CN109517840A

CN109517840A - Efficient transcriptional activation system in drosophila reproductive system

Info

Publication number: CN109517840A
Application number: CN201811312144.5A
Authority: CN
Inventors: 朱丽霏; 倪建泉; 徐荣刚; 毛德才; 孙锦; 贾豫
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2018-11-06
Filing date: 2018-11-06
Publication date: 2019-03-26
Anticipated expiration: 2038-11-06
Also published as: CN109517840B

Abstract

The present invention relates to field of biotechnology, and in particular to a kind of transcriptional activation system and its application in drosophila reproductive system.The transcriptional activation system includes dCas9 protein expression element and sgRNA Expression element, contain p- transposase promoter, dCas9 albumen coded sequence, MCP albumen coded sequence and activating transcription factor coded sequence on the dCas9 protein expression element, the activating transcription factor coded sequence is located at the downstream of the dCas9 albumen coded sequence, and the MCP albumen coded sequence is located at the downstream of the dCas9 albumen coded sequence；Contain U6B promoter, sgRNA insertion point and MCP protein recognition sequences on the sgRNA Expression element.Transcriptional activation system provided by the invention, can in drosophila reproductive system activated gene expression, and activation efficiency is high.

Description

Efficient transcriptional activation system in drosophila reproductive system

Technical field

The present invention relates to field of biotechnology, and in particular to a method of high efficiency regulatory gene expression in vivo, More particularly to a kind of transcriptional activation system applied in drosophila reproductive system.

Background technique

As the genome plan of the species such as the mankind smoothly completes, function letter is converted by the sequence information of genome Breath, decodes the password of life, is of great significance for processes such as growth and development, the disease agings of the comprehensive understanding mankind.Drosophila As the one mode biology highly conserved with the mankind, have many advantages, such as, the research achievement in drosophila is also applied for the mankind In, and the limitation of life ethics can be got rid of, therefore become the idealized model biology of biomedical research.In drosophila reproductive system Highly conserved because having, the cell quantities such as germline stem cell and position are determining and are easy to mark and cell-cell interaction machine The features such as system more understands, and become the ideal model for carrying out internal stem cell and reproductive development research.Regulate and control target gene Expression is the most commonly seen technological means of life science, main by reducing gene function and enhancing gene function.In fruit In fly reproductive system, the technological means for reducing gene function be have been relatively mature, mainly include transgenosis perturbation technique and CRISPR/Cas9 gene editing technology etc., but the technological means for enhancing gene expression is also relatively limited, often limits correlative study Carry out in a deep going way.

Existing technology depends on traditional Gal4/UAS system, by expressing the purpose base carried by foreign vector Because of coded sequence.Although this mode can express target gene, clone's step of target gene in specific organization or organ Rapid cumbersome, the period is longer, cannot simulate the expression pattern of gene itself and false positive results often occurs, and cannot mistake simultaneously Multiple genes are expressed, cannot achieve extensive building and screening operation in genome range.And by existing CRISPR/Cas9 When transcriptional activation system is applied to reproductive system, controlling gene expression efficiency is limited.Therefore, in reproductive system gene swash Living and high efficient expression also requires further improvement.

Summary of the invention

The present invention is directed to solve at least some of the technical problems in related technologies, the transcription of gene is improved Activation efficiency.For this purpose, the present invention provides a kind of transcriptional activation system, using CRISPR/dCas9 System-mediated drosophila life Efficiently activating genes of interest is grown in system, so that the regulation of gene is realized, to improve the efficiency of the transcriptional activation of gene, especially Its efficiency that can be used to improve the transcriptional activation of gene in drosophila reproductive system.

The present inventor has found in the course of the research: the digestion activity position of Cas9 albumen in CRISPR/Cas9 system (become dCas9) after point mutation, if the C-terminal in dCas9 merges activating transcription factor appropriate, in the effect of sgRNA Under, the transcriptional activation in situ of target gene may be implemented.And the particularity of reproductive system expression is combined, to CRISPR/Cas9 system System is transformed, can it is simple and quick it is efficient realize reproductive system in gene transcriptional activation and expression.

Therefore, the present invention is constructed for the deficiency for enhancing gene expression ways in reproductive system in existing drosophila FlySAMG system is able to solve following problem as transcriptional activation system: 1, gene overexpression operates in drosophila reproductive system Cumbersome problem；2, the problem of multiple genes cannot be activated in drosophila reproductive system simultaneously；3, swash in existing system reproductive system The problem of gene low efficiency living；4, in drosophila reproductive system the transcriptional activation strain of genome range construction and screening problem.

Specifically, the present invention provides the following technical scheme that

According to the first aspect of the invention, the present invention provides a kind of transcriptional activation system, the transcriptional activation system packets DCas9 protein expression element and sgRNA Expression element are included, is started on the dCas9 protein expression element containing p- transposase Son, dCas9 albumen coded sequence, MCP albumen coded sequence and activating transcription factor coded sequence, the activating transcription factor Coded sequence is located at the downstream of the dCas9 albumen coded sequence, and the MCP albumen coded sequence is located at the dCas9 albumen The downstream of coded sequence；Position is inserted into containing U6B promoter, MCP protein recognition sequences and sgRNA on the sgRNA Expression element Point.MCP (MS2coating protein) albumen coded sequence can encode MCP albumen, which can identify that sgRNA is expressed MS2 structure on element, so that dCas9 protein expression element under the guidance of sgRNA Expression element, is identified and is integrated to On target gene.And the activating transcription factor coded sequence contained on dCas9 protein expression element can encoding transcription activation because Son, p- transposase promoter can start expression of the dCas9 protein expression element in reproductive system, to realize activation purpose The target of the expression of gene.Transcriptional activation system provided by the invention using the transcriptional activation on dCas9 protein expression element because The expression of sub- coded sequence and p- transposase promoter controlling gene in reproductive system, and using U6B as promoter regulation SgRNA expression, so that the expression quantity of sgRNA is higher, to improve the activation efficiency of transcriptional activation system.SgRNA in the present invention SgRNA insertion point and MCP protein recognition sequences are respectively positioned on the downstream of U6B promoter in Expression element.It is provided using the present invention Transcriptional activation system, it is particularly possible to be applied to the transcriptional activation of gene in drosophila reproductive system, it is easy to operate, and swash Work is more efficient, is convenient for the operation of extensive high throughput.

According to an embodiment of the invention, above-described transcriptional activation system may further include following technical characteristic:

In some embodiments of the invention, the activating transcription factor coded sequence is selected from VP64 coded sequence, P65 is compiled Code at least one of sequence or HSF1 coded sequence.VP64 coded sequence can encode V64 albumen (herpes simplex virus egg The tetramer of white matter VP16), P65 coded sequence can encode P65 albumen (NF-kB trans-activation subunit), HSF1 coded sequence HSF1 albumen (the human heat shock factor 1) can be encoded, these albumen can be used as activating transcription factor, the table of activated gene It reaches.

In some embodiments of the invention, the activating transcription factor coded sequence includes VP64 coded sequence, P65 volume Code sequence and HSF1 coded sequence, the VP64 coded sequence are located at the upstream of the MCP albumen coded sequence, the VP64 Contain self cleavage peptide T2A coded sequence, the MCP albumen coded sequence between coded sequence and the MCP albumen coded sequence Downstream be followed successively by the P65 coded sequence and the HSF1 coded sequence.DCas9 albumen coded sequence can encode dCas9 Albumen is had VP64 albumen in the C-terminal fusion of dCas9 albumen, and is further melted using self cleavage peptide T2A in the downstream of MCP albumen Conjunction has P65 and HSF1 activating transcription factor.Self cleavage peptide T2A be it is a kind of can in translation skill self splicing small peptide, Its enable to dCas9 protein expression element after generating whole mRNA can self cleavage at two independent parts, dCas9- VP64 and MCP-P65-HSF1 can stablize the activation efficiency for improving transcriptional activation system.Herein, self cleavage peptide can also claim For autothermic cracking peptide.

In some embodiments of the invention, the VP64 coded sequence is SEQ ID NO:21, the code sequence of the P65 It is classified as SEQ ID NO:26, the HSF1 coded sequence is SEQ ID NO:27, and the self cleavage peptide T2A coded sequence is SEQ ID NO:23。

In some embodiments of the invention, the primer sequence for expanding the P65 coded sequence is SEQ ID NO: 24 and SEQ ID NO:25.

In some embodiments of the invention, the primer sequence for expanding the HSF1 coded sequence is SEQ ID NO: 27 and SEQ ID NO:28.

In some embodiments of the invention, the dCas9 protein expression element and the sgRNA Expression element pass through matter Grain carrier connection.The dCas9 protein expression element and the sgRNA Expression element are connected on same plasmid vector.To It can guarantee in organism while play a role.

In some embodiments of the invention, the plasmid vector is pNP plasmid.

In some embodiments of the invention, the MCP albumen coded sequence is SEQ ID NO:22.

In some embodiments of the invention, the MCP protein recognition sequences are MS2 sequence.

In some embodiments of the invention, further comprise: at least two gypsy genes, the gypsy gene difference Positioned at the upstream of the dCas9 protein expression element and the sgRNA Expression element；Ftz intron sequences and K10polyA sequence Column, the ftz intron sequences and the K10polyA sequence are located at the downstream of the dCas9 protein expression element.The present invention Open gene system at least containing there are two gypsy gene, gypsy gene is located at dCas9 protein expression element and sgRNA The both ends of Expression element are used to shield the interference and factor of insertion surrounding genes as insulator, to help two expression members The high efficient expression of part.Ftz intron sequences are located at the downstream of dCas9 protein expression element, for enhancing translation.K10 polyA The downstream that sequence is located at dCas9 protein expression element is conducive to the termination of regulatory transcription.In a specific embodiment, K10polyA sequence is located at the downstream of the ftz intron sequences.

In some embodiments of the invention, the nucleic acid sequence of the gypsy gene is SEQ ID NO:5；In the ftz It is SEQ ID NO:6 containing subsequence；The K10polyA sequence is SEQ ID NO:44.

In some embodiments of the invention, the transcriptional activation system further comprises: 10 × UAS sequence, 10 × UAS Sequence is located at the upstream of the p- transposase promoter.Under its driving that may assist in tissue and organ specificity Gal4, in spy Fixed histoorgan and stage of development realize the transcriptional activation of target gene.

In some embodiments of the invention, the transcriptional activation system further comprise antibiotic marker genes, Vermilion gene and attB gene；Wherein marker gene of the vermilion gene as screening transgenic drosophila.AttB base Because being used for the genome of transcriptional activation system site-directed integration to drosophila.Antibiotic marker genes, vermilion gene and AttB gene is located at except gypsy gene, is used to help the construction and screening of transcriptional activation system in vivo, without influencing The expression of dCas9 protein expression element and sgRNA Expression element.

In some embodiments of the invention, the antibiotic marker genes are ampicillin resistance gene, the ammonia The nucleic acid sequence of parasiticin resistant gene is SEQ ID NO:7；The nucleic acid sequence of the vermilion gene is SEQ ID NO:8；The nucleic acid sequence of the attB gene is SEQ ID NO:9.

In a kind of specific example of the invention, the transcriptional activation system includes: dCas9 protein expression element, described DCas9 protein expression element includes p- transposase promoter and dCas9 albumen coded sequence, the dCas9 albumen coded sequence Positioned at the downstream of the p- transposase promoter, the downstream of the dCas9 albumen coded sequence be followed successively by VP64 coded sequence, from Shear peptide T2A coded sequence, MCP albumen coded sequence, P65 coded sequence and HSF1 coded sequence；SgRNA Expression element, institute It states and contains U6B promoter, MS2 sequence and sgRNA insertion point on sgRNA Expression element；At least two gypsy genes, it is described Gypsy gene is located at the upstream of the dCas9 protein expression element and the upstream of the sgRNA Expression element；Ftz is included Subsequence, the ftz intron sequences are located at the downstream of the dCas9 protein expression element；K10polyA sequence, it is described K10polyA sequence is located at the downstream of the ftz intron sequences；Antibiotic marker genes, vermilion gene and attB base Cause.On this basis, the transcriptional activation system can further include 10 × UAS sequence, and 10 × UAS sequence is located at The upstream of the p- transposase promoter.

In a kind of specific example of the invention, the transcriptional activation system is as shown in Figure 1.

According to the second aspect of the invention, the present invention provides a kind of method for constructing transcriptional activation system, the transcriptions Activation system is the transcriptional activation system according to first aspect present invention, which comprises

(1) include using plamid vector construction dCas9 albumen coded sequence the first recombinant plasmid；

(2) activating transcription factor coded sequence is connected on first recombinant plasmid, building obtains the second recombination matter Grain, wherein the activating transcription factor coded sequence is located at the downstream of the dCas9 albumen coded sequence；

(3) sgRNA Expression element is connected on second recombinant plasmid, building obtains transcriptional activation system.

According to an embodiment of the invention, the method for building transcriptional activation system described above may further include following skill Art feature:

In some embodiments of the invention, step (1) further comprises:

Cas9 albumen coded sequence is connected on plasmid vector by (1-1), and building obtains including Cas9 encoding histone sequence The third recombinant plasmid of column；

(1-2) is mutated the Cas9 albumen coded sequence on the third recombinant plasmid, obtains comprising State the first recombinant plasmid of dCas9 albumen coded sequence.

In some embodiments of the invention, the plasmid vector is pNP plasmid.

In some embodiments of the invention, the primer sequence for expanding the Cas9 albumen coded sequence is SEQ ID NO:1 and SEQ ID NO:2, the Cas9 albumen coded sequence are SEQ ID NO:19.

In some embodiments of the invention, step (1-1) is by way of homologous recombination by the Cas9 encoding histone Sequence is connected on the plasmid vector.

In some embodiments of the invention, in step (1-2) using reverse complemental mutant primer SEQ ID NO:11 and SEQ ID NO:12 is mutated the base that the 10th amino acids are encoded on the Cas9 albumen coded sequence, utilizes reverse mutual Mutant primer SEQ ID NO:13 and SEQ ID NO:14 is mended to the alkali of the 840th amino acids in the Cas9 albumen coded sequence Base is mutated.

In some embodiments of the invention, in step (2) by way of homologous recombination by the activating transcription factor Coded sequence is connected on first recombinant plasmid；

In some embodiments of the invention, sgRNA Expression element is connected by way of homologous recombination in step (3) Onto second recombinant plasmid.

In some embodiments of the invention, non-containing stent sequence and U6B promoter 3 ' on the sgRNA Expression element Region sequence is translated, contains MCP protein recognition sequences in the stent sequence.

In some embodiments of the invention, the stent sequence is SEQ ID NO:34, the sequence of the U6B promoter For SEQ ID NO:36,3 ' the non-translational region sequence of U6B promoter is SEQ ID NO:37.

According to the third aspect of the invention we, the present invention provides a kind of methods of prepare transgenosis drosophila, comprising:

(a) the sgRNA sequence for targeting target gene is imported into transcription described in first aspect present invention any embodiment In activation system, the transcriptional activation system of targeting target gene is obtained；

(b) the transcriptional activation system introducing containing targeting target gene is obtained into transgenosis fruit into drosophila embryos Fly.

In some embodiments of the invention, step (a) further comprises: (a-1) carries out the transcriptional activation system Digestion processing, obtains the transcriptional activation system by digestion processing；(a-2) by the targeting target gene after annealed pairs SgRNA sequence is connected in the sgRNA insertion point of the transcriptional activation system by digestion processing, so as to will be described The sgRNA sequence of targeting target gene is imported into the transcriptional activation system.

According to the fourth aspect of the invention, the present invention provides a kind of sides of gene expression in activation drosophila reproductive system Method, comprising: the sgRNA sequence for targeting target gene is imported into transcriptional activation system, obtains the transcription of targeting target gene Activation system, the transcriptional activation system are the transcriptional activation system according to first aspect present invention；By the targeting mesh The transcriptional activation system introducing of gene is marked into drosophila embryos, obtains transgenic fly；By the transgenic fly and Gal4 work Have drosophila hybrid culture, drosophila offspring is obtained, to activate the expression of target gene in drosophila reproductive system.

It is obtained by the present invention to have the beneficial effect that the method for the invention for preparing transcriptional activation system is easy to operate；The present invention Provided transcriptional activation system activation efficiency is high；Multiple genes can be activated simultaneously；It is convenient for the operation of extensive high throughput.

Detailed description of the invention

Fig. 1 is the schematic diagram of the transcriptional activation system flySAMG constructed according to one embodiment of present invention.

Fig. 2 is the schematic diagram of the drosophila ovary germarium constructed according to one embodiment of present invention, and wherein A is represented Drosophila ovary germarium (control represents control) is compareed, B represents the drosophila ovary germarium (dpp of dpp gene transcriptional activation Activation represents dpp gene activation), C represents the drosophila ovary germarium (tkv of tkv gene transcriptional activation Activation represents tkv gene activation), D represents the drosophila ovary germarium (bam of bam gene transcriptional activation Activation represents bam gene activation).

Specific embodiment

The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached The embodiment of figure description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.

Herein, term " transcriptional activation system " can also be expressed as " transcriptional activation carrier ", it is intended that being capable of activated gene The carrier or system or component of transcription.

Herein, term " coded sequence " refers to encode the sequence of one section of protein product, also refers to express The DNA nucleic acid sequence of one section of protein product also also refers to the RNA nucleic acid sequence of one section of protein product of expression.

DCas9 protein expression element described herein refers to the Expression element of expression dCas9 albumen, the sgRNA table Refer to that the Expression element that sgRNA is inserted into containing sgRNA insertion point, " Expression element " refer to make up to element The unit of corresponding function is played for a functional unit.DCas9 protein expression element can refer to expression dCas9 albumen Nucleic acid sequence, sgRNA Expression element can refer to the nucleic acid sequence of the sgRNA sequence of insertion targeting target gene.Wherein, institute DCas9 albumen is stated compared with Cas9 albumen, does not have nuclease, but still can identify specific sgRNA (single-guide RNA), still is able in conjunction with specific DNA sequence dna.According to an embodiment of the invention, the dCas9 Albumen is compared with Cas9 albumen, and the 10th amino acid sports A by D, and the 840th amino acid sports A by H.

Herein, when describe nucleic acid sequence between positional relationship when, form of presentation " upstream " " downstream " refer to according to The sequence of duplication or the transcription of nucleic acid sequence, the nucleic acid sequence positioned at upstream are located at downstream closer to 5 ' ends of Expression element Nucleic acid sequence closer to Expression element 3 ' end.The relationship in this " upstream " " downstream " corresponds respectively to expressed albumen N-terminal and C-terminal.It should be noted that this " upstream " " downstream " relationship does not necessarily imply that between two nucleic acid sequences directly It is connected, which only represents a kind of relative positional relationship.It completely can be between " upstream " and the nucleic acid sequence in " downstream " Other nucleic acid sequences are inserted into, as long as other nucleic acid sequences of the insertion have no effect on the row for being inserted into the function of nucleic acid sequence Make and expresses.

Herein, when describing the connection relationship of two nucleic acid sequences, unless " being connected directly " refers to two nucleic acid sequences Directly it is connected by 3 ' -5 ' phosphodiester bonds between column, and other nucleic acid sequences cannot be inserted into." connected " not necessarily refer to two Nucleic acid sequence cannot be inserted between a nucleic acid sequence, as long as other nucleic acid sequences of the insertion do not influence to be inserted into nucleic acid sequence Function enforcement and expression.

Different nucleic acid sequences shown herein, such as VP64 coded sequence (SEQ ID NO:21), P65 coded sequence (SEQ ID NO:26), gypsy gene order (SEQ ID NO:5), as just being capable of preferable example, it is intended that can express Corresponding albumen or the nucleic acid sequence for playing corresponding function.Those skilled in the art can according to need, and carry out to nucleic acid sequence These replacements of corresponding function or partial replacement are perhaps played as long as replacing perhaps partial replacement and can express corresponding albumen It is also contained in protection scope of the present invention.Nucleic acid sequence is shown according to the sequence at the end of sequence 5 ' to 3 ' ends generally in the art Out.

CRISPR/Cas9 system is current most popular gene editing system, and Cas9 albumen is under the guidance of sgRNA Target gene can be specifically identified and be cut, realizes precisely efficient gene editing.By the digestion activity site of Cas9 albumen After being mutated (dCas9), dCas9 still can be identified and binding purpose gene under the guidance of sgRNA, but not into Row cutting.If the C-terminal in dCas9 albumen merges transcriptional activation domain appropriate, so that it may realize the original position of target gene Transcriptional activation.

The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument Production firm person is not specified in device, and being can be with conventional products that are commercially available.

In the examples below, during constructing dCas9 protein expression element and sgRNA Expression element, Wo Menjun Obtain purpose dCas9 protein expression element and sgRNA Expression element by means of plasmid vector, therefore in described below What dCas9 protein carrier and sgRNA carrier indicated is to prepare the mistake in dCas9 protein expression element and sgRNA Expression element Journey object.

In the course of the research, we express the activator protein dCas9-VP64 of two fusions using self cleavage peptide T2A simultaneously And MCP-p65-HSF1, and expressed in the promoter promoter downstream p-transposase.Because U6B promoter has more High activity, so we control the expression of sgRNA2.0 using U6B promoter.There are two contain MS2 for tool on sgRNA2.0 The loop-stem structure of point, specifically can be identified and be combined by MCP albumen, and realizes and recruit MCP-p65-HSF1 activating transcription factor Purpose.Therefore, the transcriptional activation activity of tri- transcription factors of VP64, p65 and HSF1 is combined in the system.Use process In, we are by the vector integration of the carrier of sgRNA2.0 and expression dCas9-VP64-T2A-MCP-p65-HSF1 at a carrier FlySAMG, and gypsy sequence is inserted respectively in the upstream of dCas9activator and the downstream of sgRNA2.0, to reduce Influence of other sequential elements to expressions of both, and enhance the transcription of gene.It can be convenient land productivity in such a way that digestion connects Multiple sgRNA are expressed simultaneously with this system, realize while activating the purpose of multiple target gene.In addition, in flySAMG carrier Further include the ampicillin resistance gene for screening positive plasmid, selects the genetic screening label of purpose transgenic fly (vermilion gene) helps the attB sequence for constructing fixed point transgenic fly, helps dCas9activator transcription and turns over The ftz sequence and K10polyA sequence translated.

The application is using Gal4/UAS binary expression system, the expression of activating genes of interest in drosophila reproductive system, 10 × UAS sequence is contained in the upstream of p-transposase promoter promoter, in this way tissue and organ specificity Gal4's It, can be in the transcriptional activation of specific histoorgan and stage of development realization target gene under driving.Only need turning for flySAMG After the single-cross of gene drosophila and specific Gal4 tool drosophila, so that it may realize that the transcription of target gene in offspring drosophila swashs It is living.

Embodiment one

CRISPR/Cas9 system is current most popular gene editing system, and Cas9 albumen is under the guidance of sgRNA The specific position of genome can be cut, but after the digestion activity site of Cas9 albumen is mutated, institute's shape At dCas9 still can identify under the guidance of sgRNA and simultaneously combine specific gene, but genome not cut. As a result, through dCas9 in conjunction with specific DNA sequence dna, and recruit the activating transcription factor for capableing of activated gene expression, Ke Yiyong To study the transcript and expression situation of gene expression.

The method for preparing dCas9 albumen is present embodiments provided, is included the following steps:

1, the clone of Cas9 albumen coded sequence

Reference literature Ren X, Sun J, Housden B E, et al.Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9[J] .Proceedings of the National Academy of Sciences of the United States of America, 2013,110 (47): the record of 19012-7. passes through PCR amplification using the non-cas9 carrier recorded in document Cas9 albumen coded sequence is obtained, wherein the primer utilized is respectively

NLS-Cas9-F(SEQ ID NO:1):

5’-AGAAGCGGAAGGTCGGTATCCACGGTGTCCCAGCAGCCATGGACAAGAAGTAC TCCATTGGGCT- 3’

NLS-Cas9-R(SEQ ID NO:2):

5’-TCTTAGCTTGACCAGCTTTCTTAGTAGCAGCAGGACGCTTGTCTCCACCGAGCT GAGAGAGG-3’

Cas9 albumen coded sequence is obtained using primer amplification as above, obtained Cas9 albumen coded sequence still has The activity of nucleic acid shearing enzyme.

2, plasmid vector skeleton is cloned

The plasmid backbone of flySAMG carrier is from pNP vector modification.Wherein, articles of reference " An efficient and multiple target transgenic RNAi technique with low toxicity in Drosophila, Nature Communications 9, Article number:4160 (2018) " obtains pNP carrier.With The mode of PCR clones the segment needed on pNP carrier, used PCR primer are as follows:

NLS-pNP-F1(SEQ ID NO:3):

5’-GAAAGCTGGTCAAGCTAAGAAAAAGAAAAATTGTTGGCATCAGGTAGGCATCA CA-3’

pNP-NLS-R1(SEQ ID NO:4):

5’-GATACCGACCTTCCGCTTCTTCTTTGGGGCCATGGTGGCGgtaccTCTAGACTTTG GTATGCGTCTTGTGATTCAAAG-3’

In amplified production obtained containing gypsy gene, ftz intron sequences, ampicillin resistance gene, Vermilion gene, attB gene, SV40polyA sequence and 10 × UAS sequence, shown below gypsy gene, ftz Intron sequences, ampicillin resistance gene, vermilion gene, attB gene and 10 × UAS sequence and DSCP starting Subsequence, and SV40polyA sequence and DSCP promoter sequence are replaced in subsequent processing, therefore are not shown.

Gypsy gene order (SEQ ID NO:5) is as follows:

TTGGCCACGTAATAAGTGTGCGTTGAATTTATTCGCAAAAACATTGCATATTTTCGG CAAAGTAAAA TTTTGTTGCATACCTTATCAAAAAATAAGTGCTGCATACTTTTTAGAGA AACCAAATAATTTTTTATTGCATACC CGTTTTTAATAAAATACATTGCATACCCTCTTTTA ATAAAAAATATTGCATACTTTGACGAAACAAATTTTCGTT GCATACCCAATAAAAGATT ATTATATTGCATACCCGTTTTTAATAAAATACATTGCATACCCTCTTTTAATAAAG AATAT TGCATACGTTGACGAAACAAATTTTCGTTGCATACCCAATAAAAGATTATTATATTGCAT ACCTTTTCT TGCCATACCATTTAGCCGATCAATTCTGCTCGGCAACAGTATATTTGTGGT GTGCCAACCAACAAC

Ftz intron sequences (SEQ ID NO:6) are as follows:

CTAGTTCTGATCTGCTAGACAATTGTTGGCATCAGGTAGGCATCACACACGATTAA CAACCCCTAAA AATACACTTTGAAAATATTGAAAATATGTTTTTGTATACATTTTTGATA TTTTCAAATAATACGCAGTTATAAAA CTCATTAGCTAACCCATTTTTTCTTTGCTTATGC TTACAGATTGCAAAGAACTAGAG

Phenalgin penicillin resistance sequence (SEQ ID NO:7) is as follows:

ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTT CCTGTTTTTG CTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTG GGTGCACGAGTGGGTTACATCGAACTGG ATCTCAACAGCGGTAAGATCCTTGAGAGT TTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTC TGCTATGTGGCG CGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTC TCAG AATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATG ACAGTAAGAGAATTATGCAGT GCTGCCATAACCATGAGTGATAACACTGCGGCCAACT TACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACC GCTTTTTTGCACAACATGG GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCA AA CGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATT AACTGGCGAACTACT TACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCG GATAAAGTTGCAGGACCACTTCTGCGCTCGGC CCTTCCGGCTGGCTGGTTTATTGCTG ATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACT GGGGCCAGA TGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGAT GAACGAA ATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAA

Vermilion gene order (SEQ ID NO:8) is as follows:

ATTTATTTTGTTATGTTATATGTATTATATGTCAGACATAAAGAAAAGGAACACATCA AATGTGATA ACAAAGACTAAACAAGTAATTTTATTACACCAAAACGACAAAACAGTAG GCAGAACAAACAACGCATAGCCAAAC ATTGACGAATTGGATACCCTGCCGATTGTCA GACACTTTTGTTGATCAGTTTCTTGCGAATGGTCTCGTCCAGCG GTGGAATCGCCTCG CGGGGAATCAGAAAAGTGGACAGATTGAACAGATCCAGAAACACCTTGTACCGATCA CTG AAACCAAAAAAAAACAAAGGGAGAACAGTTTGAGTTCATTGATCCCCGATATAA TCACATCTGCGATGATCACCT GAGAGTGGAGCGCAGATATTGATAACCAGACGAGCCA CCAGTGCCCAACTGTTGCGATCCAATCATGCGTTGCAC CATGATCACGTGATTGTCTGC GGCGGGAATAGAAAGTATTTGGTTAGGAAAACCAGTCTTAAACATAAGATATAT TTATA AAAGAGTATCAAAGAATGCAATACTTACATCTCCACTTGGTTATTAACGAGTCGATGTC CATGAGCAGG GTGAGCAACTGGTGTGGTTGGCTGAACCTGGGTTCATCCCTATAGAA GGTGATCATGATGGCTCCCTGAAGGGCA CGATGGCTAAACCGGCGATCCCCACGACG CACCAGTGCATCGTGCACTGCCGGATCAAAGATGGAGCGATACACC TCGCGTCGCTTC TCAATGTCCATGAGGCGGTAGTTTTTCGCCTTCTCCACGGGCTCCTCCATGGCGCTCTG TAC CTGCGCCTCCAGGAATCGATCGACGCTCTCCTGAAACTTGGCCCAGAAGTTGAA GCCACTCTCCTCCAGTCCGGG CGTCCTCTCCAGCCATCGCTGCACTAGCTCCAGTAGC GAGGGATCTTTCTCCGAGTTGCGAATCGAGTTCCGCGC CTCCTCGTCGCTAAAGACAT CCGAGTACTTCTGGTTGTATCTCACCCGCTGCTCTGTCAGAACTCCCAGCTTGTT CTCG ATCAAACGGAACTGCAGCGACTGAAAACCAGATGCGGGTGCCAGGTACTTGCGGAA GTCCATGAAGTCTA GCGGGGTCATGGTCTCCAGAATGGGCACTTGGTCCACCAGGAG CTGTACAAAGGAAGTTATAAACGGATTTTGGT AAGAGATTCAGAAAGCACTCACTTTT AGAATCAGAACCACTCGGTTCAGTCGCTTGACAATCTCCAGCGTCTTGG TTTCATCGA TGACCTCTGCATCCAACATGTCTCGTATGGAGTCGAACTCAAAGATGATCTGCTTGAA CCAAAGC TCGTAGGCTGTGGCGAAGGTACTTAAATGCCATTGAGTGTTGTCATCAAAG TTGTAAACCTACTCACCCTGGTGC GTGATGATGAACAGATGCTCATCGTGCACGGGTC GCTTGTCCTCCTCGGACAGCATACACTGGGCATCCAGCAGT TTGTCCAGCATCAGATA CTCTCCATAGATTTTGCCCACTTCCGTGGTTAATGGCACCGCCGAATCATCGTGATCGT TTCTGTATGGGTTTGAATTGAATCGCAGAACTGAAGATCGATTGGCATTCCTGGACAG CACGTGCTGGTGCTCAC CCGTTTCCTGCATAGGGACAGCTCATGGTGCACAGCTCAGA TCAGATCGTGACTCCTCGACCGGCGGATGCTGGC GAACTGATCTCCGCCAGCGGACC GGAGATGAGACCCCAGCGAACCGATAACAGAGCGAGAGAGCTCCAGTTCCGA CTGAT TGCACAGTCGGTGATCTGGGCGATGGGCACTGCCAGATAGGCTGGGAATTATCAATCA CTTGAGGTGAAAGTGCGGCGCACACAAAT

AttB sequence (SEQ ID NO:9) is as follows:

GCTGCATCCAACGCGTTGGGAGCTCTCCGGATCAATTCGGCTTCACGTACCGTCG ACGATGTAGGTC ACGGTCTCGAAGCCGCGGTGCGGGTGCCAGGGCGTGCCCTTGGGC TCCCCGGGCGCGTACTCCACCTCACCCATC TGGTCCATCATGATGAACGGGTCGAGGT GGCGGTAGTTGATCCCGGCGAACGCGCGGCGCACCGGGAAGCCCTCG CCCTCGAAAC CGCTGGGCGCGGTGGTCACGGTGAGCACGGGACGTGCGACGGCGTCGGCTGGTGCG GATACGCG GGGCAGCGTCAGCGGGTTCTCGACGGTCACGGCGGGCATGTCGACAAGC CGAATTGATCCACTAGAAGGCCTAATT C

10 × UAS (SEQ ID NO:10):

GCAGGTCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGT CCTCCGAGCGGA GTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGACTCCC GCGGTCGGAGTACTGTCCTCCGAGCGGAGT ACTGTCCTCCGAGCGGAGTACTGTCCTC CGAGCGGAGTAC

3, the building of pNP-dCas9

The product that step 1 and step 2 obtain is attached by way of homologous recombination, product converts Escherichia coli DH5 α, selects positive colony, and correct plasmid is sequenced and is denoted as pNP-Cas9.

The nuclease shear active of Cas9 depends on two structural domains: RuvC and HNH, the two structural domains are each responsible for cutting Two chains of DNA chain are cut, and the two structural domains can be inactivated individually by artificial point mutation.By will be in Cas9 albumen 10th D (i.e. aspartic acid, Asp) sports A (i.e. alanine, Ala), and the Cas9D10A mutant of acquisition is shown as RuvC inactivation, HNH still behave as activity；By the way that the H (i.e. histidine, His) of the 840th in Cas9 albumen is sported A, The Cas9H840A mutant of acquisition shows as HNH inactivation, and RuvC is still active, and the Cas9 of both mutant still has There is the activity of nuclease, shear action can be played to targeting sequence.When RuvC and HNH is in inactivated state simultaneously, Cas9 To not have nuclease, and become dCas9 (dead Cas9).

The D of the part the tenth of pNP-Cas9 plasmid expression Cas9 albumen is mutated into A in the following manner as a result, the 840 H are mutated into A to form the dCas9 of no digestion activity.

Using pNP-Cas9 as template, PCR amplification is carried out with the mutant primer of a pair of of reverse complemental.PCR product Dpn I enzyme Bacillus coli DH 5 alpha is directly converted after cutting, monoclonal is selected and shakes bacterium, is extracted plasmid order-checking and is identified to obtain correct mutant plasmid.Institute The primer used is respectively as follows:

D10A-F (SEQ ID NO:11):

5’-GAAGTACTCCATTGGGCTCGcTATCGGCACAAACAGCGTC-3’

D10A-R (SEQ ID NO:12):

5’-GACGCTGTTTGTGCCGATAgCGAGCCCAATGGAGTACTTC-3’

H840A-F (SEQ ID NO:13):

5’-TCCGACTACGACGTGGATgcTATCGTGCCCCAGTCTTTTC-3’

H840A-R (SEQ ID NO:14):

5’-GAAAAGACTGGGGCACGATAgcATCCACGTCGTAGTCGGA-3’

With two BbsI restriction enzyme sites (BbsI 1703 and BbsI in the above identical mutational formats mutation Cas9 2149), so as to subsequent application, obtained carrier is named as pNP-dCas9.Used primer is respectively as follows:

BbsI-dCas9-1-F (SEQ ID NO:15):

5’-ACCGTGAAACAGCTCAAAGAgGACTATTTCAAAAAGATTG-3’

BbsI-dCas9-1-R (SEQ ID NO:16):

5’-CAATCTTTTTGAAATAGTCcTCTTTGAGCTGTTTCACGGT-3’

BbsI-dCas9-2-F (SEQ ID NO:17):

5’-TTCTGGCCAGGGGGACAGTCTgCACGAGCACATCGCTAAT-3’

BbsI-dCas9-2-R (SEQ ID NO:18):

5’-ATTAGCGATGTGCTCGTGcAGACTGTCCCCCTGGCCAGAA-3’

Wherein, the dCas9 coded sequence in pNP-dCas9 carrier obtained (SEQ ID NO:19) is as follows:

ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGTGTCCCAGCAGCCATG GACAAGAAGTACT CCATTGGGCTCGCTATCGGCACAAACAGCGTCGGCTGGGCCGTC ATTACGGACGAGTACAAGGTGCCGAGCAAAA AATTCAAAGTTCTGGGCAATACCGAT CGCCACAGCATAAAGAAGAACCTCATTGGCGCCCTCCTGTTCGACTCCG GGGAGACG GCCGAAGCCACGCGGCTCAAAAGAACAGCACGGCGCAGATATACCCGCAGAAAGAA TCGGATCTGC TACCTGCAGGAGATCTTTAGTAATGAGATGGCTAAGGTGGATGACTCTT TCTTCCATAGGCTGGAGGAGTCCTTT TTGGTGGAGGAGGATAAAAAGCACGAGCGCC ACCCAATCTTTGGCAATATCGTGGACGAGGTGGCGTACCATGAA AAGTACCCAACCAT ATATCATCTGAGGAAGAAGCTTGTAGACAGTACTGATAAGGCTGACTTGCGGTTGATC TA TCTCGCGCTGGCGCATATGATCAAATTTCGGGGACACTTCCTCATCGAGGGGGACC TGAACCCAGACAACAGCGA TGTCGACAAACTCTTTATCCAACTGGTTCAGACTTACAA TCAGCTTTTCGAAGAGAACCCGATCAACGCATCCGG AGTTGACGCCAAAGCAATCCT GAGCGCTAGGCTGTCCAAATCCCGGCGGCTCGAAAACCTCATCGCACAGCTCCC TGG GGAGAAGAAGAACGGCCTGTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCC AACTTTAAATCTA ACTTCGACCTGGCCGAAGATGCCAAGCTTCAACTGAGCAAAGAC ACCTACGATGATGATCTCGACAATCTGCTGG CCCAGATCGGCGACCAGTACGCAGACC TTTTTTTGGCGGCAAAGAACCTGTCAGACGCCATTCTGCTGAGTGATA TTCTGCGAGT GAACACGGAGATCACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGCGCTATGATGAG CACCAC CAAGACTTGACTTTGCTGAAGGCCCTTGTCAGACAGCAACTGCCTGAGAAG TACAAGGAAATTTTCTTCGATCAG TCTAAAAATGGCTACGCCGGATACATTGACGGCG GAGCAAGCCAGGAGGAATTTTACAAATTTATTAAGCCCATC TTGGAAAAAATGGACGG CACCGAGGAGCTGCTGGTAAAGCTTAACAGAGAAGATCTGTTGCGCAAACAGCGCAC TTTCGACAATGGAAGCATCCCCCACCAGATTCACCTGGGCGAACTGCACGCTATACTC AGGCGGCAAGAGGATTT CTACCCCTTTTTGAAAGATAACAGGGAAAAGATTGAGAAA ATCCTCACATTTCGGATACCCTACTATGTAGGCCC CCTCGCCCGGGGAAATTCCAGATT CGCGTGGATGACTCGCAAATCAGAAGAGACCATCACTCCCTGGAACTTCGA GGAAGT CGTGGATAAGGGGGCCTCTGCCCAGTCCTTCATCGAAAGGATGACTAACTTTGATAAA AATCTGCCTA ACGAAAAGGTGCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAG TTTATAACGAGCTCACCAAGGTCAAAT ACGTCACAGAAGGGATGAGAAAGCCAGCAT TCCTGTCTGGAGAGCAGAAGAAAGCTATCGTGGACCTCCTCTTCA AGACGAACCGGA AAGTTACCGTGAAACAGCTCAAAGAGGACTATTTCAAAAAGATTGAATGTTTCGACTC TGTT GAAATCAGCGGAGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGAT CTCCTGAAAATCATTAAAGAC AAGGACTTCCTGGACAATGAGGAGAACGAGGACATT CTTGAGGACATTGTCCTCACCCTTACGTTGTTTGAAGAT AGGGAGATGATTGAAGAAC GCTTGAAAACTTACGCTCATCTCTTCGACGACAAAGTCATGAAACAGCTCAAGAGGC GCCGATATACAGGATGGGGGCGGCTGTCAAGAAAACTGATCAATGGGATCCGAGACA AGCAGAGTGGAAAGACAA TCCTGGATTTTCTTAAGTCCGATGGATTTGCCAACCGGAA CTTCATGCAGTTGATCCATGATGACTCTCTCACCT TTAAGGAGGACATCCAGAAAGCA CAAGTTTCTGGCCAGGGGGACAGTCTGCACGAGCACATCGCTAATCTTGCAG GTAGC CCAGCTATCAAAAAGGGAATACTGCAGACCGTTAAGGTCGTGGATGAACTCGTCAAA GTAATGGGAAGG CATAAGCCCGAGAATATCGTTATCGAGATGGCCCGAGAGAACCAA ACTACCCAGAAGGGACAGAAGAACAGTAGG GAAAGGATGAAGAGGATTGAAGAGGG TATAAAAGAACTGGGGTCCCAAATCCTTAAGGAACACCCAGTTGAAAAC ACCCAGCT TCAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTGGAT CAGGAACT GGACATCAATCGGCTCTCCGACTACGACGTGGATGCTATCGTGCCCCAGT CTTTTCTCAAAGATGATTCTATTGA TAATAAAGTGTTGACAAGATCCGATAAAAATAGA GGGAAGAGTGATAACGTCCCCTCAGAAGAAGTTGTCAAGAA AATGAAAAATTATTGG CGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAAG G CTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCAGGCTTCATCAAAAGGCAGCTT GTTGAGACACGCCAGATCA CCAAGCACGTGGCCCAAATTCTCGATTCACGCATGAAC ACCAAGTACGATGAAAATGACAAACTGATTCGAGAGG TGAAAGTTATTACTCTGAAGT CTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTGAGAGAGA TCAA CAATTACCACCATGCGCATGATGCCTACCTGAATGCAGTGGTAGGCACTGCACTTATCA AAAAATATCCC AAGCTTGAATCTGAATTTGTTTACGGAGACTATAAAGTGTACGATGTT AGGAAAATGATCGCAAAGTCTGAGCAG GAAATAGGCAAGGCCACCGCTAAGTACTTC TTTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTG GCCAATGGAGAGAT TCGGAAGCGACCACTTATCGAAACAAACGGAGAAACAGGAGAAATCGTGTGGGACA AGGG TAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCAGGTGAACATCG TTAAAAAGACCGAAGTACAGAC CGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAA GGAACAGCGACAAGCTGATCGCACGCAAAAAAGATTGGGA CCCCAAGAAATACGGC GGATTCGATTCTCCTACAGTCGCTTACAGTGTACTGGTTGTGGCCAAAGTGGAGAAAG GGAAGTCTAAAAAACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGC GATCAAGCTTCGAAAAAA ACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGG TCAAAAAAGACCTCATCATTAAGCTTCCCAAGTACT CTCTCTTTGAGCTTGAAAACGG CCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAGCTGG CAC TGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGG TCTCCCGAAGAT AATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTACCTT GATGAGATCATCGAGCAAATAAGCGAATTC TCCAAAAGAGTGATCCTCGCCGACGCTA ACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCC ATCAGGGAGCA GGCAGAAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTC AAGTA CTTCGACACCACCATAGACAGAAAGCGGTACACCTCTACAAAGGAGGTCCTG GACGCCACACTGATTCATCAGTC AATTACGGGGCTCTATGAAACAAGAATCGACCTCT CTCAGCTCGGTGGAGACAAGCGTCCTGCTGCTACTAAGAA AGCTGGTCAAGCTAAGA AA

4, the acquisition of transcriptional activation domain and its catenation sequence

VP64, T2A and MCP three synthesize from GENEWIZ (Suzhou, China) together, and the sequence of synthesis is (SEQ ID NO:20):

5’-GTCAAGCTAAGAAAAAGAAACAATTCGGAGGAGGTGGAAGCGGAGGAGGAGGAA GCGGAGGAGG AGGTAGCGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGGTTCC GGACGGGCTGACGCATTGGACGATTTTGA TCTGGATATGCTGGGAAGTGACGCCCTCG ATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTT TGACCTCGACATG CTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACCAATTCGGAA GCG GAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGA CCTATGGCTTCAAACTTTACTC AGTTCGTGCTCGTGGACAATGGTGGGACAGGGGATG TGACAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAG AGTGGATCAGCTCCAACTC ACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCCCAGAAGA GAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACAGTGGGCGGAG TCGAACTGCCTGTCGCCG CTTGGAGGTCCTACCTGAACATGGAGCTCACTATCCCAAT TTTCGCTACCAATTCTGACTGTGAACTCATCGTGA AGGCAATGCAGGGGCTCCTCAAA GACGGTAATCCTATCCCTTCCGCCATCGCCGCTAACTCAGGTATCTACAGCG CTGGAGG AGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCGGACCTAAGAAAAAG AGGAAGGTGGCGGCCGCTCAATTG-3’

Wherein corresponding VP64 sequence, T2A sequence and MCP sequence difference are as follows:

VP64 (SEQ ID NO:21):

GGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTG ACGCCCTCGATG ATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGAC CTCGACATGCTCGGCAGTGACGCCCTTG ATGATTTCGACCTGGACATGCTGATTAAC

MCP (SEQ ID NO:22):

ATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGATG TGACAGTGGCTC CTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCCAACTC ACGGAGCCAGGCCTACAAGGTGACATGCA GCGTCAGGCAGTCTAGTGCCCAGAAgAG AAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACAG TGGGCGGAGT CGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAGCTCACTATCCCAATT TTCGCT ACCAATTCTGACTGTGAACTCATCGTGAAGGCAATGCAGGGGCTCCTCAAAG ACGGTAATCCTATCCCTTCCGCC ATCGCCGCTAACTCAGGTATCTAC

T2A (SEQ ID NO:23):

GGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAA TCCTGGACCT

P65 is obtained from PCR on plasmid Addgene 63798, primer used in PCR are as follows:

P65-F (SEQ ID NO:24):

5’-AGGTGGCGGCCGCTCAATTGCCTTCAGGGCAGATCAGCAACC-3’

P65-R (SEQ ID NO:25):

5’-GCTTCCACCTCCTCCCTGCCCACTAGAGGAAATCTGTGAC-3’

P65 sequence (SEQ ID NO:26) obtained is as follows as a result:

CCTTCAGGGCAGATCAGCAACCAGGCCCTGGCTCTGGCCCCTAGCTCCGCTCCAG TGCTGGCCCAGA CTATGGTGCCCTCTAGTGCTATGGTGCCTCTGGCCCAGCCACCTGC TCCAGCCCCTGTGCTGACCCCAGGACCAC CCCAGTCACTGAGCGCTCCAGTGCCCAA GTCTACACAGGCCGGCGAGGGGACTCTGAGTGAAGCTCTGCTGCACC TGCAGTTCGA CGCTGATGAGGACCTGGGAGCTCTGCTGGGGAACAGCACCGATCCCGGAGTGTTCAC AGATCTG GCCTCCGTGGACAACTCTGAGTTTCAGCAGCTGCTGAATCAGGGCGTGTC CATGTCTCATAGTACAGCCGAACCA ATGCTGATGGAGTACCCCGAAGCCATTACCCGG CTGGTGACCGGCAGCCAGCGGCCCCCCGACCCCGCTCCAACT CCCCTGGGAACCAGC GGCCTGCCTAATGGGCTGTCCGGAGATGAAGATTTCTCAAGCATCGCTGATATGGACT T TAGTGCCCTGCTGTCACAGATTTCCTCTAGTGGGCAG

HSF1 is obtained from PCR on plasmid Addgene 61426, used PCR primer are as follows:

HSF1-F (SEQ ID NO:27):

5’-GGCAGGGAGGAGGTGGAAGCGGCTTCAGCGTGGACACC-3’

HSF1-R(SEQ ID NO:28):

5’-CCTACCTGATGCCAACAATTctagTTTGCTCTAGTCCTAGgCTAGGAGACAGTGGG GTCCTTGGC-3’

HSF1 sequence (SEQ ID NO:29) obtained is as follows as a result:

GGCTTCAGCGTGGACACCAGTGCCCTGCTGGACCTGTTCAGCCCCTCGGTGACCG TGCCCGACATGA GCCTGCCTGACCTTGACAGCAGCCTGGCCAGTATCCAAGAGCTCCT GTCTCCCCAGGAGCCCCCCAGGCCTCCCG AGGCAGAGAACAGCAGCCCGGATTCAG GGAAGCAGCTGGTGCACTACACAGCGCAGCCGCTGTTCCTGCTGGACC CCGGCTCCG TGGACACCGGGAGCAACGACCTGCCGGTGCTGTTTGAGCTGGGAGAGGGCTCCTACT TCTCCGAA GGGGACGGCTTCGCCGAGGACCCCACCATCTCCCTGCTGACAGGCTCGG AGCCTCCCAAAGCCAAGGACCCCACT GTCTCC

5, the building of pNP-dCas9-VP64-T2A-MCP-p65-HSF1 plasmid

PCR reaction, used primer are carried out by template of pNP-dCas9 are as follows:

PNP-dCas9-F (SEQ ID NO:30): 5 '-AATTGTTGGCATCAGGTAGGCATC-3 '

PNP-dCas9-R (SEQ ID NO:31): 5 '-TTTCTTTTTCTTAGCTTGACCAGCTTTCTTAGT-3 '

With the segment VP64-T2A-MCP of synthesis after amplified production is recycled, PCR product p65 sequence and HSF1 sequence are together Homologous recombination connection is carried out, correct plasmid is sequenced in selection after converting Escherichia coli.Due to there is the digestion of a BbsI in HSF1 The restriction enzyme site is carried out same sense mutation, used primer for the ease of subsequent experiment by site in aforementioned manners are as follows:

BbsI-HSF-F (SEQ ID NO:32):

5’-GGGCTGTCCGGAGATGAAGAtTTCTCAAGCATCGCTGATA-3’

BbsI-HSF-R (SEQ ID NO:33):

5’-TATCAGCGATGCTTGAGAAaTCTTCATCTCCGGACAGCCC-3’

The resulting plasmid name containing dCas9 albumen are as follows: pNP-dCas9-VP64-T2A-MCP-p65- HSF1。

Embodiment two

For sgRNA carrier, using the expression of U6B promoter control sgRNA, U6B promoter can in drosophila whole body, including It is expressed in reproductive system, and does not have tissue specificity.And by the expression of U6B promoter control sgRNA, sgRNA can be made Expression quantity it is higher.

Reference literature Ren X, Sun J, Housden B E, et al.Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9[J] .Proceedings of the National Academy of Sciences of the United States of America, 2013,110 (47): 19012-7. obtains sgRNA carrier by transformation using U6b-sgRNA-short plasmid.Tool Body construction method is as follows:

Firstly, the sgRNA holder part of U6B-sgRNA-short plasmid is replaced with following stent sequence (SEQ ID NO:34):

5’-GTTTTAGAGCTAGGCCAACATGAGGATCACCCATGTCTGCAGGGCCTAGCAAGT TAAAATAAGG CTAGTCCGTTATCAACTTGGCCAACATGAGGATCACCCATGTCTGCAG GGCCAAGTGGCACCGAGTCGGTGCTTTT T-3’。

Replaced stent sequence includes that can recruit MCP albumen by the MS2 sequence that MCP albumen identifies, MS2, this Sample can be enrolled into specific site promotion base together with the transcriptional activation domain p65 and HSF1 of MCP protein fusion Because of transcription, and there are also the blank that one section can identify size with BbsI digestion between U6B promoter and 3 ' UTR for stent sequence Sequence, about 680bp, with the sgRNA oligonucleotides that 20bp can be inserted after BbsI digestion.

The size when the intervening sequence that one section of spacer is added in the C-terminal of 3 ' UTR of U6B (noncoding region) is convenient for digestion simultaneously Identification.

Wherein the sequence of spacer is (SEQ ID NO:35):

5’-ACTAGCGTAATATATAGACAATGGTTTTCCGTTGACGTACATACATCTGACGTGT GTTTATTTA GACATAATAGTTATGTTTTCACATCTTTTTAATGTTCGCTTAATGCGTATGC ATACAAAATTTTTAATTTTCAAC ACAGTTGTTTTTGTTTTCATC-3’。

So constitute plasmid U6B-sgRNA2.0.

Wherein U6B promoter sequence (SEQ ID NO:36) are as follows:

GTTCGACTTGCAGCCTGAAATACGGCACGAGTAGGAAAAGCCGAGTCAAATGCC GAATGCAGAGTCT CATTACAGCACAATCAACTCAAGAAAAACTCGACACTTTTTTACC ATTTGCACTTAAATCCTTTTTTATTCGTTA TGTATACTTTTTTTGGTCCCTAACCAAAAC AAAACCAAACTCTCTTAGTCGTGCCTCTATATTTAAAACTATCAA TTTATTATAGTCAAT AAATCGAACTGTGTTTTCAACAAACGAACAATAGGACACTTTGATTCTAAAGGAAATT T TGAAAATCTTAAGCAGAGGGTTCTTAAGACCATTTGCCAATTCTTATAATTCTCAACT GCTCTTTCCTGATGTTG ATCATTTATATAGGTATGTTTTCCTCAATACTTC

3 ' UTR sequence of U6B (SEQ ID NO:37) are as follows:

TTGCTCACCTGTGATTGCTCCTACTCAAATACAAAAACATCAAATTTTCTGTCAAT AAAGCATATTT ATTTATATTTATTTTACAGGAAAGAATT

The building of three flySAMG carrier of embodiment

1, the integration of dCas9 carrier and sgRNA2.0 carrier

The part of U6B-sgRNA2.0 expression sgRNA and spacer is cloned while drawing in design with the mode of PCR Restriction enzyme site NheI and SpeI, used primer are added respectively in N-terminal and C-terminal when object are as follows:

SgRNA2.0-F (SEQ ID NO:38):

5’-AAACTCATCAATGTATCTTAACTAGTGATGAAAACAAAAACAACTGTGTTGAAA AT-3’

SgRNA2.0-R (SEQ ID NO:39):

5’-GCACACTTATTACGTGGCCAGAGCTCTGCTAGCTTGTTCGACTTGCAGCCTGAA ATACG-3’

The portion for the pNP-dCas9-VP64-T2A-MCP-p65-HSF1 that embodiment one is prepared by way of PCR Sub-sequence is expanded as carrier framework, and used primer is

FlySAM2.0-F (SEQ ID NO:40): 5 '-TGGCCACGTAATAAGTGTGCGTT-3 '

FySAM2.0-R (SEQ ID NO:41): 5 '-TGGAACCAGACATGATAAGATACATTGATGAGT-3 '

Then two kinds of PCR products are subjected to the carrier flySAM2.0 after homologous recombination is integrated.

PVALIUM22 carrier (wherein, pVALIUM22 carrier reference literature " A genome- is cloned with the mode of PCR simultaneously Scale shRNA rescource for transgenic RNAi in Drosophila, Nat Methods.2011 May； 8 (5): 405-407.doi:10.1038/nmeth.1592 " is obtained) in K10polyA sequence, followed by SpeI and AvrII carries out digestion connection, and the SV40 sequence in flySAM2.0 is replaced with K10polyA sequence.K10poly A sequence can Enhance the stability and expression of transcript in reproductive system.

K10-F (SEQ ID NO:42):

5’-AGCCAAGGACCCCACTGTCTCCTAGGTCTGATCTGCTAGACAATTGTTGGCA-3’

K10-R (SEQ ID NO:43):

5’-GTTTTGTTTTCATCACTAGTCCAATCCGCCGCACCCTCAGCTCCAA-3’

Wherein K10ployA sequence (SEQ ID NO:44) is as follows:

TAACATTATACCTAAACCCATGGTCAAGAGTAAACATTTCTGCCTTTGAAGTTGAG AACACAATTAA GCATCCCCTGGTTAAACCTGACATTCATACTTGTTAATAGCGCCATAA ACATAGCACCAATTTCGAAGAAATCAG TTAAAAGCAATTAGCAATTAGCAATTAGCAA TAACTCTGCTGACTTCAAAACGAGAAGAGTTGCAAGTATTTGTA AGGCACAGTTTATA GACCACCGACGGCTCATTAGGGCTCGTCATGTAACTAAGCGCGGTGAAACCGAATTG AAC ATATAGTGGAATTATTATTATCAATGGGGAAGATTTAACCCTCAGGTAGCAAAGTA ATTTAATTGCAAATAGAGA GTCCTAAGACTAAATAATATATTTAAAAATCTGGCCCTTTG ACCTTGCTTGTCAGGTGCATTTGGGTTCAATCGT AAGTTGCTTCTATATAAACACTTTC CCCATCCCCGCAATAATGAAGAATACCGCAGAATAAAGAGAGATTTGCAA CAAAAAAT AAAGGCATTGCGAAAACTTTTTATGGGGGATCATTACACTCGGGCCTACGGTTACAAT TCCCAGCC ACTTAAGCGACAAGTTTGGCCAACAATCCATCTAATAGCTAATAGCGCAA TCACTGGTAATCGCAAGAGTATATA GGCAATAGAACCCATGGATTTGACCAAAGGTAA CCGAGACAATGGAGAAGCAAGAGGATTTCAAACTGAACACCC ACAGTGCTGTGTACT ACCACTGGCGCGTTTGGGAGCTCACTGGCCTGATGCGTCCTCCGGGCGTTTCAAGCCT G CTTTACGTGGTATACTCCATTACGGTCAACTTGGTGGTCACCGTGCTGTTTCCCTTGA GCTTGCTGGCCAGGCTG CTGTTCACCACCAACATGGCCGGATTGTGCGAGAACCTGA CCATAACTATTACCGATATTGTGGCCAATTTGAAG TTTGCGAATGTGTACATGGTGAGG AAGCAGCTCCATGAGATTCGCTCTCTCCTAAGGCTCATGGACGCTAGAGCC CGGCTGG TGGGCGATCCCGAGGAGATTTCTGCCTTGAGGAAGGAAGTGAATATCGCACAGGGCA CTTTCCGCAC CTTTGCCAGTATTTTCGTATTTGGCACTACTTTGAGTTGCGTCCGCGTG GTCGTTCGCCCGGATCGAGAGCTCCT GTATCCGGCCTGGTTCGGCGTTGACTGGATGC ACTCCACCAGAAACTATGTGCTCATCAATATCTACCAGCTCTT CGGCTTGATAGTGCAG GCTATACAGAACTGCGCTAGTGACTCCTATCCGCCTGCGTTTCTCTGCCTGCTCACGGG TCATATGCGTGCTTTGGAGCTGAGGGTGCGGCGGATTGG

Then, the p-transposase promoter in pVALIUM22 carrier is cloned also with the mode of PCR, then benefit Digestion is carried out with KpnI with EcoRV to connect, and the DSCP promoter in upper step carrier is replaced with into p- transposase promoter (p- transposase promoter).DSCP promoter is changed to p-transposase promoter, p- can be utilized The expression of transposase promoter promotor gene in reproductive system, so that expression efficiency is higher.

Wherein primer used in PCR amplification p-transposase promoter is respectively as follows:

P-trans-F (SEQ ID NO:45):

5’-CCAAGCTTGATATCATCGATCTCGAGGCTGCATCCAACGCGTTGGGAGCTCTCC GGATCAATTCGGCTTCAGGCACAGTCG-3’

p-trans-R(SEQ ID NO:46):

5’-GGCCATGGTGGCGGTACCAATGAACAGGACCTAAC-3’

Pcr amplification product is connected in carrier obtained in the previous step, then proceedes to obtain using KpnI cutting previous step Carrier, be then connected into the oligonucleotide fragment formed by following primer annealing, through sequence verification and ultimately form flySAMG Carrier, as shown in Figure 1.

Annealing-F(SEQ ID NO:47):5’-CCGCCCGGGGATCAGAATTGAGATCTGTGGTAC-3’

Annealing-R(SEQ ID NO:48):5’-CACAGATCTCAATTCTGATCCCCGGGCGGGTAC-3’

It is incorporated by SEQ ID NO:47 and SEQ ID NO:48 oligonucleotide sequences formed of annealing and is utilized SEQ ID NO:45 and SEQ ID NO:46 is as in the obtained amplified production of primer, as p-transposase promoter sequence. Wherein p-transposase promoter sequence (SEQ ID NO:49) is as follows:

AGCCGTAGCTTACCGAAGTATACACTTAAATTCAGTGCACGTTTGCTTGTTGAGAG GAAAGGTTGTG TGCGGACGAATTTTTTTTTGAAAACCGGTGATAGAGCCTGAACCAG AAAAGATAAAAGAAGGCTATACCAGTGGG AGTACACAAACAGAGTAAGTTTGAATAG TAAAAAAAATCATTTATGTAAACAATAACGTGACTGTGCGTTAGGTC CTGTTCATTGGT ACCCGCCCGGGGATCAGAATTGAGATCTGT

As seen from Figure 1, constructed flySAMG carrier (i.e. transcriptional activation system) includes two Expression elements, i.e., DCas9 protein expression element and sgRNA Expression element are also used to screen positive plasmid containing AmpR；AttB is used to entirely to carry The site attP on body site-directed integration to drosophila gene group；Vermilion is the marker gene of a screening transgenic drosophila；Two A insulator gypsy is located at the upstream of dCas9 protein expression element and sgRNA Expression element, to guarantee sgRNA table Up to the high efficient expression of element and dCas9 protein expression element；Two 5 X UAS are the binding sites of Gal4, under the action of Gal4 Start the expression of downstream gene；DCas9 Expression element includes dCas9 albumen coded sequence, is followed successively by VP64 code sequence downstream Column, T2A coded sequence, MCP albumen coded sequence, P65 code sequence sequence and HSF1 coded sequence form dCas9-VP64- T2A-MCP-P65-HSF1 Expression element, and the Expression element is using p-transposase promoter as promoter, can be with Start the expression of this dCas9-VP64-T2A-MCP-P65-HSF1 of downstream transcription, introne ftz sequence and K10polyA sequence position In the downstream of the Expression element, the stabilization and high efficient expression of this transcript can be helped；SgRNA Expression element is with U6B promoter Start the expression of sgRNA2.0, sgRNA2.0 can be connected on carrier between two Bbs I restriction enzyme sites, sgRNA MS2 structure containing the identification of MCP albumen on scafford (sgRNA stent sequence).

The activation of target gene in 4 drosophila reproductive system of embodiment

Dpp, tkv and bam are important for maintaining the self-renewing of drosophila ovarian germinal stem cell and differentiation to have the function of, The abnormal expression of any one gene can all generate the exception of germline stem cell and reproductive system, generate apparent genetic defect. In order to verify the validity of flySAMG system, the transcriptional activation that we construct these three genes according to the methods below turns base Because of drosophila, and have activated these three genes respectively in drosophila reproductive system.

1, the building of the flySAMG carrier of dpp, tkv and bam is activated

5 ' the UTR sequences of dpp, tkv and bam are searched in flybase (http://flybase.org/), and in each base Because 5 ' UTR upstream find PAM sequence (NGG), select close to higher 20 nucleotide of the CG content of the upstream of NGG as sgRNA.By the antisense base sequences (anti-sense oligo) and its reverse complementary sequence (sense oligo) of sgRNA It is individually placed to the corresponding position of following primer, wherein ttcg and aaac is the viscosity end after BbsI digestion flySAMG carrier respectively End, after the primer annealing of different genes, connect with the carrier after BbsI digestion:

5’-ttcg“anti-sense oligo”-3’

5’-aaac“sense oligo”-3’

Obtain six primers for three different genes:

Dpp-sg-F (SEQ ID NO:50): 5 '-ttcgCGTCCAAAGCGGCCGAGGCA-3 '

Dpp-sg-R (SEQ ID NO:51): 5 '-aaacTGCCTCGGCCGCTTTGGACG-3 '

Tkv-sg-F (SEQ ID NO:52): 5 '-ttcgCGTACGTACATATGGTGGGG-3 '

Tkv-sg-R (SEQ ID NO:53): 5 '-aaacCCCCACCATATGTACGTACG-3 '

Bam-sg-F (SEQ ID NO:54): 5 '-ttcgTACAATTATACACACTGATT-3 '

Bam-sg-R (SEQ ID NO:55): 5 '-aaacAATCAGTGTGTATAATTGTA-3 '

It after three pairs of primers of synthesis are annealed respectively, is connect with the flySAMG carrier after BbsI digestion, converts large intestine bar Bacterium DH5 α, selects positive colony, and correct plasmid, respectively flySAMG-dpp, flySAMG-tkv and flySAMG- is sequenced bam。

2, the acquisition of transgenic fly

It is y sc that 3 flySAMG carriers obtained in the previous step are injected into genotype respectively by way of microinjection v nanos-integrase；In the drosophila embryos of attP2,25 degrees Celsius of cultures are put in, and final by way of genetic integration Obtain homozygous transgene transcription activation drosophila.

3, influence of the activation dpp, tkv or bam to drosophila reproductive system

By transgenic fly obtained in step 2 and nanos-Gal4 tool drosophila hybrid, 25 DEG C are incubated at, humidity is 60% incubator, and observe the phenotype of offspring's female Drosophila ovary germarium.

As a result as shown in Fig. 2, wherein A represents control drosophila ovary germarium (control drosophila is does not carry out transgenosis in Fig. 2 The drosophila of processing and nanos-Gal4 tool drosophila hybrid female descendant obtained), B, C and D represent specific gene transcription The drosophila ovary germarium of activation.Wherein grey dot (1B1 signal) shown in figure label is germline stem cell.From figure In as can be seen that in control, containing there are two germline stem cells in normal germarium, and activate BMP signal path it is crucial because Sub- dpp and tkv can then inhibit the atomization of germline stem cell, generate the phenotype (B and C in Fig. 2) that germline stem cell increases； Germline stem cell can be promoted to break up in advance when activating crucial differentiation factor bam, eventually lead to the whole of germline stem cell and lose (D in Fig. 2).This result and these genes are known that be overexpressed phenotype identical, it was demonstrated that flySAMG transcriptional activation system exists In drosophila reproductive system can special, efficiently activating genes of interest expression, and generate corresponding phenotype.

In the description of the present invention, it is to be understood that, term " center ", " longitudinal direction ", " transverse direction ", " length ", " width ", " thickness ", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom" "inner", "outside", " up time The orientation or positional relationship of the instructions such as needle ", " counterclockwise ", " axial direction ", " radial direction ", " circumferential direction " be orientation based on the figure or Positional relationship is merely for convenience of description of the present invention and simplification of the description, rather than the device or element of indication or suggestion meaning must There must be specific orientation, be constructed and operated in a specific orientation, therefore be not considered as limiting the invention.

In addition, term " first ", " second " are used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance Or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be expressed or Implicitly include at least one this feature.In the description of the present invention, the meaning of " plurality " is at least two, such as two, three It is a etc., unless otherwise specifically defined.

In the present invention unless specifically defined or limited otherwise, fisrt feature in the second feature " on " or " down " can be with It is that the first and second features directly contact or the first and second features pass through intermediary mediate contact.Moreover, fisrt feature exists Second feature " on ", " top " and " above " but fisrt feature be directly above or diagonally above the second feature, or be merely representative of First feature horizontal height is higher than second feature.Fisrt feature can be under the second feature " below ", " below " and " below " One feature is directly under or diagonally below the second feature, or is merely representative of first feature horizontal height less than second feature.

In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any It can be combined in any suitable manner in a or multiple embodiment or examples.In addition, without conflicting with each other, the technology of this field The feature of different embodiments or examples described in this specification and different embodiments or examples can be combined by personnel And combination.

Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, modifies, replacement and variant.

Claims

1. a kind of transcriptional activation system, which is characterized in that the transcriptional activation system include dCas9 protein expression element and SgRNA Expression element,

Contain p- transposase promoter, dCas9 albumen coded sequence, MCP encoding histone sequence on the dCas9 protein expression element Column and activating transcription factor coded sequence, the activating transcription factor coded sequence are located at the dCas9 albumen coded sequence Downstream, the MCP albumen coded sequence are located at the downstream of the dCas9 albumen coded sequence；

Contain U6B promoter, sgRNA insertion point and MCP protein recognition sequences on the sgRNA Expression element.

2. transcriptional activation system according to claim 1, which is characterized in that the activating transcription factor coded sequence is selected from At least one of VP64 coded sequence, P65 coded sequence or HSF1 coded sequence；

Optionally, the activating transcription factor coded sequence includes VP64 coded sequence, P65 coded sequence and HSF1 code sequence Column, the VP64 coded sequence are located at the upstream of the MCP albumen coded sequence, the VP64 coded sequence and the MCP egg Contain self cleavage peptide T2A coded sequence between white coded sequence, the downstream of the MCP albumen coded sequence is followed successively by the P65 Coded sequence and the HSF1 coded sequence；

Optionally, the VP64 coded sequence is SEQ ID NO:21, and the coded sequence of the P65 is SEQ ID NO:26, institute Stating HSF1 coded sequence is SEQ ID NO:27, and the coded sequence of the self cleavage peptide T2A is SEQ ID NO:23；

Optionally, the primer sequence for expanding the P65 coded sequence is SEQ ID NO:24 and SEQ ID NO:25；

Optionally, the primer sequence for expanding the HSF1 coded sequence is SEQ ID NO:27 and SEQ ID NO:28.

3. transcriptional activation system according to claim 1 or 2, which is characterized in that the dCas9 protein expression element and institute SgRNA Expression element is stated to connect by plasmid vector；

Optionally, the plasmid vector is pNP plasmid；

Optionally, the MCP albumen coded sequence is SEQ ID NO:22；

Optionally, the MCP protein recognition sequences are MS2 sequence.

4. transcriptional activation system described in any one of claim 1 to 3, which is characterized in that further comprise:

At least two gypsy genes, the gypsy gene are located at the dCas9 protein expression element and the sgRNA table Up to the upstream of element,

Ftz intron sequences, the ftz intron sequences are located at the downstream of the dCas9 protein expression element；

K10polyA sequence, the K10poly A sequence are located at the downstream of the dCas9 protein expression element；

Optionally, the gypsy gene order is SEQ ID NO:5；The ftz intron sequences are SEQ ID NO:6；It is described K10polyA sequence is SEQ ID NO:44；

Optionally, further comprise: 10 × UAS sequence, 10 × UAS sequence are located at the upper of the p- transposase promoter Trip；

Optionally, 10 × UAS sequence is SEQ ID NO:10；

Optionally, further comprise: antibiotic marker genes, vermilion gene, attB gene；

Optionally, the antibiotic marker genes are ampicillin resistance gene, the core of the ampicillin resistance gene Acid sequence is SEQ ID NO:7, and the vermilion gene order is SEQ ID NO:8, and the attB gene order is SEQ ID NO:9。

5. a kind of transcriptional activation system characterized by comprising

DCas9 protein expression element, the dCas9 protein expression element include p- transposase promoter and dCas9 encoding histone Sequence, the dCas9 albumen coded sequence are located at the downstream of the p- transposase promoter, the dCas9 albumen coded sequence Downstream be followed successively by VP64 coded sequence, self cleavage peptide T2A coded sequence, MCP albumen coded sequence, P65 coded sequence and HSF1 coded sequence；

SgRNA Expression element contains U6B promoter, sgRNA insertion point, MS2 sequence and U6B on the sgRNA Expression element 3 ' non-translational region sequences；

At least two gypsy genes, the gypsy gene are located at the upstream of the dCas9 protein expression element and described The upstream of sgRNA Expression element；

K10polyA sequence, the K10polyA sequence are located at the downstream of the ftz intron sequences；

Antibiotic marker genes, vermilion gene and attB gene；

Optionally, the transcriptional activation system further comprises: 10 × UAS sequence, and 10 × UAS sequence is located at the p- and turns The upstream of seat enzyme promoters；

Optionally, the transcriptional activation system is as shown in Figure 1.

6. a kind of method for constructing transcriptional activation system according to any one of claims 1 to 5 characterized by comprising

(2) activating transcription factor coded sequence being connected on first recombinant plasmid, building obtains the second recombinant plasmid, Described in activating transcription factor coded sequence be located at the downstream of the dCas9 albumen coded sequence；

7. according to the method described in claim 6, it is characterized in that, step (1) further comprises:

Cas9 albumen coded sequence is connected on plasmid vector by (1-1), and building obtains including Cas9 albumen coded sequence Third recombinant plasmid；

(1-2) is mutated the Cas9 albumen coded sequence on the third recombinant plasmid, obtains including described First recombinant plasmid of dCas9 albumen coded sequence；

Optionally, the plasmid vector is pNP plasmid；

Optionally, the primer sequence for expanding the Cas9 albumen coded sequence is SEQ ID NO:1 and SEQ ID NO:2, The dCas9 albumen coded sequence is SEQ ID NO:19；

Optionally, the Cas9 albumen coded sequence is connected to the plasmid by way of homologous recombination and carried by step (1-1) On body；

Optionally, utilize reverse complemental mutant primer SEQ ID NO:11 and SEQ ID NO:12 to described in step (1-2) The base that the 10th amino acids are encoded on Cas9 albumen coded sequence is mutated, and reverse complemental mutant primer SEQ ID is utilized NO:13 and SEQ ID NO:14 is mutated the base of the 840th amino acids in the Cas9 albumen coded sequence；

Optionally, the activating transcription factor coded sequence is connected to described by way of homologous recombination in step (2) On one recombinant plasmid；

Optionally, sgRNA Expression element is connected to second recombinant plasmid by way of homologous recombination in step (3) On.

8. method according to claim 6 or 7, which is characterized in that on the sgRNA Expression element containing stent sequence and 3 ' non-translational region sequence of U6B promoter contains MCP protein recognition sequences in the stent sequence；

Optionally, the stent sequence is SEQ ID NO:34, and the sequence of the U6B promoter is SEQ ID NO:36, described 3 ' non-translational region sequence of U6B promoter is SEQ ID NO:37.

9. a kind of method of prepare transgenosis drosophila characterized by comprising

(a) the sgRNA sequence for targeting target gene is imported into transcriptional activation system according to any one of claims 1 to 5 In, obtain the transcriptional activation system of targeting target gene；

(b) the transcriptional activation system introducing of the targeting target gene is obtained into transgenic fly into drosophila embryos；

Optionally, step (a) further comprises:

(a-1) the transcriptional activation system is subjected to digestion processing, obtains the transcriptional activation system by digestion processing；

(a-2) the sgRNA sequence of the targeting target gene after annealed pairs is connected to turn by digestion processing In the sgRNA insertion point for recording activation system, so that the sgRNA sequence of the targeting target gene is imported into described turn It records in activation system.

10. a kind of method of gene expression in activation drosophila reproductive system characterized by comprising

The sgRNA sequence for targeting target gene is imported into transcriptional activation system according to any one of claims 1 to 5, Obtain the transcriptional activation system of targeting target gene；

By the transcriptional activation system introducing of the targeting target gene into drosophila embryos, transgenic fly is obtained；

By the transgenic fly and the drosophila hybrid culture of Gal4 tool, drosophila offspring is obtained, to activate drosophila reproductive system The expression of middle target gene.