CN110468132A - A kind of sgRNA and transgene expression vector, expression strain, screening technique - Google Patents
A kind of sgRNA and transgene expression vector, expression strain, screening technique Download PDFInfo
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Abstract
The invention belongs to domestic silkworm gene engineering and biological engineering's technical field, a kind of sgRNA and transgene expression vector, expression strain, screening technique are disclosed.Using CRISPR/Cas9 system, the strain that expression let-7Complex/Cluster (let-7C) knocks out sgRNA used is initially set up, which is hybridized with specific Cas9 expression strain, realizes knockout of the let-7C in domestic natural silk gland；SgRNA expression strain and middle division of silkgland, posterior division of silkgland Cas9 expression strain are hybridized respectively, the strain and let-7C posterior division of silkgland that acquisition let-7C is specifically knocked out in middle division of silkgland specifically knock out strain.The present invention knocks out domestic natural silk gland let-7 by CRISPR/Cas9, can promote the development of sericterium, increases the expression of silk-fibroin and improves the silk yield of silkworm.
Description
Technical field
The invention belongs to induced mutations breeding technical field more particularly to a kind of sgRNA and transgene expression vector, expression
Strain, screening technique, the specially sgRNA of let-7microRNA complex/cluster and its transgene expression vector,
The knockout Screening of Strains Against method of let-7complex/cluster.
Background technique
Common traditional breeding way includes induced mutations and crossbreeding.Induced mutations breeding, which refers to, utilizes chemical mutagenesis
Or the means of physical mutagenesis handle biology, lure that biological heredity character morphs into, and filter out valuable mutation
Body.Although induced mutations improve inhereditary feature variation frequency, Mutation induction be it is nondirectional, favorable variation is few, breeding
Process needs to handle mass mutation kind or material, heavy workload.And crossbreeding refers between different cultivars or relationship pass in kind
It is to be hybridized to obtain the breeding method of hybrid vigour between closer different plant species.Crossbreeding can make excellent between different cultivars
Gesture character is constantly accumulated in Hybrid, but hybridization and screening are both needed to take considerable time, the correlated traits of acquisition is hybridizing
It is unstable in offspring.Silkworm is a kind of important economic insects, has the ability for quickly, largely synthesizing silk-fibroin.Sericterium is family
The vitals of silkworm synthesis and secretion silk-fibroin, fast growth and largely synthesize silk-fibroin during silkworm five-age larva.Silkworm
After the advent of genome frame diagram and fine map, people are just expected that by the crucial base of manipulation sericterium development and silk protein synthesis
Cause measures high cultivated silkworm breed variety to obtain spinning.With the development of molecular biology and technique for gene engineering, it is expected to compile using gene
The means of collecting manipulate these genes to improve silkworm economic characters.
CRISPR/Cas9 is a kind of defense mechanism that bacterium and archeobacteria obtain in long-term evolution journey, to resist virus
Or exogenous DNA invasion.Exogenous DNA passes through local sequence complementary pairing and CRISPR is integrated, and CRISPR is just transcribed and formed pre-
CrRNA, the compound that the latter cuts into crRNA, crRNA and trans-actingcrRNA (tracrRNA) formation again can draw
It leads Cas9 albumen pair and the exogenous DNA double-strand of crRNA complementary pairing is sheared.Based on this principle, the sgRNA that manually synthesizes
CrRNA/tracrRNA compound is substituted to guide Cas9 endonuclease to carry out fixed point cutting to exogenous DNA.In vivo
Nonhomologous end repair mechanism reparation under, the double-stranded DNA cut open reconnects, but will appear base deletion, insertion
With displacement etc..For encoding gene, these variation meeting maximum probabilities lead to the frameshift mutation of gene, to reach the mesh of gene knockout
's.Since sgRNA design is simple, synthesis is quick, answered extensively with advantages, CRISPR/Cas9 such as Cas9 joint efficiency height
Used in gene functional research and breed improvement.
CRISPR/Cas9 gene editing system is made of two parts: sgRNA and endonuclease Cas9.In sgRNA
Stent sequence (sgRNA scaffold) is a relatively conservative Duan Xulie, can be in conjunction with Cas9.The site of CRISPR/Cas9 is special
The opposite sex knocks out the sgRNA spacer sequence for depending on that 20nt is about in sgRNA, it is mutually recruited with target site on genome
Pair distinguished sequence.The sgRNA spacer for designing genome specific site, can be achieved with CRISPR/Cas9 to specific site
Specific knockdown.SgRNA spacer needs to meet the Format Series Lines of G (N19) NGG.By online software, so that it may find mesh
Sequence in meet the sgRNA spacer of condition.Designed sgRNA sequence can by transcription synthesize, then with it is commercialized
Cas9 albumen hybrid injection can realize that specific gene knocks out.
Solve the difficulty of above-mentioned technical problem:
Can find regulation sericterium development and silk protein synthesis important gene be by genetic engineering innovate cultivated silkworm breed variety,
It improves silk performance and improves the premise of silk yield.There are a protein coding genes up to ten thousand in domestic silkworm gene group, it is numerous that there are also types
More, substantial amounts non-coding RNAs.To be found from these huge genes and non-coding RNA group can promote sericterium development and
The key or important factor of silk protein synthesis need to carry out a large amount of experimental study.
MicroRNA is a kind of non-coding RNA for being about 22nt, participates in every vital movement, they by with target base
Promote target mrna degradation because of the pairing of mRNA3 ' UTR reverse complemental or inhibits target gene translation.If microRNA can be released to exist
Negative regulation effect in sericterium, will likely promote the development of sericterium and the expression of silk-fibroin.However, microRNA and albumen
Encoding gene is different, without triplet genetic code, cannot generate frameshift mutation, the loss of single base or mutation are likely to insufficient
To influence the generation of microRNA, it is knocked out and preferably deletes entire microRNA maturation body sequence in the genome.
MicroRNA is in the genome there are two types of existence forms: single microRNA and microRNA cluster (cluster).
The former refers to that the same site (locus) on genome within 10kb only generates single microRNA；The latter refers in genome
Same site within upper 10kb generates more than two microRNA, these microRNA often have co-expression mode and class
As biological function.MicroRNA cluster (cluster) is knocked out in the genome, needs to be located at microRNA on genome
The multipair sgRNA of the preceding body position cluster plays a role simultaneously, guides Cas9 albumen by the both ends cluster on genome
Sequence between microRNA, which is cut away, can be only achieved the purpose for knocking out microRNA cluster.
Solve the meaning of above-mentioned technical problem:
The meaning of the technology of the present invention is: realizing let-7microRNA in silkworm middle division of silkgland and posterior division of silkgland
Complex/cluster (let-7C) is integrally knocked out.This method also and is not introduced into new there is no modifying encoding gene
Foreign gene deletes let-7CmiRNAs in domestic silkworm silk gland cell just with CRISPR/Cas9 technology, relieves them
Negative regulation effect to sericterium development and silk protein synthesis, realizes the increase of domestic natural silk gland length and the raising of spinning amount.Tool
It sees to body, after the let-7C for knocking out middle division of silkgland, the middle section of middle division of silkgland and back segment expand, but surface is in rough tooth
Shape, middle division of silkgland DNA content increase 120%；After the let-7C for knocking out Bombyx mori posterior silkgland, DNA in posterior silkgland cells
Amount increases 120%, and the length of posterior division of silkgland increases 60%, and weight increases 150%, and weighing discovery silk increases after having spat silk
Add 10%.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of sgRNA and transgene expression vectors, expression product
System, screening technique.
The invention is realized in this way a kind of sgRNA, the sequence of the sgRNA are as follows: SEQ ID NO:5.
Another object of the present invention is to provide a kind of transgene expression vectors constructed by the sgRNA.
Another object of the present invention is to provide a kind of construction method of transgene expression vector, the transgenosis table
Construction method up to carrier includes:
(1) suitable sgRNA is found in the genome in let-7C and target site, meet G (N20) GG structure；
(2) sgRNA expression vector U6 promoter expresses two sgRNA simultaneously, and selection markers are selected in eyes and nerve
Specifically expressed promoter 3XP3 starts green fluorescence EGFP, and transgene carrier is to turn with lepidopterous insects piggyBac transposon
Gene basic framework.
Further, the construction method of the sgRNA transgene expression vector specifically includes:
1) sgRNA spacer needed for knocking out let-7C using on-line prediction website CCtop prediction, inputs let-7C institute
Genome sequence, the gRNA spacer in sequence is looked for the structure of G (N20) GG, avoids the higher sequence of off-target rate；
Connection after corresponding base, TGCA and AAAC is added for AarI digestion in 5 ' ends of sgRNA spacer sequence, TCCG
With AAAC for the connection after BbaI digestion；
2) allow primer annealing to form double-strand with following condition: 95 DEG C of denaturation 5min, 95 DEG C of gradient coolings are to 25 DEG C, under per second
0.1 DEG C of drop, totally 700 recycle；
3) following system is prepared, recycles pUC57 [U6-2gRNA] carrier framework after 37 DEG C of digestions overnight:
4) double-strand to be formed of sgRNA1-spacer annealing is connected on pUC57 [U6-2gRNA] carrier framework, is built into
PUC57 [U6-sgRNA1] plasmid；
5) it prepares following system, after 37 DEG C of digestion 4h, recycles pUC57 [U6-sgRNA1] carrier framework:
6) double-strand to be formed of sgRNA2-spacer annealing is connected on pUC57 [U6-sgRNA1] carrier framework, building
At pUC57 [U6, let-7C-2gRNA] plasmid；
7) it prepares following system, after 37 DEG C of digestion 4h, recycles [U6, let-7C-2gRNA] segment:
8) let-7C of recycling is knocked out into double sgRNA expression segment [U6, let-7C-2gRNA] and is connected to piggyBac
On [3xP3-EGFP] carrier, it is built into let-7C and knocks out double gRNA transgene expression vectors: pBac [3xp3-EGFP, let-7C-
2gRNA]。
Another object of the present invention is to provide a kind of sgRNA of transgene expression vector based on sgRNA building
Express Screening of Strains Against method.The sgRNA expression Screening of Strains Against method utilizes CRISPR/Cas9 system, initially sets up a table
The strain is hybridized with specific Cas9 expression strain, realizes that let-7C is in by the strain that sgRNA used is knocked out up to let-7C
Knockout in silk gland；Specifically sgRNA expression strain and middle division of silkgland, posterior division of silkgland Cas9 expression strain are carried out respectively
Hybridization, obtains strain [Δ let-7C-MSG] that let-7C is specifically knocked out in middle division of silkgland and let-7C posterior division of silkgland specifically strikes
Except strain [Δ let-7C-PSG].
Further, the sgRNA expression Screening of Strains Against method specifically includes:
The first step, double gRNA transgene carrier injections and positive individuals screening.SgRNA transgene expression plasmid is passed through aobvious
The method of microinjection is injected into the silkworm seed just given birth to, and the larva hatched that will succeed is raised to obtain G0 generation with new fresh mulberry leaf
Adult, G0 mate to obtain G1 for silkworm seed for adult.Green fluorescence screening is carried out when silkworm seed hastened the hatching of silkworms to 5-6 days；A, A# indicates G1 generation
The positive silkworm seed for the eyes green light being sieved to；B, B# indicates G1 generation positive adult；
Second step, sgRNA express strain and are hybridized with Cas9 expression strain, and screening let-7C knocks out strain；
(1) A. [let-7C-2gRNA] individual；B. [Cas9-MSG] individual；C. it expresses green light simultaneously and the individual of feux rouges is
Hybrid individual, as let-7C middle division of silkgland knock out individual [Δ let-7C-MSG]；
(2) A. [let-7C-2gRNA] individual；B. [Cas9-PSG] individual；C. it expresses green light simultaneously and the individual of feux rouges is
Hybrid individual, as let-7C posterior division of silkgland knock out individual [Δ let-7C-PSG]；
Third step, sequence variation on sequence verification genome, it was demonstrated that let-7C is knocked；It is designed in the upstream and downstream let-7C
Primer amplifies this section of sequence, by the method for sequencing, identifies the variation that domestic silkworm silk gland cell genomic DNA occurs；With it is normal
Sequence compares, and various forms of base deletions all have occurred in two different sgRNA target spots out, and 2-83 base of missing differs.
Further, the first step specifically includes:
(1) pBac [3xp3-EGFP, let-7C-2gRNA] ultrapure plasmid is extracted, the expression saved with laboratory
1:1 is mixed the ultrapure plasmid of assistant carrier pHA3PIG of piggyBac transposase in molar ratio；By the micro- note of mixed plasmid
It penetrates in the D9L silkworm seed that instrument is injected within laying eggs latter hour, then with nontoxic instant drying adhesive closed injection hole；
(2) silkworm seed after injection being put into 25 DEG C, the biochemical cultivation case of relative humidity 90% hastens the hatching of silkworms, after 9-10 days,
Silkworm larva is raised with new fresh mulberry leaf, this is G0 for larva；
(3) G0 for larva raising to adult and is selfed, obtains G1 generation oviposition, hastens the hatching of silkworms to the 5-6 days, it is electronic glimmering with macroscopic view
Light microscope carries out the screening of transgenic positive individual；Use EGFP fluorescin for reporter gene, so embryo's eyes in G1 generation
Green light is positive individuals, is named as [let-7C-2gRNA]；
(4) G1 generation raising carries out green fluorescence screening to adult compound eye, the positive individuals screened are used for adult again
Subsequent hybrid experiment.
Further, the second step specifically includes:
1) [let-7C-2gRNA] strain is expressed into strain [Cas9-MSG] and posterior division of silkgland with middle division of silkgland Cas9 respectively
Cas9 expression strain [Cas9-PSG] is hybridized；
2) the obtained F1 generation silkworm seed of hybridization is put into 25 DEG C, hastens the hatching of silkworms in the biochemical cultivation case of relative humidity 90%, hastens the hatching of silkworms
When to 5-6 days, reporter gene screening is carried out with the electronic fluorescence microscope of macroscopic view；F1 generation embryo eyes simultaneously green light and feux rouges
As let-7C middle division of silkgland knocks out individual [Δ let-7C-MSG] and let-7 posterior division of silkgland knocks out individual [Δ let-7C-
PSG]。
Further, the third step specifically includes:
(1) [the Δ let-7C-MSG] that screens and [Δ let-7C-PSG] larva are raised with new fresh mulberry leaf to five ages,
Dissection obtains sericterium material；
(2) tissue DNA extracts kit is utilized, silk gland cell total DNA is extracted；
(3) design primer expands target site sequence, and is connected on pMD-19-Tsimple carrier and is sequenced；
(4) sequence is compared with BioEidt software, identifies base deletion number.
Another object of the present invention is to provide a kind of sgRNA expression Screening of Strains Against methods to improve domestic natural silk gland hair
Educate and spinning amount in application, obtain let-7C after the strain [Δ let-7C-MSG] and let-7C that middle division of silkgland specifically knocks out
Portion's sericterium specifically knocks out strain [Δ let-7C-PSG].
In conclusion advantages of the present invention and good effect are as follows: people remain desirable to through genetic engineering to silkworm
Sericterium is transformed to improve silk yield, improvement cultivated silkworm breed variety, however but suffers from no ideal molecular target and science
Genetic engineering means.The present invention realizes let-7Cluster miRNAs (let-7, miR-100 and miR-2795) in silk for the first time
The whole of gland different parts (middle division of silkgland and posterior division of silkgland) knocks out.There is no carry out to any protein coding gene for this method
Modifications and changes are crossed, also and new foreign gene is not introduced into, but is deleted in domestic silkworm silk gland cell using CRISPR/Cas9 technology
In addition to whole miRNAs of let-7Cluster, relieves let-7C and the negative regulation of sericterium development and silk protein synthesis is acted on,
Realize the raising that silkworm produces silk amount.Since sericterium can complete tissue resolution after silkworm completes spinning immediately, until disappearing completely
Lose, thus in sericterium let-7C miRNAs knockout can't be hereditary to offspring, will not because of the gene exchange between species,
Other species are had an impact.So this technology is upper safe and reliable in production, more advantage.
Detailed description of the invention
Fig. 1 is sgRNA expression Screening of Strains Against method flow diagram provided in an embodiment of the present invention.
Fig. 2 is sgRNA transgene expression vector building schematic diagram provided in an embodiment of the present invention.
Fig. 3 is sgRNA expression Screening of Strains Against method flow diagram provided in an embodiment of the present invention.
Fig. 4 is that let-7C provided in an embodiment of the present invention knocks out Screening of Strains Against method schematic diagram.
Fig. 5 is knockout form identification schematic diagram provided in an embodiment of the present invention.
Fig. 6 is sericterium Phenotypic Observation schematic diagram after let-7C provided in an embodiment of the present invention is knocked out.
Fig. 7 is provided in an embodiment of the present invention to sericterium length, weight and cocoon weight schematic diagram for statistical analysis.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with embodiment, to this hair
It is bright to be further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and do not have to
It is of the invention in limiting.
The present invention utilizes CRISPR/Cas9 system, initially sets up the strain that an expression let-7C knocks out sgRNA used,
The strain is hybridized with specific Cas9 expression strain, realizes knockout of the let-7C in domestic natural silk gland；Specifically by sgRNA
Expression strain is hybridized respectively with middle division of silkgland, posterior division of silkgland Cas9 expression strain, obtains let-7C in middle division of silkgland
The strain [Δ let-7C-MSG] and let-7C posterior division of silkgland specifically knocked out specifically knocks out strain [Δ let-7C-PSG]；These product
The silkworm of system, sericterium development and spinning amount are all changed.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
As shown in Figure 1, sgRNA provided in an embodiment of the present invention expression Screening of Strains Against method the following steps are included:
S101:sgRNA design and transgene expression vector building；
S102: double gRNA transgene carrier injections and positive individuals screening；
S103:sgRNA expresses strain and is hybridized with Cas9 expression strain, and screening let-7C knocks out strain；
S104: sequence variation on sequence verification genome, it was demonstrated that let-7C is knocked；
S105: at five age of larva, dissecting sericterium, observes phenotype and takes pictures；
S106: length and weight detecting are carried out to the 5-6 days five ages sericterium；After silkworm is placed on small straw bundles to spin cocoons, cocoon is counted again.
Application principle of the invention is further described with reference to the accompanying drawing.
SgRNA provided in an embodiment of the present invention expression Screening of Strains Against method the following steps are included:
Step 1: sgRNA design and transgene expression vector building (as shown in Figure 2):
(1) suitable sgRNA is found in the genome in let-7C and target site, meet G (N20) GG structure；
(2) sgRNA expression vector U6 promoter expresses two sgRNA simultaneously, and selection markers are selected in eyes and nerve
Specifically expressed promoter 3XP3 starts green fluorescence EGFP, and transgene carrier is to turn with lepidopterous insects piggyBac transposon
Gene basic framework；
It specifically includes:
1) it is struck using on-line prediction website CCtop (https: //crispr.cos.uni-heidelberg.de/) prediction
Except sgRNA spacer needed for let-7C.The genome sequence (about 3000bp) where let-7C is inputted, with G (N20) GG's
Structure looks for the gRNA spacer in sequence, avoids the higher sequence of off-target rate；Add in 5 ' ends of sgRNA spacer sequence
Enter corresponding base (TGCA and AAAC are used for the connection after AarI digestion, and TCCG and AAAC are for the connection after BbaI digestion),
Send Hua Da gene chemical synthesis primer:
2) allow primer annealing to form double-strand with following condition: 95 DEG C of denaturation 5min, 95 DEG C of gradient coolings are to 25 DEG C, under per second
0.1 DEG C of drop, totally 700 recycle；
3) following system is prepared, pUC57 [U6-2gRNA] is recycled after 37 DEG C of digestions overnight, and (this carrier is that double gRNA expression carry
Body) carrier framework:
4) double-strand to be formed of sgRNA1-spacer annealing is connected on pUC57 [U6-2gRNA] carrier framework, is built into
PUC57 [U6-sgRNA1] plasmid；
5) it prepares following system, after 37 DEG C of digestion 4h, recycles pUC57 [U6-sgRNA1] carrier framework:
6) double-strand to be formed of sgRNA2-spacer annealing is connected on pUC57 [U6-sgRNA1] carrier framework, building
At pUC57 [U6, let-7C-2gRNA] plasmid；
7) it prepares following system, after 37 DEG C of digestion 4h, recycles [U6, let-7C-2gRNA] segment:
8) let-7C of recycling is knocked out into double sgRNA expression segment [U6, let-7C-2gRNA] and is connected to piggyBac
On [3xP3-EGFP] carrier, it is built into let-7C and knocks out double gRNA transgene expression vectors: pBac [3xp3-EGFP, let-7C-
2gRNA]。
Step 2: double gRNA transgene carrier injections and positive individuals screening (as shown in Figure 3):
SgRNA transgene expression plasmid is injected into the silkworm seed just given birth to by the method for microinjection, will be succeeded
The larva of hatching is raised to obtain G0 for adult with new fresh mulberry leaf, and G0 mates to obtain G1 for silkworm seed for adult.Silkworm seed hasten the hatching of silkworms to
Green fluorescence screening is carried out at 5-6 days.A, A# indicates the positive silkworm seed for the eyes green light that G1 generation is sieved to.B, B# indicates G1 generation sun
Property adult；It specifically includes:
(1) pBac [3xp3-EGFP, let-7C-2gRNA] ultrapure plasmid is extracted, the expression saved with laboratory
1:1 is mixed the ultrapure plasmid of assistant carrier pHA3PIG of piggyBac transposase in molar ratio；By the micro- note of mixed plasmid
It penetrates in the D9L silkworm seed that instrument is injected within laying eggs latter hour, then with nontoxic instant drying adhesive closed injection hole；
(2) silkworm seed after injection being put into 25 DEG C, the biochemical cultivation case of relative humidity 90% hastens the hatching of silkworms, after 9-10 days,
Silkworm larva is raised with new fresh mulberry leaf, this is G0 for larva；
(3) G0 for larva raising to adult and is selfed, obtains G1 generation oviposition, hastens the hatching of silkworms to the 5-6 days, it is electronic glimmering with macroscopic view
Light microscope (Olympus MVX10) carries out the screening of transgenic positive individual.Because using EGFP fluorescin for reporter gene,
So embryo's eyes green light is positive individuals in G1 generation, it is named as [let-7C-2gRNA]；
(4) G1 generation raising carries out green fluorescence screening to adult compound eye, the positive individuals screened are used for adult again
Subsequent hybrid experiment.
Hybridized step 3: sgRNA expresses strain with Cas9 expression strain, screening let-7C knocks out strain (such as Fig. 4 institute
Show):
(1) [let-7C-2gRNA] individual；B. [Cas9-MSG] individual；C. it is miscellaneous for expressing the individual of green light and feux rouges simultaneously
Individual is handed over, as let-7C middle division of silkgland knocks out individual [Δ let-7C-MSG]；
(2) [let-7C-2gRNA] individual；B. [Cas9-PSG] individual；C. it is miscellaneous for expressing the individual of green light and feux rouges simultaneously
Individual is handed over, as let-7C posterior division of silkgland knocks out individual [Δ let-7C-PSG]；
It specifically includes:
1) [let-7C-2gRNA] strain is expressed into strain [Cas9-MSG] and posterior division of silkgland with middle division of silkgland Cas9 respectively
Cas9 expression strain [Cas9-PSG] is hybridized；
2) the obtained F1 generation silkworm seed of hybridization is put into 25 DEG C, hastens the hatching of silkworms in the biochemical cultivation case of relative humidity 90%, hastens the hatching of silkworms
When to 5-6 days, reporter gene screening is carried out with the electronic fluorescence microscope (Olympus MVX10) of macroscopic view.F1 generation embryo's eyes are same
When green light and feux rouges be let-7C middle division of silkgland knock out individual [Δ let-7C-MSG] and let-7 posterior division of silkgland knock out it is a
Body [Δ let-7C-PSG].
Step 4: sequence variation on sequence verification genome, it was demonstrated that let-7C is knocked (as shown in Figure 5).
In let-7C upstream and downstream design primer, this section of sequence is amplified, by the method for sequencing, identifies domestic silkworm silk gland cell
The variation that genomic DNA occurs.Compared with normal sequence, various forms of alkali are all had occurred in two different sgRNA target spots out
Base missing, 2-83 base of missing differ；
It specifically includes:
(1) [the Δ let-7C-MSG] that screens and [Δ let-7C-PSG] larva are raised with new fresh mulberry leaf to five ages,
Dissection obtains sericterium material；
(2) tissue DNA extracts kit (Omega, D3396-02) is utilized, extracts silk gland cell total DNA；
(3) design primer expands target site sequence, and is connected on pMD-19-Tsimple carrier and is sequenced.
SEQIDNO:3: > let-7C-KO-F:GTCAACGCGCCTAAAGAA；SEQ ID NO:4 > let-7C-KO-R:
TTTAGTGGTCTTGTGAGGAATG；
(4) sequence is compared with BioEidt software, identifies base deletion number.
Step 5: dissecting sericterium at five age of larva, observing phenotype and taking pictures (as shown in Figure 6):
(1) after [let-7C-2gRNA] individual hybridizes with [Cas9-MSG] individual, there are four kinds of different light emitting species, it is right
Their middle division of silkgland it was found that: obviously expand, show in saw with rear portion in the middle part of [Δ let-7C-MSG] individual middle division of silkgland
Detent projection；
(2) after [let-7C-2gRNA] individual hybridizes with [Cas9-PSG] individual, there are four kinds of different light emitting species, it is right
Their posterior division of silkgland it was found that: the obvious elongated, thickening of [Δ let-7C-PSG] individual posterior division of silkgland shows to be serrated convex
It rises, amount of crimp increases；
It specifically includes:
1) [the Δ let-7C-MSG] that screens and [Δ let-7C-PSG] larva are raised with new fresh mulberry leaf to five ages
5-6 days to the 6th day；
2) dissection sericterium observes phenotype and is taken pictures with camera.
Step 6: carrying out length and weight detecting to the 5-6 days five ages sericterium；After silkworm is placed on small straw bundles to spin cocoons, unite again to cocoon
It counts (as shown in Figure 7):
(1) [Δ let-7C-PSG] individual posterior division of silkgland length increases 60%；B. the individual rear portion [Δ let-7C-PSG]
Sericterium weight increases 100%；
(2) [Δ let-7-PSG] individual silk cocoon weight increases 12%；D. [Δ let-7C-MSG] individual silk cocoon weight subtracts
Lack 15%；
It specifically includes:
1) [the Δ let-7C-MSG] that screens and [Δ let-7C-PSG] larva are raised with new fresh mulberry leaf to five ages
5-6 days to the 6th day；
2) dissection posterior division of silkgland measures its length, weight；
3) [Δ let-7C-MSG] and [Δ let-7C-PSG] larva raising to the spinning for continuing to screen is cocoond；Larva
It pupates, silk cocoon weight is weighed and compared after terminating spinning.
Prove that (specific embodiment/experiment/emulation/Pharmacological Analysis/is able to demonstrate that the front of the invention is real for part
Test data, evidence material, probation report, business data, research and development evidence, business associate evidence etc.)
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Southwest University
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<213>artificial sequence (Artificial Sequence)
<400> 2
tccggtcagc tcggaaagtt agcaaacgct aactttccga gctgac 46
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<213>artificial sequence (Artificial Sequence)
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gtcaacgcgc ctaaagaa 18
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<213>artificial sequence (Artificial Sequence)
<400> 4
tttagtggtc ttgtgaggaa tg 22
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<213>artificial sequence (Artificial Sequence)
<400> 5
aagtgatccg tggcttataa gcatcggtgg ttcagtggta gaatgctcgc ctgccacgcg 60
ggcggcccgg gttcgattcc cggccgatgc agcgcacaag ttcggatcta cgttttagag 120
ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 180
tcggtgcaac aaaggaaacc tgatcatgta gatcgaatgg actctaaatc cgttcagccg 240
ggttagattc ccggggtttc cggtcagctc ggaaagttag cgttttagag ctagaaatag 300
caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 360
ttttctagaa caattttata acatac 386
Claims (10)
1. a kind of sgRNA, which is characterized in that the sequence of the sgRNA are as follows: SEQ ID NO:5.
2. a kind of transgene expression vector of the building of the sgRNA as described in claim 1.
3. a kind of construction method of transgene expression vector as claimed in claim 2, which is characterized in that the transgene expression carries
The construction method of body includes:
(1) suitable sgRNA is found in the genome in let-7C and target site, meet G (N19) NGG structure；
(2) sgRNA expression vector U6 promoter expresses two sgRNA simultaneously, and selection markers are selected in eyes and neural specific
The promoter 3XP3 of expression starts green fluorescence EGFP, and transgene carrier is using lepidopterous insects piggyBac transposon as transgenosis
Basic framework.
4. the construction method of transgene expression vector as claimed in claim 3, which is characterized in that the transgene expression vector
Construction method specifically include:
1) sgRNA spacer needed for knocking out let-7C using on-line prediction website CCtop prediction, inputs where let-7C
Genome sequence looks for the gRNA spacer in sequence with the structure of G (N20) GG, avoids the higher sequence of off-target rate；In
5 ' ends of sgRNA spacer sequence are added corresponding base: TGCA and AAAC is used for the connection after Aar I digestion, TCCG and
AAAC is used for the connection after BbaI digestion；
2) primer annealing is allowed to form double-strand: 95 DEG C of denaturation 5min with following condition, 95 DEG C of gradient coolings are to 25 DEG C, decline per second
0.1 DEG C, totally 700 recycle；
3) following system is prepared, recycles pUC57 [U6-2gRNA] carrier framework after 37 DEG C of digestions overnight；
4) double-strand to be formed of sgRNA1-spacer annealing is connected on pUC57 [U6-2gRNA] carrier framework, is built into
PUC57 [U6-sgRNA1] plasmid；
5) it prepares following system, after 37 DEG C of digestion 4h, recycles pUC57 [U6-sgRNA1] carrier framework；
6) double-strand to be formed of sgRNA2-spacer annealing is connected on pUC57 [U6-sgRNA1] carrier framework, is built into
PUC57 [U6, let-7C-2gRNA] plasmid；
7) it prepares following system, after 37 DEG C of digestion 4h, recycles [U6, let-7C-2gRNA] segment；
8) let-7C of recycling is knocked out into double sgRNA expression segment [U6, let-7C-2gRNA] and is connected to piggyBac [3xP3-
EGFP] on carrier, it is built into let-7C and knocks out double gRNA transgene expression vectors: pBac [3xp3-EGFP, let-7C-
2gRNA]。
5. a kind of let-7C sericterium based on sgRNA transgene expression vector described in claim 2 knocks out Screening of Strains Against method,
It is characterized in that, using CRISPR/Cas9 system, the strain that an expression let-7C knocks out sgRNA used is initially set up, by the product
System is hybridized with specific Cas9 expression strain, realizes knockout of the let-7C in domestic natural silk gland；SgRNA is specifically expressed into product
System is hybridized respectively with middle division of silkgland, posterior division of silkgland Cas9 expression strain, is obtained let-7C and is specifically struck in middle division of silkgland
The strain [Δ let-7C-MSG] and let-7C posterior division of silkgland removed specifically knock out strain [Δ let-7C-PSG].
6. let-7C as claimed in claim 5 knocks out Screening of Strains Against method, which is characterized in that the let-7C knocks out strain sieve
Choosing method specifically includes:
The first step, double gRNA transgene carrier injections and positive individuals screening, pass through micro- note for sgRNA transgene expression plasmid
Shooting method is injected into the silkworm seed just given birth to, and the new fresh mulberry leaf of larva that success is hatched is raised, and obtains G0 for adult, G0 at
Worm mates to obtain G1 for silkworm seed；Green fluorescence screening is carried out when silkworm seed hastened the hatching of silkworms to 5-6 days；A, the eyes that A# indicates that G1 generation is sieved to are sent out
The positive silkworm seed of green light；B, B# indicates G1 generation positive adult；
Second step, sgRNA express strain and are hybridized with Cas9 expression strain, and screening let-7C knocks out strain；
(1) A. [let-7C-2gRNA] individual；B. [Cas9-MSG] individual；C. the individual of green light and feux rouges is expressed simultaneously as hybridization
Individual, as let-7C middle division of silkgland knock out individual [Δ let-7C-MSG]；
(2) A. [let-7C-2gRNA] individual；B. [Cas9-PSG] individual；C. the individual of green light and feux rouges is expressed simultaneously as hybridization
Individual, as let-7C posterior division of silkgland knock out individual [Δ let-7C-PSG]；
Third step, sequence variation on sequence verification genome, it was demonstrated that let-7C is knocked；In let-7C upstream and downstream design primer,
This section of sequence is amplified, by the method for sequencing, identifies the variation that domestic silkworm silk gland cell genomic DNA occurs；With normal sequence
Compare, various forms of base deletions all have occurred at two different sgRNA target spots, 2-83 base of missing differs.
7. let-7C posterior division of silkgland as claimed in claim 6 knocks out Screening of Strains Against method, which is characterized in that the first step tool
Body includes:
(1) pBac [3xp3-EGFP, let-7C-2gRNA] ultrapure plasmid is extracted, is turned with the expression piggyBac that laboratory saves
1:1 is mixed the ultrapure plasmid of assistant carrier pHA3PIG of seat enzyme in molar ratio；Mixed plasmid is injected into microinjection instrument
In the D9L silkworm seed laid eggs within latter hour, then with nontoxic instant drying adhesive closed injection hole；
(2) silkworm seed after injection is put into 25 DEG C, the biochemical cultivation case of relative humidity 90% hastens the hatching of silkworms, after 9-10 days, with new
Fresh mulberry leaf raises silkworm larva, this is G0 for larva；
(3) G0 for larva raising to adult and is selfed, obtains G1 generation oviposition, hastens the hatching of silkworms to the 5-6 days, it is aobvious with the electronic fluorescence of macroscopic view
Micro mirror carries out the screening of transgenic positive individual；Use EGFP fluorescin for reporter gene, so embryo's eyes greening in G1 generation
Light is positive individuals, is named as [let-7C-2gRNA]；
(4) raising of G1 generation carries out green fluorescence screening to adult compound eye, the positive individuals screened are for subsequent to adult again
Hybrid experiment.
8. let-7C middle division of silkgland as claimed in claim 6 knocks out Screening of Strains Against method, which is characterized in that the second step tool
Body includes:
1) [let-7C-2gRNA] strain is expressed into strain [Cas9-MSG] and posterior division of silkgland Cas9 with middle division of silkgland Cas9 respectively
Expression strain [Cas9-PSG] is hybridized；
2) the obtained F1 generation silkworm seed of hybridization is put into 25 DEG C, hastens the hatching of silkworms in the biochemical cultivation case of relative humidity 90%, hastens the hatching of silkworms to 5-6
It when, carry out reporter gene screening with the electronic fluorescence microscope of macroscopic view；Green light and feux rouges are F1 generation embryo eyes simultaneously
Let-7C middle division of silkgland knocks out individual [Δ let-7C-MSG] and let-7 posterior division of silkgland knocks out individual [Δ let-7C-PSG].
9. let-7C middle division of silkgland as claimed in claim 6 knocks out Screening of Strains Against method, which is characterized in that the third step tool
Body includes:
(1) [the Δ let-7C-MSG] that screens and [Δ let-7C-PSG] larva are raised with new fresh mulberry leaf to five ages, dissection
Obtain sericterium material；
(2) tissue DNA extracts kit is utilized, silk gland cell total DNA is extracted；
(3) design primer expands target site sequence, and is connected on pMD-19-T simple carrier and is sequenced；
(4) sequence is compared with BioEidt software, identifies base deletion number.
10. one kind let-7C sericterium as described in claim 5~9 any one knocks out Screening of Strains Against method and is improving domestic natural silk gland
Application in development and spinning amount, which is characterized in that obtain strain [the Δ let-7C- that let-7C is specifically knocked out in middle division of silkgland
MSG] and let-7C posterior division of silkgland specifically knock out strain [Δ let-7C-PSG].
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| CN114540421A (en) * | 2022-03-04 | 2022-05-27 | 西南大学 | Controllable editing method for silkworm MSG and PSG expression genes |
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