CN113943360A - Aquatic bioprotein molecule for improving mechanical property of silk and application method thereof - Google Patents

Aquatic bioprotein molecule for improving mechanical property of silk and application method thereof Download PDF

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CN113943360A
CN113943360A CN202111202154.5A CN202111202154A CN113943360A CN 113943360 A CN113943360 A CN 113943360A CN 202111202154 A CN202111202154 A CN 202111202154A CN 113943360 A CN113943360 A CN 113943360A
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silk
gene
protein
leu
mechanical properties
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CN113943360B (en
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钟伯雄
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Zhejiang University ZJU
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Abstract

The invention discloses an aquatic bioprotein molecule for improving mechanical properties of silk and an application method thereof. The method adopts single or multiple barnacle viscous protein genes to construct an exogenous gene vector, integrates barnacle viscous protein sequences into a silkworm genome by utilizing a molecular biology technology, finally obtains a novel silkworm variety with excellent performance of the composite silk capable of being stably inherited, and produces the composite silk by utilizing the novel silkworm variety. The invention finds the application of aquatic bioprotein molecules in improving the performance of silk and develops novel high-performance composite silk.

Description

Aquatic bioprotein molecule for improving mechanical property of silk and application method thereof
Technical Field
The invention relates to a method for constructing and applying a functional protein molecule, in particular to a method for manufacturing high-performance composite silk by using aquatic organism barnacle viscous protein molecules and protein molecules formed by connecting a plurality of genes in series and using the protein sequence.
Background
Traditional silk fibers are mainly applied to the textile industry, and with the improvement of processing technology and the development of interdisciplines, many functional silk fibers are developed at present, so that the application fields are expanded, such as application to wound healing, biosensors, energy collection and the like.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to develop a barnacle adhesive protein molecule, a protein molecule artificial design method formed by connecting a plurality of genes of the barnacle adhesive protein molecule in series, a synthetic single-chain or poly-chain protein molecule structure and a method for developing novel high-performance composite filaments by using the artificial protein molecule.
The invention specifically designs barnacle viscous protein molecules and protein molecules formed by connecting a plurality of genes in series, and obtains silkworm varieties which can produce composite silks containing single-or multi-polymer protein molecular components and fibroin components and have excellent performance by using a molecular biology technology.
The invention greatly improves the economic value of silk, creates a new situation of silkworm mulberry production, and has great economic benefit and wide application prospect.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the aquatic bioprotein molecule for improving mechanical property of silk:
the aquatic organism protein molecule is a protein sequence formed by connecting a single barnacle adhesive protein gene or a plurality of barnacle adhesive protein genes in series.
The protein sequence formed by connecting a plurality of barnacle adhesive protein genes in series is formed by repeatedly connecting the protein sequences of the barnacle adhesive protein genes for a plurality of times.
The protein sequence formed by connecting a plurality of barnacle adhesive protein genes in series is formed by repeating and connecting 2-32 times.
The protein sequence of the single barnacle adhesive protein gene is SEQ ID NO.2, and the DNA alkali sequence is SEQ ID NO. 1.
And II, application of aquatic organism protein molecules in gene editing and transgenic silkworms.
In particular to the application of the compound silk for gene editing and transgenic silkworm production to improve mechanical and mechanical properties.
Thirdly, a production method of the composite silk for improving mechanical and mechanical properties, which comprises the steps of biosynthesizing a target gene sequence by a gene engineering method, then constructing an exogenous gene vector by adopting a molecular biology technology, introducing the exogenous gene vector into silkworm eggs through microinjection and integrating the exogenous gene vector into silkworm genomes, carrying out multi-generation cultivation on silkworms, screening G1 generation positive individuals through a fluorescence microscope, mating the G1 generation positive individuals with wild type or selfing the positive individuals to obtain G2 generation, screening the positive individuals after the G3 generation, and reserving seeds by adopting the selfing of the positive individuals until the G8 generation breeds a stably inherited variety; the method comprises the steps of producing secreted silk by G8 generation silkworms to serve as composite silk, obtaining the composite silk with improved mechanical properties, wherein the target gene sequence is a gene base sequence corresponding to a protein sequence formed by connecting a single barnacle adhesive protein gene or a plurality of barnacle adhesive protein genes in series according to claim 1, and finally obtaining a novel silkworm variety with stable inheritance and excellent composite silk properties.
The positive individuals refer to individuals showing fluorescence.
The wild type refers to an individual cultivated by silkworm eggs without introduced foreign gene vectors.
The exogenous gene vector corresponding to the polyprotein molecule is a transgenic vector with piggyBac as a framework or a homologous recombination vector applied to gene editing.
The exogenous gene vector comprises an exogenous gene expression frame and a fluorescence screening marker gene expression frame; the exogenous gene expression frame comprises exogenous genes and promoters of the expression frame, the exogenous genes are gene base sequences corresponding to a single barnacle viscous protein gene or a protein sequence formed by connecting a plurality of genes in series as claimed in claim 1, and the promoters of the exogenous gene expression frame adopt the promoters of genes such as silk fibroin heavy chain, silk fibroin light chain, silk fibroin P25 or sericin;
the fluorescent screening marker gene expression frame comprises a fluorescent screening marker gene and a promoter of the expression frame, the fluorescent screening marker gene adopts a green fluorescent protein Gene (GFP) or a red fluorescent protein gene (DsRed), and the promoter of the fluorescent screening marker gene expression frame adopts an IE-1, A3 or 3xP3 promoter.
The composition of the composite silk comprises a single barnacle adhesive protein or a protein molecule component and a fibroin component which are formed by connecting a plurality of genes in series. The single-poly protein molecule designed and constructed by the invention is expressed in the composite filament, and the mechanical and mechanical properties of the composite filament are enhanced.
Barnacles (barnacles) are a class of crustaceans, and Barnacle mucin is a class of secreted proteins that function in vitro and are therefore defined in the classification as "in vitro mucin". The barnacle adhesive protein is composed of 5 different proteins, and is divided into the following proteins according to the molecular weight: mucin (cp)100, cp68, cp52, cp20, cp 19. These proteins are located at the natural adhesive junction between the barnacle calcareous substrate and the external matrix, and these five proteins are unique among the underwater adhesion proteins, and no homologous protein is found in the existing databases. The 5 kinds of barnacle adhesive proteins have low similarity of main structures, different molecular weights and different amino acid composition preferences. On the basis, barnacle adhesive proteins are classified into 3 types: hydrophobins Mrcp100k and Mrcp52 k; hydrophilic proteins Mrcp20k rich in charged amino acids, and hydrophilic proteins Mrcp19k and Mrcp68k rich in Ser, Thr, Gly, Ala, Val and Lys residues. Mrcp100k and Mrcp52k are adhesive proteins involved in the self-assembly and curing process, and their complexes form the major adhesion domains of barnacles to the external matrix, and are the major components of barnacle adhesive proteins.
The research of the invention finds that the stress performance, particularly the strain performance of the recombinant silk formed by the barnacle adhesive protein Mrcp100k is obviously improved, and the novel composite silk can be developed to expand the new application of the silk.
The invention creatively constructs barnacle adhesive protein molecules to obtain a protein sequence capable of improving the mechanical property of the composite silk of silkworms.
The invention discloses a method for forming a single-or multi-polymer protein molecular structure and a new application thereof, and develops various novel high-performance composite filaments.
The invention has the beneficial effects that:
the invention develops a method for artificially designing and synthesizing functional genes and improving the mechanical property of the composite silk of the silkworms, obtains a plurality of functional genes, and obviously enhances the mechanical property of the composite silk by utilizing the genes.
Detailed Description
The present invention will be further described with reference to the following examples.
The examples of the invention are as follows:
example 1
Optimizing a haploid sequence of barnacle viscous protein molecules Mrcp-100k according to silkworm codon preference, wherein the gene base and the protein sequence of the barnacle viscous protein molecules Mrcp-100k established after optimization are shown as SEQ ID No.1 and SEQ ID No.2, biologically synthesizing a target gene sequence by a genetic engineering method, adopting a silkworm fibroin light chain promoter as an expression frame promoter, further adopting a red fluorescent protein marker gene expression frame of an IE-1 promoter together, constructing a transgenic vector by adopting a molecular biology technology, introducing the transgenic vector into silkworm eggs through microinjection, and integrating the transgenic vector into silkworm genomes.
Screening G1 positive individuals through a fluorescence microscope, mating the positive individuals with wild type to obtain G2 generations, selfing and reserving seeds for the positive individuals after the G3 generations, and breeding into stable genetic varieties after the G8 generations. The G2 substitute material is adopted to verify that the foreign gene is successfully introduced into the silkworm genome through a PCR experiment. Fluorescence quantitative PCR detection shows that the expression of spider silk genes in silkworm varieties is obvious. A Western blot method is adopted to prove that the composite silkworms contain bands of barnacle adhesive protein molecules Mrcp-100k protein with expected sizes.
The mechanical properties of the composite silk from silkworm cocoons are tested, and the mechanical properties of the composite silk in stress and strain are obviously higher than those of the wild type silk.
Comparative analysis result of mechanical property of single-sequence composite filament of barnacle adhesive protein molecule Mrcp-100k
Figure BDA0003305347790000031
Example 2
Adopting a 32-fold repeated tandem sequence (the gene base and the protein sequence of a single-fold sequence are shown as SEQ ID NO.1 and SEQ ID NO.2) of a barnacle adhesive protein molecule Mrcp-100k sequence, optimizing according to the preference of silkworm codons, biologically synthesizing a target sequence by a genetic engineering method, adopting a silkworm fibroin heavy chain promoter as an expression frame promoter, further adopting a green fluorescent protein marker gene expression frame of an A3 promoter together, adopting a molecular biology technology to construct a homologous recombination vector, introducing the homologous recombination vector into silkworm eggs through microinjection, and integrating the homologous recombination vector into a silkworm genome.
Screening positive individuals of G1 generations by a fluorescence microscope, self-mating the positive individuals to obtain G2 generations, and after G3 generations, adopting the positive individuals to self-reserve seeds until G8 generations to breed stable genetic varieties. The G2 substitute material is adopted to verify that the foreign gene is successfully introduced into the silkworm genome through a PCR experiment. Fluorescent quantitative PCR detection shows that the expression of barnacle adhesive protein molecular genes in silkworm varieties is obvious. A Western blot method is adopted to prove that the silkworm composite silk contains bands of repeated tandem proteins with expected sizes.
The mechanical properties of the composite silk from the silkworm cocoons are tested, and the mechanical properties of stress and strain are obviously higher than those of the wild type.
Comparative analysis result of mechanical property of 32-fold poly-tandem repeat sequence composite filament of barnacle adhesive protein molecule Mrcp-100k
Figure BDA0003305347790000041
Example 3
The method comprises the steps of adopting a 4-fold repeated series sequence of a barnacle adhesive protein molecule Mrcp-100k sequence, optimizing according to silkworm codon preference, biosynthesizing a target sequence by a genetic engineering method, adopting a silkworm fibroin P25 protein promoter as an expression frame promoter, further adopting a green fluorescent protein marker gene expression frame of an IE-1 promoter together, constructing a transgenic vector by adopting a molecular biology technology, introducing the transgenic vector into silkworm eggs through microinjection, and integrating the transgenic vector into a silkworm genome.
Screening G1 positive individuals by a fluorescence microscope, self-mating the positive individuals to obtain G2 generations, and after G3 generations, adopting the positive individuals to self-breed seeds until G8 generations to breed stable genetic varieties. The G2 substitute material is adopted to verify that the foreign gene is successfully introduced into the silkworm genome through a PCR experiment. Fluorescent quantitative PCR detection shows that the barnacle adhesive protein gene expression in silkworm variety is obvious. A Western blot method is adopted to prove that the composite silkworms contain the bands of barnacle adhesive protein molecules repeated tandem proteins with expected sizes.
The mechanical properties of the composite silk from the silkworm cocoons are tested, and the mechanical properties of stress and strain are obviously higher than those of the wild type.
Comparative analysis result of mechanical property of 4-fold poly-tandem repeat sequence composite filament of barnacle adhesive protein molecule Mrcp-100k
Figure BDA0003305347790000051
It can be seen from the above embodiments that the method of the present invention can be used to artificially design functional genes with improved comprehensive mechanical properties of silk, and the method for improving the properties of silk has the advantages of strong stability, high efficiency and low cost, and can improve economic benefits.
The foregoing detailed description is intended to illustrate and not limit the invention, which is intended to be within the spirit and scope of the appended claims, and any changes and modifications that fall within the true spirit and scope of the invention are intended to be covered by the following claims.
The sequence related by the invention is as follows:
SEQ ID NO.1：
name: DNA base sequence of barnacle adhesive protein gene
The source is as follows: artificial Sequence (Artificial Sequence)
CATCGGCCGTCATTTGAGCGCCGATGCTGCGGTTGTCTTAGAAGTCCAGTAGCAGCAGACTTAGATGACGATGAAATCGGGATGCTACGAGAGTATGTAAAAAAGCAAGGTGTAATGCATTATGAGTCATTATCTGATATATCGTTAAAGGCTATCTTTCGAAACAAGTTATTAAATAATTTCCCAGAGGAGGTGCCAGCAACCCGAGATGGGGTACTACAAGTGATAACAGAGTCCTTGGGTTCATTAACGGACTCAGTAGTGCCAAGTGTGTCACAGTGTGGTCAGATCGCAGGGTACCTACAGAAGTCAGTGCCAGCTTTAGCACAAGGTGGTTTTAACGTAGACCTAAAGTCACTCGTGTCATCAGCATCAGTATTACTACATCAACGAGGGGTAACGGTGAATACTGACGAGCTAAATATATTTTTAAAATATGGTCTAATCAATTACTTAAAGTCAACTGTATATCAATCATCATACAGCATGCTTAGGCAACTAATTGTAACTCTAGACTATCTTGACCATGAGCTACCAGTAATTCTAGACTACGAGGAGCTTATAGCAGTACGACTTGCACTAAAGAAGAAGTTTGATACTTCAGTAGACATCTTTAAAAATAGATACCAATTAGCTATACAATCTTACAAGGCAAATAGAAATCTATTACTAGATAGTTTTCGAACGATGGCATACCGAGGGCCAAAGTACGAGATGTATCTACAAGAGGCAATCAGGGAGACAATAAATATCTTTCCAAGTATATCACCATCAACTGTGCGCATAGTATTTAACAATCTACAATTATCAAATACTGGTAGTGGGATGGTATCACCATTAGATCTTCTAGCAATGGTAACTACTCCGGTACTAGACGATGATTTGAAGTCAATCACTAAGGTATACGCAGAGAGACTTTACAATAAAATGCCAGGTTGCTACATGGGTCAAGAGGTAGAGATTCAGGAAATGTATTTTTTATTCTTAGTAGGTATTTTGTCACAGGGTATTCAGCCACTTAATCAAATGGCTATTTACGAGTTGTTTATATATCATTCAACTATCTACTTTAGGTCTTCATGTGCATACACGGTAGACGATTACTTTTTGTTTATTAGTCGAGTAGTAAGACCAAACATTCCATTAGGTTCAAAACATTTTAAGATAATTTCATTTGACCATTCAGTAGTAATCGAGAATATATTGGTGCCAGAGCCGTGGAGATCAAATTACGAGAAGAGTCGAGATACTATAATAAAGCGACTCGTAGGTCTACAAGGGTCATCAGACCAAATTACCAAGCGATTAATCGAGGGTGGTGGTGAGAAGGGTTACATAAAGAACATTGTAAATTTGAAGCCAGCAATAACTCCACGACCACAACCAACGTATGACGCATTTGAGTACCAAAATGTACTATTGTCATCACAACATATGCAAAGGGTAGCATTTGAATTGGCAAAGAGATTTGAGGGGCTAAAGGAGCCATCTTTTCGGCTACCACTACTTAAAATTCTTGTACGCGCAAACTTAGTAACTGACACTGGGGACAAGGCTGCAGCAGCATTTCTTCGACTTTTTCAAGGTCTTCCAGTGTTTTCACGACCATCAAGTCTATCATTTATAGTGGAGCAACTAAGGGAGTATCGACTGCAAACGACAAAGGCTCAGATTAAGGCAGCTTTAGATCAATTTTTCGTAGCAACGAAGTGCCTAGGGTATGTAATCCCGCAGCAGAAGATACCATCAATCTTTTTGGCAACGGTCGGTCGATATTTGTCAACGCTGCCAACTATTCCAAAGCAACCATTTGACTATAACTTTCTGGAGTATCTGCGATATTCACTAGCAAGTATAATCGAGCATCTGCCAGCTGTAGGGTCACAATCAGTGATAGACGATTATGCAATGTATAAAATCTTTTCAATTTTTGGGCATACTAAGTTAAGTATATATGCAAAGCGAATCATAATCAAGTATATTAATGAGTATAAGTTACTACCAAAGGCAGCGCAAAACGTCCCACTGTTAGTGCAATATCAACAACTGATGGAGTCAATGGTGTCTAAGTGTTCAGTATCTTCATTTATACTATCAAAGAAACAACTTACTACGATTCAATCTGATCTGTATAAGTCAAGACGCATCAGGATTGAGTTGTCATTGTTGGTCGATATAAATTATATGGCTTATTTTGCGGTATGCCAATCTGGTGCATATACAGCGGTGATGAACAGATATGTGTATCAATCAATTATTTCATATACGCAAACGGTCCGAAAACCAAATTATTATTCTGCAGAGTTTTTCCGAATCTTGGTAGAATCATCAAAGGGGTCAAAGCTGCCAGTGTCACGACCGCCATTAGTCCAATATCGGCGAACGCCAAAGCGATTGTGCTATATAGTTCCAGGTATCGTATTATATAGGGAGCAATTAAGACAACTGGTAACTTTAATAAGGCCACGATTTACGTTTGTGTCTATGCGCAACATCCGATCAATTGTAGCGCATACTATTCTTATACTGCGAGCAAGATATTCAATTACTCAGAATAATTGCTATGGGCATCTGACTAAGTATTATAATGGGATACCAATAAATGCATTAGGAGCTTTTGACGCATATAATCTTCTGCAAACGTTAAGAGTGCAACCAAAGCGGGCAGCAATCTCAAGTGTTGGGATACAATCAGCGATGGCAGAGTTATATATGCATATGCGACATCTACAAATGCCATTTCCAAGCGATAACGACGTACGAATAACCGTATTAAGAAATTGCTTATCTGCGTATTCATCTAAGGGAATGTATCGAAATGTTCCATTTGGTCGACGATTTTTCGCATTTCTGAATACGTATCTGCCGATGAGGCGAACTGCACCAAACAAGCGATGCATGCGATATAAGAAGTCATTTAGATGC
SEQ ID NO.2：
Name: protein sequence of barnacle adhesive protein gene
The source is as follows: artificial Sequence (Artificial Sequence)
HRPSFERRCCGCLRSPVAADLDDDEIGMLREYVKKQGVMHYESLSDISLKAIFRNKLLNNFPEEVPATRDGVLQVITESLGSLTDSVVPSVSQCGQIAGYLQKSVPALAQGGFNVDLKSLVSSASVLLHQRGVTVNTDELNIFLKYGLINYLKSTVYQSSYSMLRQLIVTLDYLDHELPVILDYEELIAVRLALKKKFDTSVDIFKNRYQLAIQSYKANRNLLLDSFRTMAYRGPKYEMYLQEAIRETINIFPSISPSTVRIVFNNLQLSNTGSGMVSPLDLLAMVTTPVLDDDLKSITKVYAERLYNKMPGCYMGQEVEIQEMYFLFLVGILSQGIQPLNQMAIYELFIYHSTIYFRSSCAYTVDDYFLFISRVVRPNIPLGSKHFKIISFDHSVVIENILVPEPWRSNYEKSRDTIIKRLVGLQGSSDQITKRLIEGGGEKGYIKNIVNLKPAITPRPQPTYDAFEYQNVLLSSQHMQRVAFELAKRFEGLKEPSFRLPLLKILVRANLVTDTGDKAAAAFLRLFQGLPVFSRPSSLSFIVEQLREYRLQTTKAQIKAALDQFFVATKCLGYVIPQQKIPSIFLATVGRYLSTLPTIPKQPFDYNFLEYLRYSLASIIEHLPAVGSQSVIDDYAMYKIFSIFGHTKLSIYAKRIIIKYINEYKLLPKAAQNVPLLVQYQQLMESMVSKCSVSSFILSKKQLTTIQSDLYKSRRIRIELSLLVDINYMAYFAVCQSGAYTAVMNRYVYQSIISYTQTVRKPNYYSAEFFRILVESSKGSKLPVSRPPLVQYRRTPKRLCYIVPGIVLYREQLRQLVTLIRPRFTFVSMRNIRSIVAHTILILRARYSITQNNCYGHLTKYYNGIPINALGAFDAYNLLQTLRVQPKRAAISSVGIQSAMAELYMHMRHLQMPFPSDNDVRITVLRNCLSAYSSKGMYRNVPFGRRFFAFLNTYLPMRRTAPNKRCMRYKKSFRC。
Sequence listing
<110> Zhejiang university
<120> aquatic bioprotein molecule for improving mechanical property of silk and application method thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 2925
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
catcggccgt catttgagcg ccgatgctgc ggttgtctta gaagtccagt agcagcagac 60
ttagatgacg atgaaatcgg gatgctacga gagtatgtaa aaaagcaagg tgtaatgcat 120
tatgagtcat tatctgatat atcgttaaag gctatctttc gaaacaagtt attaaataat 180
ttcccagagg aggtgccagc aacccgagat ggggtactac aagtgataac agagtccttg 240
ggttcattaa cggactcagt agtgccaagt gtgtcacagt gtggtcagat cgcagggtac 300
ctacagaagt cagtgccagc tttagcacaa ggtggtttta acgtagacct aaagtcactc 360
gtgtcatcag catcagtatt actacatcaa cgaggggtaa cggtgaatac tgacgagcta 420
aatatatttt taaaatatgg tctaatcaat tacttaaagt caactgtata tcaatcatca 480
tacagcatgc ttaggcaact aattgtaact ctagactatc ttgaccatga gctaccagta 540
attctagact acgaggagct tatagcagta cgacttgcac taaagaagaa gtttgatact 600
tcagtagaca tctttaaaaa tagataccaa ttagctatac aatcttacaa ggcaaataga 660
aatctattac tagatagttt tcgaacgatg gcataccgag ggccaaagta cgagatgtat 720
ctacaagagg caatcaggga gacaataaat atctttccaa gtatatcacc atcaactgtg 780
cgcatagtat ttaacaatct acaattatca aatactggta gtgggatggt atcaccatta 840
gatcttctag caatggtaac tactccggta ctagacgatg atttgaagtc aatcactaag 900
gtatacgcag agagacttta caataaaatg ccaggttgct acatgggtca agaggtagag 960
attcaggaaa tgtatttttt attcttagta ggtattttgt cacagggtat tcagccactt 1020
aatcaaatgg ctatttacga gttgtttata tatcattcaa ctatctactt taggtcttca 1080
tgtgcataca cggtagacga ttactttttg tttattagtc gagtagtaag accaaacatt 1140
ccattaggtt caaaacattt taagataatt tcatttgacc attcagtagt aatcgagaat 1200
atattggtgc cagagccgtg gagatcaaat tacgagaaga gtcgagatac tataataaag 1260
cgactcgtag gtctacaagg gtcatcagac caaattacca agcgattaat cgagggtggt 1320
ggtgagaagg gttacataaa gaacattgta aatttgaagc cagcaataac tccacgacca 1380
caaccaacgt atgacgcatt tgagtaccaa aatgtactat tgtcatcaca acatatgcaa 1440
agggtagcat ttgaattggc aaagagattt gaggggctaa aggagccatc ttttcggcta 1500
ccactactta aaattcttgt acgcgcaaac ttagtaactg acactgggga caaggctgca 1560
gcagcatttc ttcgactttt tcaaggtctt ccagtgtttt cacgaccatc aagtctatca 1620
tttatagtgg agcaactaag ggagtatcga ctgcaaacga caaaggctca gattaaggca 1680
gctttagatc aatttttcgt agcaacgaag tgcctagggt atgtaatccc gcagcagaag 1740
ataccatcaa tctttttggc aacggtcggt cgatatttgt caacgctgcc aactattcca 1800
aagcaaccat ttgactataa ctttctggag tatctgcgat attcactagc aagtataatc 1860
gagcatctgc cagctgtagg gtcacaatca gtgatagacg attatgcaat gtataaaatc 1920
ttttcaattt ttgggcatac taagttaagt atatatgcaa agcgaatcat aatcaagtat 1980
attaatgagt ataagttact accaaaggca gcgcaaaacg tcccactgtt agtgcaatat 2040
caacaactga tggagtcaat ggtgtctaag tgttcagtat cttcatttat actatcaaag 2100
aaacaactta ctacgattca atctgatctg tataagtcaa gacgcatcag gattgagttg 2160
tcattgttgg tcgatataaa ttatatggct tattttgcgg tatgccaatc tggtgcatat 2220
acagcggtga tgaacagata tgtgtatcaa tcaattattt catatacgca aacggtccga 2280
aaaccaaatt attattctgc agagtttttc cgaatcttgg tagaatcatc aaaggggtca 2340
aagctgccag tgtcacgacc gccattagtc caatatcggc gaacgccaaa gcgattgtgc 2400
tatatagttc caggtatcgt attatatagg gagcaattaa gacaactggt aactttaata 2460
aggccacgat ttacgtttgt gtctatgcgc aacatccgat caattgtagc gcatactatt 2520
cttatactgc gagcaagata ttcaattact cagaataatt gctatgggca tctgactaag 2580
tattataatg ggataccaat aaatgcatta ggagcttttg acgcatataa tcttctgcaa 2640
acgttaagag tgcaaccaaa gcgggcagca atctcaagtg ttgggataca atcagcgatg 2700
gcagagttat atatgcatat gcgacatcta caaatgccat ttccaagcga taacgacgta 2760
cgaataaccg tattaagaaa ttgcttatct gcgtattcat ctaagggaat gtatcgaaat 2820
gttccatttg gtcgacgatt tttcgcattt ctgaatacgt atctgccgat gaggcgaact 2880
gcaccaaaca agcgatgcat gcgatataag aagtcattta gatgc 2925
<210> 1
<211> 975
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
His Arg Pro Ser Phe Glu Arg Arg Cys Cys Gly Cys Leu Arg Ser Pro
1 5 10 15
Val Ala Ala Asp Leu Asp Asp Asp Glu Ile Gly Met Leu Arg Glu Tyr
20 25 30
Val Lys Lys Gln Gly Val Met His Tyr Glu Ser Leu Ser Asp Ile Ser
35 40 45
Leu Lys Ala Ile Phe Arg Asn Lys Leu Leu Asn Asn Phe Pro Glu Glu
50 55 60
Val Pro Ala Thr Arg Asp Gly Val Leu Gln Val Ile Thr Glu Ser Leu
65 70 75 80
Gly Ser Leu Thr Asp Ser Val Val Pro Ser Val Ser Gln Cys Gly Gln
85 90 95
Ile Ala Gly Tyr Leu Gln Lys Ser Val Pro Ala Leu Ala Gln Gly Gly
100 105 110
Phe Asn Val Asp Leu Lys Ser Leu Val Ser Ser Ala Ser Val Leu Leu
115 120 125
His Gln Arg Gly Val Thr Val Asn Thr Asp Glu Leu Asn Ile Phe Leu
130 135 140
Lys Tyr Gly Leu Ile Asn Tyr Leu Lys Ser Thr Val Tyr Gln Ser Ser
145 150 155 160
Tyr Ser Met Leu Arg Gln Leu Ile Val Thr Leu Asp Tyr Leu Asp His
165 170 175
Glu Leu Pro Val Ile Leu Asp Tyr Glu Glu Leu Ile Ala Val Arg Leu
180 185 190
Ala Leu Lys Lys Lys Phe Asp Thr Ser Val Asp Ile Phe Lys Asn Arg
195 200 205
Tyr Gln Leu Ala Ile Gln Ser Tyr Lys Ala Asn Arg Asn Leu Leu Leu
210 215 220
Asp Ser Phe Arg Thr Met Ala Tyr Arg Gly Pro Lys Tyr Glu Met Tyr
225 230 235 240
Leu Gln Glu Ala Ile Arg Glu Thr Ile Asn Ile Phe Pro Ser Ile Ser
245 250 255
Pro Ser Thr Val Arg Ile Val Phe Asn Asn Leu Gln Leu Ser Asn Thr
260 265 270
Gly Ser Gly Met Val Ser Pro Leu Asp Leu Leu Ala Met Val Thr Thr
275 280 285
Pro Val Leu Asp Asp Asp Leu Lys Ser Ile Thr Lys Val Tyr Ala Glu
290 295 300
Arg Leu Tyr Asn Lys Met Pro Gly Cys Tyr Met Gly Gln Glu Val Glu
305 310 315 320
Ile Gln Glu Met Tyr Phe Leu Phe Leu Val Gly Ile Leu Ser Gln Gly
325 330 335
Ile Gln Pro Leu Asn Gln Met Ala Ile Tyr Glu Leu Phe Ile Tyr His
340 345 350
Ser Thr Ile Tyr Phe Arg Ser Ser Cys Ala Tyr Thr Val Asp Asp Tyr
355 360 365
Phe Leu Phe Ile Ser Arg Val Val Arg Pro Asn Ile Pro Leu Gly Ser
370 375 380
Lys His Phe Lys Ile Ile Ser Phe Asp His Ser Val Val Ile Glu Asn
385 390 395 400
Ile Leu Val Pro Glu Pro Trp Arg Ser Asn Tyr Glu Lys Ser Arg Asp
405 410 415
Thr Ile Ile Lys Arg Leu Val Gly Leu Gln Gly Ser Ser Asp Gln Ile
420 425 430
Thr Lys Arg Leu Ile Glu Gly Gly Gly Glu Lys Gly Tyr Ile Lys Asn
435 440 445
Ile Val Asn Leu Lys Pro Ala Ile Thr Pro Arg Pro Gln Pro Thr Tyr
450 455 460
Asp Ala Phe Glu Tyr Gln Asn Val Leu Leu Ser Ser Gln His Met Gln
465 470 475 480
Arg Val Ala Phe Glu Leu Ala Lys Arg Phe Glu Gly Leu Lys Glu Pro
485 490 495
Ser Phe Arg Leu Pro Leu Leu Lys Ile Leu Val Arg Ala Asn Leu Val
500 505 510
Thr Asp Thr Gly Asp Lys Ala Ala Ala Ala Phe Leu Arg Leu Phe Gln
515 520 525
Gly Leu Pro Val Phe Ser Arg Pro Ser Ser Leu Ser Phe Ile Val Glu
530 535 540
Gln Leu Arg Glu Tyr Arg Leu Gln Thr Thr Lys Ala Gln Ile Lys Ala
545 550 555 560
Ala Leu Asp Gln Phe Phe Val Ala Thr Lys Cys Leu Gly Tyr Val Ile
565 570 575
Pro Gln Gln Lys Ile Pro Ser Ile Phe Leu Ala Thr Val Gly Arg Tyr
580 585 590
Leu Ser Thr Leu Pro Thr Ile Pro Lys Gln Pro Phe Asp Tyr Asn Phe
595 600 605
Leu Glu Tyr Leu Arg Tyr Ser Leu Ala Ser Ile Ile Glu His Leu Pro
610 615 620
Ala Val Gly Ser Gln Ser Val Ile Asp Asp Tyr Ala Met Tyr Lys Ile
625 630 635 640
Phe Ser Ile Phe Gly His Thr Lys Leu Ser Ile Tyr Ala Lys Arg Ile
645 650 655
Ile Ile Lys Tyr Ile Asn Glu Tyr Lys Leu Leu Pro Lys Ala Ala Gln
660 665 670
Asn Val Pro Leu Leu Val Gln Tyr Gln Gln Leu Met Glu Ser Met Val
675 680 685
Ser Lys Cys Ser Val Ser Ser Phe Ile Leu Ser Lys Lys Gln Leu Thr
690 695 700
Thr Ile Gln Ser Asp Leu Tyr Lys Ser Arg Arg Ile Arg Ile Glu Leu
705 710 715 720
Ser Leu Leu Val Asp Ile Asn Tyr Met Ala Tyr Phe Ala Val Cys Gln
725 730 735
Ser Gly Ala Tyr Thr Ala Val Met Asn Arg Tyr Val Tyr Gln Ser Ile
740 745 750
Ile Ser Tyr Thr Gln Thr Val Arg Lys Pro Asn Tyr Tyr Ser Ala Glu
755 760 765
Phe Phe Arg Ile Leu Val Glu Ser Ser Lys Gly Ser Lys Leu Pro Val
770 775 780
Ser Arg Pro Pro Leu Val Gln Tyr Arg Arg Thr Pro Lys Arg Leu Cys
785 790 795 800
Tyr Ile Val Pro Gly Ile Val Leu Tyr Arg Glu Gln Leu Arg Gln Leu
805 810 815
Val Thr Leu Ile Arg Pro Arg Phe Thr Phe Val Ser Met Arg Asn Ile
820 825 830
Arg Ser Ile Val Ala His Thr Ile Leu Ile Leu Arg Ala Arg Tyr Ser
835 840 845
Ile Thr Gln Asn Asn Cys Tyr Gly His Leu Thr Lys Tyr Tyr Asn Gly
850 855 860
Ile Pro Ile Asn Ala Leu Gly Ala Phe Asp Ala Tyr Asn Leu Leu Gln
865 870 875 880
Thr Leu Arg Val Gln Pro Lys Arg Ala Ala Ile Ser Ser Val Gly Ile
885 890 895
Gln Ser Ala Met Ala Glu Leu Tyr Met His Met Arg His Leu Gln Met
900 905 910
Pro Phe Pro Ser Asp Asn Asp Val Arg Ile Thr Val Leu Arg Asn Cys
915 920 925
Leu Ser Ala Tyr Ser Ser Lys Gly Met Tyr Arg Asn Val Pro Phe Gly
930 935 940
Arg Arg Phe Phe Ala Phe Leu Asn Thr Tyr Leu Pro Met Arg Arg Thr
945 950 955 960
Ala Pro Asn Lys Arg Cys Met Arg Tyr Lys Lys Ser Phe Arg Cys
965 970 975

Claims (10)

1. An aquatic bioprotein molecule for improving mechanical properties of silk is characterized in that:
the aquatic organism protein molecule is a protein sequence formed by connecting a single barnacle adhesive protein gene or a plurality of barnacle adhesive protein genes in series.
2. The aquatic bioprotein molecule capable of improving mechanical properties of silk according to claim 1, wherein:
the protein sequence formed by connecting a plurality of barnacle adhesive protein genes in series is formed by repeatedly connecting the protein sequences of the barnacle adhesive protein genes for a plurality of times.
3. The aquatic bioprotein molecule capable of improving mechanical properties of silk according to claim 1 or 2, wherein:
the protein sequence formed by connecting a plurality of barnacle adhesive protein genes in series is formed by repeating and connecting 2-32 times.
4. The aquatic bioprotein molecule capable of improving mechanical properties of silk according to claim 1, wherein:
the protein sequence of the single barnacle adhesive protein gene is SEQ ID NO. 2.
5. The application of the aquatic bioprotein molecule for improving mechanical properties of silk as claimed in claim 1, wherein the aquatic bioprotein molecule comprises: application in gene editing and transgenic silkworms.
6. The use of the aquatic bioprotein molecule with enhanced mechanical properties of claim 5, wherein: the application of the compound silk in gene editing and transgenic silkworm production for improving mechanical and mechanical properties.
7. A method for producing compound silk to improve mechanical property includes such steps as biosynthesizing target gene sequence by gene engineering, constructing exogenous gene carrier by molecular biology technique, introducing exogenous gene carrier into silkworm egg by microinjection, culturing silkworm for multiple generations, screening G1 generation positive individuals by fluorescence microscope, mating G1 generation positive individuals with wild type or positive individuals to obtain G2 generation, screening positive individuals after G3 generation, selfing, breeding until G8 generation to obtain stable genetic variety, and using G8 generation silkworm to produce secreted silk: the target gene sequence is the gene base sequence corresponding to the protein sequence formed by connecting a single barnacle adhesive protein gene or a plurality of barnacle adhesive protein genes in series according to claim 1, finally, a novel silkworm variety with excellent performance of the composite silk capable of being stably inherited is obtained, and the novel silkworm variety of the G8 generation is utilized to produce and secrete silk to be used as the composite silk, so that the composite silk with improved mechanical properties is obtained.
8. The method for producing the composite yarn with improved mechanical properties as claimed in claim 7, wherein: the exogenous gene vector is a transgenic vector with piggyBac as a framework or a homologous recombination vector applied to gene editing.
9. A method for producing a composite yarn with improved mechanical properties according to claim 7 or 8, wherein: the exogenous gene vector comprises an exogenous gene expression frame and a fluorescence screening marker gene expression frame; the exogenous gene expression frame comprises exogenous genes and promoters of the expression frame, the exogenous genes are gene base sequences corresponding to a single barnacle viscous protein gene or a protein sequence formed by connecting a plurality of genes in series as claimed in claim 1, and the promoters of the exogenous gene expression frame adopt the promoters of genes such as silk fibroin heavy chain, silk fibroin light chain, silk fibroin P25 or sericin;
the fluorescent screening marker gene expression frame comprises a fluorescent screening marker gene and a promoter of the expression frame, the fluorescent screening marker gene adopts a green fluorescent protein Gene (GFP) or a red fluorescent protein gene (DsRed), and the promoter of the fluorescent screening marker gene expression frame adopts an IE-1, A3 or 3xP3 promoter.
10. The method for producing the composite yarn with improved mechanical properties as claimed in claim 7, wherein: the composition of the composite silk comprises a single barnacle adhesive protein or a protein molecule component and a fibroin component which are formed by connecting a plurality of genes in series.
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