CN114525305B - Recombinant expression vector for automatically deleting exogenous DNA of first generation germ cells of transgenic silkworms and preparation method and application thereof - Google Patents

Abstract

The invention discloses a recombinant expression vector for automatically deleting exogenous DNA of a first generation germ cell of a transgenic silkworm, a preparation method and application thereof, wherein a transgenic activation vector for regulating GAL4 gene expression by a gonad specific expression gene promoter of the silkworm and a transgenic effect vector for regulating downstream FLP gene expression by a UAS element are firstly constructed, then the transgenic activation vector and the effect vector are respectively transformed into the silkworm, single copy transgenic silkworms are selected to establish an activation line and an effect line, the activation line and the effect line silkworms are hybridized to obtain a double-transgenic first generation silkworm, and finally the offspring silkworms without target exogenous DNA sequences in genome are obtained, thereby realizing the expression of exogenous useful proteins in specific tissues or organs of the transgenic silkworms and avoiding biological safety hazards caused by preservation of the transgenic offspring silkworm.

Description

Recombinant expression vector for automatically deleting exogenous DNA of first generation germ cells of transgenic silkworms and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, relates to a recombinant expression vector for automatically deleting exogenous DNA of a first generation germ cell of a transgenic silkworm, and a preparation method thereof, and also relates to application of the recombinant expression vector in automatically deleting exogenous DNA in the first generation germ cell of the transgenic silkworm.

Background

The safety research of transgenic organisms in the scientific community is mainly focused on the safety evaluation research of transgenic plants, particularly transgenic crops, transgenic aquatic animals such as fishes and shellfishes, and large animals such as transgenic poultry and livestock in the fields of food, medicine, agriculture and the like, and the report of the agricultural insect safety transgenic technology research with important scientific research and economic value is limited. In recent years, as the transgenic technology is gradually and widely applied to the basic research of silkworm (Bombyx mori) gene function identification and the application research field of variety improvement, more and more researchers realize that the biosafety problem of transgenic silkworms is a key problem which cannot be ignored in the popularization and application process of new varieties of transgenic silkworms.

The silkworm is used as a non-edible economic insect, and the transgenic safety problem of the silkworm is mainly embodied in the aspect of transgenic operation technical safety. The main method for creating transgenic silkworms at present is still based on a transposon (mainly piggyBac transposon) mediated technology for randomly integrating exogenous genes in genome. Unexpected negative effects such as unpredictable gene expression changes, endogenous gene structural disruption, growth and development limitation of transgenic individuals, and the like caused by random integration of transposons can seriously affect the safety of transgenic silkworms.

In addition, the transgenic silkworms established based on the transposon subsystem also have the ecological environment potential safety hazard problem of 3 aspects: (1) The non-target gene sequences such as screening marker genes, transposon frameworks and the like are reserved in the genome of the transgenic silkworms; (2) Unstable integration of transposons in the silkworm genome; (3) The transgenic silkworm seeds carrying exogenous genes have uncontrollability in the actual large-scale popularization and application process. In early researches, chinese patent No. zl201310181854.X established a strategy for achieving precise site-directed integration/replacement of exogenous genes at predetermined chromosomal sites (target sites) of transgenic silkworm genome based on a recombinant enzyme-mediated cassette exchange (phiC 31-RMCE) reaction of phiC31/att system in combination with piggyBac transposon system. The strategy can not only overcome the biological potential safety hazard that unexpected negative effects possibly caused by piggyBac transposon random integration bring adverse effects to the growth and development of transgenic silkworms, but also help to eliminate the effect of transposon random integration position and insertion mutation and the influence of non-target gene sequences on target gene expression and gene function research to a certain extent. In 2012, the university of southwest Long and the like successfully realize the fixed-point deletion of the individual genome level screening marker genes of silkworms by utilizing an FLP/FRT site-specific recombination system. Recently, long et al confirm that embryo microinjection of FLP recombinase mRNA and hybridization of introduction of FLP recombinase into a continuous expression mode can effectively improve the fixed-point deletion efficiency of FRT site anchored screening marker genes in the genome of transgenic silkworms, and further establish an inducible fixed-point deletion technology of the transgenic silkworms screening marker genes based on an FLP/FRT system. In addition, the Chinese invention patent with the patent number ZL2015155652. X discloses a method for realizing stable integration, expression and accurate replacement of exogenous target genes in the genome of silkworms by utilizing a composite piggyBac recombinant vector, thereby effectively overcoming the safety problem of transgenic silkworms caused by random integration of transposons and the ecological environment safety hidden trouble caused by the instability of transposon integration and the retention of non-target gene sequences.

In conclusion, the safety transgenic strategy established by combining the composite piggyBac transposition system and different site-specific recombination systems can solve the safety of transgenic silkworms and the ecological environmental potential safety hazard brought by the safety of the transgenic silkworms to a certain extent. However, the production and ecological environment safety problems which may occur in the actual large-scale popularization and application process of transgenic silkworm seeds carrying exogenous genes are not solved effectively at present. As is well known, silkworm industry only uses the first filial generation (F1) silkworm seeds of the domestic silkworm daily line stock and the Hua line stock to carry out large-area production, silkworm farmers cannot carry out strict management and control on silkworm seeds in the process of raising silkworms in rural areas, uncontrollable diffusion of transgenic silkworm seeds cannot be prevented, and therefore, transgenic silkworms can only be raised in a small scale in a transgenic silkworm house strictly managed in a laboratory or a silkworm farm at present. In addition, although the traditional technology of 'dry cocoon reeling' of silk fabric processing enterprises can bake and kill F1 generation silkworm chrysalis through the 'cocoon drying' process, in recent years, nearly 90% of silk reeling enterprises in China represented by Guangxi have developed the 'fresh cocoon reeling' technology without the 'cocoon drying' process, and the technology has the development trend of continuously keeping heat and even expanding. Therefore, if the transgenic F1 cocoons are treated by the technology of 'fresh cocoon reeling', the ecological environmental potential safety hazard caused by the generated transgenic F1 fresh cocoon pupas cannot be avoided.

Therefore, the problem of safety control of transgenic silkworm species is still one of the most fundamental and urgent important problems to be solved at present, both in the large-scale feeding of silkworms and in the processing process of silkworm cocoons. The problem directly affects the safety evaluation of the approved application and popularization of the transgenic silkworms and prevents the large-scale popularization and application and industrialized development of the transgenic silkworms. However, no effective strategy and method report is provided for the safety control problem of transgenic silkworm seeds at home and abroad at present.

Disclosure of Invention

In view of the above, one of the objects of the present invention is to provide a recombinant expression vector for automatically deleting exogenous DNA of a germ cell of a hybrid generation of transgenic silkworms; the second purpose of the invention is to provide a preparation method of the recombinant expression vector; the invention also aims to provide a method for automatically deleting exogenous DNA of the first generation germ cells of the hybrid generation of the transgenic silkworms by utilizing the recombinant expression vector and application thereof.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. the recombinant expression vector for automatically deleting exogenous DNA of the first generation germ cell of the transgenic silkworm is a binary transgenic expression vector, and comprises a transgenic activation vector for regulating and controlling GAL4 protein expression by a silkworm gonad specific expression gene promoter and a transgenic effector vector for regulating and controlling downstream FLP gene expression by a UAS element; wherein the transgene activating vectors comprise 2 different transgene effector vectors, and the number of the transgene effector vectors is 1.

Preferably, the transgenic activating vector comprises a vector skeleton, an activating element expression cassette and a target gene expression cassette.

Further preferably, the vector skeleton is a piggyBac recombinant vector pBac {3×P3-EGFPIF } containing a silkworm green eye fluorescence screening marker (3×P3-EGFP); the activating original expression frame comprises a silkworm gonad tissue specific gene promoter RSHP1p (with a nucleotide sequence of SEQ ID NO. 1) or Nanosp (with a nucleotide sequence of SEQ ID NO. 2), an optimized GAL4 protein gene (GAL 4NF kappa B) coding sequence (with a nucleotide sequence of SEQ ID NO. 3) and an SV40 terminator sequence (with a nucleotide sequence of SEQ ID NO. 4); the target gene expression frame is formed by fusing a red fluorescent protein (DsRed) gene which is started by a bombyx mori silk fibroin heavy chain promoter (Fibhp) with a light chain binding site sequence (LBS) (the nucleotide sequence is SEQ ID NO. 5).

Further preferably, the transgenic activating vector further comprises loxP site (SEQ ID NO. 6) and lox2272 site (SEQ ID NO. 7) for anchoring the activating original expression cassette, 2 orthographic attP sites (SEQ ID NO. 8) for anchoring the target gene expression cassette and 2 orthographic FRT sites (SEQ ID NO. 9) for anchoring the activating original expression cassette and the target gene expression cassette simultaneously.

Preferably, the transgenic effector vector comprises a vector backbone, an effector element expression cassette and a target gene expression cassette.

Further preferably, the vector backbone is piggyBac recombinant vector pBac {3×P3-DsRedaf } containing a silkworm red eye fluorescent screening marker (3×P3-DsRed); the effector element expression frame comprises an upstream activating sequence (Upstream activator sequence, UAS) element (the nucleotide sequence is SEQ ID NO. 10), an FLP gene coding sequence (the nucleotide sequence is SEQ ID NO. 11) and an SV40 terminator sequence (the nucleotide sequence is SEQ ID NO. 4); the target gene expression frame is formed by fusing a light chain binding site sequence (LBS) of an Enhanced Green Fluorescent Protein (EGFP) gene which is started by a bombyx mori silk fibroin heavy chain promoter (Fibhp) (the nucleotide sequence is SEQ ID NO. 12).

Further preferred, the transgenic effector vector further comprises 2 co-aligned attP sites (SEQ ID NO. 8) for anchoring the expression cassette of the gene of interest and 2 co-aligned FRT sites (SEQ ID NO. 9) for simultaneously anchoring the expression cassette of the effector and the expression cassette of the gene of interest.

2. The preparation method of the recombinant expression vector comprises the following steps:

(A) Preparation of transgenic activating vector: the FRT, loxP, RSHP p, GAL4nfκ B, SV40, lox2272, attP, fibhp, dsRed, LBS, attP and FRT fragments are sequentially ligated to the pSLfa1180fa vector, or the FRT, loxP, nanosp, GAL nfκ B, SV40, lox2272, attP, fibhp, dsRed, LBS, attP and FRT fragments are sequentially ligated to the pSLfa1180fa vector, by means of seamless ligation or cleavage ligation; recovering FRT-loxP-RSHP1P-GAL4NF kappa B-SV40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT or FRT-loxP-Nanosp-GAL4NF kappa B-SV40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT expression frame fragments by AscI/FseI double digestion, and connecting the fragments to an AscI/FseI double digestion pBac { 3X P3-EGFPaf } vector framework to obtain the FRT-loxP-RSHP-SV-RSHP-SV-SVK-SRP-FRT vector;

(B) Transgenic effector vector preparation: FRT, UAS, FLP, SV40, attP, fibhp, EGFP, LBS, attP and FRT were sequentially ligated into pSLfa1180fa vector; recovering FRT-UAS-FLP-SV40-attP-Fibhp-EGFP-LBS-attP-FRT expression frame fragments through AscI/FseI double-enzyme digestion, and connecting the FRT-UAS-FLP-SV40-attP-Fibhp-EGFP-LBS-attP-FRT expression frame fragments to an AscI/FseI double-enzyme digested pBac {3×P3-DsRedaf } vector skeleton to obtain a silkworm transgenic effect vector pBac {3×P3-DsRed; FRT-UAS-FLP-SV40-attP-Fibhp-EGFP-LBS-attP-FRT }.

Preferably, the specific method of step (a) is as follows:

(a-1) ligating FRT, loxP, RSHP p, GAL4nfκ B, SV40, lox2272, attP, fibhp, dsRed, LBS, attP and FRT fragments sequentially to pSLfa1180fa vector by means of seamless ligation or cleavage ligation; recovering FRT-loxP-RSHP1P-GAL4NF kappa B-SV40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT expression frame fragments through AscI/FseI double digestion, and connecting the fragments to an AscI/FseI double digestion pBac {3×P3-EGFPaf } vector skeleton to obtain a silkworm transgenic activation vector pBac {3×P3-EGFP; FRT-loxP-RSHP1p-GAL4 NFkB-SV 40-lox 2272-attP-Fibhp-DsRed-LBS-attP-FRT.

(B-2) ligating FRT, loxP, nanosp, GAL nfκ B, SV40, lox2272, attP, fibhp, dsRed, LBS, attP and FRT fragments sequentially to pSLfa1180fa vector by means of seamless ligation or enzymatic ligation; recovering FRT-loxP-Nanosp-GAL4NF kappa B-SV40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT expression frame fragments through AscI/FseI double digestion, and connecting the fragments to an AscI/FseI double digestion pBac {3 XP 3-EGFPaf } vector skeleton to obtain a silkworm transgenic activation vector pBac {3 XP 3-EGFP; FRT-loxP-Nanosp-GAL4 NFkB-SV 40-lox 2272-attP-Fibhp-DsRed-LBS-attP-FRT.

3. The recombinant expression vector is applied to the realization of the automatic deletion of exogenous DNA of the first generation germ cells of the hybrid generation of transgenic silkworms.

4. The method for preparing the first-filial generation double-transgenic silkworms by utilizing the recombinant expression vector comprises the following specific steps:

(1) Firstly, transforming the recombinant piggyBac transgenic vector (comprising 2 different transgenic activating vectors and 1 transgenic effector vector) and auxiliary plasmid pHA3PIG into a non-diapause or diapause-relieved G0 generation silkworm egg, feeding the hatched G0 generation silkworm to a chemical moth, backcrossing the G0 generation silkworm moth to produce a seed to obtain a G1 generation silkworm egg, screening single copy transgenic silkworm in the G1 generation silkworm, and then singly feeding the single copy transgenic silkworm by taking a moth area as a unit to establish 2 different transgenic activating lines and 1 transgenic effector line;

(2) The single copy transgene inserted active line silkworms obtained in the step (1) are hybridized with single copy transgene inserted effector line silkworms to obtain first filial generation silkworms, and double transgene silkworms simultaneously containing 2 eye fluorescent markers (3×P3-EGFP and 3×P3-DsRed) are screened to obtain first filial generation double transgene silkworms;

(3) And (3) feeding the first-filial generation double-transgenic silkworms obtained in the step (2) to the chemical moths, and carrying out selfing seed production on the first-filial generation double-transgenic male and female moths or backcross seed production on the first-filial generation double-transgenic silkworms to obtain second-filial generation silkworms. Because the gonad specific expression gene promoter regulates and controls the GAL4 protein to specifically express in germ cells of the hybrid generation double-transgenic silkworms, the FLP recombinase is activated to express and automatic deletion of DNA sequences of an activation original expression frame, an effect original expression frame and a target gene expression frame between the FRT site pairs arranged in the same direction is realized, the finally obtained hybrid second-generation silkworms genome does not contain the activation original expression frame, the effect original expression frame and the target gene expression frame, and the biological potential safety hazard that the exogenous target gene possibly escapes in the passage process is ensured to be eliminated; in non-germ cells, the gonad specific expression gene promoter has no promoter activity and can not regulate GAL4 gene expression, so that FLP recombinase expression is inhibited, thus the exogenous target gene is reserved in the non-germ cells, and finally, the acquisition of the transgenic product expressed by the hybrid generation double transgenic silkworms and the realization of the economic character of the transgenic product are ensured.

The invention has the beneficial effects that:

the invention firstly constructs a transgenic activating vector for regulating GAL4 protein expression by a gonad specific expression gene promoter of the silkworm and a transgenic effector vector for regulating downstream FLP gene expression by UAS elements, then respectively converts the transgenic activating vector and the effector vector into the silkworm, screens single copy transgenic silkworms to establish an activating line and an effector line, and hybridizes the activating line and the effector line silkworms to obtain double-transgenic hybrid first-generation silkworms. The specific analysis is as follows:

1) The invention modifies the traditional GAL4 sequence, and optimally designs the GAL4 sequence according to the sequence codon preference of the silkworm silk gland expression endogenous gene in a silkworm genome sequence database, so that the artificially modified GAL4 gene (GAL 4NF kappa B) is more beneficial to activating the expression of UAS downstream FLP genes in the gonad of a double-transgenic silkworm individual;

2) According to the invention, the silkworm is used as an animal mode for the first time, a relatively perfect silkworm safe transgenic operation technology system based on a site-specific recombination system and a GAL4/UAS system is established, a novel first-filial generation transgenic silkworm species is created by utilizing the technology, exogenous target genes are deleted from a first-filial generation germ cell genome, and the exogenous target genes are reserved in a first-filial generation non-germ cell genome, so that the purposes of simultaneously ensuring the realization of target economic characters and maximally eliminating the biological potential safety hazards caused by escape phenomenon of transgenes in the passage process are finally achieved;

3) The transgenic activation line and effect line silkworm materials established by the invention can be used for effectively eliminating potential safety hazards caused by non-target gene retention and researching the influence on exogenous gene expression, and can be used for realizing: establishing a novel transgenic activation line silkworm material for regulating and controlling GAL4 protein expression by other tissue-induced promoters in a Cre recombinase-mediated cassette exchange (Cre-RMCE) mode, or establishing a novel effect line and/or activation line silkworm material for accurately replacing other exogenous target genes with original target genes in a phiC31 integrase-mediated cassette exchange (phiC 31-RMCE) mode so as to meet the application research requirements of basic research of gene function identification and industrial development of transgenic silkworms;

4) The invention realizes the strategy of establishing the safe transgenic technology of silkworms, is applicable to other lepidoptera insects, and has important reference value for the safe transgenic technology research of other animal and plant species.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention is illustrated in the following drawings.

FIG. 1 is a schematic representation of the structure of 2 different transgene activating vectors and 1 transgene effector vector.

FIG. 2 shows the transgene structure (A) and positive transgene silkworm eye fluorescence and transgene cocoon fluorescence expression (B) in the genome of 2 transgene activating lines and 1 transgene effector line silkworms.

FIG. 3 shows the detection of the specificity of expression (A) of FLP and GAL4 genes (action 3 gene as reference gene) in tissues different from that of transgenic silkworms of the active line and the effector line by RT-PCR, and the detection of the relative expression levels of FLP gene (B) and GAL4 gene (C) in the spermary, ovary and other tissues of transgenic silkworms of the active line and the effector line by qRT-PCR.

FIG. 4 is a safe transgenic strategy for automatically deleting exogenous DNA of F1 germ cells of transgenic silkworms based on FLP/FRT site-specific recombination system and GAL4/UAS system.

FIG. 5 shows experimental procedures (A) of F1 double transgenic silkworms obtained by hybridizing an activation line with an effect line silkworms and screening the F2 silkworms for individuals positive for site-specific deletion reaction, and F2 double transgenic silkworms and F2 transgenic silkworm eye fluorescence and transgenic silkworm cocoon fluorescence phenotypes (B) with complete deletion, incomplete deletion and no deletion.

FIG. 6 is a schematic diagram of the transgene structure (A) and a PCR electrophoretogram (B) for molecular verification of FLP recombinase-mediated deletion of a silkworm genome site-specific gene by genomic PCR.

FIG. 7 shows the experimental principle of the establishment of other activation lines and/or effect lines of transgenic silkworms via Cre-RMCE (A) and phiC31-RMCE (B and C).

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

The experimental procedure, in which specific conditions are not noted in the examples, is generally carried out according to conventional conditions, for example, those described in the guidelines for molecular cloning experiments (third edition, J. Sam Brookfield et al), or according to the manufacturer's recommendations.

The non-diapause strain 305 (Bai Jian) of silkworm used in the examples of the present invention was stored by the genomic library of silkworm at southwest university. The vectors pUC57-T-simple (Long et al genetics hybridization of highly active exogenous functional proteins into silk-based materials using, "light-cloning" strategy. Matter 2021,4,2039-2058), pSLfa1180fa (Horn C, wimmer EA.A versatile vector set for animal transgenesis. Dev Genes Evol2000, 210:630-637), pBac {3 XP 3-DsRedaf } (Horn C, wimmer EA.A versatile vector set for animal transgenesis. Dev Genes Evol2000, 210:630-637), pBac {3 XP 3-EGFPaf } (Horn C, wimmer EA.A versatile vector set for animal transgenesis. Dev Genes Evol2000, 210:630-637) and pHA3PIG (Tamura et al Germing transformation of the silkworm Bombyx mori L.using apiggyBac transposon-driven vector Nathnote 2000, 18:81) were all stored in the laboratory.

Example 1

Construction of 2 different transgenic activation vectors and 1 transgenic Effector vector

RSHP1p promoter sequence (SEQ ID NO. 1), nanosp promoter sequence (SEQ ID NO. 2), GAL4NF kappa B gene coding sequence (SEQ ID NO. 3), SV40 terminator sequence (SEQ ID NO. 4), fibhp-DsRed-LBS sequence (SEQ ID NO. 5), loxP site (SEQ ID NO. 6), lox2272 site (SEQ ID NO. 7), attP site (SEQ ID NO. 8), FRT site (SEQ ID NO. 9), UAS element sequence (SEQ ID NO. 10), FLP gene coding sequence (SEQ ID NO. 11) and Fibhp-EGFP-LBS sequence (SEQ ID NO. 12) are all synthesized by Nanj Genscript, china.

The transgene activating vector pBac {3×P3-EGFP; FRT-loxP-RSHP1p-GAL4 NFkB-SV 40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT } (abbreviated as pBac [ R1p:: GAL4-Gr ]) was prepared as described in the technical scheme of the above-mentioned invention, genScript company sequentially connected FRT, loxP, RSHP p, GAL4 NFk B, SV40, lox2272 and attP to pUC57-T-simple vector by seamless connection to obtain recombinant vector pUC { FRT-loxP-RSHP1p-GAL4 nfkB-SV 40-lox2272-attP }; fibhp, dsRed, LBS, attP and FRT are sequentially connected to pUC57-T-simple vector in a seamless connection manner, and a recombinant vector pUC { Fibhp-DsRed-LBS-attP-FRT }, which contains a Fibhp-DsRed-LBS-attP-FRT expression frame, is obtained; the FRT-loxP-RSHP1p-GAL4NF kappa B-SV40-lox2272-attP } vector is recovered by AscI/HindIII double digestion of pUC { FRT-loxP-RSHP1p-GAL4NF kappa B-SV40-lox2272-attP expression cassette fragment, which is ligated to pSLfa1180fa vector to obtain pSL { FRT-loxP-RSHP1p-GAL4NF kappa B-SV40-lox2272-attP }; the pUC { Fibhp-DsRed-LBS-attP-FRT } vector was digested double by HindIII/SalI, and the Fibhp-DsRed-LBS-attP-FRT expression cassette fragment was recovered and ligated to the pSL { FRT-loxP-RSHP1p-GAL4 NFkB-SV 40-lox2272-attP } vector to obtain pSL { FRT-loxP-RSHP1p-GAL4 NFkB-SV 40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT }; finally, the AscI/FseI double-digested pSL { FRT-loxP-RSHP1P-GAL4NF kappa B-SV40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT } vector was recovered and the FRT-loxP-RSHP1P-GAL4NF kappa B-SV40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT expression cassette fragment was ligated to the AscI/FseI double-digested pBac {3 XP 3-EGFPaf } vector backbone to obtain the silkworm transgene activating vector pBac [ R1P:: GAL4-Gr ] (FIG. 1).

The transgene activating vector pBac {3×P3-EGFP; FRT-loxP-Nanosp-GAL4 NFkB-SV 40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT } (abbreviated as pBac [ Nsp:: GAL4-Gr ]) was prepared as described in the above-described patent technical scheme, and GenScript company sequentially connected FRT, loxP, nanosp, GAL NFk B, SV40, lox2272 and attP to pUC57-T-simple vector by seamless connection to obtain recombinant vector pUC { FRT-loxP-Nanosp-GAL4 NFkB-SV 40-lox2272-attP }, which contains FRT-lox-Nanosp-GAL 4 NFkB-SV 40-lox2272-attP expression cassette; the pUC { FRT-loxP-Nanosp-GAL4NFκB-SV40-lox2272-attP } vector was digested with AscI/HindIII, and the FRT-loxP-Nanosp-GAL4NFκB-SV40-lox2272-attP expression cassette fragment was recovered and ligated into pSLfa1180fa vector to obtain pSL { FRT-loxP-Nanosp-GAL4NFκB-SV40-lox2272-attP }; the pUC { Fibhp-DsRed-LBS-attP-FRT } vector was digested with HindIII/SalI to recover the Fibhp-DsRed-LBS-attP-FRT expression cassette fragment, which was ligated to the pSL { FRT-loxP-Nanosp-GAL4 NFkB-SV 40-lox2272-attP } vector to obtain pSL { FRT-loxP-Nanosp-GAL4 NFkB-SV 40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT }; finally, the AscI/FseI double-digested pSL { FRT-loxP-Nanosp-GAL4NF kappa B-SV40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT } vector was recovered and the FRT-loxP-Nanosp-GAL4NF kappa B-SV40-lox2272-attP-Fibhp-DsRed-LBS-attP-FRT expression cassette fragment was ligated to the AscI/FseI double-digested pBac {3 XP 3-EGFPaf } vector backbone to obtain the silkworm transgene activating vector pBac [ Nsp: GAL4-Gr ] (FIG. 1).

The transgene effector vector pBac {3×P3-DsRed; the preparation process of FRT-UAS-FLP-SV40-attP-Fibhp-EGFP-LBS-attP-FRT (abbreviated as pBac [ UAS:: FLP-Rg ]) is as described in the technical scheme of the invention, genScript company connects FRT, UAS, FLP, SV and attP to pUC57-T-simple vector in sequence by seamless connection mode to obtain recombinant vector pUC { FRT-UAS-FLP-SV40-attP }, which contains FRT-UAS-FLP-SV40-attP expression frame; fibhp, EGFP, LBS, attP and FRT are sequentially connected to a pUC57-T-simple vector in a seamless connection mode, so that a recombinant vector pUC { Fibhp-EGFP-LBS-attP-FRT }, which contains a Fibhp-DsRed-LBS-attP-FRT expression frame, is obtained; the pUC { FRT-UAS-FLP-SV40-attP } vector was digested with AscI/HindIII double, and the FRT-UAS-FLP-SV40-attP expression cassette fragment was recovered and ligated to pSLfa1180fa vector to obtain pSL { FRT-UAS-FLP-SV40-attP }; the pUC { Fibhp-EGFP-LBS-attP-FRT } vector was digested double by HindIII/SalI, and the Fibhp-EGFP-LBS-attP-FRT expression cassette fragment was recovered and ligated to the pSL { FRT-UAS-FLP-SV40-attP } vector to obtain pSL { FRT-UAS-FLP-SV40-attP-Fibhp-EGFP-LBS-attP-FRT }; finally, the AscI/FseI double-digested pSL { FRT-UAS-FLP-SV40-attP-Fibhp-EGFP-LBS-attP-FRT } vector was recovered and the FRT-UAS-FLP-SV40-attP-Fibhp-EGFP-LBS-attP-FRT expression cassette fragment was ligated to the AscI/FseI double-digested pBac {3 XP 3-DsRedaf } vector backbone to obtain the silkworm transgene effector vector pBac [ UAS: FLP-Rg ] (FIG. 1).

As shown in FIG. 1, each of the constructed 2 transgenic activating vectors contains a promoter 3×P3-initiated green fluorescent protein (EGFP) gene expression cassette (3×P3-EGFP) which is to be used as a screening marker of positive transgenic silkworms for the specific expression of the green fluorescent protein in the eyes and nerves of silkworms; the constructed 1 transgenic effector vector contains a promoter 3×P3-promoted red fluorescent protein (DsRed) gene expression frame (3×P3-DsRed), and the red fluorescent protein specifically expressed by the eyes and nerves of silkworms is used as a screening marker of positive transgenic silkworms.

Example 2

Preparation of transgenic silkworms

Recombinant vector pBac [ R1p:: GAL4-Gr was used as the starting material for the non-diapause silkworm strain 305]、pBac[Nsp::GAL4-Gr]Or pBac [ UAS:: FLP-Rg]Mixing with auxiliary plasmid pHA3PIG according to a molar ratio of 1:1, injecting into non-diapause G0 generation silkworm eggs, sealing with nontoxic glue, and hatching in an environment with 25 ℃ and relative humidity of 85%; breeding the hatched G0-generation silkworms with mulberry leaves until the silkworms are transformed, producing seeds by backcrossing the obtained G0-generation silkworms with wild 305, and collecting G1-generation silkworm eggs; by usingAnd (3) screening the positive transgenic silkworms with green or red fluorescence of eyes or nervous systems in the G1 generation silkworm eggs by using an electric macroscopic fluorescence microscope. Table 1 shows the screening statistics of transgenic vector injection and positive transgenic silkworms.

TABLE 1 transgenic vector injection and screening statistics of positive transgenic silkworms

Positive transgenic silkworm genomes obtained by screening were extracted, and finally 2 transgenic silkworm active lines (R1 p:: GAL4-Gr and Nsp:: GAL4-Gr, respectively, from pBac [ R1p:: GAL4-Gr ] and pBac [ Nsp:: GAL4-Gr ] transposition insertions) containing single copy transgene insertions in the genome and 1 transgenic silkworm effect line (UAS::: FLP-Rg, from pBac [ UAS:: FLP-Rg ] transposition insertions) were obtained by inverse PCR identification (Table 2). FIG. 2 shows a schematic representation of the transgene structure in the genome of transgenic and effector silkworms (FIG. 2, A), the transgene silkworms' fluorescent screening markers (3 XP 3-DsRed (denoted by "R") and 3 XP 3-EGFP (denoted by "G") and the fluorescence in transgenic cocoon sheets (FIG. 2, B), representing that they contain the target gene expression cassettes to be deleted (Fibhp-EGFP (denoted by "G") and Fibhp-DsRed (denoted by "R").

Table 2 inverse PCR identifies the transgene construct at the silkworm genome insertion site.

Example 3

Detection of tissue-specific expression of GAL4 gene regulated by RSHP1p and Nanosp promoters in active line and effector line silkworms

Primers were designed as shown in Table 3, and the specificity of GAL4 gene and FLP gene expressed in different tissues of the active line and the effector line transgenic silkworms was identified by RT-PCR and qRT-PCR. As shown in FIG. 3A, GAL4 gene mRNA was specifically expressed only in the testis of silkworms of 2 activation lines (R1 p:: GAL4-Gr and Nsp:: GAL 4-Gr), but not in other tissues; because the effect line UAS is that the FLP-Rg silkworm genome does not contain a GAL4 gene expression frame, the expression of GAL4 gene mRNA is not detected in each tissue of the UAS; since the genome of the silkworms of 2 activation lines (R1 p:: GAL4-Gr and Nsp:: GAL 4-Gr) did not contain the FLP gene sequence, and the FLP gene was not expressed in the tissues of the effector line UAS:: FLP-Rg silkworms, the expression of the FLP gene mRNA was not detected in the tissues of both the activation line and the effector line silkworms. The qRT-PCR results again confirm that the FLP gene is not expressed in each tissue of the active line and the effector line silkworms (FIG. 3, B), and in addition, the qRT-PCR results further show that the expression level of GAL4 gene mRNA in R1p: GAL4-Gr silkworm testis is significantly higher than that of Nsp: GAL4-Gr silkworm (FIG. 3, C). The experimental results show that both RSHP1p and Nanosp are testis-specific expression promoters, which can mediate tissue-specific expression of GAL4 gene in the spermary of transgenic silkworms. Wherein RSHP1p mediates GAL4 gene expression with significantly higher activity than Nanosp.

TABLE 3 RT-PCR and qRT-PCR primer sequences

Example 4

Automatic deletion experiment of exogenous DNA of hybrid generation (F1) germ cell target by hybridization of activated line and effector line silkworms

FIG. 4 shows the principle of the safe transgenic strategy proposed by the invention for realizing the specific deletion of exogenous target genes in the genome of germ cells of F1 transgenic silkworms, while retaining the exogenous target genes in the genome of non-germ cells: (1) establishing a transgenic activation system containing a silkworm Gonad specific expression gene promoter (Gonad-specific promoter, GSp) in a genome for regulating and controlling a GAL4 protein gene expression frame (GSp-GAL 4) and a transgenic effect system containing a downstream FLP protein gene expression frame (UAS-FLP) regulated and controlled by UAS elements; (2) hybridizing the activation line with the effect line silkworm, and obtaining F1 double-transgenic silkworm with the genome simultaneously containing GSp-GAL4 and UAS-FLP expression frames through eye fluorescence screening (3 xP 3-DsRed and 3 xP 3-EGFP); (3) in F1 double-transgenic silkworm germ cells, GSp regulates and controls GAL4 gene tissue specificity (sperm and ovum) expression, so as to activate FLP recombinase expression and mediate FRT site pair recombination reaction, and finally, exogenous target genes (Fibhp-EGFP and Fibhp-DsRed) are deleted at specific fixed points of germ cell tissue (only 1 FRT site is reserved); GSp has no promoter activity in non-germ cells and cannot regulate GAL4 gene expression, so that FLP recombinase expression is inhibited, and therefore exogenous target genes (Fibhp-EGFP and Fibhp-DsRed) are reserved in the non-germ cells; (4) backcrossing the F1 double-transgenic silkworms with non-transgenic silkworms to finally obtain F2 silkworms without exogenous target gene sequences in the genome.

To achieve the above strategy, F1 silkworms are obtained by crossing the activation line R1p:: GAL4-Gr (or NSp:: GAL 4-Gr) obtained in example 2 with the effect line UAS::: FLP-Rg silkworms according to the flow of FIG. 5A; f1 double transgenic silkworms F1-GrRg (R1 p:: GAL4& UAS:: FLP) (or NSp:: GAL4& UAS:: FLP) were obtained by eye fluorescence screening, and then orthogonal (F1-GrRg ×WT) and reverse (F1-GrRg ×WT) to non-transgenic silkworms (WT), positive individuals (FIG. 5B) who developed site-specific deletion reactions by eye fluorescence and cocoon fluorescence screening in F2-generation silkworms were counted for the efficiency of complete deletion (F2-G, F-R and F2-GR), incomplete deletion (F2-GrR and F2-GRg) and no deletion (F2-GrRg, F2-Gr and F2-Rg).

The number of F2 silkworms with different fluorescent phenotypes obtained by hybridizing F1-GrRg with WT is shown in Table 4. The results show 2F 1-GrRg (R1 p:: GAL 4)&UAS:: FLP and NSp:: GAL4&UAS: FLP) and WTF2 transgenic silkworms obtained by hybridization only have 3 fluorescent phenotypes which are not deleted (F2-GrRg, F2-Gr and F2-Rg), which indicates that no site-specific deletion event occurs in the eggs of 2F 1-GrRg; F1-GrRg->(R1p::GAL4&UAS: FLP) and WT were hybridized to obtain F2 transgenic silkworms with only 3 fluorescent phenotypes with complete deletions (F2-G, F2-R and F2-GR), indicating F1-GrRg->(R1p::GAL4&UAS: FLP) complete site-specific deletion events occurred in sperm; F1-GrRg->(Nsp::GAL4&UAS: FLP) hybridized with WT, while having all 8 fluorescent phenotypes of complete deletion (F2-G, F2-R and F2-GR), incomplete deletion (F2-GrR and F2-GRg) and no deletion (F2-GrRg, F2-Gr and F2-Rg), indicating F1-GrRg->(Nsp::GAL4&UAS: FLP) sperm has complete deletion (probability of 13.73-80.3%), incomplete deletion (12.42-17.72%) and undeleted event.

TABLE 4 statistics of the number of F2 silkworms having different fluorescent phenotypes

Example 5

Genome PCR validation of FLP recombinase-mediated target gene deletion event

Genome PCR primers were designed (Table 5), and FIG. 6A shows a schematic structure of molecular verification of the FLP recombinase-mediated bombyx mori genome site-specific gene deletion event by genome PCR. As shown in FIG. 6B, in the genome of each tissue cell of 2 different activation line silkworms and corresponding 2F 1-GrRg silkworms (testis, ovary and other tissues), 1 band of 1239-bp PCR product can be obtained by amplification of specific primer pair GAL4-g-F/GAL4-g-R, while in wild type, effector line and 2F 2-GR silkworms genome, the corresponding band can not be amplified; in the genome of each tissue cell of effector line silkworms and 2F 1-GrRg silkworms (testis, ovary and other tissues), 1 1369-bp PCR product band can be obtained by utilizing specific primer pair FLP-g-F/FLP-g-R, but in the genome of wild type, 2 activation lines and 2F 2-GR silkworms, the corresponding band can not be amplified; in the testis tissue of 2F 1-GrRg silkworms and the cell genome of each tissue of 2F 2-GR silkworms (testis, ovary and other tissues), 1 PCR product band of 272-bp after deletion can be obtained by using a specific primer pair Marker-F/Marker-R in an amplification way, and in the ovary and other tissues of wild type, 2 activation lines and 2F 1-GrRg silkworms, the corresponding band cannot be amplified. Subsequently, all F2-GR positive individuals with deletion were sequenced and analyzed by comparison with the pre-deletion sequences, confirming that FLP recombinase mediated a precise site-specific recombination reaction between 2 FRT sites in the F2-GR positive individual genome.

TABLE 5 genomic PCR primer sequences

Example 6

Strategy for preparing other novel transgenic silkworm materials by using active line and effector line silkworms

The experimental results of the embodiment prove that the specific expression of GAL4 protein in the gonad (testis) of the F1-GrRg silkworm is mediated by utilizing a gonad specific promoter, the FLP recombinase is activated to mediate the site-specific recombination reaction between FRT site pairs, the deletion of the anchored exogenous target gene sequence by the FRT site is further mediated, and finally positive individuals with the exogenous target gene sequence completely deleted in the genome are obtained by screening in F2. In addition, the activation line and effect line silkworm materials established by the invention not only can achieve the aim of ensuring the safety of transgenes and realizing the target economic character simultaneously by mediating the tissue-specific deletion of the target exogenous DNA sequence in the F1 double-transgenic silkworm germ cell genome through hybridization, but also can be used for realizing: a novel transgenic activation line silkworm material (figure 7, A) for regulating and controlling GAL4 protein expression by other tissue inducible promoters (inducible promoter, IP) is established by a Cre-RMCE mode, or a novel effect line and/or activation line silkworm material (figure 7, B-C) for accurately replacing other exogenous Target genes (Target Gene) with original Target genes is established by a phiC31-RMCE mode, so that the application research needs of basic research of Gene function identification and industrial development of transgenic silkworms are met.

Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> university of southwest

<120> recombinant expression vector for automatically deleting exogenous DNA of first generation germ cells of transgenic silkworms, and preparation method and application thereof

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1613

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

aacgcccaga ggctgtagtg cgagccttgc aagttctcga agtcagagaa aaagttgaaa 60

gggaaagaag aaggccagca ccggacctag actccggtga gcaagctctt gtgcgtgaaa 120

tgtgcaacgt agtttggggg aagttagaag acgcaccacc acaagcaccg cctcgttgaa 180

aatatcttta tttaacgttc tgctatcgag tgtcaataaa agtagaccaa gtttagttgt 240

atagaccagt gcgataggaa tccgacttga ttgaggtcac acaacatggc ctgtcgcatg 300

ctgtgaccta cagaaggaaa ttaagcgttg accttgttta tcaatccaaa cataacagta 360

aataaaataa aaacacatac atatatactg atgacagaca ttgtgatgtc cttaatacaa 420

atgaatgaaa ctgaatagta caaaattaga ttttaaaatt aggttaagat aattcttaaa 480

aaagctaagt agggattata aaatacatgg cctttttgta aactttacaa attatttcta 540

ttttcgtgtt gctaatgtat tgaaaagaaa tcggtctgct cttagaggct atacgttgtt 600

cttgctgaaa gtctgtgatc gagagtggtt ttattaatcg tctttaattg ttgcttcttg 660

ccagtcaaac acatacttgt caataataaa cttgatgttc agtttgctct gtatctagtc 720

agtgtccatt tacttactgc atatagggcc cgtcgtcatt ttaatactgt tttaatgctt 780

gctttttgta tttaaaaata tattaaggaa gtagttttaa agagttatta atttatttga 840

ttgaaaataa agttaattat taattgtttt tattcgtatg atttatttca accaatcttt 900

taggttatta ctaaaagaga atgaacaaat attaattaaa taataattaa atgaatttct 960

taaagaagat aataatatct acgtgagttt tactgttaga atcacaacgt cgtagcagca 1020

taaaacttat ttatgtttga tacattccca gatgtcagta agtgtattat ctgtgcatgt 1080

cagtaaaatg tctgtcaatg acccgccaaa ttaaaatttc atagtgattc attttcttcg 1140

gacaggtttt gattgagttc ccagcgtatt catttacaca taccgaataa acttcttggt 1200

gtcttttgta attatttaat tggtatagta ttcaaatgat gcatttgtta cagacttcct 1260

tttcttcttg ctcttatccc atattatatg gggtcgggac acaatgtcgt cttatctagg 1320

gatttggcat gagttttaga aacaaggcag acgttcctga ggtcaaccat acatgggtta 1380

tatctccagt agtacgagta tatccattgc aacgttccgt agcgagggat tatcatgttt 1440

cttcttttga tgcttgaagt aaaagacact tcatccgctt tttacttcta tgatttcaaa 1500

tttcgatttt caattaacac attttaaaaa taaaaatatt gcattttatt cgtttttctg 1560

tattttcaca ttacagtatt gacgaaaaaa gaatggtcga agaaaaggga gag 1613

<210> 2

<211> 1923

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

tggttgtaat gtaggtattg gtacaaatat tatatttgta cttaacgcgt tatacctatt 60

gattgggtca agcgcgatat gcgagacttc ttttttattt tatttagatg ggtggacgag 120

ctcacagccc acctggtgtt aagcagctat cggagcccat agacacctac aacgtaaatg 180

agccaccaac cttgacatat aagttctaaa gtctcaacaa ggtctcaagt atagttacaa 240

cggctgcccc acccttcaaa ccgaaaccca ttactgcttc acggcagaaa taggcagggt 300

ggtggtaggt acctacccgt gcagactcac aagaggtgaa caaaataaaa gacaaatata 360

tagcaaatac ataaatagtt tttttcgata ttttagggtt ataatattaa tttctggcgt 420

ttcaaatact ttcgttttcg tggtctattg tttactaaag cacgtgtggc gtcatctagc 480

gttgagtagt acaaacagga ttacacgtgc caaaattgtc ggtaggtacc tttgaagctg 540

aagccgaaaa gtcggcgcct ttttaagtta aagacaaaaa ggaaccgccc tttgaatgcg 600

tttgccgaaa gtttgtatat ttttaatgaa ctcaaaacac aagcattcga tccatcgtgg 660

tgtgctgacg tgcaacccaa atcgtgatta catttaataa aaggtaagaa acaaattaaa 720

taagcacacc tacttaatga gaaatttcgc taattttata attatgaatt aaattaatag 780

tttttataat aattagcttc aaatttaaaa gtaaataaca acacgttgat tttttcttaa 840

tgtgactgca cagtgggaag gcgttccaag gcgaagctaa ctatattatg tattatagct 900

gctattatgt gttaagtgct atatattgat agataatgtt cgcgggatgc ctacaatcac 960

taccaagtta atgccagtga tttctattta tcaaatcgta tagaacatat ttccagtttc 1020

ctatactatg gcttaatgaa gcttatacaa tcatagtctt atttcaacct ttttaaatta 1080

cgccttacca tccagtagtc tagtgccgga cttagctcat tcccataaat cctatctaaa 1140

tctatattta gttgttatta aattagcata gattaataca ctatttagta aaatcgtttt 1200

catgtgagat tagtccctca ttgctaattg cgtttcagga ttttttaaca atcataagtt 1260

aaactgctag ccaccgcgta tttattaacc atataaataa tgcaaaaaag tagcactggc 1320

tagtcagata atagcatgaa tatgttaggt attttctaaa tatactacag taggtactag 1380

atttttgtta aattccactg aataaaattg tttcaattat tgtgttaata aaaaagtctg 1440

ttcaaccact agttttaaca ctactcctta attttgatta aattttgtca tgaacgcgta 1500

aatgttcaag acataatatt tatttatggt attagatacc tatcgttaat ggcgtccata 1560

aaattaatat tgagctgtat tttaatgatg gtatcagtat aatgattatt taaatcggca 1620

ctgaattttt ttagtatttt tttggtagta agtacttttc ttacacataa attgttgatt 1680

tttttgagtc tatcgtctca tatgtataaa aagttgattc aaaataagaa tgcttccggg 1740

tgctatcgga tgttcggtca ttagaatata attcctaata atattctact tcattaaagt 1800

cattaaaatt taaagtgcct aaatttttgt ttaaagtaca tacgtagtaa aaacgtaaat 1860

gaatgtgtaa gtcatgtttt tattatgtac attacttatc ttttcagata aaataataaa 1920

aca 1923

<210> 3

<211> 1002

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atgaagctac tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag 60

tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac 120

tctcccaaaa ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg 180

ctagaaagac tggaacagct atttctactg atttttcctc gagaagacct tgacatgatt 240

ttgaaaatgg attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat 300

aatgtgaata aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta 360

acattgagac agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt 420

caaagacagt tgactgtatc ggagctccct tcgggtcaga taagcaatca agcgttagcc 480

ctcgccccct cctccgcacc tgtattagcc caaacaatgg tcccctcatc agcgatggtc 540

ccactggcac aacctccagc tccagcacca gtactcacac ctggtccacc acaaagttta 600

tcagcacctg tgccaaaatc gacacaggcg ggtgaaggaa ctctgagtga ggctctgttg 660

cacttgcagt tcgacgcaga tgaagacttg ggtgcgctct taggaaactc caccgatcca 720

ggcgtgttca cggatctcgc ttcggttgac aacagtgaat ttcaacagct tctgaatcag 780

ggtgtgtcta tgagccattc cacagccgag ccaatgctta tggaataccc ggaggcaata 840

acaagactgg ttactggatc acagcgccct ccagacccag ctccaacccc tttaggaacg 900

agcggtttac ctaatggact ttccggcgat gaggacttct catctatcgc tgatatggac 960

ttttcagcct tgctctctca aattagctcc tcgggccagt aa 1002

<210> 4

<211> 236

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc 60

tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt 120

ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 180

catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaa 236

<210> 5

<211> 2843

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gttgtacaaa actgccacac gcattttttt ctccactgta ggttgtagtt acgcgaaaac 60

aaaatcgttc tgtgaaaatt caaacaaaaa tattttttcg taaaaacact tatcaatgag 120

taaagtaaca attcatgaat aatttcatgt aaaaaaaaaa tactagaaaa ggaatttttc 180

attacgagat gcttaaaaat ctgtttcaag gtagagattt ttcgatattt cggaaaattt 240

tgtaaaactg taaatccgta aaattttgct aaacatatat tgtgttgttt tggtaagtat 300

tgacccaagc tatcacctcc tgcagtatgt cgtgctaatt actggacaca ttgtataaca 360

gttccactgt attgacaata ataaaacctc ttcattgact tgagaatgtc tggacagatt 420

tggctttgta tttttgattt acaaatgttt ttttggtgat ttacccatcc aaggcattct 480

ccaggatggt tgtggcatca cgccgattgg caaacaaaaa ctaaaatgaa actaaaaaga 540

aacagtttcc gctgtcccgt tcctctagtg ggagaaagca tgaagtaagt tctttaaata 600

ttacaaaaaa attgaacgat attataaaat tctttaaaat attaaaagta agaacaataa 660

gatcaattaa atcataatta atcacattgt tcatgatcac aatttaattt acttcatacg 720

ttgtattgtt atgttaaata aaaagattaa tttctatgta attgtatctg tacaatacaa 780

tgtgtagatg tttattctat cgaaagtaaa tacgtcaaaa ctcgaaaatt ttcagtataa 840

aaaggttcaa ctttttcaaa tcagcatcag ttcggttcca actctcaaga tgagagtcaa 900

aacctttgtg atcttgtgct gcgctctgca ggtgagttaa ttattttact attatttcag 960

aaggtggcca gacgatatca cgggccacct gataataagt ggtcgccaaa acgcacagat 1020

atcgtaaatt gtgccatttg atttgtcacg cccggggggg ctacggaata aactacattt 1080

atttatttaa aaaatgaacc ttagattatg taacttgtga tttatttgcg tcaaaagtag 1140

gcaagatgaa tctatgtaaa tacctgggca gacttgcaat atcctatttc accggtaaat 1200

cagcattgca atatgcaatg catattcaac aatatgtaaa acaattcgta aagcatcatt 1260

agaaaataga cgaaagaaat tgcataaaat tataaccgca ttattaattt attatgatat 1320

ctattaacaa ttgctattgc ctttttttcg caaattataa tcattttcat aacctcgagg 1380

tagcattctg ttacatttta atacattggt atgtgattat aacacgagct gcccactgag 1440

tttctcgcca gatcttctca gtgggtcgcg ttaccgatca cgtgatagat tctatgaagc 1500

actgctcttg ttagggctag tgttagcaaa ttctttcagg ttgagtctga gagctcacct 1560

acccatcgga gcgtagctgg aataggctac cagctaatag gtagggaaaa caaagctcga 1620

aacaagctca agtaataaca acataatgtg accataaaat ctcgtggtgt atgagataca 1680

attatgtact ttcccacaaa tgtttacata attagaatgt tgttcaactt gcctaacgcc 1740

ccagctagaa cattcaatta ttactattac cactactaag gcagtatgtc ctaactcgtt 1800

ccagatcagc gctaacttcg attgaatgtg cgaaatttat agctcaatat tttagcactt 1860

atcgtattga tttaagaaaa aattgttaac attttgtttc agtatgtcgc ttatacaaat 1920

gcaaatctag aatggtgcgc tcctccaaga acgtcatcaa ggagttcatg cgcttcaagg 1980

tgcgcatgga gggcaccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc 2040

gcccctacga gggccacaac accgtgaagc tgaaggtgac caagggcggc cccctgccct 2100

tcgcctggga catcctgtcc ccccagttcc agtacggctc caaggtgtac gtgaagcacc 2160

ccgccgacat ccccgactac aagaagctgt ccttccccga gggcttcaag tgggagcgcg 2220

tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg 2280

gctgcttcat ctacaaggtg aagttcatcg gcgtgaactt cccctccgac ggccccgtaa 2340

tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg 2400

tgctgaaggg cgagatccac aaggccctga agctgaagga cggcggccac tacctggtgg 2460

agttcaagtc catctacatg gccaagaagc ccgtgcagct gcccggctac tactacgtgg 2520

actccaagct ggacatcacc tcccacaacg aggactacac catcgtggag cagtacgagc 2580

gcaccgaggg ccgccaccac ctgttcctgg gatccaaaaa ctgtggaatt cctagaagac 2640

aactagttgt taaattcaga gcactgcctt gtgtgaattg ctaattttta atataaaata 2700

acccttgttt cttacttcgt cctggataca tctatgtttt ttttttcgtt aataaatgag 2760

agcatttaag ttattgtttt taattacttt tttttagaaa acagatttcg gattttttgt 2820

atgcatttta tttgaatgta cta 2843

<210> 6

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ataacttcgt atagcataca ttatacgaag ttat 34

<210> 7

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ataacttcgt ataaagtatc ctatacgaag ttat 34

<210> 8

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

ccccaactgg ggtaaccttt gagttctctc agttggggg 39

<210> 9

<211> 48

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttc 48

<210> 10

<211> 372

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ctgcaggtcg gagtactgtc ctccgagcgg agtactgtcc tccgagcgga gtactgtcct 60

ccgagcggag tactgtcctc cgagcggagt actgtcctcc gagcggagac tctagcgagc 120

gccggagtat aaatagaggc gcttcgtcta cggagcgaca attcaattca aacaagcaaa 180

gtgaacacgt cgctaagcga aagctaagca aataaacaag cgcagctgaa caagctaaac 240

aatctgcagt aaagtgcaag ttaaagtgaa tcaattaaaa gtaaccagca accaagtaaa 300

tcaactgcaa ctactgaaat ctgccaagaa gtaattattg aatacaagaa gagaactctg 360

aatagggaat tg 372

<210> 11

<211> 1272

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

atgccacaat ttggtatatt atgtaaaaca ccacctaagg tgcttgttcg tcagtttgtg 60

gaaaggtttg aaagaccttc aggtgagaaa atagcattat gtgctgctga actaacctat 120

ttatgttgga tgattacaca taacggaaca gcaatcaaga gagccacatt catgagctat 180

aatactatca taagcaattc gctgagtttc gatattgtca ataaatcact ccagtttaaa 240

tacaagacgc aaaaagcaac aattctggaa gcctcattaa agaaattgat tcctgcttgg 300

gaatttacaa ttattcctta ctatggacaa aaacatcaat ctgatatcac tgatattgta 360

agtagtttgc aattacagtt cgaatcatcg gaagaagcag ataagggaaa tagccacagt 420

aaaaaaatgc ttaaagcact tctaagtgag ggtgaaagca tctgggagat cactgagaaa 480

atactaaatt cgtttgagta tacttcgaga tttacaaaaa caaaaacttt ataccaattc 540

ctcttcctag ctactttcat caattgtgga agattcagcg atattaagaa cgttgatccg 600

aaatcattta aattagtcca aaataagtat ctgggagtaa taatccagtg tttagtgaca 660

gagacaaaga caagcgttag taggcacata tacttcttta gcgcaagggg taggatcgat 720

ccacttgtat atttggatga atttttgagg aattctgaac cagtcctaaa acgagtaaat 780

aggaccggca attcttcaag caataaacag gaataccaat tattaaaaga taacttagtc 840

agatcgtaca ataaagcttt gaagaaaaat gcgccttatt caatctttgc tataaaaaat 900

ggcccaaaat ctcacattgg aagacatttg atgacctcat ttctttcaat gaagggccta 960

acggagttga ctaatgttgt gggaaattgg agcgataagc gtgcttctgc cgtggccagg 1020

acaacgtata ctcatcagat aacagcaata cctgatcact acttcgcact agtttctcgg 1080

tactatgcat atgatccaat atcaaaggaa atgatagcat tgaaggatga gactaatcca 1140

attgaggagt ggcagcatat agaacagcta aagggtagtg ctgaaggaag catacgatac 1200

cccgcatgga atgggataat atcacaggag gtactagact acctttcatc ctacataaat 1260

agacgcatat ag 1272

<210> 12

<211> 2886

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gttgtacaaa actgccacac gcattttttt ctccactgta ggttgtagtt acgcgaaaac 60

aaaatcgttc tgtgaaaatt caaacaaaaa tattttttcg taaaaacact tatcaatgag 120

taaagtaaca attcatgaat aatttcatgt aaaaaaaaaa tactagaaaa ggaatttttc 180

attacgagat gcttaaaaat ctgtttcaag gtagagattt ttcgatattt cggaaaattt 240

tgtaaaactg taaatccgta aaattttgct aaacatatat tgtgttgttt tggtaagtat 300

tgacccaagc tatcacctcc tgcagtatgt cgtgctaatt actggacaca ttgtataaca 360

gttccactgt attgacaata ataaaacctc ttcattgact tgagaatgtc tggacagatt 420

tggctttgta tttttgattt acaaatgttt ttttggtgat ttacccatcc aaggcattct 480

ccaggatggt tgtggcatca cgccgattgg caaacaaaaa ctaaaatgaa actaaaaaga 540

aacagtttcc gctgtcccgt tcctctagtg ggagaaagca tgaagtaagt tctttaaata 600

ttacaaaaaa attgaacgat attataaaat tctttaaaat attaaaagta agaacaataa 660

gatcaattaa atcataatta atcacattgt tcatgatcac aatttaattt acttcatacg 720

ttgtattgtt atgttaaata aaaagattaa tttctatgta attgtatctg tacaatacaa 780

tgtgtagatg tttattctat cgaaagtaaa tacgtcaaaa ctcgaaaatt ttcagtataa 840

aaaggttcaa ctttttcaaa tcagcatcag ttcggttcca actctcaaga tgagagtcaa 900

aacctttgtg atcttgtgct gcgctctgca ggtgagttaa ttattttact attatttcag 960

aaggtggcca gacgatatca cgggccacct gataataagt ggtcgccaaa acgcacagat 1020

atcgtaaatt gtgccatttg atttgtcacg cccggggggg ctacggaata aactacattt 1080

atttatttaa aaaatgaacc ttagattatg taacttgtga tttatttgcg tcaaaagtag 1140

gcaagatgaa tctatgtaaa tacctgggca gacttgcaat atcctatttc accggtaaat 1200

cagcattgca atatgcaatg catattcaac aatatgtaaa acaattcgta aagcatcatt 1260

agaaaataga cgaaagaaat tgcataaaat tataaccgca ttattaattt attatgatat 1320

ctattaacaa ttgctattgc ctttttttcg caaattataa tcattttcat aacctcgagg 1380

tagcattctg ttacatttta atacattggt atgtgattat aacacgagct gcccactgag 1440

tttctcgcca gatcttctca gtgggtcgcg ttaccgatca cgtgatagat tctatgaagc 1500

actgctcttg ttagggctag tgttagcaaa ttctttcagg ttgagtctga gagctcacct 1560

acccatcgga gcgtagctgg aataggctac cagctaatag gtagggaaaa caaagctcga 1620

aacaagctca agtaataaca acataatgtg accataaaat ctcgtggtgt atgagataca 1680

attatgtact ttcccacaaa tgtttacata attagaatgt tgttcaactt gcctaacgcc 1740

ccagctagaa cattcaatta ttactcatca ttaccactac taaggcagta tgtcctaact 1800

cgttccagat cagcgctaac ttcgattgaa tgtgcgaaat ttatagctca atattttagc 1860

acttatcgta ttgatttaag aaaaaattgt taacattttg tttcagtatg tcgcttatac 1920

aaatgcaaat ctagaatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 1980

ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 2040

ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 2100

gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 2160

cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 2220

gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 2280

gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 2340

aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 2400

gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 2460

agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 2520

ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 2580

cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 2640

gagctgtaca agggatccaa aaactgtgga attcctagaa gacaactagt tgttaaattc 2700

agagcactgc cttgtgtgaa ttgctaattt ttaatataaa ataacccttg tttcttactt 2760

cgtcctggat acatctatgt tttttttttc gttaataaat gagagcattt aagttattgt 2820

ttttaattac ttttttttag aaaacagatt tcggattttt tgtatgcatt ttatttgaat 2880

gtacta 2886

Claims (3)

1. The recombinant expression vector for automatically deleting exogenous DNA of the first generation germ cells of the transgenic silkworms is characterized by being a binary transgenic expression vector, and comprises a transgenic activation vector for regulating and controlling GAL4 protein expression by a gonad specific expression gene promoter of the silkworms and a transgenic effect vector for regulating and controlling downstream FLP gene expression by UAS elements;

the transgenic activating vector comprises a vector framework, an activating element expression frame and a target gene expression frame; the vector skeleton is a piggyBac recombinant vector pBac {3×P3-EGFPaf } containing a silkworm green eye fluorescence screening marker; the activating element expression frame comprises a silkworm gonad tissue specific gene promoter RSHP1p or Nanosp, an optimized GAL4 protein gene coding sequence and an SV40 terminator sequence; the target gene expression frame is formed by fusing a red fluorescent protein gene initiated by a bombyx mori silk fibroin heavy chain promoter with a light chain binding site sequence; the transgene activating vector forms 2 different transgene activating vectors through promoters RSHP1p and Nanosp;

the transgenic activating vector also comprises loxP sites and lox2272 sites for anchoring the activating element expression frame, 2 attP sites which are arranged in the same direction for anchoring the target gene expression frame, and 2 FRT sites which are arranged in the same direction for anchoring the activating element expression frame and the target gene expression frame;

the transgenic effector vector comprises a vector framework, a effector element expression frame and a target gene expression frame; the vector skeleton is a piggyBac recombinant vector pBac {3×P3-DsRedaf } containing a silkworm red eye fluorescence screening marker; the response element expression frame comprises an upstream activating sequence UAS element, an FLP gene coding sequence and an SV40 terminator sequence; the target gene expression frame is formed by fusing a light chain binding site sequence of enhanced green fluorescent protein genes which are started by a bombyx mori silk fibroin heavy chain promoter;

the transgenic effector vector further comprises 2 co-directionally arranged attP sites for anchoring the target gene expression cassette and 2 co-directionally arranged FRT sites for simultaneously anchoring the effector element expression cassette and the target gene expression cassette;

wherein, the nucleotide sequence of the coding sequence of the optimized GAL4 protein gene is SEQ ID NO.3; the nucleotide sequence of the promoter RSHP1p is SEQ ID NO.1; the nucleotide sequence of the promoter Nanosp is SEQ ID NO.2; the nucleotide sequence of the SV40 terminator sequence is SEQ ID NO.4; the nucleotide sequence of the target gene expression frame is SEQ ID NO.5.

2. The recombinant expression vector of claim 1, which is used for automatically deleting exogenous DNA of a first filial generation germ cell of a transgenic silkworm.

3. The method for preparing the first filial generation double-transgenic silkworms by using the recombinant expression vector as defined in claim 1, which comprises the following specific steps:

(1) Firstly, transforming the recombinant expression vector and the helper plasmid pHA3PIG of claim 1 into a non-diapause or diapause-relieved G0 generation silkworm egg, feeding the hatched G0 generation silkworm to a chemical moth, backcrossing the G0 generation silkworm moth to produce a seed to obtain a G1 generation silkworm egg, screening single-copy transgenic silkworms in the G1 generation silkworm, and then, separately feeding the silkworms by taking a moth region as a unit to establish 2 different transgenic activated lines and 1 transgenic effect line;

(2) The single copy transgene inserted active line silkworms obtained in the step (1) are hybridized with single copy transgene inserted effector line silkworms to produce hybrid generation silkworms, and double transgene silkworms simultaneously containing 2 eye fluorescent markers are screened to obtain the hybrid generation double transgene silkworms;

(3) Raising the first-filial generation double-transgenic silkworms obtained in the step (2) to chemical moths, and carrying out selfing seed production on the first-filial generation double-transgenic male and female moths or backcross seed production on the first-filial generation double-transgenic silkworms to obtain second-filial generation silkworms; because the gonad specific expression gene promoter regulates and controls the GAL4 protein to specifically express in germ cells of hybrid generation double-transgenic silkworms, the FLP recombinase is activated to express and automatic deletion of DNA sequences of an activating element expression frame, a response element expression frame and a target gene expression frame between the FRT site pairs arranged in the same direction is realized, the finally obtained hybrid second-generation silkworms genome does not contain the activating element expression frame, the response element expression frame and the target gene expression frame, and the biological potential safety hazard that the exogenous target gene possibly escapes in the passage process is ensured to be eliminated; in non-germ cells, the gonad specific expression gene promoter has no promoter activity and can not regulate GAL4 gene expression, so that FLP recombinase expression is inhibited, thus the exogenous target gene is reserved in the non-germ cells, and finally, the acquisition of the transgenic product expressed by the hybrid generation double transgenic silkworms and the realization of the economic character of the transgenic product are ensured.