A transgenic silkworm strain stably expressing scorpion toxin peptide and a construction method thereof

Abstract

The present application relates to the field of silkworm transgenic and genetic breeding technology, and particularly relates to a transgenic silkworm strain stably expressing scorpion venom peptide and a construction method thereof. Specifically, the present application comprises the following steps: (1) synthesis of scorpion venom peptide gene; (2) construction of piggyBac transgenic vector; (3) microinjection of silkworm eggs; (4) rearing of G0 generation silkworms; (5) silkworm rearing and screening of transgenic positive individuals; (6) identification of protein expression product of exogenous scorpion venom peptide gene; and (7) determination of genetic stability of exogenous gene in silkworm genome and establishment of transgenic silkworm strain stably expressing scorpion venom peptide. The present application introduces the antibacterial peptide gene into silkworm for heterologous expression, so as to enhance the disease resistance of silkworm and improve the economic benefits of breeding.

Description

Technical Field

[0001] This invention relates to the field of silkworm transgenic and genetic breeding technology, specifically to a transgenic silkworm strain that stably expresses scorpion venom peptide and its construction method. Background Technology

[0002] Like other insects, silkworms lack an adaptive immune system and rely solely on innate immunity to defend against pathogens, making them susceptible to various pathogens such as bacteria, fungi, viruses, and microspores. Silkworms raised on artificial feed, in particular, have weaker immunity and are more vulnerable to pathogen infection, severely limiting economic benefits. Therefore, it is urgent to explore effective strategies to enhance the immune and disease-resistant capabilities of silkworms.

[0003] Scorpion venom antimicrobial peptides, a key component of the scorpion's innate immune system, are a class of cationic, amphiphilic, disulfide-free polypeptide molecules typically composed of 10–100 amino acid residues with an α-helix structure. They exhibit broad-spectrum inhibitory or killing effects against pathogenic microorganisms such as viruses, bacteria, and fungi. Scorpion venom antimicrobial peptides possess a unique bactericidal mechanism and are less prone to inducing drug resistance, making them one of the most ideal antibiotic alternatives. Scorpion venom peptides (scorpion venom peptides) are derived from the emperor scorpion (Scorpion scorpion venom peptides). Pandinus imperator This active antimicrobial peptide was isolated from the venom of a scorpion. The peptide consists of 75 amino acids, contains three disulfide bonds, and has a molecular weight of 8350 Da. The amino acid sequence of the scorpion venom peptide is unique; its N-terminal region is similar to some cecropins, while its C-terminal region is similar to some defensins. Studies have shown that the scorpion venom peptide has significant inhibitory effects on a variety of pathogens, including bacteria such as Bacillus subtilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa, as well as some fungal species. Furthermore, this type of peptide has shown potential for application in malaria control and intervention in specific types of viral infections.

[0004] Transferring antimicrobial peptide genes into plants and animals not only enhances the host's disease resistance but also holds promise for achieving efficient production of antimicrobial peptides through transgenic plant and animal platforms, thus possessing significant scientific research value and broad application prospects. In recent years, silkworm transgenic technology has developed rapidly, with various operational methods established. Among these, the microinjection method using the piggyBac vector is the most mature and has been successfully applied in multiple laboratories. It is noteworthy that transgenic silkworms possess a sound biosafety foundation. Through long-term artificial domestication, silkworms have lost their ability to survive independently in the wild; adult flight capabilities have degenerated, dispersal behavior is restricted, and they are unlikely to hybridize with wild relatives under natural conditions, effectively avoiding the ecological risk of transgenic components drifting into natural populations. Therefore, we believe that constructing silkworm strains capable of efficiently expressing scorpion venom peptide genes holds promise for improving the disease resistance of silkworms and has excellent prospects for industrial development. Summary of the Invention

[0005] The purpose of this invention is to address the prominent problem of low immunity and susceptibility to pathogenic microorganisms in silkworms by providing a technical solution that enhances the disease resistance of silkworms by constructing a silkworm strain that efficiently expresses scorpion venom peptide genes and utilizes it to produce scorpion venom peptides, thereby comprehensively improving the overall economic benefits and safety of silkworm farming.

[0006] The objective of this invention is achieved through the following technical solution: The first aspect of this invention provides a method for constructing a transgenic silkworm strain that stably expresses scorpion venom peptide, comprising the following steps: (1) Synthesis of scorpion venom peptide gene: Based on the codon preference of silkworm, the scorpion venom peptide gene Scorpine was optimized and synthesized, and its nucleotide sequence is shown in SEQ ID NO.1; (2) Construction of the piggyBac transgenic vector: using Kpn I and Hind III. The pLX-Red vector and the synthesized scorpion venom peptide gene were digested with double enzymes. After the target fragment was recovered by electrophoresis, it was ligated and transformed to obtain the piggyBac transgenic plasmid pLX-Red-Scorpine, the nucleotide sequence of which is shown in SEQ ID NO.2. (3) Microinjection of silkworm eggs: Adjust the hatching conditions to induce the silkworm strain "Haoyue B" to lay non-diapause eggs, and use them as recipients to perform microinjection of the piggyBac transgenic plasmid described in step (2); the injected silkworm eggs continue to hatch until hatching; (4) Rearing of G0 generation silkworms: After the silkworm eggs are hatched, they are raised to obtain G1 generation individuals; (5) Screening of G1 generation silkworms for hatching and transgenic positive individuals: The G1 generation silkworms were subjected to hatching treatment, and after hatching, the larvae were collected and positive individuals were screened. (6) Identify the protein expression product of the exogenous scorpion venom peptide gene: Detect the protein level of positive individuals to verify the expression of exogenous scorpion venom peptide; (7) Genetic stability of exogenous genes in the silkworm genome: Molecular identification was performed on transgenic lines that were continuously passaged to the F4 generation to assess the genetic stability of exogenous genes in transgenic silkworms; and transgenic silkworm lines that stably express scorpion venom peptides were established.

[0007] Furthermore, the greening conditions described in step (3) are a temperature of 25 ℃ and a relative humidity of 80%.

[0008] Further, the microinjection in step (3) specifically involves diluting the amplified and purified pLX-Red-Scorpine plasmid and A3 helper plasmid to 350-450 ng / μL respectively, mixing them in equal amounts, and then introducing them into the fertilized egg through microinjection, injecting 10-20 nL into each egg.

[0009] Further, step (4) specifically involves the silkworm eggs being hatched and harvested, and then the silkworms being raised in groups under normal conditions. After the larvae spin cocoons, they enter the pupal stage under standard protective conditions. After the adults emerge, they mate and produce eggs to obtain G1 generation silkworm eggs.

[0010] Furthermore, the greening conditions described in step (5) are 25 ℃ and 80% relative humidity.

[0011] Furthermore, in step (5), the screening of positive larvae specifically involves raising larvae to the second instar using conventional methods and then using a stereofluorescence microscope to screen for positive individuals that express red fluorescent protein throughout their bodies.

[0012] Further, in step (5), genomic DNA of positive larval individuals is extracted for PCR amplification and identification. The PCR amplification primer sequences are shown in SEQ ID NO.3-SEQ ID NO.4.

[0013] Further, step (7) specifically involves using transgenic silkworm larvae that have been continuously passaged to the F4 generation as material, extracting total RNA and genomic DNA from their tissues for subsequent molecular identification; designing specific primers based on the mRNA sequence of Scorpine, and detecting the transcriptional expression of the exogenous scorpion venom peptide gene by sqRT-PCR; and simultaneously using genomic DNA as a template, amplifying and identifying the flanking sequences of the exogenous gene using thermal asymmetric staggered PCR technology to determine its chromosomal insertion site.

[0014] Furthermore, the nucleotide sequences of the specific primers are shown in SEQ ID NO.5-SEQ ID NO.6.

[0015] Furthermore, the specific primers and random degenerate primer sequences used in the thermal asymmetric staggered PCR technique are shown in SEQ ID NO.7-SEQ ID NO.14.

[0016] The beneficial effects of this invention are: Scorpine is derived from the emperor scorpion (Scorpion scorpion venom peptide). Pandinus imperator An active antimicrobial peptide was isolated and identified from scorpion venom. Studies have shown that this peptide has broad-spectrum inhibitory activity against various bacteria, including Bacillus subtilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa, as well as some fungi. Furthermore, scorpion venom peptides have shown potential application value in malaria vector control and intervention for specific viral infections. Based on this, this invention transfers the antimicrobial peptide gene into silkworms for heterologous expression, aiming to enhance the disease resistance of silkworms and thus improve the economic benefits of silkworm farming. Attached Figure Description

[0017] Figure 1 Screening of positive silkworm larvae for red fluorescent reporter genes; Figure 2 Genomic PCR detection of exogenous Scorpine gene in transgenic silkworms; Figure 2 (A) in the image represents the agarose gel electrophoresis of the PCR products; Figure 2 (B) in the diagram represents the sequencing results of the amplified product, with the Scorpine gene coding region marked in red. Figure 3 Mass spectrometry was used to identify Scorpine protein in transgenic silkworms. Figure 3 (A) represents proteomics data; MH+[Da] indicates the molecular weight of the peptide with a single positive charge. Figure 3 (B) in the image represents the Scorpine-encoded peptide segment (highlighted in red) matched based on the initial amino acid sequence. Figure 4 Identification of Scorpine gene expression in transgenic silkworms; Figure 5 Tail-PCR was used to identify the genomic insertion site of the transgenic silkworm exogenous vector; Figure 5 (A) in the diagram is a schematic of Tail-PCR; Figure 5 (B) is a Tail-PCR electrophoresis image; M: DNA marker; R: right end of the vector; L: left end of the vector; Figure 5 (C) in the figure represents the R-terminal sequencing and comparison results; Figure 5 (D) in the figure represents the L-terminal sequencing and comparison results; Figure 5 (E) in the figure represents the insertion site of the transgenic vector on the silkworm chromosome. Detailed Implementation

[0018] The present invention will be further described in detail through the following embodiments, but the content of the present invention is not limited thereto.

[0019] Example 1:

[0020] 1. Synthesis of the scorpion venom peptide gene: Based on the codon usage preferences of the silkworm, the scorpion venom peptide gene sequence was optimized. Subsequently, the IE2 promoter, Kozak sequence, and sequence encoding a eukaryotic phosphate transport-related peptide were sequentially ligated upstream of the optimized gene in the 5' to 3' direction, and a 6×His tag coding sequence and SV40 sequence were fused downstream of it. To facilitate subsequent cloning, scorpion venom peptides were introduced at both ends of this expression cassette. Kpn I and Hind The synthesized fragment was cleaved at the Sma I (Blunt) site of the pUC57 vector to obtain the recombinant plasmid pUC57-Scorpine. The complete synthetic sequence of Scorpine is shown in SEQ ID NO.1.

[0021] 2. Construction of the piggyBac transgenic vector: using restriction endonucleases... Kpn I and Hind III. The piggyBac vector pLX-Red and pUC57-Scorpine plasmids were cut and separated by electrophoresis. The 7.4 kb vector fragment and the 639 bp scorpion venom peptide expression cassette fragment were recovered, respectively. They were ligated and transformed into competent cells. After spot picking and sequencing to confirm that they were correct, the piggyBac transgenic plasmid pLX-Red-Scorpine was obtained. Its nucleotide sequence is shown in SEQ ID NO.2.

[0022] 3. Microinjection of silkworm eggs: By adjusting the hatching conditions, the silkworm strain "Haoyue B" was induced to lay non-diapause eggs. The purified pLX-Red-Scorpine plasmid and A3 helper plasmid were diluted to 400 ng / μL, mixed in equal volumes, and then injected into the fertilized eggs via microinjection, 15 nL per egg. After injection, the injection well was sealed with quick-drying adhesive, and the eggs were placed in an incubator at 25℃ and 80% relative humidity until hatching.

[0023] 4. G0 generation transgenic silkworm rearing: After the silkworm eggs hatch and are harvested, they are reared in groups under conventional conditions. Once the larvae spin cocoons, they enter the pupal stage under standard protective conditions. After the adults emerge, they are mated and used to produce G1 generation silkworm eggs.

[0024] 5. Screening for transgenic positive individuals: G1 generation silkworm eggs were induced to hatch using conventional methods, then collected and fed until the 2nd instar. Individuals exhibiting red fluorescence were screened using a stereofluorescence microscope. Figure 1They were raised until they formed cocoons, and after emerging as adults, they were mated to produce offspring, and genomic DNA was extracted from the positive moths. Primers were designed (SEQ ID NO.3: F: 5'-TTCCTGTGTTGCTAACCGCA-3'; SEQ ID NO.4: R: 5'-GAGGAGTGCCACATTTGCAT-3') for PCR identification of genomic DNA (50 µL reaction system: 2 µL template DNA, 5 µL 10× PCR buffer, 8 µL 2 mmol / L dNTPs, 1 µL 10 µmol / L primer F, 1 µL 10 µmol / L primer R, 1 µL Taq enzyme, and 32 µL UP water). The PCR amplification program was as follows: initial denaturation at 94 ℃ for 30 s; then 32 cycles of "94 ℃ 35 s → 55 ℃ 35 s → 72 ℃ 45 s" were run; after the reaction, the reaction was extended at 72 ℃ for 1 min, and finally cooled to 4 ℃. The reaction was terminated, and the amplified product was sequenced for identification. Sequence alignment results showed that the amplified product was completely identical to the synthesized Scorpine gene sequence, confirming that the scorpion venom peptide gene had been successfully inserted into the silkworm genome. Figure 2 ).

[0025] 6. Identification of the protein expression product of the exogenous scorpion venom peptide gene: Protein was extracted from positive silkworm pupae, and the recombinant Scorpine protein was isolated and purified using a His-tagged protein purification kit, and then identified by mass spectrometry. Mass spectrometry analysis showed that the amino acid sequence of the obtained peptide was consistent with the theoretical sequence of Scorpine. Figure 3 ).

[0026] 7. Genetic stability of exogenous genes in the silkworm genome: Using transgenic silkworm larvae that have been continuously passaged to the F4 generation as material, total RNA and genomic DNA were extracted from their tissues for subsequent molecular identification. Specific primers were designed based on the scorpine mRNA sequence (SEQ ID NO.5: F: 5'-CGGATGGATTAACGAAGAGAAGAT-3'; SEQ ID NO.6: R: 5'-TGATGGTGATGATGATAAGACAGAG-3'), and the transcriptional expression of the exogenous scorpion venom peptide gene was detected by sqRT-PCR (50 µL reaction system: 2 µL cDNA template, 5 µL 10× PCR buffer, 8 µL 2 mmol / L dNTPs, 1 µL 10 µmol / L primer F, 1 µL 10 µmol / L primer R, 1 µL Taq enzyme, and 32 µL UP water). The PCR amplification program was as follows: initial denaturation at 94 ℃ for 5 min; then 32 cycles of "94 ℃ 30 s → 55 ℃ 40 s → 72 ℃ 2 min"; after the reaction, a further extension at 72 ℃ for 10 min. (The reaction was terminated by cooling to 4 °C for 1 minute).

[0027] The results showed that transgenic silkworms could stably express the exogenous Scorpine gene ( Figure 4 Simultaneously, the flanking sequences of the exogenous gene were amplified and identified using thermal asymmetric staggered PCR (Tail-PCR). The specific primers (SP) and random degenerate primers (AD / AP) used in this method are as follows: SEQ ID NO.7: SP1: 5'-TGACACTTACCGCATTGACAAGCACG-3'; SEQ ID NO.8: SP2: 5'-CAAGCGGCGACTGAGATGTCCTAAATG-3'; SEQ ID NO.9: SP3: 5'-CGGATTCGCGCTATTTAGAAAGAGAGAGC-3'; SEQ ID NO.10: AD1: 5'-ACGATGGACTCCAGAGCGGCCGC(G / C / A)N(G / C / A)NNNGGAA-3'; SEQ ID NO.11: AD2: 5'-ACGATGGACTCCAGAGCGGCCGC(G / C / T)N(G / C / T)NNNGGTT-3'; SEQ ID NO.12:AD3:5'- ACGATGGACTCCAGAGCGGCCGC(G / C / A)(G / C / A)N(G / C / A)NNNCCAA-3'; SEQ ID NO.13:AD4:5'-ACGATGGACTCCAGAGCGGCCGC(G / C / T)(G / A / T)N(G / C / T)NNNCGGT-3'; SEQ ID NO.14:AP:5'- ACGATGGACTCCAGAG-3'。

[0028] A three-round nested PCR strategy was adopted, and the specific process is as follows: First round PCR: SP1 and two random primers (AD1 and AD3, or AD2 and AD4) were used for the first round PCR. The PCR program was: 94 ℃ 2 min → 95 ℃ 1 min, 5 high-specificity reactions (94 ℃ 15 s → 63 ℃ 1 min → 72 ℃ 2 min), 1 low-specificity reaction (94 ℃ 15 s → 30 ℃ 3 min → increase to 72 ℃ at 0.2 ℃, 72 ℃ 2 min), 15 thermally asymmetric large cycles (94 ℃ 5 s → 63 ℃ 1 min → 72 ℃ 2 min, 94 ℃ 5 s → 63 ℃ 1 min → 72 ℃ 2 min, 94 ℃ 15 s → 44 ℃ 1 min → 72 ℃ 2 min), 72 ℃ 2 min. Second round PCR: The first round PCR product was diluted 10 times and used as the template for the second round PCR. Primers SP2 and AP were used. The PCR program was: 15 thermal asymmetric cycles (94 ℃ 5 s → 63 ℃ 1 min → 72 ℃ 2 min, 94 ℃ 5 s → 63 ℃ 1 min → 72 ℃ 2 min, 94 ℃ 15 s → 44 ℃ 1 min → 72 ℃ 2 min), 72 ℃ 2 min. The third round of PCR: The second round PCR product was diluted 10-fold and used as the template for the third round of PCR. Primers SP3 and AP were used. The PCR program was: 15 thermal asymmetric cycles (94 ℃ 5 s → 63 ℃ 1 min → 72 ℃ 2 min, 94 ℃ 5 s → 63 ℃ 1 min → 72 ℃ 2 min, 94 ℃ 15 s → 44 ℃ 1 min → 72 ℃ 2 min), 72 ℃ 5 min. The reaction volume for each of the three rounds of PCR was 30 µL, including 4 µL template DNA, 3 µL 10× PCR buffer, 2 µL 2 mmol / L dNTPs, 1 µL 10 µmol / L SP, 1 µL 10 µmol / L AD or AP, 0.2 µL Taq enzyme, and 18.8 µL UP water. The chromatin insertion site was determined to be located at Chr5:13854236-13854241 ( Figure 5 Sequence analysis confirmed that the region is located in a gene intergenic spacer and does not involve any known regulatory elements, indicating that the insertion site is genetically safe.

Claims

1. A method for constructing a transgenic silkworm strain stably expressing scorpion venom peptide, characterized in that, Includes the following steps: (1) Based on the codon preference of silkworm, the scorpion venom peptide gene Scorpine was optimized and synthesized, and its nucleotide sequence is shown in SEQ ID NO.1; (2) Use Kpn I and Hind III. The pLX-Red vector and the synthesized scorpion venom peptide gene were digested with double enzymes. After the target fragment was recovered by electrophoresis, the fragment was ligated and transformed to obtain the piggyBac transgenic plasmid pLX-Red-Scorpine, the nucleotide sequence of which is shown in SEQ ID NO.

2. (3) Adjust the incubation conditions to induce the silkworm strain "Haoyue B" to lay non-diapause eggs, and use them as recipients to perform microinjection of the piggyBac transgenic plasmid described in step (2); the injected silkworm eggs continue to incubate until hatching; (4) After the silkworm eggs are hatched, they are raised to obtain G1 generation individuals; (5) The G1 generation silkworms were subjected to priming treatment until hatching, and the larvae were collected and positive individuals were screened. (6) Protein levels were measured in positive individuals to verify the expression of exogenous scorpion venom peptides; (7) Molecular identification was performed on the transgenic lines that were continuously passed down to the F4 generation to assess the genetic stability of the exogenous gene in the transgenic silkworm and to construct a transgenic silkworm line that stably expresses scorpion venom peptide.

2. The construction method as described in claim 1, characterized in that, The microinjection in step (3) specifically involves diluting the amplified and purified pLX-Red-Scorpine plasmid and A3 helper plasmid to 350-450 ng / μL, mixing them in equal amounts, and then introducing them into fertilized eggs via microinjection, injecting 10-20 nL into each egg.

3. The construction method as described in claim 1, characterized in that, Step (4) involves the silkworm eggs hatching and ant harvesting, followed by group rearing of silkworms under normal conditions. After the larvae spin cocoons, they enter the pupal stage under standard protective conditions. After the adults emerge, they mate and produce eggs to obtain G1 generation silkworm eggs.

4. The construction method as described in claim 1, characterized in that, Step (5) involves screening positive larvae by raising the larvae to the second instar using conventional methods and then screening for positive individuals that express red fluorescent protein throughout their bodies using a stereofluorescence microscope.

5. The construction method as described in claim 1, characterized in that, Step (5) Extract genomic DNA from positive larval individuals for PCR amplification and identification. The PCR amplification primer sequences are shown in SEQ ID NO.3-SEQ ID NO.

4.

6. The construction method as described in claim 1, characterized in that, Step (7) specifically involves using transgenic silkworm larvae that have been continuously passaged to the F4 generation as material, extracting total RNA and genomic DNA from their tissues for subsequent molecular identification; designing specific primers based on the mRNA sequence of Scorpine, and detecting the transcriptional expression of the exogenous scorpion venom peptide gene by sqRT-PCR; and simultaneously using genomic DNA as a template, amplifying and identifying the flanking sequences of the exogenous gene using thermal asymmetric staggered PCR technology to determine its chromosomal insertion site.

7. The construction method as described in claim 6, characterized in that, The nucleotide sequences of the specific primers are shown in SEQ ID NO.5-SEQ ID NO.

6.

8. The construction method as described in claim 6, characterized in that, The specific primers and random degenerate primer sequences used in the thermal asymmetric staggered PCR technique are shown in SEQ ID NO.7-SEQ ID NO.14.

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