CN115820736B - Application of sericin Ser4 of family in improving silk performance and method thereof - Google Patents
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Abstract
The invention discloses an application of sericin Ser4 of silkworm in improving silk performance and a method thereof, wherein complete Ser4 protein with molecular weight more than 250kDa is expressed into a silk gum layer through seamless cloning and homologous recombination, the raised transgenic strain improves the improvement of silk mechanical property, silk of the overexpressed Ser4 strain is more hydrophobic, the degumming rate of silk cocoons is reduced, and the invention is hopeful to be applied to the production of multi-silk sericin silk products.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of silkworm sericin Ser4 in improving silk performance, and also relates to a method for improving silkworm sericin performance by utilizing the silkworm sericin Ser 4.
Background
Silk is the earliest and most widely used natural animal protein fiber for humans. Silkworm can spit different silk fibers at different stages of growth and development. The silkworm larva is subjected to four molting before the silkworm larva is subjected to cocoon preparation in the upper cluster to form five-instar larva. Silkworms secreted from the beginning and end of each age before five ages are collectively referred to as small silks. The silk and silk cocoon silk are composed of sericin and silk fibroin. Silk is the central component of silk and accounts for about 70-80% of the weight of silk. Sericin is a gelatinous globulin, which is present at the periphery of silk and occupies about 20-30% of the total amount of silk. In recent decades, the silk industry has been impacted by synthetic fibers with excellent properties, resulting in a continuous decrease in their yield. Therefore, many scholars have been devoted to develop silks with more excellent comprehensive properties.
Along with the release of the silkworm genome map and the establishment of a silkworm molecular breeding technical system, a molecular and technical foundation is laid for silk genetic improvement and material innovation research. In recent years, silk surrounding performance and silkworm genetic manipulation have become hot spots for silk protein research. In 2003, after a Japanese scholars established a silkworm transgenic technology system with piggyBac transposon as a core, researchers began to construct a genetic operating system capable of accurately regulating and controlling the genes of silkworms, directionally modifying the performances of fibroin and silk, wherein the genetic operating system comprises a gene editing system and a transgenic over-expression system capable of expressing genes in various tissues of silkworms in an increasing manner. In 2013 Zhang et al reported a new editing gene CRISPR/Cas9 system that relies on the base complementary pairing principle to recognize specific base sequences and uses endonucleases to cleave target genes. Compared with ZFN and TALEN, the specificity of the system is greatly improved. At present, CRISPR/Cas9 systems have also found wide application in a number of species including silkworms. The transgenic over-expression technology is also a method capable of effectively improving the properties of silk glands and silk. Different from the gene editing technology, the transgenic over-expression technology only recombining and expressing functional exogenous target proteins in specific tissues of silkworms is mild, and has no great influence on the physique of the silkworms and the characteristics of silk, and researchers successfully reform the performance of the silk and endow the silk with new biological functions by virtue of the genetic operating systems, so that the application field of the silk is expanded. Therefore, the influence of sericin on silk performance is researched by using a CRISPR/Cas9 gene editing system and a silk gland transgenic incremental expression system, and the method has important significance for obtaining high-performance silk.
Disclosure of Invention
Therefore, one of the purposes of the invention is to provide an application of sericin Ser4 of family silk in improving silk performance, wherein the invention utilizes the specific sericin of small silk to express in sericin, so as to obtain silk with better mechanical performance; the second purpose of the invention is to provide a method for improving the performance of silkworm silk; it is a further object of the present invention to provide silk of improved properties obtainable by said process.
In order to achieve the above purpose, the present invention provides the following technical solutions:
The application of the silkworm sericin Ser4 in improving the silkworm silk performance is that the amino acid sequence of the silkworm sericin Ser4 is shown as SEQ ID NO. 5.
Preferably, the silk performance is silk mechanical performance or hydrophobicity.
2. A method for improving silkworm silk performance comprises over-expressing silkworm sericin Ser4 in silkworm, and obtaining silk with improved performance.
Preferably, the over-expression method is to construct a transgenic vector containing sericin Ser4 gene of silkworm, then inject the transgenic vector into silkworm, feed G0 generation, mate and spawn, and fluorescence screen G1 generation silkworm eggs to successfully obtain sericin4 transgenic positive individuals.
Preferably, the transgenic vector contains promoters Ser1 and Hr3 enhancers of sericin gene specifically expressed in middle silk gland of silkworm and Ser1PA terminator.
3. The silk with improved performance is prepared by the method.
Preferably, the silk has Ser4 protein expression.
In the invention, preferably, the strength and Young's modulus of the silk are improved.
The invention has the beneficial effects that: the invention breeds sericin gene functional defect, silkworm spinning is reduced, and mechanical property is reduced homozygous strain by knocking out Ser4 gene; the silk fibroin in the small silkworm period can influence the mechanical properties of silk. Further, through seamless cloning and homologous recombination, the complete Sericin4 protein with the molecular weight of more than 250kDa is expressed into a silk cocoon Sericin layer, and the bred strain has the following characteristics:
1) The sericin Ser4 in the period of the young silkworms can be determined for the first time to influence the silk performance;
2) The improvement of the mechanical property of silk is realized under the condition of not transferring exogenous protein;
3) The silk of the over-expressed Ser4 strain is more hydrophobic, the degumming rate of the silkworm cocoons is reduced, and the over-expressed Ser4 strain is expected to be applied to the production of multi-silk sericin silk products.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 shows the acquisition and detection of transgenic lines (A: ser4 gene gRNA design; B: schematic representation of overexpressed sgRNA vector; C: hybridization of overexpressed gRNA lines with overexpressed Cas9 silkworm lines, obtaining heterozygotes of various editing forms);
FIG. 2 shows the acquisition and detection of sericin4 knockout line (A: ser4 knockout homozygous line edit form; B: transcription level detection; C: silk immunoblotting detection; D: silk gland SDS-PAGE detection);
FIG. 3 shows the mechanical property test of small silk of the strain gRNA-Ser4 and Sericiin 4 -/- (stress-strain curve of small silk of strain A. GRNA-Ser 4; stress-strain curve of small silk of strain B. Sericiin 4 -/-; average stress-strain curve of strain C. GRNA-Ser4 and Sericiin 4 -/-; comparison of silk strength and ductility of strain D. GRNA-Ser4 and Sericiin 4 -/-; analysis of significant difference: p < 0.0001);
FIG. 4 shows cloning of the full-length sericin gene and construction of the transgene vector (A. Full-length sericin gene cloning schematic; B. Sericin4 transgene vector construction. Red box represents the plasmid tested correctly).
FIG. 5 shows the schematic representation of the transgenic vector and the screening of positive individuals (A. Sericiin 4 transgenic vector; B. Positive individuals; 3xp3 is an eye-specific promoter; dsRed is a red fluorescent protein; SV40 is the stop codon sequence of sericin gene; ser1 is the promoter region of sericin1 gene; ser4 is the sericin gene coding sequence; ser1PA is the poly A termination sequence of sericin1 gene).
FIG. 6 shows the detection of the transcription level of the Sericiin 4 gene (WT: D9L line; OE: overexpressing Ser4 line).
FIG. 7 shows the detection of Ser4 protein in silk glands and silk (A: silk gland SDS-PAGE; B: silk immunoblots; WT: D9L strain; OE: overexpressing Ser4 strain).
FIG. 8 shows morphological observations of transgenic lines silk gland, cocoon and pupa (A: silk gland; B: cocoon; C: male pupa; D: female pupa).
FIG. 9 shows the mechanical property measurements of WT and Over-sericin4 silk (A: WT silk stress strain curve; B: over-sericin4 silk stress strain curve; C: WT and Over-sericin silk average stress strain curve).
FIG. 10 shows silk strength and Young's modulus (A: stress; B: strain; C: young's modulus; D: toughness).
FIG. 11 shows the WT and Over-sericin strain silk IR spectrum analysis (A: spectrum of single silk FTIR; B-C: deconvolution analysis of amide I band of silk FTIR spectrum, (. Smallcircle.) raw data, (. Smallcircle.) Total fit curve, (- -) Gaussian function fit peak; D. Beta-sheet structure content of each strain silk).
FIG. 12 shows different sericin proteins.
Fig. 13 shows the variation of cocoon degumming rate at different times (significant difference analysis: unlabeled p.gtoreq.0.05, < p <0.05, < p < 0.01).
FIG. 14 is a graph showing individual fiber contact angle analysis (A: single fiber contact angle; B: contact angle statistics).
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
Example 1
The sequences of the exons sericin and sericin were entered in the CCtop on-line prediction website (https:// crispr. Cos. Uni-heidelberg. De /), and the guide gRNA in the sequences was searched for in the structure of G (N20) GG or G (N19) GG, according to the website hint, to avoid some sites with high miss rate and poor specificity (FIG. 1A). After adding corresponding cleavage sites and terminal bases, primers were synthesized, and specific sequences were as follows:
KO-sericin4-F：5’-TTCATGGTGGTTCTTCTTTG-3’(SEQ ID NO.1)；
KO-sericin4-R：5’-CAAAGAAGAACCACCATGAA-3’(SEQ ID NO.2)；
Then construct the guide RNA transgenic over-expression vector of sericin gene (B in FIG. 1). 3×P3 is a silkworm eye specific promoter, EGFP is green fluorescent protein, and SV40 is a terminator; u6 is a systemic promoter, followed by a sericin gRNA sequence, and TTTTT is a terminator. And (3) feeding the silkworm G0 generation to the metaplastic moth by injecting the constructed gRNA transgenic overexpression vector, carrying out fluorescence screening 6-7 days after mating and spawning, and selecting an individual with eye specific green fluorescence as a gRNA transgenic individual. The resulting gRNA-Ser4 line was crossed with transgenic line individuals overexpressing Cas9 protein, resulting in the F1 generation (C in fig. 1). Selecting silkworm individuals with eyes emitting red and green fluorescence at the same time, namely knocking out the F1 generation of the sericin gene chimera. The genome of the skin sloughed off in the F1 generation positive silkworm pre-pupation stage is extracted, the sequences of ko-Sericin 4-JC-F5'-CTGTGTGTTTGTACCGTATGTTCTAAGGT-3' (SEQ ID NO. 3) and ko-Sericin 4-JC-R5'-TCTCAGTGGAGTCGGGCATGCCCATTGG-3' (SEQ ID NO. 4) are used as primers to detect the sequence near the PAM locus of the Sericin4 gene, the PCR product glue is recovered and then is sent to sequencing analysis, and as a result, various editing forms are found to appear in the PAM region sequencing of the Sericin4 gene (in figure 1C).
And selecting an individual with the sericin gene of which the main editing form is deletion of 1 base for selfing to obtain an F2 generation. The F2 generation selects individuals with single or no light eyes (no edit will continue to be made, and the experiment will be developed with single green eyes only). Screening individuals carrying green fluorescence only in the F2 generation, feeding fresh mulberry leaves until eclosion, extracting the genome of silkworm skin, and marking. After PCR amplification with the primers designed above, the mutant forms were detected again by sequencing. And (5) continuing to select individuals with the same mutation form for selfing to obtain F3 generation. The procedure was repeated until a Sericin4 knockout homozygous mutant was obtained, the homozygous genome was sequenced as a single peak, and the sequence of the Sericin4 -/- genome was deleted for one base (a in fig. 2).
To further confirm the success of the Ser4 protein knockout, four-age silk gland-extracted RNA was collected, and qPCR detection was performed after cDNA inversion, which showed a significant decrease in sericin gene transcript levels (B in FIG. 2). And (5) silk western blot analysis. The results showed that no band appeared in the silk of the Sericin4 -/- line and only bands in the gRNA-Ser4 line, indicating that Sericin4 was successfully knocked out (C in fig. 2). At the same time, SDS-PAGE detection is performed by dissecting silk glands of four-year old silkworms, and the result shows that in the Sericin4 -/- strain, two protein bands of Ser4 disappear (D in FIG. 2).
Example 2
To investigate whether different Sericin compositions contribute to the mechanical properties, the mechanical properties of silk in the Sericin4 -/- strain were examined separately. The four-year-old final silk was chosen for the experiment, and the study results showed that the mechanical properties of the Sericin4 -/- strain silk were far lower than those of the control gRNA-Ser4 strain silk (A-C in FIG. 3). Both silk strength and ductility were significantly reduced after knocking out Ser4 protein, with a 32.5% decrease in strength and a 46.5% decrease in ductility (D in fig. 3). Experimental results show that the mechanical properties of the silk are obviously reduced by knocking out Ser4 protein, which indicates that Ser4 protein may be very important to the properties of the silk.
Since the full-length CDS of sericin gene is close to 7000bp, and the repetitive region of 4000 bp is included, direct cloning and sequencing are very difficult. Therefore, by utilizing the CDS sequence of sericin gene and the piggyBac transgenic vector sequence, a homology arm is designed, the sericin gene coding region (SEQ ID NO. 5) sequence is divided into three sections, the homology arms of 20 bases are respectively designed, and PCR sequencing is respectively carried out by using silkworm embryonic cDNA as a template for amplification (A in FIG. 4). After successful sequencing, the sequence is connected by using a seamless cloning kit, and the sequence is successfully cloned to a transitional vector containing a promoter (Ser 1) of a Sericin1 gene specifically expressed in the middle silk gland of silkworms, an Hr3CQ enhancer and a Ser1PA terminator, so that pSL1180[ Hr3CQ Ser1 Sericin Ser1PA ] is obtained. Connecting an expression frame after single enzyme digestion of the transition vector by AscI with a basic expression vector piggyBac [3xp3 DsRed SV40] of the silkworm transgene after dephosphorylation to obtain a final transgene expression vector piggyBac [3xp3 DsRed SV40; hr3 Ser1 Sericiin 4 Ser1PA ], designated as Over-Sericin4 (A in FIG. 5). Positive clones were successfully detected using both the sericin non-repeat designed detection primer and DsRed sequencing primer, and the final cleavage verification showed that the sericin4 transgenic overexpression vector was successfully constructed (B in fig. 4). The constructed transgenic vector is extracted to inject ultrapure plasmid into silkworms, and after breeding G0 generation and mating and spawning of the moth, the silkworm eggs of the G1 generation are further subjected to fluorescent screening to successfully obtain sericin4 transgenic positive individuals (B in fig. 5), and the transgenic positive individuals are named as Over-sericin strain.
Example 3
Extracting silk gland RNA of five-age 5 th day silkworms of the Over-sericin4 strain and the D9L control strain, reversing the RNA into cDNA, and performing qPCR detection. The results showed that in each transgenic line, the expression of Sericiin 4 was significantly up-regulated (FIG. 6), whereas no expression of Sericiin 4 was detected in the WT control group, indicating that Sericiin 4 was indeed expressed only in the silkworm larval stage.
The above experiment 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 of the five-year-old 7 th day Over-Sericin4 strain and the D9L control strain were collected, protein quantification was performed by dissolving the silk glands and silk with lithium thiocyanate, followed by SDS-PAGE and western blot analysis. Subsequent experiments were performed with OE-12 and OE-20 lines that were highly expressed at the transcriptional level. The results show that bands of Ser4 protein appear in both transgenic lines, and that Ser4 protein accounts for about 6% of the whole fibroin as calculated from gray scale values (A in FIG. 7). Western blot results also showed that Ser4 protein was successfully overexpressed into silk (B in FIG. 7).
The silk gland morphology and development in the Over-sericin4 line were observed. The results showed that the silk gland of the Over-sericin4 line had no significant change 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 gland of bombyx mori. Further, the changes in cocoons of the Over-sericin4 strain were compared with those of the wild-type D9L strain, and cocoons of the Over-sericin strain were not significantly changed in size, shape, color, and the like (B in FIG. 8). Fig. 8C and 8D are comparisons of male and female pupae, respectively, in the two lines. No obvious difference exists between the sizes of the male pupa and the female pupa.
Mechanical property analysis is further carried out on the Over-sericin4 strain silk. The results show that the mechanical properties of the Ser4 silk over-expressed are better than those of the wild-type D9L strain (FIG. 9). Further analysis of silk mechanical property parameters found that both the strength and Young's modulus of the Over-sericin4 strain silk were significantly improved, the strength was improved by 25%, the Young's modulus was improved by 15%, and toughness and ductility were not significantly changed (FIG. 10).
The structure of silk fibres determines the properties and infrared spectroscopy (FTIR) is currently the most widely studied method for analysing silk secondary structures. We performed secondary structural analysis on WT and Over-sericin strain silk using FTIR. The results of the spectrogram show that the amide I, amide II and amide III regions of the infrared spectrum of the monofilament have good resolution, which lays a foundation for the subsequent calculation of the content of silk secondary structures (A in FIG. 11). Of the three amide bond absorption bands, amide I was most widely used by researchers, and therefore by deconvolution analysis of the amide I band, it was found that the Over-sericin strain had a significant prominence Over the control silk peak pattern at the β -sheet position around 1700cm -1 (B-C in fig. 11). The secondary structure content in each silk is calculated according to the peak area, and the result shows that the average beta-sheet content of the Over-sericin4 strain is 36.1 percent, which is higher than 32.7 percent of the control; the content of beta-turn structure is 21.8% higher than that of the control; the spiral and random coil content was 42.1% below the control, 47.3% (D in fig. 11).
At present, the silk of the households is considered to be a semi-crystalline high polymer. Among them, the beta-sheet structure is considered to be a major factor affecting the mechanical properties of silk. That is, the higher the silk beta-sheet structure content, the higher the strength is relatively. The result of the silk secondary structure analysis is also consistent with the mechanical property test result, which shows that Ser4 protein can help silk form more beta-sheet structures, thereby improving the mechanical property of silk.
According to the hydrophilic-hydrophobic prediction, ser4 protein is the most hydrophobic sericin (FIG. 12), and then the degumming rate and the hydrophilic-hydrophobic property of the silk of the Over-sericin strain are changed. First, the degumming rates of the silkworm cocoons of the control and transgenic strains were examined for different degumming times. The results show that after 15min and 30min of degumming, the degumming rate of the Over-sericin4 strain silk is obviously lower than that of the control D9L strain silk; and after degumming for 1 hour, the degumming rate of the Over-sericin line silk and the control are not obviously different, which indicates that the sericin content of the Over-sericin silk is basically the same as that of the control silk (figure 13). These results indicate that Ser4 protein resulted in poor sericin hydrophilicity, reduced degumming rate, but did not affect sericin content.
To further demonstrate that overexpression of Ser4 results in more hydrophobic silk, hydrophilic-hydrophobic analysis was performed on individual silks of different strains using an individual fiber contact angle meter. The results in FIG. 14 show that the Over-sericin4 silk is exposed to a greater angle after water drops to the silk, indicating that the transgenic silk is more hydrophobic; the average contact angle of different strain silks is counted, the contact angle of Over-sericin4 silks is obviously larger than that of control silks, and the expression of Ser4 proves that the silk cocoon filaments are more hydrophobic.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (6)
1. The application of the specific over-expression of the sericin ser4 in the silk sericin layer in improving the silk performance of the silkworm is characterized in that: the nucleotide sequence of the sericin Ser4 of the bombyx mori is shown as SEQ ID NO. 5.
2. The use according to claim 1, characterized in that: the silk performance is silk mechanical performance or hydrophobicity.
3. A method for improving the performance of silkworm silk is characterized in that: and regulating and expressing the Sericin Ser4 in silkworms by using a promoter of a Sericin1 gene, wherein the obtained silk of the transgenic positive individual is the silk with improved performance.
4. A method of improving silk performance according to claim 3, wherein: the over-expression method comprises the steps of constructing a transgenic vector containing a promoter of a Sericiin 1 gene for regulating and controlling Sericin Ser4 of a silkworm, injecting the transgenic vector into the silkworm, breeding G0 generation, mating and spawning by using a metaplastic moth, and successfully obtaining Sericin4 transgenic positive individuals by fluorescent screening of G1 generation silkworm eggs.
5. A method of improving silk performance according to claim 3, wherein: the transgenic vector contains a promoter of a Sericin1 gene specifically expressed by a silkworm middle silk gland, an Hr3 enhancer and a Ser1PA terminator.
6. A silk product with improved properties obtained by the method according to any one of claims 3 to 5.
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| CN112359061A (en) * | 2020-11-16 | 2021-02-12 | 西南大学 | Method for improving mechanical property of silk fiber and product thereof |
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| 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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| WO2021055594A1 (en) * | 2019-09-20 | 2021-03-25 | Cue Biopharma, Inc. | T-cell modulatory polypeptides and methods of use thereof |
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