A smart design gene BT3AES and its applications

Abstract

This invention provides an intelligently designed gene BT3AES and its application, belonging to the fields of genetic engineering and biointelligence. The nucleotide sequence of the BT3AES gene is shown in SEQ ID No. 1. In this invention, the toxic natural proteins Cry8Ea, Sip1Aa, and Cry3Aa to coleopteran pests are intelligently designed, and a randomly combined gene library is constructed. After screening and identification, the optimal insecticidal gene BT3AES is obtained, which can express the above three proteins in corn leaf, root, and other tissue cells. This gene has a significant insecticidal effect on coleopteran pests such as the two-spotted leaf beetle, and the co-expression of multiple protein genes has a better insecticidal effect than any single natural protein, achieving a synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and bio-intelligence technology, and in particular relates to the design of an intelligent design gene BT3AES assisted by bioinformatics technology and its application. Background Technology

[0002] Coleoptera pests cause significant damage to agriculture and forestry. Most are soil-dwelling insects that primarily live underground, feeding on the underground parts of crops, including germinating seeds and the roots and stems of seedlings. Their feeding behavior often results in large-scale seedling loss and gaps in rows, and negatively impacts crop yield and quality in the later stages of crop development. Traditional pesticide control of coleoptera pests requires deep tillage, necessitating substantial manpower and resources, and contradicts the widespread adoption of no-till farming techniques.

[0003] Various insecticidal proteins derived from Bacillus thuringiensis and their artificially modified derivatives are currently the main sources of insecticidal functional genes in transgenic crops. However, most well-known insecticidal genes are developed for lepidopteran pests, even though coleopteran pests are no less damaging to agriculture than lepidopteran pests. Currently, the cryoproteins that have been identified as resistant to coleopteran pests are mainly cry1AB, cry3, cry7, cry8, cry18, cry23, cry34, cry37, and cry43, concentrated in the cry3 and cry8 insecticidal protein families. However, possibly due to the developmental process of beetles themselves, no single insecticidal protein can achieve the complete lethality of Cry1AB against lepidopteran pests. This means that single-species insecticidal proteins may need to be hybridized or crop rotation to avoid stress selection of resistant pest mutants in the field. To prevent insecticidal genes in genetically modified crops from becoming ineffective due to pest resistance evolution in a short period of time, multiple insecticidal proteins are co-expressed to address this prominent issue. This effectively kills and reduces the damage to crops caused by coleopteran pests in the field, while simultaneously increasing the difficulty for target pests to evolve resistance. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a smart design gene BT3AES and its application. Unlike previous natural or artificially modified genes that encode a single insecticidal protein, this invention uses bioinformatics technology to assist in the design of an artificially encoded gene that co-expresses three insecticidal proteins, thereby effectively improving the insecticidal activity and insecticidal spectrum of transgenic crops while preventing the evolution of resistance in target pests.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a smart design gene BT3AES, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0007] Preferably, the BT3AES gene is co-expressed by three insecticidal proteins, Cry8Ea, Sip1Aa, and Cry3Aa, mediated by two LP4 / 2a self-cleaving peptides.

[0008] Preferably, the amino acid sequence of the LP4 / 2a self-cleaving peptide is shown in SEQ ID No. 2; the amino acid sequence of Cry8Ea is shown in SEQ ID No. 3; the amino acid sequence of Sip1Aa is shown in SEQ ID No. 4; and the amino acid sequence of Cry3Aa is shown in SEQ ID No. 5.

[0009] The present invention also provides an amino acid sequence encoded by the above-mentioned gene BT3AES.

[0010] Preferably, the amino acid sequence encoded by the BT3AES is shown in SEQ ID No. 6.

[0011] The present invention also provides an expression vector containing the above-mentioned gene BT3AES.

[0012] The present invention also provides an application of the above-mentioned gene BT3AES, amino acid sequence or expression vector in the cultivation of insecticidal transgenic plants.

[0013] Preferably, the plant includes corn.

[0014] Preferably, the insect includes Coleoptera pests.

[0015] More preferably, the Coleoptera pests include one or more of the following: two-spotted leaf beetle, grub, iron beetle, and wireworm.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention provides a smart-designed gene BT3AES and its applications. The BT3AES gene enables high-level co-expression of three insecticidal proteins—Cry8Ea, Sip1Aa, and Cry3Aa—in maize mesophyll cells. This BT3AES gene not only avoids the risk of target pests evolving tolerance to its insecticidal proteins but also effectively improves insecticidal activity and spectrum. Furthermore, bioactivity testing results of the BT3AES gene in transgenic materials show that it has significant insecticidal effects against the two-spotted leaf beetle, achieving a synergistic effect. Attached Figure Description

[0018] Figure 1 A schematic diagram of the gene structure of insecticidal proteins in different intelligent design genes;

[0019] Figure 2 The expression levels of different genes in the maize coleoptile cell-free expression system;

[0020] Figure 3 Schematic diagram of plant expression vectors for the BT3AES, Cry8Ea, Sip1Aa, and Cry3Aa genes;

[0021] Figure 4 The expression levels of BT3AES, Cry8Ea, Sip1Aa, and Cry3Aa in different tissues of different transgenic plants. Detailed Implementation

[0022] This invention provides a smart design gene BT3AES that co-expresses three insecticidal proteins in transgenic plant cells, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0023] In this invention, the BT3AES gene is preferably co-expressed by three insecticidal proteins—Cry8Ea, Sip1Aa, and Cry3Aa—mediated by an LP4 / 2a self-cleaving peptide. More preferably, the nucleotide sequence of the BT3AES gene contains Cry8Ea at positions 1-3495, an LP4 / 2a self-cleaving peptide at positions 3496-3579, Sip1Aa at positions 3580-4563, an LP4 / 2a self-cleaving peptide at positions 4564-4647, and Cry3Aa at positions 4648-6606. The BT3AES gene of this invention increases the expression levels of the insecticidal proteins.

[0024] In this invention, the higher-order structures formed by self-cleaving peptides during translation create steric hindrance to the ribosomal peptidyl transferase center, preventing the formation of normal peptide chain linkages. However, the ribosome can still continue translating downstream proteins, thus forming a principle similar to the action of proteolytic enzymes that cis-"cleave" the two proteins. This allows for the co-expression of multiple insecticidal proteins within a single gene expression frame constructed using only a common promoter and terminator in plant cells. Furthermore, because different types of insecticidal proteins are combined and arranged, the expression level of this intelligently designed gene in the host cell may be influenced by transcriptional and post-transcriptional gene expression regulation. This invention performed cell-free simulated plant cell expression on artificially designed genes with six possible combinations of Cry8Ea, Sip1Aa, and Cry3Aa proteins, obtaining the combination with the highest expression level, namely the BT3AES gene nucleotide sequence shown in SEQ ID No. 1. Insect resistance was determined by transiently expressing the natural genes of Cry8Ea, Sip1Aa, and Cry3Aa proteins and the BT3AES gene in maize leaves. The two-spotted leaf beetle was used as the test insect for insect resistance identification. The results showed that the BT3AES gene had a higher insecticidal effect than the natural gene expressing only one protein.

[0025] In this invention, the method for preparing the BT3AES gene includes the following steps:

[0026] 1) First, design and synthesize six possible combinations of gene sequences encoding three insecticidal coleopteran proteins, namely Cry8Ea, Sip1Aa, and Cry3Aa. Construct plant expression vectors and achieve cell-free expression of the above recombinant proteins through a maize coleoptile cell-free expression system. Simulate the expression of the above genes in maize cells and infer the optimal combination based on this.

[0027] 2) Construct three insecticidal protein encoding genes of Coleoptera, namely Cry8Ea, Sip1Aa, and Cry3Aa, and candidate genes for optimal arrangement and combination. Perform recombinant protein expression and insecticidal effect analysis. Based on the analysis results, further determine the optimal arrangement and combination of gene BT3AES.

[0028] 3) Codon optimization and gene genetic stability analysis: Based on the best analysis results, the optimized gene sequence is synthesized into a whole gene using chemical synthesis and stored in a cloning vector.

[0029] The BT3AES gene of this invention is a co-expression gene encoding three artificially designed insecticidal proteins: Cry8Ea, Sip1Aa, and Cry3Aa. These three insecticidal proteins are linked by an LP4 / 2a self-splicing peptide, thereby achieving co-expression of transcribed mRNA in maize cells under the drive of a common promoter. Expression tests were conducted on all possible combinations of the three proteins, and the arrangement with the highest total expression level was selected, namely the nucleotide sequence arrangement of the BT3AES gene as shown in SEQ ID No. 1. Compared with traditional insecticidal protein genes, the BT3AES gene of this invention can simultaneously express three insecticidal proteins and increases the expression level of insecticidal proteins in cells, significantly improving the insecticidal activity of transgenic maize. Insecticidal experiments have shown that maize plants transgenic with the BT3AES gene exhibit better insecticidal activity against various Coleoptera pests than existing insecticidal genes.

[0030] The present invention also provides an amino acid sequence encoded by the above-mentioned gene BT3AES, which is an unstable amino acid sequence that, after translation by the cellular ribosome, is immediately self-cleaved into the three insecticidal proteins Cry8Ea, Sip1Aa, and Cry3Aa mediated by the above-mentioned two LP4 / 2a self-cleaving peptides.

[0031] In this invention, the amino acid sequence of the LP4 / 2a self-cleaving peptide is shown in SEQ ID No. 2; the amino acid sequence of Cry8Ea is shown in SEQ ID No. 3; the amino acid sequence of Sip1Aa is shown in SEQ ID No. 4; and the amino acid sequence of Cry3Aa is shown in SEQ ID No. 5. The amino acid sequence encoded by BT3AES is shown in SEQ ID No. 6.

[0032] The present invention also provides an expression vector containing the above-mentioned gene BT3AES.

[0033] This invention increases the expression level of the BT3AES gene in transgenic materials through artificial modification of the insecticidal protein and optimization of its plant expression vector without affecting its original insecticidal function. The preferred backbone of the expression vector includes pCAMBIA3301.

[0034] The present invention also provides the above-mentioned gene BT3AES, its amino acid sequence and expression vector, and its application in the cultivation of insecticidal transgenic plants.

[0035] In this invention, the plant preferably includes corn. The insect preferably includes coleopteran pests. More preferably, the coleopteran pests include one or more of the following: two-spotted leaf beetle, grub, iron beetle, and wireworm.

[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] Design and expression evaluation of co-expression genes for three insect-resistant proteins

[0039] (1) All possible combinations of the three insecticidal proteins Cry8Ea, Sip1Aa, and Cry3Aa, which have good insecticidal effects on Coleoptera, were listed. A self-splicing peptide LP4 / 2a was added between each pair of proteins. The resulting artificial amino acid sequences were reverse-engineered using Gene Designer 2.0 software, yielding a total of six artificially designed genes, named btCelo1, btCelo2, btCelo3, btCelo4, btCelo5, and btCelo6, with the following amino acid sequences: Figure 1 As shown, the amino acid sequences of the LP4 / 2a self-cleaving peptides in btCelo1, btCelo2, btCelo3, btCelo4, btCelo5, and btCelo6 are shown in SEQ ID No. 2; the amino acid sequence of Cry8Ea is shown in SEQ ID No. 3; the amino acid sequence of Sip1Aa is shown in SEQ ID No. 4; and the amino acid sequence of Cry3Aa is shown in SEQ ID No. 5.

[0040] The amino acid sequence of the LP4 / 2a self-cleaving peptide is as follows:

[0041] SNAADEVATLLNFDLLKLAGDVESNPGP.

[0042] The amino acid sequence of Cry8Ea is as follows:

[0043]

[0044] The amino acid sequence of Sip1Aa is as follows:

[0045] MKNSKKLKRKILACGAIASISTTLVTPLPTLALADQINTSELEKDAKVDTAILEWKVPLFKATEIYGKNIVVPSGYEFRSLTSHGFNTSYKNLEYHQFSVEADGSPIITNSNNIFVFKTTLTNNTDQEQTLSTNSFSKMISNSVTHSTTHGFKFGTKASAKFNI PFVGETGIELSAEYNFSDTSSETSSESFTYIATPQNIKVPAHSSVEVVVSLDTVKANGNVKLLAKMSGEDMGSFNYKSTTGGIGKSYVYNKSFNSLVTYASKIEKLQNISANPDGKTINIIGSGKYEAEYGTEFNVTVTPIDKNGKSVDEGYTYNVKPEITKEK.

[0046] The amino acid sequence of Cry3Aa is as follows:

[0047] .

[0048] (2) After completing the intelligent design of the above artificial genes, mRNA stability was improved. Using mature miRNAs from the mirBase database as a library, the above six genes were used as bait genes. The psRobot_tar module of the psRobot (V1.2) software was used to predict and obtain information on all potential miRNA targets. Gene Designer 2.0 software was used to adjust the codons of these genes according to the principle of amino acid codon degeneracy to eliminate potential targets that may be regulated by miRNAs.

[0049] (3) A T7 promoter was added upstream of each of the six artificially designed genes, and a T7 terminator was added downstream. The sequence structure is as follows: Figure 1As shown in Table 1. The above sequence was synthesized into a complete sequence using chemical synthesis. Then, PCR was performed in vitro using the T7 promoter (TAATACGACTCACTATAGGG, SEQ ID No. 7) forward single-stranded primer and the T7 terminator (GCTAGTTATTGCTCAGCGG, SEQ ID No. 8) forward single-stranded primer. The reaction system is shown in Table 1. The reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 72℃ extension for 5 min, 10 cycles; 72℃ final extension for 15 min; and storage at 16℃.

[0050] Table 1 PCR reaction system

[0051]

[0052] (4) The PCR reaction products were detected by 0.8% agarose gel electrophoresis and the fragments were recovered. Then, using the recovered fragments as templates, mRNA transcription was performed according to Table 2. The mixture was incubated at 37°C for 3 h and heated at 75°C for 10 min after the reaction. The mRNA of the gene to be tested was successfully obtained, diluted to 100 ng / μL, and stored at -20°C for later use.

[0053] Table 2 In vitro transcription reaction system

[0054]

[0055] (5) A cell-free expression system for maize coleoptiles was established as follows: After fully absorbing water, maize seeds of Zheng 58 were cultured at 28°C in the dark until the coleoptile length reached 5-8 cm. The coleoptiles were carefully cut off with a scalpel, and an equal volume of 19.5% sucrose solution was added. The mixture was then slowly ground into a homogenate using a mortar and pestle on crushed ice. The mixture was initially filtered through four layers of gauze, then filtered again through an 800-mesh cell sieve, and finally centrifuged at 12000g for 5 minutes to completely remove cells and cell debris. To ensure that this cell-free expression system could accurately reflect the post-transcriptional and translational regulation of exogenous gene mRNA in maize cells and its impact on the final expression level of exogenous genes in maize cells, the components of the system were not excessively optimized. The maize leaf sheath extract was directly expressed cell-free or stored at 4°C for no more than 48 hours.

[0056] (6) Take the mRNA of the gene to be tested obtained by in vitro transcription and add it to 0.2 mL of freshly prepared corn coleoptile cell-free expression system reaction solution at a ratio of 1:50. To facilitate subsequent statistics, perform about 30 technical repetitions and react at 25°C for 6 h. Then, terminate the reaction by treating it in a boiling water bath for 5 min.

[0057] (7) Mouse polyclonal antibodies were prepared using Cry8Ea, Sip1Aa, and Cry3Aa proteins expressed in prokaryotes, respectively, and detected by ELISA. The expression levels of exogenous proteins obtained from cell-free expression in maize leaf sheaths were statistically analyzed. Results are shown below. Figure 2 btCelo3 showed the highest level of exogenous protein expression across all combinations. Therefore, it was selected as a candidate gene for this invention and named the BT3AES gene, wherein the nucleotide sequence of the BT3AES gene is shown in SEQ ID No. 1, and the amino acid sequence of the BT3AES gene is shown in SEQ ID No. 6.

[0058] The nucleotide sequence of the BT3AES gene is as follows:

[0059]

[0060] The amino acid sequence of the BT3AES gene is as follows:

[0061]

[0062] Example 2

[0063] Construction of plant expression vectors for intelligently designed BT3AES gene and acquisition of transgenic plants

[0064] (1) The GUS gene driven by the 35S promoter in the commercial vector pCAMBIA3301 was replaced by a plant expression vector designed with bioinformatics assistance. The vectors encoding the BT3AES gene and the respective Cry8Ea, Sip1Aa, and Cry3Aa proteins were named pCAMBIA3301-BT3AES, pCAMBIA3301-Cry8Ea, pCAMBIA3301-Sip1Aa, and pCAMBIA3301-Cry3Aa, respectively. Their vector maps are shown below. Figure 3 As shown, the nucleotide sequence of the BT3AES gene is shown in SEQ ID No. 1, the amino acid sequence of the BT3AES gene is shown in SEQ ID No. 6, the amino acid sequence of Cry8Ea is shown in SEQ ID No. 3, the amino acid sequence of Sip1Aa is shown in SEQ ID No. 4, and the amino acid sequence of Cry3Aa is shown in SEQ ID No. 5. For specific sequences, please refer to the sequences shown in Example 1.

[0065] (2) The above-mentioned plant expression vectors were transformed into Agrobacterium EHA105 using the freeze-thaw method and used as the engineered bacteria for genetic transformation. Mature maize B73 seeds were used as recipient materials and genetic transformation was carried out according to the maize germinating embryo genetic transformation method. The transformed seeds germinated and grew to the three-leaf and one-heart stage in nutrient soil. They were screened with 0.3% glufosinate solution to obtain resistant plants. Transgenic plants that were positive by PCR were retained.

[0066] (3) Transgenic plants were cultured until the above-mentioned transgenic maize was harvested. Seeds were harvested and sown in an artificial climate chamber. Specific primers designed for the upstream and downstream promoters and terminators of the target gene (35sF: AGACCAAAGGGCAATTGAG, SEQ ID No. 9; NosR: ATTTATCCTAGTTTGCGCGC, SEQ ID No. 10) were used to identify the progeny plants by PCR-seq. The PCR reaction products corresponding to the PCR-positive plants were recovered using a PCR purification and recovery kit from TransGen Biotech Ltd. and then sequenced by Shanghai Bioengineering Sequencing Co., Ltd. using specific primers 35sF and NosR. The nucleotide sequences of the transgenic BT3AES maize were identified as shown in SEQ ID No. 1, the amino acid sequences of the transgenic BT3AES maize were shown in SEQ ID No. 6, the amino acid sequences of the transgenic Cry8Ea maize were shown in SEQ ID No. 3, the amino acid sequences of the transgenic Sip1Aa maize were shown in SEQ ID No. 4, and the amino acid sequences of the transgenic Cry3Aa maize were shown in SEQ ID No. 5.

[0067] Then, using the method described in Example 1, ELISA was performed on the leaves, silks, and roots of maize at the five-leaf-one-heart stage to determine the expression levels of the exogenous gene in the tissues of maize primarily attacked by coleopteran pests. The results are as follows: Figure 4 As shown.

[0068] Example 3

[0069] The toxicity of different tissues of transgenic BT3AES maize, transgenic Cry8Ea maize, transgenic Sip1Aa maize and transgenic Cry3Aa maize obtained in Example 2 to the two-spotted firefly beetle was determined.

[0070] (1) Toxicity determination of transgenic maize leaves to adult two-spotted leaf beetle

[0071] Five genetically modified maize plants were selected as experimental groups: BT3AES transgenic maize, Cry8Ea transgenic maize, Sip1Aa transgenic maize, and Cry3Aa transgenic maize. A non-transgenic control, B73, served as the control group, for a total of five treatment groups. Each treatment group consisted of 10 plants. Leaves from the lower part of fresh leaves (approximately 20 cm) at stage V10 were randomly selected from each plant, cut with sterilized scissors, and laid flat at the bottom of the insect rearing net cage (length × width × height: 25 × 15 × 20 cm). Each cage contained 20 adult two-spotted leaf beetles, constituting one biological replicate. Ten biological replicates were created for each treatment group, meaning each experimental group used one tested maize plant. The insect rearing net cages were placed in an artificial climate incubator with a temperature of 29 ± 1℃, relative humidity of 50%, and a photoperiod of 16 L:8 D. Leaves from the same source (the same type of tested maize) were replaced daily. The number of surviving adults was recorded on day 10 and statistically analyzed using SPASS. The results are shown in Table 3.

[0072] Table 3. Results of toxicity assays on adult two-spotted leaf beetles from different transgenic maize leaves.

[0073]

[0074] Note: The data in Table 3 are mean ± standard error; different letters in the same column indicate significant differences between different materials (P<0.05).

[0075] Table 3 shows that the BT3AES gene-transgenic maize leaves were significantly more toxic to the two-spotted leaf beetle than other treatments, achieving a synergistic effect.

[0076] (2) Toxicity determination of transgenic corn silks to adult two-spotted leaf beetle

[0077] The transgenic maize materials from Example 2—BT3AES, Cry8Ea, Sip1Aa, and Cry3Aa—were selected as experimental groups, and the non-transgenic control maize B73 was selected as the treatment group, for a total of five treatment groups. Each treatment group consisted of 10 plants. Unpolluted silks were randomly selected from a single plant, cut with sterilized scissors, and laid flat at the bottom of the insect rearing net cage (length × width × height: 25 × 15 × 20 cm). Each cage contained 20 adult two-spotted leaf beetles, constituting one biological replicate. Each treatment group had 10 biological replicates, meaning each experimental group used one tested maize plant. The insect rearing net cages were placed in an artificial climate incubator with a temperature of 29 ± 1℃, relative humidity of 50%, and a photoperiod of 16 L:8 D. Silks from the same source (the same type of tested maize) were replaced daily. After 10 days of rearing, the number of surviving adults was recorded and statistically analyzed using SPASS. The results are shown in Table 4.

[0078] Table 4. Results of toxicity assays of different transgenic corn silks to adult two-spotted leaf beetles.

[0079]

[0080] Note: Data in the table are mean ± standard error; different letters in the same column indicate significant differences between different materials (P<0.05).

[0081] Table 4 shows that the transgenic BT3AES corn silks were significantly more toxic to the two-spotted leaf beetle than other treatment groups, exhibiting a synergistic effect.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the intelligent design gene BT3AES, the amino acids encoded by the gene BT3AES, or an expression vector containing the gene BT3AES in the cultivation of insecticidal transgenic plants, characterized in that, The gene BT3AES The nucleotide sequence is shown in SEQ ID No. 1; the plant is corn; the insect is the two-spotted leaf beetle.

2. The application according to claim 1, characterized in that, The gene BT3AES It is formed by the co-expression of three insecticidal proteins, Cry8Ea, Sip1Aa, and Cry3Aa, mediated by two LP4 / 2a self-cleaving peptides.

3. The application according to claim 2, characterized in that, The amino acid sequence of the LP4 / 2a self-cleaving peptide is shown in SEQ ID No. 2; the amino acid sequence of Cry8Ea is shown in SEQ ID No. 3; the amino acid sequence of Sip1Aa is shown in SEQ ID No. 4; and the amino acid sequence of Cry3Aa is shown in SEQ ID No. 5.

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