Super-strong and super-tough colored non-fading pollution-free colored imitated spider silk and preparation method thereof

Abstract

The invention discloses super-strong and super-tough colored non-fading pollution-free colored imitated spider silk and a preparation method thereof. According to the method, a super-strong and super-tough spider-silk-imitated silkworm variety is used as a basal disc, a polymer formed by connecting one or more fluorescent protein genes is used as an exogenous gene, and the exogenous gene and a spider-silk-imitated silkworm endogenous silk protein gene form a fusion protein gene in a homologous recombination manner by adopting a gene editing technology; the novel cobweb-imitated silkworm variety is obtained through multi-generation breeding, and cobweb-imitated silkworms which are excellent in mechanical performance, free of dyeing, colorful under visible light, stable and uniform in color, free of fading during silk reeling refining and high-temperature exposure and free of pollution are produced. According to the invention, the serious problems of environmental pollution, uneven coloring and fading for a long time caused by printing and dyeing of the super-strong and super-tough imitated spider silk are solved, the quality of the imitated spider silk is greatly improved, the imitated spider silk becomes a novel super-strong and super-tough material comprehensively matched with the properties of pure nature, no pollution, reproducibility, degradability and the like, and the production of the imitated spider silk completely conforms to the concept of green, environment-friendly and sustainable development.

Description

Technical Field

[0001] The present invention relates to a method for making colored silk, in particular to a super-strong and super-tough, self-colored, non-fading, and pollution-free colored spider silk imitation and its preparation method. Background Art

[0002] Mulberry silk (domestic silk) is a natural protein fiber, composed of two proteins, fibroin and sericin, secreted by the silk gland organs of domestic silkworm larvae. Fibroin is synthesized and secreted by the posterior silk gland, accounting for 70-80% of the silk, which is the core part of the silk. It is composed of fibroin heavy chain protein (FibH), fibroin light chain protein (FibL chain), and fibroin P25 glycoprotein (P25) in a molar ratio of 6:6:1. Sericin is synthesized and secreted by the middle silk gland, accounting for 20-30% of the silk. The main component is sericin 1 (Ser1), which wraps around the outer layer of fibroin and plays an adhesive role. It is easily soluble in hot water and is mostly dissolved and removed during the reeling process.

[0003] Mulberry silk can be continuously regenerated by raising silkworms and can be naturally degraded. The degradation products are polypeptides and amino acids, which are very environmentally friendly materials. However, most of the current domestic silkworm varieties for production are white cocoon silk varieties, and special processes are required for dyeing in the later processing, which causes environmental pollution. Moreover, the color fastness of mulberry silk is poor and it is easy to fade.

[0004] In order to eliminate the environmental pollution caused by the dyeing and processing of mulberry silk, currently, the following three technologies and methods are mainly tried in production to solve the problem.

[0005] I. Preparation of colored silk by the method of feeding additives

[0006] During the large feeding period of the fifth instar of domestic silkworms, copper sodium chlorophyll, nano-pigments, etc. are added to mulberry leaves or feed to form colored silk. However, in this method, the pigments are mainly attached to the sericin layer, and very little color remains after reeling. Moreover, high-concentration dyes damage the normal growth and development and health of domestic silkworms, increasing the mortality rate of silkworms. More seriously, a large amount of pigments are mixed in the remaining mulberry leaves or feed eaten by silkworms, causing greater environmental pollution.

[0007] II. Preparation of colored silk by natural colored silk silkworm varieties

[0008] In the early days, the silk of domestic silkworm varieties was originally colorful. Pigment substances such as carotenoids and flavonoids in mulberry leaves are absorbed by the digestive system of silkworms, penetrate into the blood through the digestive tract, are further transported to the silk gland, and finally participate in the synthesis, secretion, and deposition of silk in the silk protein to form colored silk.

[0009] Currently, through the improvement of wild-type colored cocoon varieties, cocoon color varieties such as golden cocoons, green cocoons, brown cocoons, and red cocoons have been successfully cultivated. However, the pigments of natural colored silk silkworm varieties are mainly deposited in the sericin layer and will be lost during the reeling process. Moreover, the colors of colored silk in different batches are inconsistent, and the product quality is unstable.

[0010] III. Preparation of Colored Silk by Transgenic Technology

[0011] The preparation of colored silk by transgenic technology includes three technical routes.

[0012] The first is to use the transgenic technology mediated by piggyBac transposon to randomly insert fluorescent protein genes into the silkworm genome to obtain colored silk, such as green fluorescent cocoons, blue fluorescent cocoons, red fluorescent cocoons, etc.

[0013] The second is to use CRISPR gene editing technology to change the gene functions of the silkworm itself to accumulate and transport pigments, so as to increase the content of natural pigments in the silkworm and thus form colored silk, such as dark brown cocoons. This method is similar to the mechanism of forming colored silk in natural colored cocoon varieties.

[0014] The third is to introduce exogenous genes encoding pigment synthase into silkworm cells, so that the silkworm can produce pigments that it could not originally synthesize to form colored silk, such as pink cocoons. This method is similar to the mechanism of preparing colored silk by the pigment feeding method.

[0015] For the colored silk prepared by the above three transgenic technologies, most of the pigments are deposited on the sericin layer, or cannot form a constant molar ratio with the endogenous silk protein. Their common disadvantage is that the pigments will be lost during the reeling process and the color is unstable. Moreover, the purpose of some technologies is to use transgenic technology to make the silk gland of the silkworm produce fluorescent protein, requiring the fluorescent protein to be easily dissolved and extracted. In this process, colored cocoons will be produced, and a large amount of fluorescent protein can be extracted after the cocoons are hydrolyzed. In addition, the phenomenon of gene silencing in transgenic technology will lead to unstable gene expression in offspring and color deviation in silk of different batches.

[0016] Spider silk is the protein silk fiber with the most excellent mechanical properties in nature. Its strength is 5 times that of steel, known as "biosteel", its toughness is 3 times that of Kevlar, and its elongation is 15 times that of Kevlar. Spiders have 7 silk-producing organs such as large sac-shaped glands, small sac-shaped glands, and flagelliform glands, and can spin 7 different functional spider silks.

[0017] Because spiders have the characteristics of aggression and cannibalism, and the individual silk-spinning amount is small, it is impossible to obtain a large amount of spider silk by breeding methods. By using transgenic technology to introduce spider silk genes into silkworms, transgenic silkworms can be used to mass-produce spider silk-like silk and create new materials that are super strong, super tough, renewable, and degradable.

[0018] However, when used as a new textile material, imitation spider silk also has the same problems as domestic silk, such as environmental pollution caused by post-printing and dyeing processing, poor silk fastness, and easy fading. Using the three technologies and methods of domestic silkworms introduced above cannot solve the problem either. Summary of the Invention

[0019] In order to solve the problems existing in the printing and dyeing of imitation spider silk mentioned in the background technology, the purpose of the present invention is to develop a method for preparing super-strong, super-tough, and colored imitation spider silk with a super-strong and super-tough imitation spider silk silkworm variety as the base, using gene editing technology to construct a fusion protein of exogenous fluorescent protein and endogenous silk protein, which does not require dyeing, has its own color under visible light, and will not fade during reeling and refining, at 100°C high temperature, or under sunlight exposure, thus enhancing the imitation spider silk to become a new material that fully matches the properties of natural, pollution-free, renewable, and biodegradable materials.

[0020] The solution of the present invention is to use a super-strong and super-tough imitation spider silk silkworm variety as the base, and use a single fluorescent protein gene or a polymer fluorescent protein gene connected by several fluorescent protein genes as the exogenous gene. Using gene editing technology, the fluorescent protein gene is integrated into the endogenous silk protein gene of the silkworm through homologous recombination to form a fusion gene of silk protein and fluorescent protein, and a fusion protein of fluorescent protein + silk protein is obtained. After multiple generations of breeding, a new silkworm variety that can stably inherit and express the fusion gene is finally obtained. The imitation spider silk of this variety not only has excellent mechanical properties, but also does not require dyeing, and the color of the imitation spider silk can be seen with the naked eye. Also, because the fluorescent protein and the endogenous silk protein become a fusion protein, they participate in the formation of silk with a constant molar ratio, firmly exist in the entire imitation spider silk, are evenly distributed, and will not fall off during reeling and refining operations or high-temperature exposure to sunlight. Therefore, the color of its imitation spider silk is uniform and stable, and will never fade.

[0021] To achieve the above purpose, the steps of the specific technical solution adopted by the present invention are as follows:

[0022] The method is that a super-strong and super-tough imitation spider silk silkworm variety can be used as the base, and a single fluorescent protein gene or a polymer composed of several fluorescent protein genes is used as the exogenous gene. The exogenous gene is introduced into the imitation spider silk silkworm variety through homologous recombination and forms a fusion protein gene with the endogenous silk protein gene of the imitation spider silk silkworm variety. Then, through multiple generations of breeding of the silkworm, a silkworm variety that can stably inherit and express the fusion protein gene is finally obtained. Using the silkworm variety to produce imitation spider silk, the imitation spider silk is super-strong and super-tough, does not require dyeing, has its own color under visible light, the color is stable and uniform, and will not fade during reeling and refining, at 100 0 high temperature or under sunlight exposure.

[0023] The silk-spider-mimicking silkworm variety mentioned above is a transgenic silkworm variety with spider silk genes already bred, or a transgenic silkworm variety with spider silk genes bred by simultaneously introducing spider silk genes using transgenic technology when introducing fluorescent protein genes into silkworms.

[0024] The super-strong and super-tough silk-spider-mimicking silkworm variety mentioned above is bred by using the transgenic technology mediated by piggyBac transposons or by using homologous recombination technology.

[0025] The method for breeding silkworm varieties by homologous recombination is to use the homologous recombination technology of TALEN or CRISPR gene editing to construct a fusion protein gene of a fluorescent foreign gene and a silk protein gene endogenous to silkworms, and then breed a new type of silkworm variety that stably inherits, synthesizes, and secretes the fusion protein. The silk-spider-mimicking silk with the fusion protein is synthesized and secreted by the new type of silkworm variety.

[0026] The spider silk genes mentioned above refer to the genes corresponding to the spider silk proteins secreted by the major ampullate gland, minor ampullate gland, flagelliform gland, aggregate gland, pyriform gland, alveolate gland, and tubular gland of spiders.

[0027] The foreign gene is a fluorescent protein gene for introducing silk components with self-color or a multimeric fluorescent protein gene composed of 2 - 8 types of the same or different fluorescent protein genes connected. The fluorescent protein gene is one of the genes of green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), orange fluorescent protein (OFP) or its mutant.

[0028] The silk protein genes endogenous to silkworms mentioned above are the fibroin heavy chain gene (FibH), fibroin light chain gene (FibL), fibroin P25 gene (P25), or sericin 1 gene (Ser1) of silkworms.

[0029] The method is to construct a homologous recombination vector of the foreign gene through molecular biology techniques, introduce the homologous recombination vector into silkworm eggs by microinjection, integrate the homologous recombination into specific endogenous silk protein genes of silkworms, screen for G1-generation individuals positive for the marker gene through a fluorescence microscope or the naked eye, cross the G1-generation positive individuals with wild types or self-cross the positive individuals to obtain the G2-generation, start screening all positive individuals and self-crossing them to save seeds from the G3-generation, conduct multi-generation screening and breeding of silkworms, and breed a variety with stable inheritance and expression by the G8-generation. The composite silk synthesized and secreted by it is silk-spider-mimicking silk, and the silk-spider-mimicking silk has the properties of being super-strong, super-tough, self-colored, and non-fading.

[0030] The exogenous gene vector described above is a homologous recombination exogenous gene vector applied to TALEN or CRISPR gene editing. The vector includes an endogenous gene homologous arm + exogenous gene + endogenous gene homologous arm, or an endogenous gene homologous arm + exogenous gene + fluorescent screening marker gene expression cassette + endogenous gene homologous arm.

[0031] The fluorescent screening marker gene expression cassette includes a promoter of the expression cassette and a fluorescent screening marker gene. The promoter of the expression cassette is an IE-1, A3 or 3xP3 promoter, and the fluorescent screening marker gene uses a green fluorescent protein gene (EGFP) or a red fluorescent protein gene (DsRed).

[0032] The silkworm described above secretes spider silk-like silk, and the spider silk-like silk contains components with inherent colors. The components with inherent colors include a fluorescent silk fusion protein expressed by a fusion gene formed by fusing a fluorescent protein gene of one of green fluorescent protein, red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, cyan fluorescent protein or orange fluorescent protein and its mutants, or a multimeric fluorescent protein gene composed of 2-8 fluorescent proteins of the same or different species and an endogenous silk protein gene, or a complex of a fluorescent silk fusion protein and other non-fluorescent silk proteins.

[0033] The fluorescent silk fusion protein or complex exists firmly in the whole spider silk-like silk at a constant molar ratio, is evenly distributed, does not fall off during the operations of reeling and refining or high-temperature exposure to the sun, and the spider silk-like silk has an inherent color under visible light without dyeing, without causing environmental pollution.

[0034] The method of introducing the homologous recombination vector into silkworm eggs by microinjection described above is to introduce a single homologous recombination vector formed by fusing an exogenous fluorescent protein gene and an endogenous silk protein gene, or to introduce multiple homologous recombination vectors formed by fusing multiple exogenous fluorescent protein genes and multiple endogenous silk protein genes simultaneously.

[0035] The spider silk-like silk of the silkworm bred by introducing multiple fusion protein gene homologous recombination vectors by microinjection shows a composite color under the synergistic action of multiple fluorescent proteins.

[0036] The obtained silkworm varieties include pure breeds and hybrid varieties between different color pure breeds. The spider silk-like silk of the hybrid variety shows a composite color under the synergistic action of the fluorescent proteins of the two pure breeds.

[0037] The present invention creatively constructs a fusion gene by combining a fluorescent protein gene with an endogenous silk protein gene, which not only enables the fused fluorescent protein to participate in the construction of silk protein at a constant molar ratio and be evenly distributed in the entire silk to maintain a stable content, but also is permanently connected to the silk protein and will not separate. Even during the reeling and refining operation, the fluorescent protein fused with the silk protein will not fall off; and it is synthesized, secreted, spun, and cocooned together with the super-strong and super-tough spider silk protein to form super-strong and super-tough imitation spider silk.

[0038] The present invention has the following beneficial effects:

[0039] The present invention solves the serious problems of environmental pollution, uneven coloring and fading over time caused by printing and dyeing in the production of imitation spider silk, greatly improves the quality of imitation spider silk, and becomes a super-strong and super-tough new material that fully matches the properties of pure natural, pollution-free, renewable and degradable. It makes the production of imitation spider silk fully comply with the concept of green environmental protection and sustainable development, creates a new situation for the production of imitation spider silk, and has great economic benefits and broad application prospects.

[0040] The present invention is a method for artificially designing spider silk protein and improving the performance of spider silk, and develops the color development function of exogenous fluorescent protein, so that the spider silk will show color under visible light without dyeing, and the color is uniform and stable, and will not fade even under high temperature exposure, eliminating environmental pollution caused by printing and dyeing; and can also produce colorful and gorgeous spider silk with the help of multiple types of fusion fluorescent proteins and different types of fusion fluorescent proteins in different ratios. This invention can greatly improve the quality of spider silk and create good economic benefits. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with the embodiments.

[0042] Embodiments of the present invention are as follows:

[0043] Example 1

[0044] The exogenous gene yellow fluorescent protein (YFP) gene sequence optimized according to the silkworm codon preference was biosynthesized by genetic engineering methods, the red fluorescent protein gene (DsRed) was used as the marker gene, the IE-1 promoter was used as the marker gene expression frame promoter, and the spider macrocystis gland silk MaSp1 gene silk fibroin light chain (FibL) was used as the endogenous targeting gene to construct a homologous recombination vector containing the silk fibroin light chain gene homology arm sequence + exogenous gene yellow fluorescent protein (YFP) gene + gene end sequence + IE-1 promoter + red fluorescent protein marker gene (DsRed) + polyA + silk fibroin light chain gene homology arm sequence for TALEN gene editing technology.

[0045] The design of TALEN targeting the FibL locus and the detection method for the expression of the foreign gene yellow fluorescent protein (YFP) are as follows:

[0046] 1. Design of TALEN targeting the FibL locus

[0047] The foreign gene yellow fluorescent protein (YFP) is site-specifically integrated at the rear of FibL, and the TALEN cleavage site is selected in the downstream region of the FibL terminator. First, genomic DNA is extracted from the transgenic silkworm variety that has been bred to carry the spider major ampullate silk MaSp1 gene, with the strength and toughness increased by 2.7 and 3.3 times respectively compared to silk. According to the silkworm genomic sequence in NCBI, primers for amplifying the FibL genome are designed. Using the above-extracted silkworm genomic DNA as a template, DNA fragments in the upstream and downstream regions of the FibL terminator are amplified. After amplification, an appropriate amount of the product is taken for agarose gel electrophoresis verification. Samples with the expected band size are directly sent for sequencing to obtain the genomic DNA sequence of this region. Then, the design of the TALEN targeting site is carried out, and the 2 target sites with the highest scores are selected for the construction of the subsequent targeting vector.

[0048] 2. Construction of the TALEN targeting vector

[0049] The TALEN targeting system includes the left-binding and right-binding plasmids. The construction methods of the two binding plasmids are the same. The following plasmid construction introduced is for the unilateral-binding TALEN plasmid, and the construction of the other binding plasmid is exactly the same as this step. The Golden Gate TALEN and TAL Effector Kit 2.0 is used for the construction of the TALEN plasmid. The specific operation steps are as follows:

[0050] (1) According to the designed targeting sequence, the modular plasmids corresponding to the first 10 bases and the backbone vector pFUS_A are sequentially selected according to the recognition principle of RVD and bases; then, according to the base recognition principle, the modular plasmids corresponding to the 11th to N - 1 (N is the length of the left or right binding sequence) bases and the backbone vector pFUS_B#N - 1 are sequentially selected.

[0051] (2) The following two reaction systems are respectively prepared in the ultra-clean workbench:

[0052] System 1:

[0053]

[0054] System 2:

[0055]

[0056] (3) Gently mix the above system and set the reaction program in a PCR instrument (37 °C, 5 min; 16 °C, 10 min) for 10 cycles; 50 °C, 5 min; 80 °C, 5 min.

[0057] (4) Plasmid - safe nuclease treatment:

[0058] 20 μL of the above reaction system

[0059] 1 μL of 10 mM ATP

[0060] 1 μL of plasmid safe nuclease

[0061] Gently mix the above reaction system, and then incubate in a 37 °C constant temperature water bath for 1 h. This step is used to remove the unligated DNA fragments in the reaction of step (3);

[0062] (5) Take 5 μL of the above reaction solution and transform it in DH5α competent cells for culture;

[0063] (6) Verify by colony PCR and select positive transformants;

[0064] (7) Culture the correct clones, and use a plasmid extraction kit to extract the pFUS_A plasmid (pA) after ligating the module fragment and the pFUS_B#N - 1 plasmid (pB) after ligating the module fragment respectively;

[0065] (8) Select the module plasmid corresponding to the last base (the Nth base) according to the base recognition principle, and ligate it with the two ligated fragments above. Prepare the following reaction system:

[0066]

[0067] (9) Set the reaction program in a PCR instrument: (37 °C, 5 min; 16 °C, 10 min) for 10 cycles; 37 °C, 15 min; 80 °C, 5 min.

[0068] (10) Take 5 μL of the above reaction solution and transform it in DH5α competent cells for culture;

[0069] (11) Verify by colony PCR and select positive transformants;

[0070] (12) Culture the correct clones, extract the plasmid and verify by sequencing to obtain the TALEN plasmid.

[0071] 3. Preparation of TALEN mRNA by in vitro transcription

[0072] Perform single digestion of the constructed TALEN plasmid with XbaⅠ and purify the digestion product by phenol - chloroform. Subsequently, using the linearized TALEN as a template, use mMESSAGE mMACHINETM Use the T7 Transcription Kit to prepare mRNA by in vitro transcription.

[0073] 4. Detection of the activity of the targeting site

[0074] Microinject the above-mentioned TALEN mRNA into the silkworm eggs within 8 h after oviposition of the transgenic silkworm variety expressing spider major ampullate silk MaSp1. The injection volume for each silkworm egg is about 5 nL. The injection system is prepared as follows:

[0075] Final concentration of TALEN-FL mRNA: 250 ng / μL

[0076] Final concentration of TALEN-FR mRNA: 250 ng / μL

[0077] The injected silkworm eggs are incubated in a constant temperature incubator at 25 °C and a relative humidity of 90%.

[0078] Extract the genome of the above-injected silkworm eggs. Using the genome as a template, amplify the DNA sequence near the targeting site, directly sequence the amplification product for verification, and judge the cleavage activity of the constructed TALEN targeting plasmid according to the sequencing peak map.

[0079] 5. Construction of transgenic lines and identification of positive individuals

[0080] Respectively perform standard phenol-chloroform purification on the above-obtained exogenous gene homologous recombination plasmid and the TALEN plasmid with high cleavage activity. Dissolve the plasmid DNA with 1×PBS solution and measure the concentration. Subsequently, prepare the injection system:

[0081] Final concentration of TALEN-FL mRNA: 250 ng / μL

[0082] Final concentration of TALEN-FR mRNA: 250 ng / μL

[0083] Final concentration of exogenous gene homologous recombination plasmid: 300 ng / μL

[0084] The above reagents were microinjected into the silkworm eggs 4 hours after oviposition of transgenic silkworms with the MaSp1 gene of the major ampullate gland silk of spiders. After injection, they were incubated in a constant temperature incubator at 25°C and 90% relative humidity for about 10 days. The larvae were fed with fresh mulberry leaves until they spun cocoons. The G0 generation adults were crossed with transgenic silkworms with the MaSp1 gene of the major ampullate gland silk of spiders to produce seeds. Positive individuals expressing the red fluorescent protein marker gene (DsRed) were selected from the first instar larvae of the G1 generation under a fluorescence microscope. The G1 generation positive larvae were fed with fresh mulberry leaves until they spun cocoons. The G1 generation positive adults were crossed with transgenic silkworms with the MaSp1 gene of the major ampullate gland silk of spiders to produce seeds. Genomic DNA was extracted from the adults after seed production, and genotype amplification verification was carried out using the genomic DNA as a template to detect the expected position of the exogenous gene homology in the silkworm genome.

[0085] 6. Detection of gene transcription level

[0086] When the G2 generation larvae verified to be precisely homologous were reared to the 3rd day of the 5th instar, the silk gland tissues were dissected, total RNA was extracted, and cDNA was synthesized using a reverse transcription kit. Real-time fluorescence quantitative analysis was carried out using the cDNA sample as a template to detect the transcription level of the gene. The silkworm GAPDH gene was selected as the internal reference gene, and 3 independent biological replicates were set for each sample.

[0087] 7. Detection of protein expression level

[0088] The silk gland tissues or cocoon shells were cooled with liquid nitrogen and then ground into fine powder in a tissue grinder. Then, the silk gland and cocoon shell proteins were extracted with a protein extraction buffer (1:200, mass / volume), and protein immunoblotting (Western blot) analysis was used to detect the specificity and size of the target protein.

[0089] The above experimental results proved that using the G2 generation as the material, it was verified by PCR experiment that the exogenous gene had been successfully introduced into the silkworm genome. Fluorescence quantitative PCR detection showed that the expression of the yellow fluorescent protein (YFP) gene in the silkworm variety was significant. Western blot method proved that the composite silk of silkworms contained bands of the fusion protein with the expected size. From the G3 generation, all positive individuals were screened and self-crossed to save seeds, and a stable genetic variety was bred by the G8 generation. The traits such as the whole cocoon weight and cocoon layer weight of the G8 generation of homologous recombinant silkworms were the same as those of the transgenic silkworm variety with the MaSp1 gene of the major ampullate gland silk of spiders, and there were no significant biological differences. The cocoons were visibly yellow and the color was uniform. The cocoons were spider silk-like containing spider silk + yellow fluorescent protein (YFP) and fibroin light chain protein fusion protein, and were reeled using the following method:

[0090] 1. Put the yellow positive cocoons into boiling water and boil for 6 minutes, press up and down to expel the air in the cocoon shell;

[0091] 2. Then, quickly transfer the silkworm cocoons into a low-temperature water bath at 60°C for 3 minutes, and press up and down to ensure that boiling water fully enters the cocoon shells; then quickly put the silkworm cocoons into boiling water at 100°C for 3 minutes, and press up and down to ensure that boiling water fully enters the cocoon shells, soak the cocoons, and make the sericin swell and soften;

[0092] 3. Then, gradually cool the temperature of the water bath in the air to 80°C, about 15 minutes or so, to further soften the sericin and achieve the purpose of proper cocoon cooking for the inner and outer cocoon layers; after the cooking process is completed, put it into a beaker containing a water bath at about 65°C and extract the silk.

[0093] 4. Further put the silk into boiling water at 100°C for 30 minutes, change the water 3 times, and remove the sericin as much as possible.

[0094] After cocoon cooking, reeling, and refining, the strength and toughness of the artificial spider silk have no significant difference from those of the transgenic silkworm silk with the MaSp1 gene of the major ampullate gland of the spider. Under visible light, the yellow color of the artificial spider silk remains stable without fading, and the whole artificial spider silk with a length of about 1000 m shows blue color, and the color distribution is uniform.

[0095] The artificial spider silk is further baked at 100°C for 12 hours, and the yellow color remains stable without fading.

[0096] Example 2

[0097] By means of genetic engineering, a dimer gene sequence of two blue fluorescent protein (BFP) genes optimized according to the codon preference of Bombyx mori is biosynthesized. The enhanced green fluorescent protein gene (EGFP) is used as a marker gene, and the A3 promoter is used as the promoter of the marker gene expression cassette. Using the fibroin light chain (FibL) of Bombyx mori as an endogenous target gene, a homologous recombination vector for CRISPR gene editing technology containing the homologous arm sequence of the fibroin light chain gene + dimer of two blue fluorescent protein (BFP) genes + gene ending sequence + A3 promoter + green fluorescent protein marker gene (EGFP) + polyA + homologous arm sequence of the fibroin light chain gene is constructed.

[0098] The design of the CRISPR targeting the FibL locus and the detection method for the expression of the exogenous gene dimer of the blue fluorescent protein (BFP) gene are as follows:

[0099] The Cas9 sequence is from Streptococcus pyogenes and has been optimized for human codons. The transcription of the Cas9 protein is initiated by the SP6 promoter.

[0100] After streaking the cryopreserved bacteria corresponding to the Cas9 plasmid on an Amp-resistant plate, invert the plate and culture it in a 37°C incubator for 12 - 14 h. Pick a single colony and transfer it to 5 mL of LB liquid medium with Amp resistance, and culture it in a shaker at 37°C for 8 - 12 h. Pipette 10 μL of the bacterial solution and transfer it to 5 mL of LB liquid medium with Amp resistance, and culture it in a shaker at 37°C for 8 - 12 h. Extract the Cas9 plasmid from 2 mL of the bacterial solution, linearize the plasmid by enzymatic digestion, and purify the enzymatic digestion product.

[0101] The DNA template for in vitro transcription of the sgRNA targeting the fibroin light chain gene (FibL) was obtained by PCR.

[0102] The corresponding mRNAs of Cas9 and sgRNA were obtained by in vitro transcription, and the following injection system was prepared:

[0103] The final concentration of the blue fluorescent protein (BFP) dimer homologous recombination plasmid was 300 ng / μL

[0104] The final concentration of Cas9 mRNA was 300 ng / μL

[0105] The final concentration of sgRNA mRNA was 150 ng / μL

[0106] The prepared sample was centrifuged at 15000 rpm at 4°C for 10 min and directly used for injection.

[0107] Inject the sample into silkworm eggs within 8 h after spawning, and inject about 5 nL of the sample into each silkworm egg. The injected silkworm eggs were placed in an incubator at 25°C with a relative humidity of 80% for cultivation and hatching.

[0108] The injected silkworm eggs were fed with fresh mulberry leaves until they spun cocoons. The G0 generation adults were crossed with wild-type to produce G1 generation individuals, and positive individuals expressing the enhanced green fluorescent protein gene (EGFP) in the first instar larvae of the G1 generation were selected under a fluorescence microscope. The G1 generation positive larvae were fed with fresh mulberry leaves until they spun cocoons. The G1 generation positive adults were crossed with wild-type again to produce seeds. After seed production, the genomes of the adults were extracted, and genotype amplification verification was performed using the genome as a template to detect the expected position of the exogenous gene homology in the silkworm genome.

[0109] Extract proteins from the posterior silk gland and detect the expression of exogenous proteins using Western Blot experiments.

[0110] The above G2 generation PCR experiments proved that the exogenous gene had been successfully introduced into the silkworm genome. Western blot methods proved that the silk of the transgenic silkworm contained bands of the expected size of the fusion protein. Starting from the G3 generation, all positive individuals were screened and self-crossed to retain seeds. By the G8 generation, a stable genetic variety was developed. The cocoons of the G8 generation were visibly blue to the naked eye and had uniform colors.

[0111] Hybridize the above-mentioned variety with a transgenic silkworm variety that has been bred and contains the MiSp1 gene from the spider's small saccular gland silk, and whose strength and toughness are 2.4 and 2.9 times higher than those of silk, respectively. The hybrid silkworm cocoons are artificial spider silk containing spider silk and a fusion fibroin light chain protein of blue fluorescent protein (BFP). The silk reeling is carried out by the following method:

[0112] 1. Put the blue positive cocoons into boiling water and boil for 2 min, press up and down to expel the air in the cocoon shell;

[0113] 2. Then quickly transfer the cocoons to a low-temperature water bath at 65 - 55 °C for 2 min, press up and down to allow the boiling water to fully enter the cocoon shell; then quickly put the cocoons into boiling water at 100 °C for 2 - 3 min, press up and down to allow the boiling water to fully enter the cocoon shell, soak the cocoons, and soften and swell the sericin;

[0114] 3. Then, gradually cool the temperature of the water bath in the air to 87 °C for about 15 min, further soften the sericin to achieve the purpose of proper cocoon cooking inside and outside; after the cooking process is completed, put it into a beaker containing a water bath at about 65 °C and extract the silk.

[0115] 4. Further refine the silk with water at 50 °C, 1% alkaline protease, and pH 9 for 2 hours to basically remove the sericin.

[0116] After cocoon cooking, silk reeling, and refining, the strength and toughness of the artificial spider silk are 2.3 and 2.6 times higher than those of silk. Under visible light, the blue color of the artificial spider silk remains stable without fading. The entire artificial spider silk with a length of about 1000 m shows a blue color, and the color distribution is uniform.

[0117] The artificial spider silk is further exposed to sunlight for 7 days, and the blue color remains stable without fading.

[0118] Example 3

[0119] By means of genetic engineering, biosynthesize a spider flagelliform gland silk gene sequence optimized according to the codon preference of silkworms, use the green fluorescent protein gene (EGFP) as a marker gene, the IE-1 promoter as the promoter of the marker gene expression cassette, use the fibroin light chain (FibL) of silkworm silk as an endogenous target gene, and construct a homologous recombination vector for TALEN gene editing technology containing a homologous arm sequence of the fibroin light chain gene + spider flagelliform gland silk gene + gene ending sequence + IE-1 promoter + green fluorescent protein marker gene (EGFP) + polyA + homologous arm sequence of the fibroin light chain gene.

[0120] By means of genetic engineering, biosynthesize a tetramer gene sequence of foreign genes optimized according to the codon preference of Bombyx mori, which is a tetramer gene sequence linked by 4 orange fluorescent protein (OFP) genes. Use the red fluorescent protein gene (DsRed) as a marker gene and the 3xP3 promoter as the promoter of the marker gene expression cassette. Take the heavy chain of Bombyx mori fibroin (FibH) as the endogenous target gene, and construct a homologous recombination vector for CRISPR gene editing technology, which contains the homologous arm sequence of the fibroin heavy chain gene + the tetramer of orange fluorescent protein (OFP) gene + the gene ending sequence + the 3xP3 promoter + the red fluorescent protein marker gene (DsRed) + polyA + the homologous arm sequence of the fibroin heavy chain gene.

[0121] Inject the above-mentioned homologous recombination vector for TALEN gene editing technology and TALEN mRNA, and the homologous recombination vector for CRISPR gene editing technology and Cas9 mRNA and sgRNA mRNA into silkworm eggs within 4 hours after spawning at the same time. Inject about 8 nL of the sample into each silkworm egg. The injected silkworm eggs are placed in an incubator at 25°C and a relative humidity of 85% for cultivation and hatching. The hatched newly hatched silkworms are fed with fresh mulberry leaves until they spin cocoons. The G0 generation adults are self-crossed to produce G1 generation individuals, and positive individuals that simultaneously express the green fluorescent protein marker gene (EGFP) and the red fluorescent protein marker gene (DsRed) are selected from the G1 generation first-instar larvae under a fluorescence microscope. The G1 generation positive larvae are fed with fresh mulberry leaves until they spin cocoons. The G1 generation positive adults are self-crossed again to produce seeds. After the seed production is completed, the genomes of the adults are extracted, and genotype amplification verification is carried out using the genome as a template to detect the expected position of the foreign gene homology in the Bombyx mori genome.

[0122] The G2 generation PCR experiment detection proves that the foreign gene has been successfully introduced into the Bombyx mori genome. The Western blot method is used to prove that the composite silk of Bombyx mori contains bands of spider silk protein and fluorescent silk fusion protein with the expected sizes. Starting from the G3 generation, self-crossing and seed preservation are carried out all the time until a stable genetic variety is bred. The G8 generation silkworm cocoons are orange visible to the naked eye and the color is uniform.

[0123] The spider silk-like silkworm cocoons are placed in a 100°C warm water bath for 2 hours to soften the sericin for reeling. The orange color of the spider silk-like silk remains stable under visible light without fading. The entire length of the spider silk-like silk is about 1100 m and all shows orange with uniform color distribution. The silk is further refined with 50°C water, 1% alkaline protease, and pH 9 for 2 hours to basically remove the sericin, and the orange color remains stable without fading.

[0124] The silk does not fade even after being baked at 100°C for 24 hours or exposed to sunlight for 10 days.

[0125] The strength and toughness of the spider silk-like silk are 2.8 and 3.6 times higher than those of silk.

[0126] Example 4

[0127] The spider aggregate gland silk gene sequence optimized according to the codon preference of Bombyx mori was biosynthesized by genetic engineering methods. Using the green fluorescent protein gene (EGFP) as a marker gene and the A3 promoter as the promoter of the marker gene expression cassette, and using the Bombyx mori fibroin light chain protein (FibL) as an endogenous target gene, a homologous recombination vector for CRISPR gene editing technology was constructed, which contains the homologous arm sequence of the fibroin light chain protein gene + spider aggregate gland silk + gene ending sequence + A3 promoter + green fluorescent protein marker gene (EGFP) + homologous arm sequence of the fibroin light chain protein gene.

[0128] The exogenous gene optimized according to the codon preference of Bombyx mori - an octamer gene sequence linked by 4 red fluorescent protein (RFP) and 4 cyan fluorescent protein (CFP) genes was biosynthesized by genetic engineering methods. Using the red fluorescent protein gene (DsRed) as a marker gene and the IE-1 promoter as the promoter of the marker gene expression cassette, and using the Bombyx mori silk fibroin P25 protein (P25) as an endogenous target gene, a homologous recombination vector for TALEN gene editing technology was constructed, which contains the homologous arm sequence of the silk fibroin P25 protein gene + an octamer gene formed by linking 4 red fluorescent protein (RFP) and 4 cyan fluorescent protein (CFP) genes + gene ending sequence + IE-1 promoter + red fluorescent protein marker gene (DsRed) + homologous arm sequence of the silk fibroin P25 protein gene.

[0129] The above homologous recombination vector for CRISPR gene editing technology was co-injected with Cas9 mRNA and sgRNA mRNA into silkworm eggs within 3 h after oviposition, and each silkworm egg was injected with about 8 nL of the sample. The injected silkworm eggs were placed in an incubator at 25 °C with a relative humidity of 85% for culture and hatching.

[0130] The hatched newly hatched silkworms were reared with fresh mulberry leaves until they spun cocoons. The G0 generation adults were self-crossed to produce G1 generation individuals. Positive individuals that simultaneously expressed the green fluorescent protein marker gene (EGFP) and the red fluorescent protein marker gene (DsRed) were selected from the G1 generation first instar larvae under a fluorescence microscope. The G1 generation positive larvae were reared with fresh mulberry leaves until they spun cocoons. The G1 generation positive adults were self-crossed to produce seeds. After seed production, the genomes of the adults were extracted, and genotype amplification verification was performed using the genome as a template to detect the expected position of the exogenous gene homology in the Bombyx mori genome.

[0131] The PCR experiment of the G2 generation detected and proved that the foreign gene had been successfully introduced into the silkworm genome. The Western blot method was used to prove that the composite silk of silkworms contained bands of spider silk protein and fluorescent silk fusion protein with the expected sizes. From the G3 generation, positive individuals were used for self-crossing to retain seeds, and a variety with stable inheritance and expression was developed by the G8 generation. The imitation spider silk cocoons of the G8 generation were visibly black and had uniform color with the naked eye.

[0132] The imitation spider silk cocoons were exposed to sunlight for 1 week, and the color did not fade. They were baked at 100 °C for 24 h, and the color also did not fade.

[0133] The silk reeling was carried out by the following method:

[0134] 1. Put the positive imitation spider silk cocoons into boiling water and boil for 2 min, press up and down to expel the air in the cocoon shell;

[0135] 2. Then quickly transfer the imitation spider silk cocoons into a low-temperature water bath at 65 - 55 °C for 2 min, press up and down to make the boiling water fully enter the cocoon shell; then quickly put the imitation spider silk cocoons into boiling water at 100 °C for 2 - 3 min, press up and down to make the boiling water fully enter the cocoon shell, soak the cocoons, and soften and swell the sericin;

[0136] 3. Then, gradually cool the temperature of the water bath in the air to 87 °C, about 15 min, to further soften the sericin and achieve the purpose of proper cocoon cooking inside and outside; after the cooking process is completed, put it into a beaker with a water bath at about 65 °C and extract the imitation spider silk.

[0137] 4. Further boil the imitation spider silk in 100 °C water for 1 h, and perform the same operation 3 times to refine the imitation spider silk.

[0138] After cocoon cooking, silk reeling, refining, and then being exposed to sunlight for 10 days, the black color of the imitation spider silk remained stable under visible light without fading. The entire length of the imitation spider silk was about 1020 m and all showed black with uniform color distribution. The strength and toughness of the imitation spider silk were 2.1 and 1.6 times higher than those of silk.

[0139] Example 5

[0140] The spider flagelliform gland silk gene sequence optimized according to the codon preference of silkworms was biosynthesized by genetic engineering methods. The red fluorescent protein gene (DsRed) was used as the marker gene, and the 3xP3 promoter was used as the promoter of the marker gene expression cassette. The fibroin light chain protein gene (FibL) of silkworms was used as the endogenous target gene to construct a homologous recombination vector for CRISPR gene editing technology containing the homologous arm sequence of the fibroin light chain protein gene, the spider flagelliform gland silk gene, the expression cassette of the red fluorescent protein marker gene (DsRed), and the homologous arm sequence of the fibroin light chain protein gene.

[0141] The polygene sequence linked by four red fluorescent protein (RFP) genes optimized according to the codon preference of Bombyx mori was biosynthesized by genetic engineering methods. The green fluorescent protein gene (EGFP) was used as a marker gene, and the IE-1 promoter was used as the promoter of the marker gene expression cassette. The Bombyx mori sericin 1 (Ser1) gene was used as an endogenous target gene, and a homologous recombination vector for TALEN gene editing technology was constructed, which contained the homologous arm sequence of the sericin 1 gene, the tetramer gene sequence linked by four red fluorescent protein (RFP) genes, the green fluorescent protein marker gene expression cassette, and the homologous arm sequence gene of the sericin 1 gene.

[0142] The above-mentioned homologous recombination vector for CRISPR gene editing technology, Cas9 mRNA, sgRNA mRNA, the homologous recombination vector for TALEN gene editing technology, and TALEN mRNA were co-injected into silkworm eggs within 4 h after oviposition, and about 7 nL of the sample was injected into each silkworm egg. The injected silkworm eggs were placed in an incubator at 25 °C with a relative humidity of 85% for cultivation and hatching.

[0143] The hatched newly-hatched silkworms were fed with fresh mulberry leaves until they spun cocoons. The G0 generation adults were self-crossed to produce G1 generation individuals. Under a fluorescence microscope, G1 generation positive adults that simultaneously expressed the red fluorescent marker gene and the green fluorescent marker gene in the first-instar larvae of the G1 generation were selected for self-crossing to produce seeds. After seed production, the genomes of the adults were extracted, and genotype amplification verification was performed using the genome as a template to detect the expected position of the exogenous gene homology in the Bombyx mori genome.

[0144] The G2 generation was detected by PCR experiments to prove that the exogenous gene had been successfully introduced into the Bombyx mori genome. The Western blot method was used to prove that the composite silk of Bombyx mori contained bands of spider silk protein and fluorescent silk fusion protein with the expected sizes. The G3 larvae with correct molecular identification were reared. Starting from the G3 generation, all positive individuals were screened and self-crossed to save seeds. By the G8 generation, a colored spider silk-like Bombyx mori variety with stable inheritance and expression of the spider ampullate gland silk gene and the fluorescent silk fusion protein gene was developed. The bionic silk cocoons of the G8 generation were visibly red to the naked eye.

[0145] The spider ampullate gland silk gene sequence optimized according to the codon preference of Bombyx mori was biosynthesized by genetic engineering methods. The green fluorescent protein gene (EGFP) was used as a marker gene, and the A3 promoter was used as the promoter of the marker gene expression cassette. The Bombyx mori fibroin P25 protein gene (P25) was used as an endogenous target gene, and a homologous recombination vector for CRISPR gene editing technology was constructed, which contained the homologous arm sequence of the fibroin P25 protein gene, the spider ampullate gland silk gene, the green fluorescent protein marker gene (EGFP) expression cassette, and the homologous arm sequence gene of the fibroin P25 protein gene.

[0146] By means of genetic engineering, a polygene sequence linked with 4 green fluorescent protein (EGFP) genes optimized according to the codon preference of Bombyx mori was biosynthesized. Using the red fluorescent protein gene (DsFP) as a marker gene and the IE-1 promoter as the promoter of the marker gene expression cassette, and taking the Bombyx mori fibroin heavy chain protein (FibH) gene as an endogenous target gene, a homologous recombination vector for TALEN gene editing technology containing the homologous arm sequence of the fibroin heavy chain protein gene, the tetramer of the green fluorescent protein (EGFP) gene, the red fluorescent protein marker gene (DsFP expression cassette), and the homologous arm sequence of the fibroin heavy chain protein gene was constructed.

[0147] The above-mentioned homologous recombination vector for CRISPR gene editing technology, Cas9 mRNA, sgRNA mRNA, as well as the homologous recombination vector for TALEN gene editing technology and TALEN mRNA were simultaneously injected into silkworm eggs within 4 h after oviposition, and about 8 nL of the sample was injected into each silkworm egg. The injected silkworm eggs were placed in an incubator at 25 °C with a relative humidity of 85% for cultivation and hatching.

[0148] The hatched newly hatched silkworms were fed with fresh mulberry leaves until they spun cocoons. The G0 generation adults were self-crossed to produce G1 generation individuals. Under a fluorescence microscope, G1 generation positive adults that simultaneously expressed the red fluorescent marker gene and the green fluorescent marker gene in the first instar larvae of the G1 generation were self-crossed to produce seeds. After the seed production was completed, the genomes of the adults were extracted, and genotype amplification verification was carried out using the genome as a template to detect the expected position of the exogenous gene homology in the Bombyx mori genome.

[0149] The G2 generation was detected by PCR experiments to prove that the exogenous gene had been successfully introduced into the Bombyx mori genome. The Western blot method was used to prove that the composite silk of Bombyx mori contained bands of spider silk protein and fluorescent silk fusion protein of the expected size. The G3 larvae with correct molecular identification were reared, and starting from the G3 generation, all positive individuals were screened for self-crossing to leave seeds. By the G8 generation, a spider silk-like Bombyx mori variety with stable inheritance and expression of the spider tubuliform gland silk protein gene and the fluorescent silk fusion protein gene was bred, and the spider silk-like silkworm cocoons were green.

[0150] The above-mentioned red spider silk-like cocoon variety and green spider silk-like cocoon variety were crossed to produce a hybrid variety. The spider silk-like silkworm cocoons obtained by rearing the hybrid variety showed yellow color. After inspection, it was found that the yellow color was the result of the synergistic effect of the red and green colors, and the cocoon color of each variety was uniform.

[0151] The following method was used for reeling silk:

[0152] 1. Put the colored positive spider silk-like silkworm cocoons into boiling water and boil for 5 min, and press up and down to discharge the air in the cocoon shell;

[0153] 2. Then, quickly transfer the silk-mimicking silkworm cocoons into a low-temperature water bath at 65°C for 3 minutes, and press up and down to allow the boiling water to fully enter the cocoon shells; then quickly place the cocoons into boiling water at 100°C for 4 minutes, and press up and down to allow the boiling water to fully enter the cocoon shells, soak the cocoons, and make the sericin swell and soften.

[0154] 3. Then, gradually cool the temperature of the water bath in the air to 80°C in about 15 minutes, so that the sericin is further softened to achieve the purpose of proper cocoon cooking for the inner and outer cocoon layers; after the cooking process is completed, place it in a beaker containing a water bath at about 65°C and extract the silk-mimicking silk.

[0155] 4. Further boil the silk-mimicking silk in a 100°C sodium carbonate aqueous solution for 15 minutes, and perform the same operation 3 times in total to refine the silk.

[0156] After cocoon cooking, reeling, and refining, the original yellow color of the silk-mimicking silk remains stable under visible light without fading. The total length of each silk-mimicking silk is about 1030 m, all showing the original yellow color with uniform color distribution.

[0157] All the yellow silk-mimicking silk is exposed to sunlight for 10 days without color fading. After baking at 100°C for 48 hours, the color also does not fade.

[0158] The strength and toughness of the silk-mimicking silk are 3.5 and 3.9 times higher than those of silk.

[0159] Example 6

[0160] By genetic engineering methods, biosynthesize the spider piriform gland silk protein gene sequence optimized according to the codon preference of Bombyx mori, and construct a vector for piggyBac-mediated transgenic technology. The vector contains 2 expression cassettes, the promoter of the Bombyx mori fibroin light chain protein gene (FibL) + the piriform gland silk protein gene expression cassette, and the IE-1 promoter + the enhanced green fluorescent protein marker gene (EGFP) expression cassette.

[0161] By genetic engineering methods, biosynthesize a polygene sequence connected by 4 red fluorescent protein (RFP) genes optimized according to the codon preference of Bombyx mori, and construct a homologous recombination vector for TALEN gene editing technology containing the homologous arm sequence of the fibroin P25 protein gene and the tetramer of the red fluorescent protein (RFP) gene.

[0162] Inject the above piggyBac transgenic vector, transgenic auxiliary plasmid, homologous recombination vector for TALEN gene editing technology, and TALEN mRNA into silkworm eggs within 4 hours after oviposition at the same time. Inject about 7 nL of the sample into each silkworm egg, and place the injected silkworm eggs in an incubator at 25°C and a relative humidity of 85% for cultivation and hatching.

[0163] The hatched silkworms were fed with fresh mulberry leaves until they spun silk and made cocoons. The G0 adults were self-pollinated to produce G1 individuals. The positive G1 adults expressing both green and red fluorescence were selected under a fluorescence microscope from the first-instar larvae of the G1 generation for self-pollination to produce seeds. The genome of the adults after seed production was completed was extracted, and genotype amplification verification was performed using the genome as a template to detect the expected position of the homologous exogenous genes in the silkworm genome.

[0164] The G2 generation was tested by PCR to prove that the exogenous gene had been successfully introduced into the silkworm genome, and the Western blot method was used to prove that the silkworm composite silk contained the expected size of spider silk protein and fluorescent silk fusion protein bands. The G3 larvae with correct molecular identification were raised, and all positive individuals were screened from the G3 generation and self-pollinated to keep seeds. The G8 generation was bred to breed with stable genetic expression of spider piriform gland silk genes. The G8 generation can be seen with naked eyes. The imitation spider silk cocoons are red.

[0165] The red imitation spider silk cocoon variety is reeled in the following method:

[0166] 1. Put the colorful positive imitation spider silk cocoon into boiling water and cook for 5 minutes, press up and down to expel the air in the cocoon shell;

[0167] 2. Then quickly move the cocoon into a 65°C low-temperature water bath for 3 minutes, pressing up and down to allow the boiling water to fully enter the cocoon shell; then quickly put the cocoon into 100°C boiling water for 4 minutes, pressing up and down to allow the boiling water to fully enter the cocoon shell, infiltrate the cocoon, and make the sericin swell and soften;

[0168] 3. Then, gradually cool the temperature of the water bath pot to 80°C in the air for about 15 minutes, so that the sericin can be further softened and the inner and outer cocoon layers can be properly cooked. After the cooking process is completed, put it into a beaker filled with a water bath at about 65°C to extract the imitation spider silk.

[0169] 4. The imitation spider silk was further boiled in a 100°C sodium carbonate aqueous solution for 15 min. The same operation was repeated 3 times to purify the silk.

[0170] After boiling cocoons, reeling and refining, the original red color of the imitation spider silk remains stable under visible light without fading. The total length of each imitation spider silk is about 1030m, and it shows the original red color with even color distribution.

[0171] All red imitation spider silks will not fade after being exposed to sunlight for 10 days or baked at 100℃ for 48 hours.

[0172] The strength and toughness of spider silk are 2.5 and 2.9 times higher than those of silk.

[0173] Comparative Example 1:

[0174] Using the TALEN gene editing technology, a homologous recombination vector containing the fibroin P25 protein gene homologous arm sequence + 3xP3 promoter + green fluorescent protein marker gene (EGFP) + polyA + fibroin P25 protein gene homologous arm sequence was injected together with TALEN mRNA into silkworm eggs within 2 hours after oviposition of the already developed silkworm variety transgenic for spider piriform gland silk protein gene. Approximately 5 nL of the sample was injected into each silkworm egg. The injected silkworm eggs were placed in an incubator at 25 °C with a relative humidity of 85% for culture and hatching. The hatched newly hatched silkworms were fed with fresh mulberry leaves until they spun cocoons.

[0175] The G0 generation adults were crossed with the already developed silkworm variety transgenic for spider piriform gland silk protein gene to produce G1 generation individuals. Since the 3xP3 promoter only initiates the specific expression of the green fluorescent protein marker gene (EGFP) in the nervous system, the eyes of the G1 generation first instar larvae showed green fluorescence under a fluorescence microscope. However, because there was no exogenous fluorescent protein gene fused with the endogenous silk protein gene to form a fluorescent silk protein gene, the silk did not contain the fluorescent fusion protein and the cocoons had no color.

[0176] By the G8 generation, although the green fluorescent protein marker gene (EGFP) had been successfully inserted behind the fibroin P25 protein gene of the silkworm, and a variety with stable inheritance of the larval eyes and expression of the green fluorescent protein gene (EGFP) was developed, the cocoons still had no color all the time. This indicates that the fusion of the exogenous fluorescent protein gene with the endogenous silk protein gene to form a fluorescent silk protein gene is the basis for the silk to become colored silk.

[0177] Comparative Example 2:

[0178] A transgenic vector mediated by the piggyBac transposon was constructed. The vector contained the left and right arms of the piggyBac transposon, the expression cassette of the green fluorescent protein marker gene initiated by the IE1 promoter, and the expression cassette of the exogenous red fluorescent protein gene (RFP) initiated by the promoter of the fibroin heavy chain gene of the silkworm. The structure was the left arm of the piggyBac transposon + IE-1 promoter + green fluorescent protein marker gene (EGFP) + polyA + promoter of the fibroin heavy chain gene of the silkworm + red fluorescent protein gene (RFP) + polyA sequence of the fibroin heavy chain gene of the silkworm + the right arm of the piggyBac transposon.

[0179] Mix the above-mentioned transgenic vector and the pHA3PIG helper plasmid capable of providing piggyBac transposase at a concentration ratio of 2:1, with a total concentration of 400 ng / μl. Then, use the microinjection method to introduce it into the fertilized eggs within 2 hours after oviposition of the silkworm variety that has been bred with the spider flagelliform gland silk protein gene. The total volume of introduction is 7 nl. Raise the microinjected silkworm eggs at 25°C and 85% humidity until they become adults, and hybridize and pass on the generation with the silkworm variety that has been bred with the spider flagelliform gland silk protein gene to obtain the G1 generation. After the G1 generation of transgenic silkworm larvae hatched from the transgenic experiment, obtain the transgenic silkworms that are positive for the expression of the EGFP marker gene by observing through a fluorescence microscope, raise them to adults, and the transgenic silkworms self-cross and pass on the generation, which is the G2 generation. From the G2 generation onwards, all transgenic silkworms are raised with single-moth breeding. Observe through a fluorescence stereomicroscope during the egg stage, select the transgenic silkworms that are positive for the EGFP marker gene, raise them to adults, and mate within the same moth area.

[0180] At the G2 generation, using the genomic DNA of the posterior silk gland of the transgenic silkworms on the 3rd day of the 5th instar as a template, use Inverse PCR to amplify the inserted fragment of the transgenic vector in the silkworm genome, and perform cloning, sequencing, and chromosomal localization analysis. The results show that the insertion site is at 18403561 on the 4th chromosome of the silkworm, proving that the transposon has been inserted into the silkworm genome.

[0181] Starting from the G4 generation, select the moth areas with a pure green fluorescence phenotype for breeding, use the silkworm moths within the same moth area to mate, and breed a new variety of transgenic silkworms with homozygous green fluorescent protein genes and posterior silk gland cells capable of synthesizing and secreting red fluorescent protein by the G8 generation.

[0182] Extract the posterior silk gland and cocoon silk protein of the above-mentioned silkworms as materials, and use SDS-PAGE electrophoresis and Western blot techniques to analyze the expression of the red fluorescent protein in the transgenic silkworms. As a result, specific protein bands consistent with the expected molecular weight size are obtained.

[0183] The research results prove that the new variety of transgenic silkworms can synthesize and secrete red fluorescent protein in the posterior silk gland cells, and this protein can enter the silkworm cocoon along with the behavior of spinning and cocooning. The cocoon is visibly red to the naked eye.

[0184] Reel silk using the following method:

[0185] 1. Put the red positive silkworm cocoons into boiling water and boil for 2 min, press up and down to expel the air in the cocoon shell. The water in the water bath pot turns red and the red color of the silkworm cocoons fades;

[0186] 2. Then quickly transfer the silkworm cocoons into a low-temperature water bath at 65 - 55°C for 2 min, press up and down to make the boiling water fully enter the cocoon shell. The water in the water bath pot turns red and the red color of the silkworm cocoons fades further;

[0187] 3. Quickly put the cocoon into boiling water at 100 °C for 2 - 3 minutes, press up and down to make the boiling water fully enter the cocoon shell, soak the cocoon, and make the sericin swell and soften, and the red color of the cocoon further fades;

[0188] 4. Then, gradually cool the temperature of the water bath in the air to 87 °C for about 15 minutes, and the sericin is further softened to achieve the purpose of proper cocoon cooking for the inner and outer cocoon layers; after the cooking process is completed, put it into a beaker with a water bath at about 65 °C and extract the silk.

[0189] 5. Boil the silk in water at 100 °C for 1 hour, and repeat the same operation 3 times to refine the silk. Each time the silk is refined, the color of the silk fades once.

[0190] The results prove that during the cocoon cooking, reeling, and refining processes, because the exogenous fluorescent protein adheres to the silk and does not fuse with the silk protein molecule to form a fusion protein, and does not participate in the formation of silk at a constant molar ratio and firmly exists in the whole silk, the exogenous fluorescent protein is continuously dissolved and disappears, and the red color of the silk continuously fades.

[0191] The above specific embodiments are used to explain and illustrate the present invention, rather than limiting the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing super-strong, super-tough, self-colored, non-fading, pollution-free colored imitation spider silk, characterized by: The method comprises the following steps: using a fluorescent protein gene or a polymer connected with several fluorescent protein genes as an exogenous gene, introducing the exogenous gene into a spider silk-like silkworm variety through homologous recombination, and forming a fusion protein gene with an endogenous silk protein gene of the spider silk-like silkworm variety, breeding the silkworms for multiple generations to finally obtain a silkworm variety that can stably inherit and express the fusion protein gene, and using the silkworm variety to produce spider silk. The spider silk is super strong and tough, does not need to be dyed, has its own color under visible light, and has stable and uniform color.

2. The method for preparing the super-strong, super-tough, self-colored, non-fading, pollution-free colored imitation spider silk according to claim 1, characterized in that: The spider silk-mimicking silkworm variety is a bred spider silk gene-transformed silkworm variety, or a spider silk gene-transformed silkworm variety bred by simultaneously introducing spider silk genes into silkworms using transgenic technology when fluorescent protein genes are introduced into silkworms.

3. The method for preparing the super-strong, super-tough, self-colored, non-fading, pollution-free colored imitation spider silk according to claim 1, characterized in that: The homologous recombination is to use the homologous recombination technology of TALEN or CRISPR gene editing to form a fusion protein gene with the fluorescent exogenous gene and the endogenous silk protein gene of the silkworm, and then cultivate a new silkworm variety that stably inherits, synthesizes and secretes the fusion protein, and the new silkworm variety synthesizes and secretes the spider silk containing the fusion protein.

4. The method for preparing the super-strong, super-tough, self-colored, non-fading, pollution-free colored imitation spider silk according to claim 2, characterized in that: The spider silk gene refers to the gene corresponding to the spider silk protein secreted by the spider's macrocystic gland, microcystic gland, whip gland, polycystic gland, piriform gland, grape gland and tubular gland.

5. A method for preparing a super-strong, super-tough, self-colored, non-fading, pollution-free colored imitation spider silk according to claim 1 or 3, characterized in that: The exogenous gene is a fluorescent protein gene for introducing a silk component with its own color, or a polymer fluorescent protein gene composed of 2-8 fluorescent protein genes of the same or different types. The fluorescent protein gene is a gene of green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), orange fluorescent protein (OFP) or a mutant thereof.

6. A method for preparing a super-strong, super-tough, self-colored, non-fading, pollution-free colored imitation spider silk according to claim 1 or 3, characterized in that: The silk protein gene endogenous to Bombyx mori is the silk fibroin heavy chain gene (FibH), the silk fibroin light chain gene (FibL), the silk fibroin P25 gene (P25) or the sericin 1 gene (Ser1) of Bombyx mori.

7. The method for preparing the super-strong, super-tough, self-colored, non-fading, pollution-free colored imitation spider silk according to claim 1, characterized in that: The method comprises the following steps: constructing a homologous recombination vector of an exogenous gene by molecular biological technology, introducing the homologous recombination vector into silkworm eggs by microinjection, integrating the homologous recombination into a specific endogenous silk protein gene of the silkworm, obtaining G1 generation marker gene positive individuals by screening under a fluorescent microscope or with the naked eye, mating the G1 generation positive individuals with wild types or self-pollinating the positive individuals to obtain the G2 generation, screening all positive individuals from the G3 generation and self-pollinating to retain seeds, performing multi-generation screening and cultivation on the silkworms, and breeding a variety with stable inheritance and expression to the G8 generation.

8. The method for preparing the super-strong, super-tough, self-colored, non-fading, pollution-free colored imitation spider silk according to claim 7, characterized in that: The exogenous gene vector is a homologous recombination exogenous gene vector used for TALEN or CRISPR gene editing, and the vector includes endogenous gene homology arms + exogenous genes + endogenous gene homology arms, or endogenous gene homology arms + exogenous genes + fluorescent screening marker gene expression frame + endogenous gene homology arms.

9. The method for preparing the super-strong, super-tough, self-colored, non-fading, pollution-free colored imitation spider silk according to claim 7, characterized in that: The homologous recombination vector is introduced into the silkworm egg by microinjection, which is a single homologous recombination vector that introduces an exogenous fluorescent protein gene fused with an endogenous silk protein gene, or multiple homologous recombination vectors that simultaneously introduce multiple exogenous fluorescent protein genes fused with multiple endogenous silk protein genes.

10. A super strong and tough, self-colored, non-fading, pollution-free colorful imitation spider silk, characterized by: Prepared by the preparation method described in any one of claims 1 to 9.

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