CN116515896A - Application of OsFID gene in improving fungal disease resistance of plants - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of an OsFID gene in improving fungal disease resistance of rice. The invention discloses a rice OsFID protein related to plant antifungal diseases, and a coding gene and application thereof. The technical scheme for solving the technical problems is that the invention provides the application of the OsFID gene in improving plant disease resistance, and experiments prove that the OsFID gene can be highly induced to be expressed by fungal pathogens, and in addition, the over-expression plant of the rice transformed with the OsFID gene has obvious resistance to rice blast and false smut. The invention provides new effective selection for raising crop disease resistance variety by using genetic engineering means.

Description

Application of OsFID gene in improving fungal disease resistance of plants

Technical Field

The invention relates to the field of molecular biology and transgenic plants, in particular to application of an OsFID gene in improving the resistance of plants to fungal diseases.

Background

Rice is one of the main grain sources for human beings, and the yield of the rice restricts the development of human beings. Plants live in environments where exposure to a variety of microorganisms coexist, many of which are pathogenic. Plants are capable of secreting large amounts of secreted proteins into the apoplast space under pathogenic bacterial infection conditions. The protease SAP1 secreted by arabidopsis thaliana, SAP2 can be induced under pathogenic bacteria infection conditions, and the protease activity of the protease is utilized to directly participate in inhibiting pathogenic bacteria infection. It is explained that apoplast protein plays an important role in plant disease resistance. However, whether other rice extracellular secretion proteins participate in the tolerance of plants to fungal diseases is known very little, and the research reports on the function identification of the rice extracellular secretion proteins are very little, and the complexity and the specificity of the extracellular secretion proteins in the plant disease resistance process are reflected to a certain extent, so that the novel plant disease resistance proteins are continuously identified, the related genes and the molecular functions of the genes are determined, the comprehensive disclosure of the mechanism of plants for preventing the infection of rice blast is facilitated, the deep disclosure of the molecular mechanism of plants for preventing the disease in a broad spectrum is facilitated, and the theoretical basis is provided for rice variety improvement screening for resisting rice blast.

We have found that rice secretion OsFID1 responds to fungal pathogen infection. The OsFID1 deletion rice mutant has obviously reduced fungal resistance, and over-expression of OsFID1 obviously enhances the resistance to rice blast fungus and false smut. The OsFID1 is involved in the disease resistance process of rice to rice blast and false smut.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the resistance of rice to rice blast and false smut.

In order to solve the technical problems, the purpose of the invention can be realized by the following technical scheme:

the application of the OsFID gene in improving the fungal disease resistance of plants is provided, wherein the nucleotide sequence of the OsFID gene is shown as SEQ ID NO. 1.

The protein for improving rice blast resistance and false smut resistance provided by the invention is named OsFID1, is shown as SEQ ID No.2 below, and consists of 258 amino acids.

In order to facilitate rice transgenic overexpression of the OsFID1 gene, a FLAG protein tag shown as SEQ ID NO.3 can be connected to the carboxyl end of the protein shown as SEQ ID NO.2 to form a fusion protein shown as SEQ ID NO. 4.

The super-expression vector is pCAMBIA1300, as shown in figure 1, and is named pCAMBIA1300-OsFID1-Flag.

The competence of the eukaryotic transformed agrobacterium is GV3101.

The invention provides application of OsFID1 protein in regulating and controlling plant disease resistance.

The invention provides the function of improving the disease resistance of transgenic plants by OsFID1 protein.

The invention provides an application of OsFID1 protein in a plant breeding process.

The invention provides the change of mRNA level of rice related genes after rice blast, false smut and bacterial blight infection, as shown in figure 2, and quantitative primers are shown in table 1.

TABLE 1 quantitative primer sequences for action and related genes

In the above application, the protein is shown in FIG. 3 for improving plant disease resistance.

In the above application, the plant is monocot rice NiP (Oryza sativa L. Spp. Japonica, varNipponbare, AAgenome), and the pathogenic bacteria are Pyricularia oryzae RB22, pyricularia oryzae HWD-2, and Xanthomonas PXO99.

The meaning of the invention is that: the invention provides a gene for improving rice blast resistance and false smut resistance of rice, and encoding protein and application thereof, and the gene can be overexpressed in wild rice NIP to improve the rice blast resistance and false smut resistance of rice, thereby laying a theoretical foundation for plant resistance breeding screening and having potential application value for molecular breeding.

Drawings

FIG. 1 is the map information of the transgenic rice overexpressing OsFID1 gene (pCAMBIA 1300-OsFID 1-Flag).

FIG. 2 shows the mRNA level changes of OsFID and its related genes after inoculation of wild type rice NIP with fungi (Pyricularia oryzae and Uygur autonomous rice U.virens) and bacteria (Xanthomonas Xao). The result shows that the OsFID1 gene can be highly induced to express after the fungal pathogen infects rice, and the OsFID1 gene is not induced by bacterial diseases. OsCYP99A2, osWRKY45, osPR5 and OsPR10 are disease-resistant marker genes.

FIG. 3 is a phenotype plot and lesion statistics of rice blast strain RB22 after spray inoculation of rice.

FIG. 4 is a phenotype map and morbidity statistics of rice inoculated by injection of the false smut strain HWD-2.

FIG. 5 is a graph showing statistics of phenotype and lesion length of rice material after inoculation with Xanthomonas.

Detailed Description

The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

1. Material

1. Rice material and variety thereof: nipponbare rice (OryzasativaL.cv.Nipponbare, NIP), nipponbare rice overexpressing OsFID gene (over-expressionOsFID, OE-OsFID, #9 and # 12), and Nipponbare rice knocked out with rice OsFID gene (CRISPR-Cas 9OsFID, osfid, osfid1 and Osfid 2). Transgenic rice materials are all from the hundred-grid gene technology (Jiangsu) company;

2. greenhouse conditions for rice growth: the temperature is 28 ℃, the humidity is 70% RH, and the photoperiod is 14h light to 10h darkness;

3. LB medium: weighing 10g of NaCl,5g of yeast extract and 10g of tryptone, adding about 900mL of ultrapure water for stirring and dissolving, adjusting the pH to 7.0-7.2 by using 1mol/L of NaOH, adding water to fix the volume to 1L, and sterilizing by high-pressure steam for 15min to obtain an LB liquid medium, wherein the LB solid medium is obtained by adding 15g/L of agar powder into the LB liquid medium and then sterilizing by high-pressure steam at high temperature;

4. kanamycin mother liquor (Kan) ⁺ 50 mg/mL): weighing 0.5g kanamycin, dissolving in 10mL of ultrapure water, filtering for sterilization, sub-packaging into small tubes, and preserving at-20 ℃;

2. cloning of OsFID1 Gene:

1. specific primers with homologous recombination of the expression vector pCAMBIA1300-OsFID1-Flag vector and forward primers are designed: 5'-TTACTTCTGCAGggtaccATGGCGCGGTGCACTTTGCT-3', reverse primer: 5'-GTCTTTGTAGTCAAGCTTCTCACGCAGCACCACCATC-3'.

2. Total RNA was extracted from rice NIP and reverse transcribed into cDNA by reverse transcriptase supplied by Novain, according to the instructions of TRIzol RNA kit;

3. amplifying the cDNA of the reverse transcription product serving as a template by using the forward and reverse primers, wherein the amplification procedure comprises the steps of pre-denaturation at 95 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 60s,35 cycles and continuous extension at 72 ℃ for 8min to obtain a PCR product;

4. the PCR product was purified using a PCR purification kit and finally ligated to the expression plasmid pCAMBIA1300 by homologous recombinase provided by Novain, and the expression recombinant plasmid pCAMBIA1300-OsFID-Flag with the target gene (SEQ ID NO. 1) was obtained.

3. RNA level change detection of related genes after wild rice NIP inoculation rice blast and false smut

The technical means used in the present embodiment are common to or fully understood by those skilled in the art.

1. Spore liquid preparation of rice blast germ:

activating rice blast bacteria on CM culture medium (namely omnipotent culture medium, taking 50mL of 20xNitratesalts nitrogen salt, 1mL of traceelents trace elements, 10g of glucose, 2g of peptone, 1g of yeast extract, 1g of casein amino acid and 1mL of vitamin solution, adding 20g of bacteria Agar after volume fixing to 1L, performing inversion culture at 28 ℃ for 5 days, inoculating the rice blast bacteria on spore-forming culture medium (taking 500g of straw, adding 4L of tap water, performing active cooking for 2 hours, then adding 160g of corn flour and 60g of bacteria Agar, performing high-temperature high-pressure sterilization), and performing inversion culture at 28 ℃. After 4 days, the spore-producing plate is taken out to scrape mycelium, and then the spore is cultivated under blue light for 4-6 days.

About 2mL of sterilized water was added to the plate with spores, the spores were scraped off, and then filtered through a filter cloth, 10. Mu.L of spore liquid was collected on a blood cell counting plate, and 10-15 spores were contained in the counting cell under observation under a microscope. Spray inoculation was performed by adding 4% gelatin to the spore liquid (200. Mu.L of 4% gelatin was added per 4mL of spore liquid).

2. Spore liquid preparation of ustilaginoidea virens:

placing ustilaginoidea virens in potato sucrose medium (150 g of peeled potato is weighed, smashed, added with about 900mL of water, boiled for 20min with strong fire, filtered with gauze, then added with 20g of sucrose, and fixed volume to 1L with water to obtain a liquid medium, adding 15g of bacteria Agar as solid, sterilizing at high temperature and high pressure, and preserving for later use.) plate, and culturing at 28 ℃. Culturing for about 6-8 days, then drawing the Aspergillus oryzae pieces, placing the pieces in potato sucrose liquid culture medium, and culturing at 28deg.C for about 5 days to obtain a large number of mycelium fragments and spores. Pouring the cultured culture solution into a tissue crusher, crushing, filtering with filter cloth, diluting with 5% potato juice (50 g potato juice, crushing, boiling with strong fire for 20min, filtering to obtain 5% potato juice), and inoculating.

3. Preparation of Xanthomonas bacteria:

xanthomonas strains were spread on inoculation NA medium (3 g cattle were weighedMixing meat extract, 1g yeast extract, 5g peptone and 10g sucrose, fixing volume to 1L, adjusting pH to 7.0-7.2 to obtain liquid culture medium, adding 15g bacteria Agar into the solid, sterilizing at high temperature, and storing at 28deg.C for 1-2 days. After yellow bacterial colonies grow on the plates, pouring 5mL of sterilizing water for bacteria scraping, collecting into a 15mL centrifuge tube, centrifuging at 4850rpm for 5min, washing with sterilizing water once, and finally adding a proper amount of sterilizing water to adjust OD ₆₀₀ After=0.5, the inoculation is to be performed.

4. Extraction of RNA:

about 30mg of the leaf blade is placed in a 1.5mL centrifuge tube containing steel balls, placed in a liquid nitrogen box for freezing, and broken by a sample grinder. The invention adopts Trizol method to extract plant tissue RNA. Kits were purchased from the biological technologies company limited of nanking nuozan. The extraction step: adding 800 mu LTrilzol into the ground sample, mixing by vortex, standing at room temperature for 10min, adding 160 mu L chloroform, mixing by inversion for 2min, centrifuging for 10min with 11600 g; centrifuging, taking the supernatant in a new 1.5mL centrifuge tube, adding equal volume of isopropanol, uniformly mixing the mixture and standing on ice for 10min; centrifuging 11600g for 10min after standing; removing supernatant, adding 75% ethanol, washing for one time, centrifuging for 5min with 11600g, removing supernatant, air drying, adding 50 μLDEPC water to dissolve RNA on ice, measuring concentration, and placing in a refrigerator at-80deg.C for use.

4. Detection of resistance to rice blast and false smut by knockout and overexpression of NIP rice OsFID1 gene

1. Rice blast fungus RB22 inoculation

1) Obtaining of Pyricularia oryzae spore suspension

Washing the activated spores with water, adding 4% gelatin, and placing in a spray can for spray inoculation; dropping 5 mu L of spores into a blood cell counting plate, and observing under a microscope, wherein about 10 spores are contained in 25 grids in the visual field;

2) Spray inoculation

Every 16 rice plants are sprayed and inoculated with about 4mL, the surfaces of the leaves are filled with fog drops, and the leaves are placed in the dark for culture; the rice is wild NIP rice, osFID1 gene knockout and overexpressed NIP rice respectively.

3) The pathogenesis of disease

Placing the spray inoculated plants in dark for 24 hours, and then transferring the plants to illumination for 3 days, observing the humidity condition of leaves every 6 hours during the cultivation, and paying attention to moisture preservation;

4) Patch statistics

The spray inoculated plants were subjected to statistical photographs of the leaf number of lesions.

2. Ustilago virens HWD-2 inoculation

1) Acquisition of Ustilago oryzae spore suspension

Ustilago virens wild type HWD-2 was cultured in PSB medium for 7d (25 ℃,220 rpm). Dispersing the bacterial liquid with a homogenizer at 15k speed, grinding for 1min, filtering with gauze to obtain mycelium spore liquid, and adjusting spore concentration to 3×10 ⁶ Each ml (prepared bacterial liquid can be stored at 4 ℃ for later use).

2) Injection inoculation

When the rice grows to the booting stage and the interval between the first leaf and the second leaf is about 5cm, the fungus liquid is injected into the ear part by a syringe, and the rice is filled until overflows. The rice is wild NIP rice, osFID1 gene knockout and overexpressed NIP rice respectively.

3) The pathogenesis of disease

The inoculated rice is transferred to a greenhouse with the temperature of 25 ℃ and the humidity of 95 percent for cultivation, and water spraying and moisture preservation are carried out every day for about 6 to 8 hours in the first week.

4) Spike statistics

About 21d after inoculation, counting the disease condition of each rice spike and taking photos for counting the disease spike.

The results are shown in fig. 3 and 4. As can be seen from the figure, after the NIP rice OsFID1 gene is knocked out (Osfid 1-1 and Osfid 1-2), the resistance of the rice to rice blast and false smut is reduced, and the resistance of the rice to rice blast and false smut can be improved after the OsFID genes (# 9 and # 12) are overexpressed. The result shows that the rice OsFID1 gene positively regulates the resistance of the rice to rice blast and false smut.

3. Xanthomonas bacteria Xoo inoculation

1) Scraping Xanthomonas from the NA culture plate by using a gun head, and re-suspending the Xanthomonas in 1.5ml of sterilized water;

2) Uniformly mixing the re-suspended bacteria liquid by a pipetting gun; transferring 800ul of bacterial liquid into a cuvette, and detecting OD by using an ultraviolet spectrophotometer ₆₀₀ ；

3) Diluting the bacterial liquid to OD with sterilized water ₆₀₀ =1.0; dipping the bacterial liquid by using scissors sterilized by 75% alcohol, and inoculating the rice leaf tips by cutting leaves;

4) Placing the plants inoculated by cutting leaves in an illumination incubator for cultivation, and paying attention to moisture preservation;

5) Statistical photographing of the leaf length was performed after two weeks of culture.

As a result, as shown in FIG. 5, it was revealed that there was no difference between the resistance of rice against xanthomonas bacteriovorus and the resistance of NIP rice and overexpressing OsFID genes (# 9 and # 12) after knocking out the OsFID1 gene of NIP rice (Osfid 1-1 and Osfid 1-2). The result shows that the rice OsFID1 gene does not play a role in the process of infecting rice by using Xanthomonas.

Claims (10)

1. The application of an OsFID gene in improving the resistance of plants to fungal diseases is disclosed, wherein the nucleotide sequence of the OsFID gene is shown as SEQ ID NO. 1.
2. 2. The use according to claim 1, wherein the plant is rice, a gramineous plant.
3. 3. The use according to claim 1, wherein the fungal disease is rice blast or false smut.
4. 4. A recombinant plasmid is characterized by comprising a gene encoding a fusion protein OsFID1-Flag shown in SEQ ID NO. 4.
5. 5. The recombinant plasmid according to claim 4, wherein the gene encoding the fusion protein OsFID1-Flag shown in SEQ ID NO.4 is obtained by PCR amplification using rice NIP genomic DNA as a template and primers shown in SEQ ID NO.5 and SEQ ID NO. 6.
6. 6. A genetically engineered bacterium comprising the OsFID gene of claim 1 or the recombinant plasmid of claim 4 or 5.
7. 7. Use of the recombinant plasmid according to claim 4 or 5 for increasing the resistance of plants to fungal diseases.
8. 8. The use according to claim 7, wherein the plant is rice belonging to the Gramineae family and the fungal disease is rice blast or false smut.
9. 9. The use of the genetically engineered bacterium of claim 6 for increasing the resistance of plants to fungal diseases.
10. 10. The use according to claim 9, wherein the plant is rice plants of the Gramineae family and the fungal disease is rice blast or false smut.