Method for improving performance of silk by combining in-vivo and in-vitro combined action of gene manipulation and physicochemical treatment

Abstract

The invention discloses a method for improving the performance of silk through the in-vivo and in-vitro combined effect of gene manipulation combined with physical and chemical treatment. According to the method, different silkworm epidermis protein genes are adopted to construct an exogenous gene vector, and the epidermis protein genes are integrated into a silkworm genome by utilizing a transgenic or gene editing molecular biological technology, so that a novel silkworm variety which can be stably inherited is finally obtained. Then a zinc metal ion cross-linking method or a substrate enzymatic reaction method is used for changing the properties of the composite filament, and the composite filament with excellent performance is produced; the exogenous gene refers to a silkworm epidermal protein gene, and can be one of RR-1, RR-2, RR-3, CPG, CPH or Glycine-rich. The mechanical property of the composite yarn obtained through treatment is remarkably enhanced after the composite yarn is subjected to chemical treatment, the quality of the silk can be greatly improved, and good economic benefits are created.

Description

Technical Field

[0001] The present invention relates to a method for constructing a functional protein molecule and combining it with metal ion crosslinking or substrate enzymatic reaction crosslinking, and in particular to a method for manufacturing high-performance composite yarn by combining epidermal protein with metal ion crosslinking or substrate enzymatic reaction crosslinking and utilizing such in vivo and in vitro combined action. Background Art

[0002] Silk fiber is the earliest natural silk material used and produced in the largest amount. It is known as the "Queen of Fibers" because of its strong hygroscopicity, comfortable feel and good breathability. Traditional silk fiber is mainly used in the textile industry. With the improvement of processing technology and the development of interdisciplinary subjects, many functional silk fibers have been developed, expanding the application field, such as wound healing, biosensors, energy harvesting, etc.

[0003] However, the tensile strength of silk is lower than that of Kevlar and nylon. In addition, silk as a textile material has defects such as easy fading and wrinkling. These problems limit the further application and promotion of silk fibers. In order to solve the problems of insufficient mechanical properties of silk fibers, researchers at home and abroad have tried to use different methods to improve the quality of silk. The main methods include physical and chemical methods, food addition methods and genetic manipulation methods.

[0004] The main physical and chemical treatment methods include heat treatment, adjusting the spinning speed, and radiation treatment; the feeding method is to add graphene, nanoparticles, or metal elements to mulberry leaves or artificial feed to feed silkworms or directly inject them into the silkworms to improve the performance of silk fibers; the genetic manipulation method is to use genetic manipulation technology to introduce foreign genes, mainly spider silk genes, into silkworms, genetically modify silkworms, cultivate new varieties, and obtain high-performance silk materials. The above-mentioned improvement methods either rely on the treatment of silk protein molecules themselves to improve silk performance, or improve silk performance by introducing foreign protein molecules, which have many problems such as cumbersome operation and poor results. Summary of the invention

[0005] In order to solve the problems existing in the background technology, the purpose of the present invention is to develop an indirect or direct combination of epidermal protein and silk protein, pre-set functional molecules in the silkworm body, and establish an in vivo and in vitro combined action to develop a new high-performance composite silk method by cross-linking the functional molecules with metal ions or with substrates.

[0006] The invention greatly improves the economic value of silk, creates a new situation in sericulture production, and has great economic benefits and broad application prospects.

[0007] In order to achieve the above object, the steps of the technical solution adopted by the present invention are as follows:

[0008] The method first uses gene manipulation to introduce exogenous genes into the silkworm genome and cultivate new transgenic silkworm varieties, and then selects and physically and chemically treats silk substances secreted by the silkworms outside the body of the silkworms, thereby improving the performance of the silk and producing composite silk with excellent performance.

[0009] The method specifically comprises the following steps: firstly using genetic manipulation techniques such as transgenic or gene editing to introduce foreign genes into the silkworm genome or precisely homologously integrate them into specific sites of the silkworm genome, then cultivating a new silkworm variety with stable inheritance, synthesizing and secreting composite silk protein, and then using an ion crosslinking method or a substrate enzymatic reaction method to perform physical and chemical treatment on the composite silk protein to change the properties of the composite silk protein.

[0010] The gene editing comprises TALEN or CRISPR.

[0011] The exogenous gene refers to a silkworm cuticle protein gene, specifically one of RR-1, RR-2, RR-3, CPG, CPH or Glycine-rich:

[0012] The gene sequence and protein sequence of the silkworm cuticle protein gene RR-1 are SEQ ID NO.1 and SEQ ID NO.2 respectively;

[0013] The gene sequence and protein sequence of the silkworm cuticle protein gene RR-2 are SEQ ID NO.3 and SEQ ID NO.4 respectively;

[0014] The gene sequence and protein sequence of the silkworm cuticle protein gene RR-3 are SEQ ID NO.5 and SEQ ID NO.6 respectively;

[0015] The gene sequence and protein sequence of the silkworm cuticle protein gene CPG are SEQ ID NO.7 and SEQ ID NO.8 respectively;

[0016] The gene sequence and protein sequence of the silkworm cuticle protein gene CPH are SEQ ID NO.9 and SEQ ID NO.10 respectively;

[0017] The gene sequence and protein sequence of the silkworm cuticle protein gene Glycine-rich are SEQ ID NO.11 and SEQ ID NO.12 respectively.

[0018] The gene manipulation is to introduce the silkworm cuticle protein gene into the silkworm genome, or precisely and homologously integrate it into the silk heavy chain gene, silk light chain gene, silk P25 gene, and sericin 1 gene of the silkworm.

[0019] The physical and chemical treatment is to further process the composite silk protein in vitro, specifically, to add zinc metal ions to the composite silk protein in vitro or during the reeling process, so that the composite silk protein molecules are cross-linked with the zinc ions, thereby improving the performance of the composite silk and obtaining a composite silk with improved mechanical properties.

[0020] The physical and chemical treatment is to further treat the composite silk protein in vitro, specifically, to add a substrate to the composite silk protein in vitro or during the reeling process for enzymatic reaction, so that the composite silk protein is cross-linked to improve the performance of the composite silk, the substrate is N-acetyl dopamine (NADA) or N-β-propionyl dopamine (NBAD), and the enzyme is laccase 2.

[0021] The present invention utilizes transgenic or gene editing technology in combination with in vitro physical and chemical treatment to enable silkworms to produce composite silk with excellent mechanical properties.

[0022] More specifically, the method is to firstly biosynthesize the target silkworm epidermal protein gene sequence as a foreign gene by genetic engineering methods, then construct a foreign gene vector by molecular biology technology, introduce the foreign gene vector into silkworm eggs by microinjection and integrate it into the silkworm genome, or precisely and homologously integrate it into a specific gene of the silkworm, cultivate the silkworm for multiple generations, screen the G1 generation positive individuals by fluorescence microscopy, mate the G1 generation positive individuals with wild types or self-pollinate the positive individuals to obtain the G2 generation, screen the positive individuals after the G3 generation and use the positive individuals for self-pollination to keep seeds, and breed a stable genetic variety to the G8 generation;

[0023] Then, G8 generation silkworms are used to produce secretory silk, which is a composite silk protein. Zinc ions are added to the silk to allow the silk protein molecules to cross-link with the zinc ions, or mixed with the substrate N-acetyl dopamine (NADA) or N-β-propionyl dopamine (NBAD), and finally laccase 2 is added to catalyze the cross-linking reaction, thereby improving the performance of the composite silk.

[0024] The target gene sequence is the gene base sequence corresponding to the silkworm cuticle protein, and finally a new silkworm variety with excellent composite silk performance that can be stably inherited is obtained.

[0025] The positive individuals are individuals that show fluorescence and correctly express the exogenous gene.

[0026] The wild type refers to individuals cultivated from silkworm eggs without the introduction of exogenous gene vectors.

[0027] The exogenous gene vector corresponding to the histone molecule is a transgenic vector using piggyBac as a skeleton or a homologous recombination vector used for TALEN or CRISPR gene editing;

[0028] The exogenous gene vector comprises an exogenous gene expression frame and a fluorescent screening marker gene expression frame;

[0029] The exogenous gene expression frame includes an exogenous gene and a promoter of the expression frame, the exogenous gene is a gene base sequence corresponding to the protein sequence of the silkworm cuticle protein gene RR-1, RR-2, RR-3, CPG, CPH or Glycine-rich as described in claim 2, and the promoter of the exogenous gene expression frame adopts the promoter of the silk fibroin heavy chain, silk fibroin light chain, silk fibroin P25 or sericin gene;

[0030] The fluorescent screening marker gene expression frame includes a fluorescent screening marker gene and a promoter of the expression frame. The fluorescent screening marker gene uses a green fluorescent protein gene (GFP) or a red fluorescent protein gene (DsRed), and the promoter of the fluorescent screening marker gene expression frame uses an IE-1, A3 or 3xP3 promoter.

[0031] The composition of the composite silk protein includes protein molecular components composed of one gene among the silkworm cuticle protein genes RR-1, RR-2, RR-3, CPG, CPH or Glycine-rich and silk protein components. The present invention adopts the combined effect of genetic manipulation and physical and chemical treatment in vivo and in vitro to enhance the mechanical properties of the composite silk.

[0032] With the development of sequencing technology, a large number of epidermal protein gene sequences have been discovered. According to the amino acid motif characteristics of epidermal proteins, they can be divided into several protein families. Most epidermal proteins contain the R&R conservative motif, and epidermal proteins with the R&R motif can be further divided into three subfamilies: RR-1, RR-2 and RR-3.

[0033] The silkworm cuticular protein genes are mostly distributed in the form of tandem clusters on chromosomes. A 2008 study speculated that there are a total of 220 cuticular protein genes in the silkworm genome, and there may even be 247 cuticular protein genes. According to their motif characteristics, they can be divided into 8 families, and the largest number of cuticular proteins belongs to the RR-2 subfamily.

[0034] The present invention has found that the stress performance, strain performance, wear resistance and wrinkle resistance of the recombinant silk composed of RR-1, RR-2, RR-3, CPG, CPH or Glycine-rich epidermal proteins and the composite silk combined with physical and chemical treatment are significantly improved, and new composite silk can be developed to expand new uses of silk.

[0035] The present invention creatively constructs composite silk with cuticle protein and silk, and then obtains a protein sequence capable of improving the mechanical properties of the composite silk through physical and chemical treatment.

[0036] The present invention has the following beneficial effects:

[0037] The present invention develops a method for artificially designing and synthesizing a functional gene for silk protein and improving the mechanical properties of composite silk from silkworms, and develops new functions of multiple genes. By utilizing this gene, the mechanical properties of the composite silk are significantly enhanced after chemical treatment. This invention can greatly improve the quality of silk and create good economic benefits. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with the embodiments.

[0039] Embodiments of the present invention are as follows:

[0040] Example 1

[0041] The silkworm cuticle protein molecule RR1 gene sequence is optimized according to the silkworm codon preference. The silkworm cuticle protein molecule RR1 gene base and protein sequences established after optimization are as SEQ ID NO.1 and SEQ ID NO.2. The target gene sequence is biosynthesized by a genetic engineering method. The silkworm fibroin light chain promoter is used as the expression cassette promoter. Together with a red fluorescent protein marker gene expression cassette using an IE-1 promoter, a transgenic vector is constructed using molecular biology technology. The vector is introduced into silkworm eggs by microinjection and integrated into the silkworm genome.

[0042] G1 positive individuals were screened by fluorescence microscopy, and the positive individuals were mated with wild types to obtain G2 generation. After G3 generation, positive individuals were self-pollinated to keep seeds, and stable genetic varieties were bred to G8 generation. Using G2 generation as material, PCR experiments verified that the exogenous gene had been successfully introduced into the silkworm genome. Fluorescence quantitative PCR detection showed that spider silk gene expression was significant in silkworm varieties. Western blot method was used to prove that the silkworm composite silk contained the expected size of silkworm cuticular protein molecule RR1 protein band.

[0043] The transgenic silkworm cocoons were incubated in deionized water at 60°C for 30 minutes, and then immersed in a solution containing 10 mM zinc metal ions and 0.5 mM Tris-HCl for 12 hours at 37°C and 150 rpm. Finally, the cocoons were washed with deionized water, and composite silk was further obtained.

[0044] At the same time, the above treatment is also performed on the silk synthesized by the wild-type silkworms.

[0045] The mechanical properties of the composite silk obtained from the above treatment were tested, and the stress and strain mechanical properties of the composite silk were significantly higher than those of the wild-type Bombyx mori silk, with the stress increased by 160% and the strain increased by 198%.

[0046] Example 2

[0047] The silkworm cuticular protein molecule RR2 gene sequence was optimized according to the silkworm codon preference. The optimized silkworm cuticular protein molecule RR2 gene base and protein sequences such as SEQ ID NO.3 and SEQ ID NO.4 were established. The target sequence was biosynthesized by genetic engineering methods. The silkworm sericin 1 gene promoter was used as the expression frame promoter. Together with the green fluorescent protein marker gene expression frame using the IE1 promoter, a homologous recombination vector was constructed using molecular biology technology. The vector was introduced into silkworm eggs by microinjection and integrated into the silkworm sericin 1 gene.

[0048] The positive individuals of G1 generation were screened by fluorescence microscopy, and the positive individuals were self-mated to obtain G2 generation. After G3 generation, the positive individuals were self-mated to keep seeds, and stable genetic varieties were bred to G8 generation. The G2 generation was used as the material and the PCR experiment verified that the exogenous gene had been successfully introduced into the silkworm genome. Fluorescence quantitative PCR detection showed that the RR2 silkworm cuticle protein molecule gene was significantly expressed in the silkworm variety. The Western blot method was used to prove that the silkworm composite silk contained a band of the expected size of the fusion protein.

[0049] The transgenic silkworm cocoons were immersed in a solution containing 100 mM zinc metal ions and 1 mM Tris-HCl for 12 h at 37° C., and then reeled. Finally, the composite silk was washed with deionized water.

[0050] At the same time, the above treatment is also performed on the silk synthesized by the wild-type silkworms.

[0051] The mechanical properties of the above composite silk were tested, and the stress and strain mechanical properties were significantly higher than those of wild-type silkworm silk. The stress was increased by 340%, the strain was increased by 203%, the wear resistance was increased by 3.5 times, and the wrinkle resistance and color fastness were significantly improved.

[0052] Example 3

[0053] The silkworm cuticle protein molecule RR3 gene sequence is used and optimized according to the silkworm codon preference. The silkworm cuticle protein molecule RR3 gene base and protein sequences established after optimization are as SEQ ID NO.5 and SEQ ID NO.6. The target sequence is biosynthesized by a genetic engineering method. The silkworm fibroin P25 protein promoter is used as an expression frame promoter. Together with a green fluorescent protein marker gene expression frame using an A3 promoter, a homologous recombination vector is constructed using molecular biology technology. The vector is introduced into silkworm eggs by microinjection and integrated into the silkworm fibroin protein P25 genome.

[0054] G1 positive individuals were screened by fluorescence microscopy, and the positive individuals were self-mated to obtain the G2 generation. After the G3 generation, the positive individuals were self-mated to keep the seeds, and the G8 generation was bred to breed stable genetic varieties. The G2 generation was used as the material and the PCR experiment verified that the exogenous gene had been successfully introduced into the silkworm genome. Fluorescence quantitative PCR detection showed that the RR3 silkworm epidermal protein gene was significantly expressed in the silkworm variety. The Western blot method was used to prove that the silkworm composite silk contained the expected size of the RR3 fusion protein molecule band.

[0055] The transgenic silkworm cocoons were immersed in a solution containing 500 mM zinc metal ions and 2 mM Tris-HCl for 18 h at a temperature of 50° C., and then reeled. Finally, the composite silk was washed with deionized water.

[0056] At the same time, the above treatment is also performed on the silk synthesized by the wild-type silkworms.

[0057] The mechanical properties of the composite yarn were tested, and the stress and strain mechanical properties were significantly higher than those of the wild type, with the stress increased by 180% and the strain increased by 158%.

[0058] Example 4

[0059] The silkworm cuticle protein molecule RR2 gene sequence is optimized according to the silkworm codon preference. The silkworm cuticle protein molecule RR2 gene base and protein sequences established after optimization are as SEQ ID NO.3 and SEQ ID NO.4. The target sequence is biosynthesized by a genetic engineering method. The silkworm fibroin P25 promoter is used as an expression frame promoter. Together with a red fluorescent protein marker gene expression frame using an IE1 promoter, a homologous recombination vector is constructed using molecular biology technology. The vector is introduced into silkworm eggs by microinjection and integrated into the silkworm fibroin P25 protein gene.

[0060] The positive individuals of G1 generation were screened by fluorescence microscopy, and the positive individuals were self-mated to obtain G2 generation. After G3 generation, the positive individuals were self-mated to keep seeds, and stable genetic varieties were bred to G8 generation. The G2 generation was used as the material and the PCR experiment verified that the exogenous gene had been successfully introduced into the silkworm genome. Fluorescence quantitative PCR detection showed that the RR2 silkworm cuticle protein molecule gene was significantly expressed in the silkworm variety. The Western blot method was used to prove that the silkworm composite silk contained a band of the expected size of the fusion protein.

[0061] The transgenic silkworm silk was immersed in a solution containing 200 mM substrate N-acetyldopamine (NADA) and 10 u laccase 2 for 12 h at 37°C to perform a cross-linking catalytic reaction to further improve the performance of the composite silk, and then reeled in the above environment.

[0062] At the same time, the above treatment is also performed on the silk synthesized by the wild-type silkworms.

[0063] The mechanical properties of the composite silk spun above were tested, and the stress and strain mechanical properties were significantly higher than those of wild-type silkworm silk, with stress increased by 400%, strain increased by 187%, wear resistance increased by 4 times, and wrinkle resistance significantly improved.

[0064] Example 5

[0065] The Bombyx mori cuticular protein molecule CPG gene sequence was optimized according to the Bombyx mori codon preference. The optimized Bombyx mori cuticular protein molecule CPG gene base and protein sequences such as SEQ ID NO.7 and SEQ ID NO.8 were established. The target sequence was biosynthesized by genetic engineering methods. The Bombyx mori sericin 1 gene promoter was used as the expression frame promoter. Together with the red fluorescent protein marker gene expression frame using the IE1 promoter, a homologous recombination vector was constructed using molecular biology technology. The vector was introduced into silkworm eggs by microinjection and integrated into the Bombyx mori sericin 1 gene.

[0066] The positive individuals of G1 generation were screened by fluorescence microscopy, and the positive individuals were self-mated to obtain G2 generation. After G3 generation, the positive individuals were self-mated to keep seeds, and stable genetic varieties were bred to G8 generation. The G2 generation was used as the material and the PCR experiment verified that the exogenous gene had been successfully introduced into the silkworm genome. Fluorescence quantitative PCR detection showed that the CPG silkworm epidermal protein molecule gene was significantly expressed in the silkworm variety. The Western blot method was used to prove that the silkworm composite silk contained a band of the expected size of the fusion protein.

[0067] The above-mentioned Bombyx mori silk was immersed in a solution containing 150 mM substrate N-β-propionyl dopamine (NBAD) and 20 u laccase 2 for 20 h at a temperature of 40° C. to perform a cross-linking catalytic reaction to further improve the performance of the composite silk.

[0068] At the same time, the above treatment is also performed on the silk synthesized by the wild-type silkworms.

[0069] The mechanical properties of the composite silk were tested, and the stress and strain mechanical properties were significantly higher than those of wild-type silkworm silk, with stress increased by 280%, strain increased by 210%, wear resistance increased by 2.8 times, and color fastness increased by 53%.

[0070] Example 6

[0071] The silkworm epidermal protein molecule CPH gene sequence is optimized according to the silkworm codon preference. The silkworm epidermal protein molecule CPH gene base and protein sequences established after optimization are as SEQ ID NO.9 and SEQ ID NO.10. The target sequence is biosynthesized by genetic engineering methods. The silkworm fibroin light chain gene promoter is used as the expression frame promoter. Together with the green fluorescent protein marker gene expression frame using the 3xP3 promoter, a homologous recombination vector is constructed using molecular biology technology. The vector is introduced into silkworm eggs by microinjection and integrated into the silkworm fibroin light chain gene.

[0072] The positive individuals of the G1 generation were screened by fluorescence microscopy, and the positive individuals were self-mated to obtain the G2 generation. After the G3 generation, the positive individuals were self-mated to keep the seeds, and the stable genetic varieties were bred to the G8 generation. The G2 generation was used as the material and the PCR experiment verified that the exogenous gene had been successfully introduced into the silkworm genome. Fluorescence quantitative PCR detection showed that the CPH silkworm epidermal protein molecule gene was significantly expressed in the silkworm variety. The Western blot method was used to prove that the silkworm composite silk contained a band of the expected size of the fusion protein.

[0073] The above-mentioned Bombyx mori silk was immersed in a solution containing 250 mM substrate N-β-propionyl dopamine (NBAD) and 30 u laccase 2 for 28 h at a temperature of 40° C. to perform a cross-linking catalytic reaction to further improve the performance of the composite silk.

[0074] At the same time, the above treatment is also performed on the silk synthesized by the wild-type silkworms.

[0075] The mechanical properties of the composite silk were tested, and the stress and strain mechanical properties were significantly higher than those of wild-type silkworm silk, with stress increased by 320%, strain increased by 214%, wear resistance increased by 3.4 times, and color fastness increased by 87%.

[0076] Example 7

[0077] The silkworm cuticle protein molecule Glycine-rich gene sequence is optimized according to the silkworm codon preference. The optimized silkworm cuticle protein molecule Glycine-rich gene base and protein sequences such as SEQ ID NO.11 and SEQID NO.12 are established. The target sequence is biosynthesized by a genetic engineering method. The silkworm fibroin heavy chain promoter is used as an expression frame promoter. Together with a green fluorescent protein marker gene expression frame using an IE1 promoter, a homologous recombination vector is constructed using molecular biology technology. The vector is introduced into silkworm eggs by microinjection and integrated into the silk fibroin heavy chain protein gene.

[0078] The positive individuals of G1 generation were screened by fluorescence microscopy, and the positive individuals were self-mated to obtain G2 generation. After G3 generation, the positive individuals were self-mated to keep seeds, and stable genetic varieties were bred to G8 generation. The G2 generation was used as the material and the PCR experiment verified that the exogenous gene had been successfully introduced into the silkworm genome. Fluorescence quantitative PCR detection showed that the Glycine-rich silkworm epidermal protein molecule gene was significantly expressed in the silkworm variety. The Western blot method was used to prove that the silkworm composite silk contained a band of the expected size of the fusion protein.

[0079] The above-mentioned Bombyx mori silk was immersed in a solution containing 50 mM substrate N-β-propionyl dopamine (NBAD) and 5 u laccase 2 for 18 h at 30° C. to perform a cross-linking catalytic reaction to further improve the performance of the composite silk.

[0080] At the same time, the above treatment is also performed on the silk synthesized by the wild-type silkworms.

[0081] The mechanical properties of the composite silk were tested, and the stress and strain mechanical properties were significantly higher than those of wild-type silkworm silk, with stress increased by 200%, strain increased by 230%, wear resistance increased by 2 times, and color fastness increased by 89%.

[0082] Comparative Example 1

[0083] The silkworm cuticle protein molecule RR1 gene sequence is optimized according to the silkworm codon preference. The silkworm cuticle protein molecule RR1 gene base and protein sequences established after optimization are as SEQ ID NO.1 and SEQ ID NO.2. The target gene sequence is biosynthesized by a genetic engineering method. The silkworm fibroin light chain promoter is used as the expression cassette promoter. Together with a red fluorescent protein marker gene expression cassette using an IE-1 promoter, a transgenic vector is constructed using molecular biology technology. The vector is introduced into silkworm eggs by microinjection and integrated into the silkworm genome.

[0084] G1 positive individuals were screened by fluorescence microscopy, and the positive individuals were mated with wild types to obtain G2 generation. After G3 generation, positive individuals were self-pollinated to keep seeds, and stable genetic varieties were bred to G8 generation. Using G2 generation as material, PCR experiments verified that the exogenous gene had been successfully introduced into the silkworm genome. Fluorescence quantitative PCR detection showed that spider silk gene expression was significant in silkworm varieties. Western blot method was used to prove that the silkworm composite silk contained the expected size of silkworm cuticular protein molecule RR1 protein band.

[0085] The mechanical properties of the composite silk obtained from the above-mentioned gene manipulation without in vitro physical and chemical treatment were tested. The stress and strain mechanical properties of the composite silk were significantly higher than those of the wild-type silkworm silk, but significantly lower than those of the composite silk after in vitro physical and chemical treatment. The stress was only increased by 70% and the strain was only increased by 86% compared with the wild-type silkworm silk. This indicates that the introduction of the exogenous gene silkworm cuticle protein molecule RR1 can significantly improve the mechanical properties of the composite silk. If further in vitro physical and chemical treatment is performed, the mechanical properties of the composite silk can be further improved. This indicates that the combined effect of the two factors in vivo and in vitro is an effective method to improve the mechanical properties of the composite silk.

[0086] Comparative Example 2

[0087] The synthetic silk secreted by wild-type silkworms was immersed in a solution containing 500 mM zinc metal ions and 2 mM Tris-HCl for 18 h at 50°C, and then reeled, and finally the composite silk was washed with deionized water.

[0088] The mechanical properties of wild-type silkworm silk after the above in vitro cross-linking reaction were tested, and the stress and strain mechanical properties were not significantly different from those of the wild type. This shows that the ability of wild-type silkworm silk to cross-link zinc metal ions is not as strong as that of silkworm cuticle proteins, resulting in little effect of in vitro cross-linking.

[0089] Comparative Example 3

[0090] The silk secreted and synthesized by the wild-type silkworm was immersed in a solution containing 200 mM substrate N-acetyldopamine (NADA) and 10 u laccase 2 for 12 h at 37° C. to perform a catalytic cross-linking reaction, and then reeled in the above environment.

[0091] The mechanical properties of the wild-type silkworm silk from the in vitro catalytic cross-linking reaction were tested, and the mechanical properties such as stress, strain, wear resistance, and color fastness were not significantly different from those of the wild-type silkworm silk. This shows that the ability of wild-type silkworm silk to catalyze cross-linking is not as strong as that of silkworm cuticle protein, resulting in little effect of in vitro catalytic cross-linking. It can be seen from the above embodiments and comparative examples that by using the method of the present invention, functional genes with improved comprehensive mechanical properties of silk were found, these functional genes were introduced or precisely homologous to the silkworm genome, and in vitro zinc metal ion cross-linking or enzyme-catalyzed cross-linking was performed. This method for improving silk performance has the advantages of strong stability, high efficiency, and low cost, and can improve economic benefits.

[0092] The above specific implementation modes are used to explain the present invention rather than to limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

[0093] The genetic sequences involved in the present invention are as follows:

[0094] SEQ ID NO.1:

[0095] Name: DNA base sequence of RR-1 epidermal protein gene

[0096] DNA type: other DNA

[0097] Source of organism: Silkworm (Bombyx Mori)

[0098]

[0099] SEQ ID NO.2:

[0100] Name: Protein sequence of RR-1 epidermal protein gene

[0101] Source of organism: Silkworm (Bombyx Mori)

[0102] MNSFKVQYVIALCVVAAVGVPVEDGKWHPYHYGDSGKYIPTDEGKYIHIPNPYIHIDNPYDGGFGPYAHDYEPYVGEATVQDQYRLILTPPKDIPFYK DGYYKNNGIKIIKQKHNYDEDKYDFDFETENKIRAEEKAVLKNANSIDEGIASKGFYEYIGPDGFMYRVDYTADENGFRPKVKRLETPYSGKWILEKVN

[0103] SEQ ID NO.3:

[0104] Name: DNA base sequence of RR-2 epidermal protein gene

[0105] DNA type: other DNA

[0106] Source of organism: Silkworm (Bombyx Mori)

[0107] ATGTGGTTCCAGGTCGCTACTCTGTGTTTAGTTGTTATTGCTGCGATGGCTCACCCACCGGCTACATCACATCAATTTCGGGTGGATCATCACGATCACCATGACCACCATCACCATGTTGACCACCACAACAATCACCATCACCACGTTGACCACGACCATCATAGCCACCATGATCACCACAGACATTTCAACCATAATGATCACCATGATCACCACGACCACCACGAACACCAAGCATATCACGGACACCATGGTCACGATGGACACCACGACCACCATGGACACCACGGACACCATGACCACCACGTAACTCATCAACAACATGGTCACTTGGACCACGCTTCGTCATACCCTTCATACCAATTTTCCTACTCAGTTGACGATCACCACACCGGTGACCACAAATCACAGTCAGAAACGCGCCATGGTGACCACGTAACAGGTGAATACTCACTCGTTGAACCCGATGGCAATGTGAGGTCTGTTCATTATAATGCTGACGACCACCACGGTTTTAACGCTGTCGTACATAACAGATTTGCCCACCACCATATCATCCCACACCACGACCACCATCACCATCACGACCATCATAATCATCACGACCACCACGAACATCACGACCATCACGACCATCACGTACATCACGACCATCACGATCACCATGACCATCACGATCATGGACACCATGAGCATCACAGTCATCACGATGGGCACCATGAACACCACCATGATCATCATCACTAASEQ ID NO.4:

[0108] Title: Protein sequence of RR-2 cuticular protein gene

[0109] Organism source: Bombyx Mori

[0110] MWFQVATLCLVVIAAMAHPPATSHQFRVDHHDHHDHHHHVDHHNNHHHHVDHDHHSHHDHHRHFNHNDHHDHHDHHEHQAYHGHHGHDGHHDHHGHHGHHDHHVTHQQHGHLDHASSYPSYQFS YSVDDHHTGDHKSQSETRHGDHVTGEEYSLVEPDGNVRSVHYNADDHHGFNAVVHNRFAHHHIIPHDHHHHHDHHNHHDHHEHHDHHDHHVHHDHHDHHDHDHGHHEHHSHHDGHHEHHHDHHH

[0111] SEQ ID NO.5:

[0112] Name: DNA base sequence of RR-3 epidermal protein gene

[0113] DNA type: other DNA

[0114] Source of organism: Silkworm (Bombyx Mori)

[0115]

[0116] SEQ ID NO.6:

[0117] Name: Protein sequence of RR-3 epidermal protein gene

[0118] Source of organism: Silkworm (Bombyx Mori)

[0119]

[0120] SEQ ID NO.7:

[0121] Name: DNA base sequence of CPG epidermal protein gene

[0122] DNA type: other DNA

[0123] Source of organism: Silkworm (Bombyx Mori) V

[0124] GCTTGTTGTAGCGAATACAAGTTCGATTGAGTAGCCAGCGTTTAGCTAATCGAAATGGCTACTATTACCAAAGTTATGTACTTAGTCGTCCTTTTGAGCAGTACCTACGCCAAACCTGGTTTTGACCAACACGATCACCATGCAACTCTTGAACACCACGATGATCACCATATCGCCAATATTGAGACAGCGCCAATAGGCCTATCTGCTCCAAGCGCTGGTGGATCTAGTTTTGCACCCAGCAGCTTCCCAGCTGGTTCGCTGCCTTCTGCTACTTTCTCCGGGGCTGGATTAATAGGGGGAGGCCATGGTGGTGGATTTGGATTCGGTGGATTTAACGGTGGCGCAGGAGGTGGCTTCGGAGGTGCCATTGGTGGACCTCAGGGTATCTTCCCCGGCGGTGATGGACCTCTTGCGGGTCTCAAATCACCAGTTATTGATGAGATCCATGGACTCGCCAACAACCATGGAGCTCCTACGCAGTATCGCATTCGTGAGGAACCCTATGAAGTATACCGTGAAGTGACCCACCGTGTGCCTCAGCCTGTGCCAGTTCCGGTACCTCACCCCGTCCAAGTTCCCGTGCCCCATCCCTACCCAGTTCAAGTTCCCGTCATAAGGAAAATCCCAGTGCCAGTAGTAAAAATCCAACACGTGAAAGTCGATAGGCCAGTGCCTTATCCCGTAGAGAAGATCATTAAGGTTCCAGTTGAGAAGGTCGTAGAAGTACCCGTGGATCATCCAGTCCCTGTTGAAAAAATAGTTGAAGTTCCAATTGTGAAACTCATCAAGGTTCCAGTGCACATTGTGAAGTCATACCCAGTTCCAGTAGTGAAGACTGTCCACCACAAGGCTCACGTCAGCCACCACCACAGCCACGGCTGGTCCGGCTGGTAA

[0125] SEQ ID NO.8:

[0126] Name: Protein sequence of CPG epidermal protein gene

[0127] Source of organism: Silkworm (Bombyx Mori)

[0128] MATITKVMYLVVLLSSTYAKPGFDQHDHHATLEHHDDHHIANIETAPIGLSAPSAGGSSFAPSSFPAGSLPSATFSGAGLIGGGHGGGFGFGGFNGGAGGGFGGAIGGPQGIFPGGDGPLAGLKSPVIDEIHGLANNHGA PTQYRIREEPYEVYREVTHRVPQPVPVPVPHPVQVPVPHPYPVQVPVIRKIPVPVVKIQHVKVDRPVPYPVEKIIKVPVEKVVEVPVDHPVPVEKIVEVPIVKLIKVPVHIVKSYPVPVVKTVHHKAHVSHHHSHGWSGW

[0129] SEQ ID NO.9:

[0130] Name: DNA base sequence of CPH epidermal protein gene

[0131] DNA type: other DNA

[0132] Source of organism: Silkworm (Bombyx Mori)

[0133]

[0134] SEQ ID NO.10:

[0135] Name: Protein sequence of CPH epidermal protein gene

[0136] Source of organism: Silkworm (Bombyx Mori)

[0137] MNALVVLFALASVTCGSLVPLAQPPHHPALVLDPHGRPLDTAEVINARALHLQAKALEHHDAHLVHAAVAPVVHSIAPAVVPAAVSHQSRVDIHSSPAIVSHAITTPVLTHSAIAHFPVLTQSTLGHEHFLCKRSVALVAPSAVSHQARVDVI SKPTLVSHAVIAPILRHSVATPFVTHAVAAPIVAVAPAAVSHQSRVDVHSSPAVVTHAISPLSAVWTGATYGAHSISHGHLLKKRSLAAVVTPVLHTAPAAVSHQSRVDVISEPAIVSHEIATPLAAAPLAHSAIWTAPVAHLGYSGLFKGLW

[0138] SEQ ID NO.11:

[0139] Name: DNA base sequence of Glycine-rich epidermal protein gene

[0140] DNA type: other DNA

[0141] Source of organism: Silkworm (Bombyx Mori)

[0142]

[0143] SEQ ID NO.12:

[0144] Name: Protein sequence of Glycine - rich cuticular protein gene

[0145] Organism source: Bombyx Mori

[0146] MKYTVILVASLAVVAFAKEEKGTPKAVEEKKQDKRGIYDIGSYGGHNFGGSDEGYGGHESYGGHEGISFGHQEGHDFGGESYNLGGHGGGEYGSSGGDWKPIASEGDYHHGHHEHHEHIKTVEVVKKVPVPYTVEKHVPYTVEKKVPYEVKVPVPQPYTVEKKVPFTVKEYVKYPVYVPEPYTVEKKVPYEVKVHVDKPYEVKVKVPTPYTVEKKIPYEVKVPVPHPYTVEKKVPVPVKYEVKVPQPYEVIKKVPYEVKVPVDRPYNVYVPKPYPVHVEKPYPVTVHKPVPYEVKVPVDKPYKVEVEKPYPVPVKVPVPKPYEPIKKIPYTVEKKVPYEVKVPIDKPYPVYKEVQVPLVKEVPYPVKYHVPIYFKKEEEHHQHHQHHGWH。

Claims

1. A method for improving silk properties by combining genetic manipulation with physical and chemical treatment in vivo and in vitro, characterized in that: The method is to firstly introduce foreign genes into the silkworm genome by gene manipulation and cultivate new transgenic silkworm varieties, and then perform physical and chemical treatment on the silk substances secreted by the silkworms outside the body of the silkworms, thereby improving the properties of the silk.

2. The method for improving silk properties by combining genetic manipulation with physical and chemical treatment in vivo and in vitro according to claim 1, characterized in that: The method specifically comprises the following steps: firstly using genetic manipulation techniques such as transgenic or gene editing to introduce foreign genes into the silkworm genome or homologously integrate them into specific sites of the silkworm genome, then cultivating a new silkworm variety with stable inheritance, synthesizing and secreting composite silk protein, and then using an ion crosslinking method or a substrate enzymatic reaction method to perform physical and chemical treatment on the composite silk protein to change the properties of the composite silk protein.

3. The method for improving silk properties by combining genetic manipulation with physical and chemical treatment in vivo and in vitro according to claim 1, characterized in that: The exogenous gene refers to a silkworm cuticle protein gene, specifically one of RR-1, RR-2, RR-3, CPG, CPH or Glycine-rich: The gene sequence and protein sequence of the silkworm cuticle protein gene RR-1 are SEQ ID NO.1 and SEQ ID NO.2 respectively; The gene sequence and protein sequence of the silkworm cuticle protein gene RR-2 are SEQ ID NO.3 and SEQ ID NO.4 respectively; The gene sequence and protein sequence of the silkworm cuticle protein gene RR-3 are SEQ ID NO.5 and SEQ ID NO.6 respectively; The gene sequence and protein sequence of the silkworm cuticle protein gene CPG are SEQ ID NO.7 and SEQ ID NO.8 respectively; The gene sequence and protein sequence of the silkworm cuticle protein gene CPH are SEQ ID NO.9 and SEQ ID NO.10 respectively; The gene sequence and protein sequence of the silkworm cuticle protein gene Glycine-rich are SEQ ID NO.11 and SEQ ID NO.12 respectively.

4. The method for improving silk properties by combining genetic manipulation with physical and chemical treatment in vivo and in vitro according to claim 1, characterized in that: The gene manipulation is to introduce the silkworm cuticle protein gene into the silkworm genome, or homologously integrate it into the silk fibroin heavy chain gene, silk fibroin light chain gene, silk fibroin P25 gene and sericin 1 gene of the silkworm.

5. The method for improving silk properties by combining genetic manipulation with physical and chemical treatment in vivo and in vitro according to claim 1, characterized in that: The physical and chemical treatment is to further treat the composite silk protein in vitro, specifically, to add zinc metal ions to the composite silk protein in vitro or during the reeling process to cross-link the composite silk protein molecules, thereby improving the performance of the composite silk.

6. The method for improving silk properties by combining genetic manipulation with physical and chemical treatment in vivo and in vitro according to claim 5, characterized in that: The physical and chemical treatment is to further treat the composite silk protein in vitro, specifically, to add a substrate to the composite silk protein in vitro or during the reeling process for enzymatic reaction, wherein the substrate is N-acetyl dopamine (NADA) or N-β-propionyl dopamine (NBAD), and the enzyme is laccase 2.

7. The method for improving silk properties by combining genetic manipulation with physical and chemical treatment in vivo and in vitro according to claim 1, characterized in that: The method comprises the following steps: firstly, a target silkworm epidermal protein gene sequence is biosynthesized as a foreign gene by a genetic engineering method, then a foreign gene vector is constructed by a molecular biological technique, the foreign gene vector is introduced into silkworm eggs by microinjection and integrated into the silkworm genome, or homologously integrated into a specific gene of the silkworm, the silkworms are cultivated for multiple generations, and positive individuals of the G1 generation are obtained by screening under a fluorescent microscope, the positive individuals of the G1 generation are mated with wild types or self-pollinated to obtain the G2 generation, positive individuals are screened after the G3 generation, and the positive individuals are self-pollinated to keep seeds, and a stable genetic variety is bred to the G8 generation; Then, G8 generation silkworms were used to produce secretory silk, and zinc ions were added to the silk to allow the silk protein molecules to cross-link with the zinc ions, or mixed with the substrate N-acetyl dopamine (NADA) or N-β-propionyl dopamine (NBAD), and finally laccase 2 was added to catalyze the cross-linking reaction, thereby improving the performance of the composite silk.

8. The method for improving silk properties by combining genetic manipulation with physical and chemical treatment in vivo and in vitro according to claim 7, characterized in that: The exogenous gene vector is a transgenic vector using piggyBac as a backbone or a homologous recombination vector used for TALEN or CRISPR gene editing; The exogenous gene vector comprises an exogenous gene expression frame and a fluorescent screening marker gene expression frame; The exogenous gene expression frame includes an exogenous gene and a promoter of the expression frame, the exogenous gene is a gene base sequence corresponding to the protein sequence of the silkworm cuticle protein gene RR-1, RR-2, RR-3, CPG, CPH or Glycine-rich as described in claim 2, and the promoter of the exogenous gene expression frame adopts the promoter of the silk fibroin heavy chain, silk fibroin light chain, silk fibroin P25 or sericin gene; The fluorescent screening marker gene expression frame includes a fluorescent screening marker gene and a promoter of the expression frame. The fluorescent screening marker gene uses a green fluorescent protein gene (GFP) or a red fluorescent protein gene (DsRed), and the promoter of the fluorescent screening marker gene expression frame uses an IE-1, A3 or 3xP3 promoter.

9. The method for improving silk properties by combining genetic manipulation with physical and chemical treatment in vivo and in vitro according to claim 7, characterized in that: The composite silk protein comprises protein molecule components constituted by one gene of silkworm cuticle protein genes RR-1, RR-2, RR-3, CPG, CPH or Glycine-rich and silk protein components.