CN113512101B - Rhizoctonia solani effector protein and application thereof in rice disease resistance - Google Patents

Abstract

The invention relates to the technical field of protein application, and particularly discloses a Rhizoctonia solani effector protein and application thereof in rice disease resistance, wherein the amino acid sequence of the Rhizoctonia solani effector protein is shown as SEQ ID No.1, and the nucleotide sequence of the Rhizoctonia solani effector protein is shown as SEQ ID No. 2. The Rhizoctonia solani effector protein can obviously improve the disease resistance of rice, and obviously improve the expression of a regulatory disease-resistant gene.

Description

Rhizoctonia solani effector protein and application thereof in rice disease resistance

Technical Field

The invention relates to the technical field of protein application, in particular to a rhizoctonia solani effector protein and application thereof in rice disease resistance.

Background

Sheath blight is a fungal disease caused by infection with rhizoctonia solani. Is one of the most common main diseases of rice, generally early rice is heavier than late rice, so that the rice grains are often not full, the rate of empty husks is increased, and plants can be seriously lodging and withered. It is commonly found in gramineae plants such as rice, wheat and millet.

The rice can occur from seedling stage to heading stage, and is most abundant before and after heading. The disease mainly harms leaf sheaths and leaves, and in severe cases, the disease invades stalks and spreads to ears. The disease spots appear on the leaf sheaths near the water surface initially, are oval and water-soaked, and then gradually spread to the upper part of the plants in a gray green or light brown shape, the disease spots are often combined into irregular shapes, the edges of the disease spots are gray brown, and the center is gray white. Sclerotia formed by entomogenous hyphae on the leaf surface can be seen with naked eyes.

At present, most of the pesticides validamycin, thiophanate-methyl and the like are directly used for preventing and treating the rice sheath blight disease, and pathogenic bacteria easily generate drug resistance, so that the significance of finding a biological means for preventing and treating the sheath blight disease is great.

Disclosure of Invention

In order to solve the technical problems, the invention provides a rhizoctonia solani effector protein and application thereof in rice disease resistance, wherein the rhizoctonia solani effector protein can inhibit rice rhizoctonia solani, remarkably improve the disease resistance of rice and remarkably improve the expression of a regulation and control disease resistance gene.

The invention provides a Rhizoctonia solani effector protein, the amino acid sequence of which is shown as SEQ ID NO.1, and the nucleotide sequence of which is shown as SEQ ID NO. 2.

The invention also provides application of the rhizoctonia solani effector protein in rice disease resistance.

The Rhizoctonia solani effector protein is used for inhibiting Rhizoctonia solani.

The invention also provides a protein solution containing the rhizoctonia solani effector protein.

Further, the concentration of the protein solution is 0.01-0.05. mu. mol/L.

The invention also provides an application of the protein solution in rice disease resistance, and the protein solution is used for inhibiting rhizoctonia solani.

Further, the protein solution is used for soaking the rice seeds until germination.

Further, the concentration of the protein solution is 0.01. mu. mol/L.

Further, the protein solution is used for spraying before tillering of rice.

Further, the concentration of the protein solution was 0.05. mu. mol/L.

Compared with the prior art, the invention has the beneficial effects that:

1. the rhizoctonia solani effector protein provided by the invention can inhibit rice rhizoctonia solani, remarkably improve the disease resistance of rice, regulate and control the improvement of disease resistance gene expression, and provide theoretical and practical bases for the research on rice disease resistance;

2. according to the invention, the rice seeds are soaked in the protein solution containing the Rhizoctonia solani effector protein until the rice seeds germinate, and then the protein solution is sprayed before tillering of the rice, so that the effect of obviously inhibiting the Rhizoctonia solani is achieved, and the disease resistance of the rice is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the effect of Rhizoctonia solani effector protein on disease resistance of indica rice in example 1 of the present invention;

wherein, the graph A shows the leaf rotting appearance and rotting area percentage statistical chart of the indica rice Huahang No. 31 after being treated by the Rhizoctonia solani effector protein;

FIG. B is a statistical chart showing the leaf rotting appearance and rotting area percentage of indica rice Yuejingsi seedling No.2 after being treated with Rhizoctonia solani effector protein;

FIG. C is a statistical chart showing the leaf rotting appearance and rotting area percentage of indica rice Shenliangyou 1173 after being treated with Rhizoctonia solani effector protein;

FIG. 2 shows the effect of the Rhizoctonia solani effector protein on japonica rice disease resistance in example 1 of the present invention;

wherein, the graph A shows the leaf rotting appearance and rotting area percentage statistical chart of the japonica rice after the japonica rice is subjected to the rhizoctonia solani effector protein treatment by the normally-raised 267;

FIG. B shows a statistical chart of leaf rotting appearance and rotting area percentage of japonica 47 treated with Rhizoctonia solani effector protein;

FIG. C shows a statistical chart of leaf rot appearance and rot area percentage of japonica rice 1705 after being treated with Rhizoctonia solani effector protein;

FIG. 3 shows the expression of the defense genes OsPR10a and OsPAL1 after treating rice with Rhizoctonia solani effector protein;

wherein, the graph A shows the expression of the defense gene OsPR10a after the rice is treated by the Rhizoctonia solani effector protein;

panel B shows the expression of the defense gene OsPAL1 after Rhizoctonia solani effector protein treatment of rice.

Detailed Description

The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

The embodiment provides a Rhizoctonia solani effector protein, the amino acid sequence of the Rhizoctonia solani effector protein is shown as SEQ ID No.1, the nucleotide sequence of the Rhizoctonia solani effector protein is shown as SEQ ID No.2, and the gene number is AG1 IA-08777.

SEQ ID NO.1：

MFFNFASIASAAILALPLVGVAANPVPHNARTEWESPKTHKVTVGAWGQLRYNPEFVHAKVGDYIKFEFHPKNHTVTESSFGKPCSAIDGGFRTGFVPVAEEDKHDLPIRKFKVTDEKPHWFYCGQIGHCPAGMVFAVNPPNEGNTFEKFQSMAKESGGK

SEQ ID NO.2：

ATGTTCTTCAACTTTGCTTCTATCGCATCCGCCGCTATTCTCGCTCTTCCTCTCGTTGGGGTTGCTGCCAACCCAGTCCCTCACAATGCACGCACCGAGTGGGAATCTCCCAAGACCCACAAAGTGACTGTTGGTGCGTGGGGACAGCTGCGATACAATCCCGAGTTCGTACATGCCAAGGTTGGAGACTACATCAAATTCGAGTTCCATCCGAAGAATCACACGGTGACCGAGAGCTCGTTCGGCAAGCCATGCAGCGCAATTGATGGAGGATTCCGCACCGGCTTTGTTCCAGTTGCAGAGGAAGACAAGCACGACCTTCCGATACGCAAATTCAAAGTTACTGATGAGAAGCCGCATTGGTTCTACTGCGGCCAGATTGGTCATTGTCCCGCAGGCATGGTGTTTGCCGTCAATCCACCCAACGAGGGAAATACGTTCGAGAAGTTCCAATCAATGGCCAAGGAATCAGGAGGAAAGTAG

Purification of bacterial blight effector protein

1. Adding 500 μ l Ni-NTA suspension into the purification column, washing the column bed with 5ml Buffer B for 4-5 times, and adding soluble protein extract;

the formula of the protein extract comprises: NaH₂PO₄.2H₂O 6.9g，NaCl 17.54g，Imidazole 17g；

2. When the protein extract flows below the upper edge of the column bed, 5ml of Buffer B is added to wash the column twice;

3. when the Buffer B flows below the upper edge of the column bed, 5ml of Buffer C is added to wash the column twice;

Buffer C：Urea 420.42g，NaH₂PO₄.2H₂O 15.6g，Tris 1.21g，Imidazole 0.34g；

4. when the Buffer C in the column bed is drained, adding 1ml of Buffer E, and eluting the target protein;

Buffer E：Urea 90.09g，NaH₂PO₄.2H₂O 3.12g，Tris 0.242g；

5. adding the eluted target protein solution into a dialysis bag, dialyzing in1 × PBS solution for 48h, and replacing the dialysate every 3-4 h;

6. after dialysis, protein concentration was determined by BCA kit, protein purity was checked by SDS-PAGE, and the cells were stored at-20 ℃.

Application of Rhizoctonia solani effector protein

1. 6 rice varieties were selected for testing, including 3 indica: huahang No. 31, Guangdong Yuejingsi No.2, Shenliangyou 1173; 3 japonica rice: tongyu 267, Ningjing 47 and Beijing 1705;

2. effect of Rhizoctonia solani Effector proteins on disease resistance of Rice

Soaking rice seeds in the protein solution with the concentration of 0.01 mu mol/L, germinating, and planting in a conventional manner. Spraying the protein solution with the concentration of 0.05 mu mol/L before tillering of rice, inoculating Rhizoctonia solani AG1IA after 24h, sampling, detecting disease resistance index, and treating with sterile water.

3. Inoculation mode (in vitro leaf inoculation method)

Cutting the part 4-5cm long in the middle of the leaf, spreading in a culture dish containing filter paper for keeping moisture, making the front surface of the leaf closely contact with the surface of the culture medium, taking a PDA agar block with 7mm diameter and hypha at the same radius of the culture dish containing Rhizoctonia solani, placing the block in the middle of the leaf in vitro, and making the side with hypha closely contact with the back axial surface of the leaf. Sealing with a sealing film, culturing at 28 ℃, and repeatedly inoculating 1 strain of leaf tissue in each rice variety, wherein each leaf is inoculated with 1 strain-carrying agar block.

4. Observing the disease resistance of the Rhizoctonia solani effector protein to rice, and detecting the expression condition of disease-resistant genes

In order to explore the influence of spraying effector protein on rice immune signals, the invention detects the expression conditions of two basic defense genes on the northern japonica rice 1705 variety by utilizing a qRT-PCR method. The specific method comprises the following steps:

(1) extraction of Total RNA

The extraction of the total RNA of the plant refers to the description of an ultra-pure RNA extraction kit (Kangji biological company, CW0597), and comprises the following specific steps:

s1, sample treatment

S1.1, organization: fully grinding 30-50mg of tissue in liquid nitrogen, adding 1ml of Buffer RLT, or adding 1ml of Buffer RLT into a tissue sample and then homogenizing;

note that: the sample volume does not exceed 10% of the Buffer RLT volume.

S1.2, cell monolayer culture: the culture medium was aspirated off, and an appropriate amount of Buffer RLT was added every 10cm²Adding 1ml Buffer RLT;

s1.3, cell suspension: cells were collected by centrifugation. Every 5X 10⁶Cells were added to 1ml Buffer RLT;

and S2, adding Buffer RLT into the sample, and repeatedly blowing for several times to fully crack the sample. Standing at room temperature for 5min to completely separate protein nucleic acid complex;

s3, adding chloroform according to the proportion that 200 mul of chloroform is added into 1ml of Buffer RLT, covering a tube cover, violently shaking for 15 seconds, and standing for 2min at room temperature;

s4, 4 ℃, 12,000rpm (. about.13,400 Xg) for 10min, at which time the sample was divided into three layers: red organic phase, middle layer and upper colorless aqueous phase, wherein RNA is mainly in the upper aqueous phase, and the upper aqueous phase is transferred to a new RNase-Free centrifuge tube;

s5, adding equal volume of 70% ethanol (prepared by RNase-free water) into the obtained aqueous solution, and reversing and uniformly mixing;

s6, the whole amount of the solution obtained in the above step was added to an adsorption column (Spin column RM) packed in a Collection Tube (Collection Tube 2 ml). If the solution can not be added at one time, the solution can be transferred for many times. Centrifuging at 12,000rpm for 20 s, pouring out waste liquid in the collecting tube, and replacing the adsorption column in the collecting tube;

s7, adding 350 μ l Buffer RW1 into the adsorption column, centrifuging at 12,000rpm for 20S, pouring the waste liquid in the collection tube, and replacing the adsorption column into the collection tube;

s8, preparing DNase I mixed solution: taking 52 ul RNase-Free Water, adding 8 ul 10 × Reaction Buffer and 20 ul DNase I (1U/ul), mixing evenly, and preparing Reaction liquid with the final volume of 80 ul;

s9, directly adding 80 μ l of DNase I mixed solution into the adsorption column, and incubating at 20-30 deg.C for 15 min;

s10, adding 350 μ l Buffer RW1 into the adsorption column, centrifuging at 10,000rpm for 1min, discarding the waste liquid, and replacing the adsorption column into the collection tube;

s11, adding 500. mu.l Buffer RW2 (checking whether absolute ethyl alcohol is added before use) into the adsorption column, centrifuging at 12,000rpm for 20S, pouring out waste liquid in the collection tube, and replacing the adsorption column into the collection tube;

s12, repeating the step 11;

s13, centrifuging at 12,000rpm for 2min, and pouring off the waste liquid in the collecting pipe. Placing the adsorption column at room temperature for several minutes to completely dry;

note that: the aim of this step is to remove the residual ethanol in the adsorption column, which affects the subsequent enzymatic reactions (digestion, PCR, etc.);

s14, placing the adsorption column into a new RNase-Free centrifuge tube (Collection tube 1.5ml), adding 30-50 μ l RNase-Free Water into the middle part of the adsorption column, standing at room temperature for 1min, centrifuging at 12,000rpm for 1min, collecting RNA solution, and storing RNA at-70 ℃ to prevent degradation;

note that: a. the volume of RNase-Free date should not be less than 30 mul, and the recovery rate is influenced by too small volume;

b. if the RNA yield is to be increased, step 14 may be repeated with 30-50. mu.l of a new RNase-Free Water;

c. if the RNA concentration is to be increased, the resulting solution may be re-applied to the adsorption column and step 14 repeated.

(2) Reverse transcription of RNA

The rice RNA inversion system is described in the specification of M-MLV reverse transcriptase of Invitrogen corporation, and comprises the following steps:

s1, adding the following components to a nuclease-free microcentrifuge tube:

Oligo dT(100μM)，1μl；

2.5mM dNTP，4μl；

Total RNA，2μg；

RNase-Free water，Up to 12μl；

s2, heating the mixture at 65 deg.C for 5min, and rapidly cooling on ice for 2 min. After a short centrifugation, the following components were added:

5 Xfirst Strand Synthesis buffer, 4. mu.l;

DTT(0.1M)，2μl；

RNase inhibitor, 1. mu.l;

s3, mixing the components gently, and incubating at 37 ℃ for 2 min;

s4, adding 1 mu l M-MLV reverse transcriptase at room temperature, gently sucking, uniformly mixing, and incubating for 50min at 37 ℃;

s5, heating at 70 ℃ for 15min to terminate the reaction.

(3) Fluorescent quantitative PCR detection of rice PR gene expression

By using

Premix Ex Taq^TMFluorescent quantitative PCR kit (Takara, RR420A), the reaction system is as follows:

reaction procedure: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, and extension at 60 ℃ for 31s for 40 cycles.

By using 2^-ΔΔCtThe method calculates the gene expression (Livak and Schmittgen,2001) and the relative expression quantity of the target gene is 2^-ΔΔCtWherein-delta Ct ═ - (delta Ct, a-delta Ct, b) (Ct: fluorescence threshold value; a: target gene; b: internal referenceA gene).

In the invention, OsActin1(Os03g0718100) is used as an internal reference gene, and OsActin1, OsPR10a (Os12g0555500) and OsPAL1(Os02g0627100) are used as primers for qRT-PCR, and specific primer information is as follows:

the primer of OsActin1 comprises OsActin1-qRT-F with the gene sequence shown as SEQ ID NO.3 and OsActin1-qRT-R with the gene sequence shown as SEQ ID NO. 4; the primer of the OsPR10a comprises OsPR10a-qRT-F with the gene sequence shown as SEQ ID NO.5 and OsPR10a-qRT-R with the gene sequence shown as SEQ ID NO. 6; the primer of the OsPAL1 comprises OsPAL1-qRT-F with the gene sequence shown as SEQ ID NO.7 and OsPAL1-qRT-R with the gene sequence shown as SEQ ID NO. 8;

SEQ ID NO.3：TCCATCTTGGCATCTCTCAG

SEQ ID NO.4：GTACCCGCATCAGGCATCTG

SEQ ID NO.5：AGCTCAAGTCACACTCGACG

SEQ ID NO.6：GCCATCCACGATGTCCTTCT

SEQ ID NO.7：CTCGAGTGCCTCAAGGAGTG

SEQ ID NO.8：GCCTCCACACTCCACTGTTA。

third, experimental results

1. As shown in FIGS. 1-2, 3 indica (FIG. 1) and japonica (FIG. 2) groups were selected for evaluation of disease resistance. After the protein solution is treated, the resistance of different varieties of rice to banded sclerotial blight is obviously improved, the disease resistance of different varieties of rice on leaves is obviously reduced, the disease area is obviously reduced compared with that of a control group, the disease resistance of the rice is obviously improved, and the expression of disease-resistant genes is obviously improved.

2. As shown in FIG. 3, the expression of the defense genes OsPR10a (Os12g0555500) and OsPAL1(Os02g0627100) was significantly induced to increase by 50-90 times after spraying the effector protein solution for 24 hours.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

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Claims (10)

1. A Rhizoctonia solani effector protein is characterized in that the amino acid sequence of the Rhizoctonia solani effector protein is shown as SEQ ID No.1, and the nucleotide sequence is shown as SEQ ID No. 2.

2. Use of the Rhizoctonia solani effector protein of claim 1 for combating rice diseases.

3. The use of a Rhizoctonia solani effector protein against diseases of rice as claimed in claim 2, wherein said Rhizoctonia solani effector protein is used to inhibit Rhizoctonia solani.

4. A protein solution comprising the rhizoctonia solani effector protein of claim 1.

5. The protein solution comprising a Rhizoctonia solani effector protein according to claim 4, wherein the concentration of the protein solution is 0.01 to 0.05 μmol/L.

6. Use of the protein solution of claim 5 for combating rice disease, wherein the protein solution is used to inhibit Rhizoctonia solani.

7. The use of the protein solution of claim 6 for disease resistance in rice, wherein the protein solution is used to soak rice seeds until germination.

8. The use of the protein solution of claim 7 for disease resistance in rice, wherein the protein solution has a concentration of 0.01 μmol/L.

9. The use of the protein solution according to claim 7 for disease resistance of rice, wherein the protein solution is sprayed before tillering of rice.

10. The use of the protein solution of claim 9 for disease resistance in rice, wherein the protein solution has a concentration of 0.05 μmol/L.

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