WO2014067299A1 - Bt toxin cry1ab-loop2-p2s having high toxicity against nilaparvata lungens and engineered bacterium - Google Patents

Abstract

Provided in the present invention are Bt toxin Cry1Ab-loop2-P2S having high toxicity against Nilaparvata lungens and an engineered bacterium. With Bravo model, a Bt Cry toxin effect model, serving as the theoretical basis, the present invention utilizes a bacteriophage peptide library display technology to screen peptide P2S that is capable of bonding with the midgut of Nilaparvata lungens; furthermore, a loop2 sequence of Domain II of Cry molecule is replaced in the sequence of the peptide by utilizing a molecular fragment replacement method, the cry1Ab-loop2-p2s toxin gene is constructed, the gene and universal expression vector pGEX-KG are connected and transformed into Escherichia coli BL21-DE2, and, after stability measurement, bonding activity measurement, and toxicity measurement, engineered bacteria 1Ab-P2S (CGMCC No: 6426) that is capable of expressing the Cry1Ab-loop2-P2S toxin having high toxic effects against Nilaparvata lungens is acquired.

Description

 Highly toxic Bt toxin Cr lAb-loop2-P2S and engineering bacteria for rice brown planthopper

BL21-DE3中， 经过稳定性测定、结合活性测定及毒力测 定， 获得了能够表达对水稻褐飞虱有高毒性作用 CrylAb-loop2-P2S毒素的工程菌 lAb-P2S, 该菌株为人工构建质粒的工程菌， 宿主为肠埃希氏菌、 Escherichia Co//) BL21 ( DE3 ) , 于 2012年 8月 13日保藏于中国微生物菌种保藏管理委员会普通微生物中心， 北京市朝阳区北 辰西路 1号院 3号， 菌株保藏号为： CGMCC NO : 6427。 所述 1οορ2序列为 Cry分子中决定受体识别的环区， 其氨基酸序列为： RRPFNIGINNQ; 所述大肠杆菌 BL21-DE3菌株由市场购得。 所述噬菌体展示技术 （phage display) 是将基因 表达与亲和选择相结合的技术， 其基本原理是将编码诱饵蛋白的 DNA片段插入噬菌体基因 组， 并使之与噬菌体外壳蛋白编码基因进行融合表达， 该重组噬菌体侵染宿主细菌后， 复制 形成大量带有杂合外壳蛋白的噬菌体颗粒，直接用于捕获靶标蛋白库中与诱饵蛋白相互作用 的蛋白质。 本发明的技术方案， 操作步骤如下： The invention relates to a toxin and an engineering bacterium which are toxic to plant pests, and particularly relates to a Bt toxin CrylAb-loop2-P2S and an engineering bacterium which are highly virulent to Hemiptera fuliginea, and belongs to the technical field of biological control. BACKGROUND OF THE INVENTION Rice is the most important food crop in China. However, rice has a large number of pests and diseases, which seriously affects the production of rice in China. The cultivation of anti-Lepidopteran and Coleoptera pest rice such as Bt gene is expected to effectively prevent the harm of such pests (Tang e/2006; Wang e/ /, 2010). At the same time, it also inevitably brings about the possibility of aggravating the harm of non-Bt insecticidal protein target insect sucking mouthparts pests such as planthoppers and leafhoppers (Chen e/, 2011). Therefore, it is extremely urgent to create and develop new insect toxins for sucking pests such as rice planthoppers. Bacillus thuringie is Bt) is the most widely used microbial insecticide in the world. Its Cry series of toxins (δ-Endotoxins) is one of the most important toxin proteins produced by Bt and has been widely used in the control of Lepidoptera, Coleoptera, Diptera pests and phytopathogenic nematodes ( Arenas e / , 2010). ^Gene is also the most widely used insecticidal gene for genetically modified crops. The genetic crops (such as corn, cotton, soybeans, potatoes, tobacco, tomatoes, rapeseed, rice, etc.) have been planted in large areas, effectively controlling the harm of lepidopteran and coleopteran pests, and becoming an important pest management. Means (Rie, 2000; Shelton i?/ /, 2002; Wang ί?/ Ζ, 2010). However, Bt and its insect toxins have no obvious toxicity to sucking mouthparts such as Hemiptera and Homoptera. So far, Bt strains which have significant toxicity to such pests have not been found. In view of the serious harm to the agricultural production caused by the sucking pests such as planthopper, leafhopper and aphid and the serious possibility of aggravating the explosive damage of sucking pests caused by the planting of genetically modified insect-resistant varieties, it is urgent to develop and target such pests. A novel resistance gene. Based on the gene, the development of a new Cry toxin engineering strain capable of producing a sucking pest by modifying the Cry toxin molecule is a research with great theoretical and practical value. SUMMARY OF THE INVENTION It is an object of the present invention to provide a Bt toxin CrylAb-l ₀₀ p2-P2S and an engineered bacterium having high virulence to the hemipteran pest rice brown planthopper. Based on the Bavo Cry toxin model Bravo model (Knowles, BH & JA Dow., 1993), the short peptide P2S which can bind to the midgut of rice brown planthopper was screened by phage short peptide library display technology; further, it will be short The peptide sequence replaces the 1οορ2 sequence of the Cry molecule Domain II by a molecular fragment replacement method to construct a toxin gene, which is linked to the universal expression vector pGEX-KG (purchased from GE Healthcare).

In BL21-DE3, through the stability measurement, binding activity assay and virulence determination, an engineering strain lAb-P2S capable of expressing CrylAb-loop2-P2S toxin with high toxicity to rice brown planthopper was obtained, which was an artificial plasmid construction project. Bacteria, hosted by Escherichia Co//) BL21 (DE3), deposited on August 13, 2012 at the General Microbiology Center of China Microbial Culture Collection Management Committee, No. 1 Beichen West Road, Chaoyang District, Beijing No. 3, strain accession number: CGMCC NO: 6427. The 1οορ2 sequence is a loop region in the Cry molecule that determines receptor recognition, and the amino acid sequence thereof is: RRPFNIGINNQ; the Escherichia coli BL21-DE3 strain is commercially available. The phage display technology is a technique for combining gene expression with affinity selection. The basic principle is that a DNA fragment encoding a bait protein is inserted into a phage genome and fused to a phage coat protein encoding gene. After the recombinant phage infects the host bacteria, it replicates to form a large number of phage particles with a hybrid coat protein, which is directly used to capture the protein in the target protein library that interacts with the bait protein. The technical solution of the present invention, the operation steps are as follows:

1. Screening of short peptides in the midgut endothelium of rice brown planthoppers Using a membrane-feeding method to obtain a phage containing a short peptide library from rice nymphs (Ph.D.-C7CTM phage display peptide library kit, New England Biolabs) Nutrient solution (containing 25% sucrose in PBS buffer). After the nymphs were fed with the phage-mixed nutrient solution for 16 hours, the phage bound to the surface of the midgut of the midgut was eluted with the elution buffer. After the obtained phage was infected with E. coli ER2738, the above-mentioned "Bio-panning" process was repeated. After 3 rounds of panning, the last round of screening of phage infected bacteria, plating culture, phage obtained from individual plaques were cultured and amplified, and single-stranded phage DNA was extracted for sequencing to obtain antigenic (Epitope) properties. And a short peptide P2S capable of forming a surface loo structure. The short peptide P2S is capable of binding to the intestinal lining of the rice brown planthopper, and the gene of the short peptide P2S has the nucleotide sequence shown in SEQ ID NO: 1. The short peptide P2S has the amino acid sequence shown as SEQ ID NO: 2. The PBS buffer is Na ₂ HP0 ₄ · 12H ₂ 0 3.473 g NaH ₂ P0 ₄ · 12H ₂ 0 0.226 g, NaCl 0.9 g is dissolved in 1 L of trihydrated water; the elution buffer is 0.2 M Glycine-HCl pH 2.2 , lmg / ml BSA; the E. coli ER2738 host strain M13 phage is included in the Ph.D.-C7C ^TM phage display peptide library kit; the Ph.D.-C7C ^TM phage The body display peptide library kit is commercially available. The biopanning refers to the binding and elution process of the phage short peptide library, as described in the Ph.D.-C7CTM phage display peptide library kit.

2. Cloning of c/jZ^ gene Using a plasmid DNA containing c/jZ^ gene as a template, a pair of primers, crylAb-F and crylAb-R, were designed, and a full-length fragment of a 3,473 bp crylAb was obtained by cloning the crylAb gene (Fig. 1). The crylAb gene was cloned into the 7/3⁄4H l and / 1 sites of the universal expression vector pGEX-KG (Fig. 2) and subjected to double enzyme digestion verification (Fig. 3). Primer: crylAb-F: 5 ' -GGATCCATGGATAACAATCCGAACAT-3 'crylAb-R: 5'-GTCGACTTACTATTCCTCCATAAGGAGTAATTC-3 '. The c/j/ gene has been published on the National Center for Biote clmology Information (NCBI). The full-length fragment of the 3483 bp c^Z^ gene was referred to the NCBI database, gene number AY395148.1.

3. Modification of crylAb gene by short peptide P2S Using the crylAb plasmid DNA obtained in step 2 as a template, a pair of primers md-F and md-R were designed, and the 1448 bp fragment to be modified in the crylAb gene sequence was amplified to obtain ^ ^ The fragment to be modified in the gene sequence (Figures 5, 6). The primers were: md-F: 5 '-CCCGGGACTAGTTGATATAATATGGGGAATTT-3 ' md-R: 5'-GGGCCCCAATTGATGTATGGAATTGTAAACCCGGG-3 '. The fragment to be engineered in the gene sequence has the nucleotide sequence as shown in SEQ ID NO: 11. Further, a pair of primers up-F, up-R were designed to amplify the 1οορ2 upstream fragment sequence (Fig. 7). Primers are: up-F: 5 ' -CCCGGGACTAGTTGATATAATATGGGGAATTT-3 '

Further, a pair of primers down-F, down-R were designed to amplify the 1οορ2 downstream fragment sequence (Fig. 7). The primers were: down-F: 5 down-R: 5 '-GGGCCCCAATTGATGTATGGAATTGTAAA-3 ' Finally, using md-F and md-R primers, the 1οορ2 upstream and downstream fragment DNA was used as a template to obtain a short peptide P2S replacement 1οορ2 Fragment gene domainll-loop2-p2s (Figures 8, 9). The fragment to be engineered in the /w/ //-/^^ y gene sequence has the nucleotide sequence shown as SEQ ID NO: 12.

4. Construction of the ^^^- toxin gene expression vector The fragment gene domainll-op2-p2s obtained in step 3 was used together with the yZ^ gene subcloning plasmid obtained in 2 with the restriction enzymes Spe I and Mim I double enzymes. After cutting, the engineered fragment was ligated back into the crylAb i granule gene using T4 ligase to obtain a toxin gene (Fig. 10, 11). The plasmid was transformed into E. coli BL21-DE3 to obtain a CrylAb_loop2-P2S toxin engineered strain lAb-P2S (CGMCC NO: 6427) capable of expressing high toxicity to rice brown planthopper.

The CrylAb-loop2-p2s toxin is toxic to rice brown planthopper, and the toxin gene has a nucleotide sequence as shown in SEQ ID NO: 3;

5. Expression and purification of toxin protein in engineered strain lAb-P2S (CGMCC NO: 6427) The engineered strain lAb-P2S obtained in step 4 (CGMCC NO: 6427) was inserted into LB medium and cultured at 37 ° C until A few stages (OD ₆ (X 0.4), adding IPTGC final concentration of 0.8 mM), induced at 16 ° C for 24 h. After cell collection and cell disruption, the CrylAb-loop2-P2S toxin protein was obtained by glutathione s-transferase (GST) tag purification (Fig. 12).

The CrylAb-loop2-p2s toxin protein is toxic to rice brown planthopper, and the CrylAb-loop2-p2s toxin protein has an amino acid sequence as shown in SEQ ID NO: 4. The LB medium is 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride is dissolved in 1 L of distilled water, and is sterilized after packaging; IPTG is isopropyl-β-D-thiogalactopyranoside (Isopropyl β -D- 1 -Thiogalactopyranoside )

6. Insecticidal activity assay The test insects were: Lepidopteran pest Plutella xylostella; Hemiptera pest, Nilaparvata lugens All chemical reagents used in the present invention were of analytical grade. The invention has the following advantages:

The present invention uses a phage short peptide library display technology to screen a short peptide P2S capable of binding to the intestinal lining of rice brown planthopper by membrane feeding method; the Bt gene is directionally transformed by the short peptide P2S screened by the present invention, and the expression vector is subjected to expression vector Construction was carried out to obtain an engineered strain lAb-P2S (CGMCC NO: 6427) capable of expressing the toxin CrylAb-loop2-P2S. The Toxicity of the Toxin Protein CrylAb-loop2-P2S Expressed by the Engineering Bacteria to Rice Brown Planthopper and the CrylAb Toxin Protein before Transformation Compared to the increase of nearly 20 times. It indicated that CrylAb-l ₀₀ p2-P2S expressed by the engineered bacteria can effectively control rice brown planthopper, and the method of screening short peptides and structuring Bt Cry toxin by phage short peptide library can effectively extend the insecticidal spectrum of Bt toxin.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a clone of the ^^ gene. Lane 1 is the crylAb gene; M is a 250 bp Marker. Figure 2 is a schematic diagram showing the construction of subclones. Figure 3 is a double restriction enzyme verification of the crylAb-linked expression vector pGEX-KG. Lane 450 is a 250 bp Marker, and Lane 2 is a crylAb-PK ΒαητΆ I and / 1 double digestion assay. Figure 4 shows the alignment of the crylAb gene sequence at NCBI. Figure 5 is a cloning of the fragment to be modified. Lane 1 is the cryalAb gene to be engineered; Lane 2 is crylAb subcloning plasmid DNA; Lane M is 250 bp Marker. Figure 6 is a double enzyme digestion verification of the fragment of the cryLAb to be engineered to the pMD-18T cloning vector. Lane 1 was verified by I and Mun I double digestion; Lane M was 250 bp Marker. Figure 7 is a cloning of the upstream and downstream fragments of CrylAb 1οορ2. Lane 1 is the 1οορ2 upstream fragment; Lane 2 is the 1οορ2 downstream fragment; M is the 250bp Marker. Figure 8 is a schematic representation of the modification of the short peptide P2S replacing CrylAb 1οορ2, the fragment shown in the figure is the 1448 bp fragment to be engineered in the ^^ gene. Figure 9 is a domainli-hop2-p2s fragment after the short peptide P2S replaces the crylAb gene 1οορ2. Lane 1 is a domain li-hop2-p2s fragment; lane M is a 250 bp Marker. Figure 10 is a schematic diagram showing the construction of the subclones of the ^^^- expression after the transformation. Figure 11 shows the transformation of ^Z^- expression subcloning after digestion. Lane 1 is the 3⁄4y expression subcloning plasmid ΒωηΆ I and double enzyme digestion; Lane 2 is the crylAb subcloning plasmid ΒωηΆ I and / I double enzyme digestion verification. Figure 12 shows the results of purification of CrylAb-loop2-P2S using GST-tagged protein. Lane 1 is CrylAb-loop2-P2S purified protein; Lane M is SDSPAGE protein Marker. Figure 13 shows the lethal concentration of the toxin protein CrylAb-loop2-P2S expressed by the engineered strain lAb-P2S (CGMCC NO: 6427) against the susceptible strain Plutella xylostella (LC _5Q ) in g/ml. Figure 14 shows the lethal concentration (LC _5Q ) of the toxin protein CrylAb-loop2-P2S expressed by the engineered strain lAb-P2S (CGMCC NO: 6427) on rice brown planthopper, in units of g/ml. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described. It should be noted that the embodiments of the present invention are illustrative only and not limiting. Example 1. Screening of a short peptide peptide in the midgut of rice brown planthopper, a phage containing a short peptide library, was obtained by a membrane-feeding method (Ph.D.-C7CTM phage display peptide library kit, New England Biolabs) Company) Nutrient solution (containing 25% sucrose in PBS buffer). After the nymphs were fed with the phage-mixed nutrient solution for 16 hours, the midgut of the nymphal nymph was dissected, washed, washed three times with PBS buffer, eluted with an elution buffer, and the phage bound to the intima of the midgut was recovered. After the obtained phage-infected bacteria are amplified, the above "Biopatming" process is repeated. After 3 rounds of panning, the last round of screening of phage infected bacteria, plating culture, phage obtained from individual plaques were cultured and amplified, and single-stranded phage DNA was extracted for sequencing to obtain antigenic (Epitope) properties. And a short peptide P2S capable of forming a surface loop structure. Example 2. Cloning of the /jZ^ gene The plasmid containing the c/jZ^ gene was used as a template to design a pair of primers, crylAb-F and crylAb-R, and cloned the crylAb gene to obtain a 3783 bp full-length fragment of crylAb (Fig. 1). The crylAb gene was cloned into the BamY I and /1 sites of the universal expression vector pGEX-KG (Fig. 2) and subjected to double enzyme digestion (Fig. 3). This gene was confirmed to be the ^7 gene by NCBI alignment (Fig. 4). Primers are: cry 1 Ab-F: 5 ' -GGATCCATGGATAACAATCCGAACAT-3 'crylAb-R: 5'-GTCGACTTACTATTCCTCCATAAGGAGTAATTC-3 '. Example 3: Modification of the crylAb gene using the short peptide P2S Using the crylAb plasmid DNA obtained in Example 2 as a template, a pair of primers md-F and md-R were designed, and the 1448 bp fragment to be modified in the crylAb gene sequence was expanded. Increase, obtain the ^ ^ gene sequence in the gene to be modified (Figures 5, 6). The primers were: md-F: 5 '-CCCGGGACTAGTTGATATAATATGGGGAATTT-3 ' md-R: 5'-GGGCCCCAATTGATGTATGGAATTGTAAACCCGGG-3 ' Further, a pair of primers up-F, up-R, and a 1οορ2 upstream fragment sequence were designed (Fig. 7). Primers are: up-F: 5 ' -CCCGGGACTAGTTGATATAATATGGGGAATTT-3 '

Further, a pair of primers down-F, down-R were designed to amplify the 1οορ2 downstream fragment sequence (Fig. 7). Primers are:

down-R: 5 '-GGGCCCCAATTGATGTATGGAATTGTAAA-3 ' Finally, md-F and md-R were used as primers to amplify the upstream and downstream fragment DNA of 1οορ2 as a template, and the short peptide P2S was replaced with the fragment gene domainll- op2-p2s of 1οορ2 ( Figure 8, 9). Example 4 Construction of a gene expression vector containing a ^Z^- 3⁄4^4^-y toxin The fragment obtained in Example 3 was used together with the c Z^ gene subcloning plasmid obtained in Example 2 with a restriction endonuclease Spe I The gene was digested with Mun I and the engineered fragment was ligated into the crylAb granule gene using T4 ligase to obtain the c/7Z^- 3⁄43⁄4^-y plasmid gene (Fig. 10). The plasmid was transformed into E. coli BL21-DE3 to form a CrylAb-loop2-P2S toxin engineering strain lAb-P2S capable of expressing high virulence to rice brown planthopper. The engineered plasmid DNA was extracted and verified by double enzyme digestion (Fig. 11) and sequenced to obtain a ^Z^-3⁄43^4^y toxin gene sequence having the nucleotide sequence shown in SEQ ID NO: 2. Example 5 Expression and purification of toxin protein in engineered strain lAb-P2S (CGMCC NO: 6427) The engineered strain lAb-P2S (CGMCC NO: 6427) was cultured in LB at 37 °C until the logarithmic phase (OD ₆ QQ) -0.5), IPTG was added to a final concentration of 0.8 mM overnight to induce protein expression, and the purification method was as described in Example 3. The purified CrylAb-loop2-P2S protein was obtained (Fig. 12). Example 6. Insecticide activity assay The test insects were: Lepidoptera pest Plutella xylostella; Nilaparvata lugens Insecticidal bioassay: Plutella xylostella: After the cabbage leaf was weighed and then immersed in the leaf method, 48 hours of investigation results; Rice planthopper: using the membrane-vibration method, 24-hour investigation results.

(1) Engineering strain lAb-P2S (CGMCC NO: 6427) expressed protein against Plutella xylostella toxicity results: As shown in Table 1, the unmodified CrylAb protein expressed by CrylAb engineering bacteria is highly toxic to Plutella xylostella, and the final lethal concentration reaches 0.88. g/ml, the CrylAb-loop2-P2S protein expressed by the engineered lAb-P2S, which recognizes the receptor loop region, was less toxic to the cabbage than the CrylAb engineered bacteria, and the final lethal concentration was 32.5 g/ml. The protein expressed by the engineered strain lAb-P2S (CGMCC NO: 6427) was the final concentration of the susceptible strain Plutella xylostella (Fig. 13).

(2) The toxicity of the engineered strain lAb-P2S (CGMCC NO: 6427) on the toxicity of rice brown planthopper: As shown in Table 2, the unmodified CrylAb protein expressed by CrylAb engineering strain is weak to rice brown planthopper, and the final concentration is 233. g/ml, and the toxicity of the toxin protein CrylAb-loop2-P2S expressed by the engineered strain lAb-P2S prepared by replacing the CrylAb 1οορ2 with the short peptide P2S which binds to the midgut of the rice brown planthopper, increased the toxicity of rice brown planthopper by nearly 20 times. The final lethal concentration was 18.75 g/ml. According to this result, the CrylAb-loop2-P2S toxin expressed by the engineering strain lAb-P2S (CGMCC NO: 6427) has a significant effect on the toxicity of rice brown planthopper. The final lethal concentration of engineered bacteria on rice brown planthopper (Fig. 14). ∞

 Table 1 Engineering bacteria lAb-P2S (CGMCC NO: 6427) toxic effect on sensitive strains of Plutella xylostella (48 hours) Sample Protein concentration (pg/ml) Repeat Total number of insects 48 hours of dead insects 48 hours corrected mortality

 0.4 3 60 21 32.71%

 0.7 3 60 28 44.85%

 CrylAb 1.0 3 60 36 58.63%

 1.5 3 60 42

 2.4 3 60 52 86.21%

 0.3 3 60 8 10.38%

 1.2 3 60 10 13.82%

 Cr lAb-loop2 2.4 3 60 13 18.99%

 -P2S 10 3 60 24 37.95%

 20.0 3 60 30 48.29%

55.0 3 60 44 72.42% Table 2 Toxicity effect of engineering strain lAb-P2S (CGMCC NO: 6427) on rice brown planthopper (24 hours) Sample protein concentration (pg ml) Repeat total number of insects 24 hours dead insects 24 hours corrected mortality

 25 3 60 9 5.56%

50 3 60 13 12.96%

CrylAb 100 3 60 17 20.37%

 200 3 60 37 57.41%

1 3 60 23 35.09%

5 3 60 27 42.11%

CrylAb-loop2 10 3 60 32 50.88%

-P2S 25 3 60 37 59.65%

 50 3 60 42 68.42%

100 3 60 50 82.46%

Claims

Claim A short peptide P2S gene characterized by having a nucleotide sequence as shown in SEQ ID NO: l; said short peptide P2S capable of binding to the intestinal lining of rice brown planthopper.  2. A short peptide P2S characterized by having the amino acid sequence as shown in SEQ ID NO: 2.  A gene of the toxin ^Ζ^-Λ3⁄43⁄4^-ί6 characterized by having a nucleotide sequence as shown in SEQ ID NO: 3 and having toxicity to rice brown planthopper.  A protein of the toxin CrylAb-o2-p2s, which has the amino acid sequence as shown in SEQ ID NO: 4 and is toxic to rice brown planthopper. 5. One pair of primers associated with the cloned yZ^ gene are crylAb-F and crylAb-R _: crylAb-F: 5 '-GGATCCATGGATAACAATCCGAACAT-3 'crylAb-R: 5'-GTCGACTTACTATTCCTCCATAAGGAGTAATTC-3'. 6. The three pairs of primers associated with the transformation of the yZ^ gene with the short peptide P2S are md-F and md-R, up-F and up-R, down-F and down-R: (1) md-F: 5 ' -CCCGGGACTAGTTGATATAATATGGGGAATTT-3 ' md-R: 5'-GGGCCCCAATTGATGTATGGAATTGTAAACCCGGG-3 '; (2) up-F: 5 '-CCCGGGACTAGTTGATATAATATGGGGAATTT-3 ' up-R : down-R: 5 '-GGGCCCCAATTGATGTATGGAATTGTAAA-3 '.  7. An engineering strain lAb-P2S, characterized in that the strain is an artificially constructed plasmid, and the host is Escherichia coli, CGMCC NO: 6427 ο  8. The engineered strain lAb-P2S according to claim 7, characterized in that it contains a ^^^- 3⁄43⁄4^^ y toxin. The engineered strain lAb-P2S according to claim 7 or 8, characterized in that the CrylAb-lo ₀ p2-P2S toxin protein obtained by the fermentation of the engineered strain 1Ab-P2S has a poisoning effect on rice brown planthopper.