Insecticidal protein tpp94aa1, its coding gene and application

Abstract

The application discloses an insecticidal protein Tpp94Aa1, an encoding gene thereof and application, and belongs to the technical field of biological control. The application provides an insecticidal protein Tpp94Aa1, and the amino acid sequence of the insecticidal protein Tpp94Aa1 is shown as SEQ ID NO. 1. Experiments prove that the protein Tpp94Aa1 shows strong lethal activity on the lepidopteran pest Ostrinia furnacalis, and the LC 50 50 is 478.443 μg / mL, and the protein Tpp94Aa1 can significantly inhibit the survival of Ostrinia furnacalis larvae; meanwhile, the protein Tpp94Aa1 shows higher virulence on Meloidogyne incognita, and the LC 50 50 is only 63.073 μg / mL, and shows excellent nematode control effect. The application provides a new toxic protein resource and an efficient biological control strategy for agricultural pest control, and has important application value and good industrialization prospect.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and in particular to an insecticidal protein Tpp94Aa1, its encoding gene and its applications. Background Technology

[0002] The corn borer, belonging to the family Pyralidae in the order Lepidoptera, is one of the most serious stalk-boring pests in corn production. Currently, in my country, the Asian corn borer (…) is the most prevalent. Ostrinia furnacalis ) and European corn borer ( Ostrinia nubilalis Two main types of pests are sorghum and sorghum, and they have a wide range of hosts. In addition to gramineous crops such as corn and sorghum, they can also harm a variety of cash crops such as cotton, sugarcane, sunflower, rice, sugar beets and beans, seriously affecting crop yield and quality.

[0003] The corn borer is a global agricultural pest, causing significant economic losses, particularly to important cash crops such as corn and sugarcane. Its larvae primarily feed by boring into plant stems, damaging vascular tissues and impairing water and nutrient transport, leading to stunted growth, reduced resistance, and in severe cases, lodging and yield reduction. Simultaneously, the mechanical damage caused by the corn borer provides entry points for various pathogens, inducing secondary diseases and further exacerbating crop disease outbreaks, posing a serious threat to agricultural production safety.

[0004] Currently, chemical pesticides remain the primary means of controlling corn borers. While chemical pesticides can reduce insect populations in a short period, long-term and excessive use can easily lead to soil and water pollution, and adversely affect non-target organisms and the ecological environment. Furthermore, the increasing resistance of pests to pesticides is causing a gradual decline in the effectiveness of traditional chemical pesticides. In addition, pesticide residues may pose potential risks to human health and the safety of agricultural ecosystems. Therefore, developing novel, highly efficient, green, safe, and environmentally friendly biological insecticides has become an important research direction in the field of green pest control in agriculture.

[0005] Bacillus thuringiensis (Bt) Bacillus thuringiensis Bt (Bacillus thuringiensis) is a class of Gram-positive bacteria capable of producing a variety of insecticidal proteins. Its toxic proteins, such as Cry and Vip, are widely used in the development of biopesticides and insect-resistant transgenic crops due to their high insecticidal activity, environmental friendliness, and safety for non-target organisms. However, the insecticidal spectrum of currently reported Tpp proteins remains relatively limited, especially regarding highly effective toxic proteins against lepidopteran pests such as the corn borer. Therefore, exploring Tpp proteins with novel insecticidal activities and excellent application potential is of great significance for enriching Bt insecticidal protein resources, delaying the development of pest resistance, and promoting the development of novel biopesticides. Summary of the Invention

[0006] The purpose of this invention is to provide an insecticidal protein Tpp94Aa1, its encoding gene, and its applications, in order to solve the problems existing in the prior art. This invention provides an insecticidal protein Tpp94Aa1, which has good toxic effects against the Asian corn borer and the bean weevil root-knot nematode. This invention provides a new toxic protein resource and an efficient biocontrol strategy for agricultural pest control, and has significant application value and good industrialization prospects.

[0007] To achieve the above objectives, the present invention provides the following solution: The present invention provides an insecticidal protein Tpp94Aa1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides the encoding gene of the insecticidal protein Tpp94Aa1, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant vector comprising the aforementioned coding gene.

[0010] The present invention also provides an engineered bacterium, wherein the engineered bacterium comprises the recombinant vector described above.

[0011] The present invention also provides the application of the insecticidal protein Tpp94Aa1, the encoding gene, the recombinant vector, or the engineered bacteria in the control of agricultural pests.

[0012] The present invention also provides the application of the insecticidal protein Tpp94Aa1, the encoding gene, the recombinant vector, or the engineered bacteria in the preparation of products for controlling agricultural pests.

[0013] Optionally, the agricultural pests include the Asian corn borer and the bean root-knot nematode.

[0014] The present invention also provides a product for controlling agricultural pests, wherein the active ingredient of the product is the insecticidal protein Tpp94Aa1.

[0015] Optionally, the agricultural pests include the Asian corn borer and the bean root-knot nematode.

[0016] The present invention also provides a method for controlling agricultural pests, including the steps of treating or feeding the agricultural pests with the insecticidal protein Tpp94Aa1. The agricultural pests mentioned include the Asian corn borer and the bean root-knot nematode.

[0017] The present invention discloses the following technical effects: This invention provides a novel Tpp-like toxic protein, Tpp94Aa1, with significant insecticidal activity. Bioactivity assays showed that Tpp94Aa1 protein exhibited excellent control effects against the Asian corn borer and the bean weevil root-knot nematode. Specifically, Tpp94Aa1 showed a high LC50 against the Asian corn borer. 50 The concentration was 478.443 μg / mL, which significantly inhibited larval survival and feeding; while it showed higher toxicity against the plant parasitic nematode *Auricularia auricula-judae*, with an LC50 of 478.443 μg / mL. 50 The concentration was 63.073 μg / mL, demonstrating good potential for nematode control.

[0018] This invention reports for the first time that Tpp-type proteins exhibit clear toxic activity against the Asian corn borer, further expanding the insecticidal spectrum of Tpp-type proteins and providing new candidate toxic protein resources for the development of novel Bt biological pesticides and the cultivation of insect-resistant transgenic crops. This protein is characterized by high efficiency, greenness, and environmental friendliness, and has significant application value and promising industrialization prospects in the field of green pest control in agriculture. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This image shows the SDS-PAGE electrophoresis results of the Tpp94Aa1 gene induced in Escherichia coli BL21(DE3). Lane 1 is the protein molecular weight standard (marker); Lane 2 is the protein sample of BL21(DE3) strain carrying the empty vector pGEX-6P-1 after IPTG induction; Lane 3 is the protein sample of BL21(DE3) strain carrying the recombinant expression vector pGEX-6P-1-Tpp94Aa1 without IPTG induction; Lane 4 is the total lysate protein obtained from BL21(DE3) strain carrying pGEX-6P-1-Tpp94Aa1 after IPTG induction; Lane 5 is the supernatant obtained by centrifugation of lysate protein after induction; Lane 6 is the precipitate obtained by centrifugation of lysate protein after induction; the red arrow points to the Tpp94Aa1 protein. Figure 2The image shows the SDS-PAGE electrophoresis results of purified Tpp94Aa1 protein; lane 1 is the pre-stained protein molecular weight standard (10-200 kDa); lane 2 is the protein sample of BL21(DE3) strain carrying the empty vector pGEX-6P-1 after IPTG induction; lane 3 is the protein sample of BL21(DE3) strain carrying pGEX-6P-1-Tpp94Aa1 without IPTG induction; lane 4 is the supernatant of ruptured protein after induction; lane 5 is the elution buffer of Tpp94Aa1 protein purified by GST affinity chromatography; the red arrow points to the Tpp94Aa1 protein. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Unless otherwise specified, the methods used in the embodiments of this invention are all conventional methods.

[0027] Unless otherwise specified, all reagents and materials used in the embodiments of this invention are commercially available products.

[0028] Preparation method of liquid LB medium: Dissolve 10 g of tryptone, 10 g of NaCl and 10 g of yeast powder in 1 L of distilled water, adjust the pH to 7.0, and autoclave at 121℃ for 20 min.

[0029] Example 1: Expression and purification of Tpp94Aa1 recombinant protein Tpp94Aa1 is derived from Bacillus thuringiensis (Bacillus thuringiensis). Bacillus thuringiensis The amino acid sequence of the Tpp insecticidal protein is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0030] SEQ ID NO.1: LEKFASIKLKKAIEWEWVNMEFKKDYIYSILNDKSQKYVDVDSNATDNGAWLDQYEWQNSDSQKWIIYPMDGGYFVLINWASGRMIDVVNNSKDSGA MLNQYEWQNSDSQLWHPEEKDENYISLQNKYSGKYADVDYNATDNGDHLNQYEWQDSDSQKWTTQEVEQFTTLPSVDIQELPPVPEYSNIDDTLPDT SDSAVTAYTLAPFFAVNDPQYDTWTVGNQANENPYYLYIKKQCWKLVKSLTLTPGETQGYDLTYGITTTDQQTASLTVSNTIGADAGLQFKDKSLGLSTQYTSELNVTISQTSEEMVQATNTHTITNKYDHEIAWSKYILVTEYHVERTNGTTVNTPWSFADINITRSVSFPPEETKSLILSETLVSSTETAR.

[0031] SEQ ID NO.2:

[0032] The coding gene for Tpp94Aa1 was cloned into the prokaryotic expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-6P-1-Tpp94Aa1. This vector was then transformed into *E. coli* BL21(DE3) competent cells to obtain the recombinant engineered strain pGEX-6P-1-Tpp94Aa1. The *E. coli* BL21(DE3) transformant pGEX-6P-1-BL21(DE3) carrying the empty vector pGEX-6P-1 served as a negative control for analyzing background protein expression in the host bacteria. The engineered strain carrying the recombinant expression vector pGEX-6P-1-Tpp94Aa1 served as the experimental group for analyzing the induced expression of the Tpp94Aa1 protein. The specific steps of protein expression are as follows.

[0033] 1. Prokaryotic expression of the insecticidal protein Tpp94Aa1 A single colony of the recombinant engineered strain pGEX-6P-1-Tpp94Aa1 was picked and inoculated into 10 mL of LB broth containing ampicillin (final concentration 100 μg / mL). The culture was incubated at 37℃ and 220 rpm with shaking for 12 h to obtain activated bacterial culture. Subsequently, the culture was transferred to 1 L of LB broth at a 1% (v / v) inoculation rate (again with ampicillin at a final concentration of 100 μg / mL) and incubated at 37℃ and 220 rpm for approximately 4 h. When the OD of the bacterial culture... 600 When the concentration reached 0.6, IPTG was added to a final concentration of 0.5 mM to induce Tpp94Aa1 protein expression. The culture was then continued at 16℃ and 120 rpm for 20 h to obtain the induced fermentation broth. The fermentation broth was centrifuged at 4℃ and 8000 rpm for 10 min, the supernatant was discarded, and the bacterial pellet was collected. The bacterial pellet was resuspended in 50 mL of 1×PBS buffer and transferred to a 100 mL beaker for later use. The bacterial cells were lysed using an ultrasonic disruption method with the following parameters: power 300 W, ultrasonic operation for 5 s, interval of 10 s, and total treatment time of 20 min, to obtain the disrupted bacterial solution.

[0034] 2. SDS-PAGE protein detection The disrupted bacterial culture was centrifuged at 4°C and 8000 rpm for 10 min. Soluble fraction (supernatant) and insoluble fraction (precipitate) were obtained after centrifugation. The precipitate was resuspended in 1×PBS buffer. The supernatant and resuspended precipitate were analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 1As shown, after IPTG induction, the recombinant strain successfully expressed the target protein Tpp94Aa1, and a distinct specific protein band appeared at the corresponding molecular weight position. Further analysis revealed that the protein was mainly distributed in the soluble fraction (supernatant). The results indicate that the Tpp94Aa1 protein can be successfully expressed in *E. coli* after IPTG induction, and exists primarily in the soluble form of the supernatant.

[0035] 3. Purification of Tpp94Aa1 recombinant protein The ultrasonically disrupted bacterial culture was centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was collected as the soluble crude protein sample and filtered through a 0.45 μm microporous membrane to remove impurities. The filtered supernatant was then placed on ice for later use. GST-tag Purification Resin (BeyoGold), pre-equilibrated with 1×PBS buffer, was loaded into an affinity chromatography column and fixed on a metal stand. The top and bottom seals of the column were opened sequentially to allow the equilibration buffer to drain naturally. The treated crude protein sample was then slowly added to the column, allowing the GST-tagged Tpp94Aa1 protein to fully bind to the affinity packing material. The eluent was collected, and a portion of the sample was reserved for subsequent SDS-PAGE analysis. After the sample had completely entered the column, the column was washed with 1–2 column volumes of 1×PBS buffer to remove unbound impurities. This washing was repeated 5 times, and a portion of the wash solution was collected for subsequent analysis. The target protein was then eluted with one column volume of Elution Buffer, repeated 6–10 times, and the eluent was collected to obtain the purified Tpp94Aa1 protein. After purification, the chromatography media was washed alternately with 5 mL of 1×PBS buffer and 5 mL of deionized water, repeated twice; finally, the media was equilibrated once with 5 mL of 20% ethanol, and the media was stored in 20% ethanol at 4°C for later use.

[0036] To evaluate the purification effect, approximately 20 μL of crude protein supernatant and purified eluent were subjected to SDS-PAGE electrophoresis analysis. The results are as follows: Figure 2 As shown, a relatively uniform band of approximately 75 kDa appeared in the purification eluent (indicated by the red arrow), while multiple protein bands were present in the crude protein supernatant, indicating that impurities in the washing buffer were effectively removed. These results demonstrate that GST affinity chromatography can efficiently purify the Tpp94Aa1 recombinant protein with high purity, making it suitable for subsequent functional and bioactivity studies.

[0037] 4. Determination of Tpp94Aa1 recombinant protein concentration The concentration of purified Tpp94Aa1 protein was determined using a BCA protein concentration assay kit (Beyotime) combined with a microplate reader. The specific steps are as follows: First, the BSA protein standard solution (25 mg / mL) was diluted with PBS buffer to prepare a BSA standard working solution with a final concentration of 0.5 mg / mL. This solution was aliquoted and stored at -20℃ for later use. Then, the diluted BSA standard solution was added to the corresponding wells of a 96-well plate at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL, respectively, and the total volume was brought to 20 μL with standard diluent to construct a standard curve. Simultaneously, an appropriate amount of the protein sample to be tested was added to the sample wells of the 96-well plate; if the sample volume was less than 20 μL, it was brought to 20 μL with standard diluent. Then, 200 μL of BCA working solution was added to each well, gently mixed, and incubated at 37℃ for 20–30 min to ensure complete colorimetric reaction. After the reaction was completed, the absorbance value A of each well at a wavelength of 562 nm was measured using a microplate reader. 562 A standard curve was plotted based on BSA standards, and the concentration of purified protein was calculated using a linear regression equation, taking into account the absorbance values ​​and corresponding volumes of the samples.

[0038] Example 2: Insecticidal bioactivity assay of Tpp94Aa1 protein To verify the insecticidal activity of the Tpp94Aa1 protein, this example uses the Asian corn borer (… Ostrinia furnacalis ) and the root-knot nematode ( Meloidogyne enterolobii As a target pest, the bioactivity of the purified Tpp94Aa1 protein was determined.

[0039] 1. Insecticidal activity assay of Tpp94Aa1 against Asian corn borer 1.1 Acquisition and Artificial Rearing of the Asian Corn Borer Asian corn borers were purchased from Henan Keyun Biological Pesticide Co., Ltd., and artificially reared under laboratory conditions using fresh corn leaves that had not been treated with pesticides. To simulate their suitable growth environment, the rearing conditions were controlled as follows: temperature 25 ± 1℃, relative humidity 60%~70%, and photoperiod of 14 h light / 10 h dark (14L:10D).

[0040] During the rearing period, the larvae's feeding and survival were observed daily, and fresh corn leaves were replaced promptly to ensure adequate nutrition and moisture in the feed. Healthy, active second-instar larvae were selected as the test source, and diseased, weak, and abnormally developed individuals were removed before the experiment to ensure the accuracy and repeatability of the bioassay results.

[0041] 1.2 Determination of bioactivity of Asian corn borer Fresh corn leaves with uniform growth, no mechanical damage, and no pesticide application were selected, cut into uniform-sized pieces, rinsed with distilled water, and air-dried for later use. Purified Tpp94Aa1 protein, digested with trypsin for 30 min, was diluted to a preset concentration, and multiple treatment concentrations were set (133.92, 267.88, 321.56, 428.63, 535.69 μg / mL). 1×PBS was used as a negative control.

[0042] Corn leaves were completely immersed in protein solutions of varying concentrations for 10–15 seconds to ensure even protein adhesion to the leaf surface. They were then removed and placed on sterile filter paper to air dry. The treated leaves were then laid flat in a perforated plastic box lined with moistened filter paper to maintain appropriate humidity. Each treatment was performed in triplicate, with each replicate containing 20 healthy, uniformly sized second-instar Asian corn borer larvae, gently placed on the leaf surface.

[0043] The bioassay conditions were controlled as follows: temperature 25 ± 1℃, relative humidity 60%–70%, and photoperiod 14L:10D. The experiment was conducted continuously for 7 days, with fresh leaves of the same treatment replaced daily to ensure that the larvae could continuously ingest the target protein at the corresponding concentration. Larval mortality was recorded daily. The criteria for determining mortality were: larvae showing no response to external touch and exhibiting obvious abnormal body coloration.

[0044] The experimental results (Table 1) show that the Tpp94Aa1 protein has a significant toxic effect on Asian corn borer larvae and can significantly inhibit larval survival and feeding.

[0045] Table 1. Insecticidal activity of Tpp94Aa1 against Asian corn borer. 2. Assay of the nematicidal activity of Tpp94Aa1 against root-knot nematode of *Auricularia auricula-judae* 2.1 Propagation and Collection of Root-knot Nematodes of the Bean Tree Second-instar larvae (J2) of the root-knot nematode of *Eriocaulon buergerianum* were inoculated into the roots of water spinach grown in sterile soil for nematode propagation and preservation. After obvious root knots formed on the water spinach roots, severely diseased root material was selected, cleaned, and nematode egg sacs were picked out with tweezers.

[0046] The oocysts were sterilized in 0.05% NaClO solution for 5 min, and then rinsed thoroughly with sterile water to remove residual NaClO. This rinsing was repeated 3 times. The sterilized oocysts were then transferred to 6-well plates, with an appropriate amount of sterile water added to each well. The plates were then incubated at 28°C for 3 days to obtain a large number of highly active second-instar larvae (J2) for subsequent experiments.

[0047] 2.2 Determination of nematicidal activity using the 96-well plate method The toxic activity of Tpp94Aa1 protein against *Auricularia auricula-judae* root-knot nematode was determined using a 96-well plate assay. Approximately 30 second-instar larvae (J2) were added to each well, along with a Tpp94Aa1 protein solution at a final concentration of 300 μg / mL. Each treatment was performed in triplicate.

[0048] A negative control group was set up (with an equal volume of 1×PBS buffer added). The 96-well plates were incubated at 28°C for 72 h, and the mortality of nematodes was observed and counted every 24 h using a stereomicroscope.

[0049] Mortality rate and adjusted mortality rate are calculated using the following formula: Mortality rate (%) = 100 × number of dead nematodes / number of dead nematodes; Corrected mortality rate (%) = 100 × (mortality rate of treatment group - mortality rate of blank control group) / (1 - mortality rate of blank control group).

[0050] The experimental results (Table 2) show that the Tpp94Aa1 protein has strong toxic activity against the root-knot nematode of the bean tree, and can significantly increase the mortality rate of the nematode, demonstrating good potential for biocontrol applications.

[0051] Table 2. Nematicidal activity assay of Tpp94Aa1 against root-knot nematodes of *Auricularia auricula-judae*. 3. Experimental Results and Toxicity Analysis Mortality rates were calculated based on the number of deceased and surviving individuals in each treatment group, and toxicity regression analysis was performed using IBM SPSS Statistics 27 software to calculate the median lethal concentration (LC50). 50 The results are shown in Table 3, along with their 95% confidence intervals.

[0052] Table 3 LC-100 for bioactivity assays 50 (μg / mL) The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An insecticidal protein Tpp94Aa1, characterized in that, The amino acid sequence of the insecticidal protein Tpp94Aa1 is shown in SEQ ID NO.

1.

2. The gene encoding the insecticidal protein Tpp94Aa1 according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The recombinant vector contains the coding gene as described in claim 2.

4. An engineered bacterium, characterized in that, The engineered bacteria comprise the recombinant vector as described in claim 3.

5. The application of the insecticidal protein Tpp94Aa1 of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, or the engineered bacteria of claim 4 in the control of agricultural pests.

6. The application of the insecticidal protein Tpp94Aa1 of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, or the engineered bacteria of claim 4 in the preparation of products for controlling agricultural pests.

7. The application as described in claim 5 or 6, characterized in that, The agricultural pests mentioned include the Asian corn borer and the bean root-knot nematode.

8. A product for controlling agricultural pests, characterized in that, The active ingredient of the product is the insecticidal protein Tpp94Aa1 as described in claim 1.

9. The product as described in claim 8, characterized in that, The agricultural pests mentioned include the Asian corn borer and the bean root-knot nematode.

10. A method for controlling agricultural pests, characterized in that, This includes the step of treating or feeding the agricultural pests with the insecticidal protein Tpp94Aa1 as described in claim 1; The agricultural pests mentioned include the Asian corn borer and the bean root-knot nematode.

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