CN108949767B - Suitable for the specific integrase phiC31 and its expression regulation system of silkworm expression and application - Google Patents
Suitable for the specific integrase phiC31 and its expression regulation system of silkworm expression and application Download PDFInfo
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Abstract
The present invention relates to a kind of specific integrase phiC31 suitable for silkworm expression and its expression regulation system and applications, the nucleotide sequence of specific integrase phiC31 is as shown in SEQ ID NO.1, the enzyme can in silkworm high efficient expression, with the site attP and the site attB joint mapping expression regulation system, the system identifies that the roll over condition between catalysis attP and attB controls promoter direction by phiC31, to realize target gene in the controlled expression of silkworm individual level；Strong tool is provided for the function parsing of domestic silkworm gene and material innovation research, and provides theoretical and reference in the application of other species for phiC31 integrase mediated gene expression control system.
Description
Technical field
The invention belongs to field of biotechnology, it is related to the specific integrase phiC31 suitable for silkworm expression, containing being related to
The expression regulation system and its application of expression specificity integrase phiC31.
Background technique
With the arrival of genome times afterwards comprehensively, to the magnanimity gene that genome period identifies, especially with biology
Shape or the relevant important gene of economic characters carry out function parsing, and carry out material innovation and kind using critical function gene
Upgrading has become the most important Task of current field of biology and direction.And to carry out the function of magnanimity gene
Parsing or material innovation research, urgent need are reformed and are expanded to traditional gene functional research means, wherein attract attention
Surely belong to import target gene in host using transgenic technology, carries out the function parsing of gene in individual level, and numerous
It succeeds in mode species and Important Economic class animals and plants application, biological function and material innovation in parsing important gene
Research field played an important role, and show big advantage and application potential.On the other hand, it is opened using transgenic technology
The functional study of gene is opened up to accurately illustrate the function of a gene, effective method is usually the controlled of progress target gene
Expression allows target gene to be expressed in specific developmental stage or organ-tissue, by the specifically expressed feature of its space-time or a
Body surface type, so that it is determined that the major function of the gene.Currently, it can be realized this mesh calibration method, be using from yeast
GAL4/UAS double base expression system.Successful application in drosophila isotype biology proves that GAL4/UAS system is research base
Because of maximally efficient one of the tool of function.Moreover, which can also realize different genes in same developmental stage or device
The specifically expressing of official's tissue.Thus, since foundation, using GAL4/UAS system and rely on transgenic technology to become for function base
Because of a group important means for age study gene function.Site-specific integration enzyme phiC31 belongs to serine recombination enzyme family, comes
Derived from Streptomyces Phage.PhiC31 integrase can be with intraphagic attP sequence (the phage attachment of specific recognition
Site it is integrated between the attB sequence (bacterial attachment site)) and in host genome, thus by bacteriophage
DNA is integrated into the genome of host.Studies have shown that attP and attB minimum bioactive sequence length is respectively 39bp and 34bp,
Integrase phiC31, which is recognized after attP and attB, to cut double-strand the base after the center TTG of two sequences respectively
DNA simultaneously rotates 180 °, and the attR of the attL and 37bp of 36bp are formed after reconnecting later.Since phiC31 integrase has
AttP and attB minimum bioactive sequence is short, and DNA delivered payload capability is strong, and is formed after abnormity sequence attP and attB exchange integration
AttL and attR will not be identified that the outstanding advantages such as integration again occur for catalysis by phiC31, which is including plant, feeding
It is widely used in the site-specific reorganizing research of multiple species such as newborn animal, insect, and in genetic manipulation and hereditary work
It played an important role in journey research, become the powerful tool for carrying out genetic manipulation.
By artificial feeding and domestication in more than 5000 years, silkworm not only became a kind of economic insects that is important and having characteristic,
And with the development of modern molecular biology, silkworm also becomes internationally recognized Lepidoptera model insects.In China, with ancient times
Planting the spun silk industry that develops of Sang Yangcan has brilliant history, it is carried forward for Chinese nation's economic development and culture is made that and holds
Continuous tremendous contribution.Nevertheless, since traditional silkworm breeding technology, cocoon fibre manufacturing technique etc. limit, nearly over half a century
Since China cultivate in silkworm excellent variety, yield and never have acquirement important breakthrough in terms of spun silk quality trait.
It is prompted from the existing successful experience to organism renovation and utilization, fundamentally to overcome the inherent shortcoming of silk, restore silk
Textile fabric market life, develop novel and multifunctional silk material, promote single silk industry to more extensive non-thin,tough silk
Silk industry extension, must just utilize modern molecular Genetic Manipulative Technology, carry out function to silkworm important economical trait related gene
Parsing and genetic modification.With asking in succession for silkworm full-length genome frame diagram, fine figure, genetic chip and mapping genetic variations
Generation provides molecular target for the breed improvement and material innovation research of silkworm.And to realize the function parsing to domestic silkworm gene
It is innovated with material, to silkworm Genetic Manipulative Technology system, more stringent requirements are proposed and challenge.Therefore, it is badly in need of establishing domestic silkworm gene
Expression regulation system provides strong tool for the function parsing of domestic silkworm gene and material innovation research.
Summary of the invention
In view of this, one of the objects of the present invention is to provide the specific integrase phiC31 for being suitable for silkworm expression；
The second object of the present invention is to provide the Gene expression regulation system containing the specific integrase phiC31；Of the invention
The third purpose is to provide application of the Gene expression regulation system in the regulation expression of target gene of silkworm individual level.
To realize above-mentioned expression purpose, the invention provides the following technical scheme:
1, suitable for the specific integrase phiC31 of silkworm expression, the nucleotides sequence of the specific integrase phiC31
Column are as shown in SEQ ID NO.1.
2, the Gene expression regulation system containing the specific integrase phiC31, the Gene expression regulation system packet
Include the expression vector of expression specificity integrase phiC31 gene and the site attP that arranges in opposite directions containing 2 and the site attB
Expression vector.
The expression system identifies that the roll over condition between catalysis attP and attB controls promoter direction by phiC31,
To realize target gene in the controlled expression of silkworm individual level.
Preferably, the specific integrase phiC31 base of the expression vector of the expression specificity integrase phiC31 gene
Because 3 ' end fusions have SV40 nuclear localization signal, the nucleotide sequence of the SV40 nuclear localization signal such as SEQ ID NO.2 the 1815th
Position~1836 shown in.
Preferably, the expression vector of the expression specificity integrase phiC31 gene is constructed by following methods: by SEQ
Sequence shown in ID NO.2 is connected to BamHI the and NotI digestion for being building up to psl1180 [hr3CQ-Act4P-DsRed-Ser1PA]
At site.
It is furthermore preferred that the expression vector in the site attP arranged in opposite directions containing 2 and the site attB is by following methods
Building: using silkworm Act4 promoter sequence as template, sequence shown in SEQ ID NO.3 and SEQ ID NO.4 is that primer carries out PCR
Then it is former to be connected into the replacement of psl1180 [Act4P-DsRed-SV40] carrier by amplification after BamHI and SalI digestion for amplified production
Some Act4 promoter forward direction sequences, then be building up in the site AscI of pBac [3xp3EGFPaf] carrier by the site AscI,
It is formed transgene expression vector pBac [attB-reversedAct4P-DsRed-SV40,3xp3EGFP].
3, application of the Gene expression regulation system in the regulation expression of target gene of silkworm individual level.
The beneficial effects of the present invention are: the present invention provides be suitable for silkworm expression specific integrase phiC31,
This can in silkworm high efficient expression, which is constructed by conjunction with attP and attB phiC31 identification catalysis attP and attB
Between roll over condition control promoter direction, to realize target gene in the controlled expression of silkworm individual level；For silkworm base
The function parsing of cause and material innovation research provide strong tool, and control system for phiC31 integrase mediated gene expression
It unites in the application offer theory of other species and reference.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out
Illustrate:
Fig. 1 be phiC31 be catalyzed promoter overturning gene expression control system schematic diagram (A: report carrier system,
DsRed reporter gene is not expressed；B:phiC31 is catalyzed the recombination exchange between attB and attP, overturns promoter；C: promoter
Start the expression of downstream reporter gene after overturning).
Fig. 2 is that phiC31 is catalyzed the overturning of silkworm Act4 promoter and the expression of control report gene in silkworm BmE cell
(A: report carrier system, DsRed reporter gene are not expressed；B:phiC31 transient expression vector schematic diagram；C:phiC31NLS wink
When expression vector schematic diagram；D-E: the BmE cell white light figure and fluorogram of single-turn report carrier；F-G: corotation report carrier and
The BmE cell white light figure and fluorogram of phiC31 transient expression vector；H-I: corotation report carrier and phiC31NLS transient expression
The BmE cell white light figure and fluorogram of carrier).
Fig. 3 is the Molecular Detection (A: genome that phiC31 is catalyzed the overturning of silkworm Act4 promoter in silkworm BmE cell
PCR detection and design of primers schematic diagram；B: Genomic PCR electrophoresis detection, S1-S3 are with corotation report carrier and phiC31NLS
The BmE cellular genome of transient expression vector is the PCR product of template；C: the feature expanded for Act4-R/Red-R primer sets
Sequence；D: the characteristic sequence expanded for Act4-F/Red-R primer sets).
Fig. 4 be transgenic bombyx mori building and Fluirescence observation (A: reporting system transgenic bombyx mori carrier schematic diagram, ITR are
The inverted repeats of piggyBac transposon；B:phiC31NLS auxiliary system transgenic bombyx mori carrier schematic diagram；C-D report
The green fluorescence and white light of system transgenic bombyx mori moth detect figure；E-F:phiC31NLS auxiliary system transgenic bombyx mori moth
Red fluorescence and white light detection figure).
Fig. 5 is the schematic diagram that phiC31 is catalyzed that promoter overturning controls gene expression in silkworm individual level.
Fig. 6 is that phiC31 is catalyzed silkworm Act4 promoter in the Molecular Detection (A: base of the heritable overturning of silkworm individual level
Because of group PCR detection and a design of primers schematic diagram；B: Genomic PCR electrophoresis detection, F1 be reporting system transgenic bombyx mori strain with
The genes of individuals group of phiC31NLS auxiliary system transgenic bombyx mori incross F1 generation, F2 is reporting system transgenic bombyx mori
The positive individuals genome that strain and phiC31NLS auxiliary system transgenic bombyx mori incross F2 generation backcrossing screen；C: for
F1 generation hybrid individual genome is template, the characteristic sequence that Act4-R/Red-R primer sets expand；D: being F2 for positive individuals
Gene is template, the characteristic sequence that Act4-F/Red-R primer sets expand).
Fig. 7 be phiC31 be catalyzed promoter the heritable overturning control DsRed of silkworm individual level detection of expression (A-B:
White light and red fluorescence figure of the F2 for positive individuals silkworm seed period；C-D:F2 is glimmering for the white light and red of positive individuals four ages hibernating worm
Light figure；E-F: normal silkworm pupa the 8th day white light and red fluorescence figure；G-H:F2 is for positive individuals pupa the 8th day white light and red
Fluorogram；SDS-Page and Western of the I-J:F2 for red fluorescent protein content in the 8th day total protein of positive individuals pupa
Blot detection figure；K:F2 is compared for red fluorescent protein content gray scale in the 8th day total protein of positive individuals pupa).
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.
It makes (P50) greatly for examination cultivated silkworm breed variety to be saved by this laboratory, larva is in 25 DEG C of growth cabinets with man-made feeds
Raising.Cell line is Silkworm Embryo Cell Lines BmE, is trained by domestic silkworm gene group biology National Key Laboratory, Southwest China university
It supports and saves, plasmid vector pSLfa1180fa, pBac [3xp3EGFPaf], pBac [3xp3DsRedaf] are protected by this laboratory
It deposits.
Main agents and solution are formulated as follows in embodiment:
The conventional medium arrived used in the process of molecular cloning, reagent buffer etc. is referring to " Molecular Cloning:A Laboratory guide the
Three editions are translated " and " TaKaRa goods catalogue " in laboratory conventional reagent preparation method chapters and sections (S1-S11) configured.DNA is poly-
Cloning vector pMD19-T simple is sequenced in synthase Ex-Taq, LA-Taq Kit, convenient restriction restriction endonuclease, alkaline phosphatase
Carrier Kit, DNA Ligation Kit Ver.2.0, PCR kit for fluorescence quantitative SYBR premix Ex TaqTM are purchased from
TaKaRa company.Conversion competent escherichia coli cell Trans1-T1, conventional plasmid DNA extraction kit Easypure
Plasmid MiniPrep Kit is purchased from Quan Shi King Company.Ago-Gel DNA QIAquick Gel Extraction Kit Gel Extraction Mini
Kit (50) is purchased from Hua Shun biotechnology company.The ultrapure plasmid extraction kit QIA prep Spin of transgenosis injection
Miniprep Kit (50) is purchased from QIAGEN.Total RNA Kit II (50) kit is purchased from Omega Bio-Tec company.It is red
Color fluorescin polyclonal antibody anti-DsRed antibody, red fluorescent protein standard items DsRedstd are public purchased from Abcam
Department.
Embodiment 1, gene chemical synthesis
SV40 nuclear location is merged from the amino acid sequence (GI:40313242) of NCBI downloading phiC31 gene, and in its C-terminal
Signal (PKKKRKV).Coded sequence is optimized according to silkworm codon usage bias type, gene sequence is synthesized by company
Column, the both ends of two genes phiC31 and phiC31-NLS of synthesis connect BamH I and Not I restriction enzyme site, nucleotide respectively
Sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.
Embodiment 2, vector construction
PhiC31 the and phiC31-NLS gene coded sequence of synthesis is building up to by BamHI and NotI restriction enzyme site
psl1180[hr3CQ-Act4P-DsRed-Ser1PA](F.Wang,H.Xu,L.Yuan,S.Ma,Y.Wang,X.Duan,
J.Duan,Z.Xiang,Q.Xia,An optimized sericin-1expression system for mass-
producing recombinant proteins in the middle silk glands of transgenic
Silkworms, Transgenic Res 22 (5) (2013) 925-38.) in, it is respectively formed psl1180 [hr3CQ-Act4P-
PhiC31-Ser1PA] and psl1180 [hr3CQ-Act4P-phiC31NLS-Ser1PA], wherein psl1180 [hr3CQ-
Act4P-phiC31NLS-Ser1PA] nucleotide sequence is building up to pBac as shown in SEQ ID NO.3, then through the site AscI
In the site AscI of [3xp3DsRedaf] carrier, it is respectively formed transgene expression vector pBac [hr3CQ-Act4P-
PhiC31NLS-Ser1PA] and pBac [hr3CQ-Act4P-phiC31NLS-Ser1PA, 3xp3DsRed].
Site differential recombination enzyme phiC31 can be catalyzed between its specific recognition sequence attB and attP occur it is irreversible heavy
Group exchange can occur recombination under the catalysis of phiC31 and exchange and delete between the two when attB and attP is collectively aligned
DNA sequence dna, and as attB and attP reversed arrangement, recombination can occur under the catalysis of phiC31 and exchange and overturn between the two
DNA sequence dna.Pass through PCR by the attB sequence of 34bp and the attP sequence of 39bp in form head to head according to this characteristic
Amplification is fused to the end 3' and the end 5' (Fig. 1, the A) of silkworm Act4 promoter respectively, has the Act4 of attB and attP to start fusion
The sub upstream end 5' for being reversely inserted into red fluorescent protein reporter gene (DsRed), forms a report carrier psl1180
[attB-reversedAct4P-DsRed-SV40].When phiC31 is not present, Act4 promoter cannot start downstream DsRed
The transcriptional expression of reporter gene, when there are phiC31, phiC31 can be sent out between special and irreversible catalysis attB and attP
Raw recombination exchange forms attL and attR, due to being reversed arrangement between attB and attP, can overturn after recombination exchange
Reversed (Fig. 1, the B) of Act4 promoter, and complete expression cassette is formed with the DsRed reporter gene in downstream, and then start DsRed
The transcriptional expression (Fig. 1, C) of reporter gene.Therefore, DsRed reporter gene can be realized taking human as the expression of control phiC31
Controlled expression, specific construction method are as follows: using silkworm Act4 promoter sequence as template, design primer: and Act4-F (BamHI):
5'-gcggatccccccaactggg gtaacctttgagttctctcagttgggggLower stroke of tcatcttgtcacaccta-3'(
Line show the attP sequence of 39bp) (SEQ ID NO.4) and Act4-R (SalI): 5'-ccgtcgacgtgccagggcgtgc ccttgggctccccgggcgcgAccaatcctctaacaac-3'(underscore show the attB reverse complementary sequence of 34bp)
(SEQ ID NO.5).Correct sequence is sequenced in PCR, psl1180 [Act4P- is building up to by BamHI and SalI restriction enzyme site
DsRed-SV40](F.Wang,H.Xu,L.Yuan,S.Ma,Y.Wang,X.Duan,J.Duan,Z.Xiang,Q.Xia,An
optimized sericin-1expression system for mass-producing recombinant proteins
in the middle silk glands of transgenic silkworms,Transgenic Res 22(5)(2013)
Original Act4 promoter forward direction sequence is replaced in 925-38.), forms psl1180 [attB-reversedAct4P-
DsRed-SV40] report carrier, nucleotide sequence is building up to pBac as shown in SEQ ID NO.6, then through the site AscI
In the site AscI of [3xp3EGFPaf] carrier, transgene expression vector pBac [attB-reversedAct4P-DsRed- is formed
SV40,3xp3EGFP] transgene expression vector.
Site differential recombination enzyme phiC31 can be catalyzed between its specific recognition sequence attB and attP occur it is irreversible heavy
Group exchange can occur recombination under the catalysis of phiC31 and exchange and delete between the two when attB and attP is collectively aligned
DNA sequence dna, and as attB and attP reversed arrangement, recombination can occur under the catalysis of phiC31 and exchange and overturn between the two
DNA sequence dna.Pass through PCR by the attB sequence of 34bp and the attP sequence of 39bp in form head to head according to this characteristic
Amplification is fused to the end 3' and the end 5' (Fig. 1, the A) of silkworm Act4 promoter respectively, has the Act4 of attB and attP to start fusion
The sub upstream end 5' for being reversely inserted into red fluorescent protein reporter gene (DsRed), forms a report carrier psl1180
[attB-reversedAct4P-DsRed-SV40].When phiC31 is not present, Act4 promoter cannot start downstream DsRed
The transcriptional expression of reporter gene, when there are phiC31, phiC31 can be sent out between special and irreversible catalysis attB and attP
Raw recombination exchange forms attL and attR, due to being reversed arrangement between attB and attP, can overturn after recombination exchange
Reversed (Fig. 1, the B) of Act4 promoter, and complete expression cassette is formed with the DsRed reporter gene in downstream, and then start DsRed
The transcriptional expression (Fig. 1, C) of reporter gene.Therefore, DsRed reporter gene can be realized taking human as the expression of control phiC31
Controlled expression.
Embodiment 3, cell transfecting
The cell transfecting carrier of above-mentioned building is extracted using the ultrapure plasmid extraction kit of Qiagen, and according to molar ratio 1:
1 ratio by psl1180 [attB-reversedAct4P-DsRed-Ser1PA] plasmid respectively with psl1180 [hr3CQ-
Act4P-phiC31-Ser1PA] plasmid and psl1180 [hr3CQ-Act4P-phiC31NLS-Ser1PA] plasmid mixing after carry out
Cell transfecting (Fig. 2, A-C).Cell transfecting according toII Reagent standard step carries out: the day before transfection is used
The BmE cell of (> 95% survival rate) in good condition is resuspended and in bed board to 24 orifice plates by the Grace culture medium of antibiotic-free, carefully
Born of the same parents final concentration of 8 × 105/ hole.Using the Grace culture medium of antibiotic-free by volume: quality=100 μ l:1 μ g ratios are diluted wait turn
Plasmid is contaminated, press volume using the Grace culture medium of antibiotic-free: volume=100 μ l:5 μ l dilute liposome, after then diluting
Plasmid and liposome by mixing in equal volume, after being stored at room temperature 0.5h, according to the amount of DNA transfection BmE cell of every 0.5 μ g of hole, 48h
After carry out Fluirescence observation.As the result is shown: single-turn contaminates psl1180 [attB-reversedAct4P-DsRed-SV40] report carrier
Control group (Fig. 2, D-E) and corotation psl1180 [attB-reversedAct4P-DsRed-SV40] and psl1180 [hr3CQ-
Act4P-phiC31-Ser1PA] experimental group (Fig. 2, F-G) not it is observed that the fluorescence signal of DsRed reporter gene, and be total to
Turn psl1180 [attB-reversedAct4P-DsRed-SV40] and psl1180 [hr3CQ-Act4P-phiC31NLS-
Ser1PA] experimental group (Fig. 2, H-I) observe apparent red fluorescent, as a result prompt not merge NLS sequence
PhiC31 cannot effectively be catalyzed the recombination exchange between identification sequence attB and attP in bombyx mori cell, when fusion has nuclear location letter
Number phiC31NLS can be catalyzed identification sequence attB and attP between in bombyx mori cell recombination exchange, realize DsRed report
The conditionity of gene is expressed.
BmE cellular genome after being transfected 72 hours using phenol chloroform extraction designs special primer Act4P- as template
R:5'-accaatcctctaacaaccgt-3'(SEQ ID NO.3), Act4P-F:5'-tcatcttgtcacacctacatct-
3'(SEQ ID NO.4), DsRed-R:5'-tctagagattctacaggaacag-3'(SEQ ID NO.5), with Act4P-R/
DsRed-R and Act4P-F/DsRed-R is that primer combination carries out PCR amplification, and wherein Act4-R/Red-R combination is opened for expanding
The sequence that mover is not flipped, Act4-F/Red-R combination expand item for the sequence (Fig. 3, A) after expanding promoter overturning
Part: 94 DEG C initial denaturation 4 minutes, 94 DEG C be denaturalized 30 seconds, 58 DEG C anneal 30 seconds, 72 DEG C extend 60 seconds, totally 30 circulation；Last 72 DEG C
Extend 10min, 4 DEG C save, as the result is shown in the genomic templates of phiC31NLS transfection group, Act4-R/Red-R and Act4-
F/Red-R two can expand the specific band to 1500bp or so, stripe size and theoretical amplification segment one to primer combination
It causes (Fig. 3, B).The segment that recycling obtains is connected on sequencing vector and carries out sequencing identification.As the result is shown: Act4-R/Red-R
The characteristic sequence that primer sets expand be Act4 promoter reverse sequence, attP reverse sequence and DsRed forward direction sequence, as
The unturned sequence of promoter (Fig. 3, C).And the characteristic sequence that Act4-F/Red-R primer sets expand be Act4 promoter just
Sequence (Fig. 3, D) to sequence, after the overturning of attR reverse sequence and DsRed forward direction sequence, as promoter.Result above mentions
Show, nuclear localization signal NLS can assist phiC31NLS to enter silkworm BmE cell, and be catalyzed the friendship of the recombination between attP and attB
It changes, realizes overturning of the Act4 promoter in silkworm BmE cell, and then control the expression of DsRed reporter gene.
Embodiment 4, microinjection and fluorescent screening
Will by BmE cellular level verify after psl1180 [hsp70-phiC31NLS-Ser1PA] assistant carrier and
Psl1180 [attB-reversedAct4P-DsRed-SV40] report carrier further constructs silkworm transgene expression vector:
PBac [hr3CQ-Act4P-phiC31NLS-Ser1PA, 3xp3DsRed] and pBac [attB-reversedAct4P-DsRed-
Ser1PA, 3xp3EGFP], wherein DsRed report carrier band green fluorescence marks, and stablizes the assistant carrier of expression phiC31NLS
Band red fluorescence label (Fig. 4, A-B).Transgenosis table is extracted using QIAGEN Plasimd Mini Kit plasmid extraction kit
Up to carrier pBac [hr3CQ-Act4P-phiC31NLS-Ser1PA, 3xp3DsRed], pBac [attB-reversedAct4P-
DsRed-Ser1PA, 3xp3EGFP] and assistant carrier phsp70PIG plasmid, each plasmid concentration is diluted to 400ng/ μ l, and
It is mixed respectively with assistant carrier phsp70PIG plasmid by 1:1 molar ratio.By mixed plasmid injection termination of diapause
Body early embryo (2~5h after oviposition) is made greatly, then injection orifice is sealed with nontoxic glue, is sterilized through 35% formaldehyde vapor
After five minutes, 25 DEG C are placed in, is hatched in the environment of relative humidity 85%, the larva (G0 generation) of hatching is raised using man-made feeds, until
The production of hybrid seeds is selfed or is returned after adult, and the G1 of acquisition is for silkworm seed (the 7th day) in macroscopical Stereo fluorescence microscope (Olypus
MVX10, Japan) under detect, red fluorescence observation use wavelength for the exciting light of 510~550nm, and green fluorescence is observed using wave
The exciting light of a length of 460~490nm filters out the transgenosis sun in eyes or neural specific excitation green fluorescence and red fluorescence
Property moth circle, be respectively designated as reversedAct4-DsRed strain and phiC31 strain, the fluorescent screening statistics of transgenic bombyx mori
It is shown in Table 1.
The building of 1. transgenic bombyx mori of table counts
Table 1 shows that wherein DsRed reports that strain positive moth circle number is 1, positive rate 7.3%, phiC31NLS supplement
The positive moth circle number of system is 4, positive rate 14%.The raising of G1 positive individuals is made greatly to the silkworm backcrossing production of hybrid seeds, shape to the moth phase and normally
At stable transgenic bombyx mori strain, it is respectively designated as flippedA4Red strain and phiC31NLS strain (Fig. 4, C-D).
Embodiment 5, the promoter roll-over test of silkworm individual level
Whether the expression that Act4 promoter starts downstream gene can be overturn in silkworm individual level for verifying phiC31NLS,
And whether can stablize heredity after overturning.In F0 generation, is respectively provided with the flippedA4Red strain of green fluorescence label and is had
The individual of the phiC31NLS strain of red fluorescence label carries out hybridization and forms F1 generation, screens from F1 generation glimmering simultaneous with two kinds
Signal it is individual with normally make the silkworm backcrossing production of hybrid seeds greatly and give birth to F2 generation, and from F2 for the 4-8 days progress fluorescent screenings of silkworm seed phase
There are the positive individuals and moth circle number of DsRed reporter gene expression in entire silkworm seed, as a result as shown in Fig. 5 and table 2.
Table 2.phiC31NLS is catalyzed the heritable Act4 promoter overturning positive rate statistics of silkworm individual level
Table 2 shows, has DsRed reporter gene expression from 56 F2 for screening altogether in moth circle in 22 moth circles and can lose
The positive individuals of biography, positive rate 39%.
Molecule inspection further is carried out to the promoter overturning individual test that phiC31NLS is catalyzed from domestic silkworm gene group level
It surveys.Extracting F1 generation has EGFP+/DsRed+Fluorescent marker and the positive individuals domestic silkworm gene group that screens of F2 generation, using special
Detection primer carries out genomic PCR amplification, and wherein Act4-R/Red-R combines the sequence not being flipped for expanding promoter,
Act4-F/Red-R combination is for the sequence (Fig. 6, A) after expanding promoter overturning.As the result is shown: in F1 generation EGFP+/DsRed+
In the silkworm genes of individuals group template of label, Act4-R/Red-R and Act4-F/Red-R two can expand primer combination and arrive
The specific band of 1500bp or so, stripe size are consistent with theoretical amplification segment.In F2 in positive individuals domestic silkworm gene group,
The combination of Act4-R/Red-R primer fails amplification to any band, and only Act4-F/Red-R primer combination can be expanded and be arrived
The specific band of 1500bp or so, stripe size are consistent with theoretical amplification segment (Fig. 6, B).Speculate the EGFP in F1 generation+/
DsRed+In individual, phiC31NLS is persistently catalyzed the exchange of the recombination between attB and attP, makes Act4 promoter in silkworm individual
The overturning of mosaic occurs for level, i.e. promoter rollover event occurs for part Tissues of Silkworm Bombyx Moril or cell.Then, by being returned,
The promoter overturning mutation occurred in reproductive organs or cell is hereditary to offspring, makes F2 for positive individuals in genomic level
The form after promoter is all overturn is presented.The sequence of amplification is further subjected to sequencing analysis, as the result is shown: F1 generation EGFP+/
DsRed+Genes of individuals group is Act4 promoter reverse sequence, attP with the characteristic sequence that Act4-R/Red-R primer sets expand
Reverse sequence and DsRed forward direction sequence, the as unturned sequence of promoter (Fig. 6, C).And F2 is for positive individuals genome
Be Act4 promoter forward direction sequence with the characteristic sequence that Act4-F/Red-R primer sets expand, attR reverse sequence and
Sequence (Fig. 6, D) after the overturning of DsRed forward direction sequence, as promoter.Result above prompt, phiC31NLS can be in silkworms
Body level is catalyzed the recombination exchange between attP and attB, realizes heritable overturning of the Act4 promoter in silkworm individual, into
And control the expression of DsRed reporter gene.
SDS-Page and Western blot detection:
To pulverize after positive silkworm chrysalis Liquid nitrogen precooler, after be dissolved in 20mM Tris-Cl, in 8.0 buffer of pH, 4 DEG C of vibrations
2h is swung, later 12000 × rpm, 5min collects supernatant.The total protein of extraction utilizes after 12% SDS-Page electrophoretic separation
Coomassie brilliant blue is dyed.It, will using electric transferring film method by the total protein of extraction after the separation of 12%SDS-Page gel electrophoresis
On protein delivery to pvdf membrane in SDS-Page glue.Pvdf membrane is placed in the TBST buffer containing 5% skimmed milk power, 4 DEG C of mistakes
Night closing.Before immuning hybridization, cleaned pvdf membrane 3 times in room temperature using TBST, each 10min.Using containing 5% skimmed milk power
TBST is configured (Abcam) primary antibody hybridization solution of anti-RFP by 10000 times of dilution ratios, and pvdf membrane is immersed room temperature in hybridization solution and is shaken
It swings and is incubated for 2h, TBST washes film 5 times, each 5min.Using TBST by the goat-anti rabbit two of 20000 times of dilution ratio configuration HRP labels
Anti- (being purchased from green skies company) hybridization solution, the pvdf membrane after TBST is cleaned immerses in secondary antibody hybridization solution to be incubated in shaken at room temperature
2h, TBST wash film 5 times, each 5min.Pvdf membrane after cleaning is placed on clean preservative film, by ECL developing solution
(Amersham Biosciences) uniformly for drop on PDVF film surface, room temperature, which is protected from light, is incubated for 5min, utilizes Chemiscope
Series (Clinx science instruments) instrument is exposed and is imaged.The results show that in the total of positive pupa individual
Detect that red fluorescent protein, molecular size range and red fluorescent protein standard items are (Fig. 7, I-J) in the same size in albumen.By sample
The hybridising band of product group carries out gray scale with RFP mark product group and compares, and estimates the content of red fluorescent protein in positive pupa individual
About 8.1mg/g (Fig. 7, K).
By carrying out Fluirescence observation for each period of positive individuals to F2, as the result is shown: the 4th day ovum phase can be at positive
DsRed fluorescence signal is observed in the entire silkworm seed of body, and is in dotted aggregation (Fig. 7, A-B)；Larval phase and silkworm chrysalis phase it is entire
Intense red fluorescence signal (Fig. 7, C-H) is presented in individual.The result shows that the heritable overturning of phiC31NLS catalysis Act4 promoter,
And driving DsRed reporter gene in F2 for the transcriptional expression in silkworm positive individuals, the red fluorescent protein of generation is in Silkworm, Bombyx mori
Interior aggregation shows strong red fluorescence.
It is raw that the above results show that the gene expression control system of phiC31NLS catalysis promoter overturning can be used as silkworm pupa
The exploitation of object reactor, to improve the comprehensive utilization and economic value of silkworm pupa.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
<110>Southwest University
<120>suitable for the specific integrase phiC31 of silkworm expression and its expression regulation system and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1815
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atggacacgt acgcgggtgc ttacgaccgt cagtcgcgcg agcgcgagaa ttcgagcgca 60
gcaagcccag cgacacagcg tagcgccaac gaagacaagg cggccgacct tcagcgcgaa 120
gtcgagcgcg acgggggccg gttcaggttc gtcgggcatt tcagcgaagc gccgggcacg 180
tcggcgttcg ggacggcgga gcgcccggag ttcgaacgca tcctgaacga atgccgcgcc 240
gggcggctca acatgatcat tgtctatgac gtgtcgcgct tctcgcgcct gaaggtcatg 300
gacgcgattc cgattgtctc ggaattgctc gccctgggcg tgacgattgt ttccactcag 360
gaaggcgtct tccggcaggg aaacgtcatg gacctgattc acctgattat gcggctcgac 420
gcgtcgcaca aagaatcttc gctgaagtcg gcgaagattc tcgacacgaa gaaccttcag 480
cgcgaattgg gcgggtacgt cggcgggaag gcgccttacg gcttcgagct tgtttcggag 540
acgaaggaga tcacgcgcaa cggccgaatg gtcaatgtcg tcatcaacaa gcttgcgcac 600
tcgaccactc cccttaccgg acccttcgag ttcgagcccg acgtaatccg gtggtggtgg 660
cgtgagatca agacgcacaa acaccttccc ttcaagccgg gcagtcaagc cgccattcac 720
ccgggcagca tcacggggct ttgtaagcgc atggacgctg acgccgtgcc gacccggggc 780
gagacgattg ggaagaagac cgcttcaagc gcctgggacc cggcaaccgt tatgcgaatc 840
cttcgggacc cgcgtattgc gggcttcgcc gctgaggtga tctacaagaa gaagccggac 900
ggcacgccga ccacgaagat tgagggttac cgcattcagc gcgacccgat cacgctccgg 960
ccggtcgagc ttgattgcgg accgatcatc gagcccgctg agtggtatga gcttcaggcg 1020
tggttggacg gcagggggcg cggcaagggg ctttcccggg ggcaagccat tctgtccgcc 1080
atggacaagc tgtactgcga gtgtggcgcc gtcatgactt cgaagcgcgg ggaagaatcg 1140
atcaaggact cttaccgctg ccgtcgccgg aaggtggtcg acccgtccgc acctgggcag 1200
cacgaaggca cgtgcaacgt cagcatggcg gcactcgaca agttcgttgc ggaacgcatc 1260
ttcaacaaga tcaggcacgc cgaaggcgac gaagagacgt tggcgcttct gtgggaagcc 1320
gcccgacgct tcggcaagct cactgaggcg cctgagaaga gcggcgaacg ggcgaacctt 1380
gttgcggagc gcgccgacgc cctgaacgcc cttgaagagc tgtacgaaga ccgcgcggca 1440
ggcgcgtacg acggacccgt tggcaggaag cacttccgga agcaacaggc agcgctgacg 1500
ctccggcagc aaggggcgga agagcggctt gccgaacttg aagccgccga agccccgaag 1560
cttccccttg accaatggtt ccccgaagac gccgacgctg acccgaccgg ccctaagtcg 1620
tggtgggggc gcgcgtcagt agacgacaag cgcgtgttcg tcgggctctt cgtagacaag 1680
atcgttgtca cgaagtcgac tacgggcagg gggcagggaa cgcccatcga gaagcgcgct 1740
tcgatcacgt gggcgaagcc gccgaccgac gacgacgaag acgacgccca ggacggcacg 1800
gaagacgtag cggcg 1815
<210> 2
<211> 1839
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atggacacgt acgcgggtgc ttacgaccgt cagtcgcgcg agcgcgagaa ttcgagcgca 60
gcaagcccag cgacacagcg tagcgccaac gaagacaagg cggccgacct tcagcgcgaa 120
gtcgagcgcg acgggggccg gttcaggttc gtcgggcatt tcagcgaagc gccgggcacg 180
tcggcgttcg ggacggcgga gcgcccggag ttcgaacgca tcctgaacga atgccgcgcc 240
gggcggctca acatgatcat tgtctatgac gtgtcgcgct tctcgcgcct gaaggtcatg 300
gacgcgattc cgattgtctc ggaattgctc gccctgggcg tgacgattgt ttccactcag 360
gaaggcgtct tccggcaggg aaacgtcatg gacctgattc acctgattat gcggctcgac 420
gcgtcgcaca aagaatcttc gctgaagtcg gcgaagattc tcgacacgaa gaaccttcag 480
cgcgaattgg gcgggtacgt cggcgggaag gcgccttacg gcttcgagct tgtttcggag 540
acgaaggaga tcacgcgcaa cggccgaatg gtcaatgtcg tcatcaacaa gcttgcgcac 600
tcgaccactc cccttaccgg acccttcgag ttcgagcccg acgtaatccg gtggtggtgg 660
cgtgagatca agacgcacaa acaccttccc ttcaagccgg gcagtcaagc cgccattcac 720
ccgggcagca tcacggggct ttgtaagcgc atggacgctg acgccgtgcc gacccggggc 780
gagacgattg ggaagaagac cgcttcaagc gcctgggacc cggcaaccgt tatgcgaatc 840
cttcgggacc cgcgtattgc gggcttcgcc gctgaggtga tctacaagaa gaagccggac 900
ggcacgccga ccacgaagat tgagggttac cgcattcagc gcgacccgat cacgctccgg 960
ccggtcgagc ttgattgcgg accgatcatc gagcccgctg agtggtatga gcttcaggcg 1020
tggttggacg gcagggggcg cggcaagggg ctttcccggg ggcaagccat tctgtccgcc 1080
atggacaagc tgtactgcga gtgtggcgcc gtcatgactt cgaagcgcgg ggaagaatcg 1140
atcaaggact cttaccgctg ccgtcgccgg aaggtggtcg acccgtccgc acctgggcag 1200
cacgaaggca cgtgcaacgt cagcatggcg gcactcgaca agttcgttgc ggaacgcatc 1260
ttcaacaaga tcaggcacgc cgaaggcgac gaagagacgt tggcgcttct gtgggaagcc 1320
gcccgacgct tcggcaagct cactgaggcg cctgagaaga gcggcgaacg ggcgaacctt 1380
gttgcggagc gcgccgacgc cctgaacgcc cttgaagagc tgtacgaaga ccgcgcggca 1440
ggcgcgtacg acggacccgt tggcaggaag cacttccgga agcaacaggc agcgctgacg 1500
ctccggcagc aaggggcgga agagcggctt gccgaacttg aagccgccga agccccgaag 1560
cttccccttg accaatggtt ccccgaagac gccgacgctg acccgaccgg ccctaagtcg 1620
tggtgggggc gcgcgtcagt agacgacaag cgcgtgttcg tcgggctctt cgtagacaag 1680
atcgttgtca cgaagtcgac tacgggcagg gggcagggaa cgcccatcga gaagcgcgct 1740
tcgatcacgt gggcgaagcc gccgaccgac gacgacgaag acgacgccca ggacggcacg 1800
gaagacgtag cggcgcccaa gaagaagaga aaggtttag 1839
<210> 3
<211> 3955
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ccatggcagc gtcgtgaaaa gaggcaatga caaatacaaa acgacgtatg agcagacccg 60
tcgccaagac gggtctacct ctaagatgat gtcatttgtt ttttaaaact aactcgcttt 120
acgagtagaa ttctacgtgt aaaacataat caagagatga tgtcatttgt ttttcaaaac 180
caaactcgct ttacgagtag aattctacgt gtaaaacaca atcaaaagat gatgtcattc 240
gtttttcaaa accgaattta agaaatgatg tcatttgttt ttcaaaacca aactcgcttt 300
acgagcagaa ttctacgtgt aaaacacaat caagagatga tgtcatttgt ttttcaaaac 360
tgaatgatgt catttgtttt tcaaaactaa acttgctttg cgagtagaat tctacgtgta 420
aaacacagtc aagagatgat gtcatttgtt tttcaaaact gaaccggctt tacgagtaga 480
attctacttg taaaacataa tcaagagatg atgtcatttg tttttcaaaa ctgaactggc 540
tttacgagta gaattctacg tgtaaaacat aatcaagaga tgatgtcatc attaaactga 600
tgtcatttta tacacgattg ttaacatgtt taataatgac taatttgttt ttccaaatta 660
aactcgcttt acgagtagaa ttctacttgt aacgcacgat taagtatgaa tcataagctg 720
atgtcatttg ttttcgacat aaaatgttta tacaatggaa tcttcttgta aattatccaa 780
ataatataat ttatccgatt ctacgttaca tttaaattcg ttgttatcgt acaattcttc 840
aggacacgcc atgtattggt catttttagc gtgcaaccaa cgattgtatt tgacgccgtc 900
gttggattgc gtgttcaggt tggcgtacac gtgactgggc acggcttctt tttccatggg 960
acgtcgactc atcttgtcac acctacatct tactaatttc gtaagtagat ttttttttac 1020
acgtataatg tatgtattct ttccttaatt aacttatttt gaaacgaaat aaataggcta 1080
ttaatatttg gaactaggtt gcggtcaatg tcaatgtctg tctcaacttt aattcagaat 1140
gccttgtgtt ccgtagatgc tataaatcaa tcaagatgca tcttggattg ttgccaactc 1200
gcagctacaa aatttgtttc caagcctaag catagtgctg tacccgttcc cgtgtattca 1260
aatcccgtat aatagtataa tatactccgt aaatgtagtg tcactgcttg ctgaaatgat 1320
attgcaagtt ccgttgggaa tcttgccgtt atcaagcaat gcgatattag cggtatggcg 1380
ggagggggac gcgcagactc cctctgctgt attaccatat atggacacaa aacttcgtgt 1440
attgtaccct agcgcgcgat tggaggagag tctgcggcgg cggggcaggg gcgccccgat 1500
aaccggcctc atttatatag tccgccaagc gcactcacca acattccacg aagtgagctt 1560
gggtcgttgc gttgtacagc aataacgaag ctgtgcaata gcaagttaat ttatttattt 1620
ataatagaac tatttaatta aaagtaagtt attttcattg tgtcttcaaa tatattaagt 1680
gattgtgata acggttaacg gttgttagag gattggtgga tccatggaca cgtacgcggg 1740
tgcttacgac cgtcagtcgc gcgagcgcga gaattcgagc gcagcaagcc cagcgacaca 1800
gcgtagcgcc aacgaagaca aggcggccga ccttcagcgc gaagtcgagc gcgacggggg 1860
ccggttcagg ttcgtcgggc atttcagcga agcgccgggc acgtcggcgt tcgggacggc 1920
ggagcgcccg gagttcgaac gcatcctgaa cgaatgccgc gccgggcggc tcaacatgat 1980
cattgtctat gacgtgtcgc gcttctcgcg cctgaaggtc atggacgcga ttccgattgt 2040
ctcggaattg ctcgccctgg gcgtgacgat tgtttccact caggaaggcg tcttccggca 2100
gggaaacgtc atggacctga ttcacctgat tatgcggctc gacgcgtcgc acaaagaatc 2160
ttcgctgaag tcggcgaaga ttctcgacac gaagaacctt cagcgcgaat tgggcgggta 2220
cgtcggcggg aaggcgcctt acggcttcga gcttgtttcg gagacgaagg agatcacgcg 2280
caacggccga atggtcaatg tcgtcatcaa caagcttgcg cactcgacca ctccccttac 2340
cggacccttc gagttcgagc ccgacgtaat ccggtggtgg tggcgtgaga tcaagacgca 2400
caaacacctt cccttcaagc cgggcagtca agccgccatt cacccgggca gcatcacggg 2460
gctttgtaag cgcatggacg ctgacgccgt gccgacccgg ggcgagacga ttgggaagaa 2520
gaccgcttca agcgcctggg acccggcaac cgttatgcga atccttcggg acccgcgtat 2580
tgcgggcttc gccgctgagg tgatctacaa gaagaagccg gacggcacgc cgaccacgaa 2640
gattgagggt taccgcattc agcgcgaccc gatcacgctc cggccggtcg agcttgattg 2700
cggaccgatc atcgagcccg ctgagtggta tgagcttcag gcgtggttgg acggcagggg 2760
gcgcggcaag gggctttccc gggggcaagc cattctgtcc gccatggaca agctgtactg 2820
cgagtgtggc gccgtcatga cttcgaagcg cggggaagaa tcgatcaagg actcttaccg 2880
ctgccgtcgc cggaaggtgg tcgacccgtc cgcacctggg cagcacgaag gcacgtgcaa 2940
cgtcagcatg gcggcactcg acaagttcgt tgcggaacgc atcttcaaca agatcaggca 3000
cgccgaaggc gacgaagaga cgttggcgct tctgtgggaa gccgcccgac gcttcggcaa 3060
gctcactgag gcgcctgaga agagcggcga acgggcgaac cttgttgcgg agcgcgccga 3120
cgccctgaac gcccttgaag agctgtacga agaccgcgcg gcaggcgcgt acgacggacc 3180
cgttggcagg aagcacttcc ggaagcaaca ggcagcgctg acgctccggc agcaaggggc 3240
ggaagagcgg cttgccgaac ttgaagccgc cgaagccccg aagcttcccc ttgaccaatg 3300
gttccccgaa gacgccgacg ctgacccgac cggccctaag tcgtggtggg ggcgcgcgtc 3360
agtagacgac aagcgcgtgt tcgtcgggct cttcgtagac aagatcgttg tcacgaagtc 3420
gactacgggc agggggcagg gaacgcccat cgagaagcgc gcttcgatca cgtgggcgaa 3480
gccgccgacc gacgacgacg aagacgacgc ccaggacggc acggaagacg tagcggcgcc 3540
caagaagaag agaaaggttt aggcggccgc tacaactaaa cacgacttgg agtattcctt 3600
gtagtgttta agattttaaa tcttacttaa tgacttcgaa cgattttaac gataactttc 3660
tctttgttta actttaatca gcatacataa aaagccccgg ttttgtatcg ggaagaaaaa 3720
aaatgtaatt gtgttgccta gataataaac gtattatcaa agtgtgtggt tttcctttac 3780
caaagacccc tttaagatgg gcctaatggg cttaagtcga gtcctttccg atgtgttaaa 3840
tacacattta ttacactgat gcgtcgaatg tacactttta ataggatagc tccactaaaa 3900
attattttat ttatttaatt tgttgcacca aaactgatac attgacgaaa agctt 3955
<210> 4
<211> 64
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gcggatcccc ccaactgggg taacctttga gttctctcag ttgggggtca tcttgtcaca 60
ccta 64
<210> 5
<211> 59
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ccgtcgacgt gccagggcgt gcccttgggc tccccgggcg cgaccaatcc tctaacaac 59
<210> 6
<211> 1762
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gtcgacgtgc cagggcgtgc ccttgggctc cccgggcgcg accaatcctc taacaaccgt 60
taaccgttat cacaatcact taatatattt gaagacacaa tgaaaataac ttacttttaa 120
ttaaatagtt ctattataaa taaataaatt aacttgctat tgcacagctt cgttattgct 180
gtacaacgca acgacccaag ctcacttcgt ggaatgttgg tgagtgcgct tggcggacta 240
tataaatgag gccggttatc ggggcgcccc tgccccgccg ccgcagactc tcctccaatc 300
gcgcgctagg gtacaataca cgaagttttg tgtccatata tggtaataca gcagagggag 360
tctgcgcgtc cccctcccgc cataccgcta atatcgcatt gcttgataac ggcaagattc 420
ccaacggaac ttgcaatatc atttcagcaa gcagtgacac tacatttacg gagtatatta 480
tactattata cgggatttga atacacggga acgggtacag cactatgctt aggcttggaa 540
acaaattttg tagctgcgag ttggcaacaa tccaagatgc atcttgattg atttatagca 600
tctacggaac acaaggcatt ctgaattaaa gttgagacag acattgacat tgaccgcaac 660
ctagttccaa atattaatag cctatttatt tcgtttcaaa ataagttaat taaggaaaga 720
atacatacat tatacgtgta aaaaaaaatc tacttacgaa attagtaaga tgtaggtgtg 780
acaagatgac ccccaactga gagaactcaa aggttacccc agttgggggg atccatggtg 840
cgctcctcca agaacgtcat caaggagttc atgcgcttca aggtgcgcat ggagggcacc 900
gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggccac 960
aacaccgtga agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg 1020
tccccccagt tccagtacgg ctccaaggtg tacgtgaagc accccgccga catccccgac 1080
tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac 1140
ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggctgctt catctacaag 1200
gtgaagttca tcggcgtgaa cttcccctcc gacggccccg taatgcagaa gaagaccatg 1260
ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc 1320
cacaaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gtccatctac 1380
atggccaaga agcccgtgca gctgcccggc tactactacg tggactccaa gctggacatc 1440
acctcccaca acgaggacta caccatcgtg gagcagtacg agcgcaccga gggccgccac 1500
cacctgttcc tgtagcggcc gcgactctag atcataatca gccataccac atttgtagag 1560
gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat 1620
gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc 1680
atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 1740
ctcatcaatg tacttaaagc tt 1762
Claims (2)
1. a kind of expression vector of the expression specificity integrase phiC31 gene in silkworm regulates and controls target base in silkworm individual level
Because of the application of expression, it is characterised in that: the specific integration of the expression vector of the expression specificity integrase phiC31 gene
The fusion of the end of enzyme phiC31 gene 3 ' has SV40 nuclear localization signal, the expression specificity integrase phiC31 gene in silkworm
The nucleotide sequence of expression vector is as shown in SEQ ID NO.3.
2. a kind of method of the regulation expression of target gene in silkworm individual level, it is characterised in that: integrated using expression specificity
The expression vector of enzyme phiC31 gene and containing the site attP and the site attB arranged in opposite directions expression vector regulation, institute
The nucleotide sequence of the expression vector of expression specificity integrase phiC31 gene is stated as shown in SEQ ID NO.3；It is described containing
The expression vector in the site attP and the site attB that arrange in opposite directions is constructed as following methods: by nucleic acid shown in SEQ ID NO.6
After sequence AscI digestion, the segment containing the site attP and the site attB arranged in opposite directions is building up to pBac
In the site AscI of [3xp3EGFPaf] carrier.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103343135A (en) * | 2013-07-04 | 2013-10-09 | 中国农业科学院兰州兽医研究所 | Recombinant expression vector pEGFP (Plasmid Enhanced Green Florescence Protein)-DsRed-BP and application thereof to detection of activity of phiC31 integrase in mammalian cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103343135A (en) * | 2013-07-04 | 2013-10-09 | 中国农业科学院兰州兽医研究所 | Recombinant expression vector pEGFP (Plasmid Enhanced Green Florescence Protein)-DsRed-BP and application thereof to detection of activity of phiC31 integrase in mammalian cell |
Non-Patent Citations (3)
| Title |
|---|
| A novel hybrid seed system for plants;Mario Gils等;《Plant Biotechnology Journal》;20071216;第6卷(第3期);第226-235页，参见全文 * |
| Transformation vector pICH14313;《Genbank》登录号：AM887683;《Genbank》;20080318;参见序列及相关信息 * |
| 家蚕安全高效转基因技术体系的研究;龙定沛;《中国博士学位论文全文数据库 基础科学辑》;20151115;A006-23，参见第71-78页 * |
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