CN109729778B

CN109729778B - Method for improving resistance of sunflower to parasitic weed sunflower by using 5-aminolevulinic acid

Info

Publication number: CN109729778B
Application number: CN201811618953.9A
Authority: CN
Inventors: 许玲; 李娟娟; 曹梦婷; 周伟军; 杨翀; 朱金文; 王尖; 白全江
Original assignee: Zhejiang University of Technology ZJUT; Zhejiang University ZJU
Current assignee: Zhejiang University of Technology ZJUT; Zhejiang University ZJU
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2021-03-23
Anticipated expiration: 2038-12-28
Also published as: CN109729778A

Abstract

The present invention provides the use of 5-aminolevulinic acid for increasing resistance of sunflower to the parasitic weed, sunflower broomrape, and further finds that 5-aminolevulinic acid increases resistance to sunflower broomrape by promoting the synthesis of antioxidant enzymes in the sunflower plant, reducing the levels of MDA and ROS in the sunflower plant, and increasing the concentration of GSH and the GSH/GSSG ratio in the sunflower plant; the problem that the sunflower broomrape parasitizes to influence the growth of the sunflower in the planting process of the sunflower is effectively solved, the use of the herbicide is greatly reduced, and the ecological environment is protected.

Description

Method for improving resistance of sunflower to parasitic weed sunflower by using 5-aminolevulinic acid

Technical Field

The invention relates to the technical field of crop planting, in particular to a method for improving the resistance of sunflowers to the parasitic weed sunflower broomrape by utilizing 5-aminolevulinic acid.

Background

Sunflower is an important economic crop in the world and is also one of five oil crops in China. In recent decades, environmental changes bring many biotic and abiotic stress problems to crop production, and particularly, weed infection causes great harm to crop yield and quality, so that weed control is of great importance to agricultural production and environment, and plays an important role in determining whether people can meet future food production requirements.

Sunflower (Orobanchhe cumana Wallr.) is a non-photosynthetic root parasitic weed, mainly parasitizes at the root of sunflower, is connected to the tissue system of host plants through a special structure called a sucker, exhausts the nutrition and moisture of the host plants, seriously damages the yield of the host, and the earlier the parasitism is, the more the quantity is, the more the yield reduction is serious. Meanwhile, the huge broomrape seed bank in soil, the difficulty in accurately predicting the control time and the rapid appearance of new physiological broomrape of the broomrape make the current control method of the broomrape of the sunflowers unpreferable.

At present, a great number of control studies on broomrape have been reported, such as:

the invention discloses a compound organic fertilizer suitable for controlling sunflower broomrape during the growth of tobacco seedlings, which is disclosed by the invention with the application publication number of CN105777268A, and the organic fertilizer comprises the following components in parts by weight: 500 parts of decomposed organic fertilizer sheep manure, 300 parts of decomposed organic fertilizer cow manure, 2-4 parts of diammonium phosphate and 10-15 parts of potassium sulfate.

The invention patent with the publication number of CN101880633B discloses a strain Br-1 of Fusarium proliferatum and a microbial agent prepared by the strain, wherein the strain has good inhibition effect on Helianthus annuus, and the field control effect reaches 61.7-82.0%.

Plant Growth Regulators (PGRs) are often used to increase plant resistance and are important precursors for the biosynthesis of chlorophyllin porphyrins and heme, with important biological functions. Treatment of seeds with plant growth regulators can place the plant in an "activated state" that produces a defense response similar to that which resists the invasion of foreign pathogens.

At present, people utilize plant growth regulators to control sunflower broomrape, such as Yangguang 32704m (action mechanism research of sunflower broomrape infection and relief effect of exogenous salicylic acid. doctor paper, 2016) to pretreat sensitive sunflower seeds with exogenous salicylic acid, induce sunflower to generate defense reaction, and improve the resistance of sunflower to sunflower broomrape.

However, there are few plant growth regulators capable of improving the resistance of helianthus annuus to helianthus annuus, so that it is necessary to search for more plant growth regulators to solve the biological stress of helianthus annuus to helianthus annuus and improve the control effect of helianthus annuus.

Disclosure of Invention

The invention provides a method for improving the resistance of sunflower to the parasitic weed sunflower by using 5-aminolevulinic acid, which effectively relieves the problem that the sunflower parasitism influences the growth of the sunflower in the planting process of the sunflower, greatly reduces the use of herbicides and protects the ecological environment.

The specific technical scheme is as follows:

the invention discovers the new application of 5-aminolevulinic acid in improving the resistance of the sunflower to the major parasitic weed, namely the sunflower broomrape, for the first time.

The invention uses 5-aminolevulinic acid (ALA) with different concentrations to soak seeds of the sunflower, and performs co-culture on the sunflower and the broomrape through a pot experiment to simulate the action process of parasitic weeds in the field; experiments show that the sunflower seeds after seed soaking can effectively relieve the growth influence caused by parasitism of the sunflower seeds, and the plant height of the sunflower seeds is obviously improved.

Further, the 5-aminolevulinic acid increases resistance of sunflower to the parasitic weed sunflower by promoting the biomass of sunflower.

Further, the 5-aminolevulinic acid increases resistance of sunflower to the parasitic weed, helianthus annuus, by promoting synthesis of antioxidant enzymes in the sunflower plant;

the antioxidant enzyme is ascorbic acid peroxidase, superoxide dismutase or glutathione reductase.

Further, the 5-aminolevulinic acid increases resistance of sunflower to the parasitic weed sunflower broomrape by reducing the levels of MDA and ROS in the sunflower plant.

Further, the 5-aminolevulinic acid increases resistance of sunflower to the parasitic weed sunflower by increasing GSH concentration and GSH/GSSG ratio in sunflower plants.

The invention also provides a method for improving the resistance of sunflower to parasitic weed sunflower, which comprises the steps of seed soaking, germination accelerating, rooting, seedling growing and field cultivation of sunflower seeds, wherein the seed soaking process comprises the following steps: soaking sunflower seeds in an aqueous solution of 5-aminolevulinic acid; the concentration of the 5-aminolevulinic acid is 5-15 mg/L.

Further, the concentration of the 5-aminolevulinic acid is 10-15 mg/L.

Further, the concentration of the 5-aminolevulinic acid is 10 mg/L.

Further, the seed soaking time is 12-18 h.

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

the invention provides a new application of 5-aminolevulinic acid in improving the resistance of sunflower to parasitic weed sunflower, and further discovers that the resistance of the 5-aminolevulinic acid to the parasitic weed sunflower is improved by improving the biomass of the sunflower, promoting the synthesis of antioxidant enzyme in the sunflower plant, reducing the levels of MDA and ROS in the sunflower plant and improving the concentration of GSH and the ratio of GSH/GSSG in the sunflower plant, so that the problem that the sunflower parasitism influences the growth of the sunflower in the sunflower planting process is effectively relieved, the use of a herbicide is greatly reduced, and the ecological environment is protected.

Drawings

FIG. 1 is a graph of the effect of different concentrations of ALA treatment on the activity of antioxidant enzymes (SOD, POD, CAT, APX) in the roots and leaves of sunflower plants after broomrape infestation;

where the values are the mean of three biological replicates ± SE (n ═ 9), with different lower case letters indicating significant differences (P ≦ 0.05, LSD); a is SOD activity; b is POD activity; c is CAT activity; d is APX activity.

FIG. 2 is a graph of the effect of different concentrations of ALA treatment on GR, GSH/GSSG in the roots and leaves of sunflower plants after broomrape infestation;

where the values are the mean of three biological replicates ± SE (n ═ 9), with different lower case letters indicating significant differences (P ≦ 0.05, LSD); a is GR activity in roots; b is GR activity in leaves; c is GSH activity in the roots; d is GSH activity in leaves; e is GSH/GSSG in the root; f is GSH/GSSG in leaves.

FIG. 3 is a graph of the effect of different concentrations of ALA treatment on ultrastructure of sunflower mesophyll cells after broomrape infection;

wherein A is 0 mg/L; b is 5 mg/L; c is 10 mg/L; d is 15 mg/L.

FIG. 4 is a graph of the effect of different concentrations of ALA treatment on the ultrastructure of sunflower root tip cells after broomrape infection;

wherein A is 0 mg/L; b is 5 mg/L; c is 10 mg/L; d is 15 mg/L.

FIG. 5 is the effect of ALA on the expression of resistance-related genes in sunflower plants after infestation by Helianthus annuus L;

where the values are the mean of three biological replicates ± SE (n ═ 9), different lower case letters indicate significant differences (P ≦ 0.05, LSD). control represents a control group, no infestation on heliothis arborescens; o means only sunflower broomrape infestations; ALA represents only 15mg/L ALA treatment; o + ALA indicates an infestation with helianthus annuus and ALA is applied exogenously; the abscissa is a different gene; the ordinate Relative expression indicates the Relative expression level of the gene.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.

The materials adopted in the invention are as follows: the seeds of the sunflower broomrape (the highest physiological race G at present in China) and the sunflower (TK0409) are provided by plant protection of inner Mongolia farm and animal husbandry scientific research institute. Sunflower variety TK0409 was susceptible to 100% sunflower broomrape. 5-Aminolevulinic acid (ALA) was purchased from Hill Ciba Biotech Inc. The electron microscope observation is finished in the analysis and test center of Zhejiang university, wherein glutaraldehyde, buffer solution, ethanol, acetone, lead citrate, methylene blue and the like are all produced by chemical reagent company of national drug group. Osmic acid, Spurr embedding agent, uranyl acetate were produced by SPI-CHEM company.

Example 1

1 preparation of 5-aminolevulinic acid at various concentrations

Weighing 5-aminolevulinic acid in dark place, and mixing with distilled water to obtain ALA (0, 5, 10, 15, or 20 mg/L) solution.

TABLE 15 concentration gradient of Aminolevulinic acid

Note: recommended concentrations refer to the optimal concentrations obtained after the test, namely: ALA content added per 1L of water.

2 materials and methods

2.1 pretreatment and Germination of sunflower seeds

Selecting full and healthy sunflower TK0409 seeds, removing episperms, disinfecting in 70% absolute ethanol solution for 2min, washing with sterile water for several times, placing the seeds in ALA solutions with different concentrations, and soaking for 18 h; and (3) washing the seeds which fully absorb the ALA solution twice with distilled water, putting the seeds into a culture dish, culturing the seeds with two layers of filter paper containing distilled water at 25 ℃ in the dark for 48 hours, accelerating germination and rooting.

2.2 establishment of Co-culture System for sunflower and Helianthus annuus

Transplanting the germinated seeds with roots of the same length into a seedling raising pot with a matrix of peat soil and vermiculite (mass ratio of 1: 1). 200mg of broomrape seeds are uniformly mixed with 0.5kg of the matrix (the matrix soil mixed with the broomrape is placed in the middle layer, and accounts for one third of the whole pot) and used for co-culture of the sunflower and the broomrape.

The culture conditions were as follows: the illumination period is 16/8h, and the illumination intensity is 300 mu mol m^-2s^-1The day and night temperature is 24/20 ℃, the relative humidity is 60-70%, and proper amount of water is poured once every 5 days. After the cotyledon is completely earthed, the mixture is cultured with broomrape for 5 weeks, 0mg/L ALA and broomrape are blank controls, and the broomrape is not applied with ALA and is a positive control.

3. Morphological index

Morphological indices include height, fresh weight and dry weight of sunflower. Fresh samples were placed in an oven at 70 ± 5 ℃ for 5 days for dry weight measurement.

The results are shown in Table 2.

TABLE 2 influence of ALA on growth of sunflower plants after infestation of Helianthus annuus

Note: values are the mean ± SE (n ═ 6) of three biological replicates, with different lower case letters in the same column indicating significant differences (P ≦ 0.05). And represents P ≦ 0.001, P ≦ 0.01, and P ≦ 0.05.

As can be seen from table 1, after parasitizing by the sunflower broomrape, the height, fresh weight and dry weight of the sunflower plants decreased by 18%, 30% and 40%, respectively. The same treatment significantly increased the biomass of the plants under conditions where sunflower was parasitized with ALA. After the treatment with 20mg/LALA, the plant leaves die after wilting regardless of whether the plant leaves are parasitized by Libang or not. High concentrations of ALA inhibit growth and even produce toxicity.

Example 2 anti-oxidase assay

The activities of antioxidant enzymes included Ascorbate Peroxidase (APX), Catalase (CAT), Peroxidase (POD), superoxide dismutase (SOD) and Glutathione Reductase (GR), and the antioxidant enzyme activities were measured using a Muti-Mode Reader (SYNERGY HTX) instrument according to the following method.

Determination of APX enzyme

The APX activity assay was modified according to the method of Nakano and Asada (1981). The reaction system was 3mL, including 2.9mL100mM potassium phosphate buffer (pH 7.0, containing 0.1mM EDTA-Na)₂0.3mM ascorbic acid, 0.06mM H₂O₂) And 100. mu.l of the enzyme extract. The change in absorbance of the reaction mixture was measured at 290 nm for 1min, and the extinction coefficient was 2.8mM^-1cm ^-1。

Determination of CAT enzyme

CAT Activity by H₂O₂Measured by the consumption of (extinction coefficient 39.4 mM)^-1cm^-1). The reaction system consisted of 3mL, containing 2.9mL of 50mM potassium phosphate buffer (pH 7.0, 2mM EDTA-Na)₂，10mM H₂O₂) And 100. mu.l of the enzyme extract. The change in absorbance of the reaction mixture at 240nm for 1min was measured (Aebi, 1984).

Measurement of POD

POD activity was measured by guaiacol oxidation (Zhou and Leu, 1999). The reaction system consisted of 3ml, containing 50mM phosphate buffer (pH 6.0), 0.3% guaiacol, 0.4% H₂O₂And 100. mu.l of the enzyme extract. The increase in absorbance of the reaction system at 470nm within 1min was measured with an extinction coefficient of 26.6mM^-1cm^-1。

Determination of SOD enzyme

The SOD activity was measured by NBT method (Zhang et al, 2008). The reaction system consisted of 3mL in total, and contained 50mM phosphate buffer (pH7.8), 13mM L-methionine, 75. mu.M NBT, 0.1mM EDTA, 2. mu.M riboflavin, and 100. mu.l of the enzyme extract. The reaction mixture was reacted for 20min under 4000 lx conditions, and the SOD activity was calculated as 50% inhibition of photochemical reduction of NBT by colorimetry at 560nm as one enzyme activity unit.

Measurement of GR

GR activity was determined by a modification of the method described by Jiang and Zhang (2002). The reaction system was 3mL, and included 2.7mL of sodium phosphate buffer 50mM (pH7.8, containing 2mM EDTA-Na)₂) 100ul of 2.4mM NADPH, 100ul of 10mM oxidized glutathione (GSSG) and 100uL of supernatant. The blank zeroing tube was charged with 100 μ L of extract (50mM potassium phosphate buffer (pH 7.8)) and the reaction system was measured at 340nm for 10s and 190s absorbance, which was calculated as a1 and a2, Δ a ═ a1-a 2. Extinction coefficient 6.2mM^-1cm^-1。

As a result:

FIGS. 1 and 2(a), (b) show the change in antioxidant activity in roots and leaves of sunflower under different treatments.

After parasitizing by sunflower broomrape, the SOD, POD and APX increase in the leaves and roots of sunflower by 17% and 11%, 19% and 31%, 39% and 57%, respectively, compared to the control group; indicating that the production of ROS and the like is inhibited by increasing the antioxidant enzyme activity under broomrape infestation.

Compared with a positive control, exogenously applied ALA can obviously improve the activity of POD, and ALA with the concentration of 10mg/L rises by 20 percent and 81 percent respectively in the roots and leaves of sunflower.

Under the same conditions, the GR activity is also obviously enhanced compared with that of a positive control, and the 15mg/L ALA content is reduced along with the increase of the ALA concentration. Exogenous application of ALA did not have a large effect on the activity of CAT on the parasitized sunflowers.

Overall, adversity stress from sunflower broomrape infestation results in rapid accumulation of antioxidant enzymes, while ALA further promotes the synthesis of antioxidant enzymes to combat herbicide damage.

Example 3 determination of MDA and ROS

The MDA and ROS are measured using a spectrophotometer (UV-5500, METASH).

Determination of MDA

Malondialdehyde was determined according to Zhou and Leul (1999) after modification by thiobarbituric acid color development. Adding 5mL of 0.5% thiobarbituric acid (TBA, prepared from 10% trichloroacetic acid) solution into 2mL of supernatant enzyme solution, reacting in a water bath at 95 ℃ for 30min, and immediately performing ice bath; centrifuging at 5000g for 10min, taking supernatant, performing color comparison at 532nm and 600nm, calculating MDA content by difference, and extinction coefficient is 155mM^-1cm^-1。

Determination of ROS

Reactive Oxygen Species (ROS) include hydrogen peroxide (H)₂O₂) Superoxide anion (O)₂ ^-) The hydroxyl group is selected from the group (a)^-OH)。

2.1 H₂O₂Measurement of (2)

H₂O₂The assay of (2 mL) of the reaction system according to the method of Velikova et al (2000), contains 0.5mL of the supernatant, 0.5mL of 10mM potassium phosphate buffer (pH 7.0) and 1mL of 1M potassium iodide. The reaction was carried out at room temperature (28 ℃ C.) for 1hr, and the absorbance at 390nm was measured. And calculating according to the standard curve.

2.2 O₂ ^-Measurement of (2)

O^2-The assay of (2) was modified according to the method of Jiang and Zhang (2001), 0.5mL of the sample extract was mixed with 1mL of 50mM sodium phosphate buffer (pH7.8) and 0.5mL of 10mM hydroxylamine hydrochloride, shaken, incubated at 25 ℃ for 1 hour, 2mL of 17mM sulfanilic acid and 2mL of 7mM 1-naphthylamine were added, mixed, incubated at 25 ℃ for 20 minutes, and the absorbance at 530nm was measured with a spectrophotometer (zeroed with a standard curve No. 1 tube solution).

2.3^-Determination of OH

^-Determination of OH was modified according to Halliwell et al (1987) by mixing 0.7mL of the supernatant solution, 3mL of 0.5% (w/v) TBA and 1mL of glacial acetic acid, boiling in a water bath for 30min, cooling to 41 ℃ and measuring the absorbance at 550nm after 10 min. Extinction coefficient of 0.28mM^-1cm^-1。

As a result:

TABLE 3 Effect of ALA on sunflower plants MDA and ROS following infestation of Helianthus annuus

Note: values are the mean ± SE (n ═ 9) of three biological replicates, with different lower case letters in the same column indicating significant differences (P ≦ 0.05). And represents P ≦ 0.001, P ≦ 0.01, and P ≦ 0.05.

As can be seen from Table 3, the parasitism of Helianthus annuus on Helianthus annuus roots H₂O₂、O₂ ^-、^-Both OH and MDA contents have an influence, in particular^-The OH content was significantly increased, 71% and 87% in the roots and leaves, respectively, compared to the control group.

Root of sunflower treated with exogenous ALA in the absence of parasitism by Helianthus annuus H₂O₂And O₂ ^-Has no significant difference with a control group (P is less than or equal to 0.05, LSD). Under the condition of parasitism by sunflower broomrape, as the concentration of exogenous ALA is gradually increased (from 5 to 15mg/L), compared with the positive control group (no ALA), MDA and O are contained in sunflower₂ ^-And^-OH content tends to decrease, while H₂O₂The content is not obviously different from that of a positive control, and the accumulation amount of MDA in the leaves is about 10 times of that of roots,^-the OH accumulation was more than 3 times that of the root.

These results indicate that under o.cumana stress, the use of ALA (10mg/L) can effectively reduce sunflower plant MDA and ROS levels.

EXAMPLE 3 determination of GSH and GSSG content

Determination of GSH + GSSG content

Determination of the GSH + GSSG content was modified according to the method of Law et al (1983) with 700. mu.L of 0.3mM NADPH and 100. mu.L of 6mM DTNB (5, 5' -dithio-2-nitrobenzoic acid), 50. mu.L of GR (10U/mL), 150. mu.L of supernatant. The change in absorbance at 412nm was measured (blank control with phosphate buffer instead of DTNB reagent), blank tube: 120ul of distilled water was substituted for the supernatant. The change in absorbance at 412nm was measured.

Determination of GSSG content

120 μ L of supernatant, 10 μ L of 2-vinylpyridine, 20 μ L of 50% triethanolamine, the solution was vortex mixed for 30s in a 25 ℃ water bath L h. Blank tube: 120ul of distilled water was substituted for the supernatant. Then measuring the change of the absorption value at 412nm to determine the content of GSSG. The GSH content is the total glutathione minus the GSSG content.

As a result:

as shown in fig. 2c-f, parasitism of helianthus annuus can significantly increase the GSH content and GSH/GSSG ratio in roots and leaves of helianthus annuus, 79% in roots, 35% in 100% of leaves, and 118% in leaves, respectively, compared to the control group.

In addition, under the action of exogenous ALA, the content of GSH and GSH/GSSG are obviously increased no matter whether sunflower broomrape exists or not. By increasing ALA concentration, both GSH content and GSH/GSSG ratio are improved, reaching maximum level at 10mg/L ALA, while ALA (15mg/L) increase reduces GSH content and GSH/GSSG ratio.

The result shows that the application of ALA (10mg/L) can effectively improve the concentration of O.cumana infected GSH and the ratio of GSH/GSSG in sunflower plants.

Example 42.6 TEM Observation of root tip and mesophyll cells

Cutting root tip (2-3mm) and leaf fragment (about 1 mm) under microscope²) Fixed in 2.5% (v/v) glutaraldehyde overnight at 4 ℃, the fixative was decanted off, and the samples were rinsed 3 times for 15min each with 0.1M sodium phosphate buffer (pH 7.4). Samples were fixed in 1% OsO4 (osmum (VIII) oxide) for 1-2h, osmic acid waste was carefully removed and washed 3 times with 0.1M PBS (pH 7.4) every 15 min. Then, every 15-20 minutes, the samples were dehydrated in a series of graded ethanol (including 30%, 50%, 70%, 80%, 90%, 95%, and 100%) and finally absolute acetone for 20 minutes. After dehydration, the sample was treated with a mixture of Spurr embedding medium and acetone (V/V. 1/1) for 1 h; the sample was treated with a mixture of Spurr embedding medium and acetone (V/V-3/1) for 3 h; samples were treated with neat embedding medium overnight at room temperature. Embedding the sample subjected to the permeation treatment, and heating at 70 ℃ overnight to obtain an embedded sample; slicing the sample in LEICA EM UC7 ultrathin microtome to obtain 70-90nm slices; staining slices with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 5-10 min; observed in a Hitachi H-7650 type transmission electron microscope.

As a result:

FIGS. 3 and 4 show the organelle structure changes of sunflower mesophyll cells and root tip cells under transmission electron microscope at different concentrations of ALA (0, 5, 10, 15mg/L) after sunflower brood infection, respectively.

Cumana infection with 0mg/L ALA treatment, Cell Wall (CW) degradation, chloroplast (Chl) swelling, thylakoid (Thy) disorganization, nucleolus (Nue) disappearance, nuclear matrix aggregation. FIG. 3(B)5mg/L ALA-treated leaf mesophyll cells retained the chloroplast structure in an elongated shape, with a lower density of stroma (G) and distinct starch granules (S). Cumana infection 10mg/L ALA treated leaf mesophyll cells Transmission Electron Microscopy (TEM) showed clear Cell Walls (CW), well-developed chloroplasts (Chl), Mitochondria (MC), nuclei (Nuc), vacuoles (Vac) and intact Cell Membranes (CM). FIG. 3(D)15mg/L ALA treatment with intact nuclei, clear Nuclear Membrane (NM), solubilized chloroplasts, unevenly distributed nuclear stroma, and incomplete Cell Wall (CW).

Therefore, a series of physiological and biochemical changes occur after broomrape is infected, the mesophyllic cell structure is obviously damaged, the resistance of the sunflower to the broomrape can be improved by applying ALA externally, and the 10mg/L ALA improvement effect is obvious.

Fig. 4(a) TEM observations of faint vacuoles (Vac) and other lysed organelles in 0mg/L ALA-treated root tip cells under o.cumana infection; fig. 4(B) TEM micrograph of non-compact nuclei (Nue), restored smooth Cell Walls (CW) of ALA-treated root tip cells under o.cumana infection; FIG. 4(C)10mg/L ALA-treated and Orobanchum postnatal root tip cells with smooth cell wall, intact mitochondria, well-defined cells and structure; fig. 4(D) o. cumana infection 15mg/L ALA-treated root tip cells lost nuclei and nucleoli, and mitochondria became fuzzy.

Thus, the results were similar in the root tip cells and the mesophyll cells, and some bacteria were found in the root tip cells infected with orobanche coerulescens and treated at 0m/L and 5mg/LALA, and it was suspected that the cells were diseased during the orobanche coerulescens process, and the specific roles thereof were to be further studied.

Example 5 extraction of RNA and qRT-PCR

Total RNA was extracted from liquid nitrogen-frozen leaves using RNAasso Plus (TaKaRa, Japan). 300ng of total RNA was reverse transcribed using TaKaRa PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect for Real Time). PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa) reagent in QuantStaudio 6Flex Real-Time PCR System (Thermo Fisher). The primers used in RT-PCR are shown in Table 4. Each treatment was repeated 3 times.

TABLE 4 fluorescent quantitative PCR specific primer sequence of growth related gene

In order to clarify the effect of ALA on the parasitized sunflower at the gene expression level, the present invention examined the relative expression levels of 10 genes associated with antioxidant enzymes, different metabolic pathways and resistance. As shown in FIG. 5, most of the genes, including some antioxidant enzyme related genes and phytohormone related genes, were up-regulated after being parasitized by Helianthus annuus. Under the condition of being parasitized by sunflower broomrape, the relative expression of most genes is improved by using the exogenous ALA, and the relative expression of Mn-SOD, lox, XTH6 and XTH9 is obviously reduced by 66 percent, 18 percent, 28 percent and 27 percent by using the exogenous ALA. It is shown that exogenous application of ALA also has an effect on the amount of gene expression.

Claims

Use of 5-aminolevulinic acid for increasing the resistance of sunflower to the parasitic weed sunflower broomrape.
2. The use of claim 1, wherein the 5-aminolevulinic acid increases resistance of the sunflower to the parasitic weed, helianthus annuus, by promoting the biomass of the sunflower.
3. The use of claim 1, wherein said 5-aminolevulinic acid increases resistance of sunflower to heliotrope by promoting synthesis of an antioxidant enzyme in the plant;

the antioxidant enzyme is peroxidase, superoxide dismutase or glutathione reductase.
4. The use of claim 1, wherein said 5-aminolevulinic acid increases resistance of sunflower to the parasitic weed helianthus annuus by reducing the levels of MDA and ROS in the plant.
5. The use of claim 1, wherein the 5-aminolevulinic acid increases the resistance of sunflower to the parasitic weed sunflower by increasing the concentration of GSH and the GSH/GSSG ratio in the plant.
6. A method for improving the resistance of sunflower to the parasitic weed sunflower by using 5-aminolevulinic acid comprises the steps of seed soaking, germination accelerating, rooting, seedling growing and field cultivation of sunflower seeds, and is characterized in that the seed soaking process comprises the following steps: soaking sunflower seeds in an aqueous solution of 5-aminolevulinic acid; the concentration of the 5-aminolevulinic acid is 5-15 mg/L.
7. The method of claim 6, wherein the concentration of 5-aminolevulinic acid is 10 to 15 mg/L.
8. The method of claim 6, wherein the concentration of 5-aminolevulinic acid is 10 mg/L.
9. The method of claim 6, wherein the seed soaking time is 12-18 hours.