CN104120127B - The oligonucleotide separated and application thereof - Google Patents

Abstract

The invention discloses an isolated oligonucleotide and its application, wherein the oligonucleotide has the nucleotide sequence shown in SEQ ID NO:1. When preparing a construct for gene knockout in Drosophila, two adjacent siRNAs among multiple siRNAs are connected through the oligonucleotide, so that the transcripts can form independent shRNAs through the cleavage mechanism of Drosophila itself , so that the respective target genes of RNAi can be simultaneously knocked out, and multiple genes can be knocked out at the same time, so as to realize the simultaneous regulation of multiple genes in Drosophila.

Description

Isolated oligonucleotides and their applications

technical field

The present invention relates to the field of biotechnology, in particular to isolated oligonucleotides and applications thereof, more specifically, to isolated nucleotides, constructs and methods for knocking out Drosophila target genes.

Background technique

RNA interference (RNA interference, RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (double-stranded RNA, dsRNA), which is highly conserved during evolution. RNAi technology can specifically reduce the expression of genes, and is highly efficient and easy to operate, so this technology has been widely used in various research fields of Drosophila.

In Drosophila research, RNAi technology is generally combined with a binary expression system to achieve tissue-specific and developmental stage-specific gene knockout in transgenic Drosophila. The binary expression system widely used in Drosophila is the GAL4-UAS system, in which the yeast transcription factor gene GAL4 is controlled by a specific promoter. In turn, GAL4 can activate the expression of another exogenously inserted gene containing an upstream activating sequence (UAS).

At present, the commonly used promoter of RNAi vector is Hsp70, and when activated by GAL4, the promoter can promote the expression of exogenously inserted genes. However, when GAL4 does not exist, the promoter can still activate the exogenously inserted gene, resulting in a certain amount of background expression of the exogenous gene, which affects the judgment of the experimental results.

The third-generation RNAi technology is transcribed from an expression vector to form short hairpin RNA (short hairpin RNA, shRNA) to mimic the naturally occurring microRNA in Drosophila. The shRNA then generates small interfering RNA (siRNA) through the microRNA pathway in Drosophila, thereby achieving gene knockout (Ni et al., Nature Methods. 2011May; 8(5):405-7.). However, this technique can only knock out a single gene. Therefore, when the function of the target gene is redundant with other genes, or when the research involves the function of multiple genes, this technology cannot well meet the needs of researchers.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. For this reason, an object of the present invention is to propose a method capable of simultaneously knocking out multiple target genes in Drosophila.

Thus, according to one aspect of the invention, the invention provides an isolated oligonucleotide. According to a specific embodiment of the present invention, the oligonucleotide has the nucleotide sequence shown in SEQ ID NO:1. The inventors found that when preparing a construct for gene knockout in Drosophila, the oligonucleotide was used to connect multiple shRNA template sequences, that is, two adjacent siRNAs among multiple siRNAs were passed through the oligonucleotide Linked together, the transcription products can be formed into independent shRNAs through the cleavage mechanism of Drosophila itself, so that the respective target genes of RNAi can be simultaneously RNAi, and then multiple genes can be knocked out at the same time to realize the simultaneous regulation of multiple genes in Drosophila. Herein, the oligonucleotide shown in SEQ ID NO: 1 is also sometimes referred to as "linker".

According to another aspect of the present invention, the present invention also provides a construct. According to an embodiment of the present invention, the construct comprises a plurality of siRNAs, two adjacent siRNAs among the plurality of siRNAs are connected by the aforementioned oligonucleotide, wherein the target genes of the plurality of siRNAs are the same or different . The inventors have surprisingly found that the construct of the present invention can not only efficiently express multiple siRNAs targeting different genes at the same time, thereby enabling simultaneous regulation of multiple genes, but also express multiple siRNAs targeting the same gene to enhance the regulation of a single gene. Knockout efficiency. Moreover, the construction of the construct is simple and convenient, the expression is efficient and stable, and it is applicable to all tissues and cells of Drosophila, so that it can be effectively used in the functional research of various Drosophila genes.

According to an embodiment of the present invention, the construct further comprises: 2×QUAS sequence; and/or 10×UAS sequence. Therefore, these two repressible binary expression systems can enrich the means of transgene expression, lineage tracing and gene function chimera analysis in Drosophila, making the regulation of expression more flexible.

According to an embodiment of the present invention, the construct further comprises a selection marker gene. Thus, it is convenient to screen positive plasmids. According to some specific examples of the present invention, the selection marker gene is an ampicillin resistance gene and/or a selection marker vermilion gene. Thus, the positive plasmid screening effect is good.

According to an embodiment of the present invention, the construct further comprises a promoter. The inventors have found that the background expression efficiency of U-promoter (i.e. U-promoter) in various tissues and organs of Drosophila is low, and the expression efficiency is high when induced. Therefore, according to an embodiment of the present invention, the promoter is U-promoter.

According to an embodiment of the present invention, the construct further comprises: genome site-specific integration attB sequence; ftz intron sequence; and SV40ployA sequence. As a result, the plasmid can be site-specifically integrated into the Drosophila genome, the translation efficiency can be enhanced, and it is beneficial to regulate the termination of transcription.

According to an embodiment of the present invention, the construct further comprises: two LoxP sequences; and an insulator gypsy sequence, wherein the insulator gypsy sequence is located between the two LoxP sequences. Thus, the gypsy sequence between LoxP can be removed under the action of Cre recombinase to achieve the purpose of regulating the expression of siRNA, and the gypsy sequence can enhance gene transcription, thereby making the regulation of gene expression more flexible, and the means of gene function analysis more diverse.

In another aspect of the present invention, the present invention also provides a method for knocking out a target gene in Drosophila. According to an embodiment of the present invention, the method utilizes the aforementioned constructs to transform Drosophila. The inventors surprisingly found that using the method of the present invention, not only can simultaneously and efficiently express multiple siRNAs targeting different genes in Drosophila, thereby enabling the simultaneous knockout of multiple genes, but also expressing multiple siRNAs targeting different genes in Drosophila. The same gene siRNA can achieve high-efficiency knockout of a single gene, and the knockout effect is good. Moreover, the method of the present invention is simple to operate and has stable results, and is applicable to all tissues and cells of Drosophila, and it is easy to regulate the gene knockout efficiency of Drosophila by using the method, and the obtained Drosophila phenotype is obvious, so it can be effectively used in Drosophila. Fly gene function studies.

According to an embodiment of the present invention, the method for knocking out a gene of interest in Drosophila of the present invention utilizes a GAL4-UAS binary expression vector system for the transformation, wherein the construct comprises 2×QUAS sequences; and/or 10×UAS sequences . Therefore, these two repressible binary expression systems can enrich the means of transgene expression, lineage tracing and gene function chimera analysis in Drosophila, making the expression regulation of this method more flexible.

According to an embodiment of the present invention, the transformation further includes: transforming the first fruit fly with the construct to obtain a first transgenic fruit fly; providing a second transgenic fruit fly capable of expressing Gal4 protein and/or Q protein; and crossing the first transgenic fruit fly with the second transgenic fruit fly to obtain offspring transgenic fruit flies in which the target gene is knocked out.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:

Fig. 1 has shown the map of the expression vector of VADUM according to one embodiment of the present invention;

Fig. 2 has shown the picture of wild type and each transgenic male fruit fly wing according to one embodiment of the present invention, wherein

Figure 2A shows a picture of a wild-type male Drosophila wing,

Figure 2B shows a picture of the wings of male Drosophila offspring of H1KD1 crossed with ms1096-Gal4,

Figure 2C shows a picture of the wings of male Drosophila offspring of H1KD2 crossed with ms1096-Gal4,

Figure 2D shows a picture of the wings of male Drosophila offspring of H1KD3 crossed with ms1096-Gal4.

detailed description

The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are only illustrative and should not be construed as limiting the present invention.

According to one aspect of the invention, the invention provides an isolated oligonucleotide. According to a specific embodiment of the present invention, the oligonucleotide has the nucleotide sequence shown in SEQ ID NO:1. Wherein, the nucleotide sequence shown in SEQ ID NO: 1 is as follows:

CGCGATGCTCCAAGGCAAAAAAAAATCAATCAAAAAACAATTTGAAAATTCCAAAAAAAAGCATAATTGAACACAAAAAGTATAAAAACGGTAACAAGTGTCTGATTTTGTTTATATCATTACATGAGCATGAAAACCAAAATTGAAAATTCTGAACTCCCGCAGTCAAGTATCCCGAAACCTCAATCAATAAAACTATATTGGCGCATTCATGCTG (SEQ ID NO: 1).

It should be noted that the oligonucleotide sequence is a spacer sequence from the Drosophila Mir2 gene cluster, which is the sequence between mir-2a-1 and mir-2b-2 in the Drosophila genome. The inventors found that the The oligonucleotide has the recognition site of Drosophila internal ribonuclease, which is used to connect multiple shRNA template sequences, that is, two adjacent siRNAs among multiple siRNAs are connected through the oligonucleotide, which can make the transcription The products form independent shRNAs through the cleavage mechanism of Drosophila itself, so that the target genes of each RNAi can be simultaneously knocked out, and multiple genes can be knocked out at the same time to realize the simultaneous regulation of multiple genes in Drosophila.

According to another aspect of the present invention, the present invention also provides a construct. According to an embodiment of the present invention, the construct comprises a plurality of siRNAs, two adjacent siRNAs among the plurality of siRNAs are connected by the aforementioned oligonucleotide, wherein the target genes of the plurality of siRNAs are the same or different . The inventors have surprisingly found that the construct of the present invention can not only efficiently express multiple siRNAs targeting different genes at the same time, thereby enabling simultaneous regulation of multiple genes, but also express multiple siRNAs targeting the same gene to enhance the regulation of a single gene. Knockout efficiency. Moreover, the construction of the construct is simple and convenient, the expression is efficient and stable, and it is applicable to all tissues and cells of Drosophila, so that it can be effectively used in the functional research of various Drosophila genes.

According to an embodiment of the present invention, the construct further comprises: 2×QUAS sequence; and/or 10×UAS sequence. Among them, it should be noted that there are currently two binary expression systems in Drosophila, Gal4/UAS and QF/QUAS systems. Their working principles are the same. Taking QF/QUAS as an example, QF is a transcriptional regulator, QUAS is the QF binding site on DNA, and 2×QUAS refers to two QF binding sites. When QF binds to QUAS, it will initiate the transcription of genes downstream of QUAS. However, the carrier of the present invention contains UAS and QUAS at the same time, so that both Gal4 and QF can be used for regulation. Therefore, these two repressible binary expression systems can enrich the means of transgene expression, lineage tracing and gene function chimera analysis in Drosophila, making the regulation of expression more flexible.

According to an embodiment of the present invention, the construct further comprises a selection marker gene. Thus, it is convenient to screen positive plasmids. According to some specific examples of the present invention, the selection marker gene is an ampicillin resistance gene and/or a selection marker vermilion gene. Thus, the positive plasmid screening effect is good.

According to an embodiment of the present invention, the construct further comprises a promoter. The inventors found that the background expression efficiency of U-promoter in various tissues and organs of Drosophila is low, and the expression efficiency is high when induced. Therefore, according to an embodiment of the present invention, the promoter is a U-promoter.

According to an embodiment of the present invention, the construct further comprises: genome site-specific integration attB sequence; ftz intron sequence; and SV40ployA sequence. As a result, the plasmid can be site-specifically integrated into the Drosophila genome, the translation efficiency can be enhanced, and it is beneficial to regulate the termination of transcription.

According to an embodiment of the present invention, the construct further comprises: two LoxP sequences; and an insulator gypsy sequence, wherein the insulator gypsy sequence is located between the two LoxP sequences. Thus, the gypsy sequence between LoxP can be removed under the action of Cre recombinase to achieve the purpose of regulating the expression of siRNA, and the gypsy sequence can enhance gene transcription, thereby making the regulation of gene expression more flexible, and the means of gene function analysis more diverse.

Wherein, according to an embodiment of the present invention, the sequence of the above elements is as follows:

attB:

GCTGCATCCAACGCGTTGGGAGCTCTCCGGATCAATTCGGCTTCACGTACCGTCGACGATGTAGGTCACGGTCTCGAAGCCGCGGTGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCGTACTCCACCTCACCCATCTGGTCCATCATGATGAACGGGTCGAGGTGGCGGTAGTTGATCCCGGCGAACGCGCGGCGCACCGGGAAGCCCTCGCCCTCGAAACCGCTGGGCGCGGTGGTCACGGTGAGCACGGGACGTGCGACGGCGTCGGCTGGTGCGGATACGCGGGGCAGCGTCAGCGGGTTCTCGACGGTCACGGCGGGCATGTCGACAAGCCGAATTGATCCACTAGAAGGCCTAATTC(SEQ ID NO：44)；

ftz intron:

CTAGTTCTGATCTGCTAGACAATTGTTGGCATCAGGTAGGCATCACACACGATTAACAACCCCTAAAAATACACTTTGAAAATATTGAAAATATGTTTTTGTATACATTTTTGATATTTCAAATAATACGCAGTTATAAAACTCATTAGCTAACCCATTTTTTTCTTTGCTTATGCTTACAGATTGCAAAGAACTAGAG (SEQ ID NO: 45);

SV40ployA:

GGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCACTGCTATACAAGAAAATTATGGAAAAATATTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGGAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGTTCCA(SEQ ID NO：46)；

LoxP:

CTAGTATAACTTCGTATAATGTATGCTATACGAAGTTATG (SEQ ID NO: 47);

gypsy:

TTGGCCACGTAATAAGTGTGCGTTGAATTTATTCGCAAAAACATTGCATATTTTCGGCAAAGTAAAATTTTGTTGCATACCTTATCAAAAAATAAGTGCTGCATACTTTTTAGAGAAACCAAATAATTTTTTATTGCATACCCGTTTTTAATAAAATACATTGCATACCCTCTTTTAATAAAAAATATTGCATACTTTGACGAAACAAATTTTCGTTGCATACCCAATAAAAGATTATTATATTGCATACCCGTTTTTAATAAAATACATTGCATACCCTCTTTTAATAAAGAATATTGCATACGTTGACGAAACAAATTTTCGTTGCATACCCAATAAAAGATTATTATATTGCATACCTTTTCTTGCCATACCATTTAGCCGATCAATTCTGCTCGGCAACAGTATATTTGTGGTGTGCCAACCAACAAC(SEQ ID NO：48)；

2×QUAS:

CGCTCGGGTAATCGCTTATCCTCGGGTAATCGCTTATCCTTAAGC (SEQ ID NO: 49);

10×UAS:

GCAGGTCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGACTCCCGCGGTCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTAC (SEQ ID NO: 50).

In another aspect of the present invention, the present invention also provides a method for knocking out a target gene in Drosophila. According to an embodiment of the present invention, the method utilizes the aforementioned constructs to transform Drosophila. The inventors surprisingly found that using the method of the present invention, not only can simultaneously and efficiently express multiple siRNAs targeting different genes in Drosophila, thereby enabling the simultaneous knockout of multiple genes, but also expressing multiple siRNAs targeting different genes in Drosophila. The same gene siRNA can achieve high-efficiency knockout of a single gene, and the knockout effect is good. Moreover, the method of the present invention is simple to operate and has stable results, and is applicable to all tissues and cells of Drosophila, and it is easy to regulate the gene knockout efficiency of Drosophila by using the method, and the obtained Drosophila phenotype is obvious, so it can be effectively used in Drosophila. Fly gene function studies.

According to an embodiment of the present invention, the method for knocking out a gene of interest in Drosophila of the present invention utilizes a GAL4-UAS binary expression vector system for the transformation, wherein the construct comprises 2×QUAS sequences; and/or 10×UAS sequences . Therefore, these two repressible binary expression systems can enrich the means of transgene expression, lineage tracing and gene function chimera analysis in Drosophila, making the expression regulation of this method more flexible.

According to an embodiment of the present invention, the transformation further includes: transforming the first fruit fly with the construct to obtain a first transgenic fruit fly; providing a second transgenic fruit fly capable of expressing Gal4 protein and/or Q protein; and crossing the first transgenic fruit fly with the second transgenic fruit fly to obtain offspring transgenic fruit flies in which the target gene is knocked out.

The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific techniques or conditions are indicated in the examples, according to the techniques or conditions described in the literature in this field (for example, refer to J. Sambrook et al., "Molecular Cloning Experiment Guide" translated by Huang Peitang, third edition, Science Press) or follow the product instructions. The reagents or instruments used, whose manufacturers are not indicated, are conventional products that can be purchased from the market, for example, they can be purchased from Illumina Corporation.

Example 1

To construct the RNAi expression vector VADUM, the specific steps are as follows:

1. Cloning of vector gene elements

1.1. Vectors used for cloning genetic elements

Vector pVALIUM20 was digested with HindⅢ and SacⅠ, ligated with the annealed synthetic MCS fragment, transformed into Escherichia coli Top10, and positive clones were selected. The correct plasmid obtained by sequencing was designated as pV-MCS, and each gene element was cloned with this plasmid.

The MCS primers used for annealing are:

MCS-F: AGCTTCAGCTAGCATGAGCTCAACTGCAGATCCGCGGAATCTAGAAGCT (SEQ ID NO: 2)

MCS-R: TCTAGATTCCGCGGATCTGCAGTTGAGCTCATGCTAGCTGA (SEQ ID NO: 3)

1.2. Clone a LoxP

Plasmid pV-MCS was digested with NheI and SacI, ligated with the annealed synthetic LoxP fragment, transformed into Escherichia coli Top10, and positive clones were selected. The correct plasmid obtained by sequencing was designated as pV-LoxP.

The LoxP primers used for annealing are:

LoxP-F1: ctagcATAACTTCGTATAATGTATGCTATACGAAGTTATGagct (SEQ ID NO: 4)

LoxP-R1: CATAACTTCGTATAGCATACATTATACGAAGTTATg (SEQ ID NO: 5)

1.3. Clone Q-UAS

Plasmid pV-LoxP was digested with PstI and SacI, ligated with the annealed synthetic Q-UAS fragment, transformed into Escherichia coli Top10, and positive clones were selected. The correct plasmid obtained by sequencing was designated as pV-Q-LoxP.

The sequences of Q-UAS primers used for annealing are:

Q-F: CGCTCGGGTAATCGCTTATCCTCGGGTAATCGCTTATCCTTAAGCTGCA (SEQ ID NO: 6)

Q-R: GCTTAAGGATAAGCGATTACCCGAGGATAAGCGATTACCCGAGCGagct (SEQ ID NO: 7)

1.4. Clone 5×UAS

Using pVALIUM20 as a template, PCR amplification was performed with primers UAS-F and UAS-R. The primer sequences are as follows:

UAS-F: AACTGCAGGTCGGAGTACTGTCCTCC (SEQ ID NO: 8)

UAS-R: AACCGCGGGAGTCTCCGCTCGGAG (SEQ ID NO: 9)

A 123-base PCR product was obtained. The PCR product was double-digested with PstⅠ and SacⅡ, ligated with the plasmid pV-Q-LoxP which had undergone the same double-digestion, transformed into Escherichia coli Top10, and positive clones were selected. The correct plasmid obtained by sequencing was designated as pV-UAS-Q-LoxP.

1.5. Clone another 5×UAS and U-promoter

Using pVALIUM3 as a template, PCR amplification was performed with primers promoter-F and promoter-R. The primer sequences are as follows:

Promoter-F: AACCGCGGTCGGAGTACTGTCCTCC (SEQ ID NO: 10)

Promoter-R: AATCTAGACTTTGGTATGCGTCTTGTGAT (SEQ ID NO: 11)

A 282-base PCR product was obtained. The PCR product was double-digested with XbaI and SacII, ligated with the plasmid pV-UAS-Q-LoxP that had undergone the same double-digestion, transformed into Escherichia coli Top10, and positive clones were selected. The correct plasmid obtained by sequencing was designated as pV-U-promoter-UAS-Q-LoxP.

2. Construction of expression vector

2.1 Construction of expression vector backbone

Vector pVALIUM20 was digested with SpeI, ligated with the annealed synthetic MCSb fragment, transformed into Escherichia coli Top10, and positive clones were selected. The correct plasmid obtained by sequencing was designated as pV-MCS2.

The MCSb primers used for annealing are:

MCSb-F: CTAG GCTAGC TCAGATCTTGGAATTCA TCTAGA CAGTA (SEQ ID NO: 12)

MCSb-R: CTAGTACTG TCTAGA TGAATTCCAAGATCTGA GCTAGC (SEQ ID NO: 13)

Use the following mutation method to remove some restriction sites of plasmid pV-MCS2 to facilitate future cloning. The enzyme cutting sites removed were EcoRⅤ, BamHI and XhoI in vermilion, XhoI between vermilion and attB, KpnI in attB, and MfeI in gypsy.

Using the plasmid pV-MCS2 as a template, a pair of reverse complementary mutation primers were used for PCR amplification. The PCR product was digested with DpnⅠ and directly transformed into Escherichia coli Top10, and the monoclonal strain was selected, and the plasmid was extracted and digested to identify the correct mutant plasmid. The mutation primers used are as follows:

EcoRⅤ-F: GGAGCGCAGATATTGATAACCAGACGAGCCACCAGTG (SEQ ID NO: 14)

EcoRⅤ-R:CACTGGTGGCTCGTCTGGTTATCAATATCTGCGCTCC (SEQ ID NO: 15)

BamHI-F: CCAGTGCCCAACTGTTGCGATCCAATCATGCGTTG (SEQ ID NO: 16)

BamHI-R:CAACGCATGATTGGATCGCAACAGTTGGGCACTGG (SEQ ID NO: 17)

XhoI-F1: CAGATCGTGACTCCTCGACCGGCGGATGCTGGCGAAC (SEQ ID NO: 18)

XhoI-R1: GTTCGCCAGCATCCGCCGGTCGAGGAGTCACGATCTG (SEQ ID NO: 19)

XhoI-F2: GATATCATCGATCTCGACGCTGCATCCAACGCGTT (SEQ ID NO: 20)

XhoI-R2: AACGCGTTGGATGCAGCGTCGAGATCGATGATATC (SEQ ID NO: 21)

KpnI-F: GGATCAATTCGGCTTCACGTACCGTCGACGATGTA (SEQ ID NO: 22)

KpnI-R: TACATCGTCGACGGTACGTGAAGCCGAATTGATCC (SEQ ID NO: 23)

MfeI-F: CCATTTAGCCGATCAATTCTGCTCGGCAACAGTATAT (SEQ ID NO: 24)

MfeI-R: ATATACTGTTGCCGAGCAGAATTGATCGGCTAAATGG (SEQ ID NO: 25)

The mutated plasmid was denoted as pVAM, which was used as the backbone to construct the expression vector.

2.2. Add expression elements U-promoter, UAS, Q-UAS and LoxP

Plasmid pV-U-promoter-UAS-Q-LoxP was digested with XbaI and NheI, and a 473-base fragment was recovered from the gel, namely U-promoter-UAS-Q-LoxP, which was ligated with the vector pVAM that was digested with the same double enzymes , transform Escherichia coli Top10, and select positive clones. The plasmid is marked as pVAM-U-promoter-UAS-Q-LoxP,

2.3. Add another LoxP

Plasmid pVAM-U-promoter-UAS-Q-LoxP was digested with NheI, treated with alkaline phosphatase, ligated with the annealed synthetic LoxP fragment, transformed into Escherichia coli Top10, and positive clones were selected. The correct plasmid obtained by sequencing was designated as pVAM-U-promoter-UAS-Q-LoxP2.

The sequences of LoxP primers used for annealing are:

LoxP-F2: CTAGTATAACTTCGTATAATGTATGCTATACGAAGTTATG (SEQ ID NO: 26)

LoxP-R2: TAGCATAACTTCGTATAGCATACATTATACGAAGTTATA (SEQ ID NO: 27)

2.4. Add gypsy between two LoxP

Using pVAM as a template, PCR amplification was performed with primers gypsy-F and gypsy-R.

Gypsy-F:TT ACTAGT TGGCCACGTAATAAGTGTGC (SEQ ID NO: 28)

gypsy-R:TT ACTAGT GTTGTTGGTTGGCACACCAC (SEQ ID NO: 29)

A 447-base PCR product was obtained. The PCR product was digested with SpeI, ligated with the plasmid pVAM-U-promoter-UAS-Q-LoxP2 that had been digested with NheI and treated with alkaline phosphatase, transformed into Escherichia coli Top10, and positive clones were selected. The correct plasmid obtained by sequencing was designated as pVAM-U-promoter-UAS-Q-LoxP2-gypsy.

2.5. Add the multiple cloning restriction site MCS1 for cloning a single hairpin DNA

Plasmid pVALIUM20 was double-digested with XbaI and SpeI, and the 730-base fragment was recovered from the gel, ligated with the same double-digested plasmid pVAM-U-promoter-UAS-Q-LoxP2-gypsy, transformed into E. coli Top10, and positive clones were selected. The correct plasmid was designated pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCS1.

2.6. Add a linker that connects multiple hairpin DNAs

Using the Drosophila genome as a template, PCR amplification was performed with primers linker-F and linker-R.

Linker-F: AA TCTAGA CGCGATGCTCAAGGCAAAAA (SEQ ID NO: 30)

linker-R:AA CCTAGG CAGCATGAATGCGCCAATAT (SEQ ID NO: 31)

A 233-base PCR product was obtained. The PCR product was double-digested with XbaⅠ and AvrⅡ, ligated with the plasmid pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCS1 that had been digested with AvrⅡ and treated with alkaline phosphatase, transformed into E. coli Top10, and positive clones were selected. The correct plasmid obtained by sequencing was designated as pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCS1-linker.

2.7. Add the multi-cloning restriction site MCS2 for cloning multiple hairpin DNA

Plasmid pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCS1-linker was digested with SpeI, treated with alkaline phosphatase, ligated with the annealed synthetic MCS2 fragment, transformed into E. coli Top10, and positive clones were selected. The correct plasmid obtained by sequencing was used as the expression vector, which was denoted as VADUM, as shown in Figure 1.

Example 2

Polycomb Group (PcG) protein is a family of proteins discovered for the first time in Drosophila that can remodel chromatin so that specific genes can be epigenetically silenced, and plays a very important role in the development process. PcG proteins function by forming different protein complexes, including two repressive complexes, Polycomb repressive complexes1and2 (PRC1and PRC2). The core PRC1 of Drosophila consists of 4 proteins, Polycomb(Pc), Sexcombs extra(Sce), Polyhomeotic(Ph) and Posterior sex combs(Psc). Due to the complexity and variability of PRC1 complex composition, its function and physiological role are not very clear. In particular, knockout of the four components of the core PRC1 alone often has no obvious phenotype, which has caused great trouble to the study of PRC1 function. The present invention can inhibit the expression of these four proteins at the same time, and obtain a phenotype different from single gene knockout, which brings convenience to the research of PRC1 function.

According to the following method, each protein in the Drosophila PRC1 complex is simultaneously inhibited using the construct of the present invention:

1. Construction of RNAi vectors that inhibit gene expression of core PRC1 components

Enter the CDS sequences of the Pc, Sce, Ph and Psc genes into the DSIR website (http://biodev.extra.cea.fr/DSIR/DSIR.html), and design 21 nucleotide siRNAs only targeting each gene. The 21-nucleotide antisense nucleotide (anti-sense oligo) sequence and its reverse complementary sequence (sense oligo) were respectively placed in the corresponding positions of the following primers to obtain 4 pairs of primers for each gene.

F: ctagcagt sense oligo tagttatattcaagcata anti-sense oligo gcg

R: aattcgc sense oligo tatgcttgaatataacta anti-sense oligo actg

Pc-F: ctagcagtCTCATCGACATCTACGAACAAtagttattcaagcataTTGTTCGTAGATGTCGATGAGgcg (SEQ ID NO: 32)

Pc-R: aattcgcCTCATCGACATCTACGAACAAtatgcttgaatataactaTTGTTCGTAGATGTCGATGAGactg (SEQ ID NO: 33)

Sce-F: ctagcagtCCGCAAGAAGCTCGTCTCCAAtagttatattcaagcataTTGGAGACGAGCTTCTTGCGGgcg (SEQ ID NO: 34)

Sce-R: aattcgcCCGCAAGAAGCTCGTCTCCAAtatgcttgaatataactaTTGGAGACGAGCTTCTTGCGGactg (SEQ ID NO: 35)

Ph-F: ctagcagtCCGGGTCAGCTGGTTTCTCTTAtagttattcaagcataTAAGAGAACCAGCTGACCCGGgcg (SEQ ID NO: 36)

Ph-R: aattcgcCCGGGTCAGCTGGTTTCTTATatgcttgaatataactaTAAGAGAACCAGCTGACCCGGactg (SEQ ID NO: 37)

Psc-F: ctagcagtCCAGTCGAATATGATGTACAAtagttattcaagcataTTGTACATCATATTCGACTGGgcg (SEQ ID NO: 38)

Psc-R: aattcgcCCAGTCGAATATGATGTACAAtatgcttgaatataactaTTGTACATCATATTCGACTGGactg (SEQ ID NO: 39)

The synthesized 4 pairs of primers were annealed, respectively ligated with the plasmid VADUM double-digested with NheI and EcoRI, transformed into Escherichia coli Top10, and positive clones were selected. The recombinant plasmids of RNAi single gene were obtained by sequencing, which were respectively marked as VADUM-Pc, VADUM-Sce, VADUM-Ph and VADUM-Psc.

Plasmid VADUM-Sce was double-digested with XbaI and SpeI, and a small fragment of 407 bases was recovered, then ligated with plasmid VADUM-Pc that was single-digested with SpeI and treated with alkaline phosphatase, transformed into Escherichia coli Top10, and positive clones were selected. The correct plasmid was designated VADUM-Pc-Sce. The plasmid VADUM-Ph was double-digested with XbaI and SpeI, and a small fragment of 407 bases was recovered, ligated with the plasmid VADUM-Pc-Sce which was single-digested with SpeI and treated with alkaline phosphatase, and transformed into E. coli Top10, which was identified as correct The plasmid for VADUM-Pc-Sce-Ph was designated as VADUM-Pc-Sce-Ph. Finally, the plasmid VADUM-Psc was double-digested with XbaI and SpeI, and a small fragment of 407 bases was recovered, ligated with the plasmid VADUM-Pc-Sce-Ph treated with SpeI single-enzyme and alkaline phosphatase, and transformed into Escherichia coli Top10, When positive clones are selected, a recombinant plasmid with simultaneous RNAi 4 genes is obtained, which is denoted as VADUM-Pc-Sce-Ph-Psc.

2. Obtaining of transgenic fruit flies

The five RNAi vectors obtained in the previous step were injected into 0-1h Drosophila (genotype ysc v nanos-integrase; attP2) embryos with a microinjector, and cultured at 25°C.

Then, the hatched male fruit flies were crossed with the fruit flies whose genotype was y sc v, and the male fruit flies whose eye color was wild-type deep red were selected from the offspring, which were the transgenic fruit flies that had been integrated into the RNAi vector. The selected male fruit flies are crossed with the fruit flies whose genotype is y sc v; Dr, e/TM3, sb, and the fruit flies with dark red eyes and short setae are selected from the offspring for selfing, and then the dark red eyes and short bristles are selected from the offspring. Drosophila with long bristles will get homozygous transgenic flies.

3. Inhibiting the expression of each component gene of core PRC1 affects the development of Drosophila

The transgenic fruit flies obtained in step 2 of this example were crossed with nos-Gal4 fruit flies, cultured at 25° C., and the phenotypes of the offspring were observed.

The results are as follows: Pc, Sce, Ph and Psc alone RNAi Drosophila showed no significant difference compared with the wild-type Drosophila; while the four genes were RNAi at the same time, the ovary of the offspring female Drosophila was significantly smaller, and Does not lay eggs. This indicates that there is redundancy in the functions of each component of the core PRC1, and the phenotype may not be shown after RNAi alone due to the effects of other components, and the obvious phenotype can only be obtained after RNAi of all components.

Example 3

Histone H1 is one of the five histones that make up eukaryotic chromatin. It not only plays an important role in the establishment and maintenance of higher-order chromosome structures, but also plays a certain role in cell proliferation and gene expression regulation. There is only one histone H1 gene in Drosophila, making it a favorable model to study its function. However, since the Drosophila histone H1 gene has 23 copies, it is difficult to obtain its mutants, and traditional RNAi methods often fail to achieve the ideal knockout efficiency. The invention can conveniently regulate the efficiency of gene knockout, which greatly facilitates the study of its function.

Using the construct of the present invention to enhance the knockout efficiency of histone H1, the specific method is as follows:

1. Construction of histone H1 RNAi vector

Design two pairs of primers against histone H1 according to the method described in Example 2, the sequence is as follows:

H1-F1: ctagcagtTTGGTACATGTTCGCAATTAAtagttattcaagcataTTAATTGCGAACATGTACCAAgcg (SEQ ID NO: 40)

H1-R1: aattcgcTTGGTACATGTTCGCAATTAAtatgcttgaatataactaTTAATTGCGAACATGTACCAAactg (SEQ ID NO: 41)

H1-F2: ctagcagtACCAGCGACAGTTGAGAAGAAtagttattcaagcataTTCTTCTCAACTGTCGCTGGTgcg (SEQ ID NO: 42)

H1-R2: aattcgcACCAGCGACAGTTGAGAAGAAtatgcttgaatataactaTTCTTCTCAACTGTCGCTGGTactg (SEQ ID NO: 43)

After the two pairs of primers were annealed, they were ligated with the plasmid VADUM double-digested with NheI and EcoRI respectively, transformed into Escherichia coli Top10, and positive clones were selected. The recombinant plasmids of RNAi histone H1 were obtained by sequencing, which were denoted as VADUM-H1-1 and VADUM-H1-2.

Plasmid VADUM-H1-2 was double-digested with XbaI and SpeI, and a small fragment of 407 bases was recovered, then ligated with plasmid VADUM-H1-1 that was digested with SpeI and treated with alkaline phosphatase, transformed into E. coli Top10, and selected Positive clone. The correct plasmid was designated VADUM-2xH1. Plasmid VADUM-2×H1 was double-digested with XbaI and SpeI, and the 814-base fragment was recovered, then ligated with plasmid VADUM-2×H1 single-digested with SpeI and treated with alkaline phosphatase, transformed into E. coli Top10, and selected Positive clone. The correct plasmid was designated VADUM-4xH1. Finally, the plasmid VADUM-4×H1 was double-digested with XbaI and SpeI, and the fragment of 1628 bases was recovered, ligated with the plasmid VADUM-4×H1 treated with SpeI single-enzyme and alkaline phosphatase, transformed into E. coli Top10, and selected Positive clone. The correct plasmid was designated VADUM-8xH1.

2. The effect of histone H1 knockout on wing development

Use the RNAi vectors VADUM-H1-1, VADUM-4×H1 and VADUM-8×H1 obtained in the previous step to obtain transgenic RNAi fruit flies according to the method described in Example 2 (that is, use a microinjector to inject The five RNAi vectors were injected into Drosophila (genotype y sc v nanos-integrase; attP2) embryos at 0-1h respectively, and cultured at 25 degrees Celsius. Then, the hatched male Drosophila and genotype y sc v Drosophila cross, select the male Drosophila whose eye color is wild-type deep red from the offspring, that is, the transgenic Drosophila that has been integrated into the RNAi vector), and record them as H1KD1, H1KD2 and H1KD3 respectively.

Three kinds of transgenic RNAi Drosophila were crossed with ms1096-Gal4, cultured at 25 degrees Celsius, and the wing phenotypes of the offspring were observed. The results are shown in Figure 2: Compared with the wings of wild-type male Drosophila (wt), the wings of male Drosophila offspring crossed between H1KD1 and ms1096-Gal4 have no significant difference; the wing size of male Drosophila offspring crossed between H1KD2 and ms1096-Gal4 is slightly Smaller, abnormal veins and slightly curled; H1KD3 and ms1096-Gal4 hybrid offspring male Drosophila significantly smaller wings, severely disordered veins and curled into a ball.

Those skilled in the art understand that traditional RNAi vectors are difficult to achieve the knockout effect of H1KD3, but the above-mentioned invention can conveniently regulate the gene knockout efficiency from weak to strong.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the invention is defined by the claims and their equivalents.

Claims (10)

1. The use of the nucleotide sequence shown in SEQ ID NO: 1 in simultaneously knocking out multiple genes of interest in Drosophila, wherein the use is represented by two adjacent siRNAs used as a knockout construct. A linker between multiple siRNAs, the target genes of the multiple siRNAs are the same or different. 2. A construct, characterized in that it comprises a plurality of siRNAs, two adjacent siRNAs in the plurality of siRNAs are connected by the nucleotide sequence shown in SEQ ID NO: 1, wherein the siRNAs of the plurality of siRNAs The target genes are the same or different. 3. The construct according to claim 2, further comprising: 2 x QUAS sequences; and/or 10 x UAS sequences. 4. The construct according to claim 2, further comprising a screening marker gene, Optionally, the selection marker gene is an ampicillin resistance gene and/or a selection marker vermilion gene. 5. The construct according to claim 2, further comprising a promoter, Optionally, the promoter is a U-promoter. 6. The construct according to claim 2, further comprising: Genome site-specific integration of attB sequence; ftz intronic sequence; and SV40ployA sequence. 7. The construct according to claim 2, further comprising: 2 LoxP sequences; and insulator gypsy sequence, Wherein, the insulator gypsy sequence is located between two LoxP sequences. 8. A method for knocking out the target gene of Drosophila, characterized in that, Transformation of Drosophila with the constructs of claims 2-7. 9 . The method according to claim 8 , wherein the transformation is performed using a GAL4-UAS binary expression vector system, wherein the construct comprises 2×QUAS sequences; and/or 10×UAS sequences. 10. The method according to claim 8, wherein said transforming further comprises: Transforming a first fruit fly with the construct to obtain a first transgenic fruit fly; providing a second transgenic fruit fly capable of expressing Gal4 protein and/or Q protein; and The first transgenic fruit fly is crossed with the second transgenic fruit fly to obtain progeny transgenic fruit flies in which the target gene is knocked out.