Application of bombyx mori BmSPP gene in preparation of silkworm larva
By knocking out the BmSPP gene in silkworms using CRISPR/Cas9 gene editing technology, a highly sensitive white-skinned silkworm variety was bred, solving the problems of unstable silkworm source and long breeding cycle, and realizing the stabilization and large-scale production of white-skinned silkworms.
Patent Information
- Application Number
- CN202511557886.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-29
- Publication Date
- 2026-01-30
Smart Images
Figure CN121428005A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering and relates to silkworms. BmSPP Application of genes in the preparation of white silkworm. Background Technology
[0002] White silkworm ( Bombyx batryticatus ) is the domesticated silkworm ( Bombyx mori ) Infection with Beauveria bassiana ( Beauveria bassiana The dried insect-fungus complex of dead silkworms is an important traditional Chinese medicine with a long history of medicinal use. It has pharmacological effects such as anticonvulsant, anticoagulant, sedative-hypnotic, antibacterial, anticancer, hypoglycemic, lipid-lowering, and beauty-enhancing effects. In recent years, with the deepening of modern pharmacological research, the pharmacological effects of silkworm larvae have been further expanded and their applications have become more widespread. Compared with traditional silk cocoon production, silkworm larvae farming only requires raising the larvae to the 5th instar, resulting in a shorter farming cycle, lower costs, greater market demand, higher market prices, and higher farming profits. Currently, silkworm larvae farming has become an emerging industry in sericulture with broad development prospects.
[0003] Traditionally, the primary source of dead silkworms (specifically, silkworms that have died naturally from infection with Beauveria bassiana during silkworm rearing) is diseased. However, this source of naturally dead silkworms is limited and unstable, necessitating the development of artificial rearing techniques. In the production of dead silkworms, screening for highly infectious Beauveria bassiana and selecting high-quality silkworm breeds are crucial. While significant progress has been made in enhancing the virulence of Beauveria bassiana, the development of high-quality silkworm breeds is rarely reported. Because different silkworm breeds have varying tolerances to Beauveria bassiana, current silkworm breeding primarily focuses on cocoon quality or resistance to the pathogen, with little research specifically targeting the rearing of dead silkworms. Consequently, the yield of artificially reared dead silkworms is low and cannot meet market demand.
[0004] Traditional hybridization breeding methods have long breeding cycles, limited genetic resources, and low efficiency. Summary of the Invention
[0005] In view of this, this invention utilizes CRISPR / Cas9 gene editing technology to achieve precise and efficient gene editing for targeted improvement, shortening the breeding cycle, reducing screening costs, and achieving breakthrough improvements that are difficult to achieve using traditional methods. The purpose of this invention is to use CRISPR / Cas9 gene editing technology to modify the silkworm signal peptidase gene (…). BmSPP The gene was knocked out, providing a method to increase the infection efficiency of Beauveria bassiana, thus making it easier to become Beauveria bassiana silkworm.
[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a silkworm BmSPP The application of genes in the preparation of white silkworms, using knockout vectors to knock out BmSPPGene expression, the silkworm BmSPP The gene sequence is shown in SEQ ID NO:3; Preferably, the knockout carrier contains knockout silkworms. BmSPP The sgRNA, the nucleic acid sequence of which is shown in SEQ ID NO:7; Furthermore, the method for preparing high-quality varieties of white-skinned silkworms involves knocking out the silkworm's... BmSPP Genes are used to breed high-quality white silkworm varieties. BmSPP The gene sequence is shown in SEQ ID NO:3; A preferred method for preparing the white-skinned silkworm variety includes the following steps: (a) Target building BmSPP sgRNA vector for genes; (b) Introduce the vector from step (a) into silkworm fertilized eggs; (c) Hybridize the sgRNA individuals obtained in step (b) with individuals expressing Cas9; (d) Filtering to obtain BmSPP Gene knockout silkworms, also known as white-skinned silkworms, are used to produce high-quality varieties. BmSPP KO strain.
[0007] Preferably, the sgRNA sequence is shown in SEQ ID NO:7; Furthermore, in the method for preparing white-stiff silkworms, the bacterial solution of Beauveria bassiana is sprayed onto the white-stiff silkworm variety prepared by the method described above, and the silkworms die after stiffening; Preferably, the concentration of the Beauveria bassiana bacterial suspension is 1×10⁻⁶. 7 conidia / mL.
[0008] The beneficial effects of this invention are as follows: This invention uses CRISPR / Cas9 gene editing technology to knock out silkworms. BmSPP Genes were used to obtain new silkworm strains. BmSPP KO ), and applied it to the production of white silkworms: 1. Significantly improves the infection efficiency of Beauveria bassiana. BmSPP KO Silkworms showed a significantly increased sensitivity to Beauveria bassiana. Experiments showed that after inoculation with the same concentration of Beauveria bassiana spore suspension, BmSPP KO The overall mortality rate of domesticated silkworms is higher than that of wild-type silkworms. BmSPP WT The survival rate is increased by 20%, and most individuals die during the larval or prepupal stage, effectively shortening the molting period.
[0009] 2. Reduce the production cost of white mulberry silkworm The mutant strain has a lower infection threshold of Beauveria bassiana, can achieve a high sclerotization rate at a lower spore concentration, reduces the dosage of the microbial agent, and reduces the production cost.
[0010] 3. Solve the industry pain point of unstable source of white mulberry silkworm The traditional natural infection method has a low and uncontrollable yield of white mulberry silkworm. The present application realizes the large-scale and stable production of white mulberry silkworm by creating high-sensitivity silkworm strains through gene editing, and fills the technical gap of high-quality silkworm breeding.
[0011] 4. Breakthrough improvement of breeding efficiency Compared with the long breeding period and low efficiency of traditional hybridization, the CRISPR / Cas9 technology realizes precise and rapid gene-directed improvement, shortens the breeding period, and provides a new path for the development of white mulberry silkworm special varieties.
[0012] Other advantages, objects and features of the present application will be explained to some extent in the following description, and to some extent, it will be obvious to those skilled in the art based on the study of the following, or can be taught from the practice of the present application. The objects and other advantages of the present application can be achieved and obtained by the following description. BRIEF DESCRIPTION OF DRAWINGS
[0013] In order to make the objects, technical solutions and advantages of the present application clearer, the preferred detailed description of the present application will be made below in combination with the drawings, in which: Figure 1 For obtaining sgRNA positive individuals and transgenic knockout lines: A, sgRNA positive individual screening, B, sgRNA insertion site detection, C, transgenic knockout line screening; Figure 2 For BmSPP KO Silkworms are more susceptible to white muscardine: A, death trend of silkworms after inoculation of white muscardine, B, proportion of death of silkworms after inoculation of white muscardine, C, death of silkworms after inoculation of white muscardine. DETAILED DESCRIPTION
[0014] The embodiments of the present application are described below by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure of the present specification. The present application can also be implemented or applied by different specific embodiments, and the details in the specification can be modified or changed based on different views and applications without departing from the spirit of the present application. It should be noted that the diagrams provided in the following examples only illustrate the basic concept of the present application in a schematic manner, and the following examples and features in the examples can be combined with each other without conflict.
[0015] The drawings are only used for exemplary illustration, and the representation is only a schematic diagram, not a physical diagram, and cannot be understood as a limitation on the present application; in order to better illustrate the embodiments of the present application, some components in the drawings will be omitted, enlarged or reduced, and do not represent the size of the actual product; it is understandable to those skilled in the art that some well-known structures and their descriptions in the drawings can be omitted.
[0016] The same or similar reference numerals in the drawings of the embodiments of the present application correspond to the same or similar components; in the description of the present application, it should be understood that if the terms 'upper', 'lower', 'left', 'right', 'front', 'back' and the like indicate the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore the terms describing the positional relationship in the drawings are only used for exemplary illustration, and cannot be understood as a limitation on the present application, for those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.
[0017] Example 1 Bombyx mori BmSPP Obtaining of the gene Taking the Bombyx mori variety dazao (The silkworm variety is stored in the Biology Research Center of the Frontiers and Interdisciplinary Research Institute of Southwest University) The fat body of the third day of the fifth instar larva was used to extract total RNA of the tissue by TRIzol reagent (Invitrogen, Carlsbad, CA), and cDNA was synthesized by M-MLV reverse transcriptase (Promega, Madison, WI).
[0018] Taking the Bombyx mori variety dazao The fat body cDNA was used as a template, and primers were designed, the upstream primer was 5'-ATGGCAGATATGGCCTCAGA-3' (SEQ ID NO: 1), and the downstream primer was 5'-TTATTCGGACTTCTTAGACTTGT-3' (SEQ ID NO: 2), PCR amplification was carried out according to the designed upstream and downstream primers, the amplification conditions were 98℃ pre-denaturation for 2 minutes; 98℃ denaturation for 10 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 60 seconds, a total of 35 cycles; finally 72℃ extension for 5 minutes, 4℃ preservation, the PCR product was identified by agarose gel electrophoresis and recovered, then connected with pEASY-Blunt vector, and pEASY- BmSPP Recombinant vector, the recombinant vector was transformed into Trans-1 competent cells, positive clones were obtained for sequencing verification, and the sequencing result showed that the full-length sequence of the gene was successfully cloned BmSPP The full-length sequence of the gene is shown in SEQ ID NO: 3, BmSPPThe full-length sequence of the gene is 1098 bp.
[0019] Example 2 BmSPP KO Obtaining of Bombyx mori The constructed pBac[3xP3-EGFP-SV40-U6-gRNA-TTTTTT] vector (see the preparation method in Feng Y-t, Yang C-y, Wu L, Wang Y-c, Shen G-w, Lin P. BmSPP is a virus resistance gene in Bombyx mori. Frontiers in Immunology. 2024. 1377270.) and the helper plasmid were mixed at a concentration of 500 ng / μL at a ratio of 1:1, and then microinjected into fresh eggs of the non-diapause line D9L (within 2 h of egg laying), to obtain the G0 generation. The G0 generation was self-crossed or crossed with wild-type Bombyx mori D9L to obtain the G1 generation. When the G1 generation eggs were incubated at room temperature for 5-6 days, the G1 generation was screened for green fluorescence under a fluorescence microscope. The presence of green fluorescence in the eyes of the sgRNA individuals (see Figure 1 A) was determined. The genomic DNA of the legs of the sgRNA individuals was extracted using a genomic DNA extraction kit (Omega, Norcross, USA), and PCR amplification was performed using the genomic DNA as a template. The upstream primer was 5'-AGCTGTCCAAGGAATGCG-3' (SEQ ID NO: 4), and the downstream primer was 5'-CGACTCGGTGCCACTTT-3' (SEQ ID NO: 5). The amplification conditions were as follows: 98℃ pre-denaturation for 2 minutes; 98℃ denaturation for 10 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 30 seconds, a total of 40 cycles; finally 72℃ extension for 5 minutes, and storage at 4℃. The PCR product was identified by agarose gel electrophoresis, and a bright band was observed at 750 bp. Sequencing was performed using the sequencing primer 5'-GTAAGACAAGGTCAGATAG-3' (SEQ ID NO: 6), and the sgRNA sequence obtained is shown in SEQ ID NO: 7 (see Figure 1 B). The obtained sgRNA individuals and Hsp90-Cas9 individuals expressing red fluorescent protein in the eyes were crossed to obtain F1 generation, and fluorescence screening was performed. The hybrid mutant individuals expressing both red fluorescent protein and green fluorescent protein in the eyes were recorded as BmSPP KO Bombyx mori (see Figure 1 C), and the individuals expressing only red fluorescent protein in the eyes were recorded as BmSPP WT Bombyx mori.
[0020] Example 3BmSPP KO Detection of susceptibility of Bombyx mori to Beauveria bassiana The obtained F1 generation silkworms were collectively reared to 5th instar, and the 5th instar silkworms were fluorescently sorted to express green and red fluorescence in the eyes at the same time BmSPP KO Bombyx mori and the eye-expressed red fluorescent BmSPP WT Bombyx mori. The flat-cultured Beauveria bassiana was made into a suspension using sterilized water containing 0.02% Tween 20 (Sigma, St. Louis, MO), and a 300-mesh nylon mesh was used to filter out excess mycelium and retain only spores to make a spore suspension, which was vortexed and homogenized, and the spore concentration was determined by a hemocytometer. The 5th instar silkworms were inoculated with the spore suspension diluted to 1×10 7 conidia / mL. The 5th instar silkworms were inoculated with the spore suspension diluted to 1×10 BmSPP KO Bombyx mori and BmSPP WT Bombyx mori, 3 groups of repeats were set up, each group of repeat had 20 larvae, and the larvae were first fed with a small amount of mulberry leaves, and after 6 hours, 2 mL of spore suspension of 1×10 7 conidia / mL was sprayed on each repeat of silkworms, and then the sprayed mulberry leaves were added to feed and kept high humidity until the silkworms were put into the cocoon, and the death of the silkworms was observed. It was found that the death trend of the two groups changed obviously after 84 hours of inoculation ( Figure 2 A), BmSPP KO Bombyx mori was more infected with Beauveria bassiana during the larval stage and pre-pupa stage, and the number of silkworms that could normally pupate was significantly reduced ( Figure 2 B), indicating that the mutant individuals BmSPP KO Bombyx mori has higher susceptibility to Beauveria bassiana and is more likely to become a white silkworm.
[0021] Example 3 Preparation of white silkworm The obtained F1 generation silkworms were collectively reared to 5th instar, and the 5th instar silkworms were fluorescently sorted to express green and red fluorescence in the eyes at the same time BmSPP KO Bombyx mori and the eye-expressed red fluorescent BmSPP WT Bombyx mori. The flat-cultured Beauveria bassiana was made into a suspension using sterilized water containing 0.02% Tween 20 (Sigma, St. Louis, MO), and a 300-mesh nylon mesh was used to filter out excess mycelium and retain only spores to make a spore suspension, which was vortexed and homogenized, and the spore concentration was determined by a hemocytometer. The 5th instar silkworms were inoculated with the spore suspension diluted to 1×10 7 conidia / mL. The 5th instar silkworms were inoculated with the spore suspension diluted to 1×10 BmSPP KO Bombyx mori and BmSPP WTEach group of silkworms was set with 3 repeats, and each repeat contained 20 larvae. The larvae were first fed with a small amount of mulberry leaves, and 6 hours later, 2 mL of 1×10 7 conidia / mL spore suspension was sprayed on each repeat of silkworms, and then the silkworms were fed with mulberry leaves sprayed with sterile water to maintain high humidity until the silkworms were put into the cocooning cage. The death of silkworms was observed, BmSPP KO The silkworms infected with Beauveria bassiana during the larval stage and pre-pupa stage were made into white bauveria silkworms (Bassiana silkworms) Figure 2 C).
[0022] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application 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 application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions, and they should all be covered in the scope of the claims of the present application.
Claims
1. Bombyx mori BmSPP The application of the gene in the preparation of white muscardine, characterized in that: Knocking out using a knockout vector BmSPP The expression of the gene, the silkworm BmSPP The sequence of the gene is shown as SEQ ID NO:
3.
2. The domesticated silkworm of claim 1 BmSPP The application of the gene in the preparation of the white muscardine worm is characterized in that: The knockout vector contains a knockout silkworm BmSPP gene, the sgRNA nucleic acid sequence of which is shown in SEQ ID NO:
7.
3. A method for preparing high-quality variety of white muscardine, characterized in that, A high-quality mulberry silk production variety is bred by knocking out a gene of the silkworm Bombyx mori, and the silkworm Bombyx mori BmSPP The sequence of the gene is shown as SEQ ID NO:
3. BmSPP The sequence of the gene is shown as SEQ ID NO:
3.
4. The method of claim 3, wherein, comprising the steps of: (a) constructing an sgRNA vector targeting BmSPP a gene; (b) introducing the vector of step (a) into a Bombyx mori fertilized egg; (c) crossing the sgRNA individual obtained in step (b) with an individual expressing Cas9; (d) screening for obtaining BmSPP Silkworm (Bombyx mori) with gene knockout, i.e. white mulberry leaf high-quality variety BmSPP KO Strains.
5. The method of claim 4, wherein, The sgRNA sequence is shown in SEQ ID NO:
7.
6. A method of preparing a white muscardine, characterized by: The method for preparing the silkworm of claim 3-5 is prepared by spraying the Beauveria bassiana liquid to the silkworm BmSPP KO The strain forms white muscardine after the silkworm is stiffened and dies.
7. The preparation method of the white muscardine according to claim 6, characterized in that: The concentration of the Beauveria bassiana bacterial solution is 1 x 10 7 conidia / mL.