CN113666994B - Ustilago oryzae effector protein and application thereof in disease resistance of rice - Google Patents
Ustilago oryzae effector protein and application thereof in disease resistance of rice Download PDFInfo
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- CN113666994B CN113666994B CN202110989605.8A CN202110989605A CN113666994B CN 113666994 B CN113666994 B CN 113666994B CN 202110989605 A CN202110989605 A CN 202110989605A CN 113666994 B CN113666994 B CN 113666994B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
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Abstract
The invention relates to the technical field of protein application, and in particular discloses false smut germ effect protein and application thereof in disease resistance of rice, wherein the amino acid sequence of the false smut germ effect protein is shown as SEQ ID NO.1, and the nucleotide sequence of the false smut germ effect protein is shown as SEQ ID NO. 2. The ustilaginoidea virens effector protein can obviously improve the disease resistance of rice, and can obviously improve the expression of regulatory disease resistance genes.
Description
Technical Field
The invention relates to the technical field of protein application, in particular to ustilaginoidea virens effector protein and application thereof in rice disease resistance.
Background
False smut, green smut, cereal smut and powdery mildew are commonly called as "high yield fruit". The disease only occurs in the ears of rice, and is harmful to some grains. The broken grain has gradually enlarged hypha blocks, and the inner and outer glumes are split to expose light yellow blocks, namely sporophores, and then are wrapped on two sides of the inner and outer glumes to form black green, and a layer of film is initially wrapped, and then is broken, so that dark green powder, namely chlamydospores of bacteria, is scattered, and black flat sclerotium is generated on two sides of the chlamydospores, and is easy to fall off when being blown by wind and rain.
The existing method for preventing false smut is generally a mode of manually avoiding diseases Tian Liuchong, deeply ploughing and burying sclerotium, removing and destroying the disease particles together during disease onset, or improving the fertilization technology. However, these methods are time-consuming and laborious, and have poor effects, and it is difficult to control the occurrence of false smut, so it is important to find a way to fundamentally prevent false smut.
Disclosure of Invention
In order to solve the technical problems, the invention provides the ustilaginoidea virens effector protein and the application thereof in rice disease resistance, wherein the ustilaginoidea virens effector protein can inhibit ustilaginoidea virens, obviously improve the rice disease resistance and obviously improve the regulation and control of the expression of disease resistance genes.
The invention provides a false smut germ 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 ustilaginoidea virens effector protein in disease resistance of rice.
The ustilaginoidea virens effector protein is used for inhibiting ustilaginoidea virens.
The invention also provides a protein solution containing the ustilaginoidea virens effector protein.
Further, the concentration of the protein solution is 0.01-0.05 mu mol/L.
The invention also provides application of the protein solution in disease resistance of rice.
Further, the protein solution is used for inhibiting ustilaginoidea virens.
Further, the protein solution is used for soaking rice seeds until sprouting, and the concentration of the protein solution is 0.01 mu mol/L.
Further, the protein solution is used for spraying before heading of rice, and the concentration of the protein solution is 0.05 mu mol/L.
Further, the protein solution is used for spraying in the flowering phase of rice, and the concentration of the protein solution is 0.05 mu mol/L.
Compared with the prior art, the invention has the beneficial effects that:
1. the ustilaginoidea virens effector protein provided by the invention can inhibit ustilaginoidea virens, obviously improve the disease resistance of rice, regulate and control the improvement of the expression of disease resistance genes, and provide theoretical and practical basis for the study of disease resistance of rice;
2. according to the invention, the protein solution containing the ustilaginoidea virens effector protein is soaked in the rice seeds until the seeds germinate, and then the rice seeds are sprayed before heading and in the flowering phase, so that the effect of obviously inhibiting ustilaginoidea virens is achieved, and the disease resistance of rice is improved.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the effect of Ustilago oryzae effector protein on disease resistance of flower 11 in rice in example 1 of the present invention;
wherein, the graph A shows the phenotype of the middle flower 11 after being treated by the ustilaginoidea virens effector protein;
FIG. B shows the change in the number of Ustilago virens after treatment of flowers 11 with Ustilago virens effector proteins;
panel C shows the change in incidence of flower 11 after treatment with ustilaginoidea virens effector protein;
FIG. 2 shows the expression of the defense genes OsPR10a and OsPAL1 after treatment of rice middle flower 11 with Ustilago oryzae effector protein;
wherein, the graph A shows the expression condition of a defense gene OsPR10a after rice is treated by the ustilaginoidea virens effector protein;
FIG. B shows the expression of the defense gene OsPAL1 after treatment of rice with Ustilago oryzae effector protein.
Detailed Description
The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified, and materials, reagents, etc. used in the examples described below are commercially available.
Example 1
The embodiment provides the ustilaginoidea virens effector protein, the amino acid sequence of the ustilaginoidea virens effector protein is shown as SEQ ID NO.1, the nucleotide sequence of the ustilaginoidea virens effector protein is shown as SEQ ID NO.2, and the gene number of the ustilaginoidea virens effector protein is UVR_00843.
SEQ ID NO.1：
MLSNTLVLAALGLAALASAQEPCGLKVAPCPTDKTCVPNAGCPNPKLCPGTCRFKNKYDSCGGKTVSPRSCKPGFECRDDPRLPESCGLACDVPGICLPKEPKRCGGFAGFACPKGLYCYYGPLTGCDPKTTSDCMGICL
SEQ ID NO.2：
ATGCTGTCCAACACCCTCGTTCTTGCTGCGCTCGGCCTCGCCGCCTTGGCCTCGGCCCAGGAACCGTGCGGTCTCAAGGTCGCCCCCTGCCCAACCGACAAGACCTGCGTTCCCAACGCCGGCTGCCCCAATCCCAAGCTGTGCCCGGGGACATGCCGCTTCAAGAACAAGTATGATTCCTGCGGCGGAAAGACCGTCTCGCCGCGCAGCTGCAAGCCTGGCTTTGAGTGTCGGGACGACCCGCGCCTGCCCGAGTCCTGCGGCCTGGCTTGCGATGTCCCGGGCATTTGCTTGCCCAAGGAGCCGAAGCGGTGCGGTGGCTTCGCCGGCTTTGCTTGTCCCAAAGGGCTGTACTGCTACTATGGGCCTTTGACTGGGTGCGATCCCAAGACTACGTCGGATTGCATGGGAATATGCCTGTAA
1. Purification of ustilaginoidea virens effector protein
1. Adding 500 μl of 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 is as follows: naH (NaH) 2 PO 4 .2H 2 O 6.9g，NaCl 17.54g，Imidazole 17g；
2. When the protein extract flows below the upper edge of the column bed, 5ml Buffer B is added to wash the column twice;
3. when 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，NaH 2 PO 4 .2H 2 O 15.6g，Tris 1.21g，Imidazole 0.34g；
4. when the Buffer C in the column bed runs out, adding 1ml Buffer E, and eluting the target protein;
Buffer E：Urea 90.09g，NaH 2 PO 4 .2H 2 O 3.12g，Tris 0.242g；
5. adding the eluted target protein solution into a dialysis bag, dialyzing in1 XPBS solution for 48 hours, and changing the dialyzate every 3-4 hours;
6. after dialysis, protein concentration was determined by BCA kit, protein purity was checked by SDS-PAGE, and stored at-20 ℃.
2. Application of ustilaginoidea virens effector protein
1. Selecting middle rice flower 11 as experimental rice;
2. influence of Ustilago oryzae effector protein on disease resistance of rice
The flower 11 seeds in rice are soaked in the protein solution with the concentration of 0.01 mu mol/L, germinated and planted in a conventional mode. Spraying the protein solution with the concentration of 0.05 mu mol/L before heading and in the flowering period of the rice, inoculating ustilago oryzae (Ustilaginoidea virens PJ) after 24 hours, sampling for disease resistance index detection, and treating a control group by using sterile water.
3. Inoculation mode
(1) Activating the ustilaginoidea virens strain and the wild strain on a PSA plate, and culturing for 10 days in a 28 ℃ incubator;
(2) Cutting fungus blocks to 100mL PS culture medium, and culturing at 28deg.C with shaking table at 180rpm for 5-7 days;
(3) Breaking mycelium in ustilaginoidea virens culture solution to form spore mycelium mixed solution, and regulating spore concentration to 1×10 with liquid PS culture medium 6 individual/mL;
(4) Selecting rice ears 5-7 days before heading, injecting 1mL of bacterial liquid into the rice ears by using an injector, and repeating 10 times for each sample;
(5) Statistical phenotype after about 30 days of inoculation: the number and incidence of false smut and the like.
4. Observing the disease resistance of rice by ustilaginoidea virens effector protein and detecting the expression condition of disease resistance genes
In order to explore the influence of spraying effector proteins on rice immune signals, the invention utilizes a qRT-PCR method to detect the expression condition of two basic defense genes on the Zhonghua 11 variety. The specific method comprises the following steps:
(1) Extraction of Total RNA
The extraction of total RNA of plants is described in reference to the specification of an ultrapure RNA extraction kit (Kangji BioCo., CW 0597), and comprises the following steps:
s1, sample treatment
S1.1, tissue: adding 1ml Buffer RLT after fully grinding 30-50mg of tissue in liquid nitrogen, or adding 1ml Buffer RLT in a tissue sample, and homogenizing;
note that: the sample volume is no more than 10% of the Buffer RLT volume.
S1.2, monolayer culture cells: sucking the culture solution, adding appropriate amount of Buffer RLT, and adding the Buffer RLT per 10cm 2 1ml Buffer RLT was added;
s1.3, cell suspension: cells were collected by centrifugation. Every 5×10 6 1ml Buffer RLT was added to the cells;
s2, repeatedly blowing for several times after adding Buffer RLT into the sample, so that the sample is fully cracked. Standing at room temperature for 5min to completely separate the protein nucleic acid complex;
s3, adding chloroform in a proportion of 200 mu l of chloroform per 1ml of Buffer RLT, covering a tube cover, shaking vigorously for 15 seconds, and standing at room temperature for 2 minutes;
s4, centrifugation is carried out at 12,000rpm (-13,400Xg) for 10min at 4℃at which time the sample is divided into three layers: a red organic phase, a middle layer and an upper colorless aqueous phase, wherein RNA is mainly in the upper aqueous phase, and the upper aqueous phase is phase-shifted into a new RNase-Free centrifuge tube;
s5, adding 70% ethanol (prepared by RNase-free water) with equal volume into the obtained aqueous phase solution, and mixing the mixture upside down;
s6, adding all the solution obtained in the previous step into an adsorption column (Spin column RM) filled into a Collection Tube (Collection Tube 2 ml). If the solution can not be added at one time, the solution can be transferred into the container for multiple times. Centrifuging at 12,000rpm for 20 seconds, pouring out the waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again;
s7, adding 350 mul Buffer RW1 into the adsorption column, centrifuging at 12,000rpm for 20 seconds, pouring out waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again;
s8, preparing DNase I mixed solution: mu.l of RNase-Free Water was taken, and 8. Mu.l of 10 Xreaction Buffer and 20. Mu.l of DNase I (1U/. Mu.l) were added thereto and mixed well to prepare a Reaction solution having a final volume of 80. Mu.l;
s9, directly adding 80 μl DNase I mixed solution into the adsorption column, and incubating for 15min at 20-30deg.C;
s10, adding 350 μl Buffer RW1 into the adsorption column, centrifuging at 10,000rpm for 1min, discarding the waste liquid, and putting the adsorption column back into the collection tube;
s11, adding 500 μl Buffer RW2 (whether absolute ethyl alcohol is added before use or not is checked) into the adsorption column, centrifuging at 12,000rpm for 20 seconds, pouring out waste liquid in the collection tube, and putting the adsorption column back into the collection tube again;
s12, repeating the step 11;
s13, centrifuging at 12,000rpm for 2min, and pouring out the waste liquid in the collecting pipe. Placing the adsorption column at room temperature for several minutes to thoroughly dry;
note that: the purpose of this step is to remove the residual ethanol from the column, which could affect subsequent enzymatic reactions (cleavage, PCR, etc.);
s14, placing the adsorption column in a new RNase-Free centrifuge tube (Collection tube 1.5 ml), adding 30-50 μl 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 preserving RNA at-70deg.C to prevent degradation;
note that: a. the volume of RNase-Free Wate should not be less than 30 μl, and the recovery rate is affected by too small volume;
b. if the RNA yield is to be increased, step 14 may be repeated with 30-50. Mu.l of the new RNase-Free Water;
c. if the RNA concentration is to be increased, the resulting solution may be re-loaded into the adsorption column and step 14 repeated.
(2) Reverse transcription of RNA
The rice RNA inversion system is described in the following manner by referring to the M-MLV reverse transcriptase of Invitrogen company:
s1, adding the following components into a nuclease-free microcentrifuge tube:
Oligo dT(100μM)，1μl；
2.5mM dNTP，4μl；
Total RNA，2μg；
RNase-Free water，Up to 12μl；
s2, after the mixture is heated at 65 ℃ for 5min, the mixture is quickly placed on ice for cooling for 2min. After brief centrifugation, the following components were added:
5 Xfirst Strand Synthesis buffer, 4. Mu.l;
DTT(0.1M)，2μl；
RNase inhibitor, 1 μl;
s3, lightly mixing the components, and incubating for 2min at 37 ℃;
s4, adding 1 mu l M-MLV reverse transcriptase at room temperature, gently sucking and beating, mixing uniformly, and incubating for 50min at 37 ℃;
s5, heating at 70 ℃ for 15min to terminate the reaction.
(3) Fluorescent quantitative PCR (polymerase chain reaction) detection of rice PR (PR) gene expression
By usingPremix Ex Taq TM Fluorescent quantitative PCR kit (Takara, RR 420A), the reaction system is as follows:
the reaction procedure: pre-denaturation at 95℃for 30s, denaturation at 95℃for 5s, extension at 60℃for 31s for 40 cycles.
By 2 -ΔΔCt Method calculates the gene expression (Livak and Schmittgen, 2001), the relative expression level of the target gene=2 -ΔΔCt Wherein- ΔΔct= - (Δct, a- Δct, b) (Ct: fluorescence threshold; a: gene of interest; b: reference gene).
In the invention, osActin1 (Os 03g 0718100) is used as an internal reference gene, and OsActin1, osPR10a (Os 12g 0555500) and OsPAL1 (Os 02g 0627100) are used as primers for qRT-PCR, wherein specific primer information is as follows:
the primers of OsActin1 comprise OsActin1-qRT-F with a gene sequence shown as SEQ ID NO.3 and OsActin1-qRT-R with a gene sequence shown as SEQ ID NO. 4; the primers of the OsPR10a comprise an OsPR10a-qRT-F with a gene sequence shown as SEQ ID NO.5 and an OsPR10a-qRT-R with a gene sequence shown as SEQ ID NO. 6; the primers of the OsPAL1 comprise an OsPAL1-qRT-F with a gene sequence shown as SEQ ID NO.7 and an OsPAL1-qRT-R with a 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。
3. experimental results
1. As shown in figure 1, we find that the disease resistance of rice is obviously improved, the quantity and incidence rate of false smut are greatly reduced, and the expression of disease resistance genes is obviously improved as apparent from the phenotype after the protein solution is treated.
2. As a result, as shown in FIG. 2, the expression of the defense genes OsPR10a (Os 12g 0555500) and OsPAL1 (Os 02g 0627100) was significantly induced to increase 7-17-fold after 24 hours of spraying of effector protein before heading.
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. It is therefore intended that the following claims be interpreted as including the 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 modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Sequence listing
<110> Shenyang agricultural university
<120> an effector protein of ustilaginoidea virens and application thereof in disease resistance of rice
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 140
<212> PRT
<213> Synthesis
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Met Leu Ser Asn Thr Leu Val Leu Ala Ala Leu Gly Leu Ala Ala Leu
1 5 10 15
Ala Ser Ala Gln Glu Pro Cys Gly Leu Lys Val Ala Pro Cys Pro Thr
20 25 30
Asp Lys Thr Cys Val Pro Asn Ala Gly Cys Pro Asn Pro Lys Leu Cys
35 40 45
Pro Gly Thr Cys Arg Phe Lys Asn Lys Tyr Asp Ser Cys Gly Gly Lys
50 55 60
Thr Val Ser Pro Arg Ser Cys Lys Pro Gly Phe Glu Cys Arg Asp Asp
65 70 75 80
Pro Arg Leu Pro Glu Ser Cys Gly Leu Ala Cys Asp Val Pro Gly Ile
85 90 95
Cys Leu Pro Lys Glu Pro Lys Arg Cys Gly Gly Phe Ala Gly Phe Ala
100 105 110
Cys Pro Lys Gly Leu Tyr Cys Tyr Tyr Gly Pro Leu Thr Gly Cys Asp
115 120 125
Pro Lys Thr Thr Ser Asp Cys Met Gly Ile Cys Leu
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<210> 2
<211> 423
<212> DNA
<213> Synthesis
<400> 2
atgctgtcca acaccctcgt tcttgctgcg ctcggcctcg ccgccttggc ctcggcccag 60
gaaccgtgcg gtctcaaggt cgccccctgc ccaaccgaca agacctgcgt tcccaacgcc 120
ggctgcccca atcccaagct gtgcccgggg acatgccgct tcaagaacaa gtatgattcc 180
tgcggcggaa agaccgtctc gccgcgcagc tgcaagcctg gctttgagtg tcgggacgac 240
ccgcgcctgc ccgagtcctg cggcctggct tgcgatgtcc cgggcatttg cttgcccaag 300
gagccgaagc ggtgcggtgg cttcgccggc tttgcttgtc ccaaagggct gtactgctac 360
tatgggcctt tgactgggtg cgatcccaag actacgtcgg attgcatggg aatatgcctg 420
taa 423
<210> 3
<211> 20
<212> DNA
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<400> 3
tccatcttgg catctctcag 20
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gtacccgcat caggcatctg 20
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agctcaagtc acactcgacg 20
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gccatccacg atgtccttct 20
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<211> 20
<212> DNA
<213> Synthesis
<400> 7
ctcgagtgcc tcaaggagtg 20
<210> 8
<211> 20
<212> DNA
<213> Synthesis
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gcctccacac tccactgtta 20
Claims (5)
1. The application of the ustilaginoidea virens effector protein in rice disease resistance is characterized in that the amino acid sequence of the ustilaginoidea virens effector protein is shown as SEQ ID NO.1, the nucleotide sequence of the ustilaginoidea virens effector protein is shown as SEQ ID NO.2, and the ustilaginoidea virens effector protein is used for inhibiting ustilaginoidea virens.
2. The application of the protein solution of the ustilaginoidea virens effector protein in rice disease resistance is characterized in that the amino acid sequence of the ustilaginoidea virens effector protein is shown as SEQ ID NO.1, the nucleotide sequence of the ustilaginoidea virens effector protein is shown as SEQ ID NO.2, and the protein solution is used for inhibiting ustilaginoidea virens.
3. The use of a protein solution of ustilaginoidea virens effector protein according to claim 2 for rice disease resistance, wherein said protein solution is used for soaking rice seeds until germination.
4. The use of a protein solution of ustilaginoidea virens effector protein according to claim 3 for rice disease resistance, wherein said protein solution is applied to rice pre-heading spray.
5. The use of a protein solution of ustilaginoidea virens effector protein according to claim 4 for rice disease resistance, wherein said protein solution is used for flowering phase spraying of rice.
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| CN110669116A (en) * | 2019-09-18 | 2020-01-10 | 中国水稻研究所 | Pathogenic factor for negatively regulating and controlling ustilaginoidea virens spore production, gene and application |
| CN110885849A (en) * | 2019-12-05 | 2020-03-17 | 沈阳农业大学 | Recombinant vector, host cell and application of Ustilaginoidea virens effector protein |
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