CN106011172A - Preparation method of bombyx mori capable of synthesizing and secreting hydrophilic sericin on posterior division of silkgland - Google Patents
Preparation method of bombyx mori capable of synthesizing and secreting hydrophilic sericin on posterior division of silkgland Download PDFInfo
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- CN106011172A CN106011172A CN201610348866.0A CN201610348866A CN106011172A CN 106011172 A CN106011172 A CN 106011172A CN 201610348866 A CN201610348866 A CN 201610348866A CN 106011172 A CN106011172 A CN 106011172A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 108010013296 Sericins Proteins 0.000 title abstract description 28
- 230000002194 synthesizing effect Effects 0.000 title abstract description 5
- 230000003248 secreting effect Effects 0.000 title abstract description 4
- 108010022355 Fibroins Proteins 0.000 claims abstract description 39
- 101100365490 Drosophila melanogaster Jon99Ci gene Proteins 0.000 claims abstract description 26
- 101150008449 ser3 gene Proteins 0.000 claims abstract description 26
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- 235000018102 proteins Nutrition 0.000 claims description 34
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0335—Genetically modified worms
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- C12N2800/00—Nucleic acids vectors
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- C12N2800/103—Plasmid DNA for invertebrates
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Abstract
The invention provides a preparation method of bombyx mori capable of synthesizing and secreting hydrophilic sericin on posterior division of silkgland. The method comprises the following steps: recombining the DNA molecule of a coding amino acid sequence SER to the bombyx mori chromosome; and screening with a fluorescent protein marker to obtain the bombyx mori capable of synthesizing and secreting hydrophilic sericin on the posterior division of silkgland, wherein a bombyx mori fibroin heavy-chain protein Fib-H gene promoter, the 5'-end signal peptide coding sequence of bombyx mori Fib-H gene, the coding sequence Ser3 of bombyx mori sericin protein SER3 and the 3'-end sequence of bombyx mori Fib-H gene are sequentially positioned from the 5' end to the 3' end of the nucleotide sequence of the DNA molecule of the coding amino acid sequence SER. In the method provided by the invention, the hydrophilic sericin protein gene specifically expressed by the middle division of silkgland of bombyx mori can be efficiently expressed and secreted on the posterior division of silkgland at the same time; and after the spinning and cocooning of the mature larvae of bombyx mori, cocoons containing hydrophilic sericin component in fibroin fiber are obtained, and the bombyx mori silk with improved water absorption ability of fiber is obtained.
Description
Technical field
The present invention relates to bioengineering field, be specifically related to a kind of posterior division of silkgland and can synthesize and secrete hydrophilic
The preparation method of the silkworm of sericin.
Background technology
Silkworms spin silk cocoons in family, and silk then can produce silk protein fiber.Sericterium is silkworm eggcase
Synthesis and the organ of secretion fibroin, a silkworm larva has 2 closely similar sericteriums, every sericterium
Divide anterior division of silkgland, middle division of silkgland and three functional areas of posterior division of silkgland.Middle division of silkgland and posterior division of silkgland are silkworms
The synthesis of silk protein and Secreting section, and anterior division of silkgland is the pipeline that fibroin output is external.
Bombyx mori posterior silkgland mainly synthesizes and secretes fibroin albumen, and this is that one has β-pleated sheet crystalline texture,
Tough and tensile and resilient protein, accounts for the 70-80% of silk total amount, is the basic of the textiles such as making silk
Raw material.Fibroin protein include fibroin light chain (light chain, Fib-L) and heavy chain (heavy chain,
Fib-H), also P25 albumen (also known as the poly-element of fiber six, fibrohexamerin, Fhx/P25) etc.
(Yamaguchi et al., 1989；Sprague 1975；Couble et al., 1983).They respectively by
Fibroin albumen gene fib-H, fib-L and p253 encode, when their expression has strict tissue and grows
Phase specificity (Couble et al., 1983；Maekawa&Suzuki 1980；Tsujimoto et al.,
1981；Tsuda et al., 1981).In Bombyx mori posterior silkgland, Fib-H, Fib-L and P25 are with 6:6:
The mol ratio composition complex of 1, as the fundamental structural unit (Inoue 2000) of fibroin, wherein Fib-H
Being connected with each other with disulfide bond with Fib-L, P25 is then with non-covalent bond hydrophobic interaction and Fib-H/Fib-L
Complex combines.P25 has the function of molecular chaperones sample, in the folding of fibroin albumen complex and non-molten
The secretion of solution property long-chained protein has important function (Tanaka et al., 1999).
Containing having more than 7 kinds of main sericins in the middle division of silkgland cell of silkworm, they are mainly by 3 positions
In silkworm the 11st linkage group Ser1, Ser2 and Ser3 gene code (Okamoto et al., 1982；
Michaille et al., 1986；1990；Takasu et al., 2007), their expression is by residing group
Knit the control of position and stage of development.The Ser1 gene 150 Intracellular transcription 4 at middle division of silkgland rear portion
Planting the sericin mRNA of different length, wherein the mRNA of 1 2.8kb is in the whole 5 instar larvae phases, only close
42 cell inner expressions of posterior division of silkgland, and 10.5,9.0 and 4.0kb 3 kinds of mRNA are only at remaining 108
In cell, respectively the different stages of development by different montage approach produce (Couble et al.,
1983).The exon alternative splicing of coding Ser1mRNA, can produce multiple at larva different growing periods
Protein variant, causes same internal, after each transcriptional events, individual gene can produce structure and
The albumen that character is different, the different montages of the primary transcript of Ser1 are also regulated and controled (Couble et by the stage of development
Al., 1983；Garelet al., 1997).At 5 initial stages in age, Ser2 gene is at all of middle division of silkgland cell
Middle expression, it expressed the front portion (Takasu et al., 2010) being limited to middle division of silkgland later；Ser3
Only the middle front part at middle division of silkgland expresses SER3 albumen (Takasu et al., 2010).
The fibroin protein of Bombyx mori posterior silkgland cell synthesis is constantly secreted to lumen of gland, and gradually by rear portion silk
Gland lumen of gland moves to middle division of silkgland lumen of gland.Silkworm larva is weaved silk during cocooing after maturation, fibroin albumen
The fibril (also referred to as fibril) that matter molecule constantly aggregates into about diameter 0.1 μm, section is irregular cycle,
And the most closely boundling forms the fibroin " core " of silkworm silk.Fibril is by crystalline fibers and amorphous cellulose molecule
Polymer, relies closely between fibril along uranium to being alternately in series, and is firmly coupled by periphery molecular side chain,
Filling without interstitial, this is the special gloss of fibroin fiber and the molecular basis that can produce ultra-fine protein fibre.
Fibroin fibril in domestic natural silk gland is successively wrapped during forming fibroin fiber and anteriorly moving
Having wrapped up in 4 layers of sericin, wherein SER3 albumen is owing to synthesizing at middle division of silkgland middle front part, is therefore wrapped in
Outer layer, directly will not contact with fibroin protein fiber.SER3 albumen has relatively low degree of crystallinity and high stream
Dynamic property.Sericin accounts for the 20%-30% of silkworm silk amount, and molecular conformation is mainly random coil, ties containing a small amount of β
Structure exists, but without αhelix, spatial configuration of molecules is loose, unordered.
The sericine of domestic natural silk gland secretion has obviously ammonia with the aminoacid composition of fibroin protein
Base acid Preference difference.Fibroin albumen is mainly made up of glycine, alanine, serine and tyrosine.
Sericin is then mainly made up of serine, aspartic acid, threonine, glycine and glutamic acid etc..
Glycine, alanine and tyrosine content in sericin are substantially less than fibroin albumen, and free ammonia
Base acid content is significantly higher than fibroin.Many arginine, lysine, glutamic acid, first is had on sericin chain
The isometric side chain amino acid of methyllanthionine, tryptophan, tyrosine, polypeptide chain surface possibly together with-OH ,-COOH,
-NH2Isopolarity hydrophilic group.Therefore, sericin has moisture absorption, moisturizing and the peculiar smell suction etc. of excellence
Effect, wherein the water absorption of sericin SER3 and mobility are significantly higher than 4 kinds of Ser1 gene translation and are close to
The internal layer sericine of fibroin.
In the filature of domestic silkworm silk textile production and the operating process such as concise, the sericin on silkworm silk surface is several
By Ex-all, wherein as the SER3 of outer layer sericin main component be eliminated more thorough.Retain sericin
Domestic silkworm silk fabric, the problem not only existing gloss difference and uneven dyeing, there is also fabric softness poor,
The problem that wearing comfort is deteriorated, therefore degumming is to improve silkworm silk gloss, the technique ensureing dyeing uniformity
Requirement.Although the water absorption of the silk fiber molecule substantially constituted with fibroin after degumming is still above cotton fiber
Nearly 30%, but the impression of taking of silk does not has cotton to absorb water.This is owing to silkworm silk is expensive, and silk is almost
Being all top-weight fabric, fiber total suction is few.To this end, mix sericine in fibroin protein,
It is expected to change fibre structure and the physicochemical property of fibroin, it is achieved change the purpose of fiber function, particularly same
Time dual-purpose fibroin and the double grading of sericin, this is at fibroin fiber remained on surface sericin, or at silkworm silk
Fiber or fabric face carry out a kind of function utilization that sericin coating cannot realize.
Summary of the invention
For solving above-mentioned technical problem, the present invention provides a kind of posterior division of silkgland can synthesize and secrete hydrophilic-silk
The preparation method of the silkworm of glue protein, the silkworm that the method is obtained can be obtained after ripe larva weaves silk and cocoons
Obtain the Bombyx bombycis containing hydrophilic-silk glue protein composition in fibroin fiber.
For achieving the above object, present invention employs techniques below scheme:
On the one hand, the invention provides a kind of posterior division of silkgland and can synthesize and secrete hydrophilic-silk glue protein
The preparation method of silkworm, comprises the following steps:
The DNA molecular of encoding amino acid sequence SER is recombinated on chromosome of mulberry silkworm, expresses, translate also
Obtain described posterior division of silkgland through screening and can synthesize and secrete the silkworm of hydrophilic-silk glue protein；
Wherein, the nucleotide sequence 5' end of the DNA molecular of described encoding amino acid sequence SER depends on to 3' end
Secondary for bombyx mori silk fibroin heavy chain protein Fib-H gene promoter, silkworm Fib-H gene 5' end signal peptide code sequence
Row, silkworm sericine SER3 coded sequence Ser3 and silkworm Fib-H gene 3' terminal sequence.
In one embodiment, silkworm sericine SER3 has the ammonia shown in SEQ ID NO:1
Base acid sequence.
Preferably, the DNA molecular of coding aminoacid sequence shown in SEQ ID NO:1 has SEQ ID
Nucleotide sequence shown in NO:2.
In another specific embodiment, aminoacid sequence SER has the aminoacid shown in SEQ ID NO:3
Sequence.
Preferably, the DNA molecular of coding aminoacid sequence shown in SEQ ID NO:3 has SEQ ID
Nucleotide sequence shown in NO:4.
In one embodiment, silkworm of being recombinated to by the DNA molecular of encoding amino acid sequence SER is dyeed
Following steps are specifically included on body:
Build the transposon vector of the DNA molecular comprising encoding amino acid sequence SER；And described swivel base is carried
Body imports in Eggs of Silkworm.
Preferably, the method that transposon vector imports Eggs of Silkworm is microinjection.
Preferably, transposon vector is the recombination Ser3 transposon vector that piggyBac transposon builds
PB-ser。
Specifically wrap it is highly preferred that piggyBac transposon builds recombination Ser3 transposon vector PB-ser
Include following steps:
Upstream 5' end in SER3 protein-encoding nucleotide sequence connects silkworm Fib-H gene promoter and silkworm
Fib-H gene 5' end signal peptide-coding sequence, the downstream 3' end in SER3 protein-encoding nucleotide sequence connects
The 3' terminal sequence of silkworm Fib-H gene obtains recombination Ser；To start with eye and nerve-specific
(sequence is such as the PiggyBac-3xP3-DsRed plasmid of sub-SV40 and red fluorescent protein DsRed element
Shown in SEQ ID NO:6) carry out AscI and FseI double digestion respectively with the Ser sequence of synthetic；Will
Piggybac-3xP3-DsRed plasmid and Ser sequence after enzyme action connect, thus obtain transposon vector
PB-Ser。
Preferably, transposon vector PB-Ser has nucleotide sequence shown in SEQ ID NO:5.
On the other hand, the present invention provides a kind of and has aminoacid sequence shown in coding SEQ ID NO:3
The transposon vector of DNA molecular.
Preferably, this transposon vector is the recombination Ser3 transposon vector that piggyBac transposon builds
PB-ser。
Preferably, this transposon vector has nucleotide sequence shown in SEQ ID NO:5.
By technique scheme, compared with prior art, the invention have the advantages that the present invention carries
Supply a kind of posterior division of silkgland can synthesize and secrete the preparation method of silkworm of hydrophilic-silk glue protein, the method
The hydrophilic-silk glue protein SER3 of silkworm middle division of silkgland specifically expressing can be enable simultaneously high at posterior division of silkgland
Efficient expression and secretion, and after silkworm maturation larva weaves silk and cocoons, it is thus achieved that containing hydrophilic in fibroin fiber
The Bombyx bombycis of sericin composition, and then obtain domestic silkworm silk and the silk fabrics that fibrous absorbent performance is improved.
Accompanying drawing explanation
Fig. 1 illustrates the recombination Ser sequence of coding SER aminoacid sequence；
Fig. 2 illustrates the transposon vector PB-Ser of recombination Ser；
Fig. 3 illustrates recombination Ser mutant silkworm and the difference of wild type silkworm.
Detailed description of the invention
Below in conjunction with the accompanying drawings the detailed description of the invention of the present invention is described in further detail.It should be understood that
Following example are merely to illustrate the present invention, and are not construed as limiting scope.
The invention provides the preparation that a kind of posterior division of silkgland can synthesize and secrete the silkworm of hydrophilic-silk glue protein
Method, recombinates on chromosome of mulberry silkworm by the DNA molecular of encoding amino acid sequence SER, then through express,
Translate and get final product.Wherein, the nucleotide sequence 5' end of the DNA molecular of encoding amino acid sequence SER is to 3'
End is followed successively by bombyx mori silk fibroin heavy chain protein Fib-H gene promoter, silkworm Fib-H gene 5' end signal peptide is compiled
Code sequence, silkworm sericine SER3 coded sequence Ser3 and silkworm Fib-H gene 3' terminal sequence.
In the preparation process in accordance with the present invention, encoding amino acid sequence SER is that one can be at silkworm rear portion silk
Gland height efficient expression, in the recombinant expressed sequence of sericine sequence SER3 of silkworm middle division of silkgland specifically expressing
Row.The DNA molecular of encoding amino acid sequence SER is recombinated on chromosome of mulberry silkworm, coded amino acid sequence
After row SER expresses in silkworm body, silkworm larva posterior silkgland cells is synthesizing the same of fibroin protein
Time can synthesize sericine SER3.Further, the sericine of posterior silkgland cells synthesis
SER3 can secrete to lumen of gland, co-exists in the fibroin protein in posterior division of silkgland lumen of gland intermolecular.Further
, the most gradually move to middle division of silkgland lumen of gland with the fibroin albumen in posterior division of silkgland lumen of gland.Further
, weave silk during cocooing after silkworm larva maturation, formed with the fibril of fibroin protein molecule and fill
Fill out between fibril, make originally without sericin charges occur between the fibroin fibril of interstitial filling.Also there is part
SER3 is in silkworm larva silking process, with the constraint of anterior division of silkgland pipeline and the extruding of orifice of spinneret, from
Overflow between fibroin fibril, be wrapped between internal layer sericin and fibroin.
In one embodiment, silkworm sericine SER3 has shown in SEQ ID NO:1
Aminoacid sequence.Shown in coding SEQ ID NO:1, the DNA molecular of aminoacid sequence has SEQ ID
Nucleotide sequence shown in NO:2.
In one embodiment, aminoacid sequence SER has the aminoacid shown in SEQ ID NO:3
Sequence.
Due to the degeneracy of codon, can exist a variety of can the aminoacid sequence of code book invention
The DNA molecular of SER.
In one embodiment, the DNA molecular of the aminoacid sequence shown in SEQ ID NO:3 is encoded
There is the nucleotide sequence shown in SEQ ID NO:4.
Those skilled in the art can obtain step according to general silkworm transgeneic procedure and transgenic silkworm,
The DNA molecular of encoding amino acid sequence SER is recombinated on chromosome of mulberry silkworm, prepares stable heredity
Posterior division of silkgland efficiently synthesize and secrete the silkworm model of hydrophilic-silk glue protein.
In one embodiment, silkworm of being recombinated to by the DNA molecular of encoding amino acid sequence SER contaminates
Comprise the following steps on colour solid: build the transposon vector of the DNA molecular comprising encoding amino acid sequence SER；
And transposon vector is imported in Eggs of Silkworm.
In another specific embodiments, take the steps by the DNA of encoding amino acid sequence SER
Molecule is incorporated on chromosome of mulberry silkworm:
1, transposon vector is built
Upstream 5' end in SER3 protein-encoding nucleotide sequence connects silkworm Fib-H gene promoter and silkworm
Fib-H gene 5' end signal peptide-coding sequence, the downstream 3' end in SER3 protein-encoding nucleotide sequence connects
The 3' terminal sequence of silkworm Fib-H gene obtains recombination Ser.To start with eye and nerve-specific
(sequence is such as the piggyBac-3xP3-DsRed plasmid of sub-SV40 and red fluorescent protein DsRed element
Shown in SEQ ID NO:6) carry out AscI and FseI double digestion respectively with the Ser sequence of synthetic.Will
Piggybac-3xP3-DsRed plasmid and Ser sequence after enzyme action connect, and form transposon vector PB-Ser.
Recombination Ser and transposon vector PB-Ser structural representation are shown in Fig. 1 and Fig. 2.
2, the silkworm of transgenic is obtained
(1), in the environment that aseptic and free nucleic acid pollutes, micro-injection method is used to inject to every silkworm seed
The transposon vector plasmid of 4000ng-5000ng；
(2) the non-toxic material envelope of the silkworm seed after injection is coated with injection orifice, then in gnotobasis, uses
26 DEG C-27 DEG C, 75%-80% relative humidity, natural lighting protection silkworm seed to hatching, after hatching larva continue
Raising with Folium Mori or man-made feeds in mutually synthermal, humidity and photoenvironment, after cocooing, larva continues to use
Identical epidemic disaster and photoenvironment are protected to adult eclosion；
(3) Bombycis mori selfing or after backcrossing, after institute's spawning and hatching, larva Folium Mori or man-made feeds are raised after sprouting wings
Supporting, investigate under larva 3-5 age, pupa time or adult stage fluorescence microscope, eye presents the individual of red fluorescence
Body, individual as transgenic G1 generation；
(4) transgenic G1 is selected to present red fluorescence for the eye of individual embryos, larva, pupa or adult
Individual subculture, efficiently synthesize to G6 for the rear posterior division of silkgland i.e. obtaining stable heredity and secrete hydrophilic-silk
The silkworm of glue protein.
Present invention also offers a kind of G1 efficiently synthesize for rear qualification posterior division of silkgland and secrete hydrophilic sericin
The method of the transgenic bombyx mori of albumen, at fluorescence microscopy Microscopic observation, the silk of tied Bombyx bombycis presents green
The larva individuality of fluorescence be posterior division of silkgland efficiently synthesize and secrete hydrophilic-silk glue protein turn Ser3 gene man
Silkworm.
Present invention also offers and a kind of there is the DNA molecular of aminoacid sequence shown in coding SEQ ID NO:3
Transposon vector.
In some embodiments, transposon vector is PB-Ser, comprises piggyBac transposase recognition sequence
PiggyBac 3'LTR and piggyBac 5'LTR, eye and neural specific promoter 3xP3, EGFP report
Accuse gene, the polyadenylic acid of SV40 adds tailer sequence, recombination Ser.Wherein piggyBac transposase
Recognition sequence lays respectively at the left end of the piggyBac 3'LTR on transposon vector and piggyBac 5'LTR's
TTAA site on right-hand member；PiggyBac transposase recognition sequence piggyBac 3'LTR and piggyBac
Be followed successively by Artificial promoters 3 × P3 between 5'LTR, EGFP reporter gene, the polyadenylic acid of SV40 add
Tailer sequence and recombination Ser.
In some preferred embodiments, transposon vector has nucleotide sequence as shown in SEQ ID NO:5.
The posterior division of silkgland that present invention also offers the stable heredity that the method for the present invention prepares efficiently synthesizes
Silkworm with secretion hydrophilic-silk glue protein SER3.
Posterior division of silkgland provided by the present invention efficiently synthesizes and secretes the silkworm of hydrophilic-silk glue protein, 5
Instar larvae phase posterior silkgland cells and posterior division of silkgland lumen of gland secretory protein are able to detect that hydrophilic sericin
Protein S ER3 composition.In some preferred embodiments, accumulation in ripe larva posterior division of silkgland lumen of gland
In secretion silk protein, sericine SER3 content is more than 5%, sericin egg in the silk protein spued
White matter SER3 content is more than 10%.
In order to be further appreciated by the present invention, the method provided the present invention below in conjunction with embodiment is carried out in detail
Explanation.
Embodiment 1
Prepare the transposon vector of recombination Ser
Silkworm Fib-H gene is connected in the upstream of sericine SER3 coding nucleotide sequence Ser3
Promoter and 5' end signal peptide-coding sequence, downstream connects the 3' terminal sequence of silkworm Fib-H gene and is recombinated
Gene Ser, its nucleotide sequence is as shown in SEQ ID NO:4.
By the family's the inactive state of the silkworm before it sheds its skin containing 3 series connection and nervous system specific transcription factor PAX-6 binding sequence group
PiggyBac-3 × P3-DsRed plasmid (sequence such as SEQ ID NO:6 of the Artificial promoters 3 × P3 become
Shown in) carry out AscI and FseI double digestion respectively with the Ser sequence of synthetic.After enzyme action
PiggyBac-3 × P3-DsRed plasmid and Ser sequence 16 DEG C of reaction overnight of T4 ligase.Then will produce
Thing carries out electrophoresis detection, and the Ser band of enzyme action is purified recovery and order-checking.Identify that sequence is correct
Being transposon vector PB-Ser, its nucleotide sequence is as shown in SEQ ID NO:5.
Concrete schematic diagram is as Figure 1-Figure 2.Fig. 1 is recombination Ser, and Fig. 2 is recombination Ser
Transposon vector.
Embodiment 2
Prepare posterior division of silkgland and can synthesize and secrete the silkworm of hydrophilic-silk glue protein
Step is obtained, by whole for recombination Ser sequence according to general silkworm transgeneic procedure and transgenic silkworm
Close the genome of silkworm N4 kind, obtain concretely comprising the following steps of Ser silkworm:
(1), in the environment that aseptic and free nucleic acid pollutes, micro-injection method is used to inject to every silkworm seed
The transposon vector plasmid of 4000ng-5000ng；
(2) the non-toxic material envelope of the silkworm seed after injection is coated with injection orifice, then in gnotobasis, with 26 DEG C
-27 DEG C, 75%-80% relative humidity, natural lighting protection silkworm seed to hatching, after hatching larva continue in phase
Raising with Folium Mori or man-made feeds in synthermal, humidity and photoenvironment, after cocooing, larva continues with identical
Epidemic disaster and photoenvironment are protected to adult eclosion；
(3) Bombycis mori selfing or after backcrossing, after institute's spawning and hatching, larva Folium Mori or man-made feeds are raised after sprouting wings
Supporting, pupa time is investigated under fluorescence microscope, and eye presents the individuality of red fluorescence, individual for transgenic G1 generation；
(4) eye selecting transgenic G1 generation individual pupa presents the individual subculture of red fluorescence, to G6
Efficiently synthesize for the rear posterior division of silkgland i.e. obtaining stable heredity and secrete the silkworm of hydrophilic-silk glue protein.
The genetically modified mutant Ser silkworm obtaining G1 to G6 generation is observed, and result is as shown in Figure 3.Its
Middle figure A is mutant Bombyx mori posterior silkgland cross section aspect graph, and the green fluorescence in the tissue of outer ring is from rear
EGFP in the recombiant protein SER of portion's silk gland cell synthesis, internal green fluorescence diagram posterior division of silkgland
The recombiant protein SER being secreted in lumen of gland after cell synthesis；Figure B is that mutant silkworm middle division of silkgland is crosscutting
Face aspect graph, green fluorescence diagram transfers to the recombiant protein of middle division of silkgland lumen of gland from posterior silkgland cells
SER；Figure C is the silk protein fiber that mutant silkworm spues, and it is fine that green fluorescence diagram is present in fibroin
The internal recombiant protein SER with surface of dimension；Figure D diagram wild type and the tied Bombyx bombycis of mutant silkworm glimmering
Light difference, has a recombiant protein SER of fluoresced green in the Bombyx bombycis fibroin of mutant, and wild type
Silk does not has the characteristic green fluorescence of SER；Corresponding wild type and mutant man in figure E pictorial image D
The visible images of the tied Bombyx bombycis of silkworm；Figure F diagram wild type and mutant silkworm 5 instar larvae eye glimmering
Light difference, the eye of the larva of mutant silkworm presents HONGGUANG fluorescence, and the eye of wild type silkworm is not in red
Light fluorescence, in figure, arrow indication is the eye locations of larva.
From Fig. 3 result, the eye of the larva of mutant Ser silkworm presents HONGGUANG fluorescence, wild type man
The eye of silkworm is not in HONGGUANG fluorescence；The silk of Ser silkworm presents green fluorescence, and the silk of wild type silkworm is not
In green-emitting fluorescent；The 5 instar larvae posterior division of silkgland lumens of gland of Ser silkworm have the green fluorescence of recombiant protein SER to deposit
, the posterior division of silkgland lumen of gland of wild type silkworm does not has the green fluorescence of recombiant protein SER.
G6 waves silk in the secretion silk protein accumulated in phase larva posterior division of silkgland lumen of gland for Ser silkworm 5 age
Glue protein matter SER3 content 4.9%-5.5%, sericine SER3 content in the silk protein spued
8.8%-10.6%.
The above is only the preferred embodiment of the present invention, is not limited to the present invention, it is noted that
For those skilled in the art, on the premise of without departing from the technology of the present invention principle,
Can also make some improvement and modification, these improve and modification also should be regarded as protection scope of the present invention.
Claims (9)
1. a posterior division of silkgland can synthesize and secrete the preparation method of silkworm of hydrophilic-silk glue protein, it is characterised in that
Comprise the following steps:
The DNA molecular of encoding amino acid sequence SER is recombinated on chromosome of mulberry silkworm, expresses, translate and obtain
Described posterior division of silkgland can synthesize and secrete the silkworm of hydrophilic-silk glue protein；
Wherein, the nucleotide sequence 5' end of the DNA molecular of described encoding amino acid sequence SER is to 3' end successively
For bombyx mori silk fibroin heavy chain protein Fib-H gene promoter, silkworm Fib-H gene 5' end signal peptide-coding sequence,
Silkworm sericine SER3 coded sequence Ser3 and silkworm Fib-H gene 3' terminal sequence.
Posterior division of silkgland the most according to claim 1 can synthesize and secrete the preparation of the silkworm of hydrophilic-silk glue protein
Method, it is characterised in that: described aminoacid sequence SER has the aminoacid sequence shown in SEQ ID NO:3.
Posterior division of silkgland the most according to claim 2 can synthesize and secrete the preparation of the silkworm of hydrophilic-silk glue protein
Method, it is characterised in that: the DNA molecular of coding aminoacid sequence shown in SEQ ID NO:3 has SEQ
Nucleotide sequence shown in ID NO:4.
Posterior division of silkgland the most according to claim 1 can synthesize and secrete the preparation of the silkworm of hydrophilic-silk glue protein
Method, it is characterised in that chromosome of mulberry silkworm of being recombinated to by the DNA molecular of encoding amino acid sequence SER is enterprising
One step comprises the following steps:
Build the transposon vector of the DNA molecular comprising encoding amino acid sequence SER；And by described transposon vector
Import in Eggs of Silkworm.
Posterior division of silkgland the most according to claim 4 can synthesize and secrete the preparation of the silkworm of hydrophilic-silk glue protein
Method, it is characterised in that: described transposon vector is that the recombination Ser3 that piggyBac transposon builds turns
Seat carrier PB-ser.
6. can synthesize and secrete the silkworm of hydrophilic-silk glue protein according to the posterior division of silkgland described in claim 4 or 5
Preparation method, it is characterised in that: described transposon vector has nucleotide sequence shown in SEQ ID NO:5.
7. one kind has the transposon vector of the DNA molecular of aminoacid sequence shown in coding SEQ ID NO:3.
Transposon vector the most according to claim 7, it is characterised in that: described transposon vector is that piggyBac turns
The recombination Ser3 transposon vector PB-ser that stand builds.
9. according to the transposon vector described in claim 7 or 8, it is characterised in that: described transposon vector has SEQ
Nucleotide sequence shown in ID NO:5.
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