CN116854791A - Application of mucin-like Orm in improving environmental resistance of Beauveria bassiana - Google Patents

Abstract

The present application relates to mucinsOrmAn application in improving the environmental resistance of beauveria bassiana belongs to the technical field of biological pesticides. The application is constructed by using a genetic engineering means on the basis of beauveria bassiana strain with excellent propertiesOrmHigh-expression beauveria bassiana strain, and the coccidiosis with greatly improved environmental stress resistance and insecticidal activity is obtainedBeauveria bassiana OE-BbOrm. The beauveria bassiana OE-Bb of the applicationOrmCompared with wild type, the expression efficiency and the content of the mucin are improved by 4.5 times, the environmental stress resistance and the insecticidal effect of the mucin are greatly improved compared with those of wild type strains, the ultraviolet resistance is particularly improved by 27%, the high temperature resistance is improved by 20%, the oxidation resistance is improved by 31%, and the half-life time LT50 of grubs is shortened by 2.8 days.

Description

Mucins-like proteinsOrmApplication in improving beauveria bassiana environmental resistance

Technical Field

The present application relates to mucinsOrmAn application in improving the environmental resistance of beauveria bassiana belongs to the technical field of biological pesticides.

Background

Pest control is a problem which needs to be solved in the current production process of various crops. The underground pests are hidden due to underground activities, are internationally recognized serious pests which are difficult to predict and control, and mainly are seedlings, root systems, tubers and the like of harmful plants. Grubs are a large group of most widely distributed, most diverse and heaviest underground pests, which not only cause yield reduction, but also can cause diseases such as root rot and the like. The data show that 86% of the damage to the underground part of the plant is caused by grubs.

At present, chemical control is often adopted for controlling grub pests in the market, and the used chemical agents are phoxim, methyl iso Liu Lin and the like, and the chemical agents are used for seed dressing and soil mixing. However, this method has many problems, firstly, the excessive use of pesticides can cause environmental problems such as air pollution, water pollution, soil hardening, etc. And secondly, the resistance of grubs to pesticides can be improved by using the same pesticide for a long time, the drug resistance is generated, and the using effect is reduced. Finally, pesticide use can affect soil microbial populations and even interfere with natural ecological balance.

At present, high-toxicity chemical agents are gradually forbidden, the high-efficiency agents capable of preventing and treating grubs are reduced, the dosage form is single, the drug resistance of grubs is gradually increased, and biological prevention and treatment are the most ideal prevention and treatment means. Beauveria bassiana belongs to the subphylum of the half-known fungus, and is an insect pathogenic fungus with wide insecticidal spectrum and strong pathogenicity, and the functions of killing insects, activating soil and the like of the strain are fully exerted by planting, breeding and transferring on the root surfaces and rhizosphere of crops, so that the yield and the output value of organic crops are improved. The beauveria bassiana is used for controlling, and the control effect of 70-85% on grubs can be achieved. And the research and comparison show that the effect of mixing soil is higher than that of spraying, beauveria bassiana is applied to the soil, so that a good control effect can be achieved, and the pesticide effect can be continued for the next year. The effective component of beauveria bassiana for preventing and treating grubs is an infectious body of spores or hyphae, and the specific principle is as follows: (1) the conidia are attached to the host wall; (2) the spores germinate under proper conditions to form hyphae, and penetrate through the body wall to enter a host blood cavity under the double functions of mechanical pressure and enzymolysis; (3) fungi utilize nutrition in the host, the budding produces a large number of blastospores, and this cellular architecture is free to disperse and invade the host immune defenses within the host's blood cavity; (4) when the host is exhausted of nutrients, the blastospores form mycelia, penetrate the tissues and body walls of the host, perform saprophytic growth and generate conidia, and complete a life cycle. The process is natural infestation, and the insects do not develop resistance. However, the prior art shows that the traditional beauveria bassiana has weak pathogenicity and low environmental stress resistance, the existing microbial inoculum formula has the problems of low germination rate, poor stability and colonization effect and the like, and has poor effect in actual field application, so that the development of the beauveria bassiana microbial inoculum is restricted.

Disclosure of Invention

Aiming at the problems of weak pathogenicity, low environmental stress resistance, low germination rate, poor stability and colonization effect and the like of the traditional beauveria bassiana, the application constructs the beauveria bassiana strain with excellent properties by using a genetic engineering method on the basis of the beauveria bassiana strain with excellent propertiesOrmThe beauveria bassiana strain with high expression can obtain beauveria bassiana OE-Bb with greatly improved environmental stress resistance and insecticidal activityOrm。

The technical scheme of the application is as follows:

mucins-like proteinsOrmApplication of mucin-like protein in improving environmental resistance of beauveria bassianaOrmThe base sequence of (C) is shown as SEQ ID No. 1.

Another object of the application is a protective composition comprising a mucin-like proteinOrmApplication of microbial inoculum of (2) in improving environmental resistance of beauveria bassiana, and mucin-like proteinOrmThe base sequence of (C) is shown as SEQ ID No. 1.

Further, the application also comprises the prevention and treatment of grubs.

Another object of the application is to protect, a method ofOrm1Over-expressing engineering bacteria by constructing mucin-like proteinOrm1Gene full-length cDNA constitutive expression vector pAN52-Orm1-Bar, transforming beauveria bassiana, screening to obtain the composition of claim 1Orm1Mucin-like over-expression strain OE-BbOrm1，Namely, isOrm1And over-expressing engineering bacteria.

Preferably, the expression vector pAN52-Orm1The mucin-like gene in Bar is derived from beauveria bassiana Bb2860.

Preferably, the screening method is to pick up normal growth single colony, extract DNA, and detect each transformant by RT-PCR using specific primer A and specific primer BOrm1And 18sRNA as an internal reference; selecting the transformant with the highest expression quantity from the strain to obtain a screened strain;

the base sequence of the specific primer A is shown as SEQ ID No.2, and specifically comprises the following steps: 5'-GGTCGGCTCTTACATCAT-3';

the base sequence of the specific primer B is shown as SEQ ID No.2, and specifically comprises the following steps: 3 '-CAAGGTCGTAGTGCGTATA-5'.

The application selects beauveria bassiana as a raw material, and performs functional research on a plurality of genes of the beauveria bassiana on the basis of researching the molecular biology of the beauveria bassiana in the earlier stage. The sphingolipid content of beauveria bassiana is found to play an important role in environmental stress reaction, the sphingolipid is an important component of cell membrane structure, and the sphingolipid and metabolic intermediate products thereof can be used as important signal molecules in cell signal transduction, cell stress reaction and cellsPlays an important regulating role in the basic activities of cells such as growth, differentiation, aging, apoptosis and the like. Sphingolipids are synthesized starting from serine and soft acyl-CoA, and are condensed by serine-palmitoyl-transferase (SPT) catalysis to produce 3-ketosphingosine and release CoA and CO ₂ . SPT is used as an initiation and rate-limiting enzyme for sphingolipid synthesis and is composed of three proteins, TSC3p-LCB2p-LCB1p, whereas the regulatory factor of the sphingolipid synthesis pathway, the serotypes mucin (orosomucoid,Orm) By combining with SPT, the regulation of SPT enzyme activity is realized, so that the balance of sphingolipids in cells is influenced. Mucins-like proteinsOrmThe regulation of serine palmitotransferase SPT activity is a key step in the sphingolipid anabolic pathway (FIG. 1) suggesting that mucoid proteins in entomopathogenic fungi @Orm) Is a possible causative agent. Construction of fungi by means of genetic engineeringOrmSuper-expression vector, and transforming beauveria bassiana to obtainOrmAn over-expressed engineering bacterium. Through screening, the beauveria bassiana OE-Bb with greatly improved environmental stress resistance and insecticidal activity is obtainedOrm。

The beneficial effects of the application are that

The beauveria bassiana OE-Bb of the applicationOrmMucin-like protein [ ]Orm) Compared with the wild type, the expression efficiency and the content of the strain are improved by 4.5 times, the environmental stress resistance and the insecticidal effect of the strain are greatly improved compared with those of the wild type strain, the specific expression of the strain is improved by 27% in ultraviolet resistance, 20% in high temperature resistance, 31% in oxidation resistance, and the half-life time LT50 of grubs is shortened by 2.8 days; the production cost is reduced by 30 percent.

Drawings

FIG. 1 is a sphingolipid anabolic pathway;

FIG. 2 is a quantitative PCR assayOrmExpression level;

FIG. 3 shows colony sizes of different beauveria bassiana test strains;

FIG. 4 shows the spore yield of different beauveria bassiana test strains;

FIG. 5 shows the infection of beauveria bassiana OE-BbOrmIs stiff to grubs;

FIG. 6 shows the results of the determination of the LT50 of grub larvae by different beauveria bassiana test strains;

FIG. 7 is a graph showing the results of the determination of environmental stress resistance of different beauveria bassiana test strains.

Detailed Description

The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Example 1

The construction and screening of the beauveria bassiana strain comprise the following steps:

1. construction of mucin-like proteinsOrm) Genomic constitutive expression vectors

(1) Designing primer 1 (5' -AAA) according to the full-length cDNA nucleic acid sequence of beauveria bassiana Bb2860 mucin-like geneGGATCCCTTTCCAATCTGGCTGTTC-3', underlined as BamHI cleavage site) and primer 2 (5' -AAA)CCATGGTGATGGTGAGGTCCGTGT-3', underlined are NcoI cleavage sites).

(2) PCR amplification 4225 bp by using beauveria bassiana Bb2860 as templateOrmThe full-length cDNA sequence of the gene is cloned between BamHI and NcoI sites of a filamentous fungus expression vector pAN52-Bar, so thatOrmThe gene is under the control of a promoter from Aspergillus nidulans 3-phosphoglyceraldehyde dehydrogenase (gpdA) gene and a terminator of tryptophan (trpC) gene to obtain a mucoid genome constitutive expression vector pAN52-Orm-Bar。

(3) pAN52-OrmCaCl for Bar expression vector ₂ Transferring into colibacillus, amplifying, extracting mass products, linearizing by SpeI, removing phosphoric acid, extracting by phenol-chloroform to obtain the linearization constitutive expression vector.

2. Establishment of spore transformation system

(1) Taking a suspension of blastospore of tube bud from a refrigerator at-80 deg.C, thawing on ice, centrifuging at 4720 Xg for 5 min at 4 deg.C, and discarding the supernatant.

(2) To the blastospore pellet was added 240. Mu.L of 50% PEG 4000, 36. Mu.L of mol/L LiAc, 25. Mu.L of 4 g/L heat denatured salmon sperm DN in this orderA. mu.L of 0.1. Mu.g/. Mu.LSpeI digested pAN52-OrmBar and 35. Mu.L of 1 mol/L dithiothreitol.

(3) After fully mixing, ice bath is carried out for 30 min, and heat shock is carried out for 20 min at 42 ℃. The blastospores were collected by centrifugation at 4720 Xg at 4℃and suspended in 0.5. 0.5 mL double distilled water to give a spore suspension.

(4) 100. Mu.L of the spore suspension was spread evenly on a Petri plate containing 200. Mu.g/mL of herbicide grass Ding Lin (Phosphinothricin, PPT) and incubated at 25℃for 6 d.

(5) The colonies grown were transferred to a multi-well plate containing 300. Mu.g/mL PPT of Chlamydia medium and cultured continuously, and colonies that could continue to grow were selected as pending transformants for further analysis.

3. Transformation and screening

After 10 days, single colony growing normally in the previous step is picked up, DNA is extracted, and the specific primer pair (5'-GGTCGGCTCTTACATCAT-3' and 3 '-CAAGGTCGTAGTGCGTATA-5') is used for detecting each transformant by RT-PCROrmAnd 18s RNA was used as an internal reference. SelectingOrmThe transformant with the highest expression quantity is the strain-OE screened- BbOrm. (FIG. 2)

Example 2

The growth capacity, spore yield, infection capacity and environmental stress resistance of beauveria bassiana are measured, and the method comprises the following steps:

1. growth ability assay

(1) Taking wild beauveria bassiana BbUJN and gene defective beauveria bassiana deltaOrmAnd beauveria bassiana over-expression transgene strain OE-BbOrmIs formulated with 0.1% tween-80 sterile water to 1 x 10 ⁸ Individual/ml spore suspension.

(2) 100 mul of bacterial liquid is sucked by a pipetting gun and evenly coated on an SDAY culture medium plate, the bacterial liquid is cultivated at a constant temperature of 25-28 ℃, the morphological characteristics of the bacterial colony are observed, the size of the bacterial colony diameter is measured by a crisscross method (figure 3) at regular intervals, and the bacterial colony diameter is observed for 15 days.

2. Sporulation quantity measurement

(1) Taking wild beauveria bassiana BbUJN and gene defective beauveria bassiana deltaOrmAnd beauveria bassiana overexpression transgeneBecause of strain OE-BbOrmIs formulated with 0.1% tween-80 sterile water to 1 x 10 ⁸ Individual/ml spore suspension.

(2) And (3) sucking 100 mu l of bacterial liquid by using a pipetting gun, uniformly coating the bacterial liquid on an SDAY culture medium plate, and culturing at a constant temperature of 25-28 ℃.

(3) Bacterial pieces having a diameter of 5. 5 mm were periodically removed from each plate with a punch, spores were washed out by ultrasonic vibration in 1 ml of 0.1% tween-80 sterile water for 10 min, and the spore concentration was measured with a hemocytometer and converted into spore yield per unit area (fig. 4).

3. Toxicity determination

(1) Taking wild beauveria bassiana BbUJN and gene defective beauveria bassiana deltaOrmAnd beauveria bassiana over-expression transgene strain OE-BbOrmIs formulated with 0.1% tween-80 sterile water to 1 x 10 ⁸ Individual/ml spore suspension.

(2) Dipping 3-year-old healthy grub larvae by using the suspension, placing the grub larvae on sterile filter paper, crawling and airing moisture on a body, then placing the grub larvae into a sterile plastic lunch box, and taking crushed wood chips as food. Each component was divided into 3 duplicate areas, 30 heads per area, each immersed for 10 seconds, observed 1 time per day after inoculation, and the death number of the test insects was recorded, and 0.1% of tween-80 sterile water was used as a control.

(3) To confirm whether beauveria bassiana is lethal, the experimental beauveria bassiana is placed in a flat plate for constant temperature and humidity culture, hyphae growth and spore production are observed, and the test is carried out by tabletting, and observing whether the test is lethal to beauveria bassiana infection under a microscope (figure 5), and finally calculating half lethal time LT of grubs ₅₀ (FIG. 6).

4. Environmental stress resistance assay

(1) Taking wild beauveria bassiana BbUJN and gene defective beauveria bassiana deltaOrmAnd beauveria bassiana over-expression transgene strain OE-BbOrmIs formulated with 0.1% tween-80 sterile water to 1 x 10 ⁸ Individual/ml spore suspension. 100 mu L of the solution is uniformly coated in a concave ring of a glass slide, and is placed on a stage of an ultraviolet radiation instrument to perform 6 gradient doses (0.1-0.5J/cm) at room temperature ² ) UV-B radiation (312)[ mu.m.), 3 slides per strain were treated as replicates per dose. After irradiation, the slides were transferred to an incubator (25.+ -. 1 ℃ and 12L: 12D) for culturing for 24 hours under a 400 Xmicroscope, the number of spores germinated and ungerminated was counted, and the spore germination rate was calculated, thereby obtaining the ultraviolet resistance of the strain (FIG. 7).

(2) Taking wild beauveria bassiana BbUJN and gene defective beauveria bassiana deltaOrmAnd beauveria bassiana over-expression transgene strain OE-BbOrmIs formulated with 0.1% tween-80 sterile water to 1 x 10 ⁸ Individual/ml spore suspension. The spore suspension test tubes of different strains are placed in a constant temperature water bath at 45 ℃ for heat shock for 90 min, 100 mu L of spore suspension is sucked every 15 min into 1 ml germination liquid (GB), the culture is carried out at 25 ℃ for 24h, the spore numbers of germination and ungermination are counted by microscopic examination, and the spore germination rate is calculated, so that the high temperature resistance of the strain is obtained (figure 7).

(3) By H ₂ O ₂ As an oxidizing agent, ddH is used ₂ O is prepared into a solution of 10 mg/mL (w/v), and the solution is filtered and sterilized by a 0.22 mu m membrane for later use. After the SDAY culture medium is sterilized at 121 ℃ under the moist heat and the high temperature, 50 mu L H is added to each 50 mL culture medium ₂ O ₂ Mixing the solutions, pouring into a flat plate, cooling for use, and blank control without H ₂ O ₂ Is a SDAY plate of (C). Taking wild beauveria bassiana BbUJN and gene defective beauveria bassiana deltaOrmAnd beauveria bassiana over-expression transgene strain OE-BbOrmIs prepared with 0.1% tween-80 sterile water to give 1000 spores/ml. The spore suspension was spread evenly on the plates, 50. Mu.L per plate, and each group 3 was repeated for a total of 18. After completion of the coating, the resulting strain was transferred to an incubator (25.+ -. 1 ℃ C. And 12L: 12D) for cultivation of 4 d, and the diameters of colonies formed on each treatment plate were measured, and 5 colonies were measured per plate, thereby obtaining the antioxidant capacity of the strain (FIG. 7).

Claims (6)

1. Mucins-like proteinsOrmImproving beauveria bassiana environmentThe use of the mucin-like protein for resistance, characterized in thatOrmThe base sequence of (C) is shown as SEQ ID No. 1.

2. Contains mucin-like proteinOrmThe application of the microbial inoculum in improving the environmental resistance of beauveria bassiana is characterized in that the mucin-like proteinOrmThe base sequence of (C) is shown as SEQ ID No. 1.

3. The use according to claim 1 or 2, characterized in that it also comprises the control of grubs.

4. Mucin-like proteinOrmAn over-expressed engineering bacterium characterized by comprising a mucin-like structureOrmGene full-length cDNA constitutive expression vector pAN52-Orm-Bar, transforming beauveria bassiana, screening to obtain the mucin-like protein according to claim 1OrmIs an over-expression strain OE-Bb of (E)Orm，Namely, isOrmAnd over-expressing engineering bacteria.

5. An engineered bacterium as in claim 4, wherein the expression vector pAN52-OrmThe mucin-like gene in Bar is derived from beauveria bassiana Bb2860.

6. The engineering bacterium according to claim 4, wherein the screening method comprises picking up single colony with normal growth, extracting DNA, and detecting each transformant by RT-PCR using specific primer A and specific primer BOrmAnd 18sRNA as an internal reference; selecting the transformant with the highest expression quantity from the strain to obtain a screened strain;

the base sequence of the specific primer A is shown as SEQ ID No.2, and specifically comprises the following steps: 5'-GGTCGGCTCTTACATCAT-3';

the base sequence of the specific primer B is shown as SEQ ID No.2, and specifically comprises the following steps: 3 '-CAAGGTCGTAGTGCGTATA-5'.