Gnrna targeting bombyx mori sericin 1 gene, raw silk preparation method and application

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a gRNA targeting Bombyx mori sericin 1 gene, a method for preparing coarse silk and application. The target sequence of the gRNA is shown as SEQ ID NO:1. The method for preparing coarse silk comprises the following steps: 1) constructing a gRNA expression vector targeting the Bombyx mori BmSer1 gene, and preparing a gRNA transgenic Bombyx mori; 2) crossing the gRNA transgenic Bombyx mori and Cas9 transgenic Bombyx mori, and screening to obtain double transgenic individuals with green fluorescence in eyes and bodies; 3) screening to obtain a heterozygous mutant Bombyx mori strain with the BmSer1 gene knocked out through typing detection; and 4) obtaining coarse silk by feeding the transgenic Bombyx mori. The genetic editing technology is used to change the genetic traits of Bombyx mori, and the obtained silk has a larger diameter, a lower sericin rate, better water resistance and durability.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a gRNA targeting the silkworm sericin 1 gene, a method for preparing coarse silk, and its application. Background Technology

[0002] Silk, also known as natural silk, is a continuous long fiber formed from the solidified silk fluid secreted by mature silkworms when they spin their cocoons. It is mainly composed of fibroin and sericin. As one of the earliest animal fibers used by humans, silk is characterized by its lightness, softness, fineness, luster, and smooth, full feel. China, as the birthplace of silk, is the world's largest silk producer, with extensive silk-producing areas including Jiangsu, Zhejiang, Sichuan, Guangdong, and Shandong provinces.

[0003] Different thicknesses of silk have different applications. Fine silk has good thermal insulation properties and is often used in the production of thermal clothing and insulation materials. Coarse silk, with its increased diameter, typically has enhanced water resistance and can be used in: 1) Waterproof materials for fishing gear and sails: Coarse silk can be used to make strong fishing lines and reduce water absorption, thus extending the lifespan of the fishing gear; it can also be added to the materials used in sails and diving suits, further improving the user experience due to its flexibility. 2) Industrial filtration materials: Coarse silk can be processed into filtration materials. Due to its coarse fibers and porous structure, it can provide preliminary filtration. 3) Medical field: Due to its poor water absorption, coarse silk can be used as surgical sutures without affecting the flow of surrounding tissue fluid. It can also be used as a tissue repair material; coarse silk scaffolds can provide space and support for cell growth, promoting tissue regeneration and repair.

[0004] Traditional silkworm breeding techniques and existing technologies focus more on the mechanical properties of silk, paying less attention to variations in silk diameter. Traditional methods of obtaining coarse silk by optimizing silkworm rearing conditions or altering feed are slow, inconsistent in their effectiveness, and yield low economic benefits. Artificial selection during the rearing process to create "new strains" of silkworms that produce coarse silk is time-consuming and labor-intensive, resulting in significant fluctuations in silk thickening rates. Using physical or chemical treatments to thicken silk may negatively impact other desirable properties of the silk, hindering further development.

[0005] CRISPR / Cas9 gene editing technology is a revolutionary molecular biology tool that utilizes bacterial CRISPR sequences and Cas9 nucleases to precisely locate and cut target gene sequences using designed guide RNAs (sgRNAs), enabling gene knockout, insertion, or replacement. CRISPR / Cas9 technology has been widely applied to modifying silk properties. For example, researchers have used this technology to produce recombinant spider silk proteins, successfully obtaining silkworm-spider chimeric silk with significantly improved mechanical properties. Another example is patent CN115820736B, which discloses the application and method of silkworm sericin protein Ser4 in improving silk performance. Through seamless cloning and homologous recombination, the complete Ser4 protein with a molecular weight exceeding 250 kDa was expressed into the sericin layer of silkworm cocoons. The resulting transgenic lines showed improved silk mechanical properties, and the silk from the Ser4-overexpressing lines was more hydrophobic, with a reduced cocoon degumming rate. For example, patent CN117802107A discloses the application of the BmEcKL1 gene in the breeding of high-silk-yield silkworm varieties. By knocking out BmEcKL1 in silkworms, the size of the middle and rear silk glands can be increased, significantly improving the total cocoon weight and cocoon layer ratio, thus obtaining male silkworm varieties with increased silk yield. Therefore, BmEcKL1 knockout can be used to create high-silk-yield mutants for the breeding of new high-silk-yield silkworm varieties. Existing technologies mostly apply CRISPR / Cas9 gene editing technology with a focus on the study of silk performance.

[0006] Sericin is an important component of silkworm cocoons, accounting for approximately 25%, and plays a role in adhering and protecting fibroin. The silkworm sericin 1 gene (BmSer1) is one of the key genes encoding sericin proteins. The gene is approximately 23.6 kb in size, located on chromosome 11 of the silkworm, and exists as a single copy in the genome. It contains nine exons and is expressed in the post- and mid-regions of the silk gland in the middle of the silkworm. It exhibits five alternative splicing patterns and encodes various proteins with molecular weights ranging from approximately 70 to 330 kDa, located in the inner layer of the sericin layer that encapsulates the silk fibers. Regulating the expression level of the sericin gene can alter the proportion of sericin proteins in the cocoon silk, thereby changing the diameter of the silk fibers after degumming. Summary of the Invention

[0007] In view of this, the present invention proposes a method for preparing coarse silkworm silk by targeting the silkworm sericin 1 gene with gRNA and its application. The present invention knocks out the silkworm BmSer1 gene using gene editing technology to obtain a BmSer1 gene knockout heterozygous mutant silkworm strain; this transgenic silkworm can stably produce coarser silk with a larger diameter. The present invention provides a feasible method for stably obtaining silk with increased diameter from a heritable perspective.

[0008] One of the objectives of this invention is to provide a gRNA that targets the BmSer1 gene in the silkworm.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A gRNA targeting the BmSer1 gene of the silkworm, the target sequence of which is shown in SEQ ID NO: 1.

[0011] The second objective of this invention is to provide a transgenic expression vector constructed from the aforementioned gRNA.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] Transgenic expression vector constructed from the aforementioned gRNA.

[0014] The transgenic expression vector is prepared as follows: sticky ends are added to both sides of the gRNA, the gRNA backbone vector is digested with BbsI enzyme, and then ligated and transformed with annealing primers to obtain the PiggyBac[3xP3-EGFP-SV40; U6-gRNA-SV40] transgenic vector; the annealing primers include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 4 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 5.

[0015] The third objective of this invention is to provide a CRISPR / Cas9 gene editing system for knocking out the BmSer1 gene in silkworms.

[0016] To achieve the above objectives, the present invention adopts the following technical solution:

[0017] A CRISPR / Cas9 gene editing system for knocking out the BmSer1 gene in silkworms, the CRISPR / Cas9 gene editing system comprising the aforementioned transgenic expression vector and Cas9 expression vector.

[0018] The fourth objective of this invention is to provide a method for preparing a heterozygous mutant silkworm strain with BmSer1 gene knockout.

[0019] To achieve the above objectives, the present invention adopts the following technical solution:

[0020] The method for preparing heterozygous mutant silkworm lines with BmSer1 gene knockout involves knocking out the BmSer1 gene in silkworms using the aforementioned CRISPR / Cas9 gene editing system, screening binary transgenic individuals, and obtaining heterozygous mutant silkworm lines.

[0021] Preferably, the method includes the following steps:

[0022] (1) Construct a gRNA expression vector targeting the BmSer1 gene of silkworm and prepare gRNA transgenic silkworms;

[0023] (2) The gRNA transgenic silkworm and the Cas9 transgenic silkworm were hybridized and screened to obtain binary transgenic individuals whose eyes and bodies simultaneously emitted green fluorescence.

[0024] (3) The binary transgenic individuals obtained in step (2) were subjected to typing tests to screen out the heterozygous mutant silkworm strain with BmSer1 gene knockout.

[0025] Preferably, in step (3), the primers used for genotyping include: a forward primer with a nucleotide sequence as shown in SEQ ID NO: 2 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 3.

[0026] The fifth objective of this invention is to provide a method for preparing coarse fiber silk.

[0027] To achieve the above objectives, the present invention adopts the following technical solution:

[0028] The method for preparing coarse fiber silk involves using the aforementioned method to prepare a heterozygous mutant silkworm strain with the BmSer1 gene knocked out, and obtaining coarse silk by feeding transgenic silkworms.

[0029] The sixth objective of this invention is to provide the application of the aforementioned gRNA, the aforementioned transgenic expression vector, and / or the aforementioned gene editing system in the cultivation of transgenic silkworm strains for coarse silk and / or the preparation of coarse silk.

[0030] The seventh objective of this invention is to provide an application of knocking out the BmSer1 gene in the breeding of transgenic silkworm lines for coarse silk and / or the preparation of coarse silk.

[0031] To achieve the above objectives, the present invention adopts the following technical solution:

[0032] Application of knocking out the BmSer1 gene in the breeding of transgenic silkworm lines for coarse silk and / or the production of coarse silk.

[0033] Preferably, the nucleotide sequence of the BmSer1 gene is shown in SEQ ID NO: 6.

[0034] As a preferred method, the BmSer1 gene in silkworms was knocked out using the aforementioned gene editing system to obtain a transgenic silkworm strain that produces coarse silk; coarse silk was obtained by raising the transgenic silkworms.

[0035] The beneficial effects of this invention are as follows:

[0036] (1) This invention uses gene editing technology to alter the genetic traits of silkworms, thereby obtaining silkworms that stably produce coarse silk through rearing. Subsequent production and development only require rearing transgenic silkworms to obtain a stable yield of coarse silk. The coarse silk obtained by this invention has a lower sericin content and a thicker diameter: the sericin content of the heterozygous mutant is approximately 13.83%, a decrease of approximately 11.37 percentage points compared to the control group; the diameter of the degummed silk from the heterozygous mutant is approximately 15.24 μm, an increase of 3.91 μm compared to the control group. This invention provides an achievable method for increasing the diameter of silk, and this method has broad application prospects in waterproof materials, fishing gear manufacturing, industrial filtration materials, and tissue repair.

[0037] (2) The coarse silk produced by this invention not only maintains the excellent luster of silk, but also has advantages in abrasion resistance and tensile strength, thus increasing durability. This coarse silk can be used to enhance the anti-immersion ability of fishing gear and fishing lines, and extend the life of fishing gear; since coarse silk has both excellent waterproofness and flexibility, it can be further used to manufacture more fitted raincoats or waterproof clothing. Attached Figure Description

[0038] Figure 1 The image shows the sequencing and mutation detection results for the knockout target site.

[0039] Figure 2 The image shows the results of sericin content analysis in the cocoons of heterozygous mutant silkworms.

[0040] Figure 3 This is a graph showing the results of diameter testing of silk fibers after degumming. Detailed Implementation

[0041] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0042] Example 1. Obtaining a heterozygous mutant silkworm strain with BmSer1 gene knockout

[0043] 1. Design of gRNA target sites for the silkworm BmSer1 gene

[0044] Download the base sequence of the silkworm BmSer1 gene (NCBI Gene ID: 101740082). Based on the base sequence of the first common exon, and following the principle of starting with G and ending with NGG, design gRNA target sites, as shown in SEQ ID NO: 1.

[0045] 2. Carrier Construction

[0046] Sticky ends (AAGT at the F end and AAAC at the R end) were added to both sides of the gRNA. After digesting the gRNA backbone vector with BbsI (see Sanyuan Ma. CRISPR / Cas9 mediated multiplex genome editing and heritable mutationnesis of BmKu70 in Bombyx mori. 2014), it was ligated with annealing primers and transformed to construct the PiggyBac[3xP3-EGFP-SV40; U6-gRNA-SV40] transgenic vector (see Yuanyuan Liu. Tissue-specific genome editing of laminA / C in the posterior silk glands of Bombyx mori. 2017). The annealing primers included gRNA(F) with the nucleotide sequence shown in SEQ ID NO: 4 and gRNA(R) with the nucleotide sequence shown in SEQ ID NO: 5.

[0047] (1) Ligation of gRNA fragments with transgenic vectors

[0048] The annealed double-stranded gRNA fragment was ligated into the PiggyBac[3xP3-EGFP-SV40; U6-gRNA-SV40] vector. The ligation system consisted of 4 μL of annealed gRNA fragment, 1 μL of vector, 0.1 μL of T4 DNA ligase, 1 μL of DNA ligase 10× Buffer, and 3.9 μL of pure water, for a total volume of 10 μL. Ligation was carried out at 16°C for 1–1.5 h.

[0049] (2) Transformation

[0050] 1) Remove Trans1-T1 competent cells from the -80℃ freezer and thaw them on ice;

[0051] 2) In a clean bench, add 10 μL of the ligation product to the competent cells that have dissolved to a milky white color, and incubate on ice for 20-25 min;

[0052] 3) Place the sample in a 42℃ metal bath for 90 seconds for heat shock, then let it stand on ice for 2-3 minutes;

[0053] 4) Add 500 μL of LB antibiotic-free liquid medium to a 1.5 mL centrifuge tube (operate in a clean bench), and incubate at 37 °C and 220 rpm for 45-60 min to activate the bacteria;

[0054] 5) In a clean bench, spread 100 μL of bacterial suspension evenly on an LB solid medium plate containing kanamycin.

[0055] 6) Incubate upright in a 37℃ constant temperature incubator for about 3-10 minutes, then invert and incubate overnight in a 37℃ incubator;

[0056] 7) In a clean bench, pick a single colony plaque into 500 μL of LB liquid medium with kanamycin resistance, and incubate at 37°C and 220 rpm in a shaker. After the bacterial solution becomes turbid, take 150 μL of the strain and send it to the company for sequencing detection. Select positive clones and extract plasmids for later use.

[0057] 3. Embryo injection and fluorescence screening

[0058] The successfully constructed PiggyBac[3xP3-EGFP-SV40; U6-Ser1gRNA-SV40] transgenic vector was mixed with the helper plasmid at a 1:1 ratio and injected into silkworm eggs via microinjection, designated as generation G0. The silkworm eggs were non-diapause eggs, and the injection was performed within 2 hours of egg laying. The injected eggs were then induced to regrow and reared until moth emergence. Generation G0 self-pollinated to produce generation G1. Generation G1 was screened for green fluorescence; those exhibiting green fluorescence in their eyes were transgenic individuals successfully expressing sgRNA.

[0059] 4. Obtaining heterozygous mutants

[0060] The obtained gRNA transgenic individuals and the obtained Cas9 transgenic individuals were crossed to obtain the offspring F1. The preparation process of Cas9 transgenic individuals is as follows: PiggyBac [IE1-EGFP-SV40; Nos-Cas9-SV40] (see JunXu. Identification of a germline-expression promoter for genome editing in Bombyxmori. 2018) was injected into silkworm eggs to obtain Cas9 transgenic individuals. Based on the presence of green fluorescence in the eyes and body, binary transgenic individuals with green fluorescence in both the eyes and body were screened. The sequences at the detection sites were genotyped using detection primers F and R, and the amplification products were sequenced for analysis. The nucleotide sequence of the forward primer (F) is shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer (R) is shown in SEQ ID NO: 3. Figure 1 As shown, if the amplified product shows overlapping peaks near the PAM motif in the sequencing peak diagram, it indicates that the knockout has been successfully completed and a heterozygous mutant silkworm strain with BmSer1 gene knockout has been successfully prepared.

[0061] Example 2. Detection of sericin content and degummed silk diameter in heterozygous mutants

[0062] 1. Silkworm cocoon degumming treatment

[0063] The transgenic silkworms obtained in Example 1 were fed to produce cocoons. The cocoons were boiled in a 1% sodium bicarbonate solution and then degummed for 1 hour. The cocoons before and after degumming were weighed and their weights calculated. The results are as follows: Figure 2 As shown, the sericin content of the control group was approximately 25.2%, while that of the heterozygous mutant was approximately 13.83%, representing a decrease of approximately 11.37 percentage points compared to the control group.

[0064] 2. Measurement of silk diameter after degumming

[0065] The diameter of the degummed silk was measured using a microscope. For example... Figure 3 As shown, the diameter of the degummed silk in the control group was approximately 11.33 μm, while the diameter of the degummed silk in the heterozygous mutant was approximately 15.24 μm. Degummed silk with a diameter increase of 3.91 μm was successfully obtained.

Claims

1. A method for preparing coarse fiber silk, characterized in that, The BmSer1 gene in silkworms was knocked out using the CRISPR / Cas9 gene editing system, and binary transgenic individuals were screened to obtain a heterozygous mutant silkworm strain. Coarse silk was obtained by feeding the transgenic silkworms. The nucleotide sequence of the BmSer1 gene is shown in SEQ ID NO:

6.

2. The preparation method according to claim 1, characterized in that, The CRISPR / Cas9 gene editing system includes a transgenic expression vector and a Cas9 expression vector; the transgenic expression vector is constructed from gRNA targeting the BmSer1 gene of the silkworm, and the target sequence of the gRNA is shown in SEQ ID NO:

1.

3. The preparation method according to claim 1, characterized in that, The preparation of the heterozygous mutant silkworm strain specifically includes the following steps: (1) Construct a gRNA expression vector targeting the BmSer1 gene of silkworm and prepare gRNA transgenic silkworms; (2) The gRNA transgenic silkworm and the Cas9 transgenic silkworm were hybridized and screened to obtain binary transgenic individuals whose eyes and bodies emitted green fluorescence at the same time; (3) The binary transgenic individuals obtained in step (2) were subjected to typing tests to screen out the heterozygous mutant silkworm strain with BmSer1 gene knockout.

4. The preparation method according to claim 3, characterized in that, In step (3), the primers used for genotyping include: a forward primer with a nucleotide sequence as shown in SEQ ID NO: 2 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

3.

5. The application of the CRISPR / Cas9 gene editing system for knocking out the BmSer1 gene in the breeding of transgenic silkworm lines producing coarse silk and / or the preparation of coarse silk, characterized in that, The nucleotide sequence of the BmSer1 gene is shown in SEQ ID NO:

6.

6. The application according to claim 5, characterized in that, The CRISPR / Cas9 gene editing system includes a transgenic expression vector and a Cas9 expression vector; the transgenic expression vector is constructed from gRNA targeting the BmSer1 gene of the silkworm, and the target sequence of the gRNA is shown in SEQ ID NO:

1.

7. The application according to claim 6, characterized in that, The transgenic expression vector is prepared as follows: sticky ends are added to both sides of the gRNA, the gRNA backbone vector is digested with BbsI enzyme, and then ligated and transformed with annealing primers to obtain the PiggyBac[3xP3-EGFP-SV40;U6-gRNA-SV40] transgenic vector; the annealing primers include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 4 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

5.

8. The application of knocking out the BmSer1 gene in the breeding of transgenic silkworm lines producing coarse silk and / or the preparation of coarse silk, characterized in that, The nucleotide sequence of the BmSer1 gene is shown in SEQ ID NO: 6.

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