GRNA of targeted bombyx mori silk glue 3 gene and preparation method and application of fine 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 3 gene, a preparation method of fine silk and application. The target sequence of the gRNA is as shown in SEQ ID NO: 1. The BmSer3 gene in the bombyx mori is knocked out by utilizing a CRISPR / Cas9 gene editing system, and the method specifically comprises the following steps: constructing a gRNA expression vector targeting the bombyx mori BmSer3 gene, and preparing a gRNA transgenic bombyx mori; hybridizing the gRNA transgenic bombyx mori and the Cas9 transgenic bombyx mori, and screening to obtain a binary transgenic individual of which the eyes and the body emit green fluorescence at the same time; and carrying out typing detection to obtain the BmSer3 gene knocked-out heterozygous mutant silkworm strain. Finally, the fine silk is obtained by feeding transgenic silkworms. Genetic characters of silkworms are changed by using a gene editing technology, and the obtained silk is thinner in diameter, higher in sericin rate and better in heat preservation and heat insulation performance.

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 3 gene, a fine silk preparation method and application. Background Art

[0002] Silk is a continuous long fiber solidified by the silk fluid secreted by mature silkworms when they make cocoons. It is also called natural silk. It is mainly composed of fibroin and sericin. Fibroin is the main body of the fiber, and sericin is coated on the outside of the fibroin, playing the role of gluing and protecting the fibroin. Silk is one of the earliest animal fibers used by humans. It is soft, slender, shiny, smooth and plump. As the birthplace of silk, China has a wide silk production area, including Jiangsu, Zhejiang, Sichuan, Guangdong and Shandong provinces. Today, China has become the world's largest silk producer, ranking among the top in the world in terms of scale and output.

[0003] The thermal conductivity and thermal stability of silk are related to the fiber diameter. As the diameter becomes thinner, the surface area of ​​the silk fiber increases, the gaps between the silk fibers decrease, and the number of pores increases, which improves its thermal insulation performance. Generally speaking, the diameter of silk is closely related to factors such as the cocoon structure, the silkworm species, the reeling process and the spinning environment. When the silk becomes thinner, it has better thermal insulation performance due to the increase in its surface area to volume ratio and the smaller gaps between them, which can open up more application areas. For example, 1) Thermal insulation clothing: fine silk can be used to make thermal insulation clothing, such as thermal insulation clothes, quilts, socks, etc. suitable for mass consumption. 2) Thermal insulation materials with skin-friendly properties for special purposes. 3) Silk micro-nano fiber aerogel: Silk micro-nano fiber aerogel has extremely low thermal conductivity and a stable three-dimensional network structure, which makes it have potential application value in the field of thermal insulation materials. This aerogel can be used for thermal management of building insulation, electronic equipment, etc.

[0004] Professor Tao Guangming of Huazhong University of Science and Technology led a research team to manufacture colorful smart fabrics using silk protein as raw material. Through freeze spinning, the researchers designed and regulated the fiber microstructure to prepare structural fibers with high internal porosity and low thermal conductivity, achieving excellent thermal insulation and warmth retention. The temperature-control phase change material was further integrated into the fiber and coated with a PDMS coating. The phase change material's ability to absorb, store and release heat was used to achieve a composite temperature-control fiber that is strong and wear-resistant, soft and waterproof, and has zero-energy intelligent temperature regulation. The microstructured fiber (MF) fabric prepared by advanced weaving technology has good bidirectional thermal insulation performance, and its thermal insulation performance increases with the increase in the number of fabric layers. The phase change material integrated inside the microstructured fiber can not only significantly slow down the rate of increase or decrease in ambient temperature, but also has good cycle stability. With the development of materials science and engineering technology, the application field of fine silk will continue to expand, bringing new growth points and innovation opportunities to the traditional silk industry.

[0005] At present, methods for changing the diameter of silk include genetic modification, chemical treatment, physical modification, feeding silkworms, etc. However, these methods all have certain defects. For example, physical modification may change the structure and mechanical properties of silk; chemical treatment will affect the glossiness and comfort of silk; feeding silkworms has an uncertain effect on the growth and development of silkworms, and it is not easy to control the diameter of silkworms. In contrast, gene editing technology provides a more convenient, precise, controllable and heritable method to modify the diameter of silkworms.

[0006] CRISPR / Cas9 gene editing technology is a revolutionary molecular biology tool that uses bacterial 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. In the prior art, patent CN110791528A discloses a method for increasing silk production and optimizing silkworm varieties through genome editing technology, including: sgRNA transgenic expression vectors of let-7 microRNA, screening of sgRNA expression strains, screening of let-7 knockout strains, identification of knockout forms, and statistical analysis of silk gland length, weight and cocoon weight after let-7 knockout. This invention uses CRISPR / Cas9 technology to delete the expression of let-7 non-coding RNA, relieve its negative regulatory effects on silk gland development and silk protein synthesis, and achieve an increase in silk production in silkworms.

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

[0008] In view of this, the present invention proposes a gRNA targeting the silkworm sericin 3 gene, a method for preparing fine silk and its application. The present invention uses gene editing technology to knock out the silkworm BmSer3 gene, change the silkworm genetic trait - silk diameter, and obtain a BmSer3 gene knockout heterozygous mutant silkworm strain; the transgenic silkworm can stably produce fine silk with a thinner diameter. The present invention provides a feasible method for changing the silk diameter, which has significant advantages and broad application prospects.

[0009] One of the purposes of the present invention is to provide a gRNA targeting the silkworm BmSer3 gene.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

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

[0012] A second object of the present invention is to provide a transgenic expression vector constructed by the aforementioned gRNA.

[0013] To achieve the above object, the present invention adopts the following technical solutions:

[0014] The transgenic expression vector constructed by the aforementioned gRNA.

[0015] Preferably, the preparation method of the transgenic expression vector is as follows: sticky ends are added to both sides of the gRNA, the gRNA backbone vector is cut with BbsI, and then connected and transformed with the annealed primer to obtain the PiggyBac [3xP3-EGFP-SV40; U6-gRNA-SV40] transgenic vector.

[0016] Preferably, the annealing primers include a forward primer having a nucleotide sequence as shown in SEQ ID NO:4 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:5.

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

[0018] A third object of the present invention is to provide a CRISPR / Cas9 gene editing system for knocking out the silkworm BmSer3 gene.

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

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

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

[0022] A fourth object of the present invention is to provide a method for preparing a BmSer3 gene knockout heterozygous mutant silkworm strain.

[0023] To achieve the above object, the present invention adopts the following technical solutions:

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

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

[0026] (1) constructing a gRNA expression vector targeting the silkworm BmSer3 gene and preparing gRNA transgenic silkworms;

[0027] (2) Crossbreeding gRNA transgenic silkworms with Cas9 transgenic silkworms to screen for dual transgenic individuals whose eyes and bodies emit green fluorescence simultaneously;

[0028] (3) Performing typing detection on the binary transgenic individuals obtained in step (2) to screen out heterozygous mutant silkworm strains with BmSer3 gene knockout.

[0029] 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.

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

[0031] 1) Add sticky ends to both sides of the gRNA, AAGT at the F end and AAAC at the R end; after using BbsI to digest the gRNA backbone vector, connect and transform it with the annealing primer to obtain the PiggyBac [3xP3-EGFP-SV40; U6-gRNA-SV40] transgenic vector; the annealing primer includes 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;

[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 the G0 generation; the injected eggs were accelerated and raised until the moths emerged, and the G0 generation was self-pollinated to produce the G1 generation. The G1 generation was screened for green fluorescence, and the transgenic individuals with green fluorescence in the eyes were those that successfully expressed the gRNA.

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

[0034] A fifth object of the present invention is to provide a method for preparing fine fiber silk.

[0035] To achieve the above object, the present invention adopts the following technical solutions:

[0036] The method for preparing fine fiber silk comprises the following steps: preparing a BmSer3 gene knockout heterozygous mutant silkworm strain by adopting the above method, and obtaining fine silk by breeding transgenic silkworms.

[0037] 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 preparing fine fiber silk or cultivating fine silk transgenic silkworm strains.

[0038] The seventh object of the present invention is to provide an application of knocking out the silkworm BmSer3 gene in preparing fine fiber silk or cultivating fine silk transgenic silkworm strains.

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

[0040] Preferably, the BmSer3 gene in silkworm is knocked out using the aforementioned gene editing system to obtain a fine silk transgenic silkworm strain; and fine silk is obtained by raising transgenic silkworms.

[0041] The beneficial effects of the present invention are:

[0042] (1) The present invention uses gene editing technology to change the genetic traits (silk diameter) of silkworms, and obtains silkworms that can produce fine silk with stable inheritance. By raising transgenic silkworms, a stable yield of fine silk can be obtained. The fine silk obtained by the present invention has a higher sericin rate and a thinner diameter: the sericin rate of the heterozygous mutant is about 38.23%, which is about 12.03 percentage points higher than that of the control group; the diameter of the degummed silk of the heterozygous mutant is about 7.8 μm, which is 3.67 μm thinner than that of the control group. The present invention provides a feasible method for changing the diameter of silk, which has significant advantages and broad application prospects.

[0043] (2) The gene editing technology used in the present invention changes the genetic traits of silkworms and does not involve the use of harmful chemicals. Compared with traditional chemical treatment methods, it is more environmentally friendly.

[0044] (3) The fine silk produced by the present invention not only maintains the excellent glossiness of silk, but also has a larger surface area and smaller gaps between them, and has better thermal insulation performance. It can be further used to make thermal insulation clothing, silk micro-nano fiber aerogel, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a diagram showing the results of knockout target site sequencing and mutation form detection.

[0046] Figure 2Statistical graph of the percentage of sericin content in heterozygous mutant cocoons.

[0047] Figure 3 This is a statistical chart of the diameter of silk fibers after degumming. DETAILED DESCRIPTION

[0048] 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.

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

[0050] 1. Design of gRNA target site for silkworm BmSer3 gene

[0051] The base sequence of the silkworm BmSer3 gene (NCBI Gene ID: 100136948) was downloaded, and the gRNA target site was designed according to the base sequence of the third exon and the principle of starting with G and ending with NGG, as shown in SEQ ID NO: 1.

[0052] 2. Vector Construction

[0053] Sticky ends (AAGT at the F end and AAAC at the R end) were added to both sides of the gRNA. The gRNA backbone vector was digested with BbsI (see SanyuanMa. CRISPR / Cas9 mediated multiplex genome editing and heritablemutagenesis of BmKu70 in Bombyx mori. 2014), and then connected and transformed with annealed primers 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), wherein the primers include 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.

[0054] (1) Connecting gRNA fragment to transgenic vector

[0055] 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, 3.9 μL of pure water, and a total system of 10 μL. The connection was carried out at 16°C for 1-1.5 hours.

[0056] (2) Conversion

[0057] 1) Take out the Trans1-T1 competent cells from the -80°C freezer and thaw them on ice;

[0058] 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;

[0059] 3) Place the sample in a 42°C metal bath for 90 seconds and place on ice for 2-3 minutes;

[0060] 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;

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

[0062] 6) Place the cells upright in a 37°C incubator for 3-10 minutes, then invert and incubate overnight in a 37°C incubator.

[0063] 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.

[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 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 gRNA.

[0066] 4. Obtaining heterozygous mutants

[0067] The obtained gRNA transgenic individuals and the obtained Cas9 transgenic individuals were hybridized to obtain the offspring F1. The process of preparing 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, binary transgenic individuals with green fluorescence in the eyes and green fluorescence in the body were screened. The sequences at the sites were typed using detection primers F and R, and the amplified products were 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. If the amplified product shows an overlapping peak near the PAM motif in the sequencing peak graph, it indicates that the knockout has occurred successfully, and the heterozygous mutant silkworm strain with BmSer3 gene knockout has been successfully prepared, as shown in FIG. Figure 1 shown.

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

[0069] 1. Cocoon degumming

[0070] 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 26.2%, and the sericin rate of the heterozygous mutant was about 38.23%, which was about 12.03 percentage points higher than that of the control group.

[0071] 2. Detection of silk diameter after degumming

[0072] The diameter of the degummed silk was measured using a microscope. Figure 3 As shown, the diameter of the degummed silk of the control group was about 11.46 μm, the diameter of the degummed silk of the heterozygous mutant was about 7.8 μm, and the degummed silk with a thinner diameter of 3.67 μm was successfully obtained.

Claims

1. A gRNA targeting the silkworm BmSer3 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 BmSer3 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 BmSer3 gene knockout heterozygous mutant silkworm strain, characterized in that: The CRISPR / Cas9 gene editing system described in claim 4 is used to knock out the BmSer3 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 BmSer3 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 BmSer3 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 fine fiber silk, characterized in that: A BmSer3 gene knockout heterozygous mutant silkworm strain is prepared by the method described in any one of claims 5 to 7, and fine 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 preparing fine fiber silk or cultivating fine silk transgenic silkworm strains.

10. Application of knocking out the silkworm BmSer3 gene in preparing fine-fiber silk or cultivating fine-silk transgenic silkworm strains.

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