CN114480410A - Transgenic method for improving mechanical property of silk by using silk protein of bagworms and silkworm variety thereof - Google Patents

Abstract

The invention provides a transgenic method for improving silk mechanical properties by using silk protein of a bagworm and a silkworm variety thereof, which construct a silkworm transgenic system for specifically expressing exogenous silk protein EvH of the bagworm, the silkworm transgenic system is started to be up-regulated at a specific part by a fibroin FibH promoter, a transgenic expression vector is based on an N-terminal sequence of the silk protein of the bagworm EvH, and the transgenic expression vector is optimized according to the preference of silkworm codons to construct transgenes. The silkworm strain with greatly improved mechanical performance of silk bred by the method is provided. The cultured silk has excellent mechanical performance.

Description

Transgenic method for improving mechanical property of silk by using silk protein of bagworms and silkworm variety thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a transgenic method for improving mechanical properties of silk by using bagworm silk protein and a silkworm variety thereof.

Background

The animal silk produced by the silk secreting insects is a natural protein fiber with excellent comprehensive performance, and has the characteristics of oxidation resistance, antibiosis, ultraviolet resistance, good biocompatibility, good biodegradability and the like. Based on the characteristics, the development and application potential of the animal silk is huge, and the animal silk is widely applied to the industries such as materials science, medical field, photoelectron and the like besides the traditional textile industry.

Spider silks have been reported to have been applied to the aerospace field and other high-precision nanometer-sized device developments among many animal silks because of their particularly excellent mechanical properties. However, due to the habits of spiders, large-scale artificial feeding is difficult, and a large amount of spider silk materials cannot be obtained.

Among organisms which are also sericultural insects, silkworms are the only economic animals which are completely domesticated by human beings, and the breeding history has been 5000 years. Although the domestic silkworm silk has low large-scale production cost, the domestic silkworm silk is not like spider silk in the aspect of mechanical property, and the development and application of the domestic silkworm silk in wider industrial fields are greatly limited.

Disclosure of Invention

Based on the technical problems in the prior art, the invention provides a transgenic method for improving the mechanical property of silk by utilizing bageworm silk protein and a silkworm variety thereof.

According to the first aspect of the technical scheme of the invention, the transgenic method for improving the mechanical property of silk by utilizing silk protein of a bageworm comprises the following steps:

step S1: constructing a silkworm transgenic system for specifically expressing exogenous bagworm silk protein EvH, wherein the gene sequence of the bagworm silk protein is shown in SEQ ID NO. 1;

step S2: the silkworm transgenic system is up-regulated at a specific part through the initiation of a fibroin fibH promoter, and the sequence of the fibroin fibH promoter is shown as SEQ ID NO. 2;

step S3: the transgenic expression vector is constructed by optimizing and synthesizing the encoding sequence of the bagworm EvH protein according to the preference of silkworm codons:

step S4: the EvH coding sequence was packaged into the subcloning vector p57S [ fibH-MCS-LBS ] which was similarly digested, and the p57S [ fibH-EvH-LBS ] vector was constructed by connecting the fibH promoter (SEQ ID NO.2) in tandem with the multiple cloning site and the LBS (binding site to fibL) sequence (SEQ ID NO. 3).

Preferably, in step S2, an expression vector is constructed based on the gene sequence SEQ ID NO.1 and the gene sequence SEQ ID NO. 2.

Preferably, the method further comprises the step of S5: the vector plasmid is subjected to single enzyme digestion by Asc I, the recovered target fragment is connected with a pBac [3 xP 3-ECFP ] skeleton vector subjected to the same enzyme digestion, the skeleton vector consists of a piggyBac right arm (SEQ ID NO.4), a 3 xP 3-ECFP (SEQ ID NO.5) and a piggyBac left arm (SEQ ID NO.6), a final expression vector pB [ fibH-EvH-LBS,3 xP 3-ECFP ] is obtained, and the vector skeleton is completed by the following steps: firstly, assembling a 3 XP 3-ECFP sequence (SEQ ID NO.5), wherein the sequence is formed by driving and Expressing Cyan Fluorescent Protein (ECFP) by a 3-fold repeated P3 promoter (eye and nerve specific promoter); then the right arm (SEQ ID NO.4) and the left arm (SEQ ID NO.6) of piggyBac are assembled at the 5 'end and the 3' end of the 3 XP 3-ECFP sequence respectively.

In addition, the transgenic method for improving the mechanical property of silk by using the silk protein of the bagworms further comprises a manufacturing step based on 305 transgenic silkworms and a molecular identification step based on EvH transgenic silkworms of the 305 strain in sequence, wherein in order to enlarge production, a transgene EvH based on the 305 strain is introduced into a QB variety, a step of observing a silk gland phenotype of the transgenic silkworms based on the QB variety EvH, a step of observing a cocoon shell phenotype of the transgenic silkworms based on the QB variety EvH and a step of detecting the mechanical property of the transgenic silkworms based on the QB variety EvH are observed.

According to the second aspect of the technical scheme of the invention, a transgenic silkworm variety with the mechanical property of silk improved by using the silk moth silk protein is provided, wherein a silkworm embryo microinjection method is adopted, namely the transgenic method for improving the mechanical property of silk by using the silk moth silk protein in any one of claims 1 to 4, and the silk gland at the rear part of a silkworm is over-expressed with the silk moth silk protein gene EvH, so that a transgenic silkworm strain is obtained.

According to a third aspect of the technical scheme of the invention, the method for obtaining the silkworm strain of the silk with improved mechanical properties comprises the following steps:

step W1, vector construction: according to the sequence codon preference of the silkworm genes in the silkworm genome sequence database, the invention carries out codon optimization design on the bag moth silk protein coding gene sequence, and the result is that the bag moth silk protein gene EvH is over-expressed in the silk gland at the rear part of the silkworm;

step W2, obtaining transgenic silkworm strain by silkworm embryo microinjection, and mixing EvH recombinant vector plasmid and transposase expression vector plasmid according to the ratio of 1: 1, mixing in proportion, microinjecting 305 early embryos 1-3h after spawning, breeding G0 generation (the first generation after injection) hatching larvae until eclosion, and then carrying out selfing or backcross seed production;

w3, breeding transgenic silkworms, detecting the obtained G1 generation (the second generation after injection) silkworm eggs under a stereoscopic fluorescence microscope about 1 day before the green-dropping stage, screening transgenic positive individuals which fluoresce in embryo eyes or nerve tissues, and feeding the individuals to adults by taking a moth ring as a unit;

step W4, a large-scale feeding method, namely hybridizing the transgenic line based on the diversification system 305 with QB for more than 3 generations to obtain a transgenic system based on QB, which is beneficial to large-scale feeding;

step W5: observing the specific character generated by the silkworm by the transgene and detecting the mechanical property.

Compared with the prior art, the transgenic method for improving the mechanical property of silk by utilizing the silk fibroin of the bagworms has the beneficial effects that:

1. the invention discloses a method for effectively improving the mechanical property of domestic silk by a transgenic technology and a domestic silkworm strain thereof, namely, the domestic silkworm strain with greatly improved mechanical property of the silk is bred by introducing exogenous bag moth silk protein EvH (SEQ ID NO. 1). 2. The silk produced by the bred silkworm strain has excellent mechanical property, is improved by 1 time compared with the silk before improvement, can be used as a good natural protein material to be widely applied to the fields of medicine, war industry, photoelectricity and the like, and has important application value.

3. The invention discloses a method for directionally introducing exogenous silk protein into a silkworm silk gland by utilizing a transgenic technology, which improves a new strategy reference for directional modification of silk.

Drawings

FIG. 1 is a positive screening graph of EvH transgene based on 305 silkworm receptor expression, positive individuals have green fluorescence in eyes;

FIG. 2 is a genomic PCR identification based on the 305 silkworm receptor;

FIG. 3 is a schematic diagram of a five-instar day 6 silk gland anatomy based on QB breed; FIG. 4 is a schematic representation of a QB variety based cocoon shell;

FIG. 5 shows the result of fluorescence quantification based on QB variety, which proves that EvH gene is successfully expressed in the posterior silk gland of silkworm;

FIG. 6 is a schematic drawing of a tensile curve based on the QB variety mechanical property testing.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments, not all embodiments, of the technical solutions. All other embodiments obtained by a person skilled in the art based on the embodiments of the present technical solution without creative efforts shall fall within the protection scope of the present invention. In addition, the scope of the present invention should not be limited to the particular structures or components or the specific parameters set forth below.

The invention provides a transgenic method for improving silk mechanical properties by using silk protein of bagworms, which utilizes a silkworm transgenic technology to over-express a silk protein gene EvH of the bagworms in silk glands at the rear part of the silkworms and proves that a silk protein gene EvH of the bagworms is successfully prepared by molecular detection. By observing the silk gland and cocoon shell of wild type QB variety and transgenic EvH, no obvious difference between silk gland and cocoon shell is found. Through fluorescent quantitative detection, EvH successful over-expression in the rear silk gland of EvH transgenic silkworms is found, and then mechanical property detection is carried out on the transgenic silkworms EvH successfully over-expressed, and the result shows that the mechanical property of silk can be greatly improved by the silk protein gene EvH of the bagworms. The QB variety is named as a practical variety for short, namely autumn white. "305" in "diversification system 305", "305 line" each refer to 305 silkworms, family Bombycidae.

The invention discloses a transgenic method for improving mechanical properties of silk by using bagworms silk protein, and further provides a silkworm transgenic system for improving mechanical properties of silk based on expression of exogenous bagworms silk protein EvH, and provides a silkworm strain for producing high-mechanical-property silk so as to expand application of the silk in various fields.

A transgenic method for improving mechanical properties of silk by utilizing silk protein of a bageworm comprises the following steps:

step S1: constructing a silkworm transgenic system for specifically expressing exogenous bagworm silk protein EvH, wherein the gene sequence of the bagworm silk protein is shown in SEQ ID NO. 1;

step S2: the silkworm transgenic system is up-regulated at a specific part through the initiation of a fibroin fibH promoter, and the sequence of the fibroin fibH promoter is shown as SEQ ID NO. 2;

step S3: the transgenic expression vector is constructed by optimizing and synthesizing the encoding sequence of the bagworm EvH protein according to the preference of silkworm codons:

step S4: the EvH coding sequence was packaged into the subcloning vector p57S [ fibH-MCS-LBS ] which was similarly digested, and the p57S [ fibH-EvH-LBS ] vector was constructed by connecting the fibH promoter (SEQ ID NO.2) in tandem with the multiple cloning site and the LBS (binding site to fibL) sequence (SEQ ID NO. 3).

In another embodiment, the transgenic method for improving the mechanical property of silk by using silk protein of bagworms of the invention comprises the following steps:

step S1: constructing a transgenic expression vector of the silk protein gene EvH of the bageworm;

step S2: performing embryo microinjection and fluorescence screening;

step S3: molecular detection of the silk protein gene EvH of the bageworm;

step S4: observing silk glands based on the QB variety introduced into the silk protein gene EvH of the bageworm;

step S5: observing cocoon shells based on the QB variety introduced with the bag-transferring moth silk protein gene EvH;

step S6: silk gland fluorescence quantitative detection based on the QB variety imported bag transfer moth silk protein gene EvH;

step S7: and (3) detecting the mechanical property of the silk introduced with the silk protein gene EvH of the bageworm based on the QB variety.

Wherein the step S1 further includes the step S1-1: a transgenic expression vector based on EvH for specifically expressing exogenous bagworm silk protein by a rear silk gland is constructed, wherein the bagworm silk protein gene is shown as SEQ ID NO.1, and the gene is subjected to codon optimization design on a bagworm silk protein gene EvH sequence according to the sequence codon preference of the silkworm gene in a silkworm genome sequence database.

Step S1-2: the optimized EvH sequence is loaded into a subcloning vector p57S [ fibH-MCS-LBS ] which is cut by the same enzyme, the subcloning is composed of a fibH promoter (SEQ ID NO.2) in series with a multiple cloning site and an LBS (binding site with fibL) sequence (SEQ ID NO.3), and the p57S [ fibH-EvH-LBS ] vector is constructed.

Step S1-3: the vector plasmid is subjected to single enzyme digestion by Asc I, the recovered target fragment is connected with a pBac [3 xP 3-ECFP ] skeleton vector subjected to the same enzyme digestion, the skeleton vector consists of a piggyBac right arm (SEQ ID NO.4), a piggyBac 3 xP 3-ECFP (SEQ ID NO.5) and a piggyBac left arm (SEQ ID NO.6), and a final expression vector pB [ fibH-EvH-LBS,3 xP 3-ECFP ] is obtained.

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1, construction of an expression vector comprising the steps of:

step S1-1: a transgenic expression vector based on EvH for specifically expressing exogenous bagworm silk protein by a rear silk gland is constructed, wherein the bagworm silk protein gene is shown as SEQ ID NO.1, and the gene is subjected to codon optimization design on a bagworm silk protein gene EvH sequence according to the sequence codon preference of the silkworm gene in a silkworm genome sequence database.

Step S1-2: the optimized EvH sequence is loaded into a subcloning vector p57S [ fibH-MCS-LBS ] which is subjected to double enzyme digestion by BamH I and Not I, and the subcloning is constructed by connecting a fibH promoter (SEQ ID NO.2) in series with a multiple cloning site and an LBS (binding site with fibL) sequence (SEQ ID NO.3) to obtain a p57S [ fibH-EvH-LBS ] vector.

Step S1-3: the vector plasmid is subjected to single enzyme digestion by Asc I, the recovered target fragment is connected with a pBac [3 xP 3-ECFP ] skeleton vector subjected to the same enzyme digestion, the skeleton vector consists of a piggyBac right arm (SEQ ID NO.4), a piggyBac 3 xP 3-ECFP (SEQ ID NO.5) and a piggyBac left arm (SEQ ID NO.6), and a final expression vector pB [ fibH-EvH-LBS,3 xP 3-ECFP ] is obtained.

Example 2 production of transgenic silkworms

Step S21 embryo microinjection and fluorescence screening:

mixing the expression vector and an auxiliary plasmid (A4Helper) respectively in equal proportion at a concentration of 450 ng/mu L (nanogram/microliter), injecting by an Eppendorf microinjector, taking a polytropic silkworm 305 (which can breed a plurality of batches of silkworm seed materials within one year) as an injection receptor, mating silkworm moths for 6 hours before injection, standing for one day at 4 ℃ (centigrade), taking out the silkworm moths to lay eggs at room temperature, taking embryos which just lay eggs for one hour, pasting the embryos on glass sheets by using paste, injecting by the Eppendorf microinjector, sealing the embryos by using nontoxic glue, sterilizing by 35% formaldehyde steam for 5 minutes, placing the embryos in an environment with a relative humidity of 25 ℃ and a relative humidity of 85%, breeding hatched G0 generation (first generation after injection) silkworm moths to the silkworm moths by using mulberry leaves, obtaining G0 generation (first generation after injection) moths, and obtaining G1 generation (second generation after injection) silkworm eggs by selfing or backcrossing, screening by an Olympus fluorescence microscope to obtain the cyan fluorescence bagworm silk protein gene EvH, and the result is shown in the attached figure 1, which proves that the transgene is successfully made. And normally preserving the seeds after breeding for one generation.

Step S22, molecular detection of silk protein gene EvH of bagworms

The genome of the positive individuals obtained in the above way, namely EvH and the wild silkworm 305, is extracted, and the extraction steps are as follows:

(1) collecting positive silkworm moth EvH and wild silkworm 305, cleaning mortar and grinding rod, and sterilizing at 180 deg.C in oven for 2-3 hr. Before the grinding operation, liquid nitrogen precooling treatment is carried out on the silkworm moths, the mortar and the grinding rod. After precooling, the silkworm moths are ground into powder and then transferred to a centrifugal tube with the volume of 1.5mL (milliliter), and the powder is stored in liquid nitrogen or at the temperature of minus 80 ℃ for standby.

(2) Add 1mL of DNA extraction Buffer to the centrifuge tube and vortex at 3000rpm to mix. RNase was added at a working concentration of 100. mu.L/mL (microliters per mL), digested in a 37 ℃ constant temperature water bath for 1 hour, then proteinase K was added, and digested in a 55 ℃ water bath overnight.

(3) Equal volume of Tris saturated phenol was added to the tube and vortexed thoroughly for 10min (min), followed by centrifugation at 13400rpm for 10min at 4 ℃ and 600. mu.L of supernatant was taken to a new tube.

(4) mu.L Tris phenol/chloroform, fully rotating and shaking for 10min, then, 4 ℃, 13400rpm centrifugation for 10min, then the supernatant is transferred to a new centrifuge tube.

(5) The supernatant was collected by thoroughly rotating and shaking chloroform in an equal volume for 10min, followed by centrifugation at 13400rpm for 10min at 4 ℃.

(6) Adding anhydrous ethanol precooled at 4 ℃ into a centrifuge tube in equal volume, slightly turning upside down until uniform white flocculent precipitate appears, and standing for 5 min.

(7) Carefully picking out the precipitate with a sterile tip, transferring to a new 1.5mL centrifuge tube, adding pre-cooled 75% ethanol at 4 deg.C, washing for 1-2 times, centrifuging at 13400rpm for 10min at 4 deg.C, and discarding the supernatant.

(8) The centrifuge tube was opened, allowed to stand at room temperature until ethanol was evaporated, and 30-50. mu.L of TE buffer was added to dissolve the DNA precipitate.

(9) Detecting DNA purity and concentration by using a spectrophotometer, carrying out agarose gel electrophoresis detection, and storing at-80 ℃ for a long time for later use.

Step S23 genomic PCR:

(1) primer5 software is used to design Primer, which is synthesized from Huada gene, and after the Primer is synthesized, ultrapure water is added to dissolve and dilute the Primer, and then the Primer is stored at 4 ℃.

(2) And (3) carrying out PCR amplification on a target fragment by taking the extracted genome as a template, wherein the reaction system comprises the following steps:

(3) the PCR amplification conditions were as follows:

(4) after the reaction was completed, 1% agarose gel was prepared, and 5. mu.L of PCR amplification product was subjected to electrophoresis. As shown in figure 2, the detection result proves that the transgenic silkworm is successfully made.

And step S24, subsequently, in order to raise the improved silkworms in a large scale, introducing a transgenic system of a 305-based diversified variety into a diapause seed QB to obtain a QB-based silk performance improvement strain.

Example 3, based on the observation of the silk gland of QB variety introduced into bagworm silk protein gene EvH:

step S31: feeding wild silkworm QB and bag-transfer moth silk protein gene EvH silkworm, namely silkworm specifically expressing cyan fluorescence in silkworm eyes to five-year old, dissecting and observing silk glands of the wild silkworm QB and bag-transfer moth silk protein gene EvH silkworm on the sixth day of five-year old (5L6D) in 1 XPBS (phosphate buffered saline), and taking pictures, wherein the results are shown in figure 3, and compared with wild control, the silk glands have no obvious change.

Example 4 cocoon observation based on introduction of QB variety into bagworm silk protein gene EvH:

step S41: feeding wild silkworm QB and bag transfer moth silk protein gene EvH silkworm, namely silkworm with blue fluorescence specifically expressed in silkworm eye, to the mounting, wherein the feeding environment is as follows: feeding in an environment with the temperature of 25 ℃ and the relative humidity of 65 percent, and mounting in an environment: the ventilation is good, the temperature is 25 ℃, then cocoons are picked and photographed, and the result is shown in figure 4, compared with wild type silkworm cocoon shells, the silk protein gene EvH cocoon shells of the bagworms have no obvious change.

Example 5, the fluorescence quantitative detection of silk gland based on the introduction of QB variety into bagworm silk protein gene EvH, comprising the following steps:

the step S5 of extracting posterior silk gland RNA further comprises the steps of:

step S51, dissecting to obtain the posterior silk gland of the 6 th day of QB and EvH five-instar larvae, respectively placing into a mortar precooled by liquid nitrogen, adding the liquid nitrogen, quickly grinding into powder, and transferring a proper amount of the powder into a new RNase-free 1.5mL centrifuge tube.

Step S52 Trizol (triazole) solution 1mL is added into the centrifuge tube and vortexed at a high speed until it is well mixed. The tube was placed on ice for 10min and centrifuged at 12500g for 10min at 4 ℃.

Step S53 transferring the supernatant to a new RNase-free 1.5mL centrifuge tube, adding 250 μ L chloroform, vortexing at high speed for 15S, standing on ice for 10min, centrifuging at 4 deg.C and 12500g for 10min, and collecting the supernatant.

Step S54 repeats the previous operation.

Step S55, the upper aqueous phase is transferred to a new rnase-free centrifuge tube, and isopropyl alcohol of equal volume is added and the mixture is fully mixed by reversing the top and bottom. Centrifuge at 12500g for 15min at 4 ℃.

Step S56 the supernatant was discarded and 1mL of 75% ethanol pre-cooled at 4 ℃ was added to wash the precipitate. Centrifuge at 13000g for 15min at 4 ℃.

Step S57 discards the supernatant, carefully sucks the liquid remaining around the precipitate with a pipette, and then dries by standing at room temperature until ethanol is evaporated.

Step S58, adding a proper amount of DEPC water into the centrifuge tube to dissolve the RNA precipitate, and standing at room temperature until the precipitate is completely dissolved.

Step S59, the purity and concentration of RNA are detected by a spectrophotometer and then diluted properly, and the RNA is stored for a long time at-80 ℃ for standby.

Step S6 in vitro reverse transcription synthesis of cDNA

According to the operating instruction of TaKaRa in vitro reverse transcription kit. The following operations are carried out:

and S61, digesting the genome, wherein the specific digestion system is as follows:

and (3) blowing and uniformly mixing the system, placing the system at room temperature for digestion for 5min, and then carrying out reverse transcription PCR.

Step S62 reverse transcription PCR system as follows;

and (4) adding the above system into the digestion product obtained in the step S21, gently blowing and uniformly mixing, placing in a PCR instrument for inversion, wherein the temperature is 37 ℃ for 15min, the temperature is 85 ℃ for 5S, and the cDNA product after inversion is stored at-20 ℃ for later use.

Step S7 real-time fluorescent quantitative PCR

Before performing the fluorescent quantitative PCR, the cDNA is diluted by 5 times and then the next operation is performed.

Quantitative primers for the gene of step S71 EvH were designed as follows:

F:CGCTGGATCTGGTGCTGGAG R:GCTCCACCAGCCGAAACGTA

the fluorescent quantitative reaction system and the amplification procedure of step S72 are as follows:

the fluorescent quantitative PCR amplification procedure was as follows:

step S73 data collection, analysis, and mapping.

After the program is finished, an Excel table containing data is exported, and the obtained data is analyzed by adopting a relative quantification method. Finally, Graph plotting was performed by using Graph Pad Prism 5, and the results are shown in FIG. 5, wherein EvH was highly expressed in the posterior silk gland of Bombyx mori, thus demonstrating that EvH was successfully expressed.

Example 6, it is based on the mechanical property test of silk with QB variety introduced into bagworm silk protein gene EvH, and it includes the following steps:

step S81, removing part of sericin of the cocoon silk, so as to facilitate subsequent silk reeling;

selecting transgenic lines based on QB as experimental groups, selecting 10 normal cocoons which are clustered, and immersing the cocoons in 100 ℃ boiling water for 30 s; immediately placing in 45 deg.C warm water for 1 min; immersing the silkworm cocoons in 100 ℃ boiling water for 30 seconds; immediately placing in 45 deg.C warm water for 1 min.

Step S82, placing the silkworm cocoons in warm water at 45 ℃ for reeling silk;

and (4) reeling the middle section of each silkworm cocoon to perform sample detection, and performing sample repetition 5 times on each silkworm cocoon. Scanning Electron Microscope (SEM) ("femina, the netherlands").

Step S83, measuring the diameter of silk;

and (3) drying the sample in a 60-degree oven for 12h, spraying gold on the sample, and then placing the sample in a voltage of 10KV, wherein the silk shape is observed under the magnification of 2500 times. The average diameter R is calculated by the Method of digital photo, pixel-ratio Method (FS-DP) [ A Simple Method for the Cross-Section Area Determination of Single Profiled Fibers and Its Application ], calculating the maximum major axis diameter L of each cocoon from the electron micrograph of each silk by using Image J, and considering the minor axis diameter as L/2, and further calculating the average diameter R by the consistent Cross-sectional Area, i.e. pi (L/4) (L/2) pi (R/2).

Step S84, testing the mechanical property of silk;

the silk was subjected to a tensile test using a single fiber strength tester [ TRAPEZIUM LITE, Shimadzu, japan ] with the diameter set to the calculated R, the tensile rate set to 10mm/min (mm per minute) and the pitch set to 10 mm. Before testing, the silk to be tested is placed in a constant temperature and humidity (25 ℃, 65%) laboratory for 24h, 50 groups of data are obtained for each silkworm cocoon, the data are imported into origin for drawing, and the average curve of 50 groups of data is calculated. Finally, the average curves of the two systems were selected and plotted, and the results are shown in FIG. 6. Compared with wild QB, the mechanical property of the silk transferred into EvH is improved by 119.0%.

The gene sequence table related by the invention is as follows:

EvH gene sequence after optimization of SEQ ID NO.1

gctgccgctgccgctgccgctgccgctgaggccgctgccgctgccgctgccgctgccgctgccgctggttccggagctggcgccggtggagctggcggttacggtgctggtgctggtgcaggagctggagctggtgccggaggcgccgctggtgctggtggagccggcggtgctggaggcgccggtggatacggcggtgcttcggtggtttacgtgggaggcggtggagctggtgccggagctggcagcggtgccggagctggctccggtgccggagctggtgctggtagcggagctggtgccggcggtgctggagccgctgccggtgctggagccggtgcaggttccggagctggctcaggttctggagctggagctggtgctggatcgggcgctggtgccggaagtggcgcaggagccggagctggcgccggtagcggagctgccggaggcgctggtgctggtgctggagccggcgctggtgctgccgctgccgctgccgctgccgctgaagccgctgccgctgccgctgccgctgccgctgccgctggttcaggtgctggtgctggtggagctggcggttatggagctggtgcaggcgctggagctggagccggtgctggaggcgctggtggagccggcggtgctggaggcgccggtggagctggcggttacggaggcgctggtgtcgtgtacgtttctgctggtggagccggtgcaggtgctggttcaggtgctggagctggttctggagcaggtgcaggtgctggatcgggtgctggtgctggcggtgctggtgccgctgccggagccggtgcaggtgccggaagtggtgctggtagcggttcaggtgcaggagcaggtgcaggcagcggagctggtgcaggctccggagctggtgctggcgccggtgctggatcaggtgctgccggaggcgccggtgctggagctggtgcaggagctgccgctgccgctgccgctgccgctgaagccgctgccgctgccgctgccgctgccgctgccgctggatctggtgctggagctggtggagctggcggttacggggctggtgcaggcgctggtgcaggtgctggagctggaggcgctggtggagctggcggtgctggaggcgctggtggagctggcggttacggaggcgctggggttgtctacgtttcggctggtggagccggtgccggtgctggttcgggagctggagctggtagtggtgcaggagccggtgctggaagtggcgccggtgctggcggtgctggcgccgctgccggcgctggtgctggtgccggatcaggtgctggttcgggaagtggtgccggagctggagctggctctggtgctgttgctggatcgggagctggtagtggtgccgccggaggcgccggtgcaggcgcaggtgctggggctgccgctgccgctgccgctgccgctgaagccgctgccgctgccgctgccgctgccgctgccgctggttcgggagccggggctggtggagctggcggttacggcgctggagcaggtgctggcgctggagctggcgctggaggcgctggtggagctggcggtgctggaggcgctggtggatatggcggtgctagtgtggtttacgttggagctggaggcgccggagcaggtgctggctcaggtgcaggtgctggctctggtgctggagctggagctggttcaggcgctggtgctggtggagctggtgccgctgccggcgctggcgctggtgctggttctggtgcaggctcaggttctggtgcaggtgctggcgcaggaagcggtgctggtgctggttcaggtgcaggagccggagccggcgctggtagcggagctgccggcggtgccggtgctggtgccggcgctgccgctgccgctgccgctgccgctgaagccgctgccgctgccgctgccgctgccgctgccgctggaggcgctggtggatacggtccttacggcggttttgcaggtgccggagctggagccggaggcgctggtggagctggcggtgctggaggcgctggtggagctggttcaacactgatcattgtcgacgaaggcggttatggaggcgctggtggagctggctcaggtgctggatctggtgtgggtgctggagctggcagcggtgcaggtgccggcggtgctggggccgctgccggtgcaggcgctggtgccggctccggtgctggaagcggctccggagcaggagctggagccggttcgggagctggagcaggaagtggtgccggtgcaggagcaggtgcgggtagcggtgcagccggaggcgctggagccggagctggtgccggaactagtgcggccgca

SEQ ID NO.2 bombyx mori fibH gene promoter sequence

cctgcgtgatcaggaaaaatgtggaaagcttaacgattttgtcacattttacttatcacaacttgtttttataataattcgcttaaatgagcagctattacttaatctcgtagtggtttttgacaaaatcagcttctttagaactaaaatatcatttttttcgtaatttttttaatgaaaaatgctctagtgttatacctttccaaaatcaccattaattaggtagtgtttaagcttgttgtacaaaactgccacacgcatttttttctccactgtaggttgtagttacgcgaaaacaaaatcgttctgtgaaaattcaaacaaaaatattttttcgtaaaaacacttatcaatgagtaaagtaacaattcatgaataatttcatgtaaaaaaaaaatactagaaaaggaatttttcattacgagatgcttaaaaatctgtttcaaggtagagatttttcgatatttcggaaaattttgtaaaactgtaaatccgtaaaattttgctaaacatatattgtgttgttttggtaagtattgacccaagctatcacctcctgcagtatgtcgtgctaattactggacacattgtataacagttccactgtattgacaataataaaacctcttcattgacttgagaatgtctggacagatttggctttgtatttttgatttacaaatgtttttttggtgatttacccatccaaggcattctccaggatggttgtggcatcacgccgattggcaaacaaaaactaaaatgaaactaaaaagaaacagtttccgctgtcccgttcctctagtgggagaaagcatgaagtaagttctttaaatattacaaaaaaattgaacgatattataaaattctttaaaatattaaaagtaagaacaataagatcaattaaatcataattaatcacattgttcatgatcacaatttaatttacttcatacgttgtattgttatgttaaataaaaagattaatttctatgtaattgtatctgtacaatacaatgtgtagatgtttattctatcgaaagtaaatacgtcaaaactcgaaaattttcagtataaaaaggttcaactttttcaaatcagcatcagttcggttccaactctcaag

SEQ ID NO.3LBS sequence

agttacggagctggcaggggatacggacaaggtgcaggaagtgcagcttcctctgtgtcatctgcttcatctcgcagttacgactattctcgtcgtaacgtccgcaaaaactgtggaattcctagaagacaactagttgttaaattcagagcactgccttgtgtgaattgctaatttttaatataaaataacccttgtttcttacttcgtcctggatacatctatgttttttttttcgttaataaatgagagcatttaagttattgtttttaattacttttttttagaaaacagatttcggattttttgtatgcattttatttgaatgtactaatataatcaattaatcaatgaattcatttatttaagggataacaataatccatgaatt

SEQ ID NO.4piggyBac right arm sequence

ccctagaaagataatcatattgtgacgtacgttaaagataatcatgcgtaaaattgacgcatgtgttttatcggtctgtatatcgaggtttatttattaatttgaatagatattaagttttattatatttacacttacatactaataataaattcaacaaacaatttatttatgtttatttatttattaaaaaaaaacaaaaactcaaaatttcttctataaagtaacaaaacttttaaacattctctcttttacaaaaataaacttattttgtactttaaaaacagtcatgttgtattataaaataagtaattagcttaacttatacataatagaaacaaattatacttattagtcagtcagaaacaactttggcacatatcaatattatgctctcgacaaataacttttttgcattttttgcacgatgcatttgcctttcgccttattttagaggggcagtaagtacagtaagtacgttttttcattactggctcttcagtactgtcatctgatgtaccaggcacttcatttggcaaaatattagagatattatcgcgcaaatatctcttcaaagtaggagcttctaaacgcttacgcataaacgatgacgtcaggctcatgtaaaggtttctcataaattttttgcgactttggaccttttctcccttgctactgacattatggctgtatataataaaagaatttatgcaggcaatgtttatcattccgtacaataatgccataggccacctattcgtcttcctactgcaggtcatcacagaacacatttggtctagcgtgtccactccgcctttagtttgattataatacataaccatttgcggtttaccggtactttcgttgatagaagcatcctcatcacaagatgataataagtataccatcttagctggcttcggtttatatgagacgagagtaaggggtccgtcaaaacaaaacatcgatgttcccactggcctggagcgactgtttttcagtacttccggtatctcgcgtttgtttgatcgcacggttcccacaatggttt

SEQ ID NO. 53 XP 3ECFP sequence

gcaaagtgaacacgtcgctaagcgaaagctaagcaaataaacaagcgcagctgaacaagctaaacaatcggggtaccgctagagtcgacggtacgatccaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctggggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacatcagccacaacgtctatatcaccgccgacaagcagaagaacggcatcaaggccaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaactctagatcataatcagccataccacatttgtag

SEQ ID NO.6piggyBac left arm sequence

agatctgacaatgttcagtgcagagactcggctacgcctcgtggactttgaagttgaccaacaatgtttattcttacctctaatagtcctctgtggcaaggtcaagattctgttagaagccaatgaagaacctggttgttcaataacattttgttcgtctaatatttcactaccgcttgacgttggctgcacttcatgtacctcatctataaacgcttcttctgtatcgctctggacgtcatcttcacttacgtgatctgatatttcactgtcagaatcctcaccaacaagctcgtcatcgctttgcagaagagcagagaggatatgctcatcgtctaaagaactacccattttattatatattagtcacgatatctataacaagaaaatatatatataataagttatcacgtaagtagaacatgaaataacaatataattatcgtatgagttaaatcttaaaagtcacgtaaaagataatcatgcgtcattttgactcacgcggtcgttatagttcaaaatcagtgacacttaccgcattgacaagcacgcctcacgggagctccaagcggcgactgagatgtcctaaatgcacagcgacggattcgcgctatttagaaagagagagcaatatttcaagaatgcatgcgtcaattttacgcagactatctttctaggg

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> university of southwest

<120> a transgenic method for improving mechanical properties of silk by using silk protein of bagworms and silkworm variety thereof

<141> 2022-03-04

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2346

<212> DNA

<213> bag moth (Eumeta Variegata)

<400> 1

gctgccgctg ccgctgccgc tgccgctgag gccgctgccg ctgccgctgc cgctgccgct 60

gccgctggtt ccggagctgg cgccggtgga gctggcggtt acggtgctgg tgctggtgca 120

ggagctggag ctggtgccgg aggcgccgct ggtgctggtg gagccggcgg tgctggaggc 180

gccggtggat acggcggtgc ttcggtggtt tacgtgggag gcggtggagc tggtgccgga 240

gctggcagcg gtgccggagc tggctccggt gccggagctg gtgctggtag cggagctggt 300

gccggcggtg ctggagccgc tgccggtgct ggagccggtg caggttccgg agctggctca 360

ggttctggag ctggagctgg tgctggatcg ggcgctggtg ccggaagtgg cgcaggagcc 420

ggagctggcg ccggtagcgg agctgccgga ggcgctggtg ctggtgctgg agccggcgct 480

ggtgctgccg ctgccgctgc cgctgccgct gaagccgctg ccgctgccgc tgccgctgcc 540

gctgccgctg gttcaggtgc tggtgctggt ggagctggcg gttatggagc tggtgcaggc 600

gctggagctg gagccggtgc tggaggcgct ggtggagccg gcggtgctgg aggcgccggt 660

ggagctggcg gttacggagg cgctggtgtc gtgtacgttt ctgctggtgg agccggtgca 720

ggtgctggtt caggtgctgg agctggttct ggagcaggtg caggtgctgg atcgggtgct 780

ggtgctggcg gtgctggtgc cgctgccgga gccggtgcag gtgccggaag tggtgctggt 840

agcggttcag gtgcaggagc aggtgcaggc agcggagctg gtgcaggctc cggagctggt 900

gctggcgccg gtgctggatc aggtgctgcc ggaggcgccg gtgctggagc tggtgcagga 960

gctgccgctg ccgctgccgc tgccgctgaa gccgctgccg ctgccgctgc cgctgccgct 1020

gccgctggat ctggtgctgg agctggtgga gctggcggtt acggggctgg tgcaggcgct 1080

ggtgcaggtg ctggagctgg aggcgctggt ggagctggcg gtgctggagg cgctggtgga 1140

gctggcggtt acggaggcgc tggggttgtc tacgtttcgg ctggtggagc cggtgccggt 1200

gctggttcgg gagctggagc tggtagtggt gcaggagccg gtgctggaag tggcgccggt 1260

gctggcggtg ctggcgccgc tgccggcgct ggtgctggtg ccggatcagg tgctggttcg 1320

ggaagtggtg ccggagctgg agctggctct ggtgctgttg ctggatcggg agctggtagt 1380

ggtgccgccg gaggcgccgg tgcaggcgca ggtgctgggg ctgccgctgc cgctgccgct 1440

gccgctgaag ccgctgccgc tgccgctgcc gctgccgctg ccgctggttc gggagccggg 1500

gctggtggag ctggcggtta cggcgctgga gcaggtgctg gcgctggagc tggcgctgga 1560

ggcgctggtg gagctggcgg tgctggaggc gctggtggat atggcggtgc tagtgtggtt 1620

tacgttggag ctggaggcgc cggagcaggt gctggctcag gtgcaggtgc tggctctggt 1680

gctggagctg gagctggttc aggcgctggt gctggtggag ctggtgccgc tgccggcgct 1740

ggcgctggtg ctggttctgg tgcaggctca ggttctggtg caggtgctgg cgcaggaagc 1800

ggtgctggtg ctggttcagg tgcaggagcc ggagccggcg ctggtagcgg agctgccggc 1860

ggtgccggtg ctggtgccgg cgctgccgct gccgctgccg ctgccgctga agccgctgcc 1920

gctgccgctg ccgctgccgc tgccgctgga ggcgctggtg gatacggtcc ttacggcggt 1980

tttgcaggtg ccggagctgg agccggaggc gctggtggag ctggcggtgc tggaggcgct 2040

ggtggagctg gttcaacact gatcattgtc gacgaaggcg gttatggagg cgctggtgga 2100

gctggctcag gtgctggatc tggtgtgggt gctggagctg gcagcggtgc aggtgccggc 2160

ggtgctgggg ccgctgccgg tgcaggcgct ggtgccggct ccggtgctgg aagcggctcc 2220

ggagcaggag ctggagccgg ttcgggagct ggagcaggaa gtggtgccgg tgcaggagca 2280

ggtgcgggta gcggtgcagc cggaggcgct ggagccggag ctggtgccgg aactagtgcg 2340

gccgca 2346

<210> 2

<211> 1126

<212> DNA

<213> silkworm (Bombyx mori)

<400> 2

cctgcgtgat caggaaaaat gtggaaagct taacgatttt gtcacatttt acttatcaca 60

acttgttttt ataataattc gcttaaatga gcagctatta cttaatctcg tagtggtttt 120

tgacaaaatc agcttcttta gaactaaaat atcatttttt tcgtaatttt tttaatgaaa 180

aatgctctag tgttatacct ttccaaaatc accattaatt aggtagtgtt taagcttgtt 240

gtacaaaact gccacacgca tttttttctc cactgtaggt tgtagttacg cgaaaacaaa 300

atcgttctgt gaaaattcaa acaaaaatat tttttcgtaa aaacacttat caatgagtaa 360

agtaacaatt catgaataat ttcatgtaaa aaaaaaatac tagaaaagga atttttcatt 420

acgagatgct taaaaatctg tttcaaggta gagatttttc gatatttcgg aaaattttgt 480

aaaactgtaa atccgtaaaa ttttgctaaa catatattgt gttgttttgg taagtattga 540

cccaagctat cacctcctgc agtatgtcgt gctaattact ggacacattg tataacagtt 600

ccactgtatt gacaataata aaacctcttc attgacttga gaatgtctgg acagatttgg 660

ctttgtattt ttgatttaca aatgtttttt tggtgattta cccatccaag gcattctcca 720

ggatggttgt ggcatcacgc cgattggcaa acaaaaacta aaatgaaact aaaaagaaac 780

agtttccgct gtcccgttcc tctagtggga gaaagcatga agtaagttct ttaaatatta 840

caaaaaaatt gaacgatatt ataaaattct ttaaaatatt aaaagtaaga acaataagat 900

caattaaatc ataattaatc acattgttca tgatcacaat ttaatttact tcatacgttg 960

tattgttatg ttaaataaaa agattaattt ctatgtaatt gtatctgtac aatacaatgt 1020

gtagatgttt attctatcga aagtaaatac gtcaaaactc gaaaattttc agtataaaaa 1080

ggttcaactt tttcaaatca gcatcagttc ggttccaact ctcaag 1126

<210> 3

<211> 391

<212> DNA

<213> silkworm (Bombyx mori)

<400> 3

agttacggag ctggcagggg atacggacaa ggtgcaggaa gtgcagcttc ctctgtgtca 60

tctgcttcat ctcgcagtta cgactattct cgtcgtaacg tccgcaaaaa ctgtggaatt 120

cctagaagac aactagttgt taaattcaga gcactgcctt gtgtgaattg ctaattttta 180

atataaaata acccttgttt cttacttcgt cctggataca tctatgtttt ttttttcgtt 240

aataaatgag agcatttaag ttattgtttt taattacttt tttttagaaa acagatttcg 300

gattttttgt atgcatttta tttgaatgta ctaatataat caattaatca atgaattcat 360

ttatttaagg gataacaata atccatgaat t 391

<210> 4

<211> 1051

<212> DNA

<213> Trichoplusia ni (Trichoplusia ni)

<400> 4

ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc 60

atgtgtttta tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt 120

tattatattt acacttacat actaataata aattcaacaa acaatttatt tatgtttatt 180

tatttattaa aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttaaa 240

cattctctct tttacaaaaa taaacttatt ttgtacttta aaaacagtca tgttgtatta 300

taaaataagt aattagctta acttatacat aatagaaaca aattatactt attagtcagt 360

cagaaacaac tttggcacat atcaatatta tgctctcgac aaataacttt tttgcatttt 420

ttgcacgatg catttgcctt tcgccttatt ttagaggggc agtaagtaca gtaagtacgt 480

tttttcatta ctggctcttc agtactgtca tctgatgtac caggcacttc atttggcaaa 540

atattagaga tattatcgcg caaatatctc ttcaaagtag gagcttctaa acgcttacgc 600

ataaacgatg acgtcaggct catgtaaagg tttctcataa attttttgcg actttggacc 660

ttttctccct tgctactgac attatggctg tatataataa aagaatttat gcaggcaatg 720

tttatcattc cgtacaataa tgccataggc cacctattcg tcttcctact gcaggtcatc 780

acagaacaca tttggtctag cgtgtccact ccgcctttag tttgattata atacataacc 840

atttgcggtt taccggtact ttcgttgata gaagcatcct catcacaaga tgataataag 900

tataccatct tagctggctt cggtttatat gagacgagag taaggggtcc gtcaaaacaa 960

aacatcgatg ttcccactgg cctggagcga ctgtttttca gtacttccgg tatctcgcgt 1020

ttgtttgatc gcacggttcc cacaatggtt t 1051

<210> 5

<211> 867

<212> DNA

<213> Victoria jellyfish (Aequorea victoria)

<400> 5

gcaaagtgaa cacgtcgcta agcgaaagct aagcaaataa acaagcgcag ctgaacaagc 60

taaacaatcg gggtaccgct agagtcgacg gtacgatcca ccggtcgcca ccatggtgag 120

caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 180

aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 240

gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 300

caccctgacc tggggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 360

cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 420

cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 480

catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 540

gtacaactac atcagccaca acgtctatat caccgccgac aagcagaaga acggcatcaa 600

ggccaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 660

ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 720

cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 780

gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaactctaga 840

tcataatcag ccataccaca tttgtag 867

<210> 6

<211> 679

<212> DNA

<213> Trichoplusia ni (Trichoplusia ni)

<400> 6

agatctgaca atgttcagtg cagagactcg gctacgcctc gtggactttg aagttgacca 60

acaatgttta ttcttacctc taatagtcct ctgtggcaag gtcaagattc tgttagaagc 120

caatgaagaa cctggttgtt caataacatt ttgttcgtct aatatttcac taccgcttga 180

cgttggctgc acttcatgta cctcatctat aaacgcttct tctgtatcgc tctggacgtc 240

atcttcactt acgtgatctg atatttcact gtcagaatcc tcaccaacaa gctcgtcatc 300

gctttgcaga agagcagaga ggatatgctc atcgtctaaa gaactaccca ttttattata 360

tattagtcac gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga 420

acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa 480

tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat 540

tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga 600

cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac 660

gcagactatc tttctaggg 679

Claims (6)

1. A transgenic method for improving the mechanical property of silk by utilizing silk protein of a bageworm is characterized by comprising the following steps:

step S1: constructing a silkworm transgenic system for specifically expressing exogenous bagworm silk protein EvH, wherein the gene sequence of the bagworm silk protein is shown in SEQ ID NO. 1;

step S2: the silkworm transgenic system is up-regulated at a specific part through the initiation of a fibroin fibH promoter, and the sequence of the fibroin fibH promoter is shown as SEQ ID NO. 2;

step S3: the transgenic expression vector is constructed by optimizing and synthesizing the encoding sequence of the bagworm EvH protein according to the preference of silkworm codons:

step S4: the EvH coding sequence was packaged into the subcloning vector p57S [ fibH-MCS-LBS ] which was similarly digested, and the p57S [ fibH-EvH-LBS ] vector was constructed by connecting the fibH promoter (SEQ ID NO.2) in tandem with the multiple cloning site and the LBS (binding site to fibL) sequence (SEQ ID NO. 3).

2. The transgenic method for improving silk mechanical properties by using silk fibroin of bagworms according to claim 1, characterized in that in step S2, an expression vector is constructed based on the gene sequence SEQ ID No.1 and the gene sequence SEQ ID No. 2.

3. The transgenic method for improving silk mechanical properties by using silk fibroin of bagworms according to claim 2, characterized by further comprising the step of S5: the vector plasmid is subjected to single enzyme digestion by Asc I, the recovered target fragment is connected with a pBac [3 xP 3-ECFP ] skeleton vector subjected to the same enzyme digestion, the skeleton vector consists of a piggyBac right arm (SEQ ID NO.4), a 3 xP 3-ECFP (SEQ ID NO.5) and a piggyBac left arm (SEQ ID NO.6), a final expression vector pB [ fibH-EvH-LBS,3 xP 3-ECFP ] is obtained, and the vector skeleton is completed by the following steps: firstly, assembling a 3 XP 3-ECFP sequence (SEQ ID NO.5), wherein the sequence is formed by driving and Expressing Cyan Fluorescent Protein (ECFP) by a 3-fold repeated P3 promoter (eye and nerve specific promoter); then the right arm (SEQ ID NO.4) and the left arm (SEQ ID NO.6) of piggyBac are assembled at the 5 'end and the 3' end of the 3 XP 3-ECFP sequence respectively.

4. The transgenic method for improving the mechanical properties of silk by using silk protein of bagworms according to claim 2, further comprising a production step of 305-based transgenic silkworms and a molecular identification step of EvH-based transgenic silkworms of 305-line, wherein for the purpose of expanding production, a transgene EvH of 305-line-based is introduced into the QB variety, a silk gland phenotype observation step of QB-based variety EvH-based transgenic silkworms, a cocoon shell phenotype observation step of QB-based variety EvH-based transgenic silkworms and a mechanical property detection step of QB-based variety EvH-based transgenic silkworms are observed.

5. A transgenic silkworm variety with a silk mechanical property enhanced by Sacha sinensis Silk protein, which is characterized in that it is obtained by overexpressing Sacha sinensis Silk protein gene EvH in the posterior silk gland of silkworm to obtain a transgenic silkworm strain by using DNA injection means, namely the transgenic method for enhancing silk mechanical property by Sacha sinensis Silk protein according to any one of claims 1 to 4.

6. A method for obtaining a silkworm strain of silk with improved mechanical properties, comprising the steps of:

step W1, vector construction: according to the sequence codon preference of the silkworm genes in the silkworm genome sequence database, the invention carries out codon optimization design on the bag moth silk protein coding gene sequence, and the result is that the bag moth silk protein gene EvH is over-expressed in the silk gland at the rear part of the silkworm;

step W2, obtaining transgenic silkworm strain by silkworm embryo microinjection, and mixing EvH recombinant vector plasmid and transposase expression vector plasmid according to the ratio of 1: 1, mixing in proportion, microinjecting 305 early embryos 1-3h after spawning, breeding G0 generation (the first generation after injection) hatching larvae until eclosion, and then carrying out selfing or backcross seed production;

w3, breeding transgenic silkworms, detecting the obtained G1 generation (the second generation after injection) silkworm eggs under a stereoscopic fluorescence microscope about 1 day before the green-dropping stage, screening transgenic positive individuals which fluoresce in embryo eyes or nerve tissues, and feeding the individuals to adults by taking a moth ring as a unit;

step W4, a large-scale feeding method, namely hybridizing the transgenic line based on the diversification system 305 with QB for more than 3 generations to obtain a transgenic system based on QB, which is beneficial to large-scale feeding;

step W5: observing the specific character generated by the silkworm by the transgene and detecting the mechanical property.

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