CN115029373B

CN115029373B - Application of destruxin CFEM85 protein and method for improving resistance of plants to gray mold and aphids

Info

Publication number: CN115029373B
Application number: CN202210693952.0A
Authority: CN
Inventors: 农向群; 蔡霓; 刘蓉; 涂雄兵; 王广君; 张泽华
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2022-06-19
Filing date: 2022-06-19
Publication date: 2023-11-17
Anticipated expiration: 2042-06-19
Also published as: CN115029373A

Abstract

The application relates to the field of agricultural and forestry pest control, in particular to application of destruxin CFEM85 protein and a method for improving resistance of plants to gray mold and aphids. The application infiltrates active metarhizium anisopliae CFEM85 protein into the back of tobacco leaves, and the allergic necrosis is observed by trypan blue staining; meanwhile, a transient expression vector is constructed, transient expression of tobacco is transformed by agrobacterium, the plant resistance activation reaction is verified, aphids and gray mold are inoculated, and the induction effect of the plant resistance is evaluated. The results show that the protein injection and the transient expression show that the metarhizium anisopliae CFEM85 protein can activate anaphylactic reaction of tobacco and improve the resistance of the tobacco to ash mold and aphid. The application verifies that the metarhizium anisopliae CFEM85 protein has the function of activating plant resistance, and provides reference for the subsequent research and utilization of the protein function.

Description

Application of destruxin CFEM85 protein and method for improving resistance of plants to gray mold and aphids

Technical Field

The application relates to the field of agricultural and forestry pest control, in particular to application of destruxin CFEM85 protein and a method for improving resistance of plants to gray mold and aphids.

Background

Metarhizium anisopliae is an important entomopathogenic fungus used as a biological pesticide for controlling various agricultural and forestry pests. The living mode is various, and insect parasitism, soil rotting and plant endophytic living can be realized. A great deal of previous research has focused on their pathogenicity, lethal mechanism and biological control of hosts, and in recent years researchers have focused on their interactions with plants. Many scientific reports prove that the metarhizium anisopliae can be planted in various host plant tissues, improve the nutrition absorption and the resistance level of plants, explore how to activate the resistance level of the plants under the condition of friendly reciprocity with the plants, thereby preventing and controlling diseases and insect pests, and having important significance for realizing continuous prevention and control of the metarhizium anisopliae when the metarhizium anisopliae is applied.

The CFEM (common in several fungal extracellular membrane protein) protein domain has about 60 amino acids, typically 8 cysteines. Unlike other cysteine-rich proteins, this protein domain is present only in fungi and is common to the extracellular membrane proteins of fungi and is therefore known. Different CFEM sources have different functions, some participate in suppressing immunity, some contribute to maintaining the structure of fungi, and the diversity of functions is related to the sources and the protein structure. In verticillium dahliae Verticillium dahliae, vdSCP76,77 is capable of inhibiting cell necrosis of tobacco triggered by effectors, inhibiting tobacco immunity. In plant pathogens, CFEM protein was studied as an exciton, the wheat leaf rust fungus Puccinia triticina CFEM effector PTTG 08198 enhanced the cell death of wheat, promoting Reactive Oxygen Species (ROS) accumulation. In Pyricularia oryzae Magnaporthe oryzae, pth11 is a G-protein coupled receptor-like protein with 7 transmembrane domains. After Pth11 gene knockout, the hydrophobic surface attachment cells are weakened and cannot penetrate the infected rice leaves. This suggests that Pth11 is related to the integrity and hydrophobicity of the cell wall. Botrytis cinerea BcCFEM1 was significantly up-regulated in early bean She Qinran and Agrobacterium-mediated transient tobacco expression showed leaf chlorosis in Nicotiana benthamiana. However, the function of such proteins has not been reported in the study of the interaction of entomopathogenic fungi with plants.

Disclosure of Invention

The application aims to provide application of the Metarrhizium anisopliae CFEM85 protein.

It is a further object of the present application to provide a method for increasing the resistance of plants to botrytis cinerea and aphids.

The application provides the following application of the Metarrhizium anisopliae CFEM85 protein:

activating allergic reactions in plants;

enhancing the resistance of plants to botrytis cinerea; or (b)

Enhancing the resistance of the plant to aphids,

wherein the amino acid sequence of the metarhizium anisopliae CFEM85 protein is shown as SEQ ID NO. 1 or 2.

SEQ ID NO:1：

SEQ ID NO. 2 (sequence of Metarhizium anisopliae CFEM85 protein after removal of the Signal peptide)

According to the use of the application, the plant is tobacco.

The method for improving the resistance of plants to botrytis cinerea and aphids comprises the step of expressing a gene encoding the metarhizium anisopliae CFEM85 protein in the plants, wherein the amino acid sequence of the metarhizium anisopliae CFEM85 protein is shown as SEQ ID NO. 1.

The method for improving the resistance of plants to botrytis cinerea and aphids according to the application, wherein the plants are tobacco.

The method for improving the resistance of plants to botrytis cinerea and aphids, provided by the application, wherein the nucleotide sequence of the coding gene of the metarhizium anisopliae CFEM85 protein is shown as SEQ ID NO. 3 or 4.

SEQ ID NO:3：

SEQ ID NO. 4 (coding sequence of Metarrhizium anisopliae CFEM85 protein with signal peptide removed)

The application takes metarhizium anisopliae (Metarhizium anisopliea) cDNA as a sample, clones to obtain CFEM85 gene, constructs a recombinant expression vector after sequencing verification and comparison and analysis are correct, uses an escherichia coli prokaryotic expression system to perform in vitro expression, and obtains active protein after protein renaturation. Metarhizium anisopliae secreted proteins (CFEM 85) containing CFEM domains have a typical CFEM structure, which contains 8 conserved cysteines in the CFEM domain. Penetrating and injecting active metarhizium anisopliae CFEM85 protein into the back of tobacco leaves, and staining trypan blue to observe allergic necrosis; meanwhile, a transient expression vector is constructed, transient expression of tobacco is transformed by agrobacterium, the plant resistance activation reaction is verified, and the relevant gene of the tobacco resistance is detected by RT-qPCR. The results show that the protein injection and the transient expression show that the metarhizium anisopliae CFEM85 protein can activate anaphylactic reaction of tobacco and improve the resistance of the tobacco to ash mold and aphid. The application verifies that the metarhizium anisopliae CFEM85 protein has the function of activating plant resistance, and provides reference for the subsequent research and utilization of the protein function.

Drawings

FIG. 1 is a diagram of full-length amplification of the CFEM85 gene of Metarrhizium anisopliae and vector construction electrophoresis, wherein lanes: m: DL2000 plus DNA marker, left 1: CFEM85 full length, middle 1: PET-21b-CFEM85, right 1: pYBA-CFEM85;

FIG. 2-1 is a SDS-PAGE diagram of CFEM85 protein, wherein M: a marker;1: cell disruption supernatant, 2: precipitating cell disruption solution;

FIG. 2-2 is a denaturing SDS-PAGE map of CFEM85 protein, wherein M: marker,1: crushing liquid supernatant, 2: buffer I wash, 3: bufferA wash, 4: first buffer C wash, 5: second buffer C wash, 6:8M urea wash 1,7:8M urea wash 2,8:8M urea wash 3;

FIGS. 2-3 are SDS-PAGE patterns of CFEM85 protein purification, wherein M: marker,1: protein dialysate, 2: protein fluid penetration, 3:20Mm imidazole, 4:30Mm imidazole, 5:50Mm imidazole, 6:150Mm imidazole elution 1,7: eluting 2 with 150Mm imidazole;

FIG. 3-1 shows the results of CFEM85 recombinant protein treatment of tobacco, wherein CK:20mM Tris-HCl; and (3) treatment: 200ug/mL CFEM85 protein treatment;

FIG. 3-2 shows the results of CFEM85 treatment of tobacco with pYBA1132, wherein: CK: GFP, treatment: pYBA1132, CFEM85;

FIG. 4 shows the results of DAB staining after CFEM85 recombinant protein treatment of tobacco;

FIG. 5 shows the results of trypan blue staining after CFEM85 recombinant protein treatment of tobacco;

fig. 6 shows the results of tobacco cell callus detection, wherein CK:20mM Tris-HCl, treatment: CFEM85 protein treatment;

fig. 7 shows that transient expression of CFEM85 induces plant resistance, wherein after a.12h infiltration of MaCFEM85 into tobacco, leaf gray mold spot size at day 2, b. aphid feeds on average the number of offspring of 24h, 48h, 72h of transient expression of MaCFEM85, the values are mean ± standard deviation, n=20.f, different letters on the column represent significant differences between different treatments at 0.05 level, P <0.05.

Detailed Description

Test plants and fungi:

the test strain is Metarhizium anisopliae (Metarhizium anisopliae) Ma9 strain, and the test plant is Nicotiana benthamiana (Nicotiana benthamiana). Inoculating Metarrhizium anisopliae on PDAY culture medium plate, culturing in constant temperature incubator at 26deg.C for two weeks, collecting spores, and preserving at 4deg.C.

EXAMPLE 1 cloning, expression and purification of the Metarrhizium anisopliae CFEM85 Gene

1. Gene cloning

The newly collected spore powder was dissolved in 0.1% Tween-80 and inoculated into hypha-producing liquid medium for 3d at 28 ℃. The mycelia were filtered by a vacuum pump, and then immediately frozen in liquid nitrogen with the dried mycelia, and then total RNA was extracted as follows. The sample RNA was reverse transcribed into cDNA using a reverse transcription kit 5×all-In-One RT Master mix (abm Co.).

The open reading frame (Open reading frame, ORF) of the CFEM85 gene was cloned using design amplification full-length specific primers CFEM85-F and CFEM85-R (as shown in Table 1 below), with a full length of 513bp and a stop codon TAA as shown in FIG. 1. The gene codes 170 amino acids, and the theoretical molecular weight of the protein is 16.3KD.

And (3) performing agarose gel electrophoresis detection on the PCR product, recovering a target strip, connecting the recovered and purified PCR product with a PMD19-T carrier, placing the mixture at 16 ℃ for connection for 10 hours, converting escherichia coli competent Tans1-T1, and culturing. And selecting positive transformants, extracting plasmids from bacterial solutions of the positive transformants, sending to a company for sequencing, checking whether the sequences are correct, and determining that CFEM85 has a signal peptide cleavage site at 18 AA. After removing the signal peptide sequence, adding a front connector sequence to form a primer 21b-CFEM85-F at the 5 'end of the gene, adding a connector sequence to form a primer 21b-CFEM85-R at the 3' end, carrying out double digestion on the pET-21b vector by EcoRI and SalI, and constructing a prokaryotic expression vector by using a seamless cloning kit. Similarly, a linker sequence is added at the 5 'front end of the complete open reading frame of the CFEM85 gene to form a primer pYBA-CFEM85-F, a linker sequence is added at the 3' end to form a primer pYBA-CFEM85-R, and the Pyba-1132 vector is subjected to EcoRI and SalI double digestion and then is subjected to prokaryotic expression vector construction by using a seamless cloning kit. Then transferred into BL21 (DE 3) and Trelif respectively ^TM 5a (Beijing Optimago Co.) competent, through the growth of the resistance plate overnight, after picking up the monoclonal, through bacterial liquid PCR and sequencing, get the positive clone of successful recombination. And extracting pYBA-CFEM85 recombinant plasmid, and preparing agrobacterium transformation.

TABLE 1 specific primers

。

Expression and purification of CFEM85 protein

Recombinant positive clones were subjected to 37℃with IPTG induction at 0.5mM, rotation speed 200rpm, and induction time 4h. CFEM85 protein was successfully induced for expression. The recombinant protein from which the signal peptide was removed was approximately 17kDa (FIG. 2-1), and after protein denaturation, CFEM85 protein was found to be largely dissolved in the supernatant under 8M urea washing (FIGS. 2-2, line6,7, 8). After dialysis and Ni column purification, a large amount of CFEM85 protein was eluted at 150mM imidazole, and after desalting, the protein solution was dissolved in 20mM Tris-HCl (pH 8.0) and placed at-20℃for use, and SDS-PAGE patterns of CFEM85 protein purification were shown in FIGS. 2-3.

Recombinant proteins comprising the signal peptide were also purified.

Example 2 functional study

The purified recombinant protein of example 1 or was used in the following study.

Cfem85 protein induces a tobacco cell necrosis response

The CFEM85 protein is diluted to 200 mug/mL, the back of the plant leaf is gently scratched by a 1mL disposable syringe needle, the front corresponding position of the injured leaf is pressed by a finger, and the diluted CFEM85 protein is injected from the wound of the back of the plant leaf by the syringe without the needle, and the treatment is repeated three times for five times. The injected plants were returned to the greenhouse for further cultivation, after 24 hours, the cell necrosis of the leaves of the plants was observed and photographed. After the plant leaves are observed and recorded, a part of the leaves are dyed, and the other part of the leaves are sheared, and then liquid nitrogen is quickly frozen, and the sample is stored at-80 ℃.

As shown in fig. 3-1, significant tobacco cell chlorosis was observed 24h after injection of tobacco leaves with CFEM85 protein after expression purification. After 5 days, a marked allergic necrosis was observed at the protein injection site.

As shown in FIG. 3-2, the CFEM85 was constructed as a transient expression vector pYBA-CFEM85, and transformed into Agrobacterium, and GFP was used as a control, and after 48 hours of injection of tobacco, the leaves of the treatment group wilted and became necrotic, while the leaves of the control group did not become necrotic significantly.

Cfem85 protein induces a burst of living ROS in tobacco

Plant Reactive Oxygen Species (ROS) mainly include: h ₂ O ₂ ，O ^2- And hydroxyl radicals. Wherein the peroxide is H ₂ O ₂ Can be stained brown by DAB, and can be used for detecting the accumulation of CFEM85 protein induced plant ROS.

ROS burst was detected using Diaminobenzidine (DAB) tissue staining: respectively picking up tobacco leaves treated by recombinant protein and buffer solution for 24 hours, washing with distilled water, putting into DAB-HCL staining solution (1 mg/mL, PH=3.8), vacuumizing to submerge the staining agent into tobacco cells, and incubating for 1-2 hours under the dark room temperature condition. Pouring out the dyeing liquid, soaking the leaves in 95% ethanol, bathing in boiling water for 10min, repeating for 1-2 times to remove chlorophyll completely, and observing.

The control buffer treated tobacco leaves were white in color by DAB staining, whereas the CFEM85 protein treated tobacco leaves were observed to be significantly brick-red like material accumulation (FIG. 4), indicating that recombinant protein CFEM85 treatment of tobacco was able to bind DAB with active oxygen (mainly hydrogen peroxide) produced by the tobacco to produce reddish brown material.

Cfem85 protein induces tobacco allergic necrosis

Trypan blue dye solution is a cell reactive dye, and is commonly used for detecting the integrity of cell membranes and detecting whether the cells survive. Living cells will not be stained blue, whereas dead cells will be stained pale blue. As shown in fig. 5, tobacco leaves in the CFEM85 protein-treated area were stained with blue spots compared to the control group, indicating that after 24 hours of treatment with CFEM85 protein, the tobacco cells were subjected to cell death, indicating that CFEM85 protein could excite allergic necrosis of tobacco.

CFEM85 protein induces tobacco callose accumulation

Tobacco leaf callose accumulation in the test using aniline blue staining: tobacco leaves treated for 24 hours by recombinant protein CFEM85 are taken and placed in a solution of acetic acid/ethanol=1/3 (V/V) for decolorization, and after the leaves are completely decolorized, the tobacco leaves are placed in a solution of 0.07M dipotassium hydrogen phosphate-aniline blue (0.1%) for dyeing overnight, and finally cut into small blocks and are observed under a fluorescence microscope.

Callose is glucan combined by beta-1, 3-bond, and plays an important role in regulating life activities such as screen metabolism, gametophyte development and the like of plants. When plants are stressed by the external adverse environment or invaded by pathogens, callose can be rapidly synthesized in sieve tube molecules and deposited on the surface of a sieve plate or in sieve holes to be blocked; the increased physical defenses prevent pathogenic infestation or disease spread. CFEM85 protein-treated tobacco leaves were chlorophyll-removed and then stained with aniline blue, and spot fluorescence was observed under a fluorescence microscope (fig. 6), namely callose. It is demonstrated that tobacco callose accumulates and defenses are activated after CFEM85 protein treatment of tobacco.

5. Transient expression of Metarrhizium anisopliae CFEM85 (MaCFEM 85) improves tobacco resistance to tobacco and ash mold

To explore the possible involvement of MaCFEM85 in the defense response against pathogens, it was examined whether overexpression of tobacco by MaCFEM85 would increase resistance to botrytis.

One week prior to the start of the bioassay, 150 adult aphids were inoculated onto 3 benthamiana plants, 50 per plant. After 72h, all adults were removed with a soft brush, nymphs continued to feed for 4 days, and then transferred to agrobacterium-infiltrated b.

To evaluate resistance to myzus persicae, gray mold, agrobacterium harboring PYBA-GFP, PYBA-MaCFEM85 were grown in LB at 28℃for 36 hours. Cells were washed three times in infiltration buffer (10 mM MgCl ₂ 10mM MES, 100. Mu.M acetokringnone, pH 5.6) to OD ₆₀₀ =0.6. Bacteria were infiltrated into the fully deployed leaves with a 1ml needleless syringe, three leaves per plant, and three plants per treatment were administered. There were 12 plants per experiment.

In terms of aphid performance measurement, after 12 hours of agroinfection, 20 adult aphids were applied to one infected leaf in the cage, each treatment was repeated 5 times, and the death rate of aphids and the number of newly produced nymphs were recorded daily for 3 consecutive days.

In the disease resistance measurement of the botrytis cinerea, after the newly cultivated botrytis cinerea is infected by agrobacterium for 12 hours, the newly cultivated botrytis cinerea is inoculated into a bacterial cake with the diameter of 5mm, and the growth surface of hypha is close to the infiltration area of the surface of the blade. The inoculated leaves and plants were placed under conditions of humidity above 70% and covered with plastic film in trays at 22 ℃ to promote disease occurrence. After 48h, disease progression in the leaf inoculation test was estimated by measuring lesion size.

After the constructed vector is transiently expressed in the raw tobacco leaves, the lesion on the aCFEM85 infiltrated leaf is obviously smaller than that of a control plant infiltrated by the eGFP vector (a graph in FIG. 7), and the size of the lesion is reduced by about 30% at 2 dpi. These data indicate that transient expression of MaCFEM85 in nicotiana benthamiana enhances resistance to botrytis cinerea.

Influence on the Aphis persicae population after transient expression of MaCFEM85

To investigate the effect of post-transient expression of MaCFEM85 on the myzus persicae population. MaCFEM85 was expressed transiently into tobacco leaves. The fertility of aphids was monitored by using the expression of Green Fluorescent Protein (GFP) as a control. 12h after infiltration, 20 peach adults were attached to each infiltrated leaf, exposing the infiltrated area to aphids. After 3d, mortality of aphid adults and larvae was recorded and the nymphs born every day were knocked out. The results showed that after macem 85 infiltration of the benthamiana plants, the average number of adult offspring per aphid was significantly lower than eGFP infiltrated benthamiana at 24h, 48h, 72h (panel b in fig. 7). It was demonstrated that transient expression of MaCFEM85 in Nicotiana benthamiana enhanced aphid resistance.

The above embodiments are only for understanding the technical solution of the present application, and do not limit the protection scope of the present application.

Sequence listing

<110> institute of plant protection of national academy of agricultural sciences

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Claims

1. The following applications of the metarhizium anisopliae CFEM85 protein:

activating allergic reactions in plants;

enhancing the resistance of plants to botrytis cinerea; or (b)

Enhancing the resistance of the plant to aphids,

the amino acid sequence of the metarhizium anisopliae CFEM85 protein is shown as SEQ ID NO. 1 or 2, and the plant is tobacco.

2. A method for improving the resistance of plants to botrytis cinerea and aphids, which is characterized by comprising the step of expressing a gene encoding a metarhizium anisopliae CFEM85 protein in plants, wherein the amino acid sequence of the metarhizium anisopliae CFEM85 protein is shown as SEQ ID NO. 1, and the plants are tobacco.

3. The method for improving the resistance of plants to botrytis cinerea and aphids according to claim 2, characterized in that the nucleotide sequence of the coding gene of the metarhizium anisopliae CFEM85 protein is shown in SEQ ID No. 3 or 4.