Insecticidal protein and use thereof

Abstract

Provided are a novel protein, a nucleic acid molecule encoding the protein, an expression cassette containing the nucleic acid molecule, and a use thereof in cultivation of insect-resistant plants.

Description

Insecticidal protein and its application Technical Field

[0001] The present invention relates to the field of plant biotechnology. Specifically, it relates to an insect-resistant protein, a nucleic acid molecule encoding the protein, an expression cassette containing the nucleic acid molecule, and uses thereof. By introducing a construct containing the expression cassette into a plant, plant materials with high resistance to Lepidoptera insects such as fall armyworm and corn borer can be produced. Background Art

[0002] Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces insecticidal crystalline proteins, known as Bt proteins. Due to their specificity against various pests and relative safety against non-target organisms, Bt proteins are widely used in agriculture and biological control.

[0003] In the research process of insecticide proteins, a large number of proteins need to be tested. For example, the quantitative evaluation of the toxicity of insecticide proteins requires the use of the half-lethal concentration (LC50). 50 ) test, which requires 3-8 concentration gradients of biological test data for calculation, and each test insect requires at least 1mg of protein. The insect resistance spectrum screening requires testing different test insects on this basis, and the data obtained from the protein test of the same batch of expression is more reliable. In addition, when verifying the safety of insect-resistant proteins, a large amount of protein is also needed to carry out relevant experiments. The protein required for the oral acute toxicity test is 2g, and the protein required for the test of the effect on large fleas is 1g. However, since most Bt proteins are crystalline proteins, their expression efficiency is relatively low. Therefore, obtaining Bt proteins with higher expression efficiency will be of great value in the preparation of pesticides and the development of insect-resistant crops.

[0004] Due to the complexity of insecticidal protein structures and mechanisms of action, even a single amino acid difference at a key site can affect the protein's insecticidal activity against target insects. Therefore, new proteins derived from the fusion of different structural domains and amino acid mutations may exhibit differences in their spectrum of activity, activity, production efficiency, and stability. Precisely testing the efficacy of these proteins and screening for insecticidal proteins that excel in all aspects is commercially valuable.

[0005] The original bacterial Bt gene contains many elements that cause unstable expression in eukaryotes, such as plant-like poly(A) signal sequences, intron cleavage signal sites, and AT-rich sequences, which can lead to instability in the gene-encoded mRNA. Compared to plant genes, the GC content and codon usage frequency of the original bacterial Bt gene differ significantly, making it unsuitable for efficient expression in plants. After the original Bt gene was transformed into plants, the expression level of the Bt insecticidal crystal protein (ICP) was generally low, resulting in poor insect resistance. The toxin protein content was as low as 0.001% of the soluble protein or even undetectable levels, making it difficult to meet the requirements of production applications. Since 1990, research on Bt gene modification has been increasingly advanced, and the expression levels of modified Cry1Ab and Cry1Ac in transgenic cotton have been significantly improved, with the insecticidal protein content reaching 0.05-0.1% of the total soluble protein in the plant, achieving the desired insect resistance and making the application of transgenic Bt crops in agricultural production possible.

[0006] Codon optimization requires comprehensive consideration of multiple factors to achieve optimal results, including host codon preference, mRNA secondary structure, restriction enzyme sites, and GC content. Simply adjusting synonymous codons in a foreign gene to codons that are highly expressed and frequently used in host cells does not necessarily improve protein translation efficiency; in fact, the effect can be counterproductive. Therefore, codon optimization is not a simple experiment. Furthermore, nucleic acid sequence optimization in plants, especially monocots, is more challenging and complex. First, plants predate animals and have genomes much larger than those of microorganisms. Plant genomes have a long and complex evolutionary history, characterized by high heterozygosity, highly repetitive sequences, and complex polyploidy. Second, research on plant genomes began relatively late, making it more challenging. Consequently, existing data on plant genome structure and gene expression is limited. Consequently, it is uncertain what coding method will achieve the highest functional efficiency for a given foreign protein.

[0007] Using overexpression vectors to drive the expression efficiency of insecticidal genes in plants can greatly enhance the insecticidal activity of insecticidal proteins. A common strategy involves using endogenous plant overexpression promoters (such as the Ubiquitin promoter endogenous to maize and rice) or the 35S overexpression promoter derived from the mosaic virus (CaMV) to drive insecticidal gene expression. Using endogenous promoters in target plants to drive insecticidal gene expression can present challenges such as interference between similar regulatory elements and difficulty distinguishing endogenous and exogenous sequences during molecular identification. Therefore, more diverse insecticidal expression cassettes with diverse functions have potential applications in the development of insect-resistant plant germplasm. Summary of the Invention

[0008] The purpose of the present invention is to provide an insect-resistant protein, a nucleic acid molecule encoding the protein, and an expression cassette containing the nucleic acid molecule. By transferring the nucleic acid molecule or the expression cassette into a plant, the transgenic plant can efficiently express the insecticidal protein, thereby protecting it from insect invasion.

[0009] To achieve the above objectives, the present invention obtained an excellent insecticidal protein LC85-AC through protein domain fusion and amino acid mutation testing; through extensive bioinformatics analysis and specific experimental testing, a nucleic acid molecule with the best insecticidal effect in plants was obtained; on this basis, multiple expression cassettes were constructed and transformed into corn and rice, and the expression cassette with the highest insecticidal activity was screened through insect-resistant trait identification.

[0010] The present invention provides a protein, characterized in that the amino acid sequence of the protein is shown as SEQ ID NO.6.

[0011] The present invention also provides a nucleic acid molecule, characterized in that the nucleic acid molecule encodes the above protein;

[0012] In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.16.

[0013] The present invention also provides an expression cassette, characterized in that the expression cassette is formed by sequentially connecting a promoter, a nucleic acid molecule encoding the above protein, and a terminator.

[0014] In some embodiments, the nucleotide sequence of the promoter is shown as SEQ ID NO.28, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.16, and the nucleotide sequence of the terminator is shown as SEQ ID NO.23.

[0015] The present invention also provides a vector, characterized in that the vector comprises the above-mentioned nucleic acid molecule or the above-mentioned expression cassette.

[0016] The present invention also provides a host cell, characterized in that the host cell contains the above-mentioned nucleic acid molecule, or the above-mentioned expression cassette, or the above-mentioned vector; the host cell is a microbial cell or a non-renewable animal or plant cell.

[0017] The present invention also provides a method for producing transgenic plants, characterized in that the above-mentioned nucleic acid molecule, or the above-mentioned expression cassette, or the above-mentioned vector, or the above-mentioned host cell is transformed into a plant to obtain a plant cell expressing the above-mentioned protein, and then the transformed plant cell is cultured into a transgenic plant.

[0018] The present invention also provides the use of the above-mentioned protein, or the above-mentioned nucleic acid molecule, or the above-mentioned expression cassette, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned method in insect resistance or preparation of insect-resistant preparations or cultivation of insect-resistant plants; wherein the insect resistance includes resistance to any one of fall armyworm, cotton bollworm, Asian corn borer, oriental armyworm, beet armyworm, Spodoptera litura, black cutworm, and two-spotted worm.

[0019] The present invention also provides a method for protecting plants from insect attack, comprising providing at least one transgenic plant cell in the diet of a target insect, wherein the transgenic plant cell comprises the aforementioned nucleic acid molecule, expression cassette, or vector in its genome and expresses the aforementioned protein; target insects that ingest the transgenic plant cell are inhibited from further ingesting the plant; the target insects are resistant to any one of the following: fall armyworm, cotton bollworm, Asian corn borer, oriental armyworm, beet armyworm, Spodoptera litura, black cutworm, and two-spotted worm; and the plant is corn or rice.

[0020] The beneficial effects of the present invention are as follows: an excellent insecticidal protein LC85-AC was obtained through a large number of protein domain fusion and amino acid mutation tests; a nucleic acid molecule with the best insecticidal effect in corn plants was obtained through a large number of bioinformatics analyses and specific experimental tests; on this basis, a variety of expression cassettes were constructed and transformed into corn and rice, and the expression cassette with the highest insecticidal activity was screened out through insect-resistant trait identification. The transgenic plants produced thereby have a high resistance level to Lepidoptera insects such as the fall armyworm, corn borer, oriental armyworm and Spodoptera litura, can effectively inhibit the reproduction and damage of pests, and protect the growth and development of crops.

[0021] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Three different versions of the third domain of Cry1Ka

[0023] Figure 2 Physical map of expression vector pBWA(V)HS-LC85-Ac-bar, taking pA5 as an example.

[0024] Figure 3. Insect resistance of pA5-transgenic corn. A: pA5-transgenic corn; B: corn recipient control B104.

[0025] Figure 4 is a schematic diagram of the structure of the expression cassette.

[0026] Figure 5 Physical map of the expression vector pCAMBIA3300-LC85-Ac-bar, taking P9 as an example.

[0027] Figure 6 Insect resistance of LC85ZM09 and B104. A: LC85ZM09 plant leaves; B: B104 leaves. DETAILED DESCRIPTION

[0028] The following definitions and methods are provided to better define this application and to guide those skilled in the art in practicing this application. Unless otherwise noted, terms are to be understood according to conventional usage by those skilled in the relevant art. All patent documents, academic papers, industry standards, and other publications cited herein are hereby incorporated by reference in their entirety.

[0029] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Any modifications or substitutions to the methods, steps, or conditions of the present invention, without departing from the spirit and substance of the present invention, are intended to be within the scope of this application. Unless otherwise specified, the examples are based on conventional experimental conditions, such as those described in Sambrook et al. (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or the conditions recommended by the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are conventional commercially available reagents, and the techniques used in the examples are conventional means well known to those skilled in the art.

[0030] Example 1 Screening of highly effective insecticidal proteins

[0031] 1. Design and modification of insecticidal proteins

[0032] Cry1Be2 is a Bt protein with moderate insecticidal activity against the fall armyworm. Modification of the third domain and C-terminal sequence of Cry1Be2 can yield novel insecticidal proteins with enhanced activity against the fall armyworm. Patent CN 107074974 A discloses a series of insecticidal proteins fused with the first and second domains of Cry1Be2 at the N-terminus, the third domains of Cry1Ka and Cry1Ca, and various C-termini.

[0033] Due to the complexity of the structure and mechanism of action of insecticidal proteins, a difference of a single amino acid at a key site will affect the insecticidal activity of the protein against target insects. Therefore, the inventors hope to systematically compare the active effects of insecticidal proteins obtained by fusion of different third domains and C-terminal sequences of Cry1Be2 at the N-terminus against various insects, in order to obtain insecticidal proteins with excellent performance in terms of anti-spectrum, activity, preparation efficiency, stability, etc.

[0034] 2. Influence of different third domains

[0035] The inventors first tested the insecticidal efficacy of fusion proteins derived from Cry1Be2 linked to different Cry1Ka and Cry1Ca third domains, and then linked to the C-terminus of Cry1Be. Three different versions of the Cry1Ka third domain were selected, each differing at the terminus of the third domain (as shown in Figure 1).

[0036] The specific test methods are as follows:

[0037] Synthesize a nucleic acid molecule encoding the specified amino acid and construct it into the pET28a expression vector to obtain a protein expression vector. Transform this vector into the E. coli BL21 cell line and express the protein. The specific steps are as follows:

[0038] A single colony was inoculated into 0.5 mL of LB liquid medium and cultured at 37°C for 4 hours until the medium became turbid. IPTG (Isopropyl-β-D-thiogalactoside) was added to 100 μL of the culture medium to a final concentration of 0.8 mM. 20 μL of the culture medium was inoculated into 2 mL of LB liquid medium and cultured at 37°C for 12-16 hours as a seed culture. The seed culture was then inoculated into 250 mL of LB liquid medium to an OD600 of 0.5-0.6. IPTG (Isopropyl-β-D-thiogalactoside) was then added to a concentration of 0.8 mM and cultured under the same conditions for another 4 hours. The culture medium was centrifuged at 5000 g for 10 minutes to collect the E. coli cells, added to 30 mL of 20 mM Tris-50 mM NaCl buffer, and sonicated for protein purification.

[0039] Activity test on fall armyworm: The surface smear method was used for bioassay. First, about 1 mL of unsolidified artificial feed (about 0.5 g) was added to a 24-well plate. The plate was gently shaken to cover the bottom of the plate. After the feed solidified, different concentrations of protein solution (20 μL / well) were added. After adding, the solution was gently shaken to spread evenly on the surface of the feed. The plate was naturally air-dried in a fume hood for 1 hour. The experiment set up 6 gradient concentrations (0.05, 0.1, 0.5, 1, 2, 4 μg / cm 2 ) and a blank control (PBS buffer). Each treatment was inoculated with 24 newly hatched larvae of Spodoptera frugiperda (Spodoptera frugiperda) reared for three replicates. The larvae were cultured in an insectary at 25±2°C, a 14:10 photoperiod (L:D) h, and a relative humidity of 50-70%. Mortality was assessed after 7 days. Larvae were considered dead if their tails remained motionless, as were larvae that had not reached the second instar.

[0040] The mortality rate and adjusted mortality rate were calculated according to the following formula, and LC was calculated using GraphPad. 50 value.

[0041] The test results show that the third domain of the V2 version of Cry1Ka has the best effect on the protein activity of the Cry1Be2-Cry1Ka-Cry1Be structure.

[0042] Table 1 Different protein structures and test results

[0043] 3. The impact of different C-ends

[0044] Cry1Be2-Cry1KaV2-Cry1Be is a novel insecticide with certain insecticidal activity against fall armyworm (LC 50 =0.192μg / cm 2 ) chimeric protein, named LC85. However, the protein's expression efficiency was very low. Therefore, the inventors sought to establish a high-efficiency expression system for this protein. However, after optimizing expression conditions such as temperature, rotation speed, induction concentration, and expression time, they were still only able to obtain 5 mL of LC85 protein at a concentration of 0.2 mg / mL from 500 mL of culture medium.

[0045] The inventors further tested the expression effect and insecticidal activity by replacing the C-terminus of LC85.

[0046] First, five Bt proteins with good expression effects were screened through experiments. Through domain analysis, the C-termini of these proteins were connected to the N-terminus of LC85 and constructed into the pET28a expression vector. The new protein structures are shown in Table 2.

[0047] Table 2 Optimized chimeric protein sequence information

[0048] Comparison of expression effects: 8 μL of expression bacteria with an OD value of 0.6 was inoculated into 500 mL of LB medium and cultured at 210 rpm and 37°C. When the OD600 value of the bacteria in the shake flask reached 0.6, 0.6 mmol / L IPTG was added to induce expression for 20 hours. After expression, the bacteria were ultrasonically disrupted, centrifuged, and purified. The protein solution was then diluted to 5 mL and the concentration was measured to evaluate expression effects.

[0049] For the activity test against Spodoptera frugiperda, refer to point 2 of this example.

[0050] The test results showed that the expression effect of LC85-Ac protein was the best. Under the same conditions, the protein concentration was as high as 2.0 mg / mL. It also had similar insecticidal activity to LC85. 50 The value is 0.179 μg / cm 2The second is LC85-Ab protein. After replacing the C-terminus, its expression concentration was 1.2 mg / mL, but its insecticidal activity was weakened to 0.309 μg / cm 2 Therefore, the LC85-Ac chimeric protein can be used as a novel protein to kill Spodoptera frugiperda and develop biopesticides and Spodoptera frugiperda-resistant transgenic plants. Protein expression and insecticidal activity data are shown in Table 3.

[0051] Table 3 Expression effect and insecticidal activity of chimeric proteins

[0052] 4. Insecticidal activity of chimeric proteins against lepidopteran pests

[0053] In order to further explore the anti-insect activity of the novel chimeric protein of the present invention against Lepidoptera, the inventors conducted bioassays on Mythimna separata, Spodoptera frugiperda, Spodoptera exigua, Spodoptera litura, Helicoverpa armigera, Ostrinia furnacalis, Agrotis ypsilon, and Dichocrocis punctiferalis according to the method of Example 2, and calculated the LC 50 The results are shown in the table below.

[0054] Table 4 Insecticidal activity of chimeric proteins against lepidopteran pests

[0055] “++++” stands for LC 50 Value: 0.01~0.20(μg / cm 2 ), “+++” represents LC 50 Value: 0.21~0.50(μg / cm 2 ), “++” represents LC 50 Value: 0.51~1.0(μg / cm 2 ), “+” represents LC 50 Value: 1.1~2.0(μg / cm 2 ), “-” represents LC 50 Value>2.1(μg / cm 2 ).

[0056] Results showed that LC85-Ac exhibited the best expression, the most effective insecticide resistance, and the broadest insect spectrum. It exhibited extremely high activity against fall armyworm, cotton bollworm, and Asian corn borer, as well as high activity against oriental armyworm, beet armyworm, Spodoptera litura, and black cutworm, while exhibiting weaker activity against Spodoptera exigua. LC85-Ac achieved the best overall resistance against these pests among all tested proteins.

[0057] Example 2 Screening of Nucleic Acid Molecules Encoding Highly Effective Insecticide Proteins in Corn

[0058] Codon usage bias exists during translation in organisms. The fundamental principle of codon optimization is to replace codons in exogenous mRNA sequences with synonymous codons frequently used in host cells, ensuring a better match between the codon usage of the exogenous mRNA sequence and the host cell's codon usage bias, thereby improving protein expression. The degree of alignment is often measured using the Codon Adaptation Index (CAI). This index reflects the degree to which synonymous codons in the coding region align with the optimal codon usage of a particular species, ranging from 0 to 1 and exhibiting species-specificity. Theoretically, the closer this value is to 1, the higher the protein expression of the exogenous mRNA in the host cell. Furthermore, mRNA secondary structure has a certain impact on translation efficiency, while restriction enzyme sites can also affect the construction of expression vectors. The GC content is associated with DNA stability; a value above 70% may indirectly affect gene expression regulation.

[0059] 1. Sequence design

[0060] To obtain a coding nucleic acid molecule with better insecticidal effects for the LC85-Ac protein (amino acid sequence shown in SEQ ID NO. 6), the present invention statistically analyzed the codon usage of different genes in monocotyledonous plants such as maize. Focusing on the CAI (Codon Adaptability Index) values ​​of codons in highly expressed genes in different species, the present invention also focused on parameters such as the relative usage of synonymous codons (RSCU) and the number of effective codons (Nc). Furthermore, factors such as mRNA secondary structure, species repeat structure, rare codons, hidden splicing sites, GC content, and enzyme cleavage sites were considered. More than 50 nucleotide sequences were initially screened. Five sequences with high CAI values ​​(CAI values ​​≥ 0.75), moderate GC content (between 50% and 60%), and low Nc values ​​(Nc ≤ 25) were selected and named A1-A5, respectively. Sequence-related parameter information is shown in Table 5.

[0061] Table 5 LC85-Ac nucleic acid sequence list

[0062] 2. Acquisition of transgenic plants and identification of insect resistance

[0063] Candidate nucleic acid molecules A1-A5 were artificially synthesized and ligated to the NOS promoter and terminator, respectively, to construct five plant expression vectors pBWA(V)HS-LC85-Ac-bar (the bar gene expression cassette was used as a selection marker), numbered pA1-pA5 in sequence. The information of the T-DNA region expression cassette elements is shown in Table 6, and the vector physical map is shown in Figure 2 (taking the pA5 vector as an example).

[0064] After transforming the above expression vectors into Escherichia coli DH5α, the plasmids were extracted and then transformed into Agrobacterium tumefaciens EHA105 or LBA4404. Using Agrobacterium-mediated transfection, the five vectors were transformed into maize inbred line B104, using glufosinate-ammonium as a screening agent. Ultimately, 70 positive transgenic maize plants were obtained (vector construction and plant genetic transformation were performed using methods commonly used in the art). The insect resistance of these transgenic plants was then assessed.

[0065] Table 6 Element information of the T-DNA region of 5 expression vectors

[0066] Using maize B104 as a control, transgenic plants with superior insect resistance were screened through leaf bioassays. Detached leaves were fed to newly hatched larvae of the fall armyworm and corn borer to initially screen for insect resistance. Two replicates were set for each material. Transgenic plants with an average mortality rate exceeding 40% were preliminarily considered resistant, while the mortality rate of the negative control did not exceed 20%.

[0067] Table 7 shows the results of laboratory bioassays of transgenic maize plants transformed with five different vectors. These results show that 33 transgenic maize plants were resistant to the fall armyworm and 32 to the corn borer. Of these, 24 were resistant to both pests. Although the nucleic acid sequence A5 in the pA5 vector does not have the highest CAI value (0.92) in maize, surprisingly, the transgenic maize plants transformed with the A5 sequence showed the highest percentage of resistance to both pests, at 83.3%.

[0068] Table 7 Indoor bioassay results of different transgenic maize plants

[0069] The resistance levels of these 24 transgenic maize plants, which were resistant to both the fall armyworm and corn borer, were further evaluated. The maize plants were numbered M1-M24, with B104 used as a control. Leaf bioassays were performed indoors to screen for transgenic plants with superior insect resistance. Newly hatched larvae of the fall armyworm were fed detached maize leaves to evaluate their insect resistance. The mortality and adjusted mortality rates of the test insects were calculated using Equations 1 and 2, respectively.

[0070] The results showed that the mortality rates of fall armyworm and corn borer feeding on the leaves of these 24 transgenic corn plants were significantly higher than that of the control (Table 8 and Figure 3). Among them, the resistance levels of the 10 transgenic corn plants M15-M24 to fall armyworm and corn borer were all high, while the others were moderately resistant or resistant.

[0071] The test results show that the nucleic acid molecule sequence in the above 10 highly resistant transgenic plants is all A5, and its insecticidal effect in corn is better than that of the other four LC85-Ac gene nucleic acid molecules A1-A4.

[0072] Table 8 Indoor bioassay results of insect-resistant corn transgenic plants

[0073] The values ​​are expressed as the mean ± standard deviation of three biological replicates, and the differences between the data in the same column were analyzed using the LSD method (α = 0.05).

[0074] 3. Identification of resistance of transgenic corn M24 to other lepidopteran insects

[0075] Insect resistance bioassays were conducted indoors to test the resistance of transgenic corn M24 to other lepidopteran insects, including fall armyworm, cotton bollworm, corn borer, oriental armyworm, beet armyworm, armyworm litura, black cutworm, and Spodoptera exigua. The test results are shown in Table 9.

[0076] Table 9 Bioassay results of transgenic corn M24 on various lepidopteran insects

[0077] By optimizing the coding sequence of the insecticidal protein LC85-Ac, a nucleic acid molecule A5 (sequence: SEQ ID NO: 16) with optimal insecticidal effect in corn was obtained. After identification, the transgenic corn produced using this nucleic acid molecule has good resistance to Lepidoptera insects such as the fall armyworm, cotton bollworm, corn borer, oriental armyworm, beet armyworm, Spodoptera litura, black cutworm, and two-spotted moth.

[0078] Example 3 Screening for expression cassettes capable of improving insect resistance in gramineous crops

[0079] 1. Expression cassette design and vector construction

[0080] The insect-resistant protein LC85-Ac was used, and the amino acid sequence was shown in SEQ ID NO. 6. The LC85-Ac gene was artificially synthesized, and the nucleic acid sequence was shown in SEQ ID NO. 16. By homologous recombination, the PCR products were connected to the maize promoter ZmUbiquitin (nucleic acid sequence shown in SEQ ID NO.18), the enhanced tobacco mosaic virus promoter CaMV 35S promoter (enhanced) (nucleic acid sequence shown in SEQ ID NO.19), the Agrobacterium tumefaciens nopaline synthase gene promoter NOS promoter (nucleic acid sequence shown in SEQ ID NO.20), the Arabidopsis promoter Atubi10 (nucleic acid sequence shown in SEQ ID NO.21), the rice promoter OsUbi (nucleic acid sequence shown in SEQ ID NO.22), three promoters from millet SETIT_016868mg, SETIT_037316mg, SETIT_003209mg (nucleic acid sequences shown in SEQ ID NO.25-27) and the artificially spliced ​​promoter SETIT_003209mgI (nucleic acid sequence shown in SEQ ID NO.28), and then connected to the rice terminator GOS2 (nucleic acid sequence shown in SEQ ID NO. 23), OsLTP (nucleic acid sequence shown in SEQ ID NO. 24), or the Agrobacterium tumefaciens nopaline synthase gene terminator TNOS (nucleic acid sequence shown in SEQ ID NO. 17), resulting in a total of 13 LC85-Ac gene expression cassettes B1-B13 composed of different promoter and terminator combinations, as shown in Figure 4.

[0081] These 13 insect-resistant expression cassettes were ligated into the backbone vector pCAMBIA3300, along with the selectable marker bar gene expression cassette, to generate 13 new pCAMBIA3300-LC85-Ac-bar expression vectors P1-P13. Detailed information on the expression cassettes in the T-DNA region of these vectors is shown in Table 10.

[0082] Table 10 Element information of T-DNA regions of 13 expression vectors

[0083] 2. Acquisition of transformation events and character identification

[0084] Using expression vectors P1-P13 (physical maps of these vectors are shown in Figure 5, with the P9 vector used as an example), we transformed rice material Zhonghua 11 and maize material B104 via Agrobacterium-mediated transformation. Using glufosinate as the screening agent, we generated 120 rice transformation events and 106 maize transformation events using these 13 vectors. The T1 generation of these transformed seedlings was then identified and screened for insect resistance. All resistant materials were screened against Ostrinia nubilalis and Spodoptera frugiperda as target insects.

[0085] (1) Screening of transgenic rice for insect resistance

[0086] Using Zhonghua 11 as a negative control, transformants with high insect resistance were screened through in vitro bioassays using detached leaves. Detached leaves from seedling-stage rice were fed to newly hatched larvae of corn borer and fall armyworm to initially screen for insect resistance. Each material was replicated in pairs. Transformants with an average mortality rate exceeding 40% were preliminarily considered resistant, while the mortality rate of the negative control did not exceed 20%. The results of in vitro bioassays of transformants transformed with 13 different vectors are shown in Table 11. Twenty transformants were found to be resistant to both fall armyworm and corn borer, designated LC85OS01-LC85OS20. Four of these transformants were transformed with the P9 vector. The LC85-Ac gene in the P9 vector is driven by the SETIT_003209mgI promoter and linked to the GOS2 terminator. The rate of transformants resistant to both insects (36.4%) was higher than with other vectors. In other words, the insecticidal activity of transgenic rice plants transformed with different insect-resistant expression cassettes is different. The B9 expression cassette driven by the SETIT_003209mgI promoter and terminated by GOS2 has the highest insecticidal activity in rice plants.

[0087] Table 11 Indoor bioassay results of rice transformants with different vectors

[0088] The resistance grades of these 20 transformants were further evaluated, and the evaluation criteria are shown in Table 12.

[0089] Cut a rice leaf about 4 cm thick and place it in a culture dish. Cover the bottom of the dish with filter paper moistened with sterile water to maintain a high humidity state. Inoculate 10 newly hatched larvae of the test insects (hatched 2-12 hours) into each dish. Cultivate under the conditions of temperature 25±1°C, humidity 50%, L:D=14:10. Check the survival of the test insects on the 6th day. Larvae that do not develop to the second instar are also considered dead. Zhonghua 11 is used as a negative control. Repeat 3 times for each material. Calculate the mortality rate and corrected mortality rate of the test insects according to the following formulas 1 and 2, respectively.

[0090] Table 12 Evaluation criteria for insect resistance of transgenic insect-resistant plants in indoor bioassays

[0091] The results showed that the mortality rates of corn borers and fall armyworms feeding on the leaves of these 20 transformants were significantly higher than those in the control (Table 13). Four of the transformation events (LC85OS11, LC85OS12, LC85OS13, and LC85OS014) displayed high resistance to both insects. LC85OS08 and LC85OS09 displayed high resistance to both corn borers and fall armyworms, while LC85OS15 and LC85OS19 displayed high resistance to both fall armyworms and corn borers. The remaining transformants displayed moderate or resistant resistance to both insects. The four transformants, LC85OS11, LC85OS12, LC85OS13, and LC85OS14, which displayed high resistance to both insects, were all transformed with the B9 expression cassette (SETIT_003209mgI promoter + LC85-AC + GOS2 terminator).

[0092] Table 13 Results of indoor bioassays of 20 rice transformants resistant to both insects

[0093] The values ​​are expressed as the mean ± SD of 3 biological replicates.

[0094] (2) Screening of transgenic corn for insect resistance

[0095] Using B104 as a control, transformants with superior insect resistance were screened through indoor leaf bioassays. Detached corn leaves were used to feed newly hatched larvae of the oriental armyworm and Spodoptera litura to initially screen for insect resistance. Each material was replicated twice. Transformants with an average mortality rate exceeding 40% were preliminarily considered resistant, while the mortality rate of the negative control did not exceed 20%. The results of indoor bioassays of transformants transformed with 13 different vectors are shown in Table 14. Of these, 17 transformants were resistant to both the oriental armyworm and Spodoptera litura, designated LC85ZM01-LC85ZM17. Three of these transformants were transformed with the P9 vector. The LC85-Ac gene in the P9 vector is driven by the SETIT_003209mgI promoter and linked to the GOS2 terminator. The rate of transformants resistant to both insects (37.5%) was higher than that obtained with other vectors. In other words, the insecticidal activity of transgenic corn with different insect-resistant expression cassettes is different. The B9 expression cassette driven by the SETIT_003209mgI promoter and terminated by GOS2 has the highest insecticidal activity in corn plants.

[0096] Table 14 Indoor bioassay results of different maize transformants

[0097] The resistance levels of these 17 transformants were further evaluated. Using B104 as a reference, transformants with superior insect resistance were screened using leaf bioassays. Detached corn leaves were fed to newly hatched larvae of the oriental armyworm and Spodoptera litura to evaluate the insect resistance of the materials. The mortality rate and adjusted mortality rate of the test insects were calculated using the following formulas 1 and 2, respectively. Resistance levels were evaluated using Table 3.

[0098] The results showed that the mortality rates of oriental armyworm and Spodoptera litura fed on leaves from these 17 transformants were significantly higher than those in the control (Table 15). Three transformants, LC85ZM08, LC85ZM09, and LC85ZM10, all showed high resistance to both insects. The LC85ZM06 transformant showed high resistance to both oriental armyworm and Spodoptera litura. The LC85ZM17 transformant showed high resistance to both Spodoptera litura and oriental armyworm. The remaining transformants showed moderate or resistant resistance to both insects. All three transformants with high resistance to both insects were transformed with the B9 expression cassette (SETIT_003209mgI promoter + LC85-AC + GOS2 terminator).

[0099] Table 15 Results of indoor bioassays of 17 maize transformants resistant to both insects

[0100] The values ​​are expressed as the mean ± SD of 3 biological replicates.

[0101] The results of insect resistance identification of transgenic rice and transgenic corn showed that the transformants containing expression cassette B9 or expression vector P9 reached a high level of resistance to fall armyworm, corn borer, oriental armyworm and Spodoptera litura, and their insecticidal effect was significantly better than other expression cassettes or expression vectors.

[0102] 3. Resistance of GM corn to other lepidopteran pests

[0103] The indoor insect resistance bioassay method described in point 2 of this Example was used to test the resistance of transgenic maize varieties LC85ZM08, LC85ZM09, and LC85ZM10 to other Lepidoptera insects, including Spodoptera frugiperda, Spodoptera exigua, Helicoverpa armigera, Corn borer, Cutworm, and Spodoptera occidentalis. The test results are shown in Table 16. The results showed that transformants containing expression cassette B9 or expression vector P9 also achieved high resistance to Spodoptera frugiperda, Spodoptera exigua, Helicoverpa armigera, Corn borer, Cutworm, and Spodoptera occidentalis, demonstrating the application value of this expression cassette in controlling Lepidoptera pests.

[0104] Table 16 Bioassay results of transgenic corn on various lepidopteran insects

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention.

Claims

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

6.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the protein according to claim 1; optionally, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

16.

3. An expression cassette, characterized in that The expression cassette is composed of a promoter, a nucleic acid molecule encoding the protein according to claim 1, and a terminator connected in sequence; optionally, the nucleotide sequence of the promoter is shown in SEQ ID NO.28, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.16, and the nucleotide sequence of the terminator is shown in SEQ ID NO.

23.

4. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 2 or the expression cassette of claim 3.

5. A host cell, characterized in that The host cell contains the nucleic acid molecule of claim 2, or the expression cassette of claim 3, or the vector of claim 4; The host cell is a microbial cell or a non-renewable animal or plant cell.

6. A method for producing transgenic plants, characterized in that The nucleic acid molecule according to claim 2, or the expression cassette according to claim 3, or the vector according to claim 4, or the host cell according to claim 5 is transformed into a plant to obtain a plant cell expressing the protein according to claim 1, and then the transformed plant cell is cultured into a transgenic plant.

7. Use of the protein according to claim 1, or the nucleic acid molecule according to claim 2, or the expression cassette according to claim 3, or the vector according to claim 4, or the host cell according to claim 5, or the method according to claim 6 in resisting insects, preparing insect-resistant preparations, or cultivating insect-resistant plants; in, The insect resistance includes resistance to any one of fall armyworm, cotton bollworm, Asian corn borer, oriental armyworm, beet armyworm, Spodoptera litura, black cutworm and two-spotted armyworm.

8. A method for protecting plants from insect infestation, characterized in that: The method comprises providing at least one transgenic plant cell in the diet of a target insect, wherein the transgenic plant cell comprises the nucleic acid molecule of claim 2 or the expression cassette of claim 3 in its genome and expresses the protein of claim 1; the target insect that feeds on the transgenic plant cell is inhibited from further feeding on the plant; The target insects include any one of fall armyworm, cotton bollworm, Asian corn borer, oriental armyworm, beet armyworm, armyworm, cutworm, and two-spotted armyworm; and the plant is corn or rice.

Images