from the DraI and StuI genome libraries.

Figure 7 shows the nucleotide sequence of the sweet potato root-derived IbTIP1 gene promoter of the present invention.

<110> Korea Research Institute of Bioscience and Biotechnology <120> IbTIP1 gene derived from a root of Ipomoea batatas and a promoter          about <130> 7P-10-28 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 1065 <212> DNA <213> IbTIP1 gene derived from a root of Ipomoea batatas <400> 1 gagggcagta gtgcaactga actacgacta aattagtgtt aattatctgt ttagagttta 60 agagattttg taaatttgta aaatggcagt tccgagaatc gctattggaa gcgtgggtga 120 ggctacctcg ccggatgctc tcaaagccgc cgtggcggag ttcatttcta tgcttatatt 180 cgtcttcgcc ggctccggct ccggcatggc tttcaataag ctgacggata atggagcggc 240 cacccccgcc ggactcattt ctgcggcaat agcccacgct tttgcactct tcgtcgcagt 300 ttccgtcggc gccgacattt ccggcggtca cgtaaaccct gctgtcacat ttggcgcctt 360 cgtcggaggt cacatcaccc tcctcagaag tgttgtttat tggattgccc aattgctcgg 420 ctctgtcgtt gcttgcttgc tcctcaagtt tgccaccggt ggattggaaa caccagcatt 480 tggtctgtcg ggagtggggc cgtggaacgc ggtggttttc gagatagtga tgacattcgg 540 gctggtttac acggtgtatg ccaccgcggt tgacccgaag aagggcaaca ttgggatcat 600 tgccccgatc gccattggtt tcatagtggg tgccaacatt ctagccggcg gggccttcga 660 cggtgcatcg atgaaccccg cagtgtcctt cgggcctgcc gtggtgagct ggagctggga 720 gtgccactgg gtttactggc tcggcccgtt cttgggtgcc gccatcgccg ccttggtcta 780 ccaagtcatc ttcatttgcc agaacactca cgaacagctc cccaccacag attactaagg 840 atttccatct ctgtctgtat cattgtgcgc cggatcctaa ggggttcgag gatgttcgtt 900 ggtctttcgt tgtcttttcg atcttttgat tccccaatgt ttgtttcaag cttcttgctg 960 gctttgcctc ctccactgta aaagcatgta aaattattgt gtcttcttcc tgtttggaat 1020 caattgttga ctttcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1065 <210> 2 <211> 251 <212> PRT <213> IbTIP1 protein derived from a root of Ipomoea batatas <400> 2 Met Ala Val Pro Arg Ile Ala Ile Gly Ser Val Gly Glu Ala Thr Ser   1 5 10 15 Pro Asp Ala Leu Lys Ala Ala Val Ala Glu Phe Ile Ser Met Leu Ile              20 25 30 Phe Val Phe Ala Gly Ser Gly Ser Gly Met Ala Phe Asn Lys Leu Thr          35 40 45 Asp Asn Gly Ala Ala Thr Pro Ala Gly Leu Ile Ser Ala Ala Ile Ala      50 55 60 His Ala Phe Ala Leu Phe Val Ala Val Ser Val Gly Ala Asp Ile Ser  65 70 75 80 Gly Gly His Val Asn Pro Ala Val Thr Phe Gly Ala Phe Val Gly Gly                  85 90 95 His Ile Thr Leu Leu Arg Ser Val Val Tyr Trp Ile Ala Gln Leu Leu             100 105 110 Gly Ser Val Val Ala Cys Leu Leu Leu Lys Phe Ala Thr Gly Gly Leu         115 120 125 Glu Thr Pro Ala Phe Gly Leu Ser Gly Val Gly Pro Trp Asn Ala Val     130 135 140 Val Phe Glu Ile Val Met Thr Phe Gly Leu Val Tyr Thr Val Tyr Ala 145 150 155 160 Thr Ala Val Asp Pro Lys Lys Gly Asn Ile Gly Ile Ile Ala Pro Ile                 165 170 175 Ala Ile Gly Phe Ile Val Gly Ala Asn Ile Leu Ala Gly Gly Ala Phe             180 185 190 Asp Gly Ala Ser Met Asn Pro Ala Val Ser Phe Gly Pro Ala Val Val         195 200 205 Ser Trp Ser Trp Glu Cys His Trp Val Tyr Trp Leu Gly Pro Phe Leu     210 215 220 Gly Ala Ala Ile Ala Ala Leu Val Tyr Gln Val Ile Phe Ile Cys Gln 225 230 235 240 Asn Thr His Glu Gln Leu Pro Thr Thr Asp Tyr                 245 250 <210> 3 <211> 1600 <212> DNA <213> Promoter of IbTIP1 gene derived from a root of Ipomoea batatas <400> 3 actagtatat attactcaac ccgttaccat ccccacaccc aattatccat acatgaattc 60 acaattaagg ttttcacatt tcaattcttt ccaaaactgg aaattttggt tggttagttt 120 gtaaaatgga taagctgaaa cgtagggata ttctcatatt ctctgtagaa aaagtctaag 180 caaggagaac acaactgatc tgttatctgt acattgcaaa caaaaaggaa atttgtccct 240 ttagccgtcc aaagcaattt aattagtaaa catataacat aaacaaagca tgtccaacta 300 ctgagctgat taaaatttaa ttaatggtac tctaaaacaa tataaataat tcccttaaat 360 tttgacatat ttgcttcttg ggcaaaaagt gcattggatt attattgagg gattagtttg 420 ctaatcactt tttaaatttt taaataaatt tgtgtatatt tattagtggt gtagcaccaa 480 gtccactacc tgccacggtg cactctcaat attttatttg gcactaacaa ctcaattatc 540 gttgttaaat tgtgattaag taatgacaca tcaccaccta gattatttta aagttttgat 600 tagccaccat cttccctctc tcttttaaac aaacaaaaat tttgtggcta tggttttctt 660 gcctgcacct acatttgttc tttactttaa ataaacattt agagttacat aggaattgat 720 gtatagttaa attactgaat tgtcttgtag atctaacaac attcagaata atttctacca 780 tgggaattga tttggcaata ttgattttgt aaccatgcaa aacagcaaaa gattctaaat 840 ttgactcctc atgaaaactg tcaattggcc ttttagtttg aactggttaa ttagttatat 900 gggcactatg cacagaattc taagtttgac tcctcatgaa aactgccaat tgaccttttt 960 agtttgaacc ggttaactag ctatggacaa acttggctgg ttcgactcat cattagttac 1020 attgtagccc acccataatg atttttatgg gtggactacg atgattttta tgagggcatg 1080 caaaatttgc aattgggtgg aaaaaaaagt ttatattttt gtttaatcgg ttagtttcac 1140 tgaaaacatg tttctaatat catttttaac agacctgttt ttatggctct tcaattcttc 1200 atgtacgtaa gataaaacag taactgtaac agaagtctct attagacata cctcaccata 1260 aggtggtcgg tcgattaaat taaatttgtg agcaatgatt gagaggatgg gccccataat 1320 tttcctactt tatgttttac cggtttcact tttcatatat catcgcatcc atggtggatg 1380 cctatcatca cgttcattgc cacgtggccg tgtcgtagaa aattggaggc tagccaacca 1440 ggcaattaca agtggctcct tcatcatccg ctccttcggg tactataaat acgtgcccgt 1500 gaatacgagg aagaacaggg cagtagtgca actgaactac gactaaatta gtgttaatta 1560 tctgtttaga gtttaagaga ttttgtaaat ttgtaaaatg 1600 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for IbTIP1 <400> 4 gtcacgtaaa ccctgctgtc ac 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for IbTIP1 <400> 5 gctagaatgt tggcacccac ta 22 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GSP1 primer <400> 6 cgaggtagcc tcacccacgc ttccaat 27 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> AP1 primer <400> 7 gtaatacgac tcactatagg gc 22 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> GSP2 primer <400> 8 agatctgcga ttctcggaac tgccatttta caa 33 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AP2 primer <400> 9 actatagggc acgcgtggt 19  

Claims (19)

A sweet potato-derived IbTIP1 protein having an amino acid sequence as set forth in SEQ ID NO: 2 . A polynucleotide encoding the IbTIP1 protein of claim 1. The polynucleotide according to claim 2, which has a nucleotide sequence set forth in SEQ ID NO: 1 . The polynucleotide of claim 2, wherein the polynucleotide is highly expressed in roots. A vector comprising the polynucleotide of claim 2. A transformant transformed with the vector of claim 5. A polynucleotide showing a promoter activity for inducing expression of a root-root specific gene comprising all or part of the nucleotide sequence set forth in SEQ ID NO: 3 . The polynucleotide of claim 7, wherein said promoter activity is induced by drying, abscidic acid, low temperature, light or plant disease. The method according to claim 7, wherein the GATA which is the common sequence of the ACGTG which is a common sequence of the TATA box, the CAAT box, and the ABRE, the CNGTTR which is the common sequence of MYB recognition sites, or the CANNTG which is the common sequence of the C / TAACG / TG and MYC recognition sites, the DRE / CRT factor A polynucleotide comprising ACCGAC, W-box, TAACAGA which is a common base sequence of GARE factor, GCCACGTGGC which is an ACGT-motif, and GGTTAA sequence which is a GT1-box. A vector comprising the polynucleotide of claim 7. A transformant transformed with the vector of claim 10. 1) preparing an expression vector comprising a gene construct comprising the polynucleotide of claim 7 and a polynucleotide encoding a foreign protein operably linked with the polynucleotide; 2) introducing the expression vector into a host cell; And 3) A method for producing a transformant capable of inducing the production of a foreign protein under stress comprising the step of selecting a transformant introduced with the expression vector. The method of claim 12, wherein the stress is induced by drying, abscidic acid, low temperature, light or plant disease. The method according to claim 12, wherein the foreign protein is a method for producing a transformant, characterized in that the protein that confers stress resistance to a protein or transformant exhibiting a pharmacological effect. The method of claim 12, wherein the host cell of step 2) is selected from the group consisting of plant cells, animal cells and microorganisms. The method of claim 12, wherein the transformant is selected from the group consisting of microorganisms, plant cells, plants, and callus derived therefrom. 1) preparing an expression vector comprising a gene construct comprising the polynucleotide of claim 7 and a polynucleotide encoding a foreign protein operably linked with the polynucleotide; 2) introducing the expression vector into a host plant cell; 3) selecting a transgenic plant cell into which the expression vector is introduced; 4) preparing a callus by inducing germination of the transgenic plant cells; 5) preparing a transgenic plant by inducing differentiation of the callus; And 6) A method for high expression of the foreign protein in the root comprising the step of culturing the transgenic plant. 18. The method of claim 17, wherein the plant cell is derived from a plant selected from the group consisting of plants belonging to Cruciferaceae, Rosaceae and Rosaceae. The method of claim 17, wherein the foreign protein is a protein expressing resistance to stress to a protein or transformant exerting a pharmacological effect.

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