GRNA (guide Ribonucleic Acid) of targeted bombyx mori silk glue 1 gene, and preparation method and application of coarse silk

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a gRNA of a targeted bombyx mori silk glue 1 gene, a preparation method of coarse silk and application. The target sequence of the gRNA is as shown in SEQ ID NO: 1. The preparation method of the coarse silk comprises the following steps: 1) constructing a gRNA expression vector of a targeted bombyx mori BmSer1 gene, and preparing a gRNA transgenic bombyx mori; 2) hybridizing the gRNA transgenic silkworms and Cas9 transgenic silkworms, and screening to obtain binary transgenic individuals with eyes and bodies emitting green fluorescence at the same time; 3) performing typing detection and screening to obtain a BmSer1 gene knockout heterozygous mutant silkworm strain; and 4) feeding the transgenic silkworms to obtain coarse silk. According to the method, genetic characters of silkworms are changed by using a gene editing technology, and the obtained silk is thicker in diameter, lower in sericin rate and better in waterproofness and durability.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a gRNA targeting the silkworm sericin 1 gene, a method for preparing crude silk, and applications thereof. Background Art

[0002] Silk, also known as natural silk, is a continuous long fiber formed by the solidification of silk fluid secreted by mature silkworms when they make cocoons. It is mainly composed of fibroin and sericin. As one of the earliest animal fibers used by humans, silk is soft, slender, shiny, smooth and plump. As the birthplace of silk, China is the world's largest silk producer, with a wide range of silk production areas, including Jiangsu, Zhejiang, Sichuan, Guangdong and Shandong provinces.

[0003] The application scenarios of silk with different thicknesses are somewhat different. Fine silk has good thermal insulation properties and is mostly used in the production of thermal insulation clothing and thermal insulation materials. Coarse silk with increased diameter usually has enhanced waterproofness and can be used for: 1) Waterproof materials such as fishing gear and sails: Coarse silk can be used to make fishing lines with excellent toughness and reduce the water absorption and soaking of fishing lines, thereby extending the service life of fishing gear; it can also be added to the materials for making sails and diving suits, and its flexibility further enhances the user experience. 2) Industrial filter materials: Coarse silk can be processed into filter materials. Because its fibers are relatively thick and have a certain pore structure between each other, it can play a role in preliminary filtration. 3) Medical field: Coarse silk can be used as surgical sutures due to its poor water absorption, which does not affect 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 and promote tissue regeneration and repair.

[0004] Traditional silk and existing technologies are more focused on the mechanical properties of silk, and less on changes in silk diameter. The traditional method of obtaining coarse silk by optimizing the feeding conditions of silkworms or changing the feed is slow, unstable, and has low economic benefits. Artificial screening during the breeding process to form a "new strain" of silkworms that spit out coarse silk is time-consuming and labor-intensive, and the resulting silk thickening rate fluctuates greatly. Using physical or chemical treatments to thicken silk may affect other excellent properties of silk, which is not conducive to subsequent development.

[0005] CRISPR / Cas9 gene editing technology is a revolutionary molecular biology tool that uses bacterial-derived CRISPR sequences and Cas9 nucleases to precisely locate and cut target gene sequences by designing specific guide RNAs (sgRNAs) to achieve gene knockout, insertion or replacement. CRISPR / Cas9 technology has been widely used in the modification of silk properties. For example, existing researchers have used this technology to produce recombinant spider silk proteins and successfully obtained silkworm-spider chimeric silk with significantly improved mechanical properties. For another example, the patent with publication number CN115820736B discloses an application of silkworm sericin Ser4 in improving silk properties and a method thereof. Through seamless cloning and homologous recombination, the complete Ser4 protein with a molecular weight of more than 250 kDa is expressed in the silk layer of the cocoon. The transgenic strains bred have improved the mechanical properties of silk, and the silk of the strains overexpressing Ser4 is more hydrophobic, and the degumming rate of the cocoon is reduced. For another example, the patent with publication number CN117802107A discloses an application of the silkworm BmEcKL1 gene in the breeding of high-yield silkworm varieties. By knocking out BmEcKL1 in silkworms, the size of the middle and posterior silk glands can be increased, the total cocoon volume and cocoon layer rate can be significantly increased, and male silkworm varieties with increased silk yield can be obtained. Therefore, BmEcKL1 knockout can be used to create high-yield mutants and applied to the breeding of new high-yield silkworm varieties. The existing technology mostly focuses on the study of silk properties when applying CRISPR / Cas9 gene editing technology.

[0006] Sericin is an important component of silkworm cocoons, accounting for about 25%, and plays a role in gluing and protecting silk fibroin. The silkworm sericin 1 gene (BmSer1) is one of the key genes encoding sericin protein. The gene size is about 23.6kb, located on chromosome 11 of the silkworm, and exists in a single copy in the genome. It contains 9 exons, which are expressed in the posterior and middle regions of the silk gland in the middle of the silkworm. It has 5 selective splicing patterns and encodes a variety of proteins with molecular weights of about 70 to 330kDa, which are located in the inner layer of the sericin layer that wraps the cocoon silk fiber. Regulating the expression level of sericin genes can change the proportion of sericin protein in the cocoon silk, and thus change the diameter of the silk fiber after degumming. Summary of the invention

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

[0008] One of the purposes of the present invention is to provide a gRNA targeting the silkworm BmSer1 gene.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

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

[0011] A second object of the present invention is to provide a transgenic expression vector constructed by the aforementioned gRNA.

[0012] To achieve the above object, the present invention adopts the following technical solutions:

[0013] The transgenic expression vector constructed by the aforementioned gRNA.

[0014] The preparation method of the transgenic expression vector is as follows: sticky ends are added on both sides of the gRNA, the gRNA backbone vector is cut with BbsI, and then connected 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] A third object of the present invention is to provide a CRISPR / Cas9 gene editing system for knocking out the silkworm BmSer1 gene.

[0016] To achieve the above object, the present invention adopts the following technical solutions:

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

[0018] A fourth object of the present invention is to provide a method for preparing a BmSer1 gene knockout heterozygous mutant silkworm strain.

[0019] To achieve the above object, the present invention adopts the following technical solutions:

[0020] The method for preparing a heterozygous mutant silkworm strain with BmSer1 gene knockout utilizes the aforementioned CRISPR / Cas9 gene editing system to knock out the BmSer1 gene in silkworms, screens binary transgenic individuals, and obtains a heterozygous mutant silkworm strain.

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

[0022] (1) constructing a gRNA expression vector targeting the silkworm BmSer1 gene and preparing gRNA transgenic silkworms;

[0023] (2) Crossbreeding gRNA transgenic silkworms with Cas9 transgenic silkworms to screen for dual transgenic individuals whose eyes and bodies emit green fluorescence simultaneously;

[0024] (3) Performing typing detection on the binary transgenic individuals obtained in step (2) to screen out heterozygous mutant silkworm strains with BmSer1 gene knockout.

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

[0026] A fifth object of the present invention is to provide a method for preparing coarse fiber silk.

[0027] To achieve the above object, the present invention adopts the following technical solutions:

[0028] The method for preparing coarse fiber silk comprises the following steps: preparing a BmSer1 gene knockout heterozygous mutant silkworm strain by adopting the above method, and obtaining coarse silk by breeding transgenic silkworms.

[0029] The sixth object of the present invention is to provide an application of the aforementioned gRNA, the aforementioned transgenic expression vector and / or the aforementioned gene editing system in cultivating coarse silk transgenic silkworm strains and / or preparing coarse silk.

[0030] The seventh object of the present invention is to provide an application of knocking out the silkworm BmSer1 gene in cultivating coarse silk transgenic silkworm strains and / or preparing coarse silk.

[0031] To achieve the above object, the present invention adopts the following technical solutions:

[0032] Application of knocking out silkworm BmSer1 gene in breeding coarse silk transgenic silkworm strains and / or preparing coarse silk

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

[0034] Preferably, the BmSer1 gene in silkworm is knocked out by using the aforementioned gene editing system to obtain a coarse silk transgenic silkworm strain; and coarse silk is obtained by breeding transgenic silkworms.

[0035] The beneficial effects of the present invention are:

[0036] (1) The present invention uses gene editing technology to change the genetic traits of silkworms, so that silkworms that produce coarse silk with stable inheritance can be obtained by raising silkworms. In subsequent production and development, only transgenic silkworms need to be raised to obtain stable yields of coarse silk. The coarse silk obtained by the present invention has a lower sericin rate and a thicker diameter: the sericin rate of the heterozygous mutant is about 13.83%, which is about 11.37 percentage points lower than that of the control group; the diameter of the degummed silk of the heterozygous mutant is about 15.24 μm, which is 3.91 μm thicker than that of the control group. The present invention provides a feasible method for thickening the diameter of silk, and the method has broad application prospects in the fields of waterproof materials, fishing gear manufacturing, industrial filtration materials and tissue repair.

[0037] (2) The coarse silk produced by the present invention not only maintains the excellent glossiness of silk, but also has advantages in abrasion resistance and tensile strength, thereby increasing durability. The coarse silk can be used to enhance the anti-immersion ability of fishing gear and fishing line, thereby extending the life of fishing gear; since the coarse silk has both excellent waterproofness and flexibility, it can be further used to manufacture more fitting raincoats or waterproof clothing. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a diagram showing the results of knockout target site sequencing and mutation form detection.

[0039] Figure 2 This is the result of analyzing the sericin content in heterozygous mutant silkworm cocoons.

[0040] Figure 3 This is the test result of silk fiber diameter after degumming. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to 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 silkworm BmSer1 gene

[0044] The base sequence of the silkworm BmSer1 gene (NCBI Gene ID: 101740082) was downloaded, and the gRNA target site was designed according to the base sequence of the first common exon and the principle of starting with G and ending with NGG, as shown in SEQ ID NO: 1.

[0045] 2. Vector Construction

[0046] Sticky ends (AAGT at the F end and AAAC at the R end) were added to both sides of the gRNA. After the gRNA backbone vector was digested with BbsI (see SanyuanMa.CRISPR / Cas9 mediated multiplex genome editing and heritablemutagenesis of BmKu70 in Bombyx mori.2014), it was connected and transformed with the annealing primer to construct the PiggyBac[3xP3-EGFP-SV40; U6-gRNA-SV40] transgenic vector (see Yuanyuan Liu.Tissue-specific genome editing oflaminA / C in the posterior silk glands ofBombyxmori.2017), wherein the annealing primer includes a gRNA (F) having a nucleotide sequence as shown in SEQ ID NO: 4 and a gRNA (R) having a nucleotide sequence as shown in SEQ ID NO: 5.

[0047] (1) Connecting gRNA fragment to transgenic vector

[0048] The annealed double-stranded gRNA fragment was connected to the PiggyBac [3xP3-EGFP-SV40; U6-gRNA-SV40] vector. The connection system included: 4 μL of gRNA annealed 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. The total system was 10 μL. The connection was carried out at 16°C for 1-1.5 hours.

[0049] (2) Conversion

[0050] 1) Take out the Trans1-T1 competent cells from the -80°C freezer and thaw them on ice;

[0051] 2) Add 10 μL of the ligation product to the competent cells dissolved to milky white in a clean bench and let stand on ice for 20-25 minutes;

[0052] 3) Place the sample in a 42°C metal bath for 90 seconds and place on ice for 2-3 minutes;

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

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

[0055] 6) Place the cells upright in a 37°C incubator for 3-10 minutes, then invert and incubate overnight in a 37°C incubator.

[0056] 7) In the clean bench, pick a single clone plaque and put it into 500 μL LB liquid culture medium with kanamycin resistance, culture it at 37°C, 220 rpm shaker, wait for the bacterial solution to become turbid, take 150 μL of the strain and send it to the company for sequencing, screen the positive clones and extract the plasmid for 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 ratio of 1:1 and injected into silkworm eggs by microinjection, which were recorded as G0 generation. The silkworm eggs were non-diapause eggs and injected within 2 hours of egg laying. The injected eggs were accelerated and raised until the moths emerged. The G0 generation was self-fertilized to produce the G1 generation. The G1 generation was screened for green fluorescence. The transgenic individuals with green fluorescence in the eyes were those that successfully expressed sgRNA.

[0059] 4. Obtaining heterozygous mutants

[0060] The obtained gRNA transgenic individuals and the obtained Cas9 transgenic individuals were hybridized 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. According to the presence of green fluorescence in the eyes and green fluorescence in the body, dual transgenic individuals with green fluorescence in the eyes and green fluorescence in the body were screened. The sequence at the site was typed with detection primers F and R, and the amplified product was sequenced and analyzed. 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. As Figure 1 As shown, if the amplified product shows overlapping peaks near the PAM motif in the sequencing peak graph, it indicates that the knockout has occurred successfully, and the heterozygous mutant silkworm strain with BmSer1 gene knockout has been successfully prepared.

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

[0062] 1. Cocoon degumming

[0063] The transgenic silkworms obtained in Example 1 were raised to obtain cocoons. The cocoons were boiled with 1% sodium bicarbonate solution and degummed for 1 hour. The cocoons before and after degumming were weighed and measured. The results are shown in Figure 2 As shown, the sericin rate of the control group was about 25.2%, and the sericin rate of the heterozygous mutant was about 13.83%, which was about 11.37 percentage points lower than that of the control group.

[0064] 2. Detection of silk diameter after degumming

[0065] The diameter of the silk after degumming was measured using a microscope. Figure 3 As shown, the diameter of the degummed silk of the control group was about 11.33 μm, and the diameter of the degummed silk of the heterozygous mutant was about 15.24 μm. Degummed silk with a thickened diameter of 3.91 μm was successfully obtained.

Claims

1. A gRNA targeting the silkworm BmSer1 gene, characterized in that: The target sequence of the gRNA is shown in SEQ ID NO:

1.

2. A transgenic expression vector constructed by the gRNA according to claim 1.

3. The transgenic expression vector according to claim 2, characterized in that: The preparation method of the transgenic expression vector is as follows: sticky ends are added on both sides of the gRNA, the gRNA backbone vector is cut with BbsI, and then connected 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.

4. A CRISPR / Cas9 gene editing system for knocking out the silkworm BmSer1 gene, characterized in that: The CRISPR / Cas9 gene editing system comprises the transgenic expression vector and the Cas9 expression vector according to any one of claims 2 to 3.

5. A method for preparing a BmSer1 gene knockout heterozygous mutant silkworm strain, characterized in that: The CRISPR / Cas9 gene editing system of claim 4 is used to knock out the BmSer1 gene in silkworms, screen binary transgenic individuals, and obtain heterozygous mutant silkworm strains.

6. The method according to claim 5, characterized in that The method comprises the following steps: (1) constructing a gRNA expression vector targeting the silkworm BmSer1 gene and preparing gRNA transgenic silkworms; (2) Crossbreeding gRNA transgenic silkworms with Cas9 transgenic silkworms to screen for dual transgenic individuals whose eyes and bodies emit green fluorescence simultaneously; (3) Performing typing detection on the binary transgenic individuals obtained in step (2) to screen out heterozygous mutant silkworm strains with BmSer1 gene knockout.

7. The method according to claim 6, characterized in that In step (3), the primers used for typing detection 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.

8. A method for preparing crude fiber silk, characterized in that: A heterozygous mutant silkworm strain with BmSer1 gene knockout is prepared by the method described in any one of claims 5 to 7, and coarse silk is obtained by breeding transgenic silkworms.

9. Use of the gRNA according to claim 1, the transgenic expression vector according to any one of claims 2 to 3, and / or the gene editing system according to claim 4 in cultivating coarse silk transgenic silkworm strains and / or preparing coarse silk.

10. Application of knocking out the silkworm BmSer1 gene in breeding coarse silk transgenic silkworm strains and / or preparing coarse silk.

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