WO2016000217A1 - 分离的寡核苷酸及其应用 - Google Patents

分离的寡核苷酸及其应用 Download PDF

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WO2016000217A1
WO2016000217A1 PCT/CN2014/081427 CN2014081427W WO2016000217A1 WO 2016000217 A1 WO2016000217 A1 WO 2016000217A1 CN 2014081427 W CN2014081427 W CN 2014081427W WO 2016000217 A1 WO2016000217 A1 WO 2016000217A1
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gene
construct
sequence
fruit fly
drosophila
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倪建泉
刘鲁萍
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Tsinghua University
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Tsinghua University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/60—New or modified breeds of invertebrates
    • A01K67/61—Genetically modified invertebrates, e.g. transgenic or polyploid
    • A01K67/65—Genetically modified arthropods
    • A01K67/68—Genetically modified insects
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to the field of biotechnology, and in particular to isolated oligonucleotides and uses thereof, and more particularly, to isolated nucleotides, constructs, and methods for knocking out genes of interest in Drosophila. Background technique
  • RNA interference refers to the highly conserved, highly-specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) during evolution. RNAi technology can specifically reduce gene expression, and is highly efficient and easy to operate, so this technology has been widely used in various research fields of fruit flies.
  • RNAi technology is generally combined with a binary expression system to achieve tissue specificity in transgenic Drosophila and gene knockout at specific developmental stages.
  • the binary expression system currently widely used in Drosophila is the GAL4-UAS system, in which the yeast transcription factor gene GAL4 is controlled by a specific promoter.
  • GAL4 can activate the expression of another exogenous insertion gene containing the upstream activation sequence (UAS).
  • the promoter commonly used in RNAi vectors is Hsp70, which activates the expression of exogenously inserted genes when activated by GAL4.
  • GAL4 when GAL4 is absent, the promoter can still act to initiate the insertion of the foreign gene, so that the exogenous gene has a certain amount of background expression, which affects the judgment of the experimental results.
  • RNAi technology is the transcription of short hairpin RNA (shRNA) from the expression vector to mimic the naturally occurring microRNA in Drosophila.
  • shRNA then produces small interfering RNA (siRNA) through the microRNA pathway in Drosophila, thereby enabling gene knockout (Ni et al., Nature Methods. 2011 May; 8(5): 405-7.).
  • siRNA small interfering RNA
  • this technique can only knock out a single gene. Therefore, when the function of the gene of interest is redundant with other genes, or when research involving multiple gene functions, the technique does not satisfactorily meet the needs of the researcher. Summary of the invention
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide a means for simultaneously knocking out multiple genes of interest in fruit flies.
  • the invention provides an isolated oligonucleotide.
  • the oligonucleotide has the nucleotide sequence set forth in SEQ ID NO: 1.
  • the inventors have found that when preparing a construct for Drosophila gene knockout, the oligonucleotide is used to join multiple shRNA template sequences, ie, two adjacent siRNAs in a plurality of siRNAs are passed through the oligonucleotide.
  • the transcripts can form independent shRNAs through the scission mechanism of Drosophila itself, thereby simultaneously targeting the respective target genes of RNAi, and, in turn, simultaneously knocking out multiple genes to achieve simultaneous regulation of multiple genes in Drosophila.
  • SEQ ID NO: 1 is sometimes also used.
  • the oligonucleotide shown is called "linker".
  • the invention also provides a construct.
  • the construct comprises a plurality of siRNAs, and two adjacent siRNAs of the plurality of siRNAs are linked by the oligonucleotides described above, 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 simultaneously express multiple siRNAs for different genes at the same time, thereby enabling simultaneous regulation of multiple genes, and also capable of expressing multiple siRNAs targeting the same gene to enhance individual genes. Knock out efficiency.
  • the construction of the construct is simple and convenient, and the expression is highly efficient and stable, and is suitable for all tissues and cells of Drosophila, so that it can be effectively used for functional research of various fruit fly genes.
  • the construct further comprises: a 2xQUAS sequence; and/or a lOxUAS sequence.
  • the two suppressable binary expression systems are capable of enriching the expression of transgene expression, lineage tracking and gene functional chimerism in Drosophila, making expression regulation more flexible.
  • the construct further comprises a selection marker gene.
  • the screening marker gene is an ampicillin resistance gene and/or a screening marker vermilion gene.
  • the positive plasmid screening effect is good.
  • the construct further comprises a promoter.
  • U-promoter i.e., U-promoter
  • the promoter is U-promoter.
  • the construct further comprises: a genomic site-specific integration attB sequence; an ftz intron sequence; and an SV40 ployA sequence.
  • a genomic site-specific integration attB sequence As a result, the plasmid can be integrated into the Drosophila genome, the translation efficiency is enhanced, and the transcription termination is regulated.
  • the construct further comprises: 2 LoxP sequences; and a segregant gypsy sequence, wherein the segregant gypsy sequence is located between 2 LoxP sequences.
  • the gypsy sequence between LoxP can be removed by the action of Cre recombinase to achieve the purpose of regulating the expression of siRNA, and the gypsy sequence can enhance the transcription of the gene, thereby making the regulation of gene expression more flexible, and the means of gene function analysis. More diverse.
  • the present invention also provides a method of knocking out a gene of interest in a fruit fly.
  • the method utilizes the constructs described above to transform fruit flies.
  • the inventors have surprisingly found that, by using the method of the present invention, not only siRNAs for different genes can be efficiently expressed in Drosophila at the same time, thereby enabling simultaneous knockout of multiple genes, and also capable of expressing multiple targets in Drosophila
  • the same gene siRNA, in order to achieve efficient knockout of a single gene, knock-out effect is good.
  • the method of the invention has simple operation and stable results, and is suitable for all tissues and cells of Drosophila, and the method is easy to regulate the gene knockout efficiency of Drosophila, and the obtained phenotype of Drosophila is obvious, so that it can be effectively used for fruit. Fly gene function research.
  • the method of knocking out a Drosophila gene of interest of the present invention performs the transformation using a GAL4-UAS binary expression vector system, wherein the construct comprises a 2xQUAS sequence; and/or a lOxUAS sequence.
  • the two suppressable binary expression systems can enrich the means of transgene expression, lineage tracing and gene function chimera analysis in Drosophila, making the expression regulation of the method more flexible.
  • the transforming further comprises: transforming the first fruit fly with the construct to obtain a first transgenic fruit fly; providing a second transgenic fruit fly, the second transgenic fruit fly capable of expressing Gal4 protein And/or Q protein; and hybridizing said first transgenic fruit fly to said second transgenic fruit fly to obtain a progeny transgenic fruit fly, said gene of interest in said progeny transgenic fruit fly being knocked out.
  • Figure 1 shows a map of a VADUM expression vector in accordance with one embodiment of the present invention
  • Figure 2 shows a picture of wild-type and transgenic male fruit fly wings according to one embodiment of the present invention, wherein Figure 2A shows a picture of wild-type male fruit fly wings,
  • Figure 2B shows a picture of the cross between the HI KD1 and the msl096-Gal4 progeny.
  • Figure 2C shows a picture of the hybrid Drosophila wings of HI KD2 and msl096-Gal4.
  • Figure 2D shows a picture of the cross between the HI KD3 and the msl096-Gal4 progeny. Detailed description of the invention
  • the invention provides an isolated oligonucleotide. Concrete embodiment according to the present invention
  • the oligonucleotide sequence is a spacer sequence derived from the Drosophila Mir2 gene cluster and is a sequence between mir-2a-1 and mir-2b-2 in the Drosophila genome.
  • the inventors found that The oligonucleotide carries a recognition site for the Drosophila nuclear ribonuclease, which is used to link multiple shRNA template sequences, ie, two adjacent siRNAs in multiple siRNAs are linked by the oligonucleotide, enabling transcription
  • the product forms independent shRNAs through the scission mechanism of Drosophila itself, so that the target genes of RNAi can simultaneously knock out multiple genes at the same time, and realize simultaneous regulation of multiple genes in Drosophila.
  • the invention also provides a construct.
  • the construct comprises a plurality of siRNAs, and two adjacent siRNAs of the plurality of siRNAs are linked by the oligonucleotides described above, wherein the target genes of the plurality of siRNAs are the same or different .
  • the inventors have surprisingly discovered that the construction of the present invention The body can not only simultaneously express multiple siRNAs for different genes, but also realize simultaneous regulation of multiple genes, and can also express multiple siRNAs for the same gene to enhance the knockout efficiency of individual genes.
  • the construct is simple and convenient to construct, and the expression is highly efficient and stable, and is suitable for all tissues and cells of Drosophila, so that it can be effectively used for functional research of various fruit fly genes.
  • the construct further comprises: a 2xQUAS sequence; and/or a lOxUAS sequence.
  • a 2xQUAS sequence there are currently two binary expression systems in the fruit fly, Gal4/UAS and QF/QUAS systems. They work in the same way.
  • QF/QUAS is an example.
  • QF is a transcriptional regulator.
  • QUAS is the site of QF binding on DNA.
  • 2 X QUAS refers to two QF binding sites.
  • the carrier of the present invention contains both UAS and QUAS, so that either Gal4 or QF can be used.
  • the two suppressable binary expression systems are capable of enriching the expression of transgene expression, lineage tracing and gene function chimerism in Drosophila, making expression regulation more flexible.
  • the construct further comprises a selection marker gene.
  • the screening marker gene is an ampicillin resistance gene and/or a screening marker vermilion gene.
  • the positive plasmid screening effect is good.
  • the construct further comprises a promoter.
  • U-promoter has a low background expression efficiency in various tissues and organs of Drosophila, and is highly efficient in expression when induced.
  • the promoter is a U-promoter.
  • the construct further comprises: a genomic site-specific integration attB sequence; an ftz intron sequence; and an SV40 ployA sequence.
  • a genomic site-specific integration attB sequence As a result, the plasmid can be integrated into the Drosophila genome, the translation efficiency is enhanced, and the transcription termination is regulated.
  • the construct further comprises: 2 LoxP sequences; and a segregant gypsy sequence, wherein the segregant gypsy sequence is located between 2 LoxP sequences.
  • the gypsy sequence between LoxP can be removed by the action of Cre recombinase to achieve the purpose of regulating the expression of siRNA, and the gypsy sequence can enhance the transcription of the gene, thereby making the regulation of gene expression more flexible, and the means of gene function analysis. More diverse.
  • the present invention also provides a method of knocking out a gene of interest in a fruit fly.
  • the method utilizes the constructs described above to transform fruit flies.
  • the inventors have surprisingly found that, by using the method of the present invention, not only siRNAs for different genes can be efficiently expressed in Drosophila at the same time, thereby enabling simultaneous knockout of multiple genes, and also capable of expressing multiple targets in Drosophila
  • the same gene siRNA, in order to achieve efficient knockout of a single gene, knock-out effect is good.
  • the method of the invention has simple operation and stable results, and is suitable for all tissues and cells of Drosophila, and the method is easy to regulate the gene knockout efficiency of Drosophila, and the obtained phenotype of Drosophila is obvious, so that it can be effectively used for fruit. Fly gene function research.
  • the method of knocking out a Drosophila gene of interest of the present invention performs the transformation using a GAL4-UAS binary expression vector system, wherein the construct comprises a 2xQUAS sequence; and/or a lOxUAS sequence.
  • the two suppressable binary expression systems are capable of enriching the expression of transgene expression, lineage tracking and gene function chimera in Drosophila, making the expression regulation of the method more flexible.
  • the transforming further comprises: transforming the first fruit fly with the construct to obtain a first transgenic fruit fly; providing a second transgenic fruit fly, the second transgenic fruit fly capable of expressing Gal4 protein And/or Q protein; and hybridizing said first transgenic fruit fly to said second transgenic fruit fly to obtain a progeny transgenic fruit fly, said gene of interest in said progeny transgenic fruit fly being knocked out.
  • RNAi expression vector VADUM was constructed, and the specific steps are as follows:
  • the vector pVALIUM20 was digested with Hindlll and Sac I, ligated with the annealed synthetic MCS fragment, transformed into E. coli ToplO, and positive clones were selected. The correct plasmid was sequenced and recorded as pV-MCS, and each gene element was cloned using this plasmid.
  • the MCS primers for annealing are:
  • the plasmid pV-MCS was digested with Nhe I and Sac I, ligated with the annealed synthetic LoxP fragment, transformed into E. coli ToplO, and positive clones were selected. The correct plasmid was sequenced and recorded as pV-LoxP.
  • the LoxP primers for annealing are:
  • LoxP-Fl ctagcATAACTTCGTATAATGTATGCTATACGAAGTTATGagct (SEQ ID NO: 4)
  • LoxP-Rl CATAACTTCGTATAGCATACATTATACGAAGTTATg (SEQ ID NO: 5)
  • the plasmid pV-LoxP was digested with Pst I and Sac I, ligated with the annealed synthetic Q-UAS fragment, transformed into E. coli ToplO, and positive clones were selected. The correct plasmid was sequenced and recorded as pV-Q-LoxP.
  • the Q-UAS primer sequences for annealing are:
  • PCR amplification was performed using primers UAS-F and UAS-R.
  • the primer sequences were as follows: UAS-F: AACTGC AGGTCGGAGTACTGTCCTCC (SEQ ID NO: 8)
  • UAS-R AACCGCGGGAGTCTCCGCTCGGAG (SEQ ID NO: 9)
  • a 123 base PCR product was obtained.
  • the PCR product was digested with Pst I and Sac II, ligated with the same double-digested plasmid pV-Q-LoxP, transformed into E. coli ToplO, and positive clones were selected.
  • the correct plasmid was sequenced and recorded as pV-UAS-Q-LoxP.
  • PCR amplification was performed with primers promoter-F and promoter-R.
  • the primer sequences were 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 digested with Xba I and Sac II, ligated with the same double-digested plasmid pV-UAS-Q-LoxP, transformed into E. coli ToplO, and positive clones were selected.
  • the correct plasmid was sequenced and recorded as pV-U-promoter-UAS-Q-LoxP.
  • the vector pVALIUM20 was digested with Spe I, ligated with the annealed synthetic MCSb fragment, transformed into E. coli ToplO, and positive clones were selected. The correct plasmid was sequenced and recorded as pV-MCS2.
  • the MCSb primers for annealing are:
  • Some of the cleavage sites of plasmid PV-MCS2 were removed by the following mutations to facilitate subsequent cloning.
  • the enzyme cleavage sites removed were EcoRV, BamH I and Xho I in vermilion, Xho I between vermilion and attB, Kpn I in attB, and Mfe I in gypsy.
  • PCR amplification was carried out using a pair of reverse-complementary mutant primers using plasmid pV-MCS2 as a template. PCR product using Dpn After digestion with I, the E. coli ToplO was directly transformed, and the monoclonal mutabacterium was selected, and the plasmid was digested to identify the correct mutant plasmid.
  • the mutant primers used are as follows:
  • Xho I -F2 GATATCATCGATCTCGACGCTGCATCCAACGCGTT (SEQ ID NO: 20)
  • Xho I -R2 AACGCGTTGGATGCAGCGTCGAGATCGATGATATC (SEQ ID NO: 21)
  • Kpn I -F GGATCAATTCGGCTTCACGTACCGTCGACGATGTA
  • Kpn I -R TACATCGTCGACGGTACGTGAAGCCGAATTGATCC (SEQ ID NO: twenty three)
  • Mfe I -R ATATACTGTTGCCGAGCAGAATTGATCGGCTAAATGG (SEQ ID NO: 25)
  • the mutated plasmid was designated as pVAM, and an expression vector was constructed using this as a skeleton.
  • the vector pVAM was ligated, transformed into E. coli ToplO, and positive clones were selected.
  • the plasmid is labeled pVAM-U-promoter-UAS-Q-LoxP,
  • the plasmid pVAM-U-promoter-UAS-Q-LoxP was digested with Nhe I, treated with alkaline phosphatase, ligated with the annealed synthetic LoxP fragment, transformed into E. coli ToplO, and selected positive clones. The correct plasmid was sequenced and recorded as pVAM-U-promoter-UAS-Q-LoxP2.
  • LoxP primer sequences for annealing are:
  • PCR amplification was performed using primers gypsy-F and gypsy-R.
  • Gypsy-F TTACTAGTTGGCCACGTAATAAGTGTGC (SEQ ID NO: 28)
  • gypsy-R TTACTAGTGTTGTTGGTTGGCACACCAC (SEQ ID NO: 29)
  • a 447 base PCR product was obtained.
  • the PCR product was digested with Spe I, ligated with plasmid pVAM-U-promoter-UAS-Q-LoxP2 which was subjected to Nhe I digestion and alkaline phosphatase treatment, and transformed into E. coli ToplO, and positive clones were selected.
  • the correct plasmid was sequenced as pVAM-U-promoter-UAS-Q-LoxP2-gypsy.
  • the plasmid pVALIUM20 was digested with Xba I and Spe I, and a 730-base fragment was recovered from the gel, and ligated with the same double-digested plasmid pVAM-U-promoter-UAS-Q-LoxP2-gypsy to transform E. coli ToplO and select positive. clone.
  • the correct plasmid is designated pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCSl.
  • PCR amplification was performed using primers linker-F and linker-R.
  • Linker-F AATCTAGACGCGATGCTCAAGGCAAAAA (SEQ ID NO: 30)
  • linker-R AACCTAGGCAGCATGAATGCGCCAATAT (SEQ ID NO: 31)
  • a 233 base PCR product was obtained.
  • the PCR product was digested with Xba I and Avr ll, and ligated with the plasmid pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCS1 treated with Avrll digestion and alkaline phosphatase to transform Escherichia coli ⁇ and select positive.
  • clone The correct plasmid was sequenced and recorded as pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCSl-linker.
  • the plasmid pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCSl-linker was digested with Spe I, treated with alkaline phosphatase, ligated with the annealed synthetic MCS2 fragment, transformed into E. coli ToplO, and positive clones were selected.
  • the correct plasmid was sequenced and expressed as VADUM, as shown in Figure 1.
  • the Polycomb Group (PcG) protein is a family of proteins first discovered in Drosophila that remodels chromatin so that specific genes remain epigenetically silent, playing a very important role in development.
  • PcG proteins act by forming different protein complexes, including two inhibitory complexes, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2).
  • Drosophila core PRC1 consists of four proteins, Polycomb (Pc) Sex combs extra (Sce), Polyhomeotic (Ph) and Posterior sex combs (Psc). Due to the complexity and variability of the PRC1 complex, its function and physiological role are not well understood.
  • the four components of the core PRC1 which are knocked out alone, often have no obvious phenotype, which causes great trouble for the study of PRC1 function.
  • the expression of these four proteins can be simultaneously inhibited, and a phenotype different from that of a single gene knockout can be obtained, which facilitates the study of PRC1 function.
  • the proteins of the Drosophila PRC1 complex were simultaneously inhibited by the construct of the present invention according to the following method:
  • RNAi vector for inhibiting gene expression of core PRC1 components 1. Construction of RNAi vector for inhibiting gene expression of core PRC1 components
  • the CDS sequences of the Pc, See, Ph and Psc genes were imported into the DSIR website (http://biodev.extra.cea.fr/DSIR/DSIR.html) to design 21-nucleotide siRNAs for each gene.
  • the 21-nucleotide anti-sense oligo sequence and its sense oligo were placed in the corresponding positions of the following primers, respectively, to obtain 4 pairs of primers for each gene.
  • Pc-F ctagcagtCTCATCGACATCTACGAACAAtagttatattcaagcataTTGTTCGTAGATGTCG ATGAGgcg (SEQ ID NO: 32)
  • Pc-R aattcgcCTCATCGACATCTACGAACAAtatgcttgaatataactaTTGTTCGTAGATGTCGA
  • Ph-F ctagcagtCCGGGTCAGCTGGTTCTCTTAtagttatattcaagcataTAAGAGAACCAGCTG ACCCGGgcg (SEQ ID NO: 36)
  • Ph-R aattcgcCCGGGTCAGCTGGTTCTCTTAtatgcttgaatataactaTAAGAGAACCAGCTGA CCCGGactg (SEQ ID NO: 37)
  • Psc-F ctagcagtCCAGTCGAATATGATGTACAAtagttatattcaagcataTTGTACATCATATTCG ACTGGgcg (SEQ ID NO: 38)
  • Psc-R aattcgcCCAGTCGAATATGATGTACAAtatgcttgaatataactaTTGTACATCATATTCGA CTGGactg (SEQ ID NO: 39)
  • the synthesized 4 pairs of primers were annealed and ligated with plasmid VADUM double-digested with Nhe l and EcoR l to transform E. coli ToplO, and positive clones were selected. Recombinant plasmids of individual RNAi genes were sequenced and recorded as VADUM-Pc, VADUM-Sce, VADUM-Ph and VADUM-Psc, respectively.
  • the plasmid VADUM-Sce was digested with Xba I and Spe I, and a small fragment of 407 bases was recovered, and ligated with plasmid VADUM-Pc which was digested with Spe I and alkaline phosphatase, and transformed into E. coli ToplO. clone.
  • the correct plasmid is labeled VADUM-Pc-Sce.
  • the plasmid VADUM-Ph was digested with Xba I and P Spe I, and a small fragment of 407 bases was recovered and ligated with the plasmid VADUM-Pc-Sce which was treated with Spe I and alkaline phosphatase, and transformed into E. coli ToplO.
  • VADUM-Pc-Sce-Ph The correct plasmid was identified as VADUM-Pc-Sce-Ph.
  • the plasmid VADUM-Psc was digested with Xba I and Spe I, and a small fragment of 407 bases was recovered and ligated with the plasmid VADUM-Pc-Sce-Ph which was treated with Spe I and alkaline phosphatase to transform the large intestine.
  • Bacillus ToplO a positive clone was selected, and a recombinant plasmid of four RNAi genes was obtained, which was recorded as VADUM-Pc-Sce-Ph-Psc.
  • RNAi vectors obtained in the previous step were injected into the 0-1 h Drosophila (genotype y sc v nanos-integrase; attP2) embryos by microinjection, and cultured at 25 °C.
  • the hatched male flies are crossed with the Drosophila genotype y sc v, and the male color flies whose wild-type deep red color is selected from the progeny are transgenic flies that have been integrated into the RNAi vector.
  • the selected male fruit flies are crossed with Drosophila genotypes of y sc V ; Dr, e /TM3, S b , and the red-eyed and short-brown Drosophila are selected from the offspring, and the deep red eyes are selected from the offspring.
  • the bristles of the bristles the homozygous transgenic fruit flies.
  • transgenic fruit flies obtained in the second step of the present example were crossed with nos-Gal4 Drosophila, cultured at 25 ° C, and the phenotype of the offspring was observed.
  • Histone HI is one of the five histone proteins that make up the chromatin of eukaryotes. It not only plays an important role in the establishment and maintenance of high-level chromosome structures, but also plays a role in cell proliferation and gene expression regulation. There is only one histone HI gene in Drosophila, which is a favorable model for studying its function. However, due to the 23 copies of the Drosophila histone HI gene, it is difficult to obtain its mutant, and the traditional RNAi method often fails to achieve the desired knockout efficiency. The use of the present invention can conveniently adjust the efficiency of gene knockout and greatly facilitate the study of its function.
  • Hl-Fl ctagcagtTTGGTACATGTTCGCAATTAAtagttatattcaagcataTTAATTGCGAACATGT ACCAAgcg (SEQ ID NO: 40)
  • Hl-Rl aattcgcTTGGTACATGTTCGATTATTAAtatgcttgaatataactaTTAATTGCGAACATGT ACCAAactg (SEQ ID NO: 41)
  • Hl-F2 ctagcagtACCAGCGACAGTTGAGAAGAAtagttatattcaagcataTTCTTCTCAACTGT CGCTGGTgcg (SEQ ID NO: 42)
  • Hl-R2 aattcgcACCAGCGACAGTTGAGAAGAAtatgcttgaatataactaTTCTTCTCAACTGTC GCTGGTactg (SEQ ID NO: 43)
  • the plasmid VADUM-H1-2 was digested with Xba I and Spe I, and a small fragment of 407 base was recovered, and then ligated with plasmid VADUM-H1-1 which was treated with Spe I and alkaline phosphatase to transform Escherichia coli. ToplO, select positive clones.
  • the correct plasmid is labeled VADUM-2 X H1.
  • the plasmid VADUM-2 X H1 was digested with Xba I and P Spe I, and the fragment of 814 base was recovered, and then ligated with the plasmid VADUM-2 X H1 which was treated with Spe l and alkaline phosphatase to transform the large intestine.
  • RNAi vectors VADUM-H1-1, VADUM-4 X HI and VADUM-8 X H1 obtained in the previous step were obtained as described in Example 2 (i.e., obtained by the microinjector)
  • the five RNAi vectors were injected into 0-1 h Drosophila (genotype y sc v nanos-integrase; attP2) embryos and cultured at 25 ° C.
  • the eye color selected from the offspring is a wild-type deep red male flies, which is a transgenic fruit fly integrated into the RNAi vector, and is recorded as H1 KD1, H1 KD2 and P HI KD3, respectively.
  • Three transgenic RNAi flies were hybridized with msl096-Gal4 and cultured at 25 degrees Celsius to observe the wing phenotype of the offspring.
  • the isolated oligonucleotide of the present invention can be effectively used for the preparation of a Drosophila gene knockout construct, in particular, when preparing a construct for Drosophila gene knockout, the oligonucleotide is used for ligation a plurality of shRNA template sequences, that is, two adjacent siRNAs of a plurality of siRNAs are connected by the oligonucleotide, so that the construct comprises a plurality of siRNAs, and the construct can simultaneously knock out a plurality of genes, thereby realizing Simultaneous regulation of multiple genes in Drosophila.
  • shRNA template sequences that is, two adjacent siRNAs of a plurality of siRNAs are connected by the oligonucleotide, so that the construct comprises a plurality of siRNAs, and the construct can simultaneously knock out a plurality of genes, thereby realizing Simultaneous regulation of multiple genes in Drosophila.

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Abstract

提供了分离的寡核苷酸及其应用，其中，该寡核苷酸具有SEQ ID NO：1所示的核苷酸序列。

Description

分离的寡核苷酸及其应用
优先权信息
无 技术领域
本发明涉及生物技术领域， 具体涉及分离的寡核苷酸及其应用， 更具体地， 涉及、 分离的核苷酸、 构建体以及敲除果蝇目的基因的方法。 背景技术
RNA 干扰 (RNA interference, RNAi)是指在进化过程中高度保守的、 由双链 RNA (double-stranded RNA, dsRNA)诱发的、 同源 mRNA高效特异降解的现象。 RNAi技术 可以特异性的降低基因的表达， 并且效率高， 易于操作， 所以该技术已被广泛应用于果 蝇的各研究领域中。
在果蝇研究中， RNAi技术一般和双元表达系统相结合， 实现转基因果蝇中组织特 异性及特定发育时期的基因敲除。目前在果蝇中广泛应用的双元表达系统是 GAL4-UAS 系统， 该系统中的酵母转录因子基因 GAL4受特定的启动子控制。 反过来， GAL4可以 激活另一个包含上游激活序列 (UAS)的外源插入基因的表达。
目前 RNAi载体普遍应用的启动子是 Hsp70， 当受到 GAL4激活时， 该启动子可以 启动外源插入基因的表达。但是， 当不存在 GAL4时， 该启动子仍然能够对外源插入基 因发挥启动作用， 使得外源基因存在一定量的背景表达， 影响对实验结果的判断。
而第三代 RNAi 技术是由表达载体转录形成短发卡 RNA ( short hairpin RNA, shRNA)，来模拟果蝇体内天然存在的 microRNA。 shRNA再通过果蝇体内的 microRNA 途径产生小干扰 RNA ( siRNA) ， 从而实现基因敲除 （Ni et al.， Nature Methods. 2011 May;8(5):405-7. ) 。 但是， 该技术只能敲除单个基因。 因此， 当目的基因的功能与其它 基因存在冗余，或在涉及多个基因功能的研究时，该技术不能很好的满足研究者的需求。 发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此， 本发明的一个目的在 于提出一种能够同时敲除果蝇的多个目的基因的手段。
因而， 根据本发明的一个方面， 本发明提供了一种分离的寡核苷酸。 根据本发明的 具体实施例， 该寡核苷酸具有 SEQ ID NO: 1所示的核苷酸序列。 发明人发现， 在制备 用于果蝇基因敲除的构建体时， 将该寡核苷酸用来连接多个 shRNA模板序列， 即将多 个 siRNA中相邻的两个 siRNA通过该寡核苷酸相连， 能够使转录产物通过果蝇自身的 剪切机制形成各自独立的 shRNA, 从而同时 RNAi各自的靶基因， 进而， 能够同时敲 除多个基因， 实现对果蝇多基因的同时调控。 在本文中， 有时也将该 SEQ ID NO: 1所 示的寡核苷酸称为 " linker" 。
根据本发明的另一方面， 本发明还提供了一种构建体。 根据本发明的实施例， 该构 建体包含多个 siRNA, 所述多个 siRNA中相邻的两个 siRNA通过前面所述的寡核苷酸 相连， 其中所述多个 siRNA的靶基因相同或不同。 发明人惊奇地发现， 本发明的构建 体不仅能够同时高效表达多个针对不同基因的 siRNA,从而能够实现对多基因的同时调 控， 还能够表达多个针对同一个基因 siRNA, 以增强单个基因的敲除效率。 并且该构建 体的构建简单方便， 表达高效稳定， 适用于果蝇所有的组织和细胞， 从而能够有效用于 各种果蝇基因功能性研究。
根据本发明的实施例， 所述构建体进一步包含： 2xQUAS序列； 和 /或 lOxUAS序 列。 由此， 这两种可抑制型二元表达系统能够丰富果蝇中转基因表达、 世系追踪和基因 功能嵌合体分析的手段， 使表达的调控更加灵活。
根据本发明的实施例， 所述构建体进一步包含筛选标记基因。 由此， 便于筛选阳性 质粒。 根据本发明的一些具体示例， 所述筛选标记基因为氨苄青霉素抗性基因和 /或筛 选标记 vermilion基因。 由此， 阳性质粒筛选效果好。
根据本发明的实施例，所述构建体进一步包含启动子。发明人发现， U-promoter (即 U-启动子） 在果蝇各组织器官中的背景表达效率低， 在受到诱导时表达效率高， 因而， 根据本发明的一个实施例， 所述启动子为 U-启动子。
根据本发明的实施例， 所述构建体进一步包含： 基因组定点整合 attB序列； ftz内 含子序列； 以及 SV40 ployA序列。 由此， 能够使质粒定点整合到果蝇基因组中、 翻译 效率增强， 且有利于调控转录终止。
根据本发明的实施例， 所述构建体进一步包含： 2个 LoxP序列； 以及隔绝子 gypsy 序列， 其中， 所述隔绝子 gypsy序列位于 2个 LoxP序列之间。 由此， 能够在 Cre重组 酶的作用下去掉 LoxP之间的 gypsy序列， 达到调节 siRNA表达量的目的， 而且 gypsy 序列可以增强基因的转录， 进而使基因表达的调控更加灵活， 基因功能分析的手段更加 多样。
本发明的再一方面， 本发明还提供了一种敲除果蝇目的基因的方法。根据本发明的 实施例， 该方法利用前面所述的构建体转化果蝇。 发明人惊奇的发现， 利用本发明的方 法， 不仅能够在果蝇中同时高效表达多个针对不同基因的 siRNA, 从而能够实现多个基 因的同时敲除，还能够在果蝇中表达多个针对同一个基因 siRNA, 从而实现单个基因的 高效敲除， 敲出效果好。 并且， 本发明的方法操作简单， 结果稳定， 适用于果蝇所有的 组织和细胞， 且利用该方法易于调控果蝇的基因敲除效率， 获得的果蝇表型明显， 从而 能够有效用于果蝇基因功能研究。
根据本发明的实施例， 本发明的敲除果蝇目的基因的方法利用 GAL4-UAS双元表 达载体系统进行所述转化， 其中所述构建体包含 2xQUAS序列； 和 /或 lOxUAS序列。 由此， 这两种可抑制型二元表达系统能够丰富果蝇中转基因表达、世系追踪和基因功能 嵌合体分析的手段， 使该方法的表达调控更加灵活。 根据本发明的实施例， 所述转化进一步包括： 利用所述构建体转化第一果蝇， 以便 获得第一转基因果蝇； 提供第二转基因果蝇， 所述第二转基因果蝇能够表达 Gal4蛋白 和 /或 Q蛋白； 以及将所述第一转基因果蝇与所述第二转基因果蝇杂交， 以便获得后代 转基因果蝇， 所述后代转基因果蝇中所述目的基因被敲除。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得 明显， 或通过本发明的实践了解到。 附图说明
本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明 显和容易理解， 其中：
图 1显示了根据本发明一个实施例的 VADUM表达载体的图谱；
图 2显示了根据本发明一个实施例的野生型及各转基因雄果蝇翅膀的图片， 其中 图 2A显示了野生型雄果蝇翅膀的图片，
图 2B显示了 HI KD1与 msl096-Gal4杂交后代雄果蝇翅膀的图片，
图 2C显示了 HI KD2与 msl096-Gal4杂交后代雄果蝇翅膀的图片，
图 2D显示了 HI KD3与 msl096-Gal4杂交后代雄果蝇翅膀的图片。 发明详细描述
下面详细描述本发明的实施例， 所述实施例的示例在附图中示出。下面通过参考附 图描述的实施例是示例性的， 仅用于解释本发明， 而不能理解为对本发明的限制。
根据本发明的一个方面， 本发明提供了一种分离的寡核苷酸。根据本发明的具体实
Figure imgf000004_0001
GCATTCATGCTG(SEQ ID NO: 1)。
需要说明的是， 该寡核苷酸序列为一段来自于果蝇 Mir2基因簇的间隔序列， 是果 蝇基因组中 mir-2a-l和 mir-2b-2之间的序列， 发明人发现， 该寡核苷酸带有果蝇体内 核糖核酸酶的识别位点， 将其用来连接多个 shRNA模板序列， 即将多个 siRNA中相邻 的两个 siRNA通过该寡核苷酸相连， 能够使转录产物通过果蝇自身的剪切机制形成各 自独立的 shRNA, 从而同时 RNAi各自的靶基因， 进而， 能够同时敲除多个基因， 实 现对果蝇多基因的同时调控。
根据本发明的另一方面， 本发明还提供了一种构建体。 根据本发明的实施例， 该构 建体包含多个 siRNA, 所述多个 siRNA中相邻的两个 siRNA通过前面所述的寡核苷酸 相连， 其中所述多个 siRNA的靶基因相同或不同。 发明人惊奇地发现， 本发明的构建 体不仅能够同时高效表达多个针对不同基因的 siRNA,从而能够实现对多基因的同时调 控， 还能够表达多个针对同一个基因 siRNA, 以增强单个基因的敲除效率。 并且该构建 体的构建简单方便， 表达高效稳定， 适用于果蝇所有的组织和细胞， 从而能够有效用于 各种果蝇基因功能性研究。
根据本发明的实施例， 所述构建体进一步包含： 2xQUAS序列； 和 /或 lOxUAS序 列。 其中， 需要说明的是， 果蝇中目前有两种二元表达系统， Gal4/UAS 和 QF/QUAS 系统。 它们的工作原理是一样的， 以 QF/QUAS为例， QF是一种转录调控因子， QUAS 是 DNA上 QF结合的位点， 2 X QUAS指含有两个 QF结合位点。 当 QF结合 QUAS时， 会启动 QUAS下游基因的转录。 而本发明的载体上同时包含 UAS和 QUAS, 从而既可 以用 Gal4调控， 也可以用 QF。 由此， 这两种可抑制型二元表达系统能够丰富果蝇中转 基因表达、 世系追踪和基因功能嵌合体分析的手段， 使表达的调控更加灵活。
根据本发明的实施例， 所述构建体进一步包含筛选标记基因。 由此， 便于筛选阳性 质粒。 根据本发明的一些具体示例， 所述筛选标记基因为氨苄青霉素抗性基因和 /或筛 选标记 vermilion基因。 由此， 阳性质粒筛选效果好。
根据本发明的实施例， 所述构建体进一步包含启动子。 发明人发现， U-promoter 在果蝇各组织器官中的背景表达效率低， 在受到诱导时表达效率高， 因而， 根据本发明 的一个实施例， 所述启动子为 U-启动子。
根据本发明的实施例， 所述构建体进一步包含： 基因组定点整合 attB序列； ftz内 含子序列； 以及 SV40 ployA序列。 由此， 能够使质粒定点整合到果蝇基因组中、 翻译 效率增强， 且有利于调控转录终止。
根据本发明的实施例， 所述构建体进一步包含： 2个 LoxP序列； 以及隔绝子 gypsy 序列， 其中， 所述隔绝子 gypsy序列位于 2个 LoxP序列之间。 由此， 能够在 Cre重组 酶的作用下去掉 LoxP之间的 gypsy序列， 达到调节 siRNA表达量的目的， 而且 gypsy 序列可以增强基因的转录， 进而使基因表达的调控更加灵活， 基因功能分析的手段更加 多样。
其中， 根据本发明的实施例， 上述各元件的序列如下：
attB:
Figure imgf000005_0001
ftz内含子: ' (OS : ON αΐ DaS)3VIOV0030V033I33IOI3
Figure imgf000006_0001
:svnxoi
Figure imgf000006_0002
M αι
： AsdAg
:(乙 i :OM ai
:(9l7 :OM QI 0aS)V33IIOOI3IOIV3IVII3IVIOIVV3IV3I3VV
Figure imgf000006_0004
： id 01 AS
:(Sl :OM QI 0aS)OVOVI3VVOVVV30IIVOV3VII30IVII30II
.ΪΖ000/9Ϊ0Ζ OAV 本发明的再一方面， 本发明还提供了一种敲除果蝇目的基因的方法。根据本发明的 实施例， 该方法利用前面所述的构建体转化果蝇。 发明人惊奇的发现， 利用本发明的方 法， 不仅能够在果蝇中同时高效表达多个针对不同基因的 siRNA, 从而能够实现多个基 因的同时敲除，还能够在果蝇中表达多个针对同一个基因 siRNA, 从而实现单个基因的 高效敲除， 敲出效果好。 并且， 本发明的方法操作简单， 结果稳定， 适用于果蝇所有的 组织和细胞， 且利用该方法易于调控果蝇的基因敲除效率， 获得的果蝇表型明显， 从而 能够有效用于果蝇基因功能研究。
根据本发明的实施例， 本发明的敲除果蝇目的基因的方法利用 GAL4-UAS双元表 达载体系统进行所述转化， 其中所述构建体包含 2xQUAS序列； 和 /或 lOxUAS序列。 由此， 这两种可抑制型二元表达系统能够丰富果蝇中转基因表达、世系追踪和基因功能 嵌合体分析的手段， 使该方法的表达调控更加灵活。
根据本发明的实施例， 所述转化进一步包括： 利用所述构建体转化第一果蝇， 以便 获得第一转基因果蝇； 提供第二转基因果蝇， 所述第二转基因果蝇能够表达 Gal4蛋白 和 /或 Q蛋白； 以及将所述第一转基因果蝇与所述第二转基因果蝇杂交， 以便获得后代 转基因果蝇， 所述后代转基因果蝇中所述目的基因被敲除。 下面将结合实施例对本发明的方案进行解释。 本领域技术人员将会理解， 下面的实施 例仅用于说明本发明， 而不应视为限定本发明的范围。 实施例中未注明具体技术或条件的， 按照本领域内的文献所描述的技术或条件（例如参考 J.萨姆布鲁克等著， 黄培堂等译的《分 子克隆实验指南》， 第三版， 科学出版社） 或者按照产品说明书进行。 所用试剂或仪器未注 明生产厂商者， 均为可以通过市购获得的常规产品， 例如可以采购自 Illumina公司。
实施例 1
构建 RNAi表达载体 VADUM, 其具体步骤如下：
1、 载体基因元件的克隆
1.1、 克隆基因元件所用的载体
载体 pVALIUM20用 Hindlll和 Sac I双酶切， 与退火的合成 MCS片段连接， 转化大肠 杆菌 ToplO, 挑选阳性克隆。 测序得到正确的质粒记为 pV-MCS， 以此质粒来克隆各个基因 元件。
退火用的 MCS引物分别为：
MCS-F：
(SEQ ID NO: 2)
MCS-R:
3)
1.2、 克隆一个 LoxP
质粒 pV-MCS用 Nhe I和 Sac I双酶切， 与退火的合成 LoxP片段连接， 转化大肠杆菌 ToplO, 挑选阳性克隆。 测序得到正确的质粒记为 pV-LoxP。 退火用的 LoxP引物分别为：
LoxP-Fl : ctagcATAACTTCGTATAATGTATGCTATACGAAGTTATGagct(SEQ ID NO: 4) LoxP-Rl： CATAACTTCGTATAGCATACATTATACGAAGTTATg(SEQ ID NO: 5)
1.3、 克隆 Q-UAS
质粒 pV-LoxP用 Pst I和 Sac I双酶切， 与退火的合成 Q-UAS片段连接，转化大肠杆菌 ToplO, 挑选阳性克隆。 测序得到正确的质粒记为 pV-Q-LoxP。
退火用的 Q-UAS引物序列分别为：
ID NO: 6)
ID NO: 7)
1.4、 克隆 5xUAS
以 pVALIUM20为模板， 用引物 UAS-F和 UAS-R进行 PCR扩增， 引物序列如下： UAS-F： AACTGC AGGTCGGAGTACTGTCCTCC(SEQ ID NO： 8)
UAS-R: AACCGCGGGAGTCTCCGCTCGGAG(SEQ ID NO: 9)
得到 123碱基 PCR产物。 PCR产物用 Pst I和 Sac II双酶切，与经过同样双酶切的质粒 pV-Q-LoxP 连接， 转化大肠杆菌 ToplO , 挑选阳性克隆。 测序得到正确的质粒记为 pV-UAS-Q-LoxP。
1.5、 克隆另外 5 X UAS和 U-promoter
以 pVALIUM3为模板，用引物 promoter-F和 promoter-R进行 PCR扩增，引物序列如下： Promoter-F： AACCGCGGTCGGAGTACTGTCCTCC(SEQ ID NO: 10)
Promoter-R: AATCTAGACTTTGGTATGCGTCTTGTGAT(SEQ ID NO: 11)
得到 282碱基 PCR产物。 PCR产物用 Xba I和 Sac II双酶切， 与经过同样双酶切的质 粒 pV-UAS-Q-LoxP连接， 转化大肠杆菌 ToplO, 挑选阳性克隆。 测序得到正确的质粒记为 pV-U-promoter-UAS-Q-LoxP。
2、 表达载体的构建
2.1构建表达载体骨架
载体 pVALIUM20用 Spe I酶切，与退火的合成 MCSb片段连接，转化大肠杆菌 ToplO, 挑选阳性克隆。 测序得到正确的质粒记为 pV-MCS2。
退火用的 MCSb引物分别为：
用以下突变的方法把质粒 PV-MCS2的一些酶切位点去除， 方便以后的克隆。 去掉的酶 切位点分别是 vermilion中的 EcoRV、 BamH I和 Xho I ， vermilion和 attB之间的 Xho I ， attB中的 Kpn I， 以及 gypsy中的 Mfe I 。
以质粒 pV-MCS2为模板，用一对反向互补的突变引物进行 PCR扩增。 PCR产物用 Dpn I酶切后直接转化大肠杆菌 ToplO, 挑选单克隆摇菌， 提取质粒酶切鉴定得到正确的突变质 粒。 用到的突变引物如下：
EcoR V -R: CACTGGTGGCTCGTCTGGTTATC AATATCTGCGCTCC(SEQ ID NO: 15) BamH I -F: CCAGTGCCCAACTGTTGCGATCCAATCATGCGTTG(SEQ ID NO: 16) BamH I -R: CAACGCATGATTGGATCGCAACAGTTGGGCACTGG(SEQ ID NO: 17)
Xho I -F2: GATATCATCGATCTCGACGCTGCATCCAACGCGTT(SEQ ID NO: 20) Xho I -R2: AACGCGTTGGATGCAGCGTCGAGATCGATGATATC(SEQ ID NO: 21) Kpn I -F: GGATCAATTCGGCTTCACGTACCGTCGACGATGTA(SEQ ID NO: 22) Kpn I -R: TACATCGTCGACGGTACGTGAAGCCGAATTGATCC(SEQ ID NO: 23)
Mfe I -R: ATATACTGTTGCCGAGCAGAATTGATCGGCTAAATGG(SEQ ID NO: 25) 突变后的质粒记为 pVAM， 以此为骨架构建表达载体。
2.2、 添加表达元件 U-promoter、 UAS、 Q-UAS和 LoxP
质粒 pV-U-promoter-UAS-Q-LoxP用 Xba I禾 P Nhe I双酶切， 胶回收得到 473碱基的片 段，即 U-promoter-UAS-Q-LoxP，与用同样双酶切的载体 pVAM连接，转化大肠杆菌 ToplO, 挑选阳性克隆。 质粒记为 pVAM-U-promoter-UAS-Q-LoxP，
2.3、 添加另一个 LoxP
质粒 pVAM-U-promoter-UAS-Q-LoxP用 Nhe I酶切， 用碱性磷酸酶处理， 再与退火的 合成 LoxP 片段连接， 转化大肠杆菌 ToplO, 挑选阳性克隆。 测序得到正确的质粒记为 pVAM-U-promoter-UAS-Q-LoxP2。
退火用的 LoxP引物序列分别为：
Figure imgf000009_0001
2.4、 在两个 LoxP之间加入 gypsy
以 p VAM为模板， 用引物 gypsy-F和 gypsy-R进行 PCR扩增。
Gypsy-F: TTACTAGTTGGCCACGTAATAAGTGTGC(SEQ ID NO: 28)
gypsy-R: TTACTAGTGTTGTTGGTTGGCACACCAC(SEQ ID NO: 29)
得到 447碱基 PCR产物。 PCR产物用 Spe I酶切， 与经过 Nhe I酶切和碱性磷酸酶处 理的质粒 pVAM-U-promoter-UAS-Q-LoxP2连接， 转化大肠杆菌 ToplO, 挑选阳性克隆。 测 序得到正确的质粒记为 pVAM-U-promoter-UAS-Q-LoxP2-gypsy。
2.5、 添加克隆单个 hairpin DNA的多克隆酶切位点 MCS1
质粒 pVALIUM20用 Xba I和 Spe I双酶切， 胶回收 730碱基的片段， 与用同样双酶切 的质粒 pVAM-U-promoter-UAS-Q-LoxP2-gypsy连接， 转化大肠杆菌 ToplO, 挑选阳性克隆。 正确的质粒记为 pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCSl。
2.6、 添加连接多个 hairpin DNA的 linker
以果蝇基因组为模板， 用引物 linker-F和 linker-R进行 PCR扩增。
Linker-F： AATCTAGACGCGATGCTCAAGGCAAAAA(SEQ ID NO: 30)
linker-R: AACCTAGGCAGCATGAATGCGCCAATAT(SEQ ID NO: 31)
得到 233碱基 PCR产物。 PCR产物用 Xba I和 Avr ll双酶切， 与经过 Avrll酶切和碱 性磷酸酶处理的质粒 pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCSl 连接， 转化大肠杆菌 ΤορΙΟ , 挑选出阳性克隆。 测序得到正确的质粒记为 pVAM-U-promoter-UAS-Q -LoxP2-gypsy-MCSl- linker。
2.7、 添加克隆多个 hairpin DNA的多克隆酶切位点 MCS2
质粒 pVAM-U-promoter-UAS-Q-LoxP2-gypsy-MCSl-linker用 Spe I酶切，用碱性磷酸酶 处理， 再与退火的合成 MCS2片段连接， 转化大肠杆菌 ToplO, 挑选阳性克隆。测序得到正 确的质粒作为表达载体， 记为 VADUM, 如图 1所示。 实施例 2
Polycomb Group (PcG)蛋白是在果蝇中首次发现的一类能够重塑染色质从而使特定基 因在表观遗传上维持沉默的蛋白质家族， 在发育过程中起着非常重要的作用。 PcG蛋白通 过形成不同的蛋白复合体发挥作用， 其中包括两种抑制复合体， Polycomb repressive complexes 1 and 2 (PRC1 and PRC2)。 果蝇的核心 PRC1由 4种蛋白构成， Polycomb (Pc) Sex combs extra (Sce)、 Polyhomeotic (Ph)禾口 Posterior sex combs (Psc)。 由于 PRC1复合体组 成的复杂及多变性， 它的功能及生理作用还不是很清楚。 尤其是单独敲除核心 PRC1 的 4 种组分往往没有明显的表型，这给 PRC1功能的研究造成很大的困扰。利用本发明可以同时 抑制这 4种蛋白的表达， 得到与单个基因敲除不同的表型， 给 PRC1功能的研究带来便利。
按照下面的方法， 利用本发明的构建体同时抑制果蝇 PRC1复合体中的各蛋白：
1、 抑制核心 PRC1各组分基因表达的 RNAi载体构建
把 Pc ， See ， Ph 和 Psc 基 因 的 CDS 序 列 输 入 DSIR 网 站 ( http://biodev.extra.cea.fr/DSIR/DSIR.html ) , 设计只针对各个基因的 21核苷酸 siRNA。 把 21核苷酸 反义核苷酸 （anti-sense oligo) 序列及其反向互补序列 （sense oligo) 分别放在以 下引物的相应位置， 得到 4对分别用于各个基因的引物。
F: ctagcagt sense oligo tagttatattcaagcata anti-sense oligo gcg
R: aattcgc sense oligo tatgcttgaatataacta anti-sense oligo actg
Pc-F:ctagcagtCTCATCGACATCTACGAACAAtagttatattcaagcataTTGTTCGTAGATGTCG ATGAGgcg(SEQ ID NO: 32)
Pc-R:aattcgcCTCATCGACATCTACGAACAAtatgcttgaatataactaTTGTTCGTAGATGTCGA
TGAGactg(SEQ ID NO: 33)
Sce-F:ctagcagtCCGCAAGAAGCTCGTCTCCAAtagttatattcaagcataTTGGAGACGAGCTT CTTGCGGgcg(SEQ ID NO: 34)
Sce-R:aattcgcCCGCAAGAAGCTCGTCTCCAAtatgcttgaatataactaTTGGAGACGAGCTTC TTGCGGactg(SEQ ID NO: 35)
Ph-F:ctagcagtCCGGGTCAGCTGGTTCTCTTAtagttatattcaagcataTAAGAGAACCAGCTG ACCCGGgcg(SEQ ID NO: 36)
Ph-R:aattcgcCCGGGTCAGCTGGTTCTCTTAtatgcttgaatataactaTAAGAGAACCAGCTGA CCCGGactg(SEQ ID NO: 37)
Psc-F:ctagcagtCCAGTCGAATATGATGTACAAtagttatattcaagcataTTGTACATCATATTCG ACTGGgcg(SEQ ID NO: 38)
Psc-R:aattcgcCCAGTCGAATATGATGTACAAtatgcttgaatataactaTTGTACATCATATTCGA CTGGactg(SEQ ID NO: 39)
把合成的 4对引物退火， 分别与用 Nhe l和 EcoR l双酶切的质粒 VADUM连接， 转化 大肠杆菌 ToplO，挑选阳性克隆。测序得到 RNAi单个基因的重组质粒，分别记为 VADUM-Pc, VADUM-Sce, VADUM-Ph和 VADUM-Psc。
质粒 VADUM-Sce用 Xba I和 Spe I双酶切， 回收 407 碱基的小片段， 再与用 Spe I单 酶切和碱性磷酸酶处理的质粒 VADUM-Pc连接， 转化大肠杆菌 ToplO, 挑选阳性克隆。 正 确的质粒记为 VADUM-Pc-Sce。 质粒 VADUM-Ph用 Xba I禾 P Spe I双酶切， 回收 407 碱基 的小片段， 与用 Spe I单酶切和碱性磷酸酶处理的质粒 VADUM-Pc-Sce连接， 转化大肠杆 菌 ToplO, 经鉴定得到正确的质粒记为 VADUM-Pc-Sce-Ph。最后， 质粒 VADUM-Psc用 Xba I和 Spe I双酶切， 回收 407 碱基的小片段， 与用 Spe I单酶切和碱性磷酸酶处理的质粒 VADUM-Pc-Sce-Ph连接， 转化大肠杆菌 ToplO, 挑选阳性克隆， 就得到同时 RNAi 4个基因 的重组质粒， 记为 VADUM-Pc-Sce-Ph-Psc。
2、 转基因果蝇的获得
用显微注射仪将上一步得到的 5种 RNAi载体分别注射到 0-1 h 的果蝇（基因型为 y sc v nanos-integrase;attP2) 胚胎中， 放于 25摄氏度培养。
然后，将孵化出的雄果蝇与基因型为 y sc v的果蝇杂交， 从后代中挑选眼睛颜色是野生 型深红色的雄果蝇， 即为已整合入 RNAi载体的转基因果蝇。 挑选出的雄果蝇与基因型为 y sc V;Dr,e/TM3,Sb的果蝇杂交， 从后代中挑选深红眼且短刚毛的果蝇自交， 再从其后代中挑 选深红眼且长刚毛的果蝇， 就得到纯合的转基因果蝇。
3、 抑制核心 PRC1各组分基因表达对果蝇发育的影响
将本实施例步骤 2得到的转基因果蝇与 nos-Gal4果蝇杂交， 放于 25°C培养， 观察后代 的表型。
结果如下： Pc， See, Ph和 Psc单独 RNAi的果蝇与野生型果蝇相比， 没有显示出明显 的不同； 而 4个基因同时被 RNAi后， 后代雌果蝇的卵巢明显变小， 并且不产卵。这表明核 心 PRC1各组分的功能存在冗余， 单独 RNAi后可能由于其他组分的作用没有显示出表型， 同时 RNAi所有组分后才得到明显的表型。 实施例 3
组蛋白 HI是组成真核生物染色质的 5种组蛋白中的一种，不仅在染色体高级结构的建 立和维持方面具有重要作用， 在细胞增殖、 基因表达调控等方面也具有一定作用。 果蝇中 只有一种组蛋白 HI基因， 是研究其功能的有利模型。 但是由于果蝇组蛋白 HI 基因有 23 个拷贝， 很难得到它的突变体， 传统 RNAi方法往往达不到理想的敲除效率。利用本发明可 以方便的调节基因敲除的效率， 大大方便了其功能的研究。
利用本发明的构建体增强组蛋白 HI的敲除效率， 具体方法如下：
1、 组蛋白 HI RNAi载体的构建
按照实施例 2中所诉的方法设计两对针对组蛋白 HI的引物， 序列如下：
Hl-Fl :ctagcagtTTGGTACATGTTCGCAATTAAtagttatattcaagcataTTAATTGCGAACATGT ACCAAgcg(SEQ ID NO: 40)
Hl-Rl :aattcgcTTGGTACATGTTCGCAATTAAtatgcttgaatataactaTTAATTGCGAACATGT ACCAAactg(SEQ ID NO: 41)
Hl-F2:ctagcagtACCAGCGACAGTTGAGAAGAAtagttatattcaagcataTTCTTCTCAACTGT CGCTGGTgcg(SEQ ID NO: 42)
Hl-R2:aattcgcACCAGCGACAGTTGAGAAGAAtatgcttgaatataactaTTCTTCTCAACTGTC GCTGGTactg(SEQ ID NO: 43)
两对引物退火后， 分别与用 Nhe l和 EcoR l双酶切的质粒 VADUM连接， 转化大肠杆 菌 ToplO, 挑选阳性克隆。 测序得到 RNAi组蛋白 HI 的重组质粒， 记为 VADUM-H1-1和 VADUM-Hl-2 o
质粒 VADUM-H1-2用 Xba I和 Spe I双酶切， 回收 407 碱基的小片段， 再与用 Spe I 单酶切和碱性磷酸酶处理的质粒 VADUM-H1-1连接， 转化大肠杆菌 ToplO, 挑选阳性克隆。 正确的质粒记为 VADUM-2 X H1。 质粒 VADUM-2 X H1再用 Xba I禾 P Spe I双酶切， 回收 814 碱基的片段， 再与用 Spe l单酶切和碱性磷酸酶处理的质粒 VADUM-2 X H1连接， 转 化大肠杆菌 ToplO,挑选阳性克隆。正确的质粒记为 VADUM-4 X H1。最后，质粒 VADUM-4 X H1用 Xba I和 Spe I双酶切， 回收 1628 碱基的片段， 与用 Spe I单酶切和碱性磷酸酶处 理的质粒 VADUM-4 X H1 连接， 转化大肠杆菌 ToplO, 挑选阳性克隆。 正确的质粒记为 VADUM-8 X Hl o
2、 组蛋白 HI敲除对翅膀发育的影响
使用上一步得到的 RNAi载体 VADUM-H1-1、 VADUM-4 X HI和 VADUM-8 X H1 , 按 照实施例 2中所述的方法获得转基因 RNAi果蝇（即用显微注射仪将上一步得到的 5种 RNAi 载体分别注射到 0-1 h 的果蝇（基因型为 y sc v nanos-integrase;attP2)胚胎中， 放于 25摄氏 度培养。然后， 将孵化出的雄果蝇与基因型为 y sc v的果蝇杂交， 从后代中挑选眼睛颜色是 野生型深红色的雄果蝇， 即为已整合入 RNAi载体的转基因果蝇）， 分别记为 H1 KD1、 H1 KD2禾 P HI KD3。 将 3种转基因 RNAi果蝇分别与 msl096-Gal4杂交， 25摄氏度培养， 观察后代的翅膀 表型。 结果如图 2所示： 相对于野生型雄果蝇 （wt) 的翅膀， HI KD1与 msl096-Gal4杂交 后代雄果蝇的翅膀无明显差异； HI KD2与 msl096-Gal4杂交后代雄果蝇的翅膀大小稍微变 小， 脉络异常且轻微卷曲； H1 KD3与 msl096-Gal4杂交后代雄果蝇的翅膀明显变小， 脉络 严重紊乱且卷曲成一团。
本领域技术人员均了解， 传统的 RNAi载体很难达到 HI KD3的敲除效果， 而利用上述 的本发明可以方便地从弱到强调节基因的敲除效率。 工业实用性
本发明的分离的寡核苷酸， 能够有效地用于制备果蝇基因敲除构建体， 具体地， 在制 备用于果蝇基因敲除的构建体时， 将该寡核苷酸用以连接多个 shRNA模板序列， 即将 多个 siRNA 中相邻的两个 siRNA 通过该寡核苷酸相连， 从而使该构建体包含多个 siRNA, 进而利用该构建体能够同时敲除多个基因， 实现对果蝇多基因的同时调控。 尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人员将会理解。 根据已 经公开的所有教导， 可以对那些细节进行各种修改和替换， 这些改变均在本发明的保护范 围之内。 本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中， 参考术语"一个实施例"、 "一些实施例"、 "示意性实施例"、 "示 例"、 "具体示例"、 或 "一些示例"等的描述意指结合该实施例或示例描述的具体特征、 结 构、 材料或者特点包含于本发明的至少一个实施例或示例中。 在本说明书中， 对上述术语 的示意性表述不一定指的是相同的实施例或示例。 而且， 描述的具体特征、 结构、 材料或 者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims

权利要求书
1、 一种分离的寡核苷酸， 其具有 SEQ ID NO: 1所示的核苷酸序列。
2、 一种构建体， 其特征在于， 包含多个 siRNA, 所述多个 siRNA中相邻的两个 siRNA 通过权利要求 1所述的寡核苷酸相连， 其中所述多个 siRNA的靶基因相同或不同。
3、 根据权利要求 2所述的构建体， 其特征在于， 进一步包含：
2xQUAS序列； 禾口 /或
lO UAS序列。
4、 根据权利要求 2所述的构建体， 其特征在于， 进一步包含筛选标记基因， 任选地， 所述筛选标记基因为氨苄青霉素抗性基因和 /或筛选标记 vermilion基因。
5、 根据权利要求 2所述的构建体， 其特征在于， 进一步包含启动子，
任选地， 所述启动子为 U-启动子。
6、 根据权利要求 2所述的构建体， 其特征在于， 进一步包含：
基因组定点整合 attB序列；
ftz内含子序列； 以及
SV40 ployA序列。
7、 根据权利要求 2所述的构建体， 其特征在于， 进一步包含：
2个 LoxP序列； 以及
隔绝子 gypsy序列，
其中， 所述隔绝子 gypsy序列位于 2个 LoxP序列之间。
8、 一种敲除果蝇目的基因的方法， 其特征在于，
利用权利要求 2-7所述的构建体转化果蝇。
9、根据权利要求 8所述的方法， 其特征在于， 利用 GAL4-UAS双元表达载体系统进行 所述转化， 其中所述构建体包含 2xQUAS序列； 和 /或 lOxUAS序列。
10、 根据权利要求 8所述的方法， 其特征在于， 所述转化进一步包括：
利用所述构建体转化第一果蝇， 以便获得第一转基因果蝇；
提供第二转基因果蝇， 所述第二转基因果蝇能够表达 Gal4蛋白和 /或 Q蛋白； 以及 将所述第一转基因果蝇与所述第二转基因果蝇杂交， 以便获得后代转基因果蝇， 所述 后代转基因果蝇中所述目的基因被敲除。
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Publication number Priority date Publication date Assignee Title
WO2005060721A2 (en) * 2003-12-19 2005-07-07 University Of Massachusetts Manipulation of rna interference through modulation of armitage activity

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