Gnrna targeting bombyx mori sericin 3 gene, method for preparing fine silk and application

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a gRNA targeting Bombyx mori sericin 3 gene, a method for preparing fine silkworm silk and application. The target sequence of the gRNA is shown as SEQ ID NO:1. The application uses a CRISPR / Cas9 gene editing system to knock out BmSer3 gene in Bombyx mori, and specifically comprises the following steps: constructing a gRNA expression vector targeting the BmSer3 gene of Bombyx mori, and preparing gRNA transgenic Bombyx mori; crossing the gRNA transgenic Bombyx mori and Cas9 transgenic Bombyx mori, and screening to obtain double transgenic individuals with green fluorescence in eyes and body; through typing detection, a heterozygous mutant Bombyx mori strain with BmSer3 gene knockout is obtained. Finally, fine silkworm silk is obtained by breeding the transgenic silkworm. The application uses gene editing technology to change the genetic traits of Bombyx mori, and the obtained silkworm silk has a smaller diameter, a higher sericin rate and better heat preservation and insulation performance.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a gRNA targeting the silkworm sericin 3 gene, a method for preparing fine silk, and its application. Background Technology

[0002] Silk is a continuous, long fiber formed from the solidified silk fluid secreted by mature silkworms during cocoon spinning; it is also known as natural silk. It is mainly composed of fibroin and sericin. Fiber is the main component of the fiber, while sericin coats the fibroin, acting as an adhesive and protecting it. Silk is one of the earliest animal fibers utilized by humans, characterized by its lightness, softness, fineness, luster, and smooth, full feel. China, as the origin of silk, has extensive silk-producing areas, including provinces such as Jiangsu, Zhejiang, Sichuan, Guangdong, and Shandong. Today, China has become the world's leading silk producer, ranking among the top in both scale and output.

[0003] The thermal conductivity and thermal stability of silk are related to its fiber diameter. A finer diameter increases the surface area of ​​the silk fibers, reduces the gaps between the fibers, and increases the number of pores, thus improving its thermal insulation properties. Generally, the diameter of silk is closely related to factors such as the cocoon structure, silkworm breed, silk reeling process, and spinning environment. Finer silk fibers, due to their increased surface area to volume ratio and smaller gaps between fibers, exhibit better thermal insulation properties, opening up more application areas. For example: 1) Thermal clothing: Fine silk can be used to make thermal clothing, such as thermal underwear, quilts, and socks suitable for mass consumption. 2) Thermal insulation materials with skin-friendly properties for special applications. 3) Silk micro / nanofiber aerogels: Silk micro / nanofiber aerogels have extremely low thermal conductivity and a stable three-dimensional network structure, making them potentially valuable in the field of thermal insulation materials. These aerogels can be used for building insulation, thermal management of electronic equipment, etc.

[0004] Professor Tao Guangming and his research team at Huazhong University of Science and Technology have created colorful smart fabrics using silk fibroin as raw material. The researchers used cryo-spinning to design and control the microstructure of the fibers, preparing structural fibers with high internal porosity and low thermal conductivity, achieving excellent thermal insulation performance. Furthermore, they integrated temperature-controlled phase change materials into the fibers and coated them with PDMS, utilizing the heat absorption, storage, and release properties of the phase change materials to create composite temperature-controlled fibers that are strong and wear-resistant, soft and waterproof, and offer zero-energy intelligent temperature regulation. The microstructured fiber (MF) fabric prepared using advanced weaving technology exhibits good bidirectional thermal insulation performance, which increases with the number of fabric layers. The phase change materials integrated within the microstructured fibers not only significantly slow down the rate of increase or decrease in ambient temperature but also have good cycle stability. With the development of materials science and engineering technology, the application fields of fine silk will continue to expand, bringing new growth points and innovation opportunities to the traditional silk industry.

[0005] Currently, methods for altering silkworm silk diameter include gene modification, chemical treatment, physical modification, and supplemental feeding. However, these methods all have certain drawbacks. For example, physical modification may alter the structure and mechanical properties of silk; chemical treatment can affect the luster and comfort of silk; and supplemental feeding has uncertain effects on the growth and development of silkworms and makes it difficult to control silk diameter. In contrast, gene editing technology offers a more convenient, precise, controllable, and heritable method for modifying silkworm silk diameter.

[0006] 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. In existing technology, patent CN110791528A discloses a method for improving silkworm silk production and optimizing silkworm varieties through genome editing technology, including: sgRNA transgenic expression vectors for let-7 microRNA, screening of sgRNA expression lines, screening of let-7 knockout lines, identification of knockout forms, and statistical analysis of silk gland length, weight, and cocoon weight after let-7 knockout. This invention utilizes CRISPR / Cas9 technology to delete the expression of let-7 non-coding RNA, thereby relieving its negative regulatory effect on silk gland development and silk protein synthesis, and ultimately increasing silk production in silkworms.

[0007] The silkworm sericin 3 gene (BmSer3) is one of the key genes encoding sericin protein. It is 5.5 kb in size, located on chromosome 11 of the silkworm, and exists as a single copy in the genome. It is expressed in the anterior region of the silk gland in the middle of the silkworm and encodes a protein with a molecular weight of approximately 120 kDa, located in the outer layer of the sericin protein layer that surrounds the cocoon silk fiber. Regulating the expression level of the sericin gene can alter the proportion of sericin protein in the cocoon silk, thereby changing the diameter of the silk fiber after degumming. Summary of the Invention

[0008] In view of this, this invention proposes a method for preparing fine silk by targeting the silkworm sericin 3 gene with gRNA and its application. This invention utilizes gene editing technology to knock out the silkworm BmSer3 gene, altering the genetic trait of silk diameter, and obtaining a BmSer3 gene knockout heterozygous mutant silkworm strain; this transgenic silkworm can stably produce finer silk with a smaller diameter. This invention provides an achievable method for altering silk diameter, with significant advantages and broad application prospects.

[0009] One of the objectives of this invention is to provide a gRNA that targets the BmSer3 gene in the silkworm.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A gRNA targeting the BmSer3 gene of the silkworm, the target sequence of which is shown in SEQ ID NO: 1.

[0012] The second objective of this invention is to provide a transgenic expression vector constructed from the aforementioned gRNA.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] Transgenic expression vector constructed from the aforementioned gRNA.

[0015] Preferably, 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.

[0016] Preferably, 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.

[0017] Preferably, AAGT is added to the F terminus of the gRNA and AAAC is added to the R terminus.

[0018] A third objective of this invention is to provide a CRISPR / Cas9 gene editing system for knocking out the BmSer3 gene in silkworms.

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

[0020] A CRISPR / Cas9 gene editing system for knocking out the BmSer3 gene in silkworms, the CRISPR / Cas9 gene editing system comprising the aforementioned transgenic expression vector and Cas9 expression vector.

[0021] Preferably, the Cas9 expression vector is the PiggyBac[IE1-EGFP-SV40; Nos-Cas9-SV40] expression vector.

[0022] The fourth objective of this invention is to provide a method for preparing a heterozygous mutant silkworm strain with BmSer3 gene knockout.

[0023] To achieve the above objectives, the present invention adopts the following technical solution:

[0024] The method for preparing heterozygous mutant silkworm lines with BmSer3 gene knockout involves knocking out the BmSer3 gene in silkworms using the aforementioned CRISPR / Cas9 gene editing system, screening binary transgenic individuals, and obtaining heterozygous mutant silkworm lines.

[0025] Preferably, the method includes the following steps:

[0026] (1) Construct a gRNA expression vector targeting the BmSer3 gene of silkworm and prepare gRNA transgenic silkworms;

[0027] (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.

[0028] (3) The binary transgenic individuals obtained in step (2) were subjected to typing tests to screen out the heterozygous mutant silkworm strains with BmSer3 gene knockout.

[0029] 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.

[0030] Preferably, step (1) includes the following steps:

[0031] 1) Add sticky ends to both sides of the gRNA, with AAGT at the F end and AAAC at the R end; digest the gRNA backbone vector with BbsI, then ligate and transform it with annealing primers to obtain the PiggyBac[3xP3-EGFP-SV40; U6-gRNA-SV40] transgenic vector; the annealing primers include a forward primer with the nucleotide sequence shown in SEQ ID NO: 4 and a reverse primer with the nucleotide sequence shown in SEQ ID NO: 5;

[0032] 2) The PiggyBac[3xP3-EGFP-SV40; U6-Ser1gRNA-SV40] transgenic vector obtained in step 1) was mixed with the helper plasmid at a ratio of 1:1 and injected into silkworm eggs by microinjection, which was recorded as generation G0. After injection, the eggs were induced to grow and raised until the moths emerged. Generation G0 self-pollinated to produce generation G1. Generation G1 was screened for green fluorescence. Those with green fluorescence in their eyes were transgenic individuals that successfully expressed gRNA.

[0033] More preferably, in step 2), the silkworm eggs are non-diapause eggs; microinjection is performed within 2 hours of egg laying.

[0034] The fifth objective of this invention is to provide a method for preparing fine fiber silk.

[0035] To achieve the above objectives, the present invention adopts the following technical solution:

[0036] The method for preparing fine silk fibers involves using the aforementioned method to prepare a heterozygous mutant silkworm strain with the BmSer3 gene knockout, and obtaining fine silk by feeding transgenic silkworms.

[0037] 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 preparation of fine fiber silk or the cultivation of transgenic silkworm strains of fine silk.

[0038] The seventh objective of this invention is to provide an application of knocking out the BmSer3 gene in the preparation of fine fiber silk or the cultivation of transgenic silkworm strains that produce fine silk.

[0039] Preferably, the nucleotide sequence of the BmSer3 gene is shown in SEQ ID NO: 6.

[0040] As a preferred method, the BmSer3 gene in silkworms was knocked out using the aforementioned gene editing system to obtain a transgenic silkworm strain that produces fine silk; fine silk was then obtained by raising the transgenic silkworms.

[0041] The beneficial effects of this invention are as follows:

[0042] (1) This invention uses gene editing technology to alter the genetic traits (silk diameter) of silkworms, obtaining silkworms that stably produce fine silk. A stable yield of fine silk can be obtained by raising transgenic silkworms. The fine silk obtained by this invention has a higher sericin content and a finer diameter: the sericin content of the heterozygous mutant is approximately 38.23%, an increase of approximately 12.03 percentage points compared to the control group; the diameter of the degummed silk from the heterozygous mutant is approximately 7.8 μm, a decrease of 3.67 μm compared to the control group. This invention provides an achievable method for altering silk diameter, with significant advantages and broad application prospects.

[0043] (2) The gene editing technology used in this invention alters the genetic traits of silkworms without involving the use of harmful chemicals, and is more environmentally friendly than traditional chemical treatment methods.

[0044] (3) The fine silk obtained by the present invention not only maintains the excellent luster of silk, but also has a larger surface area and smaller gaps between them, and has better heat insulation performance. It can be further used to make thermal clothing, silk micro-nano fiber aerogel, etc. Attached Figure Description

[0045] Figure 1 The image shows the sequencing and mutation detection results for the knockout target site.

[0046] Figure 2This is a statistical chart showing the percentage of sericin content in the cocoons of heterozygous mutant silkworms.

[0047] Figure 3 This is a statistical chart showing the diameter of silk fibers after degumming. Detailed Implementation

[0048] 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.

[0049] Example 1. Obtaining a heterozygous mutant silkworm strain with BmSer3 gene knockout

[0050] 1. Design of gRNA target sites for the BmSer3 gene in silkworm

[0051] Download the base sequence of the silkworm BmSer3 gene (NCBI Gene ID: 100136948). Based on the base sequence of the third exon, and following the principle of starting with G and ending with NGG, design gRNA target sites, as shown in SEQ ID NO: 1.

[0052] 2. Carrier Construction

[0053] 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 primers included gRNA(F) with nucleotide sequences as shown in SEQ ID NO: 4 and gRNA(R) with nucleotide sequences as shown in SEQ ID NO: 5.

[0054] (1) Ligation of gRNA fragments with transgenic vectors

[0055] 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 purified water, for a total volume of 10 μL. Ligation was carried out at 16°C for 1–1.5 h.

[0056] (2) Transformation

[0057] 1) Remove Trans1-T1 competent cells from the -80℃ freezer and thaw them on ice;

[0058] 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;

[0059] 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;

[0060] 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;

[0061] 5) In a clean bench, spread 100 μL of bacterial suspension evenly on an LB solid medium plate containing kanamycin.

[0062] 6) Incubate upright in a 37℃ constant temperature incubator for about 3-10 minutes, then invert and incubate overnight in a 37℃ incubator;

[0063] 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.

[0064] 3. Embryo injection and fluorescence screening

[0065] 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 gRNA.

[0066] 4. Obtaining heterozygous mutants

[0067] 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 used for genotyping detection 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. If the amplification product shows a peak near the PAM motif in the sequencing peak diagram, it indicates that the knockout has been successfully completed, and the heterozygous mutant silkworm strain with BmSer3 gene knockout has been successfully prepared, such as... Figure 1 As shown.

[0068] Example 2. Detection of sericin content and degummed silk diameter in heterozygous mutants

[0069] 1. Silkworm cocoon degumming treatment

[0070] 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 26.2%, while that of the heterozygous mutant was approximately 38.23%, representing an increase of approximately 12.03 percentage points compared to the control group.

[0071] 2. Measurement of silk diameter after degumming

[0072] The diameter of the degummed silk was measured using a microscope. The results are as follows: Figure 3 As shown, the diameter of the degummed silk in the control group was approximately 11.46 μm, while the diameter of the degummed silk in the heterozygous mutant was approximately 7.8 μm. Degummed silk with a diameter reduced by 3.67 μm was successfully obtained.

Claims

1. A method for preparing fine silk fibers, characterized in that, The BmSer3 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. Fine silk was obtained by feeding the transgenic silkworms. The nucleotide sequence of the BmSer3 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 BmSer3 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 BmSer3 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 BmSer3 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 gRNA targeting the BmSer3 gene of silkworm, a transgenic expression vector for said gRNA, and / or a CRISPR / Cas9 gene editing system for knocking out the BmSer3 gene of silkworm in the preparation of fine-fiber silk or the cultivation of transgenic silkworm lines for fine-fiber silk, characterized in that, The target sequence of the gRNA is shown in SEQ ID NO: 1; the nucleotide sequence of the BmSer3 gene is shown in SEQ ID NO:

6.

6. The application according to claim 5, characterized in that, The transgenic expression vector is constructed from the gRNA; the CRISPR / Cas9 gene editing system includes the transgenic expression vector and the Cas9 expression vector.

7. The application according to claim 5 or 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 BmSer3 gene in the preparation of fine-fiber silk or the cultivation of transgenic silkworm lines for fine silk, characterized in that, The nucleotide sequence of the BmSer3 gene is shown in SEQ ID NO: 6.

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