KR20100122175A - A high-affinity phosphate transporter gene ospt2 isolated from rice and transgenic plants expressing the said gene - Google Patents

Abstract

PURPOSE: A high affinity phosphate transporter gene OsPT2(Oryza sativa phosphate transporter 2) isolated from rice plant is provided to enhance growth of plant. CONSTITUTION: An OsPT2 gene comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence of sequence number 2. The gene isolated from rice plant. The gene is expressed under phosphate depletion. A method for manufacturing a plant transformed by OsPT2 gene comprises: a step of constructing a recombinant vector having OsPT2 gene; a step of transforming the plant with a recombinant vector; and a step of redifferentiating the transformed plant.

Description

A high-affinity phosphate transporter gene OsPT2 isolated from rice and transgenic plants expressing the said gene}

The present invention relates to a high-affinity phosphate carrier gene OsPT2 isolated from rice, a recombinant vector comprising the gene, a plant transformed with the recombinant vector, and a method for producing the transformed plant.

Phosphoric acid is one of the essential nutrients that plants need and plays an important role in metabolic processes in plant cells such as energy transfer, signal transduction, biosynthesis of metabolites, photosynthesis and respiration, as well as important components of DNA and RNA. Along with nitrogen, plants are the most needed nutrients. The large amount of phosphoric acid required for plant growth is supplied to plants mainly in the form of fertilizers, but most of the phosphoric acid is immobilized in the form of organic matter or metal ions that are not available to plants in the soil. Thus, the amount of available phosphate available to plants is present in the soil in very small amounts. In the case of acidic soils in particular, the phosphoric acid is immobilized by excessively accumulated aluminum ions, which lowers the phosphoric acid effectiveness of the soil. Each year, a significant portion of the total farming costs are spent on fertilizer purchases to increase the effective phosphate concentration in the soil.

In addition, soil pollution such as acidification of soil and direct salt use due to excessive use of fertilizer among agricultural environmental pollution sources not only adversely affects the growth of crops, but also penetrates surface water and causes eutrophication, which is the main source of water resources. Agro-environmental pollution not only brings qualitative deterioration of agricultural production, but also threatens public health due to environmental destruction, so eliminating pollutants is a very important issue. This problem can be solved by reducing the amount of chemical fertilizer added. Various organic fertilizers have been developed and used to cope with chemical fertilizers, but there are still many problems to replace chemical fertilizers. Thus, a more fundamental solution is to reduce the use of fertilizers by improving the availability of soil phosphates in plants.

An object of the present invention is to isolate a new high-affinity phosphate carrier gene, a gene source essential for the development of new crops with improved phosphate availability, using biotechnological methods from rice and to identify its function. In addition, by expressing its genes in plants in large quantities, the transgenic plants have increased phosphorus availability.

The present invention relates to a highly affinity phosphate transporter gene OsPT2 ( Oryza sativa phosphate transporter 2 ) isolated from rice.

OsPT2, a high-affinity phosphate carrier gene of the present invention, is a new gene that has not been previously identified, and has a total sequence of 1626 bp (SEQ ID NOs: 1 and 1) and encodes 541 amino acids (SEQ ID NOs: 2 and 2). (See Example 1).

The high-affinity phosphate carrier gene OsPT2 of the present invention has an increased expression level under phosphoric acid or nitrogen deficiency conditions and an increased expression level at the root under phosphoric acid deficiency conditions (see Example 2).

In another aspect, the present invention relates to a recombinant vector comprising the high-affinity phosphate carrier gene OsPT2.

A preferred example of a backbone vector that can be used to prepare the recombinant vector is Ti, which is capable of transferring a portion of itself, a so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumefaciens. -Plasmid vector. A particularly preferred form of Ti-plasmid vector is a binary vector. Other suitable vectors that can be used to introduce the genes according to the invention into a plant host are viral vectors such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini virus and the like. For example, from an incomplete plant viral vector.

Another aspect of the invention relates to a plant transformed with a recombinant vector comprising the high affinity phosphate carrier gene OsPT2.

According to the present invention, since the high-affinity phosphate carrier gene OsPT2 promotes plant growth by increasing the availability of phosphoric acid, the transformed plant of the present invention is characterized in that the growth is improved compared to wild type plants.

Plants into which the high-affinity phosphate carrier gene OsPT2 may be introduced include food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, and sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Specialty crops, including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers, including but not limited to roses, carnations, chrysanthemums, lilies, tulips.

Another aspect of the invention comprises the steps of constructing a recombinant vector comprising a high affinity phosphate carrier gene OsPT2; And it relates to a method for producing a transformed plant comprising the step of transforming the plant with the recombinant vector.

The method may further comprise the step of regenerating the transformed plant from the tissue of the transformed plant. The method of regenerating the transformed plant from the tissue of the transformed plant can use any method known in the art.

In the present invention, it is preferable to transform using Agrobacterium tumerfaciens mediated transformation using a recombinant vector.

In one embodiment of the present invention, the coding region of the OsPT2 gene is cloned into a binary vector (pCAMBIA1300-35S) to construct a recombinant vector, and then transferred to Agrobacterium tumerfaciens EHA105 by triparental mating method. The included Agrobacterium tumerfaciens EHA105 was transformed through the in planta transformation method (see Example 4).

In the present invention, it was confirmed that the transformants of rice suspension cultured cells of the OsPT2 gene and Arabidopsis thaliana expressing the OsPT2 gene were improved in the phosphoric acid deficient condition. Based on the results, OsPT2 By expressing genes in large quantities, new plants will be able to more efficiently overcome phosphate deficiency stress.

OsPT2 genes are also useful in field crops such as cabbage and radish and other flower crops.

By improving the plant's own nutrient absorption and transporting capacity, it reduces the pollution of soil and water resources due to the large amount of chemical fertilizers, thereby restoring ecosystems, developing pleasant and eco-friendly agriculture, and increasing the yield and quality of crops. Will contribute.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Isolation and Sequence Identification of OsPT2 Gene

This experiment used Dongjin rice, a Korean cultivar. Oligonucleotide primers specific to rice-derived high affinity phosphate carrier genes ( OsPT2 ) were prepared from genomic DNA isolated from Dongjin rice, and the coding region of the gene was amplified by PCR. The amplified product was cloned into the pGemT cloning vector to confirm the sequence of the gene through sequencing. The homology of the isolated rice high affinity phosphate carrier genes with that of other plants was compared. Among these genes, about 90% of the amino acid sequences were identical. The base sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the cDNA clone of the OsPT2 gene are shown in detail in FIGS.

Example 2 Analysis of Expression Conditions of OsPT2 Gene

In general, the expression of genes with similar functions is regulated by tissue-specific or inducer-specific factors, so the expression patterns of the isolated high-affinity phosphate carrier genes were examined using RNA blot method. In this case, a 3 'UTR (untranslated region) DNA fragment having a high affinity phosphate carrier gene-specific sequence was used as a probe. Dongjin rice suspension cultured cells were incubated for 2 days in a medium rich in phosphoric acid (1 mM) and a medium lacking phosphoric acid (10 μM), and RNA was isolated using Trizol REAGENT (Invitrogen). 10 μg of RNA was electrophoresed on a 1.2% (W / V) modified formaldehyde agarose gel to attach RNA to a nylon membrane (Amersham). Nylon membranes with RNA were attached to probes labeled with [ ³² P] dATP using 20% (W / V) SDS, 20X SSPE, 100 g / L PEG (8,000 mwt), 250 mg / L heparin, and 10 ml / L Hearing. The solution was reacted with sperm DNA (10 mg / ml) at 65 ° C. overnight.

OsPT2 gene expression in rice suspension culture cells was confirmed that the difference in the amount of expression depending on the time of phosphoric acid depletion (Fig. 3A). In addition, OsPT2 , a rice high affinity phosphate carrier gene, showed an increased expression level under phosphoric acid and nitrogen deficiency conditions (FIG. 3C). On the basis of the expression profile of the cultured cell suspension results, OsPT2 gene investigated tissue-specific expression in plants of genes OsPT2 increased amount of expression in the roots of the rice plant of phosphate deficiency conditions (Fig. 3B). These results are consistent with studies in which plants express large amounts of high-affinity phosphate carriers in the roots in the phosphate-deficient environment to absorb phosphate from the soil more efficiently. The roots of plants in the phosphate-deficient environment when given to widen the surface area created a number of root hairs absorb phosphorus from the soil up to the Judging of existing studies that use is moved as a whole plant through the phloem OsPT2 the phosphate in the soil It is thought to play an important role in absorption as root hairs.

Example 3: Expression of OsPT2 Gene

OsPT2 gene for expression of recombinant protein The EcoR I and Xba I restriction enzymes were cut from the pBluescript vector and transferred under the AcNPV polyhedrin promoter of the pBacPAK9 vector. 500ng of pBacPak9- OsPAP2 and 100ng AcNPV DNA were co-cultured with sf9 cells for 5 hours and transformed, and the transformed cells were cultured in TC 100 medium for 5 days. OsPT2 recombinant protein expressed in the transgenic insect cells was electrophoresed on an SDS-PAGE gel, and as a result, it was confirmed that 59kDa recombinant protein of interest was normally made (FIG. 4).

Example 4: Arabidopsis transformed with OsPT2 gene

Since rice lives mostly in water, it is relatively weak in phosphate stress compared to other field crops. A transformant that mass-expresses OsPT2 gene in Arabidopsis thaliana , Arabidopsis thaliana , a representative model plant of dicotyledonous plants, to prove its applicability in field crops and to investigate its function in plant crops. Made. A brief look at the Arabidopsis transformant production process is as follows. Cloning the coding region of the gene OsPT2 the EcoRI and XbaI sites of the binary vector (pCAMBIA1300-35S) made by the construct is regulated by the CaMV 35S promoter which is always strongly expressed gene OsPT2 transferred into Agrobacterium tumefaciens EHA105 by triparental mating method. Arabidopsis Agrobacterium tumefaciens EHA105 containing constructs in wild-type plants was transformed by in planta transformation. After aseptically treating seeds harvested from T1 plants, germinated in B5 medium containing carbenicillin (100 mg / L) and hygromycin (20 mg / L) to select transformed plants, and expressing the expression level of OsPT2 gene in RNA blot After confirmation by the method (FIG. 5C), pure plants were obtained from the next generation. Plant growth was compared with wild-type while cultivating obedient plants in B5 liquid medium supplemented with 50 μM and 1 mM phosphoric acid. It was confirmed that the growth of transformants in phosphate deficient conditions was better compared to wild type plants as controls (FIG. 5A). In the quantitative results, it was confirmed that the growth in the transformants is better than the wild type plants. In particular, it was confirmed that the growth of about 50% in the T2 line, which had the highest expression of the OsPT2 gene, among the transformant lines investigated (FIG. 5B).

Figure 1 shows the nucleotide sequence of the high-affinity phosphate carrier OsPT2 gene of rice, the expression level is increased under phosphoric acid deficient conditions.

Figure 2 shows the amino acid sequence deduced from the cDNA of the rice high affinity phosphate carrier OsPT2 gene with increased expression in phosphoric acid deficient conditions.

Figure 3 investigated the expression pattern of OsPT2 gene in phosphate deficient conditions using suspension cultured cells. (A) In phosphate deficient condition, the expression level of genes increased rapidly at 3 hours. (B) Phosphorus deficiency and gene expression patterns of rice seedlings in the ground and the ground under strong conditions were strongly expressed in the case of root lacking phosphate. (C) Gene expression in some mineral nutrient deficient conditions was strongly expressed in phosphate and nitrogen deficient conditions. (D) Gene expression by several hormone treatments was strongly expressed in ABA and kinetin treatment.

FIG. 4 expresses OsPT2 gene using Baculovirus vector system, and recombinant protein was strongly expressed only in cells into which OsPT2 gene was introduced.

FIG. 5 compares the growth of Arabidopsis transformants with wild-type plants expressing OsPT2 genes in phosphate deficient conditions. (A, B) We compared the growth of Arabidopsis transformants and wild-type plants in phosphate deficient conditions. (C) It was confirmed that the OsPT2 gene is expressed in Arabidopsis transformants in large quantities.

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> A high-affinity phosphate transporter gene OsPT2 isolated from          rice and transgenic plants expressing the said gene <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1626 <212> DNA <213> Artificial Sequence <220> <223> OsPT2 <400> 1 atggcgcggc aggagcagca gcagcacctg caggtgctga gcgcgctgga cgcggcgaag 60 acgcagtggt accacttcac ggcgatcgtc gtcgccggca tgggcttctt caccgacgcc 120 tacgacctct tctgcatctc cctcgtcacc aagctgctcg gccgcatcta ctacaccgac 180 ctcgccaagg agaaccccgg cagcctgccg cccaacgtcg ccgcggcggt gaacggagtc 240 gcgttctgcg gcacgctggc ggggcagctc ttcttcgggt ggctcggcga caagctcggc 300 cggaagagcg tgtacgggat gacgctgctg atgatggtca tctgctccat cgcgtcgggg 360 ctctcgttct cgcacacgcc caccagcgtc atggcgacgc tctgcttctt ccggttctgg 420 ctcggattcg gcatcggcgg cgactacccg ctgtcggcga cgatcatgtc ggagtacgcc 480 aacaagaaga cccgcggcgc gttcatcgcc gccgtgttcg cgatgcaggg gttcggcatc 540 ctcgccggcg gcatcgtcac cctcatcatc tcctccgcgt tccgcgccgg gttcccggcg 600 ccggcgtacc aggacgaccg cgcgggctcc accgtccgcc aggccgacta cgtgtggcgg 660 atcatcctca tgctcggcgc catgccggcg ctgctcacct actactggcg gatgaagatg 720 ccggagacgg cgcgctacac cgccctcgtc gccaagaacg ccaagcaggc cgccgccgac 780 atgtccaagg tgctccaggt cgagatccag gaggagcagg acaagctgga gcagatggtg 840 acccggaaca gcagcagctt cggcctcttc tcccgccagt tcgcgcgccg ccacggcctc 900 cacctcgtcg gcaccgccac gacatggttc ctcctcgaca tcgccttcta cagccagaac 960 ctgttccaga aggacatctt caccagcatc aactggatcc ccaaggccaa gaccatgtcg 1020 gcgctggagg aggtgttccg catcgcgcgc gcccagacgc tcatcgccct gtgcggcacc 1080 gtcccgggct actggttcac cgtcttcctc atcgacatcg tcggccgctt cgccatccag 1140 ctgctagggt ttttcatgat gaccgtgttc atgctcggcc tcgccgtgcc gtaccaccac 1200 tggacgacga aggggaacca catcggcttc gtcgtcatgt acgccttcac cttcttcttc 1260 gccaacttcg gccccaactc caccaccttc atcgtgccgg cggagatctt cccggcgagg 1320 ctgcgttcca cctgccacgg catctcggcg gcggcgggga aggccggcgc catcatcgga 1380 tcgttcgggt tcctgtacgc ggcgcaggac ccgcacaagc ccgacgccgg gtacaaaccc 1440 gggatcgggg tgaggaactc gctgttcgtg ctcgccggat gcaacctgct cgggttcatc 1500 tgcacgttcc tcgtgccgga gtcgaagggg aagtcgctgg aggagatgtc cggcgaggcg 1560 gaggacgacg acgacgaggt ggccgccgcc ggcggtggcg ccgccgtgcg gccgcagacg 1620 gcgtag 1626 <210> 2 <211> 541 <212> PRT <213> Artificial Sequence <220> <223> OsPT2 <400> 2 Met Ala Arg Gln Glu Gln Gln Gln His Leu Gln Val Leu Ser Ala Leu   1 5 10 15 Asp Ala Ala Lys Thr Gln Trp Tyr His Phe Thr Ala Ile Val Val Ala              20 25 30 Gly Met Gly Phe Phe Thr Asp Ala Tyr Asp Leu Phe Cys Ile Ser Leu          35 40 45 Val Thr Lys Leu Leu Gly Arg Ile Tyr Tyr Thr Asp Leu Ala Lys Glu      50 55 60 Asn Pro Gly Ser Leu Pro Pro Asn Val Ala Ala Ala Val Asn Gly Val  65 70 75 80 Ala Phe Cys Gly Thr Leu Ala Gly Gln Leu Phe Phe Gly Trp Leu Gly                  85 90 95 Asp Lys Leu Gly Arg Lys Ser Val Tyr Gly Met Thr Leu Leu Met Met             100 105 110 Val Ile Cys Ser Ile Ala Ser Gly Leu Ser Phe Ser His Thr Pro Thr         115 120 125 Ser Val Met Ala Thr Leu Cys Phe Phe Arg Phe Trp Leu Gly Phe Gly     130 135 140 Ile Gly Gly Asp Tyr Pro Leu Ser Ala Thr Ile Met Ser Glu Tyr Ala 145 150 155 160 Asn Lys Lys Thr Arg Gly Ala Phe Ile Ala Ala Val Phe Ala Met Gln                 165 170 175 Gly Phe Gly Ile Leu Ala Gly Gly Ile Val Thr Leu Ile Ile Ser Ser             180 185 190 Ala Phe Arg Ala Gly Phe Pro Ala Pro Ala Tyr Gln Asp Asp Arg Ala         195 200 205 Gly Ser Thr Val Arg Gln Ala Asp Tyr Val Trp Arg Ile Ile Leu Met     210 215 220 Leu Gly Ala Met Pro Ala Leu Leu Thr Tyr Tyr Trp Arg Met Lys Met 225 230 235 240 Pro Glu Thr Ala Arg Tyr Thr Ala Leu Val Ala Lys Asn Ala Lys Gln                 245 250 255 Ala Ala Ala Asp Met Ser Lys Val Leu Gln Val Glu Ile Gln Glu Glu             260 265 270 Gln Asp Lys Leu Glu Gln Met Val Thr Arg Asn Ser Ser Ser Phe Gly         275 280 285 Leu Phe Ser Arg Gln Phe Ala Arg Arg His Gly Leu His Leu Val Gly     290 295 300 Thr Ala Thr Thr Trp Phe Leu Leu Asp Ile Ala Phe Tyr Ser Gln Asn 305 310 315 320 Leu Phe Gln Lys Asp Ile Phe Thr Ser Ile Asn Trp Ile Pro Lys Ala                 325 330 335 Lys Thr Met Ser Ala Leu Glu Glu Val Phe Arg Ile Ala Arg Ala Gln             340 345 350 Thr Leu Ile Ala Leu Cys Gly Thr Val Pro Gly Tyr Trp Phe Thr Val         355 360 365 Phe Leu Ile Asp Ile Val Gly Arg Phe Ala Ile Gln Leu Leu Gly Phe     370 375 380 Phe Met Met Thr Val Phe Met Leu Gly Leu Ala Val Pro Tyr His His 385 390 395 400 Trp Thr Thr Lys Gly Asn His Ile Gly Phe Val Val Met Tyr Ala Phe                 405 410 415 Thr Phe Phe Phe Ala Asn Phe Gly Pro Asn Ser Thr Thr Phe Ile Val             420 425 430 Pro Ala Glu Ile Phe Pro Ala Arg Leu Arg Ser Thr Cys His Gly Ile         435 440 445 Ser Ala Ala Ala Gly Lys Ala Gly Ala Ile Ile Gly Ser Phe Gly Phe     450 455 460 Leu Tyr Ala Ala Gln Asp Pro His Lys Pro Asp Ala Gly Tyr Lys Pro 465 470 475 480 Gly Ile Gly Val Arg Asn Ser Leu Phe Val Leu Ala Gly Cys Asn Leu                 485 490 495 Leu Gly Phe Ile Cys Thr Phe Leu Val Pro Glu Ser Lys Gly Lys Ser             500 505 510 Leu Glu Glu Met Ser Gly Glu Ala Glu Asp Asp Asp Asp Glu Val Ala         515 520 525 Ala Ala Gly Gly Gly Ala Ala Val Arg Pro Gln Thr Ala     530 535 540  

Claims (9)

An isolated OsPT2 ( Oryza sativa phosphate transporter 2 ) gene comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence shown as SEQ ID NO: 2. The method of claim 1, The nucleotide sequence is a gene, characterized in that consisting of the nucleotide sequence shown in SEQ ID NO: 1. The method of claim 1, Gene, characterized in that isolated from rice. The method of claim 1, Gene characterized in that the expression is induced in phosphate deficient conditions. Recombinant vector comprising the gene according to claim 1. Plant transformed with the recombinant vector according to claim 5. The method of claim 6, The plant includes rice, wheat, barley, corn, beans, potatoes, wheat, red beans, oats, sorghum crops; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Specialty crops, including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, bananas; A plant, which is selected from flowers, including roses, carnations, chrysanthemums, lilies, and tulips. Constructing a recombinant vector according to claim 5; And Method for producing a transformed plant comprising the step of transforming the plant with the recombinant vector. The method of claim 8, And regenerating the transformed plant from the tissue of the transformed plant.

Images