CN118389532A - Application of rice OsSAFM gene or protein coded by same in improving rice blast resistance of rice - Google Patents

Abstract

The present invention discloses the application of rice OsSAFM6 gene or the protein encoded by it in improving rice resistance to rice blast fungus, and relates to the technical field of rice blast prevention and control. The present invention has found through research that the protein encoded by rice gene OsSAFM6 can be used as a medicament for preventing and controlling rice blast fungus. OsSAFM6 protein has a significant inhibitory effect on rice blast fungus appressorium. When the concentration of SAFM6-GST is 0.05 μg/μL, the appressorium formation rate of rice blast fungus spores is 26.67%; when the concentration is 0.1 μg/μL, the appressorium formation rate of rice blast fungus spores is 5.33%. Therefore, the protein encoded by rice gene OsSAFM6 can be used to improve rice resistance to rice blast fungus.

Description

Application of rice OsSAFM6 gene or its encoded protein in improving rice resistance to rice blast fungus

Technical Field

The invention relates to the technical field of rice blast prevention and control, and in particular to application of rice OsSAFM6 gene or protein encoded by it in improving rice resistance to rice blast fungus.

Background technique

Rice has a long history of cultivation and is an important economic crop in many countries. Because of its high nutritional value and strong adaptability to the growing environment, it is an important source of food and nutrition security in developing countries. For rice as an important food crop, the most typical fungal disease is rice blast, which is caused by Magnaporthe oryzae. The rice blast fungus that causes rice blast can infect the above-ground tissues of rice at all growth stages of rice, causing a reduction in rice yield. In addition, the rice blast fungus has different effects on different rice resistance varieties, rice growth environment conditions, different rice growth stages, and when infecting different locations of rice.

Rice blast may occur at different stages and different parts of rice growth and development, mainly including leaf blast, ear neck blast and grain blast. Rice blast fungi mainly spread in the field through asexual reproduction and complete their infection cycle. The three-cell conidia of rice blast fungi scattered on weeds or plant residues in the field last year are spread to the surface of rice leaves through air, rain, etc. When the spores fall on the surface of hydrophobic rice leaves, they will secrete some mucus to adhere to the surface of the hydrophobic leaves. And form germ tubes. Germ tubes form attachment cells through accumulation, bending and swelling, and a large amount of melanin is deposited in the attachment cells. The attachment cells differentiate into invasion spikes, which penetrate the cuticle and cell wall of the leaf epidermis. The formed infection hyphae continuously infect adjacent cells, and the infected cells continue to produce infection hyphae and conidia, and repeatedly infect adjacent cells. In a humid environment, gray or gray-brown spindle-shaped spots will appear on rice leaves. This is the process of rice blast fungi transforming from a living trophic type to a dead trophic type. The newly produced conidia at the lesions can be spread to new host plants through wind and rain, starting a new round of infection cycle. Spraying related pesticides is currently a fast and efficient method for preventing and controlling rice blast, and it is also the most commonly used method. Generally, pesticides are applied at the end of the heading stage and the full heading stage. Tricyclazole, rice blast, etc. are more commonly used drugs for the prevention and control of rice blast, but the continuous use of chemical pesticides for a long time will make rice blast fungi resistant to drugs and pollute the surrounding air environment. In current agricultural production, antimicrobial peptides play an important role. Antimicrobial peptides are a class of naturally occurring small molecule peptide chains with strong antibacterial activity that can fight against various pathogenic microorganisms. The application of antimicrobial peptides in agricultural production is a green and efficient strategy that can effectively ensure the yield and quality of crops and is of great significance to achieving more sustainable agricultural development.

However, little is known about new agents that can effectively prevent and control rice blast. Through research, the present invention reveals the regulatory mechanism of rice antimicrobial peptides. Based on its function of inhibiting the germination of rice blast fungi, inhibiting the formation of appressorium and inhibiting the infection of rice leaves by rice blast fungi, it is effectively applied to the prevention and control of rice blast, and an efficient agent is developed to achieve effective prevention and control of rice blast.

Summary of the invention

The present invention finds that the OsSAFM6 protein encoded by the rice OsSAFM6 gene has a significant inhibitory effect on the appressorium formation of rice blast fungus and can be used as a drug to prevent and control rice blast.

The technical solution of the present invention is as follows:

The invention provides application of rice OsSAFM6 gene or protein encoded by the gene in improving resistance of rice to rice blast fungus.

The present invention also provides the use of rice OsSAFM6 gene or the protein encoded by the gene in preparing a drug against rice blast fungus.

The present invention also provides the use of rice OsSAFM6 gene or the protein encoded by it in rice breeding, and obtains rice strains resistant to rice blast fungus by screening rice plants with high expression of rice OsSAFM6 gene or high expression of protein encoded by rice OsSAFM6 gene.

The CDS nucleotide sequence of the rice OsSAFM6 gene is shown in SEQ ID No.2, and the amino acid sequence of the protein encoded by the rice OsSAFM6 gene is shown in SEQ ID No.1.

The present invention also provides an anti-rice blast fungus drug, wherein the active ingredient comprises a protein encoded by the rice OsSAFM6 gene with an amino acid sequence as shown in SEQ ID No. 1. The concentration of the protein encoded by the rice OsSAFM6 gene is 0.05-0.1πg/μL.

The purified OsSAFM6 protein (SAFM6-GST protein) has a significant inhibitory effect on the appressorium of the rice blast fungus. When the concentration of SAFM6-GST is 0.05 μg/μL, the appressorium formation rate of the rice blast fungus spores is 26.67%, and when the concentration is 0.1 μg/μL, the appressorium formation rate of the rice blast fungus spores is 5.33%.

The invention also provides application of the anti-rice blast fungus drug in preventing and controlling rice blast fungus infection.

The present invention also provides a method for constructing transgenic rice resistant to rice blast fungus, wherein the rice OsSAFM6 gene is transferred into rice plants to obtain transgenic rice highly expressing the rice OsSAFM6 gene, and the CDS nucleotide sequence of the rice OsSAFM6 gene is shown in SEQ ID No.2.

Specifically, the CDS region nucleotide sequence of the rice OsSAFM6 gene is cloned into a vector, first transferred into Agrobacterium, and then transferred into rice cells through callus transformation to obtain transgenic rice with high expression of the rice OsSAFM6 gene. The vector is the PGEX-4T-1 vector.

Beneficial effects of the present invention:

The present invention has found through research that the protein encoded by the rice gene OsSAFM6 can be used as a drug for preventing and controlling rice blast. The OsSAFM6 protein has a significant inhibitory effect on the appressorium of the rice blast fungus. When the concentration of OsSAFM6 is 0.05 μg/μL, the appressorium formation rate of the rice blast fungus spores is 26.67%, and when the concentration is 0.1 μg/μL, the appressorium formation rate of the rice blast fungus spores is 5.33%. Therefore, the protein encoded by the rice gene OsSAFM6 can be used to improve the resistance of rice to the rice blast fungus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the inhibition of rice OsSAFM6 purified protein on the appressorium formation of the rice blast fungus, wherein A is a graph showing the appressorium formation of the rice blast fungus, and B is a graph showing the statistical results of the appressorium formation.

FIG. 2 is a graph showing the inhibition of mycelial growth of rice blast fungus by purified rice OsSAFM6 protein, wherein A is a graph showing mycelial growth of rice blast fungus, and B is a graph showing the inhibition rate of rice blast fungus colonies.

FIG. 3 shows the results of inoculation of rice leaves with purified rice OsSAFM6 protein to inhibit rice blast fungus from infecting rice leaves.

Detailed ways

Example 1

Obtaining purified OsSAFM6 protein.

According to the CDS sequence of the gene OsSAFM6 (shown in SEQ ID No. 2), the primer OsSAFM6GST-F/R was designed. The primer sequence is as follows:

OsSAFM6GST-F: ggttccgcgtggatccATGAAGACCGCC;

OsSAFM6GST-R:gtcgacccgggaattcGTTCTCGCACGA.

Vector construction: Using the Nipponbare genome as a template, the CDS sequence of the gene OsSAFM6 was amplified using primers OsSAFM6GST-F/R. The amplified fragment was connected to the PGEX-4T-1 vector after BamHI and EcoRI digestion by seamless cloning technology. The connected plasmid was transformed into E. coli by heat shock method, and positive clones were selected for detection.

Induce SAFM6-GST protein expression: After the SAFM6-GST vector was sequenced correctly, the plasmid was transferred into the Escherichia coli strain BL21 by the heat shock method. The positive clones were selected and cultured at 37°C until the OD value was 0.6. They were induced at 16°C for 16 hours with an IPTG concentration of 1mM. The protein expression was then detected by Coomassie brilliant blue staining and Western-blot to confirm that the protein induction was successful.

OsSAFM6-GST protein purification:

(1) Centrifuge 150 mL of bacterial cells at 10,000 rpm for 2 min and remove the supernatant. Add 15 mL of GSTbinding/washing buffer to the precipitated bacterial mass to suspend the bacterial cells, then add 300 μL of 10 mg/mL lysozyme, 30 μL of 0.5 M MgCl ₂ , and 150 μL of 0.1 M phenylmethylsulfonyl fluoride (PMSF). Slowly shake at 4°C for 30 min.

(2) After the enzymatic hydrolysis process was completed, the bacteria were disrupted by ultrasound and then centrifuged at 4°C for 10 min at a speed of 10,000 rpm;

(3) Take 200 μL of the supernatant and mark it as Input. Transfer the remaining supernatant to the equilibrated glutathione transferase (GST) protein resin flow column. Adjust the flow rate to 7-9 s/drop. Collect 200 μL of the effluent and mark it as Flowthrough. Add 15 mL of water to the bacterial residue and take 200 μL and mark it as Bacteria pellet. The whole process is carried out in a 4°C refrigerator to prevent protein denaturation.

(4) Add 5 mL of GST binding/washing buffer and control the flow rate to 4-6 s/drop. Collect 200 μL of the effluent at the bottom of the column and label it as Wash1. Repeat this step once and collect 200 μL of the effluent and label it as Wash2.

(5) Add an appropriate amount of glutathione to 1 mL of GST elution buffer to elute the target protein, collect the eluate (the eluate contains the target protein) and mark it as Elution 1; repeat this step three times and mark them as Elution 2, Elution 3, and Elution 4 respectively;

The purified protein (sequence shown in SEQ ID No. 1) was detected by Coomassie Brilliant Blue staining and Western-blot, and then the concentration of the purified protein was determined.

Example 2

Purified OsSAFM6 protein inhibits appressorium formation of Magnaporthe oryzae.

Wild rice blast fungus strain RB22 was activated on OA medium, cultured in the dark at 25℃ for 3 days and in the light for 4 days. Sterile ddH ₂ O was added to the culture dish, and the mycelium was gently scraped with an inoculation loop to elute the rice blast fungus spores from the culture medium. The eluate was filtered through a magic filter cloth to obtain a spore suspension. The spore suspension was placed in a 2mL centrifuge tube and centrifuged at 10000rpm for 1min. The supernatant was discarded (to avoid pouring out the bottom spores), sterile ddH ₂ O was added, and the spore concentration was adjusted to no less than 1-2×10 ⁵ /mL using a hemocytometer, and the concentration of SAFM6-GST purified protein in the suspension was set to 0, 0.05 and 0.1μg/μL respectively. The spore drop was dropped on the surface of a hydrophobic glass slide, and after culturing at 25℃ for 12h, the germination of rice blast fungus spores was observed under a microscope; after 24h, the formation of rice blast fungus spores and appressorium was observed under a microscope.

As shown in Figure 1, the SAFM6-GST protein has a significant inhibitory effect on the appressorium of the rice blast fungus. When the concentration of SAFM6-GST is 0.05 μg/μL, the appressorium formation rate of the rice blast fungus spores is 26.67%, and when the concentration is increased to 0.1 μg/μL, the appressorium formation rate of the rice blast fungus spores is 5.33%. This result shows that the OsSAFM6 protein is expected to be used as an agent for the prevention and control of rice blast.

Example 3

The purified protein of OsSAFM6 inhibits the mycelial growth of rice blast fungus.

SAFM6-GST purified protein was mixed into CM-C medium, and the concentrations were set to 0 and 0.05 μg/μL, respectively. A colony with a diameter of 1 mm was cut from the activated wild rice blast fungus strain RB22 and placed on CM-C medium. After culturing in a 25°C incubator for 7 days, the colony diameter was measured and recorded.

As shown in Figure 2, SAFM6-GST protein has a certain inhibitory effect on the mycelial growth of rice blast fungus.

Example 4

Purified OsSAFM6 protein inhibits rice blast fungus from infecting rice leaves.

The wild rice blast fungus strain RB22 was activated on OA medium, and the culture conditions and the process of obtaining the spore suspension were the same as in Example 2. The spore concentration was adjusted to 1×10 ⁶ /mL using a hemocytometer, 1/10 volume of 0.1% Gelatin (final concentration of Gelatin was 0.01%, v/v) was added to the spore solution, and the concentrations of the purified SAFM6-GST protein in the suspension were set to 0 and 0.05 μg/μL, respectively, for rice blast fungus inoculation.

In vivo inoculation: Take rice blast susceptible material CO39 at the 4-leaf stage, spray 1 mL of spore mixture evenly onto the leaf surface with a spray gun, seal with PVC film and plastic wrap to maintain humidity. After 48 hours of dark culture, resume light, and culture at 22°C for 7 days to investigate the disease situation.

The inoculation conditions are shown in (Figure 3): compared with CK (SAFM6-GST purified protein concentration 0 μg/μL) and GST (GST purified protein concentration 0.05 μg/μL), the addition of 0.05 μg/μL SAFM6-GST purified protein can reduce the lesion area, indicating that OsSAFM6 can inhibit the infection of rice leaves by rice blast fungus.

Claims (10)

1. The rice OsSAFM gene or the coded protein thereof is applied to improving the rice blast resistance of rice.

2. Application of rice OsSAFM gene or coded protein thereof in preparing medicines for resisting rice blast bacteria is provided.

3. The rice OsSAFM gene or the protein coded by the gene is applied to rice breeding, and rice plants with high expression of the rice OsSAFM gene or the rice OsSAFM gene coded by the gene are screened to obtain rice plant lines resistant to rice blast bacteria.

4. The use according to any one of claims 1 to 3, wherein the nucleotide sequence of the CDS gene of rice OsSAFM is shown in SEQ ID No.2, and the amino acid sequence of the protein encoded by the rice OsSAFM gene is shown in SEQ ID No. 1.

5. A medicine for resisting rice blast is characterized in that the active ingredient comprises a protein coded by rice OsSAFM gene with an amino acid sequence shown as SEQ ID No. 1.

6. The rice blast resistant pharmaceutical composition according to claim 5, wherein said rice OsSAFM gene encodes a protein having a concentration of 0.05 to 0.1. Mu.g/. Mu.L.

7. The use of a rice blast resistant agent as claimed in claim 5 or 6 for controlling rice blast infection.

8. The use according to claim 7, wherein the rice blast resistant agent is sprayed onto the leaves of plants during use.

9. A construction method of rice blast resistant transgenic rice is characterized in that rice OsSAFM gene is transferred into rice plants to obtain transgenic rice with high expression of rice OsSAFM6 gene, and CDS nucleotide sequence of rice OsSAFM gene is shown as SEQ ID No. 2.

10. The method for constructing transgenic rice against Pyricularia oryzae according to claim 9, wherein the nucleotide sequence of CDS region of rice OsSAFM gene is cloned into vector, transferred into agrobacterium first, and transferred into rice cell by callus transformation to obtain transgenic rice with high expression of rice OsSAFM gene.