CN115197305A - Bt protein Cry53A and gene and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology and discloses a Bt protein Cry53A, wherein an amino acid sequence is shown as SEQ ID NO. 2. The Bt protein Cry53A, the gene and the application thereof provide that Cry53A protein is Bt protein, has extremely strong killing toxicity to Spodoptera frugiperda, can resist the harm of Spodoptera frugiperda to crops, reduces the use amount of pesticides, reduces the cost and reduces the environmental pollution when being used for preparing transgenic plants, and the situation that pests generate resistance to the protein is not found in the effect verification test process of the invention. Therefore, the Bt protein Cry53A has important economic value and application prospect, and is suitable for large-scale application in improving the insect resistance of plants.

Description

Bt protein Cry53A and gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a Spodoptera frugiperda-resistant protein separated and cloned from Bacillus thuringiensis, and a coding gene and application thereof.

Background

Spodoptera frugiperda, also known as fall armyworm, pseudoarmyworm and the like, belongs to a Spodoptera of Trichoplusia (Lepidotera) Spodoptera of Noctuidae, and has the characteristics of wide habitat area, strong migration capacity and reproductive capacity, wide host range, large food intake and the like.

Spodoptera frugiperda, which was originally found in america, including regions of the united states, brazil and mexico, has been recognized as a sporadic but extremely destructive agricultural pest, was documented as a pest in the state of georgia in the united states as early as 1797, spodoptera frugiperda is generally recognized as unable to overwinter in regions other than tropical and subtropical regions, but, in summer and autumn where temperatures are favorable, it can also survive in temperate zones, in 1977, spodoptera frugiperda has invaded and formed middle east populations, and based on its differences in response to sex pheromones, spodoptera frugiperda was derived from the united states-caribbean area, rather than from brazil in south america, and subsequently, spodoptera frugiperda invades into places such as africa, africa and asia, becoming a major agricultural pest endangering global food production, 2016, spodoptera frugiperda was first discovered in africa, 2018, india was first recognized as a global crop pest, and was recently recognized as a global spodoptera crop pest, or a global spodoptera insect.

Spodoptera frugiperda belongs to a polyphagic insect which can eat 76 families in total, such as Poaceae, solanaceae, brassicaceae, caryophyllaceae and Compositae, and comprises more than 300 plants, such as corn, rice, sorghum, wheat, soybean, alfalfa, barley, buckwheat, cotton, oat, millet, peanut, ryegrass, beet, sudan grass, tobacco, tomato, potato and onion, but mainly gramineous rice and corn can cause serious yield loss, since Spodoptera frugiperda occurs in Africa in 12 Africa countries, about 830-2060 million tons of corn are produced each year, which is equivalent to 0.4-1 population of grains, in Argentina in south America, the damage can cause 72% of corn, even in developed countries with good control conditions, the damage in Florida can cause 20% of corn, more seriously, only 2-3 years, the spread in nearly 100 world countries, and the loss of Bt is relatively low in North America, and the cause relatively high Bt loss.

Bacillus thuringiensis is a rod-shaped gram-positive bacterium and is listed as one of the eighteenth group of bacilli in the second category, ernst Berlinier is separated from the diseased larva of the pink borer in Suyun province of Germany again in 1911, the Bacillus thuringiensis is formally named at the moment, mattes is separated from the pink borer again in 1927 in attempt to apply Bt to biological control by human beings, a plan for controlling the pink borer is started in the U.S. in 1928, and a foundation is laid for the Bt to form a commercial product in 1929 for the first time.

Since the first commercial product Sporeine containing Bt components was sold in France for insect prevention and control in 1938, 1956, scientist Angus research determined that the main insecticidal activity of Bt comes from crystal protein contained in Bt, in 1988, mengshanda company transferred Bt protein gene into cotton to obtain the first batch of transgenic insect-resistant cotton, in 1996, mengshanda company proposed insect-resistant Bt transgenic cotton, and then Bt transgenic cotton and Bt corn were widely popularized in the world at an beyond imaginable speed, and according to ISAAA statistics, about 12% of transgenic insect-resistant crops are occupied in the crop planting area in 2019 in the world.

The bacillus thuringiensis is widely distributed, dust in the air from soil on the ground is distributed from rivers to mountains, dust in the air is distributed from Antarctic frozen soil to tropical rainforests, and the dust is distributed from plain to desert, so that bacillus thuringiensis strains and gene resources are abundant, the bacillus thuringiensis strains and gene resources are the most important insecticidal gene resources all over the world, and by 2020, according to a new classification method, scientific researches have found that 79 groups of 741 Cry proteins (https:// www.bpprc.org /), the insecticidal principle of bacillus thuringiensis mainly depends on insecticidal crystal protein (Cry) thereof, the protein is decomposed into toxic polypeptides in pests, the polypeptides are combined with specific receptor proteins of epithelial cells of the pests intestines, cell membranes are damaged, and the pests die, the epithelial cells of the human intestines have no corresponding receptor proteins, and the PH environment has large difference, so the proteins are safe for human beings and mammals, and the bacillus thuringiensis is mainly separated from the soil at present, and the self is not part of a natural ecological system of the earth environment.

The invasion development process of foreign species comprises three stages of invasion, colonization and outbreak, spodoptera frugiperda has invaded and colonizes China in 2019, the outbreak stage is entered after 2020, the prevention and control of Spodoptera frugiperda in China currently comprises chemical prevention and control, physicochemical induction and control, agricultural prevention and control, biological prevention and control and cultivation of resistant varieties, the chemical prevention and control is the most common method for preventing and controlling Spodoptera frugiperda, and pesticides such as cyfluthrin, chlorantraniliprole, cis-cypermethrin and carbosulfan are commonly used internationally to prevent and control Spodoptera frugiperda, but the excessive use of the chemical pesticides can cause the problems of increased production cost, environmental pollution, edible safety, damage to an ecological system and the like, and even pests generate drug resistance; physical and chemical trapping and control mainly refers to trapping and killing Spodoptera frugiperda imagoes by using lamp trapping, sexual trapping and food trapping technologies, but for larval stages which are harmful to crops, agricultural control mainly takes measures from the aspects of cultivation management, crop layout, variety resistance and the like so as to create a farmland ecological environment which is not beneficial to generation and harm of Spodoptera frugiperda, only part of agricultural control can be controlled, and the control cannot be fundamentally controlled; the biological control mainly refers to the control of spodoptera frugiperda by utilizing natural enemy insects, microbial pesticides, plant-derived pesticides and entomopathogenic nematodes, in China, predatory natural enemy insects of spodoptera frugiperda, such as stinkbug, yellow-banded rhinoceros sutake, southern yellow-banded stinkbug and the like, but China does not have specific parasitic natural enemies of spodoptera frugiperda, only can introduce natural enemies of the natural environment in the America, such as Nostoc exis, aphana longissima, and Scopus pervirens, and the like.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects in the prior art, the invention provides a Bt protein Cry53A, a gene and an application thereof, so as to provide a novel Spodoptera frugiperda Bt protein resource.

(II) technical scheme

In order to achieve the purpose, the invention provides the following technical scheme:

the amino acid sequence of the Bt protein Cry53A is shown in SEQ ID NO. 2.

Furthermore, the amino acid sequence of the Bt protein is the amino acid sequence of the protein with the same function expressed by the sequence shown in SEQ ID NO.2 through substitution, deletion and/or addition of one or more amino acids.

It is understood that one skilled in the art can substitute, delete and/or add one or more amino acids according to the amino acid sequence (SEQ ID No. 2) of the Cry53A protein disclosed in the invention without affecting the activity thereof to obtain a mutant sequence of the protein, wherein a transmembrane region is formed between 86 and 108, the amino acids constituting the transmembrane region protein are mostly hydrophobic amino acids, and usually, the membrane protein cannot be expressed in a prokaryotic expression system, so that the Bt protein Cry53A of the invention also comprises a Cry 53A-derived protein with the same activity as the Bt protein Cry53A by substituting, replacing and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.

A gene encoding the Bt protein Cry53A as claimed in the preceding claims, the nucleotide sequence is as follows; or the nucleotide sequence shown in SEQ ID NO.1 is substituted, deleted and/or added with one or more nucleotides, and can encode the nucleotide sequence of the same functional protein.

Furthermore, it will be appreciated that, given the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons suitable for expression in a particular species as desired.

The gene and the protein can be cloned or separated from Bt strain PMS45-2, or obtained by a DNA or peptide synthesis method.

The gene of the invention can be operably connected with an expression vector to obtain a recombinant expression vector capable of expressing the protein of the invention, and the expression vector can be further introduced into a host by a transgenic method such as an agrobacterium-mediated method, a gene gun method, a pollen tube channel method and the like to obtain a transformant of a transgenic Cry53A gene, namely a plant endangered by spodoptera frugiperda, so that the plant has insect-resistant activity.

In addition, fermentation liquor containing Cry53A protein can be obtained by fermenting the strain PMS45-2 disclosed by the invention, and the fermentation liquor can be prepared into an insecticide for preventing and controlling Spodoptera frugiperda. The skilled person can also transform the above genes into bacteria or fungi to produce the Bt protein of the invention by large-scale fermentation.

A recombinant expression vector containing the gene.

A host cell containing the above expression vector.

Further, the host cell expresses the protein.

Feed containing the Bt protein.

The Bt protein is applied to improving the Spodoptera frugiperda resistance of plants.

The application of the gene in cultivating transgenic plants, preparing pesticides or improving the insect resistance of plants.

Those skilled in the art can also transform the Cry53A gene disclosed by the invention into crops such as wheat, corn, rice, vegetables and the like which are damaged by Spodoptera frugiperda, so that the crops have corresponding insect-resistant activity, such as: utilizing degeneracy of codon, designing Cry53A gene into gene sequence with corn preferred codon, connecting the synthesized Cry53A gene sequence with vector, transferring the gene sequence into corn genome by means of agrobacterium mediation so as to obtain transgenic corn variety with Spodoptera frugiperda resistance activity

(III) advantageous effects

Compared with the prior art, the invention provides a Bt protein Cry53A, a gene and an application thereof, and has the following beneficial effects:

the Bt protein Cry53A, the gene and the application thereof provide that Cry53A protein is Bt protein, has extremely strong killing toxicity to Spodoptera frugiperda, can resist the harm of Spodoptera frugiperda to crops, reduces the use amount of pesticides, reduces the cost and reduces the environmental pollution when being used for preparing transgenic plants, and the situation that pests generate resistance to the protein is not found in the effect verification test process of the invention. Therefore, the Bt protein Cry53A has important economic value and application prospect, and is suitable for large-scale application in improving the insect resistance of plants.

Drawings

FIG. 1 shows signal peptide analysis of the Cry53A gene using a Desitai bioinformatics tool;

FIG. 2 shows a transmembrane region prediction analysis of the Cry53A gene using a Desitai bioinformatics tool;

FIG. 3 shows the comparison of amino acids after knocking out transmembrane region and modifying base of Cry53A gene with the amino acids before modification;

FIG. 4 shows a vector map of recombinant plasmid pET30a-Cry 53A;

FIG. 5 shows a restriction enzyme digestion identification map of recombinant plasmid pET30a-Cry53A, wherein Lane 1 is DNA marker, and Lane 2 is restriction enzyme digestion recombinant plasmid pET30a + Cry53A;

fig. 6 shows SDS-PAGE detection map of Cry53A gene expression in e.coli BL21 (DE 3);

wherein, the Lane M is protein marker (molecular weight from top to bottom: 160, 120, 700, 50, 40, 35, 25, 20, 10 KDa); lane 0 is control (no IPTG added); line 1 is an expression protein of E.coli BL21 (DE 3) containing a vector pET-30a, induced for 16h at 15 ℃ by IPTG; line 2 is the supernatant of E.coli BL21 (DE 3) containing the vector pET-30a after the whole bacteria is broken; line 3 is the precipitate after the whole bacteria of E.coli BL21 (DE 3) containing the vector pET-30a are broken;

FIG. 7 shows the result of SDS-PAGE analysis of purification of Cry53A protein from inclusion bodies.

Wherein, the Lane M is protein marker (molecular weight from top to bottom: 160, 120, 700, 50, 40, 35, 25, 20, 10 KDa); line 1 is supernatant obtained after the inclusion body is dissolved and centrifuged; line 2 is an effluent liquid of the supernatant liquid and the supernatant liquid after the supernatant liquid and the Ni-IDA are incubated; line 3 is the elution fraction of 50mM Imidazole; line 4-6;

FIG. 8 shows that after three days of feeding, the larvae were observed to grow to essentially two instars, whereas the feed formulated with 200ng/ml Cry53A protein, the first instars died mostly after consumption, while the larvae at 2 μ g/ml but fed all died. (Note: the scale bars in the figure are all 1 mm).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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 obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The first embodiment is as follows: analysis of original sequence and codon bias modification of Cry53A protein coding

Before prokaryotic protein expression is carried out, signal peptide prediction is carried out on a sequence, and the signal peptide is not shown in a figure 1 in the sequence after testing.

Protein sequences are analyzed by transmembrane region prediction software, and 23 amino acid residues in a region spanning from 79 to 101 through cell membranes are shown in a blue region of SEQ ID No.3, and transmembrane region sequences need to be knocked out before prokaryotic expression.

Under the condition that the amino acid sequence coded by the original Cry53A gene is not changed, the preferred codon of escherichia coli is used for optimization, the content of G + C is improved, and certain sequences which can possibly reduce expression are avoided, the modified condition is shown in a table 1, and the amino acid sequence pair before and after modification shown in a modified Cry53A gene sequence SEQ ID No.3 is shown in a figure 3.

TABLE 1 before and after reforming

Example two: cry53A gene synthesis and construction

The novel Bt gene (Cry 53A) is obtained by separating and cloning Bt bacteria PMS45-2 collected from Sichuan forest in the laboratory and modifying according to the favorite codon of escherichia coli gene, a Cry53A gene is obtained by adopting a whole-gene synthesis method, pET30a plasmid is processed by NdeI and HindIII endonuclease to be linearized, the Cry53A gene is connected to the linearized pET30a plasmid through T4 ligase to obtain recombinant plasmid pET30a-Cry53Ag (the recombinant plasmid map is shown in figure 4), then the recombinant plasmid pET30a-Cry53A is converted into DH5a to obtain a large number of recombinant plasmids, the primary identification of the recombinant plasmids is completed by an enzyme cutting method, the recombinant plasmid is processed by XbaI/XhoI endonuclease, the enzyme cutting result is detected by agarose gel electrophoresis, the recombinant plasmid after enzyme cutting is separated by electrophoresis, two strips appear, the size of the strips accords with the size of the target gene, and the sequencing is consistent with the original gene sequence, and shows that the pET30a-Cry2Ag1 vector is successfully constructed.

Example three: cry53A protein acquisition

The correct pET30a-53A vector was transferred into a recipient bacterium E.coli.BL21 (DE 3) (purchased from Beijing Quanjin Biotechnology Co., ltd.), a single clone was selected from the transformed plate, inoculated into 4mL of LB medium (containing 50. Mu.g/mL of kanamycin sulfate) to be cultured to OD600 of 0.5-0.8, and 0.5mM IPTG was added to the test tube culture solution to a final concentration, followed by induction of expression at 37 ℃.

The cells were grown up to OD600=0.8 by scale-up culture, and after induction at 15 ℃ for 16h, the cells were collected (without purification on the same day, cells were frozen at-20 ℃).

Centrifuging the induced culture solution at 12000rpm for 5min, removing the supernatant, adding PBS (sodium dodecyl sulfate) solution to resuspend the precipitate, finally adding SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) loading buffer to heat the sample at 100 ℃ for 10min, centrifuging and taking the supernatant for electrophoresis, carrying out ultrasonic lysis on the whole bacteria by using 20mM Tris (pH 8.0), 300mM NaCl,20mM Imidazole containing 1% Triton X-100,1mM DTT and 1mM PMSF, taking the supernatant and the precipitate for SDS-PAGE analysis detection, and as shown in figure 5, the Cry53A molecular weight is about 79kDa and is consistent with the predicted protein molecular weight, and the detection result shows that the protein is expressed in an inclusion body.

After the inclusion bodies were washed with 20mM Tris (pH 8.0), 300mM NaCl 1% Triton X-100,2mM EDTA, and 5mM DTT, the inclusion bodies were solubilized with 20mM Tris (pH 8.0), 300mM NaCl,8M Urea, and 2mM Imidazole buffer while equilibrating the Ni-IDA column, and finally the target protein was eluted with equilibration buffers of different concentrations of Imidazole, and each eluted fraction was collected for SDS-PAGE analysis, the results of which are shown in FIG. 7.

Relatively high purity Lane 5-6 was collected by Ni-IDA affinity chromatography, dialyzed into buffer solution [1 XPBS (pH 7.4), 4mM GSH,0.4mM GSSG,0.4M L-Arginine,1M Urea,10 Glycerol ] at 4 ℃ in a dialysis bag after treatment, renatured, and finally dialyzed into 1 XPBS (pH 7.4) and 10 Glycerol for about 6-8 hours, and after the renaturation, the supernatant was filtered with a 0.22 μm filter, aliquoted, and frozen to-80 ℃.

Example 4: cry53A protein stability test (freeze-thaw experiment) and concentration determination

Taking out the protein from a refrigerator at minus 80 ℃, placing the protein in an ice water bath for 5-10min until the protein is slowly melted, placing the melted protein in a refrigerator at 4 ℃ for 0.5h, and indicating that the protein freeze-thaw experiment is normal without abnormal phenomenon.

Protein concentration was measured using the Bradford protein concentration assay kit and the concentrations are given in table 2 below.

TABLE 2 Cry53A protein concentration

Example 5: determination of insecticidal Activity of proteins

The Cry53A protein obtained in example 3 was assayed for insecticidal activity against spodoptera frugiperda.

Diluting a protein solution to four concentration gradients of 10ng/ml, 100ng/ml, 1ug/ml and 5ug/ml by using 1 XPBS buffer solution (phosphate buffered saline), adding 1ml of each protein solution into feed, using 1ml of the 1 XPBS solution as a negative control, using clear water as a blank control, cutting the feed after solidification, putting the cut feed into a low-age larva tank, lightly inserting the pre-hatched larvae with a writing brush, repeating the steps for 3 times in each larva tank, placing the larva tanks in an illumination incubator for culture, checking the death rate of the larvae after 3d, 7d and 14d, using the writing brush to lightly touch the larva bodies, determining the larva as dead, and counting the death rate.

Mortality rate: (number of dead test insects/total number of test insects before treatment). Times.100%

LC50 was calculated using SPSS 10.0 software and the results are shown in Table 3.

TABLE 3 insecticidal Activity of Cry53A

According to the biological activity assay results of Table 3, the Cry53A expression product has a good insecticidal activity against Spodoptera frugiperda with a semilethal concentration LC50 of 0.1683. Mu.g/mL (95% confidence interval: 0.0290-0.6498. Mu.g/mL); while the negative control PBS buffer and the blank control had no insecticidal activity against Spodoptera frugiperda.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (9)

1. The Bt protein Cry53A is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.

2. The Bt protein Cry53A according to claim 1, wherein the amino acid sequence of the Bt protein is the amino acid sequence of the protein expressed by the same functional protein, which is obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown by SEQ ID NO. 2.

3. A gene for the Bt protein Cry53A according to claim 1 or 2, wherein said nucleotide sequence is as set forth in; or the nucleotide sequence shown in SEQ ID NO.1 is substituted, deleted and/or added with one or more nucleotides, and can encode the nucleotide sequence of the same functional protein.

4. A recombinant expression vector containing the gene of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. The host cell of claim 5, wherein the host cell is E.coli.

7. A pesticide containing the Bt protein of claim 1 or 2.

8. Use of the Bt protein of claim 1 or 2 for increasing insect resistance in plants.

9. Use of the gene of claim 2 for breeding transgenic plants, preparing pesticides or improving the insect resistance of plants.

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