CN108642029B - Metarhizium anisopliae protease Pr1C and its gene and application - Google Patents

Abstract

The invention belongs to the field of agricultural biotechnology, and in particular relates to metarhizium anisopliae protease Pr1C and its gene and application. The present invention obtains the extracellular protease Pr1C gene of Metarhizium anisopliae through gene cloning technology, and obtains the Pr1C protein through expression. The function verification of the protein is carried out through the method of artificially feeding locusts, and it is found that the protease Pr1C can significantly increase the virulence of Metarhizium anisopliae , to promote the efficiency of Metarhizium anisopliae in killing locusts.

Description

Metarhizium anisopliae protease Pr1C and its gene and application

technical field

The invention belongs to the field of agricultural biotechnology, and in particular relates to metarhizium anisopliae protease Pr1C and its gene and application.

Background technique

Locust is the main agricultural pest in my country. It has the characteristics of various types, serious damage, and frequent outbreaks. It often causes heavy losses to our national economy, especially agricultural production. In recent years, with the increasing trend of global warming, high-density locust swarms have appeared from time to time, and the risk of long-distance migration has intensified, seriously threatening the grain production in my country's main agricultural production areas. Existing pest control is mostly based on emergency control and chemical control, which relies too much on chemical pesticides, resulting in environmental pollution. Therefore, it is urgent to develop new green and pollution-free pest control methods.

Contents of the invention

In order to overcome the problem of relying on chemical pesticides and not being able to prevent in advance in the existing locust disease control methods, the invention provides a Metarhizium anisopliae protease Pr1C, which can significantly increase the virulence of Metarhizium anisopliae and improve the rate of killing of Metarhizium anisopliae. Locust efficiency.

The object of the present invention is to provide a metarhizium anisopliae protease Pr1C.

Another object of the present invention is to provide a gene encoding the above-mentioned Metarhizium anisopliae protease Pr1C.

Another object of the present invention is to provide a recombinant expression vector containing the gene encoding the above-mentioned metarhizium anisopliae protease Pr1C.

Another object of the present invention is to provide a recombinant strain containing the gene encoding the above-mentioned Metarhizium anisopliae protease Pr1C.

Another object of the present invention is to provide a method for preparing Metarhizium anisopliae protease Pr1C.

Another object of the present invention is to provide the use of Metarhizium anisopliae protease Pr1C.

According to a specific embodiment of the present invention, the amino acid sequence of the metarhizium anisopliae protease Pr1C of the present invention is shown in SEQ ID NO: 1:

The total number of amino acids of the above-mentioned Metarhizium anisopliae protease Pr1C is 708, and the weight average molecular weight is 75036.

According to a specific embodiment of the present invention, the nucleotide sequence of the gene encoding the above-mentioned Metarhizium anisopliae protease Pr1C is as shown in SEQ ID NO: 2:

According to a specific embodiment of the present invention, the present invention provides a recombinant expression vector comprising the gene encoding the above-mentioned metarhizium anisopliae protease Pr1C, preferably the vector is a pET-Pr1C plasmid.

According to a specific embodiment of the present invention, the present invention provides a recombinant bacterial strain comprising the gene encoding the above-mentioned metarhizium anisopliae protease Pr1C, and the bacterial strain is preferably Escherichia coli.

According to a specific embodiment of the present invention, the method for preparing Metarhizium anisopliae protease Pr1C comprises the following steps:

(1) Insert the expression vector comprising the above-mentioned metarhizium anisopliae protease Pr1C gene;

(2) Transform host cells with a recombinant expression vector comprising the gene encoding Metarhizium anisopliae protease Pr1C;

(3) Cultivate host cells to induce the expression of Metarhizium anisopliae protease Pr1C.

Metarhizium anisopliae (Metarhizium anisopliae) is a widespread entomopathogenic fungus, it can parasitize a variety of pests and invade host pests through the body surface, proliferate continuously in the pests, consume nutrients, mechanically penetrate, produce toxins, and Continuously spread among the pest populations, causing the pests to die. Metarhizium anisopliae has a certain specificity, is harmless to humans and animals, and also has the advantages of not polluting the environment, and pests will not develop drug resistance. Using it to develop microbial insecticides has a good development prospect. However, in the actual application of Metarhizium anisopliae, it takes a long time to take effect and is greatly affected by the environment.

Metarhizium anisopliae extracellular protease Pr1C according to the present invention can act on the carboxyl group of aspartic acid and glutamic acid in the insect epidermis, degrade protein, by artificially feeding locusts with Metarhizium anisopliae, produce synergistic effect, improve metarhizium anisopliae The efficiency of bacteria killing locusts can significantly increase the virulence of Metarhizium anisopliae.

Description of drawings

Fig. 1 shows the protein expression situation of zombalinase Pr1C;

Figure 2 is a line chart of locust survival rate.

Detailed ways

1. Source of tested insects

The purified population of migratory locusts used in the experiment was artificially reared population. Migratory locust eggs are hatched in an artificial climate incubator, the hatching conditions are: temperature 30±2°C, humidity 60%, light time:dark time=16h:8h. The locust gnats of the same size hatched at the same time were transferred to insect cages (60cm×50cm×60cm), and were raised with fresh wheat seedlings until adults. Raising conditions: light time: dark time=16h:8h, temperature 30±2°C, relative humidity 60%.

2. Tested strains

Metarhizium anisopliae IPPM202 has high virulence to migratory locusts in indoor bioassays.

Under the condition of 28°C, cultivate them with PDAY medium (potato 200g, agar 20g, sucrose 20g, yeast powder 5g, water 1000mL) for 15 days, scrape off the conidia and dry them and store them at 4°C. Before use, calculate the spore content and determine the spore germination rate under the hemocytometer, and process according to the effective spore content.

Example 1 Clone Metarhizium anisopliae Pr1C gene

Metarhizium anisopliae total RNA was extracted with Separation reagents were used to extract the RNA of Metarhizium anisopliae tissue samples, the reverse transcription kit was used for reverse transcription, the Pr1C gene was cloned, the specific primers were shown in Table 1, and the Metarhizium anisopliae protease with nucleotide sequence as shown in SEQ ID No.2 was obtained Pr1C gene.

Table 1 The specific primers for cloning Pr1C gene

Metarhizium anisopliae protease Pr1C gene was digested, and the digested product was inserted into pET-21b expression vector to obtain plasmid pET-Pr1C.

The ligation product was transformed into Escherichia coli DH5a strain, screened according to the marker of the recombinant vector (anti-Amp), single spot was picked, a small amount of plasmid was extracted by alkaline lysis, and preliminary identification was performed by double enzyme digestion. The plasmid pET-Pr1C was transformed into BL21(DE3) competent bacteria. SDS-PAGE electrophoresis was performed to detect the expression of the target protein, and the results are shown in Figure 1.

Embodiment 2 Measuring the survival rate of locusts after feeding Metarhizium anisopliae PR1C protein

The 3rd instar nymphs of locusts with the same growth state were selected for the experiment, and 3 treatment groups and 2 control groups were set up. The treatment groups were: PR1C + Metarhizium anisopliae strain 202 treatment, PR1C treatment, Metarhizium anisopliae strain 202 treatment; the control group was: Induced bacteria, blank group. Each treatment group and control group had 5 replicates, and each replicate had 30 locusts.

Put the selected locusts into a constant temperature incubator and cultivate them at 28°C with a light-to-dark ratio of 16h:8h. After starvation treatment for 12h, they were fed with artificial feed, and fresh wheat seedlings were replaced after 24h. The number of deaths per day was recorded. The results of the survival rate of locusts are shown in the table 2 and Figure 2:

Table 2 Comparison table of locust survival rate

According to the experimental results, the natural survival rate of the blank control group of locusts without treatment dropped from initial 84.7% to 77.3%, and the natural death rate was 7.5%; after adding PR1C alone in the feed, the survival rate of locusts dropped from 78.7% to 71.3%, the mortality rate was 7.4%, and there was no change compared with the blank group; but after feeding locusts with PR1C and Metarhizium anisopliae strains, the survival rate of locusts dropped from 71.3% to 36.7% within 5 days, and the survival rate Only half of the original, the locust survival rate was only 28% on the 10th day.

The protease Pr1C of the present invention can act on the carboxyl groups of aspartic acid and glutamic acid in the insect epidermis to degrade proteins. The protease PR1C and Metarhizium anisopliae produce a synergistic effect, which significantly improves the virulence of Metarhizium anisopliae. play an important role in pathogenicity.

sequence listing

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Claims (10)

1. The metarhizium anisopliae protease Pr1C is characterized in that the amino acid sequence is shown as SEQ ID NO: 1 is shown.

2. The destruxins Pr1C gene, characterized in that the destruxins Pr1C gene encodes the destruxins Pr1C of claim 1.

3. The metarhizium protease Pr1C gene of claim 2, wherein the sequence of the metarhizium protease Pr1C gene is as shown in SEQ ID NO: 2, respectively.

4. A recombinant expression vector comprising the destruxins Pr1C gene of claim 2.

5. A recombinant strain comprising the destruxins Pr1C gene of claim 2.

6. A process for the preparation of the destruxins Pr1C of claim 1, comprising the steps of:

(1) inserting a gene encoding the destruxins of Metarrhizium anisopliae Pr1C of claim 1 into an expression vector to obtain a recombinant expression vector;

(2) transforming a host cell with the recombinant expression vector obtained in the step (1);

(3) culturing host cells, and inducing and expressing the destruxin Pr 1C.

7. The use of the metarhizium protease Pr1C of claim 1.

8. The use of the metarhizium protease Pr1C of claim 1 for controlling locust diseases.

9. The use of the destruxins of Metarhizium anisopliae Pr1C of claim 7, wherein the destruxins of Metarhizium anisopliae Pr1C is used in combination with Metarhizium anisopliae.

10. A method for controlling locusts, which comprises the step of applying the metarhizium protease Pr1C of claim 1 in combination with metarhizium anisopliae to kill locusts.