Colored, fading-free and pollution-free colored silk and preparation method thereof

Abstract

The invention discloses a colored non-fading pollution-free colored silk and a preparation method thereof. The method comprises the following steps: by taking a fluorescent protein gene or a polymer connected by a plurality of fluorescent protein genes as an exogenous gene, forming a fusion protein gene from the exogenous gene and a silkworm endogenous silk protein gene in a homologous recombination manner by adopting a gene editing technology; the novel silkworm variety capable of stably inheriting and expressing the fusion protein gene is finally obtained through multi-generation breeding, the silk does not need to be dyed, has colors under visible light, is stable and uniform in color, does not fade in silk reeling refining and high-temperature exposure, and is free of pollution. According to the invention, the serious problems of environmental pollution, uneven coloring and fading for a long time caused by silk printing and dyeing are solved, the silk quality is greatly improved, the silk production completely conforms to the concept of green, environment-friendly and sustainable development, and the silk becomes a new material comprehensively matched with the properties of pure nature, no pollution, reproducibility, degradability and the like.

Description

Technical Field

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

[0002] Mulberry silk is a natural protein fiber composed of two proteins, fibroin and sericin, secreted by the silk gland organs of silkworm larvae. Fibroin is synthesized and secreted by the posterior silk gland, accounting for 70 - 80% of silk, which is the core part of 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 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 silk 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 silkworm breeds for production are white cocoon silk breeds, and special processes are required for dyeing during post-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 silkworms, chlorophyll copper sodium, 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 little color remains after silk reeling. Moreover, high-concentration dyes damage the normal growth and development and health of 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 breeds

[0008] In the early days, the silk of silkworm breeds 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, forming colored silk in the silk protein.

[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 silk reeling process. Moreover, the colors of colored silk in different batches are inconsistent, and the product quality is unstable.

[0010] III. Preparation of Silk by Transgenic Technology

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

[0012] The first is to use the piggyBac transposon-mediated transgenic technology 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 the 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 body, thereby forming 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 silkworms can produce pigments that they 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 silk reeling process and the color is unstable.

[0016] Moreover, the technical purpose of some is to use transgenic technology to make the silk gland of silkworms produce fluorescent proteins, requiring that the fluorescent proteins are easily dissolved and extracted. In this process, colored cocoons will be produced, and a large amount of fluorescent proteins 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 the silk of different batches. Summary of the Invention

[0017] In order to solve the problems existing in the background technology, the purpose of the present invention is to develop a method for preparing colored silk that uses gene editing technology to form a fusion protein of fluorescent protein and endogenous silk protein, does not require dyeing, has its own color under visible light, and will not fade during silk reeling, refining, exposure to high temperature of 100 °C, or sunlight exposure, thereby enhancing the silk to become a new material that fully matches the properties of being natural, pollution-free, renewable, and biodegradable.

[0018] The solution of the present invention uses a fluorescent protein gene or a polymer of several fluorescent protein genes linked together as an exogenous gene. By using gene editing technology, the exogenous 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, obtaining a fusion protein of fluorescent protein + silk protein. After multiple generations of breeding, a new silkworm variety that can stably inherit and express the fusion gene is finally obtained. The silk of this silkworm variety does not need to be dyed, and the color of the 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 at a constant molar ratio, firmly exist in the whole silk thread, are evenly distributed, and will not fall off during silk reeling, refining operations, or high-temperature exposure to sunlight. Therefore, the color of its silk is uniform and stable, and will never fade.

[0019] To achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0020] The method is to use a fluorescent protein gene or a polymer composed of several linked fluorescent protein genes as an exogenous gene, introduce the exogenous gene into the silkworm through homologous recombination, and form a fusion protein gene with the endogenous silk protein gene of the silkworm. Then, through multiple generations of breeding of the silkworm, a new silkworm variety that can stably inherit and express the fusion protein gene is finally obtained. The new silkworm variety is used to produce silk with its own color. The silk does not need to be dyed, has its own color under visible light, and the color is stable and uniform, and will not fade after silk reeling, refining, exposure to 100 °C high temperature, or sunlight exposure.

[0021] The method is to use the homologous recombination technology of TALEN or CRISPR gene editing to form a fusion protein gene between the exogenous gene and the endogenous silk protein gene of the silkworm through homologous recombination, and then cultivate and obtain a new silkworm variety with stable inheritance. The new silkworm variety synthesizes and secretes colored silk with the fusion protein, which is silk with its own color. The fusion protein and the fusion protein gene correspond to each other.

[0022] The exogenous gene is 1 fluorescent protein gene for introducing silk components with its own color or a polymer fluorescent protein gene composed of 2 - 8 identical or different types of fluorescent protein genes linked together. 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 a mutant thereof.

[0023] The endogenous silk protein gene of the silkworm is the fibroin heavy chain gene (FibH), fibroin light chain gene (FibL), fibroin P25 gene (P25) or sericin 1 gene (Ser1) of the silkworm.

[0024] Specifically, the method constructs a homologous recombination vector of exogenous genes through molecular biology techniques, introduces the homologous recombination vector into silkworm eggs by microinjection, integrates it into specific endogenous silk protein genes of silkworms through homologous recombination, and then obtains G1 generation positive individuals by screening with a fluorescence microscope or the naked eye. The G1 generation positive individuals are mated with wild-type individuals or self-crossed to obtain the G2 generation. Starting from the G3 generation, all positive individuals are screened and self-crossed to save seeds. The silkworms are screened and cultivated for multiple generations until the G8 generation to breed a variety in which the silk protein + fluorescent protein gene of silkworms is stably inherited and expressed.

[0025] The described exogenous gene vector 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 + gene ending sequence + fluorescence screening marker gene expression cassette + endogenous gene homologous arm.

[0026] The described fluorescence screening marker gene expression cassette includes a fluorescence screening marker gene promoter + fluorescence screening marker gene + polyA sequence. The promoter is the IE-1, A3, or 3xP3 promoter, and the fluorescence screening marker gene is the green fluorescent protein gene (EGFP) or the red fluorescent protein gene (DsRed).

[0027] The described silkworms secrete silk, and the silk contains components with self-colored properties. The components with self-colored properties include 1 fluorescence protein gene 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 fusion gene expression of a multi-polymer fluorescence protein gene formed by linking 2-8 fluorescence protein genes of the same or different species and an endogenous silk protein gene. The fluorescent silk fusion protein can be formed by fusing one fluorescence protein with one endogenous silk protein, or by fusing multiple fluorescence proteins with multiple endogenous silk proteins.

[0028] During the process of spinning cocoons and forming silk by the silkworm variety with self-colored silk, the fluorescent silk fusion protein participates in the formation of silk at a constant molar ratio, firmly exists in the whole silk, is evenly distributed, does not fall off during the reeling operation, and the silk has self-color under visible light without dyeing, without causing environmental pollution.

[0029] The described method of introducing the homologous recombination vector into silkworm eggs by microinjection is to introduce a single homologous recombination vector in which an exogenous fluorescent protein gene is fused with an endogenous silk protein gene, or to introduce multiple homologous recombination vectors in which multiple exogenous fluorescent protein genes are fused with multiple endogenous silk protein genes simultaneously.

[0030] The silkworm bred by introducing multiple fusion protein gene homologous recombination vectors through microinjection has silk showing complex colors under the synergistic effect of multiple fluorescent proteins.

[0031] The obtained silkworm varieties include original species and hybrid species between original species with different colors. The silk of the screened hybrid species presents complex colors under the synergistic effect of the fluorescent proteins of the two original species.

[0032] 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.

[0033] The present invention has the following beneficial effects:

[0034] The present invention solves the serious problems of environmental pollution, uneven coloring and fading over time caused by printing and dyeing in silk production, greatly improves the quality of silk, and makes silk production fully comply with the concept of green, environmental protection and sustainable development. It becomes a new material that fully matches the properties of pure natural, pollution-free, renewable and degradable, creates a new situation in silk production, and has great economic benefits and broad application prospects.

[0035] The present invention is a method for artificially designing silk protein and improving the performance of silk. It also develops the color development function of exogenous fluorescent protein, so that silk can show color under visible light without dyeing, and the color is uniform and stable, and it will not fade even under high temperature exposure, eliminating environmental pollution caused by printing and dyeing; it can also produce colorful and gorgeous 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 silk and create good economic benefits. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the embodiments.

[0037] Embodiments of the present invention are as follows:

[0038] Example 1

[0039] The exogenous gene yellow fluorescent protein (YFP) gene sequence optimized according to the codon preference of Bombyx mori was biosynthesized by genetic engineering methods. The red fluorescent protein gene (DsRed) was used as a marker gene, and the IE-1 promoter was used as the promoter of the marker gene expression cassette. The fibroin light chain (FibL) of Bombyx mori silk was used as an endogenous target gene, and a homologous recombination vector for TALEN gene editing technology containing the homologous arm sequence of the fibroin light chain gene + the exogenous gene yellow fluorescent protein (YFP) gene + the gene ending sequence + the IE-1 promoter + the red fluorescent protein marker gene (DsRed) + polyA + the homologous arm sequence of the fibroin light chain gene was constructed.

[0040] The design of TALEN targeting the FibL locus and the detection method for the expression of the exogenous gene yellow fluorescent protein (YFP) gene are as follows:

[0041] 1. Design of TALEN targeting the FibL locus

[0042] The exogenous gene of yellow fluorescent protein (YFP) was site-specifically integrated at the rear end of FibL, and the TALEN cleavage site was selected in the downstream region of the FibL terminator. The genomic DNA of Bombyx mori was extracted using a kit, and the specific operation steps are as follows:

[0043] (1) Place silkworm eggs or larvae in a sterilized 1.5 mL centrifuge tube, add 180 μL of Buffer ACL, then add 20 μL of proteinase K solution, mix well in a tissue grinder, and then incubate at 56 °C in a water bath for 1 h;

[0044] (2) Add 200 μL of Buffer CL to the system in step (1) and mix well;

[0045] (3) Add 200 μL of absolute ethanol to the solution in step (2) and mix well;

[0046] (4) Transfer all of the above solution and the translucent fibrous suspension to a filter column, let it stand at room temperature for 2 min, then centrifuge at 12,000×g for 1 min, and discard the waste liquid;

[0047] (5) Add 500 μL of CW1 solution to the filter column, centrifuge at 12,000×g for 30 s, and discard the waste liquid;

[0048] (6) Add 500 μL of CW2 solution to the filter column, centrifuge at 12,000×g for 30 s, and discard the waste liquid;

[0049] (7) Put the filter column back into the collection tube and centrifuge at 13,000×g for 2 min to remove the residual CW2 solution;

[0050] (8) Add 50 μL of sterilized water to the filtration column and centrifuge at 13,000×g for 2 min. The resulting solution is the genomic sample, which is stored in a -20°C refrigerator for future use.

[0051] Design amplification primers for the FibL genome according to the silkworm genome sequence in NCBI. Using the silkworm genome extracted above as a template, amplify the DNA fragments in the upstream and downstream regions of the FibL terminator. After amplification, take an appropriate amount of the product 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 submit the sequence to the website (https: / / tale-nt.cac.cornell.edu / ) for the design of TALEN targeting sites, and select the 2 targeting sites with the highest scores for the construction of subsequent targeting vectors.

[0052] 2. Construction of TALEN targeting vectors

[0053] 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 is for the unilateral binding TALEN plasmid, and the construction of the other binding plasmid is exactly the same as this step. Use the Golden Gate TALEN and TAL Effector Kit 2.0 to construct the TALEN plasmid. The specific operation steps are as follows:

[0054] (1) According to the designed targeting sequence, sequentially select the modular plasmids corresponding to the first 10 bases and the backbone vector pFUS_A according to the recognition principle of RVD and bases; then sequentially select 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 according to the base recognition principle.

[0055] (2) Prepare the following two reaction systems respectively in a laminar flow hood:

[0056] System 1:

[0057]

[0058] System 2:

[0059]

[0060] (3) Gently mix the above systems 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.

[0061] (4) Plasmid - safe nuclease treatment:

[0062] The above reaction system: 20 μL

[0063] 1 μL of 10 mM ATP

[0064] 1 μL of plasmid-safe nuclease

[0065] Gently mix the above reaction system, and then incubate it in a constant temperature water bath at 37 °C for 1 h. This step is used to remove the unligated DNA fragments in the reaction of step (3);

[0066] (5) Take 5 μL of the above reaction solution and transform it in DH5α competent cells;

[0067] (6) Verify by colony PCR and select positive transformants;

[0068] (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;

[0069] (8) Select the module plasmid corresponding to the last base (the Nth base) according to the base recognition principle, and ligate it with the above two ligated fragments. Prepare the following reaction system:

[0070]

[0071] (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.

[0072] (10) Take 5 μL of the above reaction solution and transform it in DH5α competent cells;

[0073] (11) Verify by colony PCR and select positive transformants;

[0074] (12) Culture the correct clones, extract the plasmid and verify by sequencing to obtain the TALEN plasmid.

[0075] 3. Preparation of TALEN mRNA by in vitro transcription

[0076] Perform single digestion of the constructed TALEN plasmid with XbaⅠ and purify the digestion product with phenol-chloroform. Subsequently, use the linearized TALEN as a template to prepare mRNA by in vitro transcription. Inject the above TALEN mRNA into silkworm eggs within 8 h after oviposition by microinjection, and the injection volume for each silkworm egg is about 5 nL. Prepare the injection system as follows:

[0077] Final concentration of TALEN-FL mRNA: 250 ng / μL

[0078] The final concentration of TALEN-FR mRNA is 250 ng / μL

[0079] The injected silkworm eggs were incubated in a constant temperature incubator at 25°C and 90% relative humidity.

[0080] 4. Detection of the activity of the targeting site

[0081] Genomic DNA was extracted from the above-injected silkworm eggs. Using the genomic DNA as a template, the DNA sequence near the targeting site was amplified, and the amplified product was directly sequenced for verification. The cleavage activity of the constructed TALEN targeting plasmid was judged according to the sequencing peak map.

[0082] 5. Construction of transgenic lines and identification of positive individuals

[0083] The above-obtained exogenous gene homologous recombination plasmid and the targeting plasmid with high TALEN cleavage activity were respectively purified by standard phenol-chloroform method. The plasmid DNA was dissolved in 1×PBS solution and its concentration was measured. Subsequently, an injection system was prepared:

[0084] The final concentration of TALEN-FL mRNA is 250 ng / μL

[0085] The final concentration of TALEN-FR mRNA is 250 ng / μL

[0086] The final concentration of the exogenous gene homologous recombination plasmid is 300 ng / μL

[0087] The silkworm eggs after microinjection 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 wild-type to breed seeds. Positive individuals expressing 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 fed with fresh mulberry leaves until they spun cocoons. The G1 generation positive adults were crossed with wild-type again to breed seeds. Genomic DNA was extracted from the adults after breeding, 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.

[0088] 6. Detection of gene transcription and expression levels

[0089] 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. Subsequently, the silk gland tissues were ground into fine powder with liquid nitrogen, total RNA was extracted using a kit, cDNA was synthesized using a reverse transcription kit, and real-time fluorescence quantitative analysis was carried out 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.

[0090] The silk gland tissue and cocoon shell were cooled with liquid nitrogen and then ground into fine powder in a tissue grinder. Subsequently, silk gland and cocoon shell proteins were extracted with protein extraction buffer (1:200, mass / volume), and Western blot technology was used to detect the specific expression of the target protein.

[0091] The above experimental results proved that the foreign gene had been successfully introduced into the silkworm genome, the expression of the yellow fluorescent protein (YFP) gene was significant, and bands of the fusion protein with the expected size were contained in the composite silk of the silkworm cocoon. Positive individuals were screened and self-crossed for seed preservation starting from the G3 generation, and a stable genetic variety was developed 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 wild-type variety, with no significant biological differences. The silkworm cocoons were visibly yellow and had uniform color.

[0092] The following method was used for reeling silk:

[0093] 1. The yellow positive silkworm cocoons were put into boiling water and boiled for 6 min, and pressed up and down to expel the air in the cocoon shell.

[0094] 2. Then, the silkworm cocoons were quickly transferred to a low-temperature water bath at 60 °C for 3 min, and pressed up and down to allow the boiling water to fully enter the cocoon shell; then the silkworm cocoons were quickly put into boiling water at 100 °C for 3 min, and pressed up and down to allow the boiling water to fully enter the cocoon shell, soak the cocoons, and soften the sericin by swelling.

[0095] 3. Then, the temperature of the water bath was gradually cooled to 80 °C in the air for about 15 min, and the sericin was further softened to achieve the purpose of proper cocoon cooking inside and outside; after the cooking process was completed, it was put into a beaker containing a water bath at about 65 °C to extract the silk.

[0096] 4. The silk was further put into boiling water at 100 °C for 30 min, and the water was changed 3 times to remove the sericin as much as possible.

[0097] After cocoon cooking, reeling, and refining, the yellow color of the silk remained stable under visible light without fading. The whole silk length of about 1000 m was yellow and had uniform color distribution.

[0098] The silk was further baked at 100 °C for 12 h, and the yellow color remained stable without fading.

[0099] Example 2

[0100] Through genetic engineering methods, a dimeric gene sequence of an exogenous gene optimized according to the codon preference of silkworm - 2 blue fluorescent protein (BFP) genes connected was biosynthesized, the green fluorescent protein gene (EGFP) was used as a marker gene, the A3 promoter was used as the marker gene expression frame promoter, and the silk fibroin light chain (FibL) was used as an endogenous targeting gene to construct a homologous recombination vector containing a silk fibroin light chain gene homology arm sequence + 2 blue fluorescent protein (BFP) gene dimers + gene end sequence + A3 promoter + green fluorescent protein marker gene (EGFP) + polyA + silk fibroin light chain gene homology arm sequence for CRISPR gene editing technology.

[0101] The design of the CRISPR targeting FibL site and the detection method of the exogenous gene blue fluorescent protein (BFP) gene dimer expression are as follows:

[0102] The Cas9 sequence is derived from Streptococcus pyogenes and has been codon-optimized for human. Transcription of the Cas9 protein is initiated by the SP6 promoter.

[0103] After streaking the frozen bacteria corresponding to the Cas9 plasmid on the Amp-resistant plate, invert the plate and culture it in a 37°C incubator for 12 to 14 hours. Pick a single colony and place it in 5mL of Amp-resistant LB liquid culture medium, and culture it in a shaker at 37°C for 8 to 12 hours. Pipette 10uL of bacterial solution and transfer it to 5mL of Amp-resistant LB liquid culture medium, and also culture it in a shaker at 37°C for 8 to 12 hours. Extract the Cas9 plasmid in 2mL of bacterial solution. The specific plasmid extraction steps are as follows:

[0104] (1) Buffer P2 and Buffer P3 are incubated in a 37°C water bath until the precipitate is completely dissolved;

[0105] (2) Take 2 mL of bacterial culture medium into an EP tube, centrifuge at 8000 × g for 2 min at room temperature, and remove the supernatant with a pipette;

[0106] (3) Add 250 μL of Buffer P1 to the EP tube and thoroughly suspend the bacteria on a shaker;

[0107] (4) After adding 250 μL of Buffer P2 to the EP tube, gently invert the tube 5 to 10 times to mix well and let it stand at room temperature for 2 to 4 minutes;

[0108] (5) After adding 350 μL of Buffer P3 to the EP tube, gently invert the tube 5 to 10 times to mix thoroughly;

[0109] (6) Centrifuge at 15,000 × g for 10 min at room temperature, transfer the supernatant to the adsorption column, and centrifuge again at 9,000 × g for 30 s at room temperature. Discard the waste liquid in the waste liquid collection tube;

[0110] (7) Add 500 μL of deproteinized solution Buffer DW1 to the adsorption column and centrifuge at 9000×g for 30 s at room temperature. Discard the waste liquid in the waste liquid collection tube;

[0111] (8) Add 500 μL of Wash Solution to the adsorption column and centrifuge at 9000×g for 30 s at room temperature. Discard the waste liquid in the waste liquid collection tube;

[0112] (9) Add 500 μL of Wash Solution to the adsorption column again and centrifuge at 9000×g for 30 s at room temperature. Discard the waste liquid in the waste liquid collection tube;

[0113] (10) Centrifuge the empty column at 9000×g for 1 min at room temperature. Replace the collection tube with a new sterilized EP tube.

[0114] (11) Add 50 μL of sterilized ddH 2 O to the center of the adsorption membrane, let it stand at room temperature for 2 min, then centrifuge at 9000×g for 1 min. Store the finally obtained plasmid in a -20°C refrigerator for later use;

[0115] After obtaining the Cas9 plasmid, linearize the plasmid by enzymatic digestion. The linearization enzymatic digestion system is as follows:

[0116]

[0117] After incubating the enzymatic digestion reaction solution in a 37°C water bath for 3 - 4 h, take 5 μL of the enzymatic digestion reaction solution for electrophoresis detection. After the electrophoresis result shows complete enzymatic digestion and a single target band, purify the enzymatic digestion product.

[0118] Design sgRNA targeting the fibroin light chain gene (FibL) and obtain the in vitro transcription template DNA by PCR.

[0119] Obtain the mRNAs corresponding to Cas9 and sgRNA by in vitro transcription and prepare the following injection system:

[0120] Final concentration of blue fluorescent protein (BFP) dimer homologous recombination plasmid: 300 ng / μL

[0121] Final concentration of Cas9 mRNA: 300 ng / μL

[0122] Final concentration of sgRNA mRNA: 150 ng / μL

[0123] Inject the injection system into silkworm eggs within 8 h after spawning, and inject about 5 nL of the sample into each silkworm egg. Put the injected silkworm eggs into an incubator at 25°C with a relative humidity of 80% for cultivation and hatching.

[0124] The injected silkworm eggs are reared with fresh mulberry leaves until they spin cocoons. The G0 generation adults are crossed with wild-type to produce G1 generation individuals, and positive individuals expressing the green fluorescent protein marker gene (EGFP) in the first instar larvae of the G1 generation are selected under a fluorescence microscope. The G1 generation positive larvae are reared with fresh mulberry leaves until they spin cocoons. The G1 generation positive adults are crossed with wild-type again to produce seeds. After seed production, 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 exogenous gene homologous in the silkworm genome.

[0125] Proteins are extracted from the posterior silk glands, and Western Blot experiments are used to detect the expression of exogenous proteins.

[0126] The above-mentioned G2 generation PCR experiments prove that the exogenous gene has been successfully introduced into the silkworm genome. Western blot method is used to prove that there are bands of fusion proteins with the expected size in the composite silk of silkworm cocoons. Starting from the G3 generation, all positive individuals are screened and self-crossed to save seeds. By the G8 generation, a stable genetic variety is developed. The G8 generation silkworm cocoons are visibly blue and the color is uniform. The silk reeling is carried out by the following methods:

[0127] 1. Put the blue positive silkworm cocoons into boiling water and boil for 2 min, press up and down to discharge the air in the cocoon shell;

[0128] 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; then quickly put the silkworm 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 make the sericin swell and soften;

[0129] 3. Then, gradually cool the temperature of the water bath in the air to 87 °C for about 15 min, and the sericin is further softened to 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 silk.

[0130] 4. The silk is further refined with water at 50 °C, 1% alkaline protease, and pH 9 for 2 hours to basically remove the sericin.

[0131] After cocoon cooking, silk reeling, and refining, the blue color of the silk remains stable under visible light without fading. The entire silk length of about 1000 m shows blue, and the color distribution is uniform.

[0132] The silk is further exposed to sunlight for 7 days, and the blue color remains stable without fading.

[0133] Example 3

[0134] The exogenous gene optimized according to the codon preference of Bombyx mori, a tetramer gene sequence in which four orange fluorescent protein (OFP) genes are ligated, was biosynthesized by genetic engineering methods. Using the red fluorescent protein gene (DsRed) as a marker gene and the 3xP3 promoter as the promoter of the marker gene expression cassette, and using the heavy chain of Bombyx mori fibroin protein (FibH) as the endogenous target gene, a homologous recombination vector for CRISPR gene editing technology was constructed, which contains the homologous arm sequence of the fibroin heavy chain gene, the tetramer of the orange fluorescent protein (OFP) gene, the marker gene expression cassette of the red fluorescent protein gene (DsRed), and the homologous arm sequence of the fibroin heavy chain gene.

[0135] The homologous recombination vector, Cas9 mRNA and sgRNA mRNA were injected into silkworm eggs within 6 h after oviposition, and about 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 80% for cultivation and hatching.

[0136] The hatched newly hatched silkworms 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 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 fed with fresh mulberry leaves until they spun cocoons. The G1 generation positive adults were crossed with wild type again to produce seeds. After mating, 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.

[0137] The G2 generation PCR experiment detected and proved that the exogenous gene had been successfully introduced into the Bombyx mori genome. The Western blot method was used to prove that the silk composite silk of the Bombyx mori cocoon contained bands of the fusion protein with the expected size. Starting from the G3 generation, all positive individuals were screened and self-crossed to save seeds until a stable genetic variety was developed. The G8 generation silkworm cocoons were orange visible to the naked eye and had uniform color. The cocoons were placed in a 100 °C warm water bath for 2 h to soften the sericin for reeling. The orange color of the silk remained stable under visible light without fading. The entire silk length of about 1200 m showed orange color with uniform color distribution. The silk was further refined with 50 °C water, 1% alkaline protease, and pH 9 for 2 h to basically remove the sericin, and the orange color remained stable without fading.

[0138] The silk did not fade after being baked at 100 °C for 24 h or exposed to sunlight for 7 days.

[0139] Example 4

[0140] By means of genetic engineering, biosynthesize an octamer gene sequence optimized according to the codon preference of Bombyx mori, which is a sequence of four red fluorescent protein (RFP) genes and four cyan fluorescent protein (CFP) genes linked together. Use the red fluorescent protein gene (DsRed) as a marker gene and the IE-1 promoter as the promoter of the marker gene expression cassette. Use the Bombyx mori fibroin P25 protein (P25) as an endogenous target gene to construct a homologous recombination vector for TALEN gene editing technology, which contains a homologous arm sequence of the fibroin P25 protein gene, an octamer gene formed by linking four red fluorescent protein (RFP) genes and four cyan fluorescent protein (CFP) genes, an expression cassette with the red fluorescent protein gene (DsRed) as a marker gene, and a homologous arm sequence of the fibroin P25 protein gene.

[0141] Inject the above homologous recombination vector together with TALEN mRNA into silkworm eggs within 2 hours after spawning, and inject about 5 nL of the sample into each silkworm egg. After the injection is completed, place the silkworm eggs in an incubator at 25 °C with a relative humidity of 85% for cultivation and hatching.

[0142] Raise the newly hatched silkworm larvae with fresh mulberry leaves until they spin cocoons. Cross the G0 generation adults with wild-type ones to breed and produce G1 generation individuals. Select positive individuals expressing the red fluorescent protein marker gene (DsRed) among the first-instar larvae of the G1 generation under a fluorescence microscope. Raise the G1 generation positive larvae with fresh mulberry leaves until they spin cocoons. Then cross the G1 generation positive adults with wild-type ones again to breed. Extract the genome from the adults after breeding is completed, and use the genome as a template for genotype amplification verification to detect the expected position of the exogenous gene homology in the Bombyx mori genome.

[0143] The G2 generation PCR experiment detected and proved that the exogenous gene has been successfully introduced into the Bombyx mori genome. The Western blot method was used to prove that the silk composite of the Bombyx mori cocoon contains a band of the fusion protein with the expected size. Starting from the G3 generation, all positive individuals were screened and self-crossed to save seeds. By the G8 generation, a stable genetic variety was bred. The G8 generation silkworm cocoons are visibly black to the naked eye, which is the result of the synergistic action of red fluorescent protein and cyan fluorescent protein, and the color is uniform.

[0144] After the silkworm cocoons are exposed to sunlight for one week, the color does not fade. After baking at 100 °C for 24 hours, the color also does not fade.

[0145] Use the following method for reeling silk:

[0146] 1. Put the black positive silkworm cocoons into boiling water and boil for 2 minutes, press up and down to expel the air in the cocoon shell;

[0147] 2. Then, quickly transfer the silkworm cocoons into a low-temperature water bath at 65 - 55°C for 2 minutes, and press up and down to allow the boiling water to fully enter the cocoon shells; then quickly put the silkworm cocoons into boiling water at 100°C for 2 - 3 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;

[0148] 3. Then, gradually cool the temperature of the water bath in the air to 87°C for 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, put it into a beaker with a water bath at about 65°C and extract the silk.

[0149] 4. Further boil the silk in boiling water at 100°C for 1 hour, and perform the same operation 3 times in total to refine the silk.

[0150] After cocoon cooking, reeling, and refining, expose it to sunlight for 10 days. Under visible light, the black color of the silk remains stable without fading. The entire length of the silk is about 1050m and shows a black color with uniform color distribution.

[0151] Example 5

[0152] By means of genetic engineering, biosynthesize a foreign gene optimized according to the codon preference of Bombyx mori - a multimer gene sequence linked with 4 red fluorescent protein (RFP) genes or 4 green fluorescent protein (GFP) genes. Use the enhanced green fluorescent protein gene (EGFP) as the marker gene and the IE-1 promoter as the promoter of the marker gene expression cassette. Use the Bombyx mori sericin 1 (Ser1) gene or the fibroin light chain protein gene (FibL) as the endogenous target gene, and construct 2 homologous recombination vectors for the TALEN gene editing technology respectively. One homologous recombination vector contains the homologous arm sequence of the sericin 1 gene, the tetramer gene sequence formed by linking 4 red fluorescent protein (RFP) genes, and the gene of the homologous arm sequence of the sericin 1 gene; the other homologous recombination vector contains the homologous arm sequence of the fibroin light chain protein gene, the tetramer gene sequence formed by linking 4 green fluorescent protein (GFP) genes, the expression cassette with the enhanced green fluorescent protein gene (EGFP) as the marker gene, and the homologous arm sequence of the fibroin light chain protein gene.

[0153] Inject the above 2 kinds of homologous recombination vectors and 2 kinds of TALEN mRNAs into silkworm eggs within 4 hours after spawning, and inject about 8 nL of the sample into each silkworm egg. After injection, put the silkworm eggs into an incubator at 25°C with a relative humidity of 85% for cultivation and hatching.

[0154] The hatched newly-hatched silkworms are fed with fresh mulberry leaves until they spin cocoons. The G0 generation adults are self-crossed or crossed with wild-type to produce G1 generation individuals. Under a fluorescence microscope, positive individuals expressing the red fluorescent protein gene, green fluorescent protein gene, or both red and green fluorescence in the first instar larvae of the G1 generation are selected. The G1 generation positive adults with red fluorescence or green fluorescence are crossed with wild-type to produce seeds, and the G1 generation positive adults expressing both red and green fluorescence are self-crossed to produce seeds. After 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 exogenous gene homology in the silkworm genome.

[0155] The G2 generation is detected by PCR experiments to prove that the exogenous gene has been successfully introduced into the silkworm genome, and the Western blot method is used to prove that the composite silk of silkworms contains bands of the fusion protein with the expected size. The G2 larvae with correct molecular identification are reared, and starting from the G3 generation, all positive individuals are screened and self-crossed to save seeds until a stable hereditary variety is developed in the G8 generation. Three cocoon-color silkworm varieties can be obtained in the G8 generation. The silkworm cocoons are visibly red, green, and yellow. After inspection, the yellow variety is a variety developed by simultaneously introducing two endogenous silk protein genes of silkworms into two homologous recombinant vectors of red and green, and yellow is the result of the synergistic effect of red and green. The cocoon color of each variety is uniform.

[0156] The red-cocoon variety and the green-cocoon variety are crossed to produce a hybrid variety, and the cocoon color of the hybrid variety obtained by rearing is yellow formed by the synergistic effect of red and green.

[0157] The following method is used for reeling silk:

[0158] 1. Put the colored positive cocoons into boiling water and boil for 5 minutes, press up and down to discharge the air in the cocoon shell;

[0159] 2. Then quickly transfer the cocoons into a low-temperature water bath at 65 °C for 3 minutes, press up and down to make the boiling water fully enter the cocoon shell; then quickly put the cocoons into boiling water at 100 °C for 4 minutes, press up and down to make the boiling water fully enter the cocoon shell, soak the cocoons, and make the sericin swell and soften;

[0160] 3. Then, gradually cool the temperature of the water bath in the air to 80 °C for about 15 minutes, and the sericin is further softened to achieve the purpose of appropriate 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.

[0161] 4. The silk is further boiled in 100 °C water for 15 minutes, and the same operation is carried out 3 times to refine the silk.

[0162] After boiling cocoons, reeling and refining, the original color of the silk remains stable under visible light without fading. Each silk strand is about 1000m in total length and shows its original color with even color distribution.

[0163] All colored silks will not fade after being exposed to sunlight for 10 days or baked at 100℃ for 48 hours.

[0164] Comparative Example 1:

[0165] The homologous recombination vector containing the homology arm sequence of the silk fibroin P25 protein gene + 3xP3 promoter + green fluorescent protein marker gene (EGFP) + polyA + homology arm sequence of the silk fibroin P25 protein gene was injected into silkworm eggs within 2 hours after egg laying using TALEN gene editing technology, and about 5nL of sample was injected into each silkworm egg. After the injection, the silkworm eggs were placed in an incubator at 25°C and a relative humidity of 85% for culture and hatching. The hatched silkworms were fed with fresh mulberry leaves until they spun silk and formed cocoons.

[0166] G0 generation adults mate with wild type to produce G1 generation individuals. Because the 3xP3 promoter only activates the specific expression of the green fluorescent protein marker gene (EGFP) in the nervous system, the eyes of the G1 generation first-instar larvae show green fluorescence under a fluorescence microscope. However, because there is no exogenous fluorescent protein gene to fuse with the endogenous silk protein gene to become a fluorescent silk protein gene, the silk does not contain fluorescent fusion protein and the cocoon has no color.

[0167] The green fluorescent positive adults of the G1 generation were then mated with the wild type to produce seeds. PCR experiments in the G2 generation proved that the green fluorescent protein marker gene (EGFP) had been successfully inserted behind the silk fibroin P25 protein gene. Starting from the G3 generation, all positive individuals were screened and self-pollinated for seed preservation. By the G8 generation, varieties with stable genetic expression of the green fluorescent protein gene (EGFP) were bred.

[0168] However, the cocoons of each generation from G2 to G8 were colorless, which indicated that the fusion of exogenous fluorescent protein gene and endogenous silk protein gene to form fluorescent silk protein gene was the basis for silk to become colored silk.

[0169] Comparative Example 2:

[0170] Construct a transgenic vector mediated by the piggyBac transposon. The vector contains the left and right arms of the piggyBac transposon, the expression cassette of the green fluorescent protein marker gene driven by the IE1 promoter, and the expression cassette of the exogenous red fluorescent protein gene (RFP) driven by the promoter of the heavy chain gene of Bombyx mori silk fibroin. The structure is the left arm of the piggyBac transposon + IE-1 promoter + green fluorescent protein marker gene + polyA + promoter of the heavy chain gene of Bombyx mori silk fibroin + red fluorescent protein gene (RFP) + polyA sequence of the heavy chain gene of Bombyx mori silk fibroin + the right arm of the piggyBac transposon.

[0171] Mix the above 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 the silkworm eggs are laid. The total volume of the injection is 7 nl. Raise the microinjected silkworm eggs at 25°C and 85% humidity until they become adults, and cross them with the wild-type variety for passaging to obtain the G1 generation. After the G1 generation of transgenic silkworm larvae hatched from the transgenic experiment are observed under a fluorescence microscope to obtain transgenic silkworms that are positive for the expression of the EGFP marker gene, raise them to adults, and let the transgenic silkworms self-cross and pass on, which is the G2 generation. From the G2 generation onwards, single-moth rearing is adopted for the transgenic silkworms. Observe under 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 them within the same moth area.

[0172] At the G2 generation, using the genomic DNA of the posterior silk gland of the transgenic silkworm on the 3rd day of the 5th instar as a template, perform Inverse PCR to amplify the inserted fragment of the transgenic vector in the silkworm genome, and conduct 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.

[0173] Starting from the G4 generation, select the moth areas with a pure green fluorescence phenotype for rearing, and use the silkworm moths within the same moth area to mate. By the G8 generation, a new variety of transgenic silkworms that are homozygous for the green fluorescent protein gene and whose posterior silk gland cells can synthesize and secrete red fluorescent protein are developed.

[0174] Extract the posterior silk gland and cocoon silk protein of the above 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 corresponding to the expected molecular weight are obtained.

[0175] 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 cocoons along with the behavior of spinning silk and cocoon formation. The cocoons are visibly red to the naked eye.

[0176] Reel silk using the following method:

[0177] 1. Place the red positive cocoons in boiling water and boil for 2 min. Press up and down to expel the air inside the cocoon shells. The water in the water bath turns red and the red color of the cocoons fades;

[0178] 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 shells. The water in the water bath turns red and the red color of the cocoons fades further;

[0179] 3. 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 shells, soak the cocoons, and make the sericin swell and soften. The red color of the cocoons fades further;

[0180] 4. Then, gradually cool the temperature of the water bath in the air to 87 °C, about 15 min. The sericin softens further 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 containing a water bath at about 65 °C and extract the silk.

[0181] 5. Boil the silk in 100 °C water for 1 h and repeat the same operation 3 times to refine the silk. Each time the silk is refined, the color of the silk fades once.

[0182] The results prove that during the cocoon cooking and reeling, and refining processes, because the exogenous fluorescent protein adheres to the silk and does not fuse with the silk protein molecules to form a fusion protein, does not participate in the formation of silk at a constant molar ratio and firmly exists in the whole silk, so the exogenous fluorescent protein is continuously dissolved and disappears, and the red color of the silk continuously fades.

[0183] The above specific embodiments are used to explain and illustrate the present invention, rather than to limit the present invention. Any modifications, equivalent replacements, 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 colored silk that is naturally colored, non-fading, and pollution-free, characterized in that: The method comprises the following steps: using a fluorescent protein gene or a polymer composed of several fluorescent protein genes as an exogenous gene, introducing the exogenous gene into silkworms through homologous recombination, and forming a fusion protein gene with an endogenous silk protein gene of the silkworms, breeding the silkworms through multiple generations to finally obtain a new silkworm variety that can stably inherit and express the fusion protein gene, and using the new silkworm variety to produce silk. The silk does not need to be dyed, has its own color under visible light, and the color is stable and uniform.

2. The method for preparing the self-colored, non-fading, pollution-free colored silk according to claim 1, characterized in that: The method is to utilize TALEN or CRISPR gene editing homologous recombination technology to homologously recombine an exogenous gene with an endogenous silk protein gene of the silkworm to form a fusion protein gene, and then cultivate a new silkworm variety with stable inheritance, so that the new silkworm variety synthesizes and secretes colored silk with the fusion protein.

3. The method for preparing the self-colored, non-fading, pollution-free colored silk according to claim 1, 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.

4. The method for preparing the self-colored, non-fading, pollution-free colored silk according to claim 1, 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.

5. The method for preparing the self-colored, non-fading, pollution-free colored silk according to claim 1, characterized in that: The method specifically 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 and integrating the homologous recombination vector into a specific endogenous silk protein gene of the silkworm, and then obtaining G1 generation positive individuals by screening with a fluorescent microscope or 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, and performing multi-generation screening and cultivation of silkworms to breed silkworm varieties with stable inheritance and expression of silk protein + fluorescent protein genes in the G8 generation.

6. The method for preparing the self-colored, non-fading, pollution-free colored silk according to claim 5, 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.

7. The method for preparing the self-colored, non-fading, pollution-free colored silk according to claim 6, characterized in that: The fluorescent screening marker gene expression frame includes a fluorescent screening marker gene promoter+a fluorescent screening marker gene+a polyA sequence, the promoter is an IE-1, A3 or 3xP3 promoter, and the fluorescent screening marker gene is a green fluorescent protein gene (EGFP) or a red fluorescent protein gene (DsRed).

8. The method for preparing the self-colored, non-fading, pollution-free colored silk according to claim 5, 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.

9. A self-colored, non-fading, pollution-free colored silk, characterized by: Prepared by the preparation method described in any one of claims 1 to 8.

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