Proteins and their use in increasing virulence of bacillus thuringiensis parasporal crystal proteins

Abstract

The present application provides a protein and its use in improving the virulence of a parasporal crystal protein of Bacillus thuringiensis, which comprises a protein having an amino acid sequence shown in SEQ ID NO: 1. The protein of the present application can effectively improve the virulence of the parasporal crystal protein, especially the insecticidal capacity, after cooperating with the parasporal crystal protein, thereby reducing the use amount of the parasporal crystal protein, lowering the insecticidal cost, improving the insecticidal efficiency, and being suitable for large-scale popularization and application.

Description

Technical Field

[0001] This invention relates to the field of biology. Specifically, this invention relates to proteins and their use in enhancing the virulence of parasporal crystal proteins of Bacillus thuringiensis. Background Technology

[0002] The initial understanding of Bacillus thuringiensis (Bt) originated in the early 20th century when Asian expert Shigetane Ishiwata extracted Sottbacteria, a type of Bacillus thuringiensis var. sot, from silkworms (Bombyx mori) suffering from damping-off disease. Berliner isolated a bacterium from the Mediterranean mealybug (Anagasta kuhniella Zeller) infecting the region of Thüing, Germany, and named it Bacillus thuringiensis after detailing its morphology and culture characteristics. The first commercially available Bt preparation, Sporeine, appeared in France in 1938, primarily targeting the Mediterranean mealybug.

[0003] A prominent feature of Bacillus thuringiensis is the production of parasporal crystal proteins during spore formation. These parasporal crystal proteins, composed of one or more proteins, possess highly specific insecticidal activity and are commonly referred to as insecticidal crystal proteins. Existing as protoxins, these proteins dissolve in an alkaline environment upon entering the digestive tract of susceptible insects and are activated by proteases into toxic peptides. These activated peptides bind to receptors on the border membrane of the insect's gut, forming pores in the cell membrane, disrupting osmotic balance, causing cell lysis, and ultimately leading to larval death.

[0004] Currently, to improve the bactericidal properties of parasporal crystal proteins, the main technical methods employed include screening and mutagenesis of highly virulent strains and de novo design of high-performance toxic proteins. However, strain mutagenesis screening is inefficient and costly, while de novo protein design technology has high requirements and struggles to meet stability and other agricultural requirements in a single step. Therefore, highly efficient, stable, and cost-effective synergistic substances are attracting increasing attention. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, this invention proposes a protein and its use in enhancing the toxicity of parasporal crystal proteins of Bacillus thuringiensis, as well as its preparation method, composition, insecticide, method for enhancing the toxicity of parasporal crystal proteins of Bacillus thuringiensis, and insecticidal method. When the protein of this invention works synergistically with the parasporal crystal protein, it can effectively enhance the toxicity of the parasporal crystal protein, especially its insecticidal ability, thereby reducing the amount of parasporal crystal protein used, lowering insecticidal costs, and improving insecticidal efficiency, making it suitable for large-scale application.

[0006] In one aspect of the invention, a protein is provided. According to an embodiment of the invention, the protein comprises a protein having the amino acid sequence shown in SEQ ID NO: 1.

[0007] While researching how to enhance the toxicity of parasporal crystal proteins, the inventors discovered that mixing Bacillus thuringiensis supernatant with parasporal crystal proteins could increase the toxicity of the parasporal crystals. Furthermore, the inventors deduced that the presence of certain proteins in the supernatant might be responsible for this effect. Further, the inventors isolated and screened the proteins in the supernatant, finding that a protein with the amino acid sequence shown in SEQ ID NO: 1 (also referred to as the "synergistic protein") itself did not have insecticidal activity, but when acting in conjunction with parasporal crystal proteins, it could significantly enhance the toxicity of the parasporal crystal proteins, especially their insecticidal ability. This reduces the amount of parasporal crystal protein used, lowers insecticidal costs, and increases insecticidal efficiency, making it suitable for large-scale application.

[0008] SVRYFANQIGHHTGKTWFFSNTHWRLQYQSSKFHWPRIRHAWYKHAFGEQNRNRHVWVLNEGYDYCCEMDWECNASALWQSAQICFLHHRASCIYEHGEPPKQRPAQDPWNHTREYRQYPSLHHDQYLEGLRTSVYRTFEHDPTLIMILHPYLAMRECPCQHLHEQHPALKIVKGCWIHYDKFNATGAPELLRSLDANLVAPYPIHVGDLEAPDDRPPQCMTGCQDIIFFYGQWLTISRMLFECFPMECSHLMCNHVEPPCQRDQNSWLPHTEMVKAWHQMGGDPSSRFHFLDKMDTDAVMNMWEYLCKYLELDDQENKDFVWAKKSNGVWYALVGQCHLSQCCLPEVYRCHCMKIRMDEMCTYDLPKKLADITAVASQSTSPPIMEVHHMFKMMDVCCCMRNQSAKDICVIVSEQIKEWCWQDEHDMRIVRTTMFNWEQTMTFSPTLHRNCVGKVVNLREMRDNALYWVEEEGEPRMDPRNFVTYIQNMPANTLFWKSSGHDMTEDLWWCQNDCGERVVRLFPLMQFYHASAKAKCRLHYYIIHRLKMFCKFLITKLYTVRPVGHFHCHSYFWQCYDMGMWHSGYGMAPQEELPQCMHESGWDGCIMILEFFIMCPTETIMDGHKAEVAEKLQDDTQECNRYQTGTAPGWNCDFNVQEQDAGSNIDKYWAPLGLWQLWIKVREIIWEHWMVLEHRYKWCCLDMRNIQFKLSTDKPRSYDECHTTCAFTGHFKDRVGWWLSFTDPEMAWHQFFGNAASPWAKMRFVIQITGYIQPKKHQKKPFSKRMAPCVPRLSWQLMGRVGPLWPCASNLRQMSLEPMATELWVDEIKWMHWRRCPLLPVPNHVKIAVLGCRPLPCVVINISRCMHDFIWYESETVTRLNNFQNYASGNYFKRPYGFPDGVFRITHWNIVKSH(SEQ ID NO:1)

[0009] In another aspect of the invention, the present invention proposes the use of the aforementioned protein in enhancing the toxicity of parasporal crystal proteins of Bacillus thuringiensis. As previously described, when a protein having the amino acid sequence shown in SEQ ID NO: 1 acts in conjunction with a parasporal crystal protein, it can significantly enhance the toxicity of the parasporal crystal protein, particularly its insecticidal ability, thereby reducing the amount of parasporal crystal protein used, lowering insecticidal costs, and improving insecticidal efficiency, making it suitable for large-scale application.

[0010] According to an embodiment of the present invention, the protein is used to enhance the insecticidal ability of parasporal crystal proteins.

[0011] In another aspect, the present invention provides a composition. According to an embodiment of the invention, the composition comprises: the aforementioned protein; and parasporal crystal protein of Bacillus thuringiensis. The synergistic effect of the synergistic protein and the parasporal crystal protein of Bacillus thuringiensis can effectively enhance the toxicity of the parasporal crystal protein, especially its insecticidal ability, thereby reducing the amount of parasporal crystal protein used, lowering insecticidal costs, and improving insecticidal efficiency, making it suitable for large-scale promotion and application.

[0012] According to an embodiment of the present invention, the mass ratio of the parasporal crystal protein to the protein is 1:(0.5-7). The inventors obtained the above-mentioned preferred ratio through numerous experiments, thereby further improving the toxicity of the parasporal crystal protein, especially its insecticidal ability.

[0013] In another aspect, the present invention provides an insecticide. According to embodiments of the present invention, the insecticide comprises the composition described above. The insecticide according to embodiments of the present invention has good insecticidal effect, high efficiency, and low dosage.

[0014] In another aspect of the invention, a method for preparing the aforementioned protein is provided. According to an embodiment of the invention, the method includes: Step 1: fermenting Bacillus thuringiensis, centrifuging the resulting fermentation broth, and collecting the supernatant; Step 2: dialyzing the supernatant and collecting the retentate; Step 3: passing the retentate through a chromatography column and collecting the fraction eluent over a predetermined time period to obtain the protein.

[0015] The enhancing protein is present in the fermentation supernatant of Bacillus thuringiensis. The supernatant is dialyzed to remove small molecules and salts, and then separated by column chromatography to obtain the protein. During column chromatography separation, the fractions collected at different times were studied. It was found that the protein containing the amino acids shown in SEQ ID NO: 1 has a good effect on enhancing the activity of parasporal crystal proteins, and its content is high with high separation degree. Therefore, the enhancing protein was selected as the target protein. Thus, the method according to the embodiments of the present invention is simple to operate, has high protein separation degree, high yield, high protein content in the fraction, and low impurities.

[0016] According to an embodiment of the present invention, the filter membrane used in the dialysis has a molecular weight cutoff of 1000-6000. This effectively removes small molecules and salts while retaining the target protein. Salt removal facilitates subsequent chromatography.

[0017] According to embodiments of the present invention, the chromatography column comprises an anion exchange column and a cation exchange column, wherein the cation exchange column is selected from GE Superose™ 6Increase, and the anion exchange column is selected from GE Capto IEX anion exchange column. Through extensive experiments, the inventors have discovered that using both cation and anion exchange columns effectively separates the target protein, resulting in a fraction with high target protein content and low impurities. Furthermore, the inventors have optimized and screened the aforementioned specific anion and cation exchange columns, achieving even better separation performance.

[0018] According to an embodiment of the present invention, step 3 includes: passing the retentate through a cation exchange column to collect a first fraction effluent over a specific time period, and then passing the first fraction through an anion exchange column to collect a second fraction effluent over a specific time period to obtain the protein. This further improves the separation effect, resulting in a fraction with high protein content, low impurities, and high yield.

[0019] According to an embodiment of the present invention, step 3 further includes: freeze-drying the collected second fraction to prepare dialysis lyophilized powder. This facilitates storage.

[0020] According to an embodiment of the present invention, step 3 further includes: freeze-drying the collected second fraction and the supernatant together to prepare a supernatant and dialysis mixed lyophilized powder. Since the parasporal crystal protein can also enhance its toxicity when acting together with the supernatant, although the effect is not as good as that of the parasporal crystal protein combined with synergistic proteins, the resulting supernatant and dialysis mixed lyophilized powder can further enhance the toxicity of the parasporal crystal protein by freeze-drying the collected fraction and supernatant together.

[0021] In another aspect, the present invention provides a method for enhancing the toxicity of parasporal crystal proteins of Bacillus thuringiensis. According to an embodiment of the present invention, the method includes: mixing the parasporal crystal protein of Bacillus thuringiensis with the aforementioned protein. The synergistic effect of the synergistic protein and the parasporal crystal protein of Bacillus thuringiensis can effectively improve the toxicity of the parasporal crystal protein, especially its insecticidal ability, thereby reducing the amount of parasporal crystal protein used, lowering insecticidal costs, and improving insecticidal efficiency, making it suitable for large-scale application.

[0022] According to an embodiment of the present invention, the mass ratio of the parasporal crystal protein to the protein is 1:(0.5-7), preferably 1:(1-4). This further enhances the toxicity of the parasporal crystal protein, particularly its insecticidal ability.

[0023] According to embodiments of the present invention, the protein is provided in the form of the dialysis lyophilized powder or the supernatant and dialysis mixed lyophilized powder obtained by the aforementioned method for obtaining the protein. This further enhances the toxicity of the parasporal crystal protein, particularly its insecticidal activity.

[0024] In another aspect, the present invention provides a method for insecticidal treatment. According to an embodiment of the present invention, the method includes applying the aforementioned insecticide to the insect to be treated. Thus, the method according to the embodiment of the present invention can effectively kill insects, reduce the amount of insecticide used, improve insecticidal efficiency, and is suitable for large-scale application.

[0025] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Detailed Implementation

[0026] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0027] Example 1

[0028] Bacillus thruingiensis aizawai_HD133 was fermented in a 50L fermenter. After 50 hours, the culture was terminated, and the fermentation broth was collected to obtain parasporal crystal protein and a protein with the amino acid sequence shown in SEQ ID NO: 1.

[0029] 1. Obtaining parasporal crystal proteins

[0030] Let the fermentation broth stand for 24 hours, centrifuge at 12,000 rpm, and collect the precipitate. Wash / centrifuge three times with physiological saline, and then freeze-dry.

[0031] 2. Obtain a protein having the amino acid sequence shown in SEQ ID NO: 1.

[0032] The fermentation broth was centrifuged at 10,000 rpm for 30 minutes at 4°C, and the supernatant was collected. The supernatant was divided into two portions. One portion was lyophilized overnight at -60°C to obtain a lyophilized supernatant powder. The other portion was dialyzed at 4°C for 24 hours with a molecular weight cutoff of 3000, using a total dilution of 10,000 times. The water was changed every 8 hours, and the retentate was collected. The retentate was filtered through a GE Superose™ 6Increase column with a mobile phase of Tris (0.05 mol), pH 7.8, selecting the 41-43 min fraction. The fraction was then filtered through a GE Capto IEX anion exchange column with a mobile phase of MOPS (0.05 mol), pH 7.3, selecting the 12 min fraction and collecting the peak. The distillate was lyophilized overnight at -60°C to obtain a lyophilized dialyzable powder. The powder was then stored in a sealed container.

[0033] Example 2

[0034] Bacillus thruingiensis aizawai_HD133 was fermented in a 50L fermenter. After 60 hours, the culture was terminated, and the fermentation broth was collected to obtain parasporal crystal protein and a protein with the amino acid sequence shown in SEQ ID NO: 1.

[0035] 1. Obtaining parasporal crystal proteins

[0036] Same as Example 1.

[0037] 2. Obtain a protein having the amino acid sequence shown in SEQ ID NO: 1.

[0038] The fermentation broth was centrifuged at 10,000 rpm for 30 minutes at 4°C, and the supernatant was collected. The supernatant was divided into two portions. One portion was lyophilized overnight at -60°C to obtain a lyophilized supernatant powder. The other portion was dialyzed at 4°C for 24 hours with a molecular weight cutoff of 4000, using a total dilution of 8000 times. The water was changed every 8 hours, and the retentate was collected. The retentate was filtered through a GE Superose™ 6Increase column with a mobile phase of Tris (0.05 mol), pH 7.8, selecting the 41-43 min fraction. The fraction was then filtered through a GE Capto IEX anion exchange column with a mobile phase of MOPS (0.05 mol), pH 7.3, selecting the 12 min fraction and collecting the peak. The distillate was lyophilized overnight at -60°C to obtain a lyophilized dialysis powder. The powder was then stored in a sealed container.

[0039] Bioactivity experiment

[0040] The following example, using Example 1, shows the bioactivity of prepared parasporal crystal protein powder (referred to as spore crystal powder), lyophilized fermentation supernatant powder, and lyophilized dialysis powder. The results of Example 2 are similar to those of Example 1 and will not be repeated here.

[0041] The reference paper for the biopotency test method is: "Study on the toxicity bioassay of Bacillus thuringiensis preparations with cotton bollworm as test insect (DOI: CNKI:SUN:ZSWF.0.1990-S1-000)".

[0042] The results are shown in Table 1. Data from groups 1, 2, and 3 show that the synergistic protein itself has low insecticidal activity. Data from groups 4 and 5 show that when the synergistic protein is mixed with spore crystal powder, the insecticidal activity of the spore crystal powder can be increased more than three times, significantly improving the toxicity of the spore crystal powder, thus reducing the amount of spore crystal powder used and lowering the cost. Since small molecules exist in the fermentation supernatant and are removed after dialysis, they are not present in the dialysis lyophilized powder. The similar potency in groups 5 and 6 indicates that the substance enhancing the toxicity of parasporal crystals is not a small molecule, but a large molecule. Dialysis to remove small molecules can further increase the content of synergistic protein in the dialysis lyophilized powder. Therefore, with a reduction in the amount of parasporal crystal protein and dialysis lyophilized powder added, a similar effect of enhanced toxicity can be achieved.

[0043] Table 1 Bioactivity

[0044]

[0045] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0046] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A protein, characterized in that, The amino acid sequence of the protein is SEQ ID NO:

1.

2. Use of the protein of claim 1 in enhancing the toxicity of the parasporal crystal protein of Bacillus thuringiensis HD133 against cotton bollworm.

3. The use according to claim 2, characterized in that, The protein is used to enhance the insecticidal ability of parasporal crystal proteins.

4. A composition, characterized in that, include: The protein of claim 1; Parasporal crystal protein of Bacillus thuringiensis HD133.

5. The composition according to claim 4, characterized in that, The mass ratio of the parasporal crystal protein to the protein is 1:(0.5~7).

6. An insecticide, characterized in that, include: The composition according to claim 4 or 5.

7. A method for preparing the protein of claim 1, characterized in that, include: Step 1: Ferment Bacillus thuringiensis HD133, centrifuge the resulting fermentation broth, and collect the supernatant; Step 2: Dialyze the supernatant and collect the retentate; Step 3: Pass the retentate through a chromatography column and collect the fraction that flows out over a predetermined time period to obtain the protein.

8. The method according to claim 7, characterized in that, The dialysis membrane used has a molecular weight cutoff of 1000-6000.

9. The method according to claim 7, characterized in that, The chromatography column includes anion exchange column and cation exchange column. The cation exchange column is selected from GE Superose™ 6 Increase, and the anion exchange column is selected from GE Capto IEX anion exchange column.

10. The method according to claim 7, characterized in that, Step 3 includes: passing the retentate through a cation exchange column to collect the first fraction that flows out over a specific time period, and then passing the first fraction through an anion exchange column to collect the second fraction that flows out over a specific time period, thereby obtaining the protein.

11. The method according to claim 10, characterized in that, Step 3 further includes: freeze-drying the collected second fraction to produce dialysis lyophilized powder.

12. The method according to claim 10, characterized in that, Step 3 further includes: freeze-drying the collected second fraction and the supernatant together to prepare a supernatant and dialysis mixed freeze-dried powder.

13. A method for enhancing the virulence of parasporal crystal proteins in Bacillus thuringiensis HD133, characterized in that, include: The parasporal crystal protein of Bacillus thuringiensis HD133 is mixed with the protein described in claim 1.

14. The method according to claim 13, characterized in that, The mass ratio of the parasporal crystal protein to the protein is 1:(0.5~7).

15. The method according to claim 13, characterized in that, The protein is provided in the form of the dialysis lyophilized powder of claim 11 or the supernatant and dialysis mixed lyophilized powder of claim 12.

16. A method for killing insects, characterized in that, include: The insecticide of claim 6 is applied to the insect to be treated, wherein the insect is a cotton bollworm.

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