CN115820736A - Application of sericin protein Ser4 in improving silk performance and method thereof - Google Patents
Application of sericin protein Ser4 in improving silk performance and method thereof Download PDFInfo
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Abstract
The invention discloses application of sericin silk Ser4 in silk performance improvement and a method thereof, wherein complete Ser4 protein with the molecular weight of more than 250kDa is expressed in a cocoon silk glue layer through seamless cloning and homologous recombination, the mechanical performance of silk is improved by a cultured transgenic strain, silk over-expressing the Ser4 strain is more hydrophobic, the degumming rate of silkworm cocoon is reduced, and the sericin silk product is expected to be applied to production of a multi-silk product.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of sericin Ser4 in improving silk performance, and further relates to a method for improving the performance of sericin Ser4 in silkworm.
Background
Silk is the earliest and most widely used natural animal protein fiber for human beings. The silkworms can spit different kinds of silk fibers at different stages of growth and development. Silkworm larvae are ecdysis for four times to form five-instar larvae before cocoons are clustered. Silkworms excreted at the beginning and end of each age before the fifth age are collectively called young silks. The small silk and the silkworm cocoon silk are both composed of silk glue and silk fibroin. The silk fibroin is the central component of silk and accounts for about 70-80% of the weight of the silk. Sericin is a colloidal globulin, and is present around silk fibroin, accounting for about 20-30% of the total silk. The silk industry has been impacted by synthetic fibers with superior properties over the last several decades, resulting in a continuous decrease in its yield. Therefore, many researchers have been working on developing silks with more excellent overall properties.
With the release of the silkworm genome map and the establishment of the silkworm molecular breeding technical system, a molecular and technical foundation is laid for the genetic improvement and material innovation research of silk. In recent years, genetic manipulation around silk performance and silkworms has become a hot spot for silk protein research. In 2003, after a Japanese scholar establishes a silkworm transgenic technical system taking piggyBac transposon as a core, researchers begin to construct a genetic operation system which can accurately regulate and control the genes of silkworms and directionally modify the performance of fibroin and silk, wherein the genetic operation system comprises a gene editing system and a transgenic overexpression system which can incrementally express the genes in each tissue of the silkworms. In 2013, zhang et al report a new editing gene CRISPR/Cas9 system, which recognizes a specific base sequence by the base complementary pairing principle and cuts a target gene by endonuclease. Compared with ZFN and TALEN, the specificity of the system is greatly improved. At present, the CRISPR/Cas9 system has also found wide application in a number of species including bombyx mori. The transgenic overexpression technology is also a method capable of efficiently modifying the performance of the silkworm silk gland and the silk. Different from a gene editing technology, the transgenic overexpression technology only recombines and expresses functional exogenous target protein in specific tissues of the silkworms, is mild and is not easy to have great influence on the physique of the silkworms and the characteristics of the silks, and researchers successfully reform the performance of the silks, endow the silks with new biological functions and expand the application field of the silks by means of the genetic operation systems. Therefore, the CRISPR/Cas9 gene editing system and the silk gland transgenic incremental expression system are utilized to research the influence of sericin on the performance of silk, and the method has important significance for obtaining high-performance silk.
Disclosure of Invention
In view of the above, one of the purposes of the present invention is to provide an application of sericin Ser4 in silk performance improvement, wherein the silk with better mechanical properties is obtained by expressing the specific sericin of small silks in sericin; the second purpose of the invention is to provide a method for improving the performance of the domestic silk; the invention also aims to provide silk with improved performance, which is prepared by the method.
In order to achieve the purpose, the invention provides the following technical scheme:
the application of the sericin protein Ser4 in improving the performance of the silkworm silk is disclosed, wherein the amino acid sequence of the sericin protein Ser4 is shown as SEQ ID No. 5.
Preferably, the silk performance is silk mechanical performance or hydrophobicity.
2. A method for improving the performance of silkworm silk comprises the steps of carrying out overexpression on silkworm sericin Ser4 in silkworms, and obtaining silk obtained from transgenic positive individuals, namely the silk with improved performance.
Preferably, the overexpression method comprises the steps of constructing a transgenic vector containing the sericin Ser4 gene of the silkworm, injecting the transgenic vector into the silkworm, feeding G0 generation, mating and oviposition of moths, and performing fluorescence screening on G1 generation silkworm eggs to successfully obtain a sericin4 transgenic positive individual.
Preferably, the transgenic vector contains a promoter Ser1, an Hr3 enhancer and a Ser1PA terminator of sericin1 gene specifically expressed by the middle silk gland of the silkworm.
3. The silk with improved performance is prepared by the method.
Preferably, the silk has Ser4 protein expression.
Preferably, the strength and Young's modulus of the silk are improved.
The invention has the beneficial effects that: according to the invention, by knocking out the Ser4 gene, a homozygous strain with sericin gene function defect, reduced silking of silkworms and reduced mechanical properties is bred; the first time proves that the sericin in the young silkworm stage of the silkworms can influence the mechanical properties of the silks. Further through seamless cloning and homologous recombination, the complete Sericin4 protein with the molecular weight of more than 250kDa is expressed in a cocoon silk glue layer, and the bred strain has the following characteristics:
1) Determining that sericin Ser4 in a young silkworm period can influence the performance of silk for the first time;
2) The mechanical property of the silk is improved under the condition of not transferring foreign protein;
3) The silk over-expressing the Ser4 strain is more hydrophobic, the degumming rate of the silkworm cocoon is reduced, and the silk over-expressing Ser4 strain is expected to be applied to the production of multi-silk glue silk products.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the transgenic line acquisition and detection (A: ser4 gene gRNA design; B: overexpression sgRNA vector schematic diagram; C: hybridization of overexpression gRNA line and overexpression Cas9 silkworm line, obtaining heterozygote of multiple editing forms);
FIG. 2 shows the acquisition and detection of sericin4 gene knockout strain (A: ser4 gene knockout homozygous strain editing form; B: transcription level detection; C: silk immunoblot detection; D: silk gland SDS-PAGE detection);
FIG. 3 shows gRNA-Ser4 and Sericin4 -/- Mechanical property detection of small silk strain (A.gRNA-Ser 4 strain small silk stress-strain curve; B.Sericin 4) -/- Strain small silk stress-strain curve; gRNA-Ser4 and Sericin4 -/- Strain mean stress strain curve; gRNA-Ser4 and Sericin4 -/- Comparing the strength and the ductility of the strain silk; significant difference analysis: * P<0.0001)；
FIG. 4 shows the cloning of full-length sericin4 gene and the construction of transgenic vector (A. Full-length sericin4 gene cloning diagram; B. Sericin4 transgenic vector construction. Red box represents the correct plasmid to be detected).
FIG. 5 shows a schematic diagram of a transgenic vector and a screening of positive individuals (A. Sericin4 transgenic vector, B. Screening of positive individuals, 3xp3 is an eye-specific promoter, dsRed is a red fluorescent protein, SV40 is a stop codon sequence of a seriin 1 gene, ser1 is a promoter region of a seriin 1 gene, ser4 is a coding sequence of the seriin 4 gene, and Ser1PA is a poly A stop sequence of the seriin 1 gene).
FIG. 6 shows the measurement of the transcriptional level of Sericin4 gene (WT: D9L strain; OE: line Ser4 overexpression).
FIG. 7 shows the detection of Ser4 protein in silk glands and silk (A: silk gland SDS-PAGE; B: silk immunoblot; WT: D9L strain; OE: line over-expressed Ser 4).
FIG. 8 shows the morphology observation of transgenic silk gland, silkworm cocoon and silkworm pupa (A: silk gland; B: silkworm cocoon; C: male pupa; D: female pupa).
FIG. 9 shows the mechanical property test of WT and Over-sericin4 silkworm cocoon silk (A: WT silk stress-strain curve; B: over-sericin4 silk stress-strain curve; C: WT and Over-sericin4 silk average stress-strain curve).
FIG. 10 shows silk strength and Young's modulus (A: stress; B: strain; C: young's modulus; D: tenacity).
FIG. 11 shows the infrared spectrum analysis of WT and Over-serin 4 silk strains (A: spectrum of single silk FTIR; B-C: deconvolution analysis of spectrum I band of silk FTIR spectrum, (. Smallcircle.) raw data, (. Smallcircle. -) fitting curve, (. Beta. -folding structure content of each strain silk).
FIG. 12 shows different sericins.
FIG. 13 shows the change in the degumming rate of cocoons at different times (significant difference analysis: unlabeled p.gtoreq.0.05,. Times.p <0.05,. Times.p < 0.01).
FIG. 14 is a single fiber contact angle analysis (A: single fiber contact angle; B: contact angle statistics).
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1
Sequences of the exons of serin 4 and serin 5 were entered into the CCtop online prediction website (https:// crispr. Cos. Uni-heidelberg. De /), and the structure of G (N20) GG or G (N19) GG was used to find guide gRNA in the sequence, according to which the website suggested avoidance of some sites with high off-target rates and poor specificity (A in FIG. 1). After adding corresponding enzyme cutting sites and terminal bases, synthesizing primers, wherein the specific sequences are as follows:
KO-sericin4-F：5’-TTCATGGTGGTTCTTCTTTG-3’(SEQ ID NO.1)；
KO-sericin4-R：5’-CAAAGAAGAACCACCATGAA-3’(SEQ ID NO.2)；
then, a guide RNA transgenic overexpression vector of the sericin4 gene was constructed (B in FIG. 1). 3xP3 is a specific promoter of the eye of the silkworm, EGFP is green fluorescent protein, and SV40 is a terminator; u6 is a systemic promoter, a sericin gRNA sequence is connected behind the promoter, and TTTTT is a terminator. The constructed gRNA transgenic overexpression vector is injected, silkworms are bred from G0 generation to moth breeding, fluorescence screening is carried out 6-7 days after mating and oviposition, and an individual with eye part specifically emitting green fluorescence is selected as a gRNA transgenic individual. The obtained gRNA-Ser4 line was crossed with individuals of transgenic line overexpressing Cas9 protein to generate F1 generation (C in fig. 1). And selecting a silkworm individual with eyes simultaneously emitting red and green fluorescence, namely a chimera F1 generation of knocking out the sericin4 gene. The genome of the skin after the F1 generation positive silkworm pupa pre-stage sloughing was extracted, the sequence near the PAM site of Sericin4 gene was detected using the sequences ko-Sericin4-JC-F:5 'CTGTGTGTTTGTACCGTATGTTCTAAGGT-3' (SEQ ID NO. 3) and ko-Sericin4-JC-R:5 'TCTCAGTGGAGTCGGGCATGCCCATTGG-3' (SEQ ID NO. 4) as primers, and PCR product gel was recovered and subjected to sequencing analysis, and as a result, it was found that various editing forms occurred in the PAM region of Sericin4 gene (C in FIG. 1).
Individuals with the main editing form of the sericin4 gene of lacking 1 base are selected for selfing to obtain F2 generation. Individuals with single or no eye light were selected in the F2 generation (editing was not resumed, and experiments were subsequently developed with only single eye green light individuals). Screening individuals carrying green fluorescence in the F2 generation, feeding the individuals with fresh mulberry leaves until emergence, and extractingThe genome of silkworm skin is marked. After PCR amplification with the primers designed above, the mutant forms were detected by sequencing again. And continuously selecting individuals with the same mutation form for selfing to obtain an F3 generation. Repeating the operation until obtaining a Sericin4 gene knockout homozygous mutant, sequencing the genome of the homozygote as a single peak, and Sericin4 -/- The genome sequence is missing one base (A in FIG. 2).
To further confirm the success of the Ser4 protein knock-out, four-year old silk gland-extracted RNA was collected, and qPCR assay was performed after cDNA inversion, showing a significant decrease in the sericin4 gene transcript level (B in fig. 2). And (3) analyzing the silk western blot. The results show that in Sericin4 -/- No bands were present in the silk of the line, only in the gRNA-Ser4 line, indicating that Sericin Sericin4 was successfully knocked out (C in FIG. 2). Meanwhile, the silk gland of the four-instar silkworm is dissected and subjected to SDS-PAGE detection, and the result shows that the silk gland of the four-instar silkworm is detected in Sericin4 -/- In the line, two protein bands of Ser4 disappeared (D in FIG. 2).
Example 2
To investigate whether different Sericin compositions contributed to mechanical properties, sericin4 was examined separately -/- Mechanical properties of silk in the strain. Four-year old silk was selected for the experiment and the results of the study showed that Sericin4 -/- The mechanical property of the silk strain is far lower than that of the silk of a control gRNA-Ser4 strain (A-C in figure 3). Both silk strength and ductility were significantly reduced after Ser4 protein knock-out, with a 32.5% reduction in strength and a 46.5% reduction in ductility (fig. 3D). The experimental result shows that the mechanical property of the small silk is obviously reduced by knocking out the Ser4 protein, which indicates that the Ser4 protein is probably very important to the performance of the silk.
As the full-length CDS of the sericin4 gene is close to 7000bp, and a repetition region of more than 4000 bp is also included, direct cloning and sequencing have great difficulty. Therefore, a homologous arm is designed by using a CDS sequence of the sericin4 gene and a piggyBac transgenic vector sequence, a coding region (SEQ ID NO. 5) sequence of the sericin4 gene is divided into three sections, homologous arms with 20 bases are respectively designed, and PCR sequencing is respectively carried out by taking silkworm embryonic-stage cDNA as a template for amplification (A in figure 4). After sequencing is successful, a seamless cloning kit is used for connection, and the continuous cloning is successfully carried out to a transition vector containing a Sericin1 gene promoter (Ser 1), an Hr3CQ enhancer and a Ser1PA terminator which are specifically expressed by the middle silk gland of the silkworm, so as to obtain pSL1180[ Hr3CQ Ser1 Sericin4 Ser1PA ]. Connecting an expression frame after the transition vector is digested by the AscI single enzyme with a dephosphorylated bombyx mori transgenic basic expression vector piggyBac [3xp3 DsRed SV40] to obtain a final transgenic expression vector piggyBac [3xp3 DsRed SV40; hr3 Ser1 Sericin4 Ser1PA ], designated Over-Sericin4 (A in FIG. 5). The detection primer designed by utilizing the sericin4 non-repetitive region and the sequencing primer of DsRed successfully detect positive clones, and the final enzyme digestion verification result shows that a sericin4 transgenic overexpression vector is successfully constructed (B in figure 4). Ultrapure plasmids extracted from the constructed transgenic vector are injected into silkworms, and the silkworms are bred for G0 generation, mated and laid eggs, and further fluorescence screening of G1 generation silkworm eggs successfully obtains a sericin4 transgenic positive individual (B in figure 5), which is named as an Over-sericin4 strain below.
Example 3
And extracting silk gland RNA of five-instar days of the Over-sericin4 strain and the D9L control strain, inverting the silk gland RNA into cDNA, and performing qPCR detection. The results showed that Sericin4 was significantly up-regulated in each transgenic line (fig. 6), whereas no expression of Sericin4 was detected in the WT control group, also indicating that Sericin4 was indeed expressed only in the young silkworm stage of the silkworm larvae.
The above experiments successfully detected the expression of Sericin4 at the transcriptional level, and to verify the presence of Ser4 at the protein level, silk glands and cocoons from the five-year-old 7 day Over-Sericin4 line and the D9L control line were collected, and silk glands and silk were solubilized with lithium thiocyanate for protein quantification, followed by SDS-PAGE and western blot analysis. Lines of OE-12 and OE-20 that are highly expressed at the transcriptional level were selected for subsequent experiments. The results showed that a band of Ser4 protein appeared in both transgenic lines, the Ser4 protein accounting for approximately 6% of the total fibroin, calculated from grey values (a in fig. 7). Western blot results also showed that Ser4 protein was successfully overexpressed in silk (B in FIG. 7).
Silk gland morphology and development were observed in the Over-sericin4 line. The results showed that there was no significant change in the silk glands of the Over-sericin4 line compared to the wild-type D9L line (a in fig. 8), indicating that the expression of Ser4 protein had no effect on the development and morphology of the silk glands of silkworms. Further, in comparison of the change of cocoons of the Over-sericin4 line and the wild-type D9L line, no significant change was observed in the cocoons of the Over-sericin4 line in terms of size, shape, color, and the like (B in fig. 8). C in FIG. 8 and D in FIG. 8 are comparisons of male and female pupae in the two lines, respectively. No matter whether male pupae or female pupae exist, the sizes of the male pupae and the female pupae are not obviously different.
Further, mechanical property analysis was performed on the Over-sericin4 strain silk. The results show that the mechanical properties of silk overexpressing Ser4 are superior to those of silk of wild type D9L strain (FIG. 9). Further analysis on the mechanical property parameters of silk shows that the strength and Young modulus of silk of the Over-sericin4 strain are both obviously improved, the strength is improved by 25%, the Young modulus is improved by 15%, and the toughness and the ductility are not obviously changed (figure 10).
The structure of silk fiber determines the performance, and infrared spectroscopy (FTIR) is the most extensive research method for analyzing the secondary structure of silk at present. We performed secondary structure analysis on WT and Over-sericin4 line silk using FTIR. The spectrum result shows that the amide I, amide II and amide III regions of the monofilament infrared spectrum have good resolution, which lays a foundation for the subsequent calculation of the content of the secondary structure of the silk (A in figure 11). Of the three amide bond absorption bands, amide I was most widely used by researchers, and was therefore found by deconvolution analysis of the amide I band at 1700cm -1 At the left and right beta-sheet positions, the Over-sericin4 line has a distinct prominence compared with the peak pattern of the control silk (B-C in FIG. 11). Calculating the content of the secondary structure in each silk according to the peak area, wherein the result shows that the average beta-sheet content of the Over-sericin4 strain is 36.1 percent and is higher than that of the contrast by 32.7 percent; the content of beta-turn structure is 21.8 percent which is also higher than 20 percent of the control; the helix and random coil content was 42.1% lower than 47.3% of the control (D in fig. 11).
At present, the silkworm silk is considered to be a semi-crystalline high molecular polymer by research. Among them, the β -sheet structure is considered to be a major factor affecting the mechanical properties of silk. That is, the higher the content of the β -sheet structure of silk, the higher the strength. The result of the secondary structure analysis of the silk also accords with the result of the mechanical property test, which shows that the Ser4 protein can help the silk to form more beta-sheet structures, thereby improving the mechanical property of the silk.
According to the hydropathic prediction, the Ser4 protein is the most hydrophobic sericin (FIG. 12), and then whether the degumming rate and the hydropathic property of the silk of the Over-sericin4 line are changed or not is judged. The degumming rates of the silkworm cocoons of different degumming time, control and transgenic lines are firstly detected. The results show that after degumming is carried out for 15min and 30min, the degumming rate of the Over-sericin4 strain silk is obviously lower than that of the contrast D9L strain silk; after 1 hour of degumming, the degumming rate of the Over-sericin4 silk was not significantly different from that of the control, which indicates that the sericin content of the Over-sericin4 silk was substantially the same as that of the control silk (fig. 13). These results indicate that Ser4 protein results in a poor hydrophilicity of sericin and a reduced degumming rate without affecting sericin content.
To further prove that overexpression of Ser4 causes silk to be more hydrophobic, hydrophilic and hydrophobic property analysis was performed on single silk of different strains by using a single fiber contact angle measuring instrument. FIG. 14 shows that the contact angle of Over-sericin4 silk after dripping water to silk is larger, which indicates that the transgenic silk is more hydrophobic; counting the average contact angle of silk of different strains, wherein the contact angle of Over-sericin4 silk is obviously larger than that of contrast silk, and proving that the expression of Ser4 leads the silkworm cocoon silk to be more hydrophobic.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (8)
1. The application of the sericin protein Ser4 in improving the performance of the silkworm silk is characterized in that: the amino acid sequence of the sericin Ser4 is shown in SEQ ID NO. 5.
2. Use according to claim 1, characterized in that: the silk performance is silk mechanical property or hydrophobicity.
3. A method for improving the performance of silkworm silk is characterized by comprising the following steps: the sericin protein Ser4 of the silkworm is over-expressed in the silkworm, and the silk obtained by the obtained transgenic positive individual is the silk with improved performance.
4. The method for improving the performance of silkworm silk according to claim 3, wherein the method comprises the following steps: the overexpression method comprises the steps of constructing a transgenic vector containing sericin Ser4, injecting the transgenic vector into silkworms, feeding G0 generation, enabling moths to mate and lay eggs, and screening G1 generation silkworm eggs by fluorescence to successfully obtain sericin4 transgenic positive individuals.
5. The method for improving the performance of silkworm silk according to claim 3, wherein the method comprises the following steps: the transgenic vector contains a promoter Ser1, an Hr3 enhancer and a Ser1PA terminator of sericin1 gene specifically expressed by middle silk glands of silkworms.
6. Silk with improved properties obtainable by the process according to any one of claims 3 to 5.
7. Silk according to claim 6, characterized in that: the silk has Ser4 protein expression.
8. Silk according to claim 6, characterized in that: the strength and Young's modulus of the silk are improved.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211572164.2A CN115820736B (en) | 2022-12-08 | 2022-12-08 | Application of sericin Ser4 of family in improving silk performance and method thereof |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6270812B1 (en) * | 2000-01-31 | 2001-08-07 | Texas Tech University | Reducing E. coli content of beef |
| CN106011172A (en) * | 2016-05-24 | 2016-10-12 | 苏州大学 | Preparation method of bombyx mori capable of synthesizing and secreting hydrophilic sericin on posterior division of silkgland |
| CN112359061A (en) * | 2020-11-16 | 2021-02-12 | 西南大学 | Method for improving mechanical property of silk fiber and product thereof |
| WO2021055594A1 (en) * | 2019-09-20 | 2021-03-25 | Cue Biopharma, Inc. | T-cell modulatory polypeptides and methods of use thereof |
| CN114457121A (en) * | 2022-03-04 | 2022-05-10 | 西南大学 | Transgenic method for directionally inhibiting silkworm fibroin gene by using BmYki and isomer thereof and silkworm variety thereof |
| CN114540421A (en) * | 2022-03-04 | 2022-05-27 | 西南大学 | Controllable editing method for silkworm MSG and PSG expression genes |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6270812B1 (en) * | 2000-01-31 | 2001-08-07 | Texas Tech University | Reducing E. coli content of beef |
| CN106011172A (en) * | 2016-05-24 | 2016-10-12 | 苏州大学 | Preparation method of bombyx mori capable of synthesizing and secreting hydrophilic sericin on posterior division of silkgland |
| WO2021055594A1 (en) * | 2019-09-20 | 2021-03-25 | Cue Biopharma, Inc. | T-cell modulatory polypeptides and methods of use thereof |
| CN112359061A (en) * | 2020-11-16 | 2021-02-12 | 西南大学 | Method for improving mechanical property of silk fiber and product thereof |
| CN114457121A (en) * | 2022-03-04 | 2022-05-10 | 西南大学 | Transgenic method for directionally inhibiting silkworm fibroin gene by using BmYki and isomer thereof and silkworm variety thereof |
| CN114540421A (en) * | 2022-03-04 | 2022-05-27 | 西南大学 | Controllable editing method for silkworm MSG and PSG expression genes |
Non-Patent Citations (1)
| Title |
|---|
| ZHAOMING DONG等: "Identification of bombyx mori sericin 4 protein as a new biological adhesive", INT J BIOL MACROMOL, 27 March 2019 (2019-03-27) * |
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