CN109517840B - Efficient transcriptional activation system in drosophila reproductive system - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a transcription activation system and application thereof in a drosophila reproductive system. The transcriptional activation system comprises a dCas9 protein expression element and a sgRNA expression element, wherein the dCas9 protein expression element comprises a p-transposase promoter, a dCas9 protein coding sequence, an MCP protein coding sequence and a transcriptional activator coding sequence, the transcriptional activator coding sequence is positioned at the downstream of the dCas9 protein coding sequence, and the MCP protein coding sequence is positioned at the downstream of the dCas9 protein coding sequence; the sgRNA expression element contains a U6B promoter, an sgRNA insertion site and an MCP protein recognition sequence. The transcription activation system provided by the invention can activate the expression of genes in the drosophila reproductive system, and has high activation efficiency.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for efficiently regulating gene expression in an organism, and especially relates to a transcription activation system applied to a drosophila reproductive system.
Background
With the successful completion of genome plans of species such as human beings, the sequence information of the genome is converted into functional information, and life passwords are decoded, so that the method has important significance for comprehensively understanding the processes of human growth and development, disease aging and the like. The fruit fly has many advantages as a model organism highly conserved with human, and the research result in fruit fly is also suitable for human, and can get rid of the limitation of ethical life, thus becoming an ideal model organism for biomedical research. The fruit fly reproductive system is an ideal model for researching stem cells and reproductive development in vivo due to the characteristics of high conservation, definite cell number and position of reproductive stem cells and the like, easy marking, clear interaction mechanism among cells and the like. Regulating the expression of a target gene is the most common technical means in the field of life science, mainly by reducing the gene function and enhancing the gene function. In the fruit fly reproductive system, technical means for reducing gene functions are mature, mainly comprising a transgenic interference technology, a CRISPR/Cas9 gene editing technology and the like, but technical means for enhancing gene expression are limited, and the deep development of related researches is limited.
The existing technology relies mainly on the traditional Gal4/UAS system, by expressing the coding sequence of the gene of interest carried by a foreign vector. Although the method can express the target gene in a specific tissue or organ, the cloning steps of the target gene are complicated, the period is long, the expression mode of the gene can not be simulated, false positive results often occur, a plurality of genes can not be simultaneously over-expressed, and large-scale construction and screening work in a genome range can not be realized. When the existing CRISPR/Cas9 transcription activation system is applied to the reproductive system, the expression efficiency of the regulatory gene is limited. Therefore, further improvements are needed for activation and high expression of genes in the reproductive system.
Disclosure of Invention
The present invention is intended to solve at least one of the technical problems of the related art to an extent that the efficiency of transcriptional activation of a gene is improved. Therefore, the invention provides a transcription activation system, which uses a CRISPR/dCas9 system to mediate to efficiently activate a target gene in the reproductive system of Drosophila, so as to realize the regulation and control of the gene, thereby improving the efficiency of the transcription activation of the gene, and particularly can be used for improving the efficiency of the transcription activation of the gene in the reproductive system of Drosophila.
The inventor of the invention finds out in the research process that: after the enzyme active site of the Cas9 protein in the CRISPR/Cas9 system is mutated (changed into dCas9), if a proper transcription activator is fused at the C end of dCas9, in-situ transcription activation of a target gene can be realized under the action of sgRNA. And the CRISPR/Cas9 system is modified by combining the specificity of reproductive system expression, so that the transcriptional activation and expression of genes in the reproductive system can be simply, quickly and efficiently realized.
Therefore, aiming at the defect of enhancing the gene expression mode in the reproductive system of the fruit fly, the invention constructs a flySAMG system as a transcription activation system, and can solve the following problems: 1. the problem of complicated gene overexpression operation in the drosophila reproductive system; 2. the problem that a plurality of genes cannot be activated simultaneously in the fruit fly reproductive system; 3. the problem of low gene activating efficiency in the existing system reproductive system; 4. the construction and screening of genome-wide transcription-activating lines in the Drosophila reproductive system.
Specifically, the invention provides the following technical scheme:
according to a first aspect of the present invention, there is provided a transcriptional activation system comprising a dCas9 protein expression element comprising a p-transposase promoter, a dCas9 protein coding sequence, an MCP protein coding sequence and a transcriptional activator coding sequence, on the dCas9 protein expression element, the transcriptional activator coding sequence being located downstream of the dCas9 protein coding sequence, and the MCP protein coding sequence being located downstream of the dCas9 protein coding sequence; the sgRNA expression element contains a U6B promoter, an MCP protein recognition sequence and an sgRNA insertion site. The MCP (MS2coating protein) protein coding sequence is capable of encoding an MCP protein that recognizes the MS2 structure on the sgRNA expression element, thereby allowing the dCas9 protein expression element to recognize and bind to the gene of interest under the guidance of the sgRNA expression element. And a transcription activator coding sequence contained on the dCas9 protein expression element can code a transcription activator, and a p-transposase promoter can start the expression of the dCas9 protein expression element in a reproductive system, so that the aim of activating the expression of a target gene is fulfilled. The transcription activation system provided by the invention utilizes the transcription activation factor coding sequence on the dCas9 protein expression element and the expression of the p-transposase promoter regulating gene in a reproductive system, and uses U6B as a promoter to regulate the expression of sgRNA, so that the expression level of the sgRNA is higher, and the activation efficiency of the transcription activation system is improved. In the sgRNA expression element, both the sgRNA insertion site and the MCP protein recognition sequence are located downstream of the U6B promoter. The transcriptional activation system provided by the invention can be particularly applied to transcriptional activation of genes in a drosophila reproductive system, is simple to operate, has higher activation efficiency, and is convenient for large-scale high-flux operation.
According to an embodiment of the present invention, the transcriptional activation system described above may further include the following technical features:
in some embodiments of the invention, the transcriptional activator coding sequence is selected from at least one of a VP64 coding sequence, a P65 coding sequence, or a HSF1 coding sequence. The VP64 coding sequence can encode V64 protein (tetramer of herpes simplex virus protein VP 16), P65 coding sequence can encode P65 protein (NF-kB transactivating subunit), HSF1 coding sequence can encode HSF1 protein (human heat shock factor 1), and these proteins can be used as transcription activator to activate gene expression.
In some embodiments of the invention, the transcriptional activator coding sequence comprises a VP64 coding sequence, a P65 coding sequence, and an HSF1 coding sequence, the VP64 coding sequence is upstream of the MCP protein coding sequence, the VP64 coding sequence and the MCP protein coding sequence contain a self-cleaving peptide T2A coding sequence therebetween, and the P65 coding sequence and the HSF1 coding sequence are downstream of the MCP protein coding sequence, in that order. The coding sequence of the dCas9 protein can code dCas9 protein, VP64 protein is fused at the C terminal of dCas9 protein, and P65 and HSF1 transcription activator are further fused at the downstream of MCP protein by using self-cleavage peptide T2A. The self-cleavage peptide T2A is a small peptide capable of self-cleavage at the translation level, which enables the dCas9 protein expression element to self-cleave into two separate parts after the whole mRNA is generated, dCas9-VP64 and MCP-P65-HSF1, and can stably improve the activation efficiency of the transcriptional activation system. Self-cleaving peptides may also be referred to herein as self-cleaving peptides.
In some embodiments of the invention, the VP64 encoding sequence is SEQ ID NO:21, the P65 encoding sequence is SEQ ID NO:26, the HSF1 encoding sequence is SEQ ID NO:27, and the self-cleaving peptide T2A encoding sequence is SEQ ID NO: 23.
In some embodiments of the invention, the primer sequences used to amplify the P65 coding sequence are SEQ ID NO. 24 and SEQ ID NO. 25.
In some embodiments of the invention, the primer sequences used to amplify the HSF1 coding sequence are 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 are linked by a plasmid vector. The dCas9 protein expression element and the sgRNA expression element are ligated to the same plasmid vector. Thereby ensuring that the two drugs can simultaneously act in the organism.
In some embodiments of the invention, the plasmid vector is a pNP plasmid.
In some embodiments of the invention, the MCP protein coding sequence is SEQ ID NO 22.
In some embodiments of the invention, the MCP protein recognition sequence is a MS2 sequence.
In some embodiments of the invention, further comprising: at least two gypsy genes located upstream of the dCas9 protein expression element and the sgRNA expression element, respectively; ftz intron sequence and a K10polyA sequence, the ftz intron sequence and the K10polyA sequence being located downstream of the dCas9 protein expression element. The transcription gene system at least comprises two gypsy genes, wherein the gypsy genes are positioned at two ends of a dCas9 protein expression element and an sgRNA expression element and are used as insulators for shielding interference and factors around an inserted gene, so that the high-efficiency expression of the two expression elements is facilitated. ftz intron sequences are located downstream of the dCas9 protein expression elements to enhance translation. The K10polyA sequence is located downstream of the dCas9 protein expression element and is useful for regulating the termination of transcription. In one embodiment, the K10polyA sequence is located downstream of the ftz intron sequence.
In some embodiments of the invention, the nucleic acid sequence of the gypsy gene is SEQ ID No. 5; the ftz intron sequence is SEQ ID NO 6; 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 located in the p-transposase promoter upstream. It can help to realize the transcriptional activation of the target gene in specific tissue organs and developmental stages driven by tissue organ specific Gal 4.
In some embodiments of the invention, the transcriptional activation system further comprises an antibiotic marker gene, a verimion gene, and an attB gene; wherein the vermileon gene is used as a marker gene for screening transgenic fruit flies. The attB gene is used for site-directed integration of the transcriptional activation system into the Drosophila genome. The antibiotic marker gene, the vermileon gene and the attB gene are positioned outside the gypsy gene and are used for assisting the construction and the screening of a transcription activation system in an organism without influencing the expression of a dCas9 protein expression element and an sgRNA expression element.
In some embodiments of the invention, the antibiotic marker gene is an ampicillin resistance gene having the nucleic acid sequence of SEQ ID NO 7; the nucleic acid sequence of the vermileon gene is SEQ ID NO. 8; the nucleic acid sequence of the attB gene is SEQ ID NO. 9.
In a specific example of the present invention, the transcription activation system includes: a dCas9 protein expression element, the dCas9 protein expression element comprising a P-transposase promoter and a dCas9 protein coding sequence, the dCas9 protein coding sequence being located downstream of the P-transposase promoter, the dCas9 protein coding sequence being downstream of the VP64 coding sequence, the self-splicing peptide T2A coding sequence, the MCP protein coding sequence, the P65 coding sequence, and the HSF1 coding sequence in that order; an sgRNA expression element comprising a U6B promoter, an MS2 sequence, and an sgRNA insertion site thereon; at least two gypsy genes, said gypsy genes being located upstream of said dCas9 protein expression element and upstream of said sgRNA expression element, respectively; ftz intron sequence, the ftz intron sequence being downstream of the dCas9 protein expression element; a K10polyA sequence, said K10polyA sequence being downstream of said ftz intron sequence; antibiotic marker gene, verimion gene and attB gene. On this basis, the transcription activation system may further include a 10 × UAS sequence, and the 10 × UAS sequence is located upstream of the p-transposase promoter.
In one specific example of the present invention, the transcriptional activation system is shown in FIG. 1.
According to a second aspect of the present invention, there is provided a method of constructing a transcriptional activation system according to the first aspect of the present invention, the method comprising:
(1) constructing a first recombinant plasmid containing a dCas9 protein coding sequence by using a plasmid vector;
(2) ligating a transcription activator coding sequence to the first recombinant plasmid to construct a second recombinant plasmid, wherein the transcription activator coding sequence is located downstream of the dCas9 protein coding sequence;
(3) and connecting the sgRNA expression element to the second recombinant plasmid to construct a transcriptional activation system.
According to an embodiment of the present invention, the method for constructing a transcription activation system as described above may further include the following technical features:
in some embodiments of the invention, step (1) further comprises:
(1-1) connecting the Cas9 protein coding sequence to a plasmid vector to construct a third recombinant plasmid containing the Cas9 protein coding sequence;
(1-2) mutating the Cas9 protein coding sequence on the third recombinant plasmid to obtain a first recombinant plasmid comprising the dCas9 protein coding sequence.
In some embodiments of the invention, the plasmid vector is a pNP plasmid.
In some embodiments of the invention, the primer sequence used to amplify the Cas9 protein coding sequence is SEQ ID NO:1 and SEQ ID NO:2, the Cas9 protein coding sequence is SEQ ID NO: 19.
in some embodiments of the invention, step (1-1) links the Cas9 protein-encoding sequence to the plasmid vector by means of homologous recombination.
In some embodiments of the invention, the base encoding amino acid 10 of the Cas9 protein coding sequence is mutated in step (1-2) with reverse complementary mutation primers SEQ ID NO:11 and SEQ ID NO:12, and the base encoding amino acid 840 of the Cas9 protein coding sequence is mutated with reverse complementary mutation primers SEQ ID NO:13 and SEQ ID NO: 14.
In some embodiments of the invention, the transcriptional activator coding sequence is ligated to the first recombinant plasmid by homologous recombination in step (2);
in some embodiments of the invention, the sgRNA expression element is ligated to the second recombinant plasmid by means of homologous recombination in step (3).
In some embodiments of the invention, the sgRNA expression element comprises a scaffold sequence comprising an MCP protein recognition sequence and a U6B promoter 3' untranslated region sequence.
In some embodiments of the invention, the scaffold sequence is SEQ ID NO 34, the U6B promoter sequence is SEQ ID NO 36, and the U6B promoter 3' untranslated region sequence is SEQ ID NO 37.
According to a third aspect of the present invention, there is provided a method of producing a transgenic fruit fly, comprising:
(a) introducing a sgRNA sequence of a target gene into the transcriptional activation system according to any embodiment of the first aspect of the present invention to obtain a transcriptional activation system of the target gene;
(b) and (3) introducing the transcription activation system containing the target gene into a drosophila embryo to obtain the transgenic drosophila.
In some embodiments of the invention, step (a) further comprises: (a-1) carrying out enzyme digestion treatment on the transcription activation system to obtain the transcription activation system subjected to enzyme digestion treatment; (a-2) connecting the sgRNA sequence of the target gene subjected to annealing pairing to the sgRNA insertion site of the transcription activation system subjected to enzyme digestion treatment, so as to introduce the sgRNA sequence of the target gene into the transcription activation system.
According to a fourth aspect of the present invention, there is provided a method of activating gene expression in the reproductive system of Drosophila comprising: introducing an sgRNA sequence of a target gene into a transcription activation system to obtain the transcription activation system of the target gene, wherein the transcription activation system is the transcription activation system according to the first aspect of the invention; introducing the transcription activation system of the target gene into a drosophila embryo to obtain a transgenic drosophila; and (3) hybridizing and culturing the transgenic drosophila and the Gal4 tool drosophila to obtain drosophila offspring, thereby activating the expression of the target gene in the drosophila reproductive system.
The beneficial effects obtained by the invention are as follows: the method for preparing the transcription activation system is simple to operate; the transcription activation system provided by the invention has high activation efficiency; can activate multiple genes simultaneously; is convenient for large-scale high-flux operation.
Drawings
FIG. 1 is a schematic diagram of a transcriptional activation system, fliSAMG, constructed according to one embodiment of the present invention.
FIG. 2 is a schematic diagram of a Drosophila ovary ovule region constructed according to an embodiment of the present invention, wherein A represents a control Drosophila ovary ovule region (control represents control), B represents a Drosophila ovary ovule region with transcriptional activation of a dpp gene (dpp activation represents dpp gene activation), C represents a Drosophila ovary ovule region with transcriptional activation of tkv gene (tkv activation represents tkv gene activation), and D represents a Drosophila ovary ovule region with transcriptional activation of a bam gene (bam activation represents bam gene activation).
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Herein, the term "transcription activation system" may also be expressed as "transcription activation vector", meaning a vector or system or component capable of activating transcription of a gene.
As used herein, the term "coding sequence" refers to a sequence capable of encoding a protein product, and may refer to a DNA nucleic acid sequence that expresses a protein product, or may refer to an RNA nucleic acid sequence that expresses a protein product.
The dCas9 protein expression element herein refers to an expression element capable of expressing dCas9 protein, the sgRNA expression element refers to an expression element capable of inserting sgRNA including an sgRNA insertion site, and the "expression element" refers to a unit capable of performing a corresponding function as a functional unit. The dCas9 protein expression element may refer to a nucleic acid sequence capable of expressing dCas9 protein, and the sgRNA expression element may refer to a nucleic acid sequence capable of inserting a sgRNA sequence targeting a target gene. Wherein the dCas9 protein has no nuclease activity compared to the Cas9 protein, but still recognizes a specific sgRNA (single-guide RNA) and is still able to bind to a specific DNA sequence. According to an embodiment of the invention, compared to the Cas9 protein, the dCas9 protein has a mutation of the amino acid at position 10 from D to a and a mutation of the amino acid at position 840 from H to a.
Herein, when describing the positional relationship between nucleic acid sequences, the expressions "upstream" and "downstream" refer to a nucleic acid sequence located upstream closer to the 5 'end of an expression element and a nucleic acid sequence located downstream closer to the 3' end of the expression element in the order of replication or transcription of the nucleic acid sequences. This "upstream" and "downstream" relationship corresponds to the N-terminus and C-terminus, respectively, of the expressed protein. It should be noted that the "upstream" and "downstream" relationship does not necessarily mean that two nucleic acid sequences are directly connected, and the expression merely represents a relative positional relationship. Other nucleic acid sequences may be inserted between the "upstream" and "downstream" nucleic acid sequences as long as the insertion of the other nucleic acid sequences does not affect the functioning or expression of the inserted nucleic acid sequence.
Herein, when describing the connection relationship of two nucleic acid sequences, unless "directly connected" means that two nucleic acid sequences are directly connected to each other via a 3 '-5' phosphodiester bond, and no other nucleic acid sequence can be inserted. "linked" does not necessarily mean that a nucleic acid sequence cannot be inserted between two nucleic acid sequences, as long as the other nucleic acid sequences inserted do not interfere with the functioning and expression of the inserted nucleic acid sequence.
The different nucleic acid sequences shown herein, such as the VP64 coding sequence (SEQ ID NO:21), the P65 coding sequence (SEQ ID NO:26), the gypsy gene sequence (SEQ ID NO:5), are only by way of a preferred example, meaning a nucleic acid sequence capable of expressing the corresponding protein or performing the corresponding function. The skilled person can substitute or partially substitute the nucleic acid sequence as required, and these substitutions or partial substitutions are also included in the scope of the present invention as long as they can express the corresponding protein or exert the corresponding function. The nucleic acid sequences are shown in the 5 'to 3' order as is common in the art.
The CRISPR/Cas9 system is the most widely used gene editing system at present, and the Cas9 protein can specifically recognize and cut a target gene under the guidance of sgRNA, so that accurate and efficient gene editing is realized. After mutating the enzymatically active site of Cas9 protein (dCas9), dCas9 still recognizes and binds the gene of interest, but does not cleave, under the guidance of sgRNA. In situ transcriptional activation of a gene of interest can be achieved if a suitable transcriptional activation domain is fused to the C-terminus of dCas9 protein.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
In the following examples, we have used plasmid vectors to obtain the dCas9 protein expression element and sgRNA expression element of interest in the process of constructing the dCas9 protein expression element and sgRNA expression element, and thus the dCas9 protein vector and the sgRNA vector represent the processes in the preparation of the dCas9 protein expression element and the sgRNA expression element in the following description.
During the course of the study, we used the self-cleaving peptide T2A to express two fused activator proteins dCas9-VP64 and MCP-p65-HSF1 simultaneously and downstream of the p-transposase promoter. Because the U6B promoter has higher activity, we used the U6B promoter to control expression of sgrna 2.0. The sgRNA2.0 has two stem-loop structures containing MS2 sites, can be specifically recognized and combined by MCP protein, and achieves the aim of recruiting MCP-p65-HSF1 transcriptional activator. Therefore, the transcriptional activation activity of three transcription factors, VP64, p65 and HSF1, is integrated in the system. In the using process, a vector of sgRNA2.0 and a vector expressing dCas9-VP64-T2A-MCP-p65-HSF1 are integrated into a vector flySAMG, and gypsy sequences are respectively inserted into the upstream of dCas9activator and the downstream of sgRNA2.0, so that the influence of other sequence elements on the expression of the two is reduced, and the transcription of genes is enhanced. The system can be conveniently used for expressing a plurality of sgRNAs simultaneously by an enzyme digestion connection mode, and the purpose of activating a plurality of target genes simultaneously is achieved. In addition, the flySAMG vector also comprises ampicillin resistance genes for screening positive plasmids, genetic screening markers (Vermilion genes) for selecting target transgenic fruit flies, attB sequences for helping to construct site-specific transgenic fruit flies, ftz sequences for helping dCas9activator to transcribe and translate and K10polyA sequences.
The application utilizes a Gal4/UAS binary expression system to activate the expression of a target gene in a drosophila reproductive system, and a 10 XUAS sequence is contained at the upstream of a p-transposase promoter, so that the transcriptional activation of the target gene can be realized in a specific tissue organ and a development stage under the drive of tissue organ specificity Gal 4. The transcriptional activation of the target gene in the offspring Drosophila can be realized only by simple crossing of the transgenic Drosophila with flySAMG and the specific Gal4 tool Drosophila.
Example one
The CRISPR/Cas9 system is the most widely used gene editing system at present, the Cas9 protein can cleave a specific position of a genome under the guidance of sgRNA, but after the active site of the Cas9 protein is mutated, the formed dCas9 can still recognize and bind to a specific gene under the guidance of sgRNA, but does not cleave the genome. Thus, dCas9 can be combined with a specific DNA sequence to recruit a transcription activator capable of activating gene expression, and can be used to study the transcription and expression of gene expression.
This example provides a process for preparing dCas9 protein comprising the steps of:
1. cloning of Cas9 protein coding sequence
With reference to Ren X, Sun J, Housden B E, et al, optimized gene encoding technology for Drosophila melanogaster using germ line-specific Cas9[ J ]. Proceedings of the National Academy of Sciences, 2013,110(47):19012-7, the Cas9 protein coding sequence was obtained by PCR amplification using the non-Cas9 vector described in the literature, wherein the primers used were respectively
NLS-Cas9-F(SEQ ID NO:1):
5’-AGAAGCGGAAGGTCGGTATCCACGGTGTCCCAGCAGCCATGGACAAGAAGTAC TCCATTGGGCT-3’
NLS-Cas9-R(SEQ ID NO:2):
5’-TCTTAGCTTGACCAGCTTTCTTAGTAGCAGCAGGACGCTTGTCTCCACCGAGCT GAGAGAGG-3’
The Cas9 protein coding sequence was amplified using the primers as above, and the resulting Cas9 protein coding sequence still had nucleolytic activity.
2. Plasmid vector backbone cloning
The plasmid backbone of the fliSAMG vector was engineered from the pNP vector. Among them, the pNP vector was obtained in the Article "An effective and multiple target transgenic RNAi technique with low sensitivity in Drosophila, Nature Communications 9, aromatic number:4160 (2018)". Cloning the required fragment on the pNP vector by means of PCR, wherein the PCR primers used 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’
the amplification product obtained contained the gypsy gene, ftz intron sequence, ampicillin resistance gene, vermile gene, attB gene, SV40polyA sequence and 10 × UAS sequence, and the gypsy gene, ftz intron sequence, ampicillin resistance gene, vermile gene, attB gene and 10 × UAS sequence and DSCP promoter sequence are shown below, while the SV40polyA sequence and DSCP promoter sequence were replaced in the subsequent processing and are not shown.
The gypsy gene sequence (SEQ ID NO:5) is as follows:
TTGGCCACGTAATAAGTGTGCGTTGAATTTATTCGCAAAAACATTGCATATTTTCGG CAAAGTAAAATTTTGTTGCATACCTTATCAAAAAATAAGTGCTGCATACTTTTTAGAGA AACCAAATAATTTTTTATTGCATACCCGTTTTTAATAAAATACATTGCATACCCTCTTTTA ATAAAAAATATTGCATACTTTGACGAAACAAATTTTCGTTGCATACCCAATAAAAGATT ATTATATTGCATACCCGTTTTTAATAAAATACATTGCATACCCTCTTTTAATAAAGAATAT TGCATACGTTGACGAAACAAATTTTCGTTGCATACCCAATAAAAGATTATTATATTGCAT ACCTTTTCTTGCCATACCATTTAGCCGATCAATTCTGCTCGGCAACAGTATATTTGTGGT GTGCCAACCAACAAC
ftz intron sequence (SEQ ID NO:6) is as follows:
CTAGTTCTGATCTGCTAGACAATTGTTGGCATCAGGTAGGCATCACACACGATTAA CAACCCCTAAAAATACACTTTGAAAATATTGAAAATATGTTTTTGTATACATTTTTGATA TTTTCAAATAATACGCAGTTATAAAACTCATTAGCTAACCCATTTTTTCTTTGCTTATGC TTACAGATTGCAAAGAACTAGAG
the ampicillin resistance sequence (SEQ ID NO:7) is as follows:
ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTT CCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTG GGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGT TTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCG CGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTC TCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATG ACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACT TACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGG GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAA CGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATT AACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCG GATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTG ATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGA TGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGAT GAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAA
the vermileon gene sequence (SEQ ID NO:8) is as follows:
ATTTATTTTGTTATGTTATATGTATTATATGTCAGACATAAAGAAAAGGAACACATCA AATGTGATAACAAAGACTAAACAAGTAATTTTATTACACCAAAACGACAAAACAGTAG GCAGAACAAACAACGCATAGCCAAACATTGACGAATTGGATACCCTGCCGATTGTCA GACACTTTTGTTGATCAGTTTCTTGCGAATGGTCTCGTCCAGCGGTGGAATCGCCTCG CGGGGAATCAGAAAAGTGGACAGATTGAACAGATCCAGAAACACCTTGTACCGATCA CTGAAACCAAAAAAAAACAAAGGGAGAACAGTTTGAGTTCATTGATCCCCGATATAA TCACATCTGCGATGATCACCTGAGAGTGGAGCGCAGATATTGATAACCAGACGAGCCA CCAGTGCCCAACTGTTGCGATCCAATCATGCGTTGCACCATGATCACGTGATTGTCTGC GGCGGGAATAGAAAGTATTTGGTTAGGAAAACCAGTCTTAAACATAAGATATATTTATA AAAGAGTATCAAAGAATGCAATACTTACATCTCCACTTGGTTATTAACGAGTCGATGTC CATGAGCAGGGTGAGCAACTGGTGTGGTTGGCTGAACCTGGGTTCATCCCTATAGAA GGTGATCATGATGGCTCCCTGAAGGGCACGATGGCTAAACCGGCGATCCCCACGACG CACCAGTGCATCGTGCACTGCCGGATCAAAGATGGAGCGATACACCTCGCGTCGCTTC TCAATGTCCATGAGGCGGTAGTTTTTCGCCTTCTCCACGGGCTCCTCCATGGCGCTCTG TACCTGCGCCTCCAGGAATCGATCGACGCTCTCCTGAAACTTGGCCCAGAAGTTGAA GCCACTCTCCTCCAGTCCGGGCGTCCTCTCCAGCCATCGCTGCACTAGCTCCAGTAGC GAGGGATCTTTCTCCGAGTTGCGAATCGAGTTCCGCGCCTCCTCGTCGCTAAAGACAT CCGAGTACTTCTGGTTGTATCTCACCCGCTGCTCTGTCAGAACTCCCAGCTTGTTCTCG ATCAAACGGAACTGCAGCGACTGAAAACCAGATGCGGGTGCCAGGTACTTGCGGAA GTCCATGAAGTCTAGCGGGGTCATGGTCTCCAGAATGGGCACTTGGTCCACCAGGAG CTGTACAAAGGAAGTTATAAACGGATTTTGGTAAGAGATTCAGAAAGCACTCACTTTT AGAATCAGAACCACTCGGTTCAGTCGCTTGACAATCTCCAGCGTCTTGGTTTCATCGA TGACCTCTGCATCCAACATGTCTCGTATGGAGTCGAACTCAAAGATGATCTGCTTGAA CCAAAGCTCGTAGGCTGTGGCGAAGGTACTTAAATGCCATTGAGTGTTGTCATCAAAG TTGTAAACCTACTCACCCTGGTGCGTGATGATGAACAGATGCTCATCGTGCACGGGTC GCTTGTCCTCCTCGGACAGCATACACTGGGCATCCAGCAGTTTGTCCAGCATCAGATA CTCTCCATAGATTTTGCCCACTTCCGTGGTTAATGGCACCGCCGAATCATCGTGATCGT TTCTGTATGGGTTTGAATTGAATCGCAGAACTGAAGATCGATTGGCATTCCTGGACAG CACGTGCTGGTGCTCACCCGTTTCCTGCATAGGGACAGCTCATGGTGCACAGCTCAGA TCAGATCGTGACTCCTCGACCGGCGGATGCTGGCGAACTGATCTCCGCCAGCGGACC GGAGATGAGACCCCAGCGAACCGATAACAGAGCGAGAGAGCTCCAGTTCCGACTGAT TGCACAGTCGGTGATCTGGGCGATGGGCACTGCCAGATAGGCTGGGAATTATCAATCA CTTGAGGTGAAAGTGCGGCGCACACAAAT
the attB sequence (SEQ ID NO:9) is as follows:
GCTGCATCCAACGCGTTGGGAGCTCTCCGGATCAATTCGGCTTCACGTACCGTCG ACGATGTAGGTCACGGTCTCGAAGCCGCGGTGCGGGTGCCAGGGCGTGCCCTTGGGC TCCCCGGGCGCGTACTCCACCTCACCCATCTGGTCCATCATGATGAACGGGTCGAGGT GGCGGTAGTTGATCCCGGCGAACGCGCGGCGCACCGGGAAGCCCTCGCCCTCGAAAC CGCTGGGCGCGGTGGTCACGGTGAGCACGGGACGTGCGACGGCGTCGGCTGGTGCG GATACGCGGGGCAGCGTCAGCGGGTTCTCGACGGTCACGGCGGGCATGTCGACAAGC CGAATTGATCCACTAGAAGGCCTAATTC
10×UAS(SEQ ID NO:10)：
GCAGGTCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGT CCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGACTCCC GCGGTCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTC CGAGCGGAGTAC
3. construction of pNP-dCas9
And (3) connecting the products obtained in the steps (1) and (2) in a homologous recombination mode, transforming the products into escherichia coli DH5 alpha, selecting positive clones, and marking the plasmids with correct sequencing as pNP-Cas 9.
The nuclease cleavage activity of Cas9 depends on two domains: RuvC and HNH, which are responsible for cleaving the two strands of the DNA strand, respectively, and which can be individually inactivated by artificial point mutations. The Cas9D10A mutant obtained by mutating D (i.e. aspartic acid, Asp) to a (i.e. alanine, Ala) at position 10 in Cas9 protein, exhibited RuvC inactivation, HNH still exhibited activity; by mutating H (namely histidine, His) at the 840 th site in the Cas9 protein to A, the obtained Cas9H840A mutant shows HNH inactivation, RuvC still has activity, and Cas9 of the two mutants still has nuclease activity and can perform a shearing action on a target sequence. When RuvC and HNH are simultaneously in an inactivated state, Cas9 will have no nuclease activity, becoming dCas9(dead Cas 9).
Thus, the pNP-Cas9 plasmid was mutated in the following manner to express D to a and H to a at position 840 of a part of the Cas9 protein to form dCas9 without enzymatic cleavage activity.
PCR amplification was performed using a pair of reverse complementary mutation primers using pNP-Cas9 as a template. After the PCR product is digested by Dpn I, directly transforming escherichia coli DH5 alpha, selecting monoclonal shake bacteria, extracting plasmid, sequencing and identifying to obtain correct mutant plasmid. The primers used were respectively:
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’
two BbsI cleavage sites (BbsI 1703 and BbsI 2149) in Cas9 were mutated in the same manner as above for subsequent use, and the resulting vector was designated pNP-dCas 9. The primers used were respectively:
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 coding sequence (SEQ ID NO:19) in the obtained pNP-dCas9 vector is as follows:
ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGTGTCCCAGCAGCCATG GACAAGAAGTACTCCATTGGGCTCGCTATCGGCACAAACAGCGTCGGCTGGGCCGTC ATTACGGACGAGTACAAGGTGCCGAGCAAAAAATTCAAAGTTCTGGGCAATACCGAT CGCCACAGCATAAAGAAGAACCTCATTGGCGCCCTCCTGTTCGACTCCGGGGAGACG GCCGAAGCCACGCGGCTCAAAAGAACAGCACGGCGCAGATATACCCGCAGAAAGAA TCGGATCTGCTACCTGCAGGAGATCTTTAGTAATGAGATGGCTAAGGTGGATGACTCTT TCTTCCATAGGCTGGAGGAGTCCTTTTTGGTGGAGGAGGATAAAAAGCACGAGCGCC ACCCAATCTTTGGCAATATCGTGGACGAGGTGGCGTACCATGAAAAGTACCCAACCAT ATATCATCTGAGGAAGAAGCTTGTAGACAGTACTGATAAGGCTGACTTGCGGTTGATC TATCTCGCGCTGGCGCATATGATCAAATTTCGGGGACACTTCCTCATCGAGGGGGACC TGAACCCAGACAACAGCGATGTCGACAAACTCTTTATCCAACTGGTTCAGACTTACAA TCAGCTTTTCGAAGAGAACCCGATCAACGCATCCGGAGTTGACGCCAAAGCAATCCT GAGCGCTAGGCTGTCCAAATCCCGGCGGCTCGAAAACCTCATCGCACAGCTCCCTGG GGAGAAGAAGAACGGCCTGTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCC AACTTTAAATCTAACTTCGACCTGGCCGAAGATGCCAAGCTTCAACTGAGCAAAGAC ACCTACGATGATGATCTCGACAATCTGCTGGCCCAGATCGGCGACCAGTACGCAGACC TTTTTTTGGCGGCAAAGAACCTGTCAGACGCCATTCTGCTGAGTGATATTCTGCGAGT GAACACGGAGATCACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGCGCTATGATGAG CACCACCAAGACTTGACTTTGCTGAAGGCCCTTGTCAGACAGCAACTGCCTGAGAAG TACAAGGAAATTTTCTTCGATCAGTCTAAAAATGGCTACGCCGGATACATTGACGGCG GAGCAAGCCAGGAGGAATTTTACAAATTTATTAAGCCCATCTTGGAAAAAATGGACGG CACCGAGGAGCTGCTGGTAAAGCTTAACAGAGAAGATCTGTTGCGCAAACAGCGCAC TTTCGACAATGGAAGCATCCCCCACCAGATTCACCTGGGCGAACTGCACGCTATACTC AGGCGGCAAGAGGATTTCTACCCCTTTTTGAAAGATAACAGGGAAAAGATTGAGAAA ATCCTCACATTTCGGATACCCTACTATGTAGGCCCCCTCGCCCGGGGAAATTCCAGATT CGCGTGGATGACTCGCAAATCAGAAGAGACCATCACTCCCTGGAACTTCGAGGAAGT CGTGGATAAGGGGGCCTCTGCCCAGTCCTTCATCGAAAGGATGACTAACTTTGATAAA AATCTGCCTAACGAAAAGGTGCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAG TTTATAACGAGCTCACCAAGGTCAAATACGTCACAGAAGGGATGAGAAAGCCAGCAT TCCTGTCTGGAGAGCAGAAGAAAGCTATCGTGGACCTCCTCTTCAAGACGAACCGGA AAGTTACCGTGAAACAGCTCAAAGAGGACTATTTCAAAAAGATTGAATGTTTCGACTC TGTTGAAATCAGCGGAGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGAT CTCCTGAAAATCATTAAAGACAAGGACTTCCTGGACAATGAGGAGAACGAGGACATT CTTGAGGACATTGTCCTCACCCTTACGTTGTTTGAAGATAGGGAGATGATTGAAGAAC GCTTGAAAACTTACGCTCATCTCTTCGACGACAAAGTCATGAAACAGCTCAAGAGGC GCCGATATACAGGATGGGGGCGGCTGTCAAGAAAACTGATCAATGGGATCCGAGACA AGCAGAGTGGAAAGACAATCCTGGATTTTCTTAAGTCCGATGGATTTGCCAACCGGAA CTTCATGCAGTTGATCCATGATGACTCTCTCACCTTTAAGGAGGACATCCAGAAAGCA CAAGTTTCTGGCCAGGGGGACAGTCTGCACGAGCACATCGCTAATCTTGCAGGTAGC CCAGCTATCAAAAAGGGAATACTGCAGACCGTTAAGGTCGTGGATGAACTCGTCAAA GTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGAGATGGCCCGAGAGAACCAA ACTACCCAGAAGGGACAGAAGAACAGTAGGGAAAGGATGAAGAGGATTGAAGAGGG TATAAAAGAACTGGGGTCCCAAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCT TCAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTGGAT CAGGAACTGGACATCAATCGGCTCTCCGACTACGACGTGGATGCTATCGTGCCCCAGT CTTTTCTCAAAGATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAAATAGA GGGAAGAGTGATAACGTCCCCTCAGAAGAAGTTGTCAAGAAAATGAAAAATTATTGG CGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAAG GCTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCAGGCTTCATCAAAAGGCAGCTT GTTGAGACACGCCAGATCACCAAGCACGTGGCCCAAATTCTCGATTCACGCATGAAC ACCAAGTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTATTACTCTGAAGT CTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTGAGAGAGATCAA CAATTACCACCATGCGCATGATGCCTACCTGAATGCAGTGGTAGGCACTGCACTTATCA AAAAATATCCCAAGCTTGAATCTGAATTTGTTTACGGAGACTATAAAGTGTACGATGTT AGGAAAATGATCGCAAAGTCTGAGCAGGAAATAGGCAAGGCCACCGCTAAGTACTTC TTTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCCAATGGAGAGAT TCGGAAGCGACCACTTATCGAAACAAACGGAGAAACAGGAGAAATCGTGTGGGACA AGGGTAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCAGGTGAACATCG TTAAAAAGACCGAAGTACAGACCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAA GGAACAGCGACAAGCTGATCGCACGCAAAAAAGATTGGGACCCCAAGAAATACGGC GGATTCGATTCTCCTACAGTCGCTTACAGTGTACTGGTTGTGGCCAAAGTGGAGAAAG GGAAGTCTAAAAAACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGC GATCAAGCTTCGAAAAAAACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGG TCAAAAAAGACCTCATCATTAAGCTTCCCAAGTACTCTCTCTTTGAGCTTGAAAACGG CCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAGCTGGCAC TGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGG TCTCCCGAAGATAATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTACCTT GATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAGTGATCCTCGCCGACGCTA ACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCCATCAGGGAGCA GGCAGAAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTC AAGTACTTCGACACCACCATAGACAGAAAGCGGTACACCTCTACAAAGGAGGTCCTG GACGCCACACTGATTCATCAGTCAATTACGGGGCTCTATGAAACAAGAATCGACCTCT CTCAGCTCGGTGGAGACAAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGA AA
4. acquisition of transcriptional activation Domain and its connecting sequence
VP64, T2A and MCP were synthesized together from GENEWIZ (Suzhou, China) in the sequence (SEQ ID NO: 20):
5’-GTCAAGCTAAGAAAAAGAAACAATTCGGAGGAGGTGGAAGCGGAGGAGGAGGAA GCGGAGGAGGAGGTAGCGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGGTTCC GGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCG ATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATG CTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACCAATTCGGAA GCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGA CCTATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGATG TGACAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCCAACTC ACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCCCAGAAGA GAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACAGTGGGCGGAG TCGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAGCTCACTATCCCAAT TTTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCAATGCAGGGGCTCCTCAAA GACGGTAATCCTATCCCTTCCGCCATCGCCGCTAACTCAGGTATCTACAGCGCTGGAGG AGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCGGACCTAAGAAAAAG AGGAAGGTGGCGGCCGCTCAATTG-3’
wherein the corresponding VP64 sequence, T2A sequence and MCP sequence are respectively as follows:
VP64(SEQ ID NO:21)：
GGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTG ACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGAC CTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAAC
MCP(SEQ ID NO:22)：
ATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGATG TGACAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCCAACTC ACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCCCAGAAgAG AAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACAGTGGGCGGAGT CGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAGCTCACTATCCCAATT TTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCAATGCAGGGGCTCCTCAAAG ACGGTAATCCTATCCCTTCCGCCATCGCCGCTAACTCAGGTATCTAC
T2A(SEQ ID NO:23)：
GGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAA TCCTGGACCT
p65 was obtained from plasmid Addgene 63798 by PCR using the following primers:
P65-F(SEQ ID NO:24)：
5’-AGGTGGCGGCCGCTCAATTGCCTTCAGGGCAGATCAGCAACC-3’
P65-R(SEQ ID NO:25)：
5’-GCTTCCACCTCCTCCCTGCCCACTAGAGGAAATCTGTGAC-3’
thus, the P65 sequence obtained (SEQ ID NO:26) was as follows:
CCTTCAGGGCAGATCAGCAACCAGGCCCTGGCTCTGGCCCCTAGCTCCGCTCCAG TGCTGGCCCAGACTATGGTGCCCTCTAGTGCTATGGTGCCTCTGGCCCAGCCACCTGC TCCAGCCCCTGTGCTGACCCCAGGACCACCCCAGTCACTGAGCGCTCCAGTGCCCAA GTCTACACAGGCCGGCGAGGGGACTCTGAGTGAAGCTCTGCTGCACCTGCAGTTCGA CGCTGATGAGGACCTGGGAGCTCTGCTGGGGAACAGCACCGATCCCGGAGTGTTCAC AGATCTGGCCTCCGTGGACAACTCTGAGTTTCAGCAGCTGCTGAATCAGGGCGTGTC CATGTCTCATAGTACAGCCGAACCAATGCTGATGGAGTACCCCGAAGCCATTACCCGG CTGGTGACCGGCAGCCAGCGGCCCCCCGACCCCGCTCCAACTCCCCTGGGAACCAGC GGCCTGCCTAATGGGCTGTCCGGAGATGAAGATTTCTCAAGCATCGCTGATATGGACT TTAGTGCCCTGCTGTCACAGATTTCCTCTAGTGGGCAG
HSF1 was obtained from PCR on plasmid Addgene 61426 using PCR primers:
HSF1-F(SEQ ID NO:27)：
5’-GGCAGGGAGGAGGTGGAAGCGGCTTCAGCGTGGACACC-3’
HSF1-R(SEQ ID NO:28):
5’-CCTACCTGATGCCAACAATTctagTTTGCTCTAGTCCTAGgCTAGGAGACAGTGGG GTCCTTGGC-3’
thus, the HSF1 sequence obtained (SEQ ID NO:29) was as follows:
GGCTTCAGCGTGGACACCAGTGCCCTGCTGGACCTGTTCAGCCCCTCGGTGACCG TGCCCGACATGAGCCTGCCTGACCTTGACAGCAGCCTGGCCAGTATCCAAGAGCTCCT GTCTCCCCAGGAGCCCCCCAGGCCTCCCGAGGCAGAGAACAGCAGCCCGGATTCAG GGAAGCAGCTGGTGCACTACACAGCGCAGCCGCTGTTCCTGCTGGACCCCGGCTCCG TGGACACCGGGAGCAACGACCTGCCGGTGCTGTTTGAGCTGGGAGAGGGCTCCTACT TCTCCGAAGGGGACGGCTTCGCCGAGGACCCCACCATCTCCCTGCTGACAGGCTCGG AGCCTCCCAAAGCCAAGGACCCCACTGTCTCC
5. construction of pNP-dCas9-VP64-T2A-MCP-p65-HSF1 plasmid
PCR was carried out using pNP-dCas9 as a template, using the following primers:
pNP-dCas9-F(SEQ ID NO:30)：5’-AATTGTTGGCATCAGGTAGGCATC-3’
pNP-dCas9-R(SEQ ID NO:31)：5’-TTTCTTTTTCTTAGCTTGACCAGCTTTCTTAGT-3’
recovering the amplified product, performing homologous recombination and connection with the synthesized fragment VP64-T2A-MCP, the PCR product p65 sequence and the HSF1 sequence, transforming the Escherichia coli, and selecting a plasmid with correct sequencing. Since HSF1 has a BbsI site, for the convenience of subsequent experiments, the site is subjected to synonymous mutation by the method, and the primers used are as follows:
BbsI-HSF-F(SEQ ID NO:32)：
5’-GGGCTGTCCGGAGATGAAGAtTTCTCAAGCATCGCTGATA-3’
BbsI-HSF-R(SEQ ID NO:33)：
5’-TATCAGCGATGCTTGAGAAaTCTTCATCTCCGGACAGCCC-3’
the plasmid thus formed containing dCas9 protein was designated: pNP-dCas9-VP64-T2A-MCP-p65-HSF 1.
Example two
Aiming at the sgRNA vector, the U6B promoter is used for controlling the expression of the sgRNA, and the U6B promoter can be expressed in the whole body of drosophila, including a reproductive system, and has no tissue specificity. And the expression of sgRNA is controlled by the U6B promoter, so that the expression level of sgRNA can be increased.
Reference is made to Ren X, Sun J, Housden B E, et al, optimized gene encoding technology for Drosophila melanogaster using germ line-specific Cas9[ J ]. Proceedings of the National Academy of Sciences, 2013,110(47):19012-7. sgRNA vectors were engineered using the U6B-sgRNA-short plasmid. The specific construction method comprises the following steps:
first, the sgRNA scaffold portion of the U6B-sgRNA-short plasmid was replaced with the following scaffold sequence (SEQ ID NO: 34):
5’-GTTTTAGAGCTAGGCCAACATGAGGATCACCCATGTCTGCAGGGCCTAGCAAGT TAAAATAAGGCTAGTCCGTTATCAACTTGGCCAACATGAGGATCACCCATGTCTGCAG GGCCAAGTGGCACCGAGTCGGTGCTTTTT-3’。
the replaced scaffold sequence comprises an MS2 sequence which can be recognized by MCP protein, MS2 can recruit the MCP protein so that a transcription activation domain p65 fused with the MCP protein and HSF1 are recruited to a specific site to promote gene transcription, and the scaffold sequence also has a blank sequence with the size identified by BbsI digestion between a U6B promoter and a 3' UTR, about 680bp, and 20bp of sgRNA oligonucleotide can be inserted after BbsI digestion.
Meanwhile, a spacer sequence of spacer is added at the C end of the 3' UTR (non-coding region) of U6B 3, so that the size of the product can be conveniently identified during enzyme digestion.
Wherein the sequence of the spacer is (SEQ ID NO: 35):
5’-ACTAGCGTAATATATAGACAATGGTTTTCCGTTGACGTACATACATCTGACGTGT GTTTATTTAGACATAATAGTTATGTTTTCACATCTTTTTAATGTTCGCTTAATGCGTATGC ATACAAAATTTTTAATTTTCAACACAGTTGTTTTTGTTTTCATC-3’。
this constituted the plasmid U6B-sgRNA 2.0.
Wherein the U6B promoter sequence (SEQ ID NO:36) is:
GTTCGACTTGCAGCCTGAAATACGGCACGAGTAGGAAAAGCCGAGTCAAATGCC GAATGCAGAGTCTCATTACAGCACAATCAACTCAAGAAAAACTCGACACTTTTTTACC ATTTGCACTTAAATCCTTTTTTATTCGTTATGTATACTTTTTTTGGTCCCTAACCAAAAC AAAACCAAACTCTCTTAGTCGTGCCTCTATATTTAAAACTATCAATTTATTATAGTCAAT AAATCGAACTGTGTTTTCAACAAACGAACAATAGGACACTTTGATTCTAAAGGAAATT TTGAAAATCTTAAGCAGAGGGTTCTTAAGACCATTTGCCAATTCTTATAATTCTCAACT GCTCTTTCCTGATGTTGATCATTTATATAGGTATGTTTTCCTCAATACTTC
the sequence of the 3' UTR of U6B 3 (SEQ ID NO:37) is:
TTGCTCACCTGTGATTGCTCCTACTCAAATACAAAAACATCAAATTTTCTGTCAAT AAAGCATATTTATTTATATTTATTTTACAGGAAAGAATT
EXAMPLE construction of the TriflySAMG vector
1. Integration of dCas9 vector with sgRNA2.0 vector
The part of U6B-sgRNA2.0 expressing sgRNA and spacer was cloned by PCR, and the primers were designed by adding enzyme cutting sites NheI and SpeI at the N-and C-termini, respectively, and the primers used were:
sgRNA2.0-F(SEQ ID NO:38)：
5’-AAACTCATCAATGTATCTTAACTAGTGATGAAAACAAAAACAACTGTGTTGAAA AT-3’
sgRNA2.0-R(SEQ ID NO:39)：
5’-GCACACTTATTACGTGGCCAGAGCTCTGCTAGCTTGTTCGACTTGCAGCCTGAA ATACG-3’
the partial sequence of pNP-dCas9-VP64-T2A-MCP-p65-HSF1 prepared in the first example was amplified as a vector backbone by PCR using primers
flySAM2.0-F(SEQ ID NO:40)：5’-TGGCCACGTAATAAGTGTGCGTT-3’
fySAM2.0-R(SEQ ID NO:41)：5’-TGGAACCAGACATGATAAGATACATTGATGAGT-3’
Then the two PCR products are subjected to homologous recombination to obtain an integrated vector flySAM2.0.
The K10polyA sequence in pVALIUM22 vector (among others, pVALIUM22 vector is obtained from "A genome-scale shRNA recovery for transducing genic RNAi in Drosophila, Nat methods.2011 May; 8(5):405-407.doi: 10.1038/nmeth.1592") was cloned by PCR, followed by digestion with SpeI and AvrII to replace the SV40 sequence in FlySAM2.0 with the K10polyA sequence. The K10poly A sequence can enhance the stability and expression level of the transcript in the reproductive system.
K10-F(SEQ ID NO:42)：
5’-AGCCAAGGACCCCACTGTCTCCTAGGTCTGATCTGCTAGACAATTGTTGGCA-3’
K10-R(SEQ ID NO:43)：
5’-GTTTTGTTTTCATCACTAGTCCAATCCGCCGCACCCTCAGCTCCAA-3’
Wherein the K10ployA sequence (SEQ ID NO:44) is as follows:
TAACATTATACCTAAACCCATGGTCAAGAGTAAACATTTCTGCCTTTGAAGTTGAG AACACAATTAAGCATCCCCTGGTTAAACCTGACATTCATACTTGTTAATAGCGCCATAA ACATAGCACCAATTTCGAAGAAATCAGTTAAAAGCAATTAGCAATTAGCAATTAGCAA TAACTCTGCTGACTTCAAAACGAGAAGAGTTGCAAGTATTTGTAAGGCACAGTTTATA GACCACCGACGGCTCATTAGGGCTCGTCATGTAACTAAGCGCGGTGAAACCGAATTG AACATATAGTGGAATTATTATTATCAATGGGGAAGATTTAACCCTCAGGTAGCAAAGTA ATTTAATTGCAAATAGAGAGTCCTAAGACTAAATAATATATTTAAAAATCTGGCCCTTTG ACCTTGCTTGTCAGGTGCATTTGGGTTCAATCGTAAGTTGCTTCTATATAAACACTTTC CCCATCCCCGCAATAATGAAGAATACCGCAGAATAAAGAGAGATTTGCAACAAAAAAT AAAGGCATTGCGAAAACTTTTTATGGGGGATCATTACACTCGGGCCTACGGTTACAAT TCCCAGCCACTTAAGCGACAAGTTTGGCCAACAATCCATCTAATAGCTAATAGCGCAA TCACTGGTAATCGCAAGAGTATATAGGCAATAGAACCCATGGATTTGACCAAAGGTAA CCGAGACAATGGAGAAGCAAGAGGATTTCAAACTGAACACCCACAGTGCTGTGTACT ACCACTGGCGCGTTTGGGAGCTCACTGGCCTGATGCGTCCTCCGGGCGTTTCAAGCCT GCTTTACGTGGTATACTCCATTACGGTCAACTTGGTGGTCACCGTGCTGTTTCCCTTGA GCTTGCTGGCCAGGCTGCTGTTCACCACCAACATGGCCGGATTGTGCGAGAACCTGA CCATAACTATTACCGATATTGTGGCCAATTTGAAGTTTGCGAATGTGTACATGGTGAGG AAGCAGCTCCATGAGATTCGCTCTCTCCTAAGGCTCATGGACGCTAGAGCCCGGCTGG TGGGCGATCCCGAGGAGATTTCTGCCTTGAGGAAGGAAGTGAATATCGCACAGGGCA CTTTCCGCACCTTTGCCAGTATTTTCGTATTTGGCACTACTTTGAGTTGCGTCCGCGTG GTCGTTCGCCCGGATCGAGAGCTCCTGTATCCGGCCTGGTTCGGCGTTGACTGGATGC ACTCCACCAGAAACTATGTGCTCATCAATATCTACCAGCTCTTCGGCTTGATAGTGCAG GCTATACAGAACTGCGCTAGTGACTCCTATCCGCCTGCGTTTCTCTGCCTGCTCACGGG TCATATGCGTGCTTTGGAGCTGAGGGTGCGGCGGATTGG
then, the p-transposase promoter in the pVALIUM22 vector was cloned also by means of PCR, followed by enzymatic ligation using EcoRV and KpnI to replace the DSCP promoter in the above vector with a p-transposase promoter. The DSCP promoter is replaced by a p-transposase promoter, so that the p-transposase promoter can be used for promoting the expression of genes in a reproductive system, and the expression efficiency is higher.
Wherein the primers used for PCR amplification of the p-transposase promoter are respectively as follows:
p-trans-F(SEQ ID NO:45)：
5’-CCAAGCTTGATATCATCGATCTCGAGGCTGCATCCAACGCGTTGGGAGCTCTCC GGATCAATTCGGCTTCAGGCACAGTCG-3’
p-trans-R(SEQ ID NO:46):
5’-GGCCATGGTGGCGGTACCAATGAACAGGACCTAAC-3’
the PCR amplification product was ligated to the vector obtained in the previous step, and then the vector obtained in the previous step was further cleaved with KpnI, followed by ligation of the oligonucleotide fragment formed by annealing of the lower primer, and sequencing was confirmed to finally form a fliSAMG vector, as shown in FIG. 1.
Annealing-F(SEQ ID NO:47):5’-CCGCCCGGGGATCAGAATTGAGATCTGTGGTAC-3’
Annealing-R(SEQ ID NO:48):5’-CACAGATCTCAATTCTGATCCCCGGGCGGGTAC-3’
The oligonucleotide sequences formed by annealing of SEQ ID NO:47 and SEQ ID NO:48 were incorporated into the amplification products obtained using SEQ ID NO:45 and SEQ ID NO:46 as primers as p-transposase promoter sequences. Wherein the p-transposase promoter sequence (SEQ ID NO:49) is as follows:
AGCCGTAGCTTACCGAAGTATACACTTAAATTCAGTGCACGTTTGCTTGTTGAGAG GAAAGGTTGTGTGCGGACGAATTTTTTTTTGAAAACCGGTGATAGAGCCTGAACCAG AAAAGATAAAAGAAGGCTATACCAGTGGGAGTACACAAACAGAGTAAGTTTGAATAG TAAAAAAAATCATTTATGTAAACAATAACGTGACTGTGCGTTAGGTCCTGTTCATTGGT ACCCGCCCGGGGATCAGAATTGAGATCTGT
as can be seen from fig. 1, the constructed flyssamg vector (i.e., transcription activation system) contains two expression elements, i.e., dCas9 protein expression element and sgRNA expression element, and also contains AmpR for screening positive plasmids; attB is used to site-directed integration of the entire vector into attP sites on the Drosophila genome; vermileon is a marker gene for screening transgenic fruit flies; the two insulators gypsy are respectively positioned at the upstream of the dCas9 protein expression element and the sgRNA expression element, so that the high-efficiency expression of the sgRNA expression element and the dCas9 protein expression element is ensured; two 5 xuAS are binding sites for Gal4, which initiates the expression of downstream genes under the action of Gal 4; the dCas9 expression element comprises a dCas9 protein coding sequence, and the downstream of the dCas9 protein coding sequence is sequentially provided with a VP64 coding sequence, a T2A coding sequence, an MCP protein coding sequence, a P65 coding sequence and an HSF1 coding sequence to form a dCas9-VP64-T2A-MCP-P65-HSF1 expression element, the expression element takes a P-transposase promoter as a promoter, can start the expression of a downstream transcript dCas9-VP64-T2A-MCP-P65-HSF1, and an intron ftz sequence and a K10polyA sequence are positioned downstream of the expression element and can help the stable and efficient expression of the transcript; the sgRNA expression element initiates the expression of sgRNA2.0 with the U6B promoter, sgRNA2.0 can be ligated into the vector between two Bbs I cleavage sites, and the sgRNA scaffold (sgRNA scaffold sequence) contains the MS2 structure recognized by the MCP protein.
Example 4 activation of genes of interest in the Drosophila reproductive System
dpp, tkv and bam have important functions for maintaining the self-renewal and differentiation of drosophila ovary germ cells, and the abnormal expression of any gene can generate the abnormality of the germ cells and the germ system, thereby generating obvious reproductive defects. To validate the flySAMG system, we constructed transcriptional-activated transgenic Drosophila with these three genes and activated the three genes separately in the Drosophila reproductive system as follows.
1. Construction of flySAMG vectors activating dpp, tkv and bam
Searching 5 'UTR sequences of dpp, tkv and bam in flybase (http:// flybase. org /), searching PAM sequences (NGG) at the upstream of 5' UTR of each gene, and selecting 20 nucleotides with higher CG content at the position close to the upstream of the NGG as sgRNA. Respectively placing antisense nucleotide sequences (anti-sense oligo) and reverse complementary sequences (sense oligo) of sgRNA at corresponding positions of the following primers, wherein ttcg and aaac are respectively sticky ends of a BbsI digested fSAMG vector, annealing primers of different genes, and connecting the annealed primers with the BbsI digested vector:
5’-ttcg“anti-sense oligo”-3’
5’-aaac“sense oligo”-3’
six primers for three different genes are obtained:
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’
and respectively annealing the three synthesized pairs of primers, connecting the primers with a flySAMG vector digested by BbsI, transforming Escherichia coli DH5 alpha, selecting a positive clone, and sequencing correct plasmids which are respectively flySAMG-dpp, flySAMG-tkv and flySAMG-bam.
2. Obtaining transgenic Drosophila
Respectively injecting the 3 flySAMG carriers obtained in the last step into the gene type of the ysc v nanometers-integrin by a microinjection mode; in the fruit fly embryo of attP2, the embryo is cultured at 25 ℃ and finally the homozygous transgenic transcriptional activation fruit fly is obtained by means of genetic integration.
3. Effect of activating dpp, tkv or bam on the Drosophila reproductive System
The transgenic fruit fly obtained in step 2 was crossed with a Nanos-Gal4 tool fruit fly, cultured in an incubator at 25 ℃ and 60% humidity, and the phenotype of the ovary ovule region of the offspring female fruit fly was observed.
The results are shown in FIG. 2, in which A in FIG. 2 represents the control Drosophila ovary ovule region (the control Drosophila is a female offspring obtained by crossing Drosophila with the nanos-Gal4 tool Drosophila without transgenic treatment), B, C and D represent the Drosophila ovary ovule region activated by transcription of a specific gene. Wherein the grey circles shown in the figure (1B1 signal) are marked as germ stem cells. As can be seen from the figure, in the control, the normal ovular zone contains two germ stem cells, while the critical factors dpp and tkv activating the BMP signaling pathway inhibit the differentiation process of the germ stem cells, resulting in the germ stem cell-increasing phenotype (B and C in FIG. 2); activation of the key differentiation factor bam promotes premature differentiation of the germ stem cells, eventually leading to total loss of the germ stem cells (D in fig. 2). This result is identical to the known over-expression phenotype of these genes, demonstrating that the flySAMG transcriptional activation system is capable of specifically and efficiently activating the expression of the gene of interest in the Drosophila reproductive system and producing the corresponding phenotype.
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting of the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Qinghua university
<120> highly efficient transcriptional activation system in Drosophila reproductive System
<130> PIDE3185443
<160> 55
<170> PatentIn version 3.5
<210> 1
<211> 64
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 1
agaagcggaa ggtcggtatc cacggtgtcc cagcagccat ggacaagaag tactccattg 60
ggct 64
<210> 2
<211> 62
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 2
tcttagcttg accagctttc ttagtagcag caggacgctt gtctccaccg agctgagaga 60
gg 62
<210> 3
<211> 55
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 3
gaaagctggt caagctaaga aaaagaaaaa ttgttggcat caggtaggca tcaca 55
<210> 4
<211> 78
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 4
gataccgacc ttccgcttct tctttggggc catggtggcg gtacctctag actttggtat 60
gcgtcttgtg attcaaag 78
<210> 5
<211> 432
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 5
ttggccacgt aataagtgtg cgttgaattt attcgcaaaa acattgcata ttttcggcaa 60
agtaaaattt tgttgcatac cttatcaaaa aataagtgct gcatactttt tagagaaacc 120
aaataatttt ttattgcata cccgttttta ataaaataca ttgcataccc tcttttaata 180
aaaaatattg catactttga cgaaacaaat tttcgttgca tacccaataa aagattatta 240
tattgcatac ccgtttttaa taaaatacat tgcataccct cttttaataa agaatattgc 300
atacgttgac gaaacaaatt ttcgttgcat acccaataaa agattattat attgcatacc 360
ttttcttgcc ataccattta gccgatcaat tctgctcggc aacagtatat ttgtggtgtg 420
ccaaccaaca ac 432
<210> 6
<211> 199
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 6
ctagttctga tctgctagac aattgttggc atcaggtagg catcacacac gattaacaac 60
ccctaaaaat acactttgaa aatattgaaa atatgttttt gtatacattt ttgatatttt 120
caaataatac gcagttataa aactcattag ctaacccatt ttttctttgc ttatgcttac 180
agattgcaaa gaactagag 199
<210> 7
<211> 861
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 7
atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240
cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300
gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360
tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540
cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840
tcactgatta agcattggta a 861
<210> 8
<211> 1880
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 8
atttattttg ttatgttata tgtattatat gtcagacata aagaaaagga acacatcaaa 60
tgtgataaca aagactaaac aagtaatttt attacaccaa aacgacaaaa cagtaggcag 120
aacaaacaac gcatagccaa acattgacga attggatacc ctgccgattg tcagacactt 180
ttgttgatca gtttcttgcg aatggtctcg tccagcggtg gaatcgcctc gcggggaatc 240
agaaaagtgg acagattgaa cagatccaga aacaccttgt accgatcact gaaaccaaaa 300
aaaaacaaag ggagaacagt ttgagttcat tgatccccga tataatcaca tctgcgatga 360
tcacctgaga gtggagcgca gatattgata accagacgag ccaccagtgc ccaactgttg 420
cgatccaatc atgcgttgca ccatgatcac gtgattgtct gcggcgggaa tagaaagtat 480
ttggttagga aaaccagtct taaacataag atatatttat aaaagagtat caaagaatgc 540
aatacttaca tctccacttg gttattaacg agtcgatgtc catgagcagg gtgagcaact 600
ggtgtggttg gctgaacctg ggttcatccc tatagaaggt gatcatgatg gctccctgaa 660
gggcacgatg gctaaaccgg cgatccccac gacgcaccag tgcatcgtgc actgccggat 720
caaagatgga gcgatacacc tcgcgtcgct tctcaatgtc catgaggcgg tagtttttcg 780
ccttctccac gggctcctcc atggcgctct gtacctgcgc ctccaggaat cgatcgacgc 840
tctcctgaaa cttggcccag aagttgaagc cactctcctc cagtccgggc gtcctctcca 900
gccatcgctg cactagctcc agtagcgagg gatctttctc cgagttgcga atcgagttcc 960
gcgcctcctc gtcgctaaag acatccgagt acttctggtt gtatctcacc cgctgctctg 1020
tcagaactcc cagcttgttc tcgatcaaac ggaactgcag cgactgaaaa ccagatgcgg 1080
gtgccaggta cttgcggaag tccatgaagt ctagcggggt catggtctcc agaatgggca 1140
cttggtccac caggagctgt acaaaggaag ttataaacgg attttggtaa gagattcaga 1200
aagcactcac ttttagaatc agaaccactc ggttcagtcg cttgacaatc tccagcgtct 1260
tggtttcatc gatgacctct gcatccaaca tgtctcgtat ggagtcgaac tcaaagatga 1320
tctgcttgaa ccaaagctcg taggctgtgg cgaaggtact taaatgccat tgagtgttgt 1380
catcaaagtt gtaaacctac tcaccctggt gcgtgatgat gaacagatgc tcatcgtgca 1440
cgggtcgctt gtcctcctcg gacagcatac actgggcatc cagcagtttg tccagcatca 1500
gatactctcc atagattttg cccacttccg tggttaatgg caccgccgaa tcatcgtgat 1560
cgtttctgta tgggtttgaa ttgaatcgca gaactgaaga tcgattggca ttcctggaca 1620
gcacgtgctg gtgctcaccc gtttcctgca tagggacagc tcatggtgca cagctcagat 1680
cagatcgtga ctcctcgacc ggcggatgct ggcgaactga tctccgccag cggaccggag 1740
atgagacccc agcgaaccga taacagagcg agagagctcc agttccgact gattgcacag 1800
tcggtgatct gggcgatggg cactgccaga taggctggga attatcaatc acttgaggtg 1860
aaagtgcggc gcacacaaat 1880
<210> 9
<211> 368
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 9
gctgcatcca acgcgttggg agctctccgg atcaattcgg cttcacgtac cgtcgacgat 60
gtaggtcacg gtctcgaagc cgcggtgcgg gtgccagggc gtgcccttgg gctccccggg 120
cgcgtactcc acctcaccca tctggtccat catgatgaac gggtcgaggt ggcggtagtt 180
gatcccggcg aacgcgcggc gcaccgggaa gccctcgccc tcgaaaccgc tgggcgcggt 240
ggtcacggtg agcacgggac gtgcgacggc gtcggctggt gcggatacgc ggggcagcgt 300
cagcgggttc tcgacggtca cggcgggcat gtcgacaagc cgaattgatc cactagaagg 360
cctaattc 368
<210> 10
<211> 182
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 10
gcaggtcgga gtactgtcct ccgagcggag tactgtcctc cgagcggagt actgtcctcc 60
gagcggagta ctgtcctccg agcggagtac tgtcctccga gcggagactc ccgcggtcgg 120
agtactgtcc tccgagcgga gtactgtcct ccgagcggag tactgtcctc cgagcggagt 180
ac 182
<210> 11
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 11
gaagtactcc attgggctcg ctatcggcac aaacagcgtc 40
<210> 12
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 12
gacgctgttt gtgccgatag cgagcccaat ggagtacttc 40
<210> 13
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 13
tccgactacg acgtggatgc tatcgtgccc cagtcttttc 40
<210> 14
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 14
gaaaagactg gggcacgata gcatccacgt cgtagtcgga 40
<210> 15
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 15
accgtgaaac agctcaaaga ggactatttc aaaaagattg 40
<210> 16
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 16
caatcttttt gaaatagtcc tctttgagct gtttcacggt 40
<210> 17
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 17
ttctggccag ggggacagtc tgcacgagca catcgctaat 40
<210> 18
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 18
attagcgatg tgctcgtgca gactgtcccc ctggccagaa 40
<210> 19
<211> 4197
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 19
atggccccaa agaagaagcg gaaggtcggt atccacggtg tcccagcagc catggacaag 60
aagtactcca ttgggctcgc tatcggcaca aacagcgtcg gctgggccgt cattacggac 120
gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata 180
aagaagaacc tcattggcgc cctcctgttc gactccgggg agacggccga agccacgcgg 240
ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag 300
gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag 360
tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg 420
gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta 480
gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa 540
tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa 600
ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca 660
tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa 720
aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc 780
ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag 840
cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc 900
gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt 960
gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag 1020
cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg 1080
cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt 1140
gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg 1200
gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc 1260
actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatactc 1320
aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc 1380
ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg 1440
tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat 1500
aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct 1560
aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag 1620
ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag 1680
cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag 1740
ctcaaagagg actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg 1800
gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac 1860
aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt 1920
acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc 1980
gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca 2040
agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt 2100
aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc 2160
tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct gcacgagcac 2220
atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc 2280
gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg 2340
gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg 2400
attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac 2460
acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac 2520
gtggatcagg aactggacat caatcggctc tccgactacg acgtggatgc tatcgtgccc 2580
cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat 2640
agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg 2700
cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct 2760
gaacgaggtg gcctgtctga gttggataaa gcaggcttca tcaaaaggca gcttgttgag 2820
acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac 2880
gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc 2940
tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat 3000
gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag 3060
cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca 3120
aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg 3180
aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc 3240
gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc 3300
cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga 3360
ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa 3420
aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta 3480
ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg 3540
ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag 3600
gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc 3660
tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt 3720
aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa 3780
aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa 3840
cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc 3900
gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg 3960
gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc 4020
ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg 4080
gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct 4140
cagctcggtg gagacaagcg tcctgctgct actaagaaag ctggtcaagc taagaaa 4197
<210> 20
<211> 824
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 20
gtcaagctaa gaaaaagaaa caattcggag gaggtggaag cggaggagga ggaagcggag 60
gaggaggtag cggacctaag aaaaagagga aggtggcggc cgctggttcc ggacgggctg 120
acgcattgga cgattttgat ctggatatgc tgggaagtga cgccctcgat gattttgacc 180
ttgacatgct tggttcggat gcccttgatg actttgacct cgacatgctc ggcagtgacg 240
cccttgatga tttcgacctg gacatgctga ttaaccaatt cggaagcgga gagggcagag 300
gaagtctgct aacatgcggt gacgtcgagg agaatcctgg acctatggct tcaaacttta 360
ctcagttcgt gctcgtggac aatggtggga caggggatgt gacagtggct ccttctaatt 420
tcgctaatgg ggtggcagag tggatcagct ccaactcacg gagccaggcc tacaaggtga 480
catgcagcgt caggcagtct agtgcccaga agagaaagta taccatcaag gtggaggtcc 540
ccaaagtggc tacccagaca gtgggcggag tcgaactgcc tgtcgccgct tggaggtcct 600
acctgaacat ggagctcact atcccaattt tcgctaccaa ttctgactgt gaactcatcg 660
tgaaggcaat gcaggggctc ctcaaagacg gtaatcctat cccttccgcc atcgccgcta 720
actcaggtat ctacagcgct ggaggaggtg gaagcggagg aggaggaagc ggaggaggag 780
gtagcggacc taagaaaaag aggaaggtgg cggccgctca attg 824
<210> 21
<211> 171
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 21
ggttccggac gggctgacgc attggacgat tttgatctgg atatgctggg aagtgacgcc 60
ctcgatgatt ttgaccttga catgcttggt tcggatgccc ttgatgactt tgacctcgac 120
atgctcggca gtgacgccct tgatgatttc gacctggaca tgctgattaa c 171
<210> 22
<211> 390
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 22
atggcttcaa actttactca gttcgtgctc gtggacaatg gtgggacagg ggatgtgaca 60
gtggctcctt ctaatttcgc taatggggtg gcagagtgga tcagctccaa ctcacggagc 120
caggcctaca aggtgacatg cagcgtcagg cagtctagtg cccagaagag aaagtatacc 180
atcaaggtgg aggtccccaa agtggctacc cagacagtgg gcggagtcga actgcctgtc 240
gccgcttgga ggtcctacct gaacatggag ctcactatcc caattttcgc taccaattct 300
gactgtgaac tcatcgtgaa ggcaatgcag gggctcctca aagacggtaa tcctatccct 360
tccgccatcg ccgctaactc aggtatctac 390
<210> 23
<211> 63
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 23
ggaagcggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctgga 60
cct 63
<210> 24
<211> 42
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 24
aggtggcggc cgctcaattg ccttcagggc agatcagcaa cc 42
<210> 25
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 25
gcttccacct cctccctgcc cactagagga aatctgtgac 40
<210> 26
<211> 552
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 26
ccttcagggc agatcagcaa ccaggccctg gctctggccc ctagctccgc tccagtgctg 60
gcccagacta tggtgccctc tagtgctatg gtgcctctgg cccagccacc tgctccagcc 120
cctgtgctga ccccaggacc accccagtca ctgagcgctc cagtgcccaa gtctacacag 180
gccggcgagg ggactctgag tgaagctctg ctgcacctgc agttcgacgc tgatgaggac 240
ctgggagctc tgctggggaa cagcaccgat cccggagtgt tcacagatct ggcctccgtg 300
gacaactctg agtttcagca gctgctgaat cagggcgtgt ccatgtctca tagtacagcc 360
gaaccaatgc tgatggagta ccccgaagcc attacccggc tggtgaccgg cagccagcgg 420
ccccccgacc ccgctccaac tcccctggga accagcggcc tgcctaatgg gctgtccgga 480
gatgaagatt tctcaagcat cgctgatatg gactttagtg ccctgctgtc acagatttcc 540
tctagtgggc ag 552
<210> 27
<211> 38
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 27
ggcagggagg aggtggaagc ggcttcagcg tggacacc 38
<210> 28
<211> 65
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 28
cctacctgat gccaacaatt ctagtttgct ctagtcctag gctaggagac agtggggtcc 60
ttggc 65
<210> 29
<211> 372
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 29
ggcttcagcg tggacaccag tgccctgctg gacctgttca gcccctcggt gaccgtgccc 60
gacatgagcc tgcctgacct tgacagcagc ctggccagta tccaagagct cctgtctccc 120
caggagcccc ccaggcctcc cgaggcagag aacagcagcc cggattcagg gaagcagctg 180
gtgcactaca cagcgcagcc gctgttcctg ctggaccccg gctccgtgga caccgggagc 240
aacgacctgc cggtgctgtt tgagctggga gagggctcct acttctccga aggggacggc 300
ttcgccgagg accccaccat ctccctgctg acaggctcgg agcctcccaa agccaaggac 360
cccactgtct cc 372
<210> 30
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 30
aattgttggc atcaggtagg catc 24
<210> 31
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 31
tttctttttc ttagcttgac cagctttctt agt 33
<210> 32
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 32
gggctgtccg gagatgaaga tttctcaagc atcgctgata 40
<210> 33
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 33
tatcagcgat gcttgagaaa tcttcatctc cggacagccc 40
<210> 34
<211> 141
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 34
gttttagagc taggccaaca tgaggatcac ccatgtctgc agggcctagc aagttaaaat 60
aaggctagtc cgttatcaac ttggccaaca tgaggatcac ccatgtctgc agggccaagt 120
ggcaccgagt cggtgctttt t 141
<210> 35
<211> 160
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 35
actagcgtaa tatatagaca atggttttcc gttgacgtac atacatctga cgtgtgttta 60
tttagacata atagttatgt tttcacatct ttttaatgtt cgcttaatgc gtatgcatac 120
aaaattttta attttcaaca cagttgtttt tgttttcatc 160
<210> 36
<211> 400
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 36
gttcgacttg cagcctgaaa tacggcacga gtaggaaaag ccgagtcaaa tgccgaatgc 60
agagtctcat tacagcacaa tcaactcaag aaaaactcga cactttttta ccatttgcac 120
ttaaatcctt ttttattcgt tatgtatact ttttttggtc cctaaccaaa acaaaaccaa 180
actctcttag tcgtgcctct atatttaaaa ctatcaattt attatagtca ataaatcgaa 240
ctgtgttttc aacaaacgaa caataggaca ctttgattct aaaggaaatt ttgaaaatct 300
taagcagagg gttcttaaga ccatttgcca attcttataa ttctcaactg ctctttcctg 360
atgttgatca tttatatagg tatgttttcc tcaatacttc 400
<210> 37
<211> 95
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 37
ttgctcacct gtgattgctc ctactcaaat acaaaaacat caaattttct gtcaataaag 60
catatttatt tatatttatt ttacaggaaa gaatt 95
<210> 38
<211> 56
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 38
aaactcatca atgtatctta actagtgatg aaaacaaaaa caactgtgtt gaaaat 56
<210> 39
<211> 59
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 39
gcacacttat tacgtggcca gagctctgct agcttgttcg acttgcagcc tgaaatacg 59
<210> 40
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 40
tggccacgta ataagtgtgc gtt 23
<210> 41
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 41
tggaaccaga catgataaga tacattgatg agt 33
<210> 42
<211> 52
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 42
agccaaggac cccactgtct cctaggtctg atctgctaga caattgttgg ca 52
<210> 43
<211> 46
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 43
gttttgtttt catcactagt ccaatccgcc gcaccctcag ctccaa 46
<210> 44
<211> 1377
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 44
taacattata cctaaaccca tggtcaagag taaacatttc tgcctttgaa gttgagaaca 60
caattaagca tcccctggtt aaacctgaca ttcatacttg ttaatagcgc cataaacata 120
gcaccaattt cgaagaaatc agttaaaagc aattagcaat tagcaattag caataactct 180
gctgacttca aaacgagaag agttgcaagt atttgtaagg cacagtttat agaccaccga 240
cggctcatta gggctcgtca tgtaactaag cgcggtgaaa ccgaattgaa catatagtgg 300
aattattatt atcaatgggg aagatttaac cctcaggtag caaagtaatt taattgcaaa 360
tagagagtcc taagactaaa taatatattt aaaaatctgg ccctttgacc ttgcttgtca 420
ggtgcatttg ggttcaatcg taagttgctt ctatataaac actttcccca tccccgcaat 480
aatgaagaat accgcagaat aaagagagat ttgcaacaaa aaataaaggc attgcgaaaa 540
ctttttatgg gggatcatta cactcgggcc tacggttaca attcccagcc acttaagcga 600
caagtttggc caacaatcca tctaatagct aatagcgcaa tcactggtaa tcgcaagagt 660
atataggcaa tagaacccat ggatttgacc aaaggtaacc gagacaatgg agaagcaaga 720
ggatttcaaa ctgaacaccc acagtgctgt gtactaccac tggcgcgttt gggagctcac 780
tggcctgatg cgtcctccgg gcgtttcaag cctgctttac gtggtatact ccattacggt 840
caacttggtg gtcaccgtgc tgtttccctt gagcttgctg gccaggctgc tgttcaccac 900
caacatggcc ggattgtgcg agaacctgac cataactatt accgatattg tggccaattt 960
gaagtttgcg aatgtgtaca tggtgaggaa gcagctccat gagattcgct ctctcctaag 1020
gctcatggac gctagagccc ggctggtggg cgatcccgag gagatttctg ccttgaggaa 1080
ggaagtgaat atcgcacagg gcactttccg cacctttgcc agtattttcg tatttggcac 1140
tactttgagt tgcgtccgcg tggtcgttcg cccggatcga gagctcctgt atccggcctg 1200
gttcggcgtt gactggatgc actccaccag aaactatgtg ctcatcaata tctaccagct 1260
cttcggcttg atagtgcagg ctatacagaa ctgcgctagt gactcctatc cgcctgcgtt 1320
tctctgcctg ctcacgggtc atatgcgtgc tttggagctg agggtgcggc ggattgg 1377
<210> 45
<211> 81
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 45
ccaagcttga tatcatcgat ctcgaggctg catccaacgc gttgggagct ctccggatca 60
attcggcttc aggcacagtc g 81
<210> 46
<211> 35
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 46
ggccatggtg gcggtaccaa tgaacaggac ctaac 35
<210> 47
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 47
ccgcccgggg atcagaattg agatctgtgg tac 33
<210> 48
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 48
cacagatctc aattctgatc cccgggcggg tac 33
<210> 49
<211> 259
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 49
agccgtagct taccgaagta tacacttaaa ttcagtgcac gtttgcttgt tgagaggaaa 60
ggttgtgtgc ggacgaattt ttttttgaaa accggtgata gagcctgaac cagaaaagat 120
aaaagaaggc tataccagtg ggagtacaca aacagagtaa gtttgaatag taaaaaaaat 180
catttatgta aacaataacg tgactgtgcg ttaggtcctg ttcattggta cccgcccggg 240
gatcagaatt gagatctgt 259
<210> 50
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 50
ttcgcgtcca aagcggccga ggca 24
<210> 51
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 51
aaactgcctc ggccgctttg gacg 24
<210> 52
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 52
ttcgcgtacg tacatatggt gggg 24
<210> 53
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 53
aaacccccac catatgtacg tacg 24
<210> 54
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 54
ttcgtacaat tatacacact gatt 24
<210> 55
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 55
aaacaatcag tgtgtataat tgta 24
Claims (29)
1. A transcriptional activation system comprising a dCas9 protein expression element and a sgRNA expression element,
the dCas9 protein expression element comprises a p-transposase promoter, a dCas9 protein coding sequence, an MCP protein coding sequence and a transcription activator coding sequence, wherein the transcription activator coding sequence is positioned at the downstream of the dCas9 protein coding sequence, and the MCP protein coding sequence is positioned at the downstream of the dCas9 protein coding sequence;
the sgRNA expression element contains a U6B promoter, an sgRNA insertion site and an MCP protein recognition sequence;
wherein the transcription activator coding sequence comprises a VP64 coding sequence, a P65 coding sequence and an HSF1 coding sequence, the VP64 coding sequence is positioned at the upstream of the MCP protein coding sequence, a self-splicing peptide T2A coding sequence is contained between the VP64 coding sequence and the MCP protein coding sequence, and the P65 coding sequence and the HSF1 coding sequence are positioned at the downstream of the MCP protein coding sequence in sequence;
the transcriptional activation system is used for transcriptional activation of genes in the drosophila reproductive system.
2. The transcriptional activation system of claim 1, wherein the VP64 encoding sequence is SEQ ID NO 21, the P65 encoding sequence is SEQ ID NO 26, the HSF1 encoding sequence is SEQ ID NO 27, and the self-cleaving peptide T2A encoding sequence is SEQ ID NO 23.
3. The transcriptional activation system of claim 2, wherein the primer sequences for amplifying the P65 coding sequence are SEQ ID NO. 24 and SEQ ID NO. 25.
4. The transcriptional activation system of claim 2, wherein the primer sequences for amplifying the HSF1 coding sequence are SEQ ID NOs 27 and 28.
5. The transcriptional activation system of claim 1, wherein the dCas9 protein expression element and the sgRNA expression element are linked by a plasmid vector.
6. The transcriptional activation system of claim 5, wherein said plasmid vector is a pNP plasmid.
7. The transcriptional activation system of claim 5, wherein the coding sequence of the MCP protein is SEQ ID NO. 22.
8. The transcriptional activation system of claim 5, wherein the MCP protein recognition sequence is MS2 sequence.
9. The transcriptional activation system according to claim 1, further comprising:
at least two gypsy genes located upstream of the dCas9 protein expression element and the sgRNA expression element, respectively,
ftz intron sequence, the ftz intron sequence being downstream of the dCas9 protein expression element;
a K10polyA sequence, the K10polyA sequence being located downstream of the dCas9 protein expression element.
10. The transcriptional activation system of claim 9, wherein said gypsy gene sequence is SEQ ID No. 5; the ftz intron sequence is SEQ ID NO 6; the K10polyA sequence is SEQ ID NO: 44.
11. The transcriptional activation system according to claim 9, further comprising: a 10 × UAS sequence, said 10 × UAS sequence being located upstream of said p-transposase promoter.
12. The transcriptional activation system of claim 11, wherein the 10 × UAS sequence is SEQ ID NO 10.
13. The transcriptional activation system according to claim 9, further comprising: antibiotic marker gene, verimion gene and attB gene.
14. The transcription activation system according to claim 13, wherein the antibiotic marker gene is ampicillin resistance gene, the nucleic acid sequence of ampicillin resistance gene is SEQ ID NO 7, the nucleotide sequence of veririon gene is SEQ ID NO 8, and the nucleotide sequence of attB gene is SEQ ID NO 9.
15. A transcriptional activation system, comprising:
a dCas9 protein expression element, the dCas9 protein expression element comprising a P-transposase promoter and a dCas9 protein coding sequence, the dCas9 protein coding sequence being located downstream of the P-transposase promoter, the dCas9 protein coding sequence being downstream of the VP64 coding sequence, the self-splicing peptide T2A coding sequence, the MCP protein coding sequence, the P65 coding sequence, and the HSF1 coding sequence in that order;
an sgRNA expression element comprising a U6B promoter, an sgRNA insertion site, an MS2 sequence, and a U6B 3' untranslated region sequence thereon;
at least two gypsy genes, said gypsy genes being located upstream of said dCas9 protein expression element and upstream of said sgRNA expression element, respectively;
ftz intron sequence, the ftz intron sequence being downstream of the dCas9 protein expression element;
a K10polyA sequence, the K10polyA sequence being downstream of the ftz intron sequence;
antibiotic marker gene, vermileon gene and attB gene;
the transcriptional activation system is used for transcriptional activation of genes in the drosophila reproductive system.
16. The transcriptional activation system according to claim 15, further comprising: a 10 × UAS sequence, said 10 × UAS sequence being located upstream of said p-transposase promoter.
17. A method for constructing the transcription activation system according to any one of claims 1 to 16, comprising:
(1) constructing a first recombinant plasmid containing a dCas9 protein coding sequence by using a plasmid vector;
(2) ligating a transcription activator coding sequence to the first recombinant plasmid to construct a second recombinant plasmid, wherein the transcription activator coding sequence is located downstream of the dCas9 protein coding sequence;
(3) and connecting the sgRNA expression element to the second recombinant plasmid to construct a transcriptional activation system.
18. The method of claim 17, wherein step (1) further comprises:
(1-1) connecting the Cas9 protein coding sequence to a plasmid vector to construct a third recombinant plasmid containing the Cas9 protein coding sequence;
(1-2) mutating the Cas9 protein coding sequence on the third recombinant plasmid to obtain a first recombinant plasmid comprising the dCas9 protein coding sequence.
19. The method of claim 17, wherein the plasmid vector is a pNP plasmid.
20. The method of claim 17, wherein the primer sequence used to amplify the Cas9 protein coding sequence is SEQ ID NO:1 and SEQ ID NO:2, the coding sequence of the dCas9 protein is SEQ ID NO: 19.
21. the method of claim 18, wherein step (1-1) links the Cas9 protein-encoding sequence to the plasmid vector by way of homologous recombination.
22. The method of claim 18, wherein the base encoding amino acid 10 of the Cas9 protein coding sequence is mutated in step (1-2) with reverse complementary mutation primers SEQ ID NO:11 and SEQ ID NO:12, and the base encoding amino acid 840 of the Cas9 protein coding sequence is mutated with reverse complementary mutation primers SEQ ID NO:13 and SEQ ID NO: 14.
23. The method according to claim 17, wherein the transcriptional activator coding sequence is ligated to the first recombinant plasmid in step (2) by means of homologous recombination.
24. The method according to claim 17, wherein the sgRNA expression elements are ligated to the second recombinant plasmid in step (3) by homologous recombination.
25. The method of any one of claims 17-24, wherein the sgRNA expression element comprises a scaffold sequence and a U6B promoter 3' untranslated region sequence, and the scaffold sequence comprises an MCP protein recognition sequence.
26. The method of claim 25, wherein the scaffold sequence is SEQ ID NO 34, the U6B promoter sequence is SEQ ID NO 36, and the U6B promoter 3' untranslated region sequence is SEQ ID NO 37.
27. A method of producing a transgenic fruit fly, comprising:
(a) introducing a sgRNA sequence targeting a target gene into the transcriptional activation system of any one of claims 1 to 16 to obtain a transcriptional activation system targeting the target gene;
(b) and (3) introducing the transcription activation system of the target gene into a drosophila embryo to obtain the transgenic drosophila.
28. The method of claim 27, wherein step (a) further comprises:
(a-1) carrying out enzyme digestion treatment on the transcription activation system to obtain the transcription activation system subjected to enzyme digestion treatment;
(a-2) connecting the sgRNA sequence of the target gene subjected to annealing pairing to the sgRNA insertion site of the transcription activation system subjected to enzyme digestion treatment, so as to introduce the sgRNA sequence of the target gene into the transcription activation system.
29. A method of activating gene expression in the reproductive system of drosophila comprising:
introducing a sgRNA sequence targeting a target gene into the transcriptional activation system of any one of claims 1 to 16 to obtain a transcriptional activation system targeting the target gene;
introducing the transcription activation system of the target gene into a drosophila embryo to obtain a transgenic drosophila;
and (3) hybridizing and culturing the transgenic drosophila and the Gal4 tool drosophila to obtain drosophila offspring, thereby activating the expression of the target gene in the drosophila reproductive system.
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