KR100925798B1 - Rice transgenic plants having enhanced ability of phosphate acquisition and methods of preparing the same - Google Patents

Abstract

PURPOSE: A rice plant transformant with enhanced absorption of phosphoric acid is provided to remove phosphate which excessively exists in a river and prevent eutrophication of soil or river. CONSTITUTION: A rice plant transformant with enhanced absorption of phosphoric acid overexpresses OsPT1 which is a phosphoric acid carrier gene of rice plant of the sequence number 1. The OsPT1 always expresses in all tissues of rice plant regardless of variation of phosphoric acid concentration.

Description

Rice transgenic plants having enhanced ability of phosphate acquisition and methods of preparing the same}

The present invention is an over-expression of a high affinity phosphate carrier gene of rice, which is always expressed irrespective of the phosphate concentration, a rice transformant having enhanced phosphate absorption capacity, a rice transformant having enhanced phosphate absorption capacity by overexpressing the high affinity phosphate carrier in rice. It relates to a method for producing a, and a method for purifying soil or water quality using the rice transformant.

A large amount of phosphoric acid required for the development of differentiation of plants is supplied to plants as fertilizers, but most of the phosphoric acid is immobilized in a form that the plant cannot directly absorb by binding to organic matter or metal ions in the soil. Each year, a significant portion of the total farming costs are spent on fertilizer purchases to increase the effective phosphorus concentration in the soil, and phosphoric acid immobilized in the soil causes eutrophication of groundwater or rivers. On the other hand, the pollution of the agricultural environment is not only a qualitative decrease in agricultural production, but also a threat to the public health due to environmental destruction, so the removal of pollutants is a very important problem.

Globally, the deterioration of soil due to industrialization and urbanization and the long-term cultivation have increased the limit of effective inorganic nutrients in the soil, while the importance of eco-friendly agricultural management has caused soil and water pollution due to the excessive use of chemical fertilizers. Regulations are expected to be tightened further. In Korea, excessive use of chemical fertilizers due to long-term cultivation of facility houses has resulted in the accumulation of large amounts of phosphoric acid in the soil, which is a major factor in stream nutrient nourishment. The future development and industrialization of agricultural life science will depend on securing useful genetic resources, separating and analyzing useful genes, and developing application technologies for these genes, thus selecting and concentrating research areas that can be competitive compared to developed countries. It is necessary to make systematic efforts to secure new useful right actors and analyze their functions.

Genomic studies based on the similarity of DNA sequences in Arabidopsis, tomatoes, potatoes, rice, barley, tobacco, and corn are now used to isolate high-affinity phosphate carrier genes and their functions. In particular, Syngenta and other global teams of researchers are focusing on specific high-affinity phosphate carriers derived from rice and potatoes that specifically react to Mycorrhiza and participate in phosphate absorption from the mycorrhiza.

After reports of tobacco cultivation cells overexpressing Arabidopsis high affinity phosphate carriers have been reported to improve phosphate uptake, development of transformants with enhanced phosphate uptake using plant high affinity phosphate carriers has been attempted. . For example, based on the similarity of DNA sequences, barley phosphate high affinity carrier gene families were isolated and some residues were overexpressed in barley, but no expected changes in the expression of enhanced phosphate uptake were observed (Rae, et al. (2004) Over-expression of a high-affinity phosphate transporter in transgenic barley plants does not enhance phosphate uptake rate.Functional Plant Biology 31, 141-148). These results indicate that although similarity of sequences exists between high affinity phosphate carrier genes, there are a great variety of expression patterns and specific functionalities. As a result similar to the above, the team has produced a transformant overexpressing each of the four high-affinity phosphate carrier genes isolated from rice, but only two types including OsPT1 specified in the present specification are directly related to rice phosphate absorption ability. It was found to affect. Therefore, it is thought that many basic studies should be preceded in order to directly apply many nutrient carrier genes, including many high-affinity phosphate carriers, which have been isolated so far to useful crops.

Recently, a Chinese research team reported successful development of rice phosphate transformants using the transcriptional regulatory gene (OsPTF1) that specifically expresses phosphate deficiency, but did not directly enhance phosphate uptake but adapted to phosphate deficiency conditions. Metabolic regulation has been reported to be resistant to phosphate deficiency. Research on the enhancement of phosphate uptake of agriculturally important crops using high affinity phosphate carriers is needed.

It is an object of the present invention to provide a rice transformant with enhanced phosphoric acid uptake by overexpressing the highly affinity phosphate carrier gene OsPT1 of rice, which is always expressed regardless of phosphoric acid concentration.

It is still another object of the present invention to provide a method for preparing a rice transformant having enhanced phosphoric acid uptake by overexpressing a high affinity phosphate carrier gene OsPT1, which is always expressed regardless of phosphoric acid concentration in rice.

Still another object of the present invention is to provide a water purification method using a rice transformant having improved phosphoric acid absorption.

The present invention provides a new rice transformant with enhanced phosphate uptake by overexpressing a rice-derived high affinity phosphate carrier gene expressed in all tissues of rice regardless of phosphate concentration in Dongjin rice, a rice cultivation variety. By agriculturally testing the function, it aims to secure useful genetic resources for developing new environmentally friendly rice varieties.

Lack of food resources due to the declining agricultural productivity due to deterioration of agricultural land and finite biological resources and industrialization is an important task for humanity. Based on recent genomic studies, important genes involved in plant phosphate uptake have been isolated and its application is being conducted through basic research.

Therefore, the present inventors have separated the rice-derived high affinity phosphate carrier genes expressed in all tissues of rice regardless of the phosphate concentration from the rice and characterized the characteristics and registered the domestic patent (Registration No. 10-0640284) In the present invention, a transformant overexpressing a specific high-affinity phosphate carrier gene, which is always expressed regardless of the phosphate concentration of rice, in a cultivated rice variety (Dongjin rice) using a CaMV 35S promoter. Among these transformants, the transformant lines in which overexpression of the introduced OsPT1 gene was confirmed were selected for two years from generation to generation, and the safety and sequence lines of the introduced genes were selected. The phosphate uptake and important agricultural traits were compared with the wild type. As a result, the present invention was confirmed by confirming that the phosphate absorption capacity of the prepared rice transformant increased more than twice compared to wild type.

This genetic property of the rice transformant produced in the present invention will be used as an important intermediate model for the new breed of rice breeding can reduce the use of chemical fertilizers in the future. In addition, the use of the excellent phosphate absorption ability of these rice transformants and the physiological characteristics of aquatic plant rice is expected to be used in the development of a new water purification method that can reduce the phosphoric acid content that causes eutrophication of rivers.

More specifically, the present invention provides a rice transformant having improved phosphate uptake by overexpressing the phosphate carrier gene OsPT1 of rice having the nucleotide sequence of SEQ ID NO: 1, and a method for producing the same. Overexpression of the gene OsPT1 can be performed by any method commonly known in the art. For example, the gene OsPT1 can be overexpressed by operably linking the gene OsPT1 with the CaMV 35S promoter (SEQ ID NO: 2), or the ubiquitin promoter accession number (AY954394).

Rice transformant according to the present invention is significantly increased the absorption of phosphoric acid, it may have the effect of preventing and purifying the soil or river eutrophication by removing the excess phosphoric acid in the river. Accordingly, another aspect of the present invention provides a method for purifying soil or water quality using a rice transformant overexpressing the phosphate carrier gene OsPT1 of rice having the nucleotide sequence of SEQ ID NO: 1.

It can be used as an intermediate model for developing excellent rice varieties with enhanced phosphoric acid absorption, and will provide agricultural biotechnology-based technology for the development of other crops with enhanced phosphoric acid absorption. In addition, it will be possible to develop environmentally friendly rice varieties that can be grown without using phosphate fertilizers in phosphate-rich soils, which are caused by overuse of chemical fertilizers. In addition, the use of aquatic plant properties and enhanced phosphate absorption capacity of rice will also be available for water purification.

The present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[ Example ]

Example  One.

The present inventors have already found OsPT1, a high-affinity phosphate carrier gene that is always expressed in all tissues of rice regardless of the change in phosphate concentration, which is already registered in Korea (No. 10-0640284, which is incorporated herein by reference). A recombinant vector construct regulated by the 35S promoter of CaMV, which is always strongly expressed, was constructed and transformed into rice using Agrobacterium tumefaciens .

Briefly, the coding region of the OsPT1 gene was directly produced in the laboratory by the above-mentioned CaMV 35S on ECoRI and HindIII cleavage sites of the binary vector pCAMBIA1300-35S (commercially available binary vector pCAMBIA1300 (CAMBIA). The recombinant vector was cloned into the XbaI site of the cloned recombinant vector) to produce a recombinant vector regulated by a CaMV 35S promoter that always strongly expresses the OsPT1 gene. Agrobacterium tumefaciens ( Agrobacterium ) using the recombinant vector prepared as described above by a triple parental mating method tumefaciens ) was transferred to EHA105 strain.

Agrobacterium-based rice transformation was well established by Toki (Toki, S (1996) Rapid and efficient Agrobacterium-mediated transformation in rice.Plant Molecular Biology Report 15, 16-21). Briefly described as follows. Callus derived from seed of Dongjin rice in callus induction medium for 5 weeks was infected with A. tumefaciens containing the recombinant vector prepared above and co-cultured in co-culture for 3 days. Then, the infected callus was selected and cultured for two weeks in a callus propagation medium containing hygromycin (50 μg / ml) and transformed to survive, and the callus surviving from the differentiation medium was differentiated into roots and leaves for two weeks. Induced and then transferred to pollen to cultivate the transformant.

In this independently regenerated rice transformant, total RNA was first isolated and Northern blot analysis was performed using DNA fragments of OsPT1 specific sequences as probes. More specifically, RNA was isolated using Trizol REAGENT (Invitrogen). 10 μg of RNA was electrophoresed on a 1.2% (W / V) denaturing formaldehyde agarose gel to attach RNA to a nylon membrane (Amersham). Nylon membranes with RNA attached a [ ³² P] dATP labeled probe (full 1.6 kbp OsPT1 gene) with 20% (W / V) SDS, 20X SSPE, 100 g / L PEG (8,000 mwt), 250 mg / L It was reacted overnight at 65 ° C with a solution containing heparin and 10 ml / L Hearing sperm DNA (10 mg / ml).

Line 2 was selected to express OsPT1 gene expression significantly higher than wild type and progressed generation for 2 years (FIG. 1 (A)). The identification of the naïve line was assayed by examining whether the hygromycin resistance phenotype by the hygromycin resistance gene used as a selection marker continued to be separated from the second generation. Seeds and wild-type seeds from these lines were germinated and transferred to the GMO packaging of Yeongam Agricultural Research Institute at 15X30 cm intervals. The phosphate concentrations in the plants were fertilized under fertilizer or fertilizer conditions, and the phosphate concentrations in plants were measured by Vanadate method. In addition, major agricultural specific surveys were carried out in accordance with the Rural Agricultural Research and Agricultural Research Standards.

As a result of the above investigation, when grown under conditions of sufficient phosphoric acid (phosphate concentration: 178 mg / kg), the rice transformants overexpressing OsPT1 showed physiological characteristics with shorter length and longer division than those of the control wild type. (Figures 1, (B) and (C)).

A comparison of important agricultural traits of overexpressing rice transformants (OsPT1-OX) is shown in Table 1 below.

TABLE 1

Increasing rice cleavage is closely related to the phosphate concentration in plants, so it is expected that rice transformants overexpressing OsPT1 will have higher phosphate concentrations in plants compared to controls. In fact, cultivation of phosphate fertilizers in fertilized or fertilized fertilizers in general cropland soil was compared with the phosphate content in plants between wild type and rice transformants overexpressing OsPT1 during splitting, harvesting, and harvesting. The transformants accumulated almost twice as much phosphoric acid in the leaves at all stages of growth regardless of fertilization conditions (FIG. 2).

However, there was no difference between wild type and rice transformants overexpressing OsPT1 in the contents of other inorganic nutrients including nitrogen and potassium. The phosphate content in the soil before the test was 178 mg / kg, but the remaining soil phosphate content after the test was 163 mg / kg in the control cultivated soil and 152 mg / kg in the soil in which the rice transformants overexpressed OsPT1 were grown. Decreased. However, the transformants overexpressing OsPT8 similar to OsPT1 were detected after cultivation similar to the control group. Soil nutrients and chemical analysis results before and after cultivation of overexpressing rice transformant (OsPT1-OX) were shown in Table 2 below. Indicated.

  TABLE 2

kind Fertilization method PH EC T-N (mg / kg) P2O5 (mg / kg) Inorganic Ingredients (cmol / kg) K Ca Mg Before the test 5.81 0.30 240 178 0.54 4.49 0.85 After the test Dongjin Rice NK 5.18 0.23 240 163 0.41 3.02 0.45 After the test OsPT1-OX NK 5.24 0.29 240 152 0.36 3.16 0.50 After the test OsPT8-OX NK 5.18 0.26 230 169 0.37 3.09 0.48

These results indicate that rice transformants overexpressing OsPT1 absorb more phosphoric acid from the soil and accumulate in plants compared to controls. As a result, it was confirmed that the rice transformants overexpressing OsPT1 exhibited increased division and increased number of sorghum. However, excessive phosphoric acid accumulation of rice transformants overexpressed by OsPT1 may be interpreted as a result of the breakdown of relative ratios with important inorganic nutrients such as nitrogen and potassium, which are absolutely necessary for plant growth.

Figure 1 shows the identification and growth specificity of the rice transformant overexpressing the high-affinity phosphate carrier of rice that is always expressed regardless of the phosphate concentration change.

Figure 2 shows the results of a comparative analysis of the phosphate content between the rice morphogens overexpressed by the high-affinity phosphate carrier of the rice always expressed regardless of wild type and phosphate concentration changes according to fertilization conditions and growth stages.

<110> DONG-A UNIVERSITY <120> Rice transgenic plants having enhanced ability of phosphate          acquisition and methods of preparing the same <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1584 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of OsPT1 <400> 1 atggcgggag ggcagctcaa cgtgctgagc acgctcgacc aggcgaagac gcaatggtac 60 cacttcatgg cgatcgtcat cgccggcatg ggcttcttca ccgacgccta cgacctcttc 120 tgcatttccc tcgtcaccaa gctgctcggc cgcatctact acaccgacga ttccaaggac 180 acccccggcg cgctcccgcc caacgtgtcg gccgccgtca ccggcgtcgc gctctgcggc 240 acgctcgccg gccagctttt cttcggatgg ctcggcgaca agctcggacg caagagcgtg 300 tatggtttca cgctgattct gatggtcgtg tgctcggtcg cgtccgggct ctcgttcggg 360 agctcggcca agggcgtcgt gtctacgctc tgcttcttcc ggttctggct cggcttcggc 420 atcggcggcg actacccgct cagcgccacc atcatgtcgg agtacgcgaa caagaggacg 480 cgcggggcct tcatcgccgc cgtgttcgcc atgcaggggt tcgggatcct cttcggcgcc 540 atcgtcgcgc tcgcggtgtc ggcggggttc cggcacgcgt acccggcgcc gtcctactcc 600 gacaaccacg ccgcgtcgct cgtcccgcag gccgactacg tgtggcgcat catcctcatg 660 ttcggcaccg tcccggcggc gctcacctac tactggcgga tgaagatgcc cgagacggcg 720 cggtacacgg cgctcatcgc ccgcaacgcg aagcaggcgg cggccgacat gtccaaggtg 780 ctgcacaccc agattgagga gagcgcggac cgcgccgaga cggtggccgt cggcggcgag 840 agctggggcc tcttctcgcg ccagttcctg cgccgccacg gcctccacct cctcgccacc 900 accagcacgt ggttcctcct cgacatcgcc ttctacagcc agaacctgtt ccagaaggac 960 atcttcagca aggtcgggtg gatcccgccg gcgaagacca tgaacgcgct cgaggagctc 1020 taccgcatcg cccgcgccca ggcgctcatc gcgctctgcg gcaccatccc gggctactgg 1080 ttcaccgtcg cattcatcga gatcatgggc aggttctgga tccagatcat gggcttcgcc 1140 atgatgacgg cgttcatgct cggcctcgcc atcccgtacc accactggac gacgccgggg 1200 caccacaccg gcttcatcgt catgtacgga ttcaccttct tcttcgcgaa cttcgggcca 1260 aacagcacca ccttcatcgt gccggcggag atatacccgg cgcggctccg gtcgacgtgc 1320 cacggcatct ccgccgccgc cgggaaggcc ggcgccatca tcggagcgtt cgggttcctg 1380 tacgcggcgc aggaccagca caagcccgag cctgggtacc ccagggggat cggcatcaag 1440 aacgcgctct tcgtgctcgc cggcacaaac ttcctcggga cgatcatgac gctgctcgtg 1500 ccggagtcca agggcatgtc gctcgaggtt atctcgcagg aggtcgccga cggcgacgac 1560 gaggaggcgg cctacccgaa gtaa 1584 <210> 2 <211> 538 <212> DNA <213> Artificial Sequence <220> <223> CaMV 35S Promoter sequence <400> 2 agtcccccgt gttctctcca aatgaaatga acttccttat atagaggaag ggtcttgcga 60 aggatagtgg gattgtgcgt catcccttac gtcagtggag atatcacatc aatccacttg 120 ctttgaagac gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc 180 atctttggga ccactgtcgg cagaggcatc ttcaacgatg gcctttcctt tatcgcaatg 240 atggcatttg taggagccac cttccttttc cactatcttc acaataaagt gacagatagc 300 tgggcaatgg aatccgagga ggtttccgga tattaccctt tgttgaaaag tctcaattgc 360 cctttggtct tctgagactg tatctttgat atttttggag tagacaagtg tgtcgtgctc 420 caccatgttg acgaagattt tcttcttgtc attgagtcgt aagagactct gtatgaactg 480 ttcgccagtc tttacggcga gttctgttag gtcctctatt tgaatctttg actccatg 538  

Claims (5)

delete delete delete delete A method of purifying soil or water, characterized by reducing the phosphoric acid of soil or stream by using a rice transformant overexpressing the phosphate carrier gene OsPT1 of the SEQ ID No. 1 and enhancing the phosphoric acid uptake from the soil.

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