CN112746085B - Method for improving mechanical property of silk fiber and product thereof - Google Patents
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Abstract
The invention discloses a method for improving mechanical properties of silk fibers and a product thereof, wherein silk with good mechanical properties is obtained by over-expressing silkworm ferritin subunits in silk glands at the front part of a silkworm and cocooning; the nucleotide sequence of the silkworm ferritin subunit is shown in SEQ ID NO. 1; or the nucleotide shown in SEQ ID NO.1 is subjected to substitution and/or deletion and/or addition of one or more bases and expresses a nucleotide sequence with the same function as the subunit of the silkworm ferritin; mechanical tensile test is carried out on the obtained silk of the transgenic silkworm, and the strength, the ductility and the toughness of the transgenic silk fiber are all improved, so that the silk mechanical property can be improved by the silkworm ferritin subunit, and the novel silk fiber is obtained.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for improving mechanical property of silk fiber, and also relates to a product prepared by the method.
Background
Silk has been widely concerned in the fields of textile industry, medicine, biotechnology, military and the like, and the modification of the silk performance is a hot spot of the scientific research of the silkworm industry due to the fiber forming mechanism of silk fiber and how to obtain more and better silk. Silk has better toughness and ductility, but weaker strength and rigidity than other materials. The silk has the defects of mechanical properties, so that the application range and the value of the silk are limited to a certain extent. The improvement on the mechanical property of the silk becomes the important point in expanding the application of the silk, improving the value of the silk and exciting the industry of the silkworm mulberry. At present, researchers have made some progress through methods such as foreign matter feeding, artificial drawing, silk post-treatment, and spider silk protein gene transfer into silkworms. However, these methods, whether by manual drawing or by various treatments of silk, are time consuming, labor intensive and costly and are not suitable for large scale industrial applications. In order to obtain a high-performance silk fiber silkworm strain capable of stably inheriting, high-performance silk needs to be produced from a source, the formation of silk fiber is determined by physiological and biochemical environments and physical factors in a spinning pipeline, calcium ion protein is over-expressed in silkworm silk glands to improve the content of calcium ions, so that the performance of the silk is changed, but the improvement of the mechanical performance of the silk is not high, and therefore, a method capable of improving the mechanical performance of the silk fiber more efficiently is needed.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for improving mechanical properties of silk fibers; the second object of the present invention is to provide silk obtained by the above method; the invention also aims to provide the application of the silkworm ferritin subunit in improving the mechanical property of silk fiber.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the method for improving the mechanical property of silk fiber comprises the steps of excessively expressing silkworm ferritin subunits in front silk glands of a silkworm, and cocooning to obtain silk with good mechanical property; the nucleotide sequence of the silkworm ferritin subunit is shown as SEQ ID NO. 1; or the nucleotide shown in SEQ ID NO.1 is subjected to substitution and/or deletion and/or addition of one or more bases and expresses a nucleotide sequence with the same function as the subunit of the silkworm ferritin.
Preferably, the method for over-expressing the silkworm ferritin subunit in the front silk gland of the silkworm is to use the silkworm epidermal protein BmCP231 promoter to regulate the expression of the silkworm ferritin subunit.
Preferably, the method for over-expressing the domestic silkworm ferritin subunit in the silk gland at the front part of the domestic silkworm comprises the steps of injecting a transgenic expression vector containing the domestic silkworm epidermal protein BmCP231 promoter for regulating the domestic silkworm ferritin subunit into silkworm eggs, culturing and screening the transgenic domestic silkworm, and cocooning to obtain the silk with good mechanical property.
Preferably, the transgenic expression vector is obtained by connecting an expression frame consisting of a bombyx mori epidermal protein BmCP231 promoter, a bombyx mori ferritin subunit and an SV40 termination signal into a multiple cloning site of a shuttle vector pSLfa1180fa to obtain a recombinant vector, then carrying out enzyme digestion on the recombinant vector by Asc I, and then connecting a pBac [3xP3-dsRed Pafm ] vector which is also subjected to enzyme digestion by Asc I.
Preferably, the method for injecting the silkworm eggs comprises the following steps: the transgenic expression vector and the auxiliary vector pHA3PIG are mixed in a molar ratio of 1:1, and the plasmid mixed solution is injected into early embryos by using a microscope and an injection instrument.
Preferably, the transgenic silkworm is obtained by sealing the injected silkworm eggs, hatching to hatch, breeding the hatched larvae into adults, selfing to obtain G1-generation silkworm eggs, and screening transgenic positive individuals expressing red fluorescence in eyes and nerves of silkworm embryos.
2. Silk obtained by the method.
Preferably, the silk contains overexpressed silkworm ferritin subunits.
Preferably, the silk breaking strain is increased by at least 43%, and the breaking strength is increased by at least 57.8%; the Young modulus is improved by at least 48.9 percent, and the toughness is improved by at least 95 percent.
3. The application of the silkworm ferritin subunit in improving the mechanical property of silk fiber, wherein the nucleotide sequence of the silkworm ferritin subunit is shown as SEQ ID NO. 1; or the nucleotide shown in SEQ ID NO.1 is subjected to substitution and/or deletion and/or addition of one or more bases and expresses a nucleotide sequence with the same function as the subunit of the silkworm ferritin.
The invention has the beneficial effects that: the invention discloses a method for preparing novel silk fibers, which expresses a silkworm ferritin subunit in a silk gland at the front part of a silkworm, and mechanical tensile test is carried out on the obtained silk of a transgenic silkworm to find that the strength, the ductility and the toughness of the transgenic silk fibers are all improved, thereby showing that the mechanical property of the obtained silk is obviously improved.
The research process of the invention adopts an overexpression vector to be injected into silkworm eggs, and researches on the positive individuals of the transgenic silkworms find that the ferritin subunits of the silkworms are overexpressed on the mRNA and protein levels, and the content of the secondary structure of the transgenic silks is found to be changed by utilizing the synchronous radiation infrared spectrum analysis. The influence of the silkworm ferritin subunit on the formation of silk fiber is shown, so that the silkworm ferritin subunit can be used for improving the mechanical property of silk.
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 acquisition of transgenic silkworms (A shows a schematic diagram of a transgenic vector and B shows a fluorescent screening diagram).
FIG. 2 shows fluorescence quantitative PCR detection of transgenic silkworms.
FIG. 3 shows Western Blot assay of transgenic silkworms.
FIG. 4 shows the results of mechanical properties of cocoon silk (A and B show the original stress-strain curves of non-transgenic cocoon silk and transgenic cocoon silk; C shows the average stress-strain curve of non-transgenic cocoon silk and transgenic cocoon silk; and D-G shows the statistical results of various mechanical property indexes).
FIG. 5 is an infrared scanning image of transgenic silkworm cocoon silk (A shows an infrared scanning image of non-transgenic cocoon silk and transgenic cocoon silk; B and C show peak separation images of amide I regions of non-transgenic cocoon silk and transgenic cocoon silk; D shows content results of each secondary structure of cocoon silk).
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.
The silkworm variety used in the embodiment is a practical variety of hibiscus, and is provided by a silkworm gene resource library of southwest university of China.
Example 1 cloning of Bombyx mori ferritin subunit Gene BGIBMGA008768
According to a CDS sequence (SEQ ID NO.1) of a BGIBMGA008768 gene in a silkworm genome database, 1 pair of upstream and downstream primers are designed. The designed primers were synthesized by committee of Biotechnology (Shanghai) Co., Ltd. The primer sequences are as follows:
Bm-FerritinH MluI-F：
(SQE ID NO.2) (the portion with a bold font is Mlu I restriction site);
Bm-FerritinH NotI-R：
(SQE ID NO.3) (Not I cleavage site in bold).
And amplifying a CDS fragment of BGIBMGA008768 by using five-day-old Hibiscus manihot tube cDNA as a template and primers Bm-Ferritinh MluI-F and Bm-Ferritinh NotI-R. The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 30 seconds; then denaturation at 98 ℃ for 10 seconds, annealing at 65 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 30 cycles; final extension at 72 ℃ for 2 min. And separating the PCR product by agarose gel electrophoresis, recovering and purifying a target fragment by using a kit gel cutting, and cloning into a vector pMD19-T Simple (TaKaRa) to obtain a recombinant vector pMD19-BGIBMGA 008768.
Example 2 construction of recombinant expression vector containing Bombyx mori BGIBMGA008768 Gene
The recombinant vector pMD19-BGIBMGA008768 is subjected to double enzyme digestion by Mlu I and Not I, a BGIBMGA008768CDS fragment is recovered, and then is connected with a vector pSL [ BmCP231-DsRed-SV40] subjected to double enzyme digestion by Mlu I and Not I (the pSL [ BmCP231-DsRed-SV40] is pSL [ containing a signal peptide BmCP231-DsRed-SV40] in CN102604954A, and the recombinant vector pSL [ BmCP231-BGIBMGA008768-SV40] is obtained. The vector pSL [ BmCP231-DsRed-SV40] is constructed by cloning a shuttle vector with pSLfa1180fa according to a literature reference method (Horn and Wimmer,2000), and the specific construction method is shown in Chinese patent ZL201210085897.3, wherein the sequence containing a signal peptide BmCP231 is shown in SQE ID NO. 8.
The recombinant vector pSL [ BmCP231-BGIBMGA008768-SV40] is cut by Asc I, the BmCP231-BGIBMGA008768-SV40 fragment is recovered, and then is connected with the vector pBac [3xP3-dsRed ] which is also cut by Asc I to obtain the recombinant vector pBac [3xP3-dsRed, BmCP231-BGIBMGA008768-SV40 ]. The vector pBac [3xP3-dsRed ] was constructed according to the literature method (Horn and Wimmer, 2000).
Example 3 obtaining of transgenic Bombyx mori overexpressing ferritin subunit in Pre-Silk gland of Bombyx mori
Mixing a recombinant vector pBac [3xP3-dsRed, BmCP231-BGIBMGA008768-SV40] and a transgenic auxiliary vector pHA3PIG for coding piggyBac transposase according to a molar ratio of 1:1, injecting early embryos (2-4 hours after egg laying and G0 generations) of practical varieties of cottonrose hibiscus (FR) by a microinjector, sealing the injected silkworm eggs with non-toxic glue, accelerating the silkworm eggs to hatch at 25 ℃, feeding the hatched larvae with mulberry leaves, carrying out self-crossing seed production after adults, detecting red fluorescence by exciting light with the wavelength of 460-490 nm under an optometry microscope (Olympus MVX10) for the silkworm eggs at 6-7 days, and screening out transgenic positive individuals specifically exciting the red fluorescence in eyes or nerves, namely obtaining transgenic silkworms excessively expressing ferritin subunits (figure 1).
Example 4 acquisition and detection of novel Silk fibers
(1) Molecular detection of ferritin subunits in transgenic silkworms:
taking front silk gland samples of five-year-old transgenic silkworms and non-transgenic silkworms on the third day respectively, quickly grinding the samples in liquid nitrogen, extracting total RNA, reversely transcribing the total RNA into cDNA, and adopting a BGIBMGA008768 specific primer [ Ferritin-qPCR-F: 5'-cgatgctgcgactgaaga-3' (SQE ID NO. 4); Ferritin-qPCR-R: 5'-ttgatgacctcccggatg-3' (SQE ID NO. 5); performing fluorescent quantitative PCR detection, and taking sw as an internal reference sw-F: 5'-ttcgtactggctcttctcgt-3' (SQE ID NO. 6); sw-R: 5'-caaagttgatagcaattccct-3' (SQE ID NO.7), ABI 7500Fast (USA), the reaction conditions are: pre-denaturation at 95 ℃ for 30s, then 40 cycles are carried out, wherein each cycle is denaturation at 95 ℃ for 3s, annealing at 60 ℃ for 30s, each tissue is subjected to 3 times of repeated experiments, and the Ct value of the target gene and the internal reference Ct mean are collected for data analysis, and the result is shown in figure 2. The results show that the BGIBMGA008768 gene is over-expressed in the anterior silk gland of the transgenic line.
Taking the front silk glands of the transgenic silkworms and the non-transgenic silkworms of the third day of five years old respectively, quickly grinding in liquid nitrogen, dissolving in RIPA lysate, standing at 4 ℃ for 1h, centrifuging to take the supernatant, measuring the total protein concentration, mixing with 5 times of sample adding buffer solution, incubating at 37 ℃ for 30 minutes, carrying out 12% SDS-polyacrylamide gel electrophoresis, and carrying out Western blot analysis by using Anti-BGIBMGA008768 antibody as a primary antibody and Tubulin antibody as an internal reference antibody after the electrophoresis is finished, wherein the result is shown in figure 3. The results show that ferritin subunits were successfully overexpressed in the anterior silk glands of the transgenic lines.
(2) Novel silk fiber obtaining and detecting method
Silkworm cocoons of transgenic silkworms and non-transgenic silkworms are reeled at a constant speed, the same sections (0-100m) are selected, and a part of silk fibers are fixed on a die for mechanical testing and are subjected to a tensile test by using a universal tester (Shimadzu). The experiment was carried out at 25 ℃ and 60% air humidity, using a 50N sensor, at a stretching speed of 1mm/min, and the load and displacement of the silk during stretching were recorded. Another part of the silk fibers was subjected to electron microscope scanning (japanese electron), and the diameters of the silk fibers were measured. The above data were used to plot stress strain curves and the results are shown in figure 4. The results show that in the transgenic lines, the silk breaking strain is 47.86%, and the breaking strength is 540.19 MPa; young's modulus5.66GPa and a toughness of 135.57MJm-3. Compared with wild type, the breaking strain of the silk is improved by 43 percent, and the breaking strength is improved by 57.8 percent; the Young's modulus is improved by 48.9 percent, and the toughness is improved by 95 percent.
In addition, after non-transgenic cocoon silks and transgenic cocoon silks are degummed and dried, synchronous infrared spectrum detection (Thermo Scientific) is carried out, the experiment is carried out at 22 ℃ and 46% of air humidity, infrared transmission scanning is adopted, the collection time is 51s, and the collection times are 256. The results are shown in FIG. 5, for the IR spectrum amide I (1600 cm)-1-1700cm-1) Carrying out peak separation treatment on the regions, and counting the content proportion of each secondary structure, wherein the data shows that the content of the beta-sheet secondary structure in the transgenic cocoon silk is increased; therefore, the novel silk fiber is obtained by the method, the secondary structure of the silk fiber is changed, and the mechanical property is better.
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 substitutions or changes made by the person skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the invention is subject to the claims.
Sequence listing
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<120> method for improving mechanical property of silk fiber and product thereof
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Claims (9)
1. The method for improving the mechanical property of the silk fiber is characterized by comprising the following steps: the silkworm ferritin subunit is excessively expressed in the silk gland at the front part of the silkworm, and the cocoon is formed to obtain the silk with good mechanical property; the nucleotide sequence of the silkworm ferritin subunit is shown in SEQ ID NO. 1.
2. The method for improving the mechanical properties of silk fibers according to claim 1, wherein the mechanical properties of the silk fibers are as follows: the method for over-expressing the silkworm ferritin subunit in the front silk gland of the silkworm is to use the silkworm epidermal protein BmCP231 promoter to regulate the expression of the silkworm ferritin subunit.
3. The method for improving the mechanical property of the silk fiber according to any one of claims 1 to 2, wherein the method comprises the following steps: the method for over-expressing silkworm ferritin subunit in silkworm front silk gland is to inject silkworm egg into transgenic expression vector containing silkworm epidermal protein BmCP231 promoter to regulate silkworm ferritin subunit, culture and screen transgenic silkworm, and obtain silk with good mechanical property.
4. The method for improving the mechanical properties of silk fibers according to claim 3, wherein the mechanical properties of the silk fibers are as follows: the transgenic expression vector is obtained by connecting an expression frame consisting of a bombyx mori epidermal protein BmCP231 promoter, a bombyx mori ferritin subunit and an SV40 termination signal into a multiple cloning site of a shuttle vector pSLfa1180fa to obtain a recombinant vector, then carrying out enzyme digestion on the recombinant vector by Asc I, and then connecting a pBac [3xP3-dsRed Pafm ] vector which is also subjected to enzyme digestion by Asc I.
5. The method for improving the mechanical properties of silk fibers according to claim 3, wherein the mechanical properties of the silk fibers are as follows: the method for injecting the silkworm eggs comprises the following steps: the transgenic expression vector and the auxiliary vector pHA3PIG are mixed in a molar ratio of 1:1, and the plasmid mixed solution is injected into early embryos by using a microscope and an injection instrument.
6. The method for improving the mechanical properties of silk fibers according to claim 3, wherein the mechanical properties of the silk fibers are as follows: the culture and screening of the transgenic silkworm is to seal the injected silkworm eggs, accelerate the hatching to hatch, breed the hatched larvae into adults, perform selfing to obtain G1 generation silkworm eggs, and screen transgenic positive individuals expressing red fluorescence in eyes and nerves of silkworm embryos to obtain the transgenic silkworm.
7. Silk obtainable by the method according to any one of claims 1 to 6.
8. Silk according to claim 7, wherein: the silk breaking strain is improved by at least 43 percent, and the breaking strength is improved by at least 57.8 percent; the Young modulus is improved by at least 48.9 percent, and the toughness is improved by at least 95 percent.
9. The application of the silkworm ferritin subunit in improving the mechanical property of silk fiber is characterized in that: the nucleotide sequence of the silkworm ferritin subunit is shown in SEQ ID NO. 1.
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