KR20020015179A - Gene encoding for a transcription factor which regulates the expression of defense related genes in rice and method for inducing the systemic acquired resistance by using this gene - Google Patents

Abstract

PURPOSE: Provided are gene encoding for a transcription factor which regulates the expression of defense related genes in rice and method for inducing the systemic acquired resistance by using this gene. In particular, provided is Oryza sativa ethylene response element binding protein(OsEREBP), a transcription factor which regulates the expression of defense related genes in rice. CONSTITUTION: The method of selecting OsEREBP comprises: treating the known systemic acquired resistance inducer, benzo-1,2,3-thiadiazole-7-carbothioic acid-S-methyl ether (BTH) to produce mRNA which is related to induction of systemic acquired resistance; transcribing the mRNA to obtain cDNA clone by using a transcriptase; selecting cDNA clone capable of being amplified by BTH treatment using the cDNA as a probe; and confirming a OsEREBP by analyzing nucleotide sequence and homology of cDNA clone. The nucleotide sequence of OsEREBP is represented by SEQ ID NO:1.

Description

Gene encoding for a transcription factor which regulates the expression of defense related genes in rice and method for inducing the systemic acquired resistance by using this gene}

The present invention relates to a transcription factor gene that regulates the expression of a group of disease defense genes in rice.

When plants are invaded by pathogens or are injured from the outside and are at risk of being invaded by pathogens, plants express themselves in a group of disease defense genes, thereby obtaining resistance to disease.

On the other hand, plants are known to express a variety of specific disease defense genes, and studies have been conducted to isolate these specific disease defense genes from plants and apply them genetically to other plants. For example, glucanase, chitinase, and the like, which are representative disease defense genes and are known as cell wall enzymes of pathogens, have been reported to improve disease resistance by individually introducing them into tobacco or rice.

On the other hand, much research has been carried out on how to express the disease defense gene group at the same time as the overall disease defense mechanism in vivo rather than individually expressing disease defense genes owned by plants. In particular, among these methods, research on the transcription factor genes that regulate the expression of the disease defense gene family has attracted attention. In this example, the potency of the transcription factors CBF and Alfin1 to regulate the expression of cold-resistant genes or salt-tolerant gene groups has been demonstrated.

On the other hand, another transcription factor EREBP (Ethylene response element binding protein) that regulates the expression of the disease defense gene group includes a cis -acting element known as a GCC box in a promoter. It is known to regulate the expression of several pathogenic gene families. Examples of pathogenic genes for which EREBP regulates expression are: PR1 (basic type PR1) and class I chitinase, osmotin (PR5) and glutathione-S-transferase (STA) in tobacco -transferase) and the like are known. In addition, in tomato, Pseudomonas astringent pv. Pti 4,5,6, known to bind to Pto, a resistance gene for tomato (Pseudomonas syringae pv.tamato), which regulates the expression of disease-resistant gene family, is a transcription factor of EREBP. ) By the method.

On the other hand, the transcription factor of the EREBP-based, etc., or Arabidopsis Italia, Nikko tiahna other bakum has the nucleotide sequence is known for the various types. However, only rice nucleotide sequences of two EREBP transcription factor genes have been reported in rice, which is used as a staple food, and no functional analysis has been reported.

Therefore, the present inventors studied to find a new transcription factor that regulates the expression of disease defense gene group in rice used as a stock, and find a new transcription factor EREBP expressed in rice, and its expression is induced by pathogens and pathogenic stimuli. It confirmed and completed this invention.

Accordingly, it is an object of the present invention to provide a transcription factor OsEREBP that regulates the expression of disease defense genes in rice ( Oryza sativa ).

1 is a homology analysis of the OsEREBP of rice. Here, AF190770 (SEQ ID NO: 2) and AF193803 (SEQ ID NO: 3) are sequences of EREBP genes reported in previously known rice.

Figure 2a is a diagram showing the expression level of OsEREBP by the external hospital in rice.

Figure 2b is a diagram showing the expression of OsEREBP in each tissue of rice.

Figure 3 is a view showing the expression of OsEREBP expression and PIP and glucanase of the present invention when treated with plants ACC, a precursor of ethylene.

The present invention provides a transcription factor gene OsEREBP ( Oryza sativa ethylene response element binding protein) of a disease resistant gene that gives resistance to rice.

Plants themselves contain genes such as disease resistance reactions, for example, hypersensitivity reactions and acquired resistance, but expression is amplified when there is a stimulus such as a pathogen or a wound.

Expression of disease defense genes that can provide such acquisition resistance is achieved by complex signaling systems in plants, but the final stage of the signaling system is that the transcription factor, a product of the transcription factor gene, binds to the promoter of the disease defense gene family. To express a group of defense genes. That is, the disease defense gene group is regulated by itself or by a regulator, and the expression of the transcription factor is amplified by the pathogenic stimulus, thereby amplifying the expression of the disease defense gene group, thereby making the plant resistant. .

In the present invention, an object of the present invention is to provide a transcription factor OsEREBP that enables expression of a group of disease defense genes in rice.

In order to select the OsEREBP gene in the present invention, first, benzo- (1,2,3) -thiadiazole-7-carbothioic acid S-methyl ether (benzo- (1,2, 3) -thiadiazole-7-carbothioic acid S-methyl ether (hereinafter referred to as 'BTH') to generate mRNAs related to induction of acquisition resistance. Then, the obtained mRNA is made into a cDNA clone using a reverse transcriptase enzyme, and this cDNA is used as a probe to select cDNA clones whose expression is amplified by BTH treatment by reverse northern blot. Through sequencing and homology analysis of the selected cDNA clone, it can be confirmed that one of the selected genes is the transcription factor OsEREBP of the EREBP system.

The reverse northern blot method used to select the OsEREBP gene of the present invention is carried out by the following procedure. ① First, BTH is treated to the root of rice grown to some extent (when BTH is applied to the root of rice, BTH is spread throughout the plant through the transport system in the plant). It induces the production of related mRNA, which separates the mRNA from the leaves of the treated rice. As a control, mRNA that is not BTH treated is also isolated. ② create a cDNA Gene Bank with the mRNA using a reverse transcriptase, to create a phage cDNA gene bank prepared the cDNA in λ phage vectors of Uni-XR vector zap and, through in vitro packaging (in vitro packaging), E. Infection with Coli amplifies the cDNA amount. Using the prepared phage cDNA gene bank, plasmid mass excision protocol was used to convert phage DNA of about 50 kb into phagemid of about 4-5 kb, and then inoculated into solid medium. Incubate to form colonies. DNA is optionally isolated from the selected colonies to produce DNA to be conjugated to the nylon membrane. ③ BTH-treated and untreated mRNAs isolated from ① are obtained using reverse transcriptase to obtain single-chain cDNA labeled with ³² P and used as a hybridization probe. ④ Sequencing of the DNA attached to the nylon membrane formed in ② and hybridization with cDNA formed in ③, comparing the radiolabeling degree of BTH-treated and untreated, and selecting a large difference, sequencing and Northern blot identification experiment Do it.

OsEREBP gene of the present invention selected by this method is a transcription factor gene that regulates the expression of the disease defense gene group in rice, its sequence is shown in SEQ ID NO: 1.

As a result of sequencing by GeneBank for the gene of SEQ ID NO: 1, two genes, AF190770 (SEQ ID NO: 2) and AF193803 (SEQ ID NO: 3), were known as EREBP transcription factor genes expressed in rice. His nucleotide sequence was reported. However, as shown in Figure 1, it can be seen that the base sequence of the EREBP of the present invention is significantly different, and thus the OsEREBP gene of the present invention can be seen that the new EREBP is expressed in rice.

On the other hand, as shown in Figures 2a and 2b, the OsEREBP gene found in the present invention is a chemical that induces acquisition resistance, such as BTH, salicylic acid (SA), or pathogenic stimuli such as wounds or rice bran blight bacteria Santomonas Expression is induced by pathogens such as Xoo and Magnapote grisea. In addition, the OsEREBP gene of the present invention is not expressed in roots or the like, but is mainly expressed in leaves, leaves, leaf veins, and the like.

In addition, as shown in Figure 3, after treating the ethylene-producing substance ACC (1-aminocyclopropane-1-carboxylic acid), OsEREBP and PIP (probenazole inducible protein) and glucanase which are representative pathogenic genes in rice Northern blotting with glucanase probe confirmed the expression level of each plant in the treatment time-dependent manner. OsEREBP production was increased in the treatment time-dependent manner, and the expression levels of glucanase and PIP, which are pathogenic genes, were also increased. You can see that. That is, the gene found in the present invention is a gene whose expression is regulated by ethylene, and shows that it is an EREBP-based transcription factor that increases the expression of the pathogenic gene group.

As a method for increasing the acquisition resistance using the OsEREBP gene of the present invention, a conventional method that can be carried out in genetic engineering is possible, for example, a plasmid having a strong and always expressed promoter present in a plant, etc. By inserting into a vector, a recombinant vector can be made, and the recombinant vector can be used to cause plant transformation. Plants transformed by this method can induce the expression of the acquisition resistance gene group even in the absence of stimuli such as pathogens can increase the acquisition resistance of the plant.

Hereinafter, the present invention will be described in more detail with reference to Examples and drawings, but the present invention is not limited thereto.

Example 1 Preparation of Nylon Film Attached DNA

Generation and Isolation of mRNA Related to Disease Resistant Expression

After sowing, 23 ppm of rice roots were treated with 1 ppm of BTH to induce mRNA production of genes involved in disease resistant expression. After 2 days of BTH treatment, the rice leaves were recovered and the recovered rice leaves were ground using liquid nitrogen. Then all RNAs expressed by BTH were extracted using the Trizol reagent from LIFE Technologies, Inc .; Maryland, U.S.A., according to the manufacturer's instructions. The extracted RNAs were used for cDNA gene bank preparation.

Creation of cDNA Gene Bank

cDNA gene banks were prepared from mRNAs isolated from BTH-treated rice, respectively.

First, using oligotex of Qiagen, only mRNAs having a poly-A terminal sequence were isolated from RNAs isolated with the trizol reagent.

Then, in the absence of RNase H, RNAs having the isolated poly-A terminal sequences were templated, and these were prepared using oligo dT primers capable of binding to reverse transcriptase and poly-A sequences. Single chain cDNA complementary to mRNAs was synthesized. Then, RNA was used as a template by adding RNase H, which selectively removes only RNA while the RNA and DNA were hybridized. CDNAs were synthesized using DNA polymerase I as a template using the obtained single-chain cDNA. The resulting cDNA was ligated with an EcoRI adapter and treated with XhoI and EcoRI to create a DNA restriction site for XhoI and EcoRI.

To prepare a cDNA gene bank with the obtained cDNAs, using Lambda ZAP II cDNA synthesis kit and Gigapack II gold packaging extract of Stratagene, Inc., California, USA , As instructed by the manufacturer.

Briefly, the cDNA obtained above was ligated to Uni-zap XR vector. The ligated vector was then in vitro packaged and transfected with E. Coli . Phage formed in the medium was quantified, and then the amount of phage was amplified and used for preservation. At this time, the number of plaques was 700,000.

Preparation of Nylon Film Attached DNA and DNA Attached Nylon Film

According to the method provided by Stratagen, the phagemid cDNA gene bank formed from the Lambda ZAP II cDNA gene bank by the plasmid massexcision method, wherein the phagemid cDNA gene bank formed is the same as the plasmid cDNA gene bank. Changed to.) DNA was isolated by randomly selecting a number of clones from the cDNA gene bank that formed colonies in the culture medium. Then, drop a DNA solution of the same kind and 200 ng of DNA obtained from the plasmid cDNA gene bank onto Amersham, Inc.'s hybond-N membrane (nylon membrane), 1.5M NaCl and The reaction was carried out in 0.5 M NaOH for 2 minutes to denature the DNA, and then neutralized by addition of 1.5 M NaCl and 0.5 M Tris-HCl (pH 8.0). Then, two DNA-attached nylon membranes were prepared by fixing to the nylon membrane by UV cross linking.

Example 2 Preparation of Probe cDNA

After sowing, 23 ppm of rice roots were treated with 1 ppm of BTH to induce mRNA production of genes involved in disease resistant expression. After 2 days of BTH treatment, the rice leaves were recovered and the recovered rice leaves were ground using liquid nitrogen. Then all RNAs expressed by BTH were extracted using the Trizol reagent from LIFE Technologies, Inc .; Maryland, U.S.A., according to the manufacturer's instructions.

In addition, RNA was also extracted from the rice not treated with BTH as a control in the same manner.

^A single chain cDNA was synthesized from 50 ug of total RNA isolated above using reverse transcriptase in the presence of ³² P labeled dCTP and oligo dT primers. The ³² P labeled single chain cDNA thus produced was used for the following hybridization.

Example 3 DNA Selection by Hybrid Formation

In each of the two BTH-treated and BTH untreated DNA-attached nylon membranes made in Example 1, the same amount of ³² P-labeled single-chain cDNA made in Example 2 (total RNA amount: 50 µg) was used as a probe. Hybrid formation was carried out at 占 폚, respectively. Then, genes with large differences in radiolabeling of these two nylon membranes were selected.

Example 4 Base Sequence Determination

The cDNA of the plasmid corresponding to the gene selected in Example 3 was determined using ABI's Big Dye Terminater to determine its base sequence, and the sequence thereof is shown in SEQ ID NO: 1.

Example 5 Homology Analysis

Homology analysis was carried out through the NCBI Blast program, and the selected gene was identified as an EREBP transcription factor, and the results of homology analysis with the EREBP transcription factor reported in rice are shown in FIG. Indicated. Figure 1 shows that even at the amino acid sequence level, there is little homology except for the DNA binding domain (DNA binding domain), ie, the amino acid sequence of 129-189 of OsEREBP, which is a typical feature of EREBP-based transcription factors. Since the OsEREBP gene of the present invention has a sequence different from other transcription factor genes (SEQ ID NO: 2 and SEQ ID NO: 3) of other EERBP systems registered in Genebank, it was confirmed that the EREBP gene of the present invention is a new OsEREBP gene expressed in rice. .

Example 6 Expression Analysis of OsEREBP

Six hours after 1 ppm BTH and 1 mM salicylic acid (SA) were applied to the roots of rice, the leaves of the rice were cut with scissors and wounded for 24 hours. Santomonas duckwood pv. After 24 hours of inoculation with Xoo 98K1 and Magnapote grisea KJ401 by the method of spraying rice leaves, leaves were sampled and RNA was extracted with Life Technologies' Trizol reagent. Then, 30 μg of total RNA was subjected to electrophoresis on an agarose gel containing formaldehyde. Electrophoresed RNA was transferred to the nylon membrane by capillary transfer and attached by UV.

In addition, the fragment obtained by cutting the OsEREBP cDNA clone selected from Example 3 with EcoRI and XhoI was reacted at 55 ° C using a Laderman labeling kit from Takara, and labeled with radioactive material. The probe was then purified by an HR spin column from PHARMACIA.

Northern blots of the isolated RNAs were performed at 65 ° C using this probe. The results are shown in Figure 2a, it can be seen that the expression of OsEREBP amplified when BTH, SA, wound, Xoo, Magnafote greece treatment.

The results of Figure 2a was confirmed that for the pathogenic bacteria and pathogenic stimulation, rice amplifies the expression of OsEREBP.

Example 7 Tissue Specificity of OsEREBP

1 ppm BTH was treated to the roots of the cultivator rice and the roots of the young rice, RNA extraction was carried out using Life Technologies' Trizol Reagent, and Northern blot was performed in the same manner as in Example 6. The results are shown in Figure 2b, the expression of OsEREBP is not expressed in the roots, mainly in the leaves, it was confirmed that the expression is reduced in the leaves of the divider.

Example 8 Northern Blot Test of Selected DNA

After sowing, the ethylene precursor ACC was treated with a soil drench application in 20 days old rice. 12, 24 and 48 hours after the treatment, the rice leaves were recovered and total RNA was isolated using Life Technologies' Trizol reagent. The isolated RNA was then electrophoresed with 1.2% formaldehyde denature agarose gel. Northern blots were then performed by hybridization with OsEREBP, and each cDNA of Probenzazole Inducing Protein (PIP) and Glucanase. The results are shown in FIG. RNA gel stained with EtBr in Figure 3 shows that the amount of RNA in each treatment is the same amount.

Referring to FIG. 3, as the ACC treatment time increases, the expression levels of OsEREBP, the disease-resistant transcription factor, and PIP and glucanase, the disease-resistant genes of the present invention, increase. These results suggest that OsEREBP of the present invention is induced by ethylene, and also OsEREBP of the present invention induces expression of pathogenic gene groups. That is, in conclusion, the DNA selected in the present invention may indicate that EREBP-based genes are expressed in rice.

OsEREBP gene of the present invention is a transcription factor capable of giving resistance to rice, its complete sequence is shown in SEQ ID NO: 1. OsEREBP gene is expressed in the leaves, leaf veins, leaves, etc., it can be seen through the present invention that the expression is not in the root. In addition, the OsEREBP of the present invention can be used to enhance the acquisition resistance to disease in rice.

Claims (2)

OsEREBP transcription factor gene of SEQ ID NO: 1 that controls the expression of disease defense gene family in rice. Method of enhancing the acquired resistance in rice using the OsEREBP transcription factor of claim 1.