CN115428684A - Method for compensating reduction of blast resistance of OsLOX3 knockout rice strain and verification method and application - Google Patents
Method for compensating reduction of blast resistance of OsLOX3 knockout rice strain and verification method and application Download PDFInfo
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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
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
The invention relates to the technical field of agricultural biological genetic engineering, in particular to a method for compensating reduction of blast resistance of an OsLOX3 knockout rice plant and a verification method and application, wherein the OsLOX3 knockout rice plant growing to a three-leaf one-heart stage is sprayed with 15-20 mL of exogenous phenylalanine solution with the concentration of 100ppm according to 100 rice plants, and the contents of linolenic acid and linoleic acid are measured by sampling for 24 hours; when the OsLOX3 knockout rice plant growing to the three-leaf one-heart stage is infected with rice blast fungi for 36-48 h, exogenous phenylalanine solution is obtained in the manner; sampling at 36h,48h,72h and 96h after spraying and inoculating rice blast fungi, and determining JA response, defense, SA signals, active oxygen and POD gene relative expression quantity; disease investigation is carried out after the rice blast fungi is inoculated for 168 hours after spraying. The method makes up the problem that the blast resistance of the OsLOX3 knock-out rice plant is reduced, so that the control time is more accurate, the method is safe and effective, and important measures are provided for the utilization and protection of rice germplasm resources.
Description
Technical Field
The invention relates to the technical field of agricultural biological genetic engineering, in particular to a method for compensating the reduction of blast resistance of an OsLOX3 knockout rice plant and application thereof.
Background
The plant hormone and other active small molecular substances play an important role in regulating and controlling plant growth and development and responding to environmental stress, and are the 'watchdog' which responds firstly when a plant responds to environmental stimuli, so that the plant can distribute limited resources to respond to the most life-threatening stress, and various stress responses are balanced, thereby ordering complex processes, jasmonic Acid (JA) is used as a core component in a plurality of plant hormones and participates in a plurality of plant hormone signals to jointly regulate plant growth and development and stress responses, for example, JA can enhance the resistance of rice to plant pathogenic fungi, bacteria and viruses from multiple aspects. Research shows that OsLOX3 is mainly involved in converting linolenic acid into 13-hydroperoxide, and linolenic acid is a synthetic precursor of jasmonic acid. Therefore, osLOX3 is not only involved in jasmonic acid synthesis but also in defense response during pathogen infection. In order to clarify the resistance of an OsLOX3 knockout rice strain and the function of OsLOX3 in JA synthesis and defense response, a regulation mechanism of OsLOX 3-mediated JA synthesis and plague resistance is further disclosed, and an OsLOX3 knockout rice strain is obtained by knocking out OsLOX3 gene in rice by using a CRISPR/Cas 9-mediated gene editing technology, and a great deal of research shows that OsLOX3 is taken as a defense related gene, the rice resistance is reduced after knockout, but the rice quality is obviously improved, mainly manifested in that the content of linolenic acid and linoleic acid in the OsLOX3 knockout rice strain is high, the germination rate of the OsLOX3 knockout rice strain is obviously improved, and meanwhile, transcriptional component analysis finds that the gene down regulation in the biosynthesis/metabolism/catalysis process of aromatic amino acid in the OsLOX3 knockout rice strain means that the content of aromatic amino acid such as phenylalanine and the like in the OsLOX3 knockout rice strain is reduced, and the amino acid plays an important role in plant resistance.
The invention provides a method for compensating the reduction of the resistance of an OsLOX3 knockout rice plant and a verification method, namely exogenous spraying phenylalanine (Phe). Although many methods for improving the resistance of exogenous phenylalanine-treated plants can effectively reduce the degree of plant diseases, no method for improving the blast resistance of OsLOX3 knockout transgenic rice has been reported for phenylalanine-treated rice.
Disclosure of Invention
The invention aims to provide a method for compensating the reduction of blast resistance of an OsLOX3 knockout rice plant and application thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention adopts the technical scheme that: the method for compensating the reduction of the blast resistance of the OsLOX3 knockout rice plant comprises the following steps:
(1) OsLOX3 knockout rice plants growing to a three-leaf one-heart stage are sprayed with 15-20 mL of exogenous phenylalanine solution with the concentration of 100ppm according to 100 rice plants, and the contents of linolenic acid and linoleic acid are sampled and determined after 24 hours;
(2) Spraying 15-20 mL of exogenous phenylalanine solution with the concentration of 100ppm according to 100 rice plants when OsLOX3 knockout rice plants growing to the three-leaf one-heart stage infect rice blast fungi for 36-48 h;
(3) Sampling at 36h,48h,72h and 96h after spraying in the step (2) and inoculating rice blast, and determining relative expression amounts of JA response genes, defense genes, SA signal genes, active oxygen genes and POD genes;
(4) And (3) after spraying in the step (2), inoculating the rice blast fungi for 168 hours, and then carrying out disease investigation.
Further, in the step (2), the time for infecting the rice blast fungi is 48 hours.
Further, in the step (2), the strain of Pyricularia oryzae is 95234I-1b.
Further, the concentration of the spore suspension of the rice blast fungus strain is 1X 10 5 One per mL.
And (3) rice blast bacterium culture and spore suspension preparation: activating the magnaporthe grisea strain on a PDA solid culture medium, putting the magnaporthe grisea strain in a constant-temperature incubator at 28 ℃ for activation culture for 4-5 days, taking activated mycelium blocks on the PDA culture medium by using a puncher, and carrying out subculture in the constant-temperature incubator at 28 ℃ for 4-5 days; using a puncher to punch fungus blocks with good edge activity of a PDA culture medium into a PDB liquid culture medium, placing the PDB liquid culture medium in a constant-temperature shaking table at 28 ℃ for 3-4 days, sucking 500uL of hypha culture fluid by a pipettor to produce spores on a prune juice culture medium, uniformly coating a coating rod, placing the obtained product in a constant-temperature illumination incubator at 28 ℃ for 3-4 days in the dark, then alternately culturing for 5-6 days in the dark and in the light/light alternating 12h, scraping surface hypha when the hypha on the prune juice culture medium is changed from white to black, flushing the spores with sterile water to prepare spore suspension, and using a blood counting plate to adjust the spore concentration of the suspension to be 1 x 10 under a microscope 5 One per mL.
Further, in the steps (1) and (2), the exogenous phenylalanine solution is a phenylalanine water solution containing 0.1% tween.
Further, the JA response gene is OsbHLH35, the defense gene is OsPR1a, the SA signal gene is OsWRKY45, the active oxygen group is OsRBohB, and the POD gene is OsPOX1.
In the invention, an OsLOX3 knockout rice plant is constructed by using a CRISPR/Cas9 mediated gene editing technology, wild rice Ilmibyeo is selected as a control, and the Ilmibyeo rice plant is a disease-resistant rice plant which can be infected by a rice blast strain 95234I-1b.
In the method, the sampling time points of an OsLOX3 knockout rice plant experimental group and an Ilmibyeo rice plant control group after exogenous phenylalanine is sprayed for 36h are 36h,48h and 72h after the rice blast fungus is inoculated; the sampling time points after spraying exogenous phenylalanine for 48h are 48h,72h and 96h after inoculating the Magnaporthe grisea. The time for disease investigation after spraying exogenous phenylalanine is 168 hours after inoculating rice blast fungi in both the experimental group and the control group.
Phenylalanine (Phe) is a major component of protein biosynthesis and of many phenylalanines, and in plants, the biosynthesis of phenylalanine begins with the conversion of the end product of the oxalate pathway (chelate) to prephenate by a chelate mutase. Phenylalanine is the major phenolic precursor, phenolic compounds have been shown to be an important factor in plants against fungal infections, accumulation of phenolic compounds by plants may be a means of passive defense, and may also be in the systemic response system induced defense against pathogen attack, the intensity of the response being partly due to the provision of the major precursor l-Phenylalanine (PHE), studies have shown that systemic application of phenolic precursors can enhance the resistance of plants to pests and thereby promote the production of phenolic substances.
The invention refines the method for improving rice blast resistance by externally spraying phenylalanine to treat rice, according to the infection process that the OsLOX3 knockout rice plant and the wild rice Ilmibyeo are infected by rice blast germs, the OsLOX3 knockout rice plant and the leaf of the wild rice Ilmibyeo are inoculated by the rice blast germs when the rice blast germs grow for 14 days, 100ppm phenylalanine is externally sprayed when the rice blast germs are inoculated for 36h and 48h respectively, rice blast disease investigation is carried out when the rice blast germs are inoculated for 168h, meanwhile, the disease index is counted, 100ppm phenylalanine effectively improves the resistance of the OsLOX3 knockout rice plant to the rice blast germs, wherein the rice blast disease control effect of the rice when the rice blast germs are treated by phenylalanine and inoculated for 48h is the best, the key time point that the phenylalanine effectively controls the rice blast is further determined, the difficult problem that the disease resistance of local rice varieties with excellent agronomic characters is reduced due to resistance gene failure and is eliminated in production can be solved, and important measures can be provided for utilization and protection of rice germplasm resources. In addition, the phenylalanine is generated in the metabolic process of the plant, and the proper amount of phenylalanine has no toxicity to the plant, does not cause residue and environmental pollution, and achieves the ecological environment safety of farmland and the safety of consumers.
The beneficial technical effects of the invention are as follows:
the method of the invention is to spray exogenous phenylalanine water solution, phenylalanine is the main component of protein biosynthesis and is also the main component of a plurality of phenylalanine compounds, phenylalanine is the main phenol precursor, the phenol compounds are proved to be the important factors for plants to resist fungal infection, the accumulation of the phenol compounds by the plants is a means of passive defense and is also in a systemic reaction system induced defense system to pathogen attack, and the strength of the reaction is partly due to the supply of the main precursor l-phenylalanine. The purpose of the Tween-20 in the components of the exogenous phenylalanine aqueous solution is to enable the exogenous phenylalanine to be better attached to the rice leaves.
The invention also provides a treatment method of exogenous phenylalanine, and experiments prove that after the rice inoculated with the pyricularia grisea for 36h and 48h is respectively treated by the exogenous phenylalanine, 168h is counted, and the length of disease spots sprayed with the exogenous phenylalanine and the relative growth amount of fungi for 48h are both smaller than 36h, so that the method makes up the reduction of the blast resistance of an OsLOX3 knockout rice strain, ensures that the control time is more accurate, is safe and effective, and provides important measures for utilization and protection of rice germplasm resources.
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, and 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 these drawings without creative efforts.
FIG. 1 is a graph showing the results of the linolenic acid content in the experimental group and the control group in example 2;
FIG. 2 is a graph showing the results of linoleic acid content in the experimental group and the control group in example 2;
FIG. 3 is a graph showing the results of observation of the phenotypes of the experimental group and the control group at different time points after the infection with Pyricularia oryzae in example 3;
FIG. 4 is a graph showing the results of disease investigation 168 hours after the test group and the control group in example 3 were infected with Pyricularia oryzae;
FIG. 5 is a graph showing the process of hypha infection after 30 to 40 days of infection of the scab of the flag leaf with rice blast fungi in the experimental group and the control group in example 3;
FIG. 6 is a graph showing the results of the synthesis of phytohormones and the expression of signal genes (JA, SA, ABA, etc.) after infection of rice blast bacteria in the experimental group and the control group in example 4;
FIG. 7 is a graph showing the observation results of the phenotype of 168 hours after the blast fungus was wounded to inoculate in the experimental group and the control group in example 5 for 36 hours and 48 hours, respectively, after the treatment with exogenous phenylalanine;
FIG. 8 is a graph showing the results of measuring the length of lesion spots 168h after the experimental group and the control group in example 5 were wounded and inoculated with Pyricularia oryzae for 36h and 48h, respectively, and treated with exogenous phenylalanine;
FIG. 9 is a graph showing the relative expression results of 168 hours of fungi after the experimental group and the control group in example 5 are wounded and inoculated with Pyricularia oryzae for 36 hours and 48 hours respectively and treated with exogenous phenylalanine;
FIG. 10 is a graph showing the results of hypha infection of the test and control groups in example 5 with exogenic phenylalanine after 30-40 days of inoculation of the scabies on the scabies for 36h and 48 h;
FIG. 11 is a graph showing the results of the synthesis of phytohormones and the expression of signal genes for defense genes, (JA, SA, ABA, etc.) after the test group and the control group in example 6 were inoculated with Pyricularia oryzae for 36h and 48h, respectively, and treated with exogenous phenylalanine.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for compensating reduction of blast resistance of an OsLOX3 knockout rice plant is characterized in that the rice plant with an OsLOX3 knockout gene grows to a three-leaf one-heart stage (14 days), 20mL of exogenous phenylalanine solution with the concentration of 100ppm is sprayed on 100 rice when the rice plant is infected with rice blast fungi for 36h or 48h, and the better effect is 48h, and the exogenous phenylalanine solution is a phenylalanine aqueous solution containing 0.1% of Tween. Wild type rice, ilmibyo, was selected as a control.
The strain of the magnaporthe grisea used for spraying is 95234I-1b, the magnaporthe grisea strain is activated on a PDA solid culture medium and is placed in a constant-temperature incubator at 28 ℃ for activation culture for 4 days, and a puncher is used for taking activated mycelium blocks to perform subculture on the PDA culture medium in the constant-temperature incubator at 28 ℃ for 4 days; using a puncher to punch fungus blocks with good edge activity of a PDA culture medium into a PDB liquid culture medium, placing the PDB liquid culture medium into a constant-temperature shaking table at 28 ℃ for culturing for 3 days, using a pipettor to suck 500uL of hypha culture solution to produce spores on a prune juice culture medium, uniformly coating a coating rod, placing the obtained product into a constant-temperature illumination incubator at 28 ℃ for dark culturing for 3 days, then alternately culturing for 12 hours in an illumination/dark environment for 5 days, scraping surface hypha when the hypha on the prune juice culture medium is changed from white to black, flushing the spores in sterile water to prepare spore suspension, and using a blood counting plate to adjust the spore concentration of the suspension to 1 x 10 under a microscope 5 one/mL.
In the examples, any methods related to the breeding of rice seedlings, the sporulation of rice blast strains, disease investigation and the statistics of disease incidence are conventional in the art.
Example 2
And (3) analyzing the physiological indexes of the OsLOX3 knockout rice strain, and taking wild rice Ilmibyeo as a control.
Treating OsLOX3 knockout rice plants by exogenous phenylalanine, and determining the contents of linolenic acid and linoleic acid:
spraying exogenous phenylalanine with the concentration of 100 mu mol/L to rice seedlings growing for 14 days, sampling and grinding after spraying for 24 hours, then weighing a proper amount of sample (0.1-1 g, 0.5g in the embodiment) in a clean imported EP tube, then using for extraction and purification, removing impurities from an extracting solution by using a CP-Sil 88 (100 m multiplied by 0.25mm multiplied by 0.25 mu m, agilent, USA) gas chromatographic column, taking a supernatant, and carrying out LC-MS analysis by sample injection; samples were separated using an Agilent gas chromatography system (Agilent 6890, agilent technologies, USA).
Mass spectrometry was performed using a quadrupole mass spectrometry detection system (Agilent 5977, agilent technologies, USA) from Aiglent, USA, and the analyte was detected in a single channel Scan (SIM) mode using an electron impact ion source (EI). And (3) data processing, namely processing mass spectrum data by using software Quant-My-Way, and extracting chromatographic peak area and retention time. And calculating the contents of the linolenic acid and the linoleic acid in the sample according to the standard curve.
The contents of linolenic acid and linoleic acid are measured and shown in a figure 1 and a figure 2, and the figures 1 and 2 show that the contents of the linolenic acid and the linoleic acid of an OsLOX3 knockout strain are obviously higher than that of a wild type, which indicates that the accumulation of the linolenic acid is caused after the OsLOX3 knockout.
Example 3
The pestilence resistance of the OsLOX3 knockout rice strain and the expression analysis of related genes are analyzed, and wild rice Ilmibyeo is used as a control.
The blast resistance of OsLOX3 knockout rice plants is identified, a rice blast spore suspension (preparation of the suspension is referred to example 1) is inoculated on rice seedling leaves in a three-leaf one-heart stage by a sprayer, spraying is continuously carried out until bacterial liquid on the rice leaves is in a water mist shape and does not drop down, disease statistics is carried out for 168h, and the disease index is referred to Xu Zhigang (2002), and the disease index =100 multiplied (the number of each stage of disease leaves is multiplied by the representative value of each stage)/(the number of total investigated leaves is multiplied by the representative value of the highest stage). In addition, the rice sheath growing for 30-40 days is inoculated with rice blast fungi by an injection method (the specification of an injector is 1 mL), hyphae are stained by a live/dead cell kit (Abbkin, KAT 1001), and the infection conditions of the hyphae after inoculation for 24h, 36h and 48h are observed.
As a result, referring to FIGS. 3, 4 and 5, FIG. 3 shows that the OsLOX3 knockout strain begins to show brown necrotic spots at 48h of inoculation of Pyricularia oryzae, while the wild type begins to show at 72h, and the wild type and knockout strain show yellow halos around the brown necrotic spots at 96h. At 144h, a connected necrotic spot appears, and in 168h, the disease statistics result shows that the disease degree of the knockout strain is higher than that of the wild type (shown in figure 4).
From the hypha invasion results (FIG. 5), the wild type and knockout lines showed that the coarse nonpathogenic primary hyphae did not expand to the adjacent cells at 24h, the pathogenic fine secondary hyphae began to grow and expand to the adjacent cells at 36h, and a large amount of secondary hyphae grew at 48h, but the secondary hyphae of knockout line # 2 was significantly larger than that of the wild type.
Example 4
Rice seedlings growing to the three-leaf one-heart stage are sampled in inoculation 0h, 24h, 36h,48h,72h and 96h for detecting defense genes, (JA, SA, ABA and the like) phytohormone synthesis and expression analysis of signal genes in OsLOX3 knockout rice plants:
(1) Extraction of RNA: the GenStar (Beijing Kang Runcheng Biotech Co., ltd.) kit is used for extracting total RNA of rice, and the operation method is shown in the kit specification.
(2) Reverse transcription of cDNA
TABLE 1 cDNA reverse transcription Components
According to the above table, the total system except RNA is prepared and then is loaded in a PCR tube, the target RNA is added, mixed gently, placed in a PCR instrument, incubated for 15min at 42 ℃, and heated for 5s at 85 ℃ to ensure that
RT/RI and gDNA Remover were inactivated and the resulting cDNA was placed in a refrigerator at 4 ℃ for subsequent use.
(3) Primers were designed based on the nucleic acid sequences of the JA synthesis/response, SA synthesis/receptor and defense-related genes, with the primer names and sequences shown in Table 2.
TABLE 2 primer sequences
(4) The real-time fluorescent quantitative PCR (qRT-PCR) was carried out using TransStart Top Green qPCR Supermix-AQ131 (Beijing Quanyujin Biotechnology Co., ltd.) kit, and the reaction system is shown in Table 3.
TABLE 3 qRT-PCR reaction System
The system was prepared as above, gently mixed and dispensed into 96-well PCR plates, each well was 19.5. Mu.L, and finally 0.5. Mu.L of cDNA was added, centrifuged at 1500rpm for 1min and then taken out, and qRT-PCR was performed in CFX96 quantitative PCR instrument, and the reaction procedure is shown in Table 4.
TABLE 4 conditions of qRT-PCR reactions
(5) qRT-PCR data analysis of genes of interest
qRT-PCR data processing with 2 -△△Ct The method comprises calculating and analyzing the expression level of the above genes, and the relative expression amount of the genes =2 -(Ct1-actinCt1)-(Ct2-actinCt2) All data were processed using an IBM SPSS Modeler 17.0 and plotted using Primer 8.0.
(6) Analysis of results
The gene expression results are shown in a figure 6, and the figure 6 shows that the relative expression quantity of the JA response gene OsbHLH35 in the rice blast fungus infected wild type and the knockout strain for 0h-96h is that the wild type is higher than that of the knockout strain, the expression quantity is remarkably up-regulated at 36h and reaches the maximum value, and the expression quantity is in a down-regulated trend after 48 h;
the expression level of the defense related gene OsPR1a in wild type is obviously higher than that of a knockout strain, and the expression level in wild type is higher at 24h, 36h and 48h, the expression level starts to be obviously reduced at 72h, and the expression level is hardly expressed in the knockout strain at 48h,72h and 96 h;
the expression level of the SA signal related gene OsWRKY45 in a wild type is obviously higher than that of a knockout strain and is highest at 36h, and the expression level is hardly expressed in the knockout strain at 72h and 96h.
The expression level of the active oxygen related gene OsRbohB in a knockout strain is obviously higher than that of a wild type, and the active oxygen related gene OsRbohB is extremely obviously up-regulated and reaches the highest level at 96 h;
the expression level of POD related gene OsPOX1 in wild type is obviously higher than that of a knockout strain, the expression level starts to be up-regulated at 72h, and the expression level reaches the highest at 96h.
Example 5
Carrying out wounding inoculation on rice leaves growing for 40 days, wherein the concentration of spore suspension is 1 multiplied by 105/mL, the diameter of a wound is 5mm, carrying out dark heat preservation (28 ℃) and moisture preservation (the humidity is more than 95%) treatment on the inoculated rice leaves for 24h, inoculating phenylalanine on the wound part respectively at the time of inoculation for 36h and 48h, measuring the length and the width of a lesion at the time of 144hpi, selecting a single lesion of the rice leaves, extracting DNA of the rice leaves by adopting a CTAB method, and detecting a relative biomass related gene primer sequence of a lesion fungus, wherein the relative biomass related gene primer sequence is shown in a table 5.
In addition, the rice sheath growing for 35 days is inoculated with the rice blast fungi by adopting an injection method, phenylalanine with the concentration of 100ppm is injected after the rice blast fungi are inoculated for 36h and 48h, the hypha infection conditions of the inoculated fungi for 36h,48h and 72h are observed after the phenylalanine is injected for 36h, and the hypha infection conditions of the inoculated fungi for 48h,72h and 96h are observed after the phenylalanine is injected for 48h.
TABLE 5 RT-PCR primer List
(1) A20.0. Mu.L PCR reaction was prepared as shown in Table 3: the reaction solution is prepared on ice, master Mix is prepared first, and then split charging is carried out.
(2) The reaction procedure for qRT-PCR is shown in Table 4.
(3) qRT-PCR data analysis of target genes
qRT-PCR data processing with 2 -△△Ct The method carries out calculation analysis on the expression level of MoPot2 gene, and the relative expression quantity of the gene =2 -(Ct1-actinCt1)-(Ct2-actinCt2) . Data were processed using an IBM SPSS Modeller 17.0 and plotted using Primer 8.0.
(4) Analysis of results
The results of the leaf-induced wounding inoculation are shown in the figures 7, 8 and 9, and the figure shows that the lesion length of phenylalanine spraying when 48h of inoculation of the rice blast fungus is obviously longer than that of exogenous jasmonic acid spraying when 36h is carried out, (figure 7 and figure 8), the relative expression amount of the fungus is higher when phenylalanine spraying is carried out for 36h, and the disease degree of a knockout strain is higher than that of a wild type strain no matter whether phenylalanine spraying is carried out for 36h or 48h (figure 9), which shows that the inducing and resisting effect of exogenous phenylalanine spraying is better when 48h of inoculation is carried out.
The hypha infection result is shown in figure 10, and figure 10 shows that although a small amount of secondary hyphae (with pathogenicity) begin to grow and expand towards adjacent cells in both wild type and knockout lines at 36h of inoculation, the primary hyphae (without pathogenicity) are coarse basically, which indicates that rice defense is not activated correspondingly at the moment, phenylalanine with the concentration of 100ppm is sprayed at the moment, a large amount of primary hyphae are remained at 48h, the hypha expansion degree in knockout lines is higher than that in wild type, and a large amount of secondary hyphae begin to grow at 72h, which indicates that the phenylalanine does not play a role at the moment; a large amount of secondary hyphae begin to appear at 48h of inoculation of the rice blast fungi, which indicates that the rice defense response is activated at the moment, and the spraying of the phenylalanine with the concentration of 100ppm can improve the rice defense capacity to a greater extent, so that a lot of primary hyphae are remained at 72h and 96h.
Example 6
Respectively spraying exogenous phenylalanine for 36h and 48h after the rice blast fungi is inoculated, and sampling for 36h,48h and 72h after the exogenous phenylalanine is sprayed for 36 h;48h after exogenous phenylalanine is sprayed, sampling at 48h,72h and 96h after inoculation, and detecting defensive genes, (JA, SA, ABA and the like) plant hormone synthesis and expression analysis of signal genes in OsLOX3 knockout rice strains, wherein the RNA extraction and related gene expression analysis method refers to example 4, the gene expression result refers to figure 11, and figure 11 shows that the relative expression amounts of the OsbHLH35, osPR1a, osWRKY45 and OsPOX1 genes after exogenous phenylalanine is sprayed at 36h are the highest in 48h of inoculation; the expression level reaches the highest after spraying exogenous phenylalanine for 48 hours after inoculation for 72 hours; compared with the gene expression result of untreated exogenous phenylalanine after inoculation (figure 6 in example 4), the gene expression results are improved, the expression quantity of exogenous phenylalanine sprayed for 48 hours is improved obviously, the expression quantity of a knockout strain # 2 is still lower than that of wild WT, and the expression result of an active oxygen related gene OsRbohB is opposite to that of other genes.
Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover in the claims the invention as defined in the appended claims.
Claims (8)
1. The method for compensating the reduction of the blast resistance of the OsLOX3 knockout rice plant and verifying the blast resistance is characterized by comprising the following steps of: the method comprises the following steps:
(1) OsLOX3 knockout rice plants growing to a three-leaf one-heart stage are sprayed with 15-20 mL of exogenous phenylalanine solution with the concentration of 100ppm according to 100 rice plants, and the contents of linolenic acid and linoleic acid are sampled and determined after 24 hours;
(2) Spraying 15-20 mL of exogenous phenylalanine solution with the concentration of 100ppm according to 100 rice plants when OsLOX3 knockout rice plants growing to the three-leaf one-heart stage infect rice blast fungi for 36-48 h;
(3) Sampling at 36h,48h,72h and 96h after spraying and inoculating the rice blast fungi, and determining relative expression amounts of JA response genes, defense genes, SA signal genes, active oxygen genes and POD genes;
(4) And (3) after spraying in the step (2), inoculating the rice blast fungi for 168 hours, and then carrying out disease investigation.
2. The method according to claim 1, wherein the time for infecting Magnaporthe grisea in step (2) is 48 hours.
3. The method according to claim 1, wherein in the step (2), the strain of Pyricularia oryzae is 95234I-1b.
4. The method of claim 3, wherein the step of removing the metal oxide layer is performed by a chemical vapor deposition methodThe concentration of the spore suspension of the rice blast fungus strain is 1 x 10 5 One per mL.
5. The method as claimed in claim 4, wherein the Pyricularia oryzae strains are activated on PDA solid culture medium, and are placed in a constant temperature incubator at 28 ℃ for activation culture for 4-5 days, and the activated mycelium blocks are taken out by a puncher and are placed on the PDA solid culture medium in the constant temperature incubator at 28 ℃ for subculture for 4-5 days; using a puncher to punch fungus blocks with good edge activity of a PDA culture medium into a PDB liquid culture medium, placing the PDB liquid culture medium into a constant-temperature shaking table at 28 ℃ for culturing for 3-4 days, using a pipettor to suck 500uL of hypha culture solution onto a prune juice culture medium for spore production, uniformly coating a coating rod, placing the coated layer into a constant-temperature illumination incubator at 28 ℃ for dark culturing for 3-4 days, then alternately culturing for 5-6 days in an illumination/dark manner for 12h, scraping surface hypha when the hypha on the prune juice culture medium is changed from white to black, flushing the spores with sterile water to prepare spore suspension, and using a blood counting plate to adjust the spore concentration of the suspension to be 1 x 10 under a microscope 5 one/mL.
6. The method of claim 1, wherein in steps (1) and (2), the exogenous phenylalanine solution is an aqueous phenylalanine solution containing 0.1% tween.
7. The method of claim 1, wherein the JA response gene is OsbHLH35, the defense gene is OsPR1a, the SA signal gene is OsWRKY45, the active oxygen group is OsRBohB, and the POD gene is OsPOX1.
8. Use of the method according to any one of claims 1 to 7 in rice breeding.
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