KR20050077118A - A phosphate transporter genes and promoters of said genes that are expressed specifically in the root hair of rice and a production method of transgenic plants using said genes - Google Patents

Abstract

The present invention provides a novel phosphate carrier gene having a nucleotide sequence of the following SEQ ID NO: 1 or SEQ ID NO: 2 and an amino acid sequence which is specifically expressed on the root hair of Oryza sativa L. It relates to a method for producing a transgenic plant having improved phosphoric acid uptake.

<SEQ ID NO 1>

<SEQ ID NO 2>

Description

Phosphate transporter genes and promoters of said genes that are expressed specifically in the root of rice root hair specific expression phosphate carrier gene, root hair specific expression promoter of the gene and the gene hair of rice and a production method of transgenic plants using said genes}

The present invention relates to a novel phosphate carrier gene that specifically expresses the root hair of Oryza sativa L., a root hair specific expression promoter of the gene, and a method for producing a transformed plant using the gene.

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 from 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 particular, in the case of acidic soils, phosphoric acid is immobilized by excessively accumulated aluminum ions, thereby lowering 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 accumulation of salt 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 pollutant of water resources. Agricultural environmental pollution not only brings down the quality of agricultural production, but also threatens the public's health due to environmental destruction, so eliminating pollutants is a very important problem. This problem can be solved by reducing the amount of chemical fertilizers added. Various organic fertilizers have been developed and used to replace chemical fertilizers, but there are still many problems. Thus, a more fundamental solution to the problem is to reduce the use of fertilizers by improving the plant's ability to absorb phosphates.

On the other hand, rice is one of the most important crops in the world, and in recent years, since the entire gene sequence has been revealed, many efforts have been made to secure a large number of useful genes and preoccupy them. Research on the absorption of inorganic nutrients from plants has been done for a long time in cultivation and plant physiological aspects, but recently, based on the molecular-level basic research on the absorption of inorganic nutrients, In order to increase the growth and yield of crops and to grow crops that can grow under extremely limited nutrients, research is being actively conducted worldwide.

The conventional research trend in this regard has been the study of phosphate deficiency stress mainly on cultivation and physiology. In the 1990s, new trends began to be developed to understand and apply the plant's response to phosphate deficiency at the molecular level. Currently, phosphate carrier genes are isolated from Arabidopsis, tomato, potato, tobacco, rice, barley, etc., and the structure and expression control characteristics of these genes are revealed. However, due to the lack of the mechanism of inducing the expression of phosphate-related genes or understanding of the molecular cell biology of phosphate carriers, new plants that have improved phosphate absorption capacity as expected by changing the phosphate absorption system of the plant by genetic manipulation have not been developed. It is not. However, there has been a report that the cell growth rate is increased in the phosphate deficient condition by the large expression of Arabidopsis high affinity phosphate carriers in tobacco suspension cells, suggesting the need for a study to improve the phosphate absorption capacity using high affinity phosphate carriers.

Accordingly, it is an object of the present invention to provide a novel phosphate carrier gene that specifically expresses rice root hairs in the absence of phosphate.

Another object of the present invention is to provide a root hair specific expression promoter of the gene.

Still another object of the present invention is to provide a method for producing a transgenic plant having improved phosphate uptake using the gene.

In accordance with the above object, in the present invention, the phosphate carrier gene OsPT1 having the nucleotide sequence and the amino acid sequence of SEQ ID NO: 1, which is specifically expressed in the root hair of Oryza sativa L. The phosphate carrier gene OsPT7 with sequence is provided.

<SEQ ID NO 1>

<SEQ ID NO 2>

According to another object, the rice root hair specific expression promoter having the following SEQ ID NO: 3 corresponding to the 1 st to 1422 nucleotide sequences of the gene OsPT1 and the following SEQ ID NO: 4 corresponding to the 1 st to 1403 base sequences of the gene OsPT7 It provides a rice root hair specific expression promoter.

<SEQ ID NO 3>

<SEQ ID NO 4>

According to another object, the present invention provides a method for producing a transformed plant having improved phosphoric acid uptake.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

Example 1 Isolation of Rice Root Hair Specific Expression Genes OsPT1 and OsPT7

(Step 1) Isolation of OsPT Gene

In the present invention, Dongyin rice ( Oryza sativa L.), a cultivated variety of Korea, was purchased and used from Yeongnam Crop Experiment Station. First, genomic DNA was isolated from Dongjin rice to prepare oligonucleotide primers specific for each rice-derived high affinity phosphate carrier gene ( OsPT ) based on the entire genome sequence of rice (see Table 1). The coding region of each gene was amplified using conventional PCR methods. Thereafter, the amplified product was cloned into a pBluescript cloning vector to confirm the nucleotide sequence of the gene through sequencing.

As a result, we compared the homology of amino acid sequences between the 10 isolated high affinity phosphate carrier genes and phylogenetic methods for the evolutionary relationship between other dicotyledonous, monocotyledonous and fungal high affinity phosphate carrier genes. It was investigated using.

The results of homology of the amino acid sequences between the high-affinity phosphate carrier genes are shown in FIG. 1 and the results of phylogenetic analysis of evolutionary relationships. In FIG. 1, twelve lines represent 12 membrane-spanning regions of the OsPT gene.

As a result of the investigation, it was found that more than 70% amino acid sequence was identical among the genes, and phylogenetically evolved into a separate group between the high affinity phosphate carriers of the fungus and the plant. In addition, it was found that these genes isolated from rice plants, which are monocotyledonous plants, have evolved separately from similar genes isolated from dicotyledonous plants.

(Step 2) RNA blotting analysis

In general, when similarly functioning genes exist in a family, expression of each gene is regulated tissue-specific or inducer-specific, so the expression patterns of the 10 high-affinity phosphate carrier genes isolated in step 1 are Was investigated using the RNA blot method.

In this case, only 3 ′ UTR DNA fragments having each gene-specific nucleotide sequence were used as probes to selectively distinguish each gene. Callus cells derived from Dongjin rice seeds were incubated for 2 days in a medium rich in phosphoric acid (1 mM) and a medium lacking phosphoric acid (10 μM), respectively, and RNA was purified using Trizol REAGENT (Invitrogen). Was separated. 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 were prepared using [ ³² P] dATP labeled probes with 20% (W / V) SDS, 20X SSPE, 100 g / L PEG (8,000 mwt), 250 mg / L heparin and 10 ml / The mixture was reacted overnight at 65 ° C with a solution containing L Hearing sperm DNA (10 mg / ml).

As a result of the experiment, two genes ( OsPT1 and OsPT7 ) among the 10 highly affinity phosphate carrier genes examined showed an increase in expression under phosphate deficient conditions (see FIG. 5A).

Therefore, intensive studies were conducted on the two high-affinity phosphate carrier genes, OsPT1 and OsPT7, which have similar expression patterns. Meanwhile, the nucleotide sequences of genomic clones of these genes are shown in detail in FIGS. 3 and 4, respectively.

In order to investigate the expression of OsPT1 and OsPT7 genes in more detail, the expression level of phosphate and phosphate deprivation time were examined. At this time, the concentration of phosphoric acid was tested in 20, 100, 250, 500, 1000μM differently, and the phosphoric acid depletion time was experimented with 0, 1, 2, 3, 4 hours.

As a result, the expression of OsPT1 and OsPT7 genes increased when the concentration of phosphoric acid was 20 μM to 250 μM, but the expression was decreased when it was 500 μM or more (see FIG. 5B). This is because rice callus cells do not receive phosphate deficiency stress when the phosphate concentration in the medium is 500 μM or more, but when it is lowered to 500 μM or less, they try to overcome the phosphate deficiency stress by expressing OsPT1 and OsPT7 genes. In addition, the expression of these genes rapidly increased when the phosphate deficiency time continued for more than 1 hour (Fig. 5C).

Based on the expression pattern of the callus cells, tissue-specific expression in the plants of OsPT1 and OsPT7 genes was examined by the same RNA blot method. As a result of investigating phosphate sufficient condition and phosphate deficiency condition in the root and stem tissue, respectively, OsPT1 and OsPT7 were expressed specifically in the roots lacking phosphate (see FIG. 6). These results are consistent with the results from other plants studied to date, in which plants express large amounts of high affinity phosphate carriers in the phosphate deficient environment in the roots to absorb phosphate from the soil more efficiently.

Example 2 Preparation of Transformed Rice and Investigation of GUS Tissue Activity of the Rice>

In order to investigate more detailed tissue-specific expression of OsPT1 gene, the promoter region of the gene was linked with the GUS reporter gene to make a transformant using Agrogbacterium , and the tissue-specific activity of GUS according to the phosphate concentration. Was carried out in accordance with conventional GUS assay.

First, the production process of the rice transformant is briefly described as follows. The promoter portion of the OsPT1 gene was combined with the ATG region of the GUS reporter gene at the EcoRI / NcoI site of the binary vector pCAMBIA1381Z (CAMBIA, 1281Z) to make a structure, and then, by using a triple parental mating method. Agrobacterium tumefaciens was transferred to the EHA105 strain. Then, the callus cells of rice capable of forming embryos were infected with the transformed Agrobacterium strain, and the transformed callus was selected in a callus growth medium containing hygromycin (50 µg / ml). To produce a completely transformed rice using a tissue culture method. The transformed rice was cultured for one day in liquid medium with sufficient phosphate concentration (1000uM) and deficient condition (35uM), and then the GUS activity of each root, stem, and leaf tissue was examined according to a conventional method.

The results are shown in FIG. 7. In FIG. 7, 1, 2, 5, and 6 represent experimental results under sufficient phosphoric acid conditions, and 3, 4, 7, and 8 represent experimental results under phosphoric acid deficient conditions.

OsPT1 Genes were specifically expressed in the root hairs of root tissues deficient in phosphoric acid. Therefore, in the case of the phosphorus-deficient environment, the roots of plants make numerous root hairs to increase the surface area and thereby maximize the absorption of phosphoric acid from the soil. Was considered to play an important role when absorbed.

<Example 3: Preparation and expression confirmation of transformed Arabidopsis pole>

Since rice lives mostly in water, it is relatively weak in phosphate stress compared to other field crops. Therefore, the mass expression of OsPT1 in Arabidopsis thaliana , a representative model plant of dicotyledonous plants, to prove its applicability in field crops of the high affinity phosphate carrier gene isolated from rice and to investigate its function in plants. Transformants were made.

A brief look at the Arabidopsis transformant production process is as follows. The coding region of the OsPT1 gene was cloned into the XbaI site of the binary vector pCAMBIA1300-35S, which was produced in our laboratory, to prepare a structure regulated by the CaMV 35S promoter, which always expresses strongly the OsPT1 gene, Agrobacterium tumefaciens EHA105 was transferred by the maturing (triparental mating) method. Then, Arabidopsis wild-type plants were transformed into Arabidopsis by the in planta transformation method using the Agrobacterium tumefaciens EHA105. After aseptic sterilization of the seeds harvested from T1 plants, the transformed plants were germinated in B5 medium containing carbenicillin (100 mg / L) and hygromycin (20 mg / L). The transgenic plants were selected from the next generation. The transgenic plants were grown in B5 liquid medium to which 35 μM and 1 mM of phosphoric acid were added, and their growth status was compared with that of wild type plants.

The results are shown in FIG. In FIG. 8, T1, T2, T4, and T6 arbitrarily indicate independently produced transformants. First, it was confirmed that the expression of the OsPT1 gene was increased in the OsPT1 transformant using a conventional RNA blot method (see FIG. 8C). In addition, it was confirmed that the growth of transformants in the phosphoric acid deficient condition is better compared to the wild type plants as a control (see Fig. 8A). In the quantitative results, it was confirmed that the growth in the transformants is better than the wild type plants. In particular, the transformants (T4), which had the highest expression of the OsPT1 gene among the four transformant lines, were found to increase about 50% in growth (see FIG. 8B).

As can be seen from the above examples, the present inventors were able to confirm that the transformants of Arabidopsis thaliana expressing the OsPT1 gene in large amounts enhance the uptake of phosphoric acid under phosphate deficient conditions. Therefore, on the basis of the above results, the rice root hair specific expression OsPT gene provided by the present invention is expected to be able to develop a new plant that overcomes phosphate deficiency stress more efficiently by mass-expressing it, and also like cabbage or radish OsPT genes in field crops and other flower crops are also expected to be useful. In addition, the present invention reduces the pollution of soil and water resources due to the use of large amounts of chemical fertilizers by improving the nutrient absorption of the plant itself, thereby greatly contributing to the restoration of ecosystems and the development of pleasant environmentally friendly agriculture, and to increasing the yield and quality of crop production. It is thought to be.

1 is a result of examining the homology to the amino acid sequence of the genes (OsPT) of 10 high affinity phosphate carriers isolated from rice. Twelve lines represent the 12 membrane-spanning regions of the OsPT gene.

2 is a result of examining the evolutionary relationship between the high affinity phosphate carrier of the OsPT gene and other dicotyledonous plants, monocotyledonous plants and fungi.

Figure 3 shows the nucleotide sequence comprising the high affinity phosphate carrier OsPT1 gene and promoter region of rice.

Figure 4 shows the nucleotide sequence comprising the high affinity phosphate carrier OsPT7 gene and promoter region of rice.

5A is a result of examining various expression patterns of OsPT1 and OsPT7 genes under phosphoric acid deficient conditions using callus cultured cells of rice. 5B and 5C show the results of examining various expression patterns of OsPT1 and OsPT7 genes according to phosphate concentration and phosphate deficiency time.

Figure 6 is a result of examining the expression patterns of tissue-specific OsPT1 and OsPT7 genes in phosphate deficient conditions.

FIG. 7 shows a structure in which the GUS reporter gene is linked to the promoter of the OsPT1 gene in order to investigate detailed tissue-specific expression patterns of the OsPT1 gene, to produce transformed rice using Agrobacterium and the activity of GUS in the transformant. This is the result of research by organization. Numbers 1, 2, 5, and 6 were the results obtained under phosphate sufficient conditions, and 3, 4, 7, and 8 were obtained under the phosphate deficient conditions.

Figure 8 is a result of comparing the growth of Arabidopsis transformant and wild type plants expressing a large amount of OsPT1 gene in phosphate deficient conditions.

Claims (5)

A phosphate carrier gene having the nucleotide sequence and the amino acid sequence of SEQ ID NO: 1 and which is specifically expressed in the root hair of Oryza sativa L. when phosphate is deficient. <SEQ ID NO 1> A phosphate carrier gene having the nucleotide sequence and the amino acid sequence of SEQ ID NO: 2 and which is specifically expressed in the root hair of Oryza sativa L. when phosphoric acid is deficient. <SEQ ID NO 2> Oryza sativ a L. root hair specific expression promoter of the gene of claim 1 having the nucleotide sequence of SEQ ID NO: 3. <SEQ ID NO 3> Oryza sativa L. root hair specific expression promoter of the gene of claim 2 having the nucleotide sequence of SEQ ID NO: 4. <SEQ ID NO 4> A method for producing a transgenic plant having enhanced phosphoric acid uptake using the gene of claim 1.