WO2019091390A1 - 一种核苷酸序列及其在增强植物抗有害生物能力中的应用 - Google Patents

一种核苷酸序列及其在增强植物抗有害生物能力中的应用 Download PDF

Info

Publication number
WO2019091390A1
WO2019091390A1 PCT/CN2018/114295 CN2018114295W WO2019091390A1 WO 2019091390 A1 WO2019091390 A1 WO 2019091390A1 CN 2018114295 W CN2018114295 W CN 2018114295W WO 2019091390 A1 WO2019091390 A1 WO 2019091390A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
coo2
plant
nucleotide sequence
atpase subunit
Prior art date
Application number
PCT/CN2018/114295
Other languages
English (en)
French (fr)
Inventor
唐克轩
赵静雅
付雪晴
刘航
潘琪芳
陈甜甜
钱虹妹
孙小芬
Original Assignee
上海交通大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海交通大学 filed Critical 上海交通大学
Priority to US16/760,447 priority Critical patent/US11208668B2/en
Publication of WO2019091390A1 publication Critical patent/WO2019091390A1/zh
Priority to AU2020100833A priority patent/AU2020100833A4/en

Links

Images

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
    • C12Y306/01003—Adenosine triphosphatase (3.6.1.3)
    • 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
    • C12N15/11—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
    • C12N15/1137—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 against enzymes
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to a technology in the field of bioengineering, in particular to a method for enhancing plant mites resistance based on V-ATPase subunit E and COO2 double gene fusion.
  • Aphids are one of the main pests in agricultural production. Aphids belong to the order of the Homoptera, including the genus Corydalis and the genus Aphididae. The body length is between 1-10mm. It is a sucking mouthparts, often clustered in leaves, top buds, tender stems, flower buds and other parts. There are many kinds of mites, and there are about 4,700 species known around the world, and about 1,100 species are distributed in China. When feeding, the aphids pierce the epidermis, parenchyma and mesophyll of the plant into the sieve through the needle, and feed the juice of the plant.
  • the needle can effectively avoid the defensive substances of the epidermal tissue of the plant and facilitate the puncture between the tissues. Smoothly absorb nutrient-rich juice from plant tissues.
  • the mites use plant sap to make the plant's nutrition worse, the growth and development are poor or even stagnant, leading to reduced yield; the honeydew secreted by the mites gathers in the leaves, stems and other parts, seriously affecting the photosynthesis and respiration of crops; in addition, the honeydew excreted by the mites It can promote the breeding of some pathogens and spread a variety of plant viruses.
  • the host plants of aphids include almost all families of angiosperms and gymnosperms, and can spread viruses among plants, which is extremely harmful.
  • Economic crops such as wheat, cotton, fruits and vegetables, and flowers have suffered greatly, causing huge economic losses to the agroforestry and horticulture industries.
  • the temperature of wheat in the main planting areas of China has increased during the period of growing, and there is a “warm winter”, which causes the mites to be harmful, and the annual economic losses caused by aphids and their transmitted viruses in wheat production. Very serious.
  • Pesticides play an important role in the fight against insect pests. However, pests gradually develop resistance to pesticides. In addition, pesticides are also prone to environmental pollution.
  • plant-mediated RNAi technology is one of the hotspots of crop insect resistance research, and its application prospect is very broad.
  • This technique is to express the dsRNA of the corresponding insect-specific gene in the host plant. After the insects eat the host plant, the corresponding gene is silenced and the expression level is decreased, causing the death of the insect, thereby controlling the pest damage. Therefore, the use of plant-mediated RNAi technology to cultivate resistant varieties can achieve the purpose of safe, long-lasting and high-efficiency control of aphids, and has important practical application value.
  • the technical problem to be solved by the present invention is to provide a safe and effective method for combating mites.
  • nucleotide sequence which, in a preferred embodiment, is selected from the group consisting of:
  • nucleotide sequence produced by derivatization, deletion, insertion or addition of one or several nucleotides of the nucleotide sequence shown in any one of SEQ ID NOS: 4-6;
  • a nucleotide sequence comprising at least 80% homology to any one of SEQ ID NOS: 4-6.
  • a second aspect of the invention provides a double stranded RNA, in a preferred embodiment consisting of a nucleotide sequence as described above and a reverse complement thereof.
  • a third aspect of the invention provides a small interfering RNA, in a preferred embodiment, a short-segment double-stranded RNA molecule produced by cleavage of double-stranded RNA as described above.
  • the small interfering RNA is 19-25 nt in length.
  • a fourth aspect of the invention provides a recombinant expression vector, in a preferred embodiment, comprising a nucleotide sequence as described above or a double stranded RNA molecule as described above.
  • the above recombinant expression vector further includes a promoter capable of transcription in a plant.
  • the promoter is the maize ubiquitin-1 promoter.
  • the maize ubiquitin-1 promoter is located upstream of the nucleotide sequence or the double stranded RNA molecule.
  • the maize ubiquitin-1 promoter has a rice intron downstream, and the nucleotide sequence of the maize ubiquitin-1 promoter fused to the rice intron (OSintron) is shown in SEQ ID NO: 7. Wherein, the position 1-108 of SEQ ID NO: 7 is the maize ubiquitin-1 promoter, and the position 1143-1621 of SEQ ID NO: 7 is the rice intron.
  • a fifth aspect of the invention provides the use of a nucleotide sequence as described above or a double-stranded RNA as described above for enhancing the pest resistance of a plant.
  • the pest is a mites.
  • the locusts are Myzus persicae, Acyrthosiphon pisum, Diuraphis noxia, Melanaphis sacchari, Sipha flava, and cotton aphid ( One or more of Aphis gossypii) and the like.
  • the plant is a dicot, a monocot or a gymnosperm; alternatively, the dicot comprises a cruciferous plant and a Solanaceae; alternatively, the plant is Arabidopsis, rice, wheat, Corn, cotton, soybeans, canola, sorghum, tobacco, chrysanthemum, greens, cabbage, radish or tomato.
  • the application includes the following steps:
  • Resistant plant seedlings are obtained by antibiotic screening.
  • a method for enhancing the resistance of a plant by double gene fusion which utilizes V-ATPase to generate energy by hydrolyzing ATP to provide energy for various life activities and aphid salivary gland enzyme gene.
  • V-ATPase to generate energy by hydrolyzing ATP to provide energy for various life activities and aphid salivary gland enzyme gene.
  • the three sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 both comprise a forward sequence portion and a complementary sequence portion, and SEQ ID NO: 1, SEQ ID NO: 2 and The three sequences of SEQ ID NO: 3 are understood to refer to the forward sequence portion thereof.
  • the forward sequence portion of SEQ ID NO: 1 is 1-60, 121-180, 241-3300, 361-420, 481-540, 601-611 (ie, the sequence shown in SEQ ID NO: 4), which is complementary
  • the sequence parts are 61-120, 181-240, 301-360, 421-480, 541-600, and 612-622.
  • the positive sequence portion of SEQ ID NO: 2 is 1-60, 121-180, 241-300, 361-420, 481-540, 601-659 (ie, the sequence shown in SEQ ID NO: 5), the complementary sequence thereof
  • the parts are 61-120, 181-240, 301-360, 421-480, 541-600, 600-718.
  • the forward sequence portion of SEQ ID NO: 3 is 1-60, 121-180, 241-3300, 361-420, 481-540, 601-660, 721-780, 841-900, 961-1020, 1081-1140 , 1201-1260, 1321-1330 (ie, the sequence shown in SEQ ID NO: 6), the complementary sequence portions are 61-120, 181-440, 301-360, 421-480, 541-600, 661-720 , 781-840, 901-960, 1021-1080, 1141-1200, 1261-1320, 1331-1340 bits.
  • the present invention is achieved by the following technical scheme:
  • the present invention relates to a gene dsRNA having anti-aphid function, which is specifically fused from the V-ATPasesubunit E and COO2 gene fragments of aphids, and the nucleotide sequence thereof is set forth in SEQ ID NO: Show.
  • SEQ ID NO: 3 which is a fragment of V-ATPase subunit E (shown by SEQ ID NO: 1), COO2 gene (SEQ ID NO: 2), and V-ATPase subunit E and COO2 gene fragments of aphid Fusion fragment gene synthesis.
  • the invention relates to a method for enhancing plant mites resistance by double gene fusion, and constructing a gene having anti-aphid function by using V-ATPase subunit E gene, COO2 gene and V-ATPase subunit E gene and COO2 gene partial sequence fusion fragment, respectively.
  • the expression vector drives the V-ATPase subunit E, the COO2 gene, and the V-ATPase subunit E gene and the COO2 fusion gene silencing vector, ie, the RNAi vector, by constructing a maize ubiquitin-1 promoter.
  • the ubiquitin-1 promoter is specifically a constitutive promoter.
  • the infecting that is, the wheat immature embryos are transferred to the Agrobacterium liquid and inoculated on the medium under dark conditions to obtain the immature embryo callus, and then the resistant plants are screened to obtain the wheat resistant seedlings. .
  • the present invention relates to a recombinant expression vector, an RNAi vector, which is constructed based on dsRNA.
  • the dsRNA includes:
  • the tamper resistance includes:
  • the experiments of the present invention demonstrate that the obtained dsRNA expressing the sequence of the aphid-related gene V-ATPase subunit E, COO2, and the V-ATPase subunit E and COO2 fusion fragment in wheat results in a lethal effect, and V-ATPase subunit E
  • the dsRNA that expresses the sequence of the cDNA with the COO2 fusion fragment has the best inhibitory effect on the aphid.
  • the insect-resistant transgenic plants obtained by genetic engineering have the advantages of being effective only for the target pests, but have no effect on the non-hazardous organisms.
  • the insect-resistant substances produced by the plants are present in the plants and will not cause environmental damage. Pollution, and low cost, is conducive to promotion.
  • the present invention is not particularly limited as to the plants suitable for the present invention as long as it is suitable for a gene transformation operation such as various crops, flower plants or forestry plants, as compared with the prior art.
  • FIG. 1 is a schematic diagram showing the construction of a pDE1005:proUBI:V-ATPase subunit E+COO2 vector; wherein, FIG. 1a is a schematic diagram of a pDE1005 vector; FIG. 1b is a schematic diagram of constructing a V-ATPase subunit E+COO2 hairpin structure; FIG. FIG. 1b is a partial structural diagram of the hairpin structure connected to the carrier;
  • FIG. 2 is a schematic diagram showing the PCR identification of the T1 generation wheat of the V-ATPase subunit E+COO2 gene driven by the ubiquitin-1 promoter in the example;
  • M Marker III
  • + pDE1005: proUBI: V-ATPase subunit E+COO2 plasmid
  • CK wild type Fileder spring wheat
  • Figure 3 is a schematic diagram showing the semi-quantitative results of the transgenic ubiquitin-1 promoter driving the V-ATPase subunit E+COO2 gene T1 wheat V-ATPase subunit E+COO2 gene;
  • M Marker III
  • + pDE1005: proUBI: V-ATPase subunit E+COO2 plasmid
  • CK wild type Fileder spring wheat
  • V-ATPase During the growth and development of aphids, V-ATPase generates energy by hydrolyzing ATP to provide energy for various life activities.
  • the salivary gland enzyme gene COO2 is also very important in the process of aphid synthetase. Therefore, in one embodiment of the present invention, a fragment of the V-ATPase and COO2 genes is used for gene fusion to construct a component for gene silencing, by which the V-ATPase and COO2 gene expression levels in the aphids are reduced, and the aphids are affected.
  • the normal growth and development to achieve the purpose of inhibiting the growth of aphids.
  • a fragment gene fusion of a V-ATPase subunit E and a non-conserved region of the COO2 gene is used to construct an element for gene silencing. Selection of non-conserved regions prevents interference with other homologous sequences other than the aphid V-ATPase subunit E and COO2.
  • the element for gene silencing is a double stranded RNA.
  • a recombinant expression vector carrying the forward and reverse sequences of the fusion gene is constructed by fusing a fragment of the V-ATPase subunit E of the aphid and a non-conserved region of the COO2 gene.
  • the recombinant expression vector is transformed into a plant (for example, by means of Agrobacterium-mediated infection), and a dsRNA producing a V-ATPase subunit E and a COO2 double gene is expressed in the plant, and the aphid feeding produces both V-ATP.
  • the V-ATPase subunit E and COO2 gene expression levels in the body were simultaneously decreased.
  • the above-mentioned RNA interference affects the normal growth and development of aphids, thereby achieving the purpose of inhibiting the growth of aphids.
  • RNA interference refers to a small double-stranded RNA that efficiently and specifically blocks the expression of a specific target gene in the body, promotes the degradation of mature mRNA, and causes the individual to exhibit a phenotype of a specific gene deletion.
  • RNA interference is a gene silencing inhibition at highly specific mRNA levels.
  • RNA Small interfering RNA
  • RNAi can be realized in wheat, which can be achieved in other plants (monocotyledonous, dicotyledonous, gymnosperm).
  • the selected V-ATPase subunit E gene fragment (SEQ ID NO: 4) was subjected to nucleic acid sequence alignment, and the result showed that the sequence was associated with Acyrthosiphon pisum V-type proton ATPase subunit E- Like sequence (NCBI accession number: NM_001162178.2) homology 100%; homology with Myzus persicae V-type proton ATPase subunit E sequence (NCBI accession number: XM_022312248.1) 95%; Diuraphis noxia V-type proton ATPase subunit E sequence (NCBI accession number: XM_015522279.1) homology 92%; with sorghum (Melanaphis sacchari) V-type proton ATPase subunit E sequence (NCBI accession number: XM_025342771 .1) 94% homology; 86% homology to the Sipha flava V-type proton ATPase
  • the selected COO2 gene fragment (SEQ ID NO: 5) was subjected to nucleic acid sequence alignment, and the result showed that the sequence was 99% homologous to the Acyrthosiphon pisum sequence (NCBI accession number: XM_001948323.3); Myzus persicae sequence (NCBI accession number: XM_022310905.1) homology 89%; homology with cotton aphid (Aphis gossypii) sequence (NCBI accession number: KJ451424.1) 89%; with sorghum (Melanaphis The sacchari) sequence (NCBI accession number: XM_025338353.1) has 80% homology.
  • the homology with the V-ATPase subunit E and COO2 gene sequences of the present invention is 80% or more, and there is an anti-aphid effect.
  • V-ATPase subunit E gene fragment, the COO2 gene fragment and the V-ATPase subunit E and COO2 double gene fragments were respectively synthesized, and the forward gene, intron sequence and reverse gene of the hairpin structure were constructed respectively, and the pDE1005 vector was constructed.
  • pDE1005 proUBI: V-ATPase subunit E
  • pDE1005 proUBI: COO2
  • pDE1005 proUBI: V-ATPase subunit E+COO2 gene silencing vector were obtained.
  • the gene was synthesized by Shanghai Shenggong Bioengineering Co., Ltd.
  • a schematic representation of the RNAi vector constructing the V-ATPase subunit E and COO2 double gene fragments is shown in Figure 1.
  • the pDE1005 vector carries a rice intron
  • the maize ubiquitin-1 promoter is located upstream of the rice intron
  • the maize ubiquitin-1 promoter and the rice intron are fused to a sequence such as SEQ ID NO:7.
  • the forward gene sequences of the hairpin structures of the constructed three RNAi vectors are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and the intron sequences are shown in SEQ ID NO: 8,
  • the complementary sequences are the reverse complement of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
  • Example 3 Agrobacterium tumefaciens-mediated V-ATPase subunit E, COO2 and V-ATPase subunit E+COO2 double gene RNAi vector transformed Fielder spring wheat
  • Immature seeds 13 to 14 days after flowering pollination (young embryo size 1.0 to 1.2 mm), disinfected with 70% alcohol for 1 to 2 minutes, 15% sodium hypochlorite for 15 minutes, and sterile water for 4 to 5 times.
  • the Agrobacterium cells were collected by centrifugation at 3500 rpm for 10 min at room temperature, and the supernatant was removed, and resuspended in a 1:2 ratio with a 1/10 WCC resuspension (ie, MS minimal medium).
  • the wheat immature embryos were transferred to Agrobacterium tumefaciens for 30 min, and the callus was transferred to sterile filter paper in a sterilized culture dish, and co-cultured for 2 d (young embryo) under dark conditions at 25 °C.
  • 2d old embryo callus was transferred to IESX1 screening medium (MS basic medium (including MS vitamin) + 30gL -1 sucrose + 2.0mg L -1 dicamba dicamba + 250mg L -1 carbenicillin Cb + 5mg L -1 glufosinate PPT, pH 5.8), cultured in the dark at 25 °C for 2 weeks, then transferred to IESX2 screening medium (MS minimal medium (with MS vitamins) + 30 g L -1 sucrose + 2.0 mg L -1 dicamba Dicamba + 250 mg L -1 carbenicillin Cb + 10 mg L -1 glufosinate PPT, pH 5.8), incubated for 2-3 weeks at 25 ° C in the dark.
  • MS basic medium including MS vitamin
  • IESX2 screening medium MS minimal medium (with MS vitamins) + 30 g L -1 sucrose + 2.0 mg L -1 dicamba Dicamba + 250 mg L -1 carbenicillin Cb + 10 mg L -1 glufosinate PPT, pH 5.8
  • MS vitamin was purchased from Sigma.
  • the seedlings as long as 2 to 3 cm were transferred to a rooting and vigorous seedling medium (1/2 MS medium (containing MS vitamin) + 20 g L -1 sucrose + 250 mg L -1 Cb + 5 mg L -1 PPT, pH 5.8). Incubate for 3 to 4 weeks at 25 ° C under light. Transplant and grow robust resistant plants into pots.
  • a rooting and vigorous seedling medium 1/2 MS medium (containing MS vitamin) + 20 g L -1 sucrose + 250 mg L -1 Cb + 5 mg L -1 PPT, pH 5.8.
  • the Agrobacterium used was Agrobacterium EHA105.
  • the MS minimal medium composition was: 4.4 g/L MS, 30 g/L sucrose, pH 5.8, wherein the MS medium was purchased from Sigma.
  • the target gene was detected using a positively identified PCR primer (pDE1005-FP: ATGACAGTTCCACGGCAGTAGATA (SEQ ID NO: 9) and intron-RP: TTTCTTGGTTAGGACCCTTTTCTCTT (SEQ ID NO: 10)).
  • pDE1005-FP ATGACAGTTCCACGGCAGTAGATA
  • intron-RP TTTCTTGGTTAGGACCCTTTTCTCTT
  • the plant expression vector is transformed into Agrobacterium tumefaciens, and the Agrobacterium tumefaciens strain containing the V-ATPase subunit E, COO2 and V-ATPase subunit E+COO2 gene overexpression vector for transforming wheat is obtained respectively.
  • the transformed Agrobacterium tumefaciens strain was used to transform wheat immature embryos to obtain transgenic wheat plants which were detected by PCR.
  • the Agrobacterium tumefaciens used was Agrobacterium tumefaciens EHA105, and the competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.
  • V-ATPase subunit E, COO2 and V-ATPase subunit E+COO2 double-gene RNAi vector T1 generation wheat was identified for resistance to aphid.
  • V-ATPase subunit E, COO2, and V-ATPase subunit E+COO2 double-gene RNAi transgenic wheat plants obtained significantly improved resistance to mites compared to wild-type controls.
  • V-ATPase subunit E, COO2 and V-ATPase subunit E+COO2 double-gene RNAi transgenic wheat plants were compared with insects, and V-ATPase subunit E+COO2 double-gene RNAi transgenic wheat plants had the best insect resistance.

Abstract

利用V-ATPase subunit E基因片段或COO2基因片段或V-ATPase subunit E基因片段和COO2基因片段构建基因RNAi载体，转入植物，在植物中表达产生V-ATPase subunit E、COO2或V-ATPase subunit E与COO2双基因的dsRNA，从而抑制蚜虫生长，增强植物抗有害生物能力。

Description

一种核苷酸序列及其在增强植物抗有害生物能力中的应用 技术领域
本发明涉及的是一种生物工程领域的技术，具体是一种基于V-ATPase subunit E和COO2双基因融合增强植物抗蚜性的方法。
背景技术
蚜虫是农业生产中的主要害虫之一。蚜虫属于同翅目，包括球蚜总科和蚜总科，体长在1-10mm之间，为刺吸式口器，常群集于叶片、顶芽、嫩茎、花蕾等部位。蚜虫种类繁多，全世界目前己知约4700余种，中国分布约1100种。蚜虫在取食时，通过口针刺破植物表皮、薄壁组织及叶肉进入到筛管，取食植物的汁液，口针能够有效避开植物表皮组织的防御性物质，利于在组织间穿刺，顺利吸食植物组织中营养丰富的汁液。蚜虫吸食植物汁液，使得植株营养恶化，生长发育不良甚至停滞，导致减产；蚜虫分泌的蜜露聚集在叶片、茎秆等部位，严重影响农作物的光合作用和呼吸作用；此外，蚜虫排泄的蜜露能够促进一些病原菌的孽生和传播多种植物病毒。
蚜虫的寄主植物几乎包括被子植物和裸子植物的松柏纲所有的科，并且能在植物间传播病毒，危害性极广。诸如小麦、棉花、蔬果、花卉等经济农作物都深受其害，给农林业和园艺业造成了巨大经济损失。特别是近年来受大气温室效应的影响，我国小麦主要种植地区小麦生育期间气温升高，出现“暖冬”，致使蚜虫危害猖獗，每年在小麦生产上因蚜虫及其传播的病毒所造成的经济损失十分严重。
由于目前我国小麦主栽品种对蚜虫的抗性都不强，如何有效防治蚜虫已成为小麦生产上的重大问题之一。农药在抵抗虫害中起着重要作用，然而害虫对农药逐渐产生抗药性，此外，农药还容易污染环境。
目前植物介导的RNAi技术作为农作物抗虫研究的热点之一，应用前景十分广阔。此技术就是通过在寄主植物中表达相应昆虫特异基因的dsRNA，昆虫食用寄主植物后，其相应的基因被沉默而表达量下降，造成昆虫的死亡，从而起到控制害虫危害的作用。因此利用植物介导的RNAi技术培育抗蚜品种能够达到安全、持久、高效防治蚜虫的目的，具有重要的实际应用价值。
因此，本领域的技术人员致力于开发一种安全有效的抗蚜虫方法。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是提供一种安全有效 的抗蚜虫方法。
本发明的一个方面提供了一种核苷酸序列，在一个优选的实施方式中，该核苷酸序列选自如下序列：
1)SEQ ID NO:4-6中任一项所示的核苷酸序列；
2)SEQ ID NO:4-6中任一项所示的核苷酸序列通过一个或几个核苷酸的取代、缺失、插入或添加衍生产生的核苷酸序列；
3)包含与SEQ ID NO:4-6中任一项具有至少80％以上同源性的核苷酸序列。
本发明的第二个方面提供了一种双链RNA，在一个优选的实施方式中，由如上所述的核苷酸序列和其反向互补序列组成。
本发明的第三个方面提供了一种小干扰RNA，在一个优选的实施方式中，由如上所述的双链RNA裂解产生的短片段双链RNA分子。优选地，所述小干扰RNA的长度为19-25nt。
本发明的第四个方面提供了一种重组表达载体，在一个优选的实施方式中，含有如上所述的核苷酸序列或如上所述的双链RNA分子。
可选地，上述重组表达载体还包括能在植物中转录的启动子。可选地，启动子为玉米ubiquitin-1启动子。玉米ubiquitin-1启动子位于所述核苷酸序列或所述双链RNA分子的上游。可选地，玉米ubiquitin-1启动子下游具有水稻内含子，玉米ubiquitin-1启动子与水稻内含子(OSintron)融合后的核苷酸序列如SEQ ID NO:7所示。其中SEQ ID NO:7的1-1080位为玉米ubiquitin-1启动子，SEQ ID NO:7的1143-1621位为水稻内含子。
本发明的第五个方面提供了如上所述的核苷酸序列或如上所述的双链RNA在增强植物抗有害生物能力中的应用。在一个优选的实施方式中，所述有害生物为蚜虫。
可选地，蚜虫为桃蚜(Myzus persicae)、豆长管蚜(Acyrthosiphon pisum)、麦双尾蚜(Diuraphis noxia)、高粱蚜(Melanaphis sacchari)、黄伪毛蚜(Sipha flava)和棉蚜(Aphis gossypii)等中的一种或多种。
可选地，植物为双子叶植物、单子叶植物或裸子植物；可选地，双子叶植物包括十字花科植物和茄科植物；可选地，所述植物为拟南芥、水稻、小麦、玉米、棉花、大豆、油菜、高粱、烟草、菊花、青菜、白菜、萝卜或番茄等。
可选地，该应用包括如下步骤：
1)构建含有如上所述的核苷酸序列或如上所述的双链RNA分子的重组表达载体；
2)将所述重组表达载体转入农杆菌，利用所述农杆菌侵染植物幼胚；
3)通过抗生素筛选得到抗性植物幼苗。
本发明的一个具体实施方式中，提供了一种双基因融合增强植物抗蚜性的方法，利用了V-ATPase通过水解ATP产生能量从而为各种生命活动提供所需能量以及蚜虫唾液腺酶类基因COO2在蚜虫合成酶过程上的重要特性，利用基因工程手段获得的抗虫转基因植物具有只对目标害虫有效，而对非危害生物没有影响的优点，植物表达产生的抗虫物质存在于植物体内，不会对环境造成污染，且成本低，利于推广。
说明：本文中，SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3三条序列都包含了正向序列部分和互补序列部分，文中出现SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3三条序列时，应理解为指代其中的正向序列部分。
SEQ ID NO:1的正向序列部分为1-60、121-180、241-300、361-420、481-540、601-611位(即SEQ ID NO:4所示的序列)，其互补序列部分为61-120、181-240、301-360、421-480、541-600、612-622位。
SEQ ID NO:2的正向序列部分1-60、121-180、241-300、361-420、481-540、601-659位(即SEQ ID NO:5所示的序列)，其互补序列部分为61-120、181-240、301-360、421-480、541-600、600-718位。
SEQ ID NO:3的正向序列部分为1-60、121-180、241-300、361-420、481-540、601-660、721-780、841-900、961-1020、1081-1140、1201-1260、1321-1330位(即SEQ ID NO:6所示的序列)，其互补序列部分为61-120、181-240、301-360、421-480、541-600、661-720、781-840、901-960、1021-1080、1141-1200、1261-1320、1331-1340位。
本发明是通过以下技术方案实现的：本发明涉及一种具有抗蚜虫功能的基因dsRNA，其具体融合自蚜虫的V-ATPasesubunit E和COO2基因片段，其核苷酸序列如SEQ ID NO:3所示。
所述的SEQ ID NO:3，通过将蚜虫的V-ATPase subunit E(SEQ ID NO:1所示)、COO2基因(SEQ ID NO:2所示)以及V-ATPase subunit E和COO2基因部分片段融合片段基因合成。
本发明涉及一种双基因融合增强植物抗蚜性的方法，用V-ATPase subunit E基因、COO2基因以及V-ATPase subunit E基因和COO2基因部分序列融合片段分别构建得到具有抗蚜虫功能的基因，通过构建表达载体并将其分别转入农杆菌，以该农杆菌侵染小麦幼胚，分别在小麦中表达产生V-ATPase subunit E、COO2基因以及V-ATPase subunit E和COO2双基因的dsRNA，从而达到趋避蚜虫的目的。
所述的表达载体，通过构建玉米ubiquitin-1启动子驱动V-ATPase subunit E，COO2基因以及V-ATPase subunit E基因和COO2融合基因沉默载体，即RNAi载体。
所述的ubiquitin-1启动子具体为：组成型启动子。
所述的侵染，即将小麦幼胚转移至农杆菌菌液中侵染后置于黑暗条件下的培养基上培养得到幼胚愈伤组织，进而经抗性再生植株筛选后得到小麦抗性苗。
本发明涉及一种重组表达载体，即RNAi载体，其基于dsRNA构建得到。
所述的dsRNA包括：
1)由SEQ ID NO:1所示的在植物中表达的核苷酸和其反向互补序列；
2)由SEQ ID NO:1所示的在植物中表达的核苷酸和其反向互补序列；
3)由SEQ ID NO:3所示的在植物中表达的核苷酸和其反向互补序列，由Seq IDNo.1所示的核苷酸和SEQ ID NO:2所示的核苷酸组成。
所述的抗蚜性包括：
1)在防治蚜虫中的应用或在制备防治蚜虫产品中的应用；
2)在促进蚜虫死亡中的应用或在制备促进蚜虫死亡产品中的应用；
3)在抑制蚜虫生长中的应用或在制备抑制蚜虫生长产品中的应用。
技术效果
本发明的实验证明所得在小麦中表达蚜虫相关基因V-ATPase subunit E、COO2以及V-ATPase subunit E和COO2融合片段表达cDNA的序列的dsRNA，导致麦蚜产生致死效应，并且V-ATPase subunit E和COO2融合片段表达cDNA的序列的dsRNA抑制蚜虫效果最佳。
与化学防治相比，利用基因工程手段获得的抗虫转基因植物具有只对目标害虫有效，而对非危害生物没有影响的优点，植物表达产生的抗虫物质存在于植物体内，不会对环境造成污染，且成本低，利于推广。
与现有技术相比，本发明对于适用于本发明的植物没有特别的限制，只要其适合进行基因的转化操作，如各种农作物、花卉植物或林业植物等。
附图说明
图1为本发明pDE1005:proUBI:V-ATPase subunit E+COO2载体构建示意图；其中，图1a是pDE1005载体的示意图；图1b是构建V-ATPase subunit E+COO2发卡结构的示意图；图1c是将图1b中的发卡结构连接到载体上后的部分结构示意图；
图2为实施例中转ubiquitin-1启动子驱动V-ATPase subunit E+COO2基因T1代小麦PCR鉴定示意图；
图中：M：Marker III；+：pDE1005:proUBI:V-ATPase subunit E+COO2质粒；CK：野生型Fileder春小麦；1-1，1-3，1-6，2-3，2-3，2-6，3-2，3-4，3-6，4-1，4-3，4-4，5-3，5-5，5-8：转ubiquitin-1启动子驱动V-ATPase subunit E+COO2基因T1代小麦植株，简称VC。
图3为实施例中转ubiquitin-1启动子驱动V-ATPase subunit E+COO2基因T1 代小麦V-ATPase subunit E+COO2基因半定量结果示意图；
图中：M：Marker III；+：pDE1005:proUBI:V-ATPase subunit E+COO2质粒；CK：野生型Fileder春小麦；1-1，1-3，1-6，2-3，2-3，2-6，3-2，3-4，3-6，4-1，4-3，4-4，5-3，5-5，5-8：转ubiquitin-1启动子驱动V-ATPase subunit E+COO2基因T1代小麦植株，简称VC。
具体实施方式
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。所采用的试剂，若无特殊说明，均为市售或公开渠道可以获得的试剂。
在蚜虫的生长发育过程中，V-ATPase通过水解ATP产生能量从而为各种生命活动提供所需能量，唾液腺酶类基因COO2在蚜虫合成酶过程中也非常重要。因此，本发明的一个具体实施方式中，采用V-ATPase和COO2基因的片段进行基因融合，构建用于基因沉默的元件，通过该元件降低蚜虫体内的V-ATPase和COO2基因表达水平，影响蚜虫的正常生长发育，达到抑制蚜虫生长的目的。
在一个具体实施方式中，采用V-ATPase subunit E和COO2基因非保守区域的片段基因融合，构建用于基因沉默的元件。选择非保守区域可以防止干扰除蚜虫V-ATPase subunit E和COO2外的其他同源序列。
在一个具体实施方式中，用于基因沉默的元件是双链RNA。
在一个具体实施方式中，通过将蚜虫的V-ATPase subunit E和COO2基因非保守区域的片段进行融合，并构建带有该融合基因的正向和反向序列的重组表达载体。该重组表达载体被转入植物(例如，可以通过农杆菌介导的侵染的方式)，在植物中表达产生V-ATPase subunit E和COO2双基因的dsRNA，蚜虫取食产生同时包括V-ATP和COO2基因的siRNA的转基因植物后，使其体内的V-ATPase subunit E和COO2基因表达水平同时下降。通过上述RNA干扰的方式影响蚜虫正常的生长发育，从而达到抑制蚜虫生长的目的。
“RNA干扰(RNA interference，RNAi)”是指一些小的双链RNA可以高效、特异地阻断体内特定靶基因的表达，促使成熟mRNA降解，使生物个体表现出特定基因缺失的表型。RNA干扰是高度特异的mRNA水平上的基因沉默抑制。
“小干扰RNA(small interfering RNA，siRNA)”是指一种短片段双链RNA分子，能够以同源互补序列的mRNA为靶目标，降解特定的mRNA，这个过程就是RNA干扰途径(RNA interference pathway)。
通过比对已经测序的不同种类蚜虫V-ATPase subunit E和COO2基因序列，可知这两个基因序列非常的保守，虽然其他双子叶、单子叶等植物的蚜虫没有被测序， 但是可以推测出这两个基因不同物种蚜虫同源性非常的高，在小麦中能实现RNAi，就能在其他植物(单子叶、双子叶、裸子植物)实现。
实施例1 V-ATPase subunit E和COO2基因序列同源性比对
利用NCBI数据库，将选择的V-ATPase subunit E基因片段(SEQ ID NO:4)进行核酸序列比对，结果显示，该段序列与豆长管蚜(Acyrthosiphon pisum)V-type proton ATPase subunit E-like序列(NCBI登录号：NM_001162178.2)同源性100％；与桃蚜(Myzus persicae)V-type proton ATPase subunit E序列(NCBI登录号：XM_022312248.1)同源性95％；与麦双尾蚜(Diuraphis noxia)V-type proton ATPase subunit E序列(NCBI登录号：XM_015522279.1)同源性92％；与高粱蚜(Melanaphis sacchari)V-type proton ATPase subunit E序列(NCBI登录号：XM_025342771.1)同源性94％；与黄伪毛蚜(Sipha flava)V-type proton ATPase subunit E序列(NCBI登录号：XM_025565024.1)同源性86％。
将选择的COO2基因片段(SEQ ID NO:5)进行核酸序列比对，结果显示，该段序列与豆长管蚜(Acyrthosiphon pisum)序列(NCBI登录号：XM_001948323.3)同源性99％；桃蚜(Myzus persicae)序列(NCBI登录号：XM_022310905.1)同源性89％；与棉蚜(Aphis gossypii)序列(NCBI登录号：KJ451424.1)同源性89％；与高粱蚜(Melanaphis sacchari)序列(NCBI登录号：XM_025338353.1)同源性80％。
根据RNAi技术原理，与本发明中V-ATPase subunit E和COO2基因序列同源性为80％以上均会有抗蚜虫效果。
实施例2 含V-ATPase subunit E和COO2双基因的RNAi载体的合成
分别合成V-ATPase subunit E基因片段，COO2基因片段以及V-ATPase subunit E和COO2双基因片段，分别构建发夹结构的正向基因，内含子序列和反向的基因，构建到pDE1005载体(购自北京拜尔迪生物技术有限公司)的多克隆位点上，分别获得pDE1005:proUBI:V-ATPase subunit E，pDE1005:proUBI:COO2和pDE1005:proUBI:V-ATPase subunit E+COO2基因沉默载体，基因由上海生工生物工程公司合成。构建V-ATPase subunit E和COO2双基因片段的RNAi载体的示意图如图1所示。
在一个可选的实施例中，pDE1005载体上自带水稻内含子，玉米ubiquitin-1启动子位于水稻内含子的上游，玉米ubiquitin-1启动子和水稻内含子构建融合之后的序列如SEQ ID NO:7所示。构建的三种RNAi载体的发夹结构的正向基因序列如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示，内含子序列如SEQ ID NO:8所示，反向互补序列为SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6的反向互 补序列。
实施例3根癌农杆菌介导V-ATPase subunit E，COO2以及V-ATPase subunit E+COO2双基因RNAi载体转化Fielder春小麦
1.1.幼胚的预培养
开花授粉后13～14d的未成熟种子(幼胚大小1.0～1.2mm)，用70％酒精表面消毒1～2min，15％的次氯酸钠灭菌15min，无菌水冲洗4～5次。
1.2.农杆菌与幼胚愈伤组织的共培养
室温下，3500rpm离心10min收集农杆菌菌体，尽去上清，用1/10WCC重悬液(即MS基本培养基)以1：2比例重悬。将小麦幼胚转移至农杆菌菌液中侵染30min，将愈伤组织转移至灭菌培养皿中的无菌滤纸上，25℃黑暗条件下共培养2d(幼胚)。
共培养2d的幼胚愈伤组织转移到IESX1筛选培养基(MS基本培养基(含MS维生素)+30gL -1蔗糖+2.0mg L -1麦草畏dicamba+250mg L -1羧苄青霉素Cb+5mg L -1草丁膦PPT，pH 5.8)，25℃黑暗培养2周，然后转移到IESX2筛选培养基(MS基本培养基(含MS维生素)+30g L -1蔗糖+2.0mg L -1麦草畏dicamba+250mg L -1羧苄青霉素Cb+10mg L -1草丁膦PPT，pH 5.8)，25℃黑暗培养2-3周。
1.3.抗性再生植株的筛选
筛选后的幼胚愈伤组织转移到IEFH培养基(MS基本培养基(含MS维生素)+20g L -1蔗糖+0.2mg L -1 2,4-D+250mg L -1Cb+5mg L -1PPT，pH＝5.8)，25℃、光照培养3～4周。其中MS维生素购自Sigma。
筛选后的成熟胚愈伤组织转移到XCFH分化培养基(MS基本培养基(不含MS维生素)+20g L -1蔗糖+10.0mg L -1B1维生素+1.0mg L -1B3维生素+1.0mg L -1B6维生素+2.0mgL -1甘氨酸+5.0mg L -1谷氨酰胺+0.2mg L -1IAA+250mg L -1Cb+5mg L -1PPT，pH 5.8)上，25℃光照培养3～4周，分化植株。
将长至2～3cm的幼苗移至生根壮苗培养基(1/2MS培养基(含MS维生素)+20g L -1蔗糖+250mg L -1Cb+5mg L -1PPT，pH 5.8)上，25℃、光照条件下培养3～4周。移栽生长健壮的抗性植株到花盆中。
其中，使用的农杆菌为农杆菌EHA105。MS基本培养基成分为：4.4g/L MS，30g/L蔗糖，pH 5.8，其中MS培养基购于sigma公司。
转基因小麦的PCR检测
利用阳性鉴定的PCR引物(pDE1005–FP：ATGACAGTTCCACGGCAGTAGATA(SEQ ID NO:9)和intron-RP：TTTCTTGGTTAGGACCCTTTTCTCTT(SEQ ID NO:10))对目的基因进行检测。结果表明，利用所设计的PCR特异引物，能扩增出特异DNA片段。而以非转化 小麦基因组DNA为模板时，没有扩增出任何片段。
本实施例将所述的植物表达载体转化根癌农杆菌，分别获得用于转化小麦的含V-ATPase subunit E、COO2以及V-ATPase subunit E+COO2基因植物过量表达载体的根癌农杆菌菌株，利用所构建的根癌农杆菌菌株转化小麦幼胚，获得经PCR检测的转基因小麦植株。
其中，使用的根癌农杆菌为根癌农杆菌EHA105，感受态细胞买自上海唯地生物技术有限公司。
实施例4 V-ATPase subunit E、COO2以及V-ATPase subunit E+COO2双基因RNAi载体T1代小麦进行抗蚜虫性鉴定。
将同一虫龄的麦蚜分别接于待测转基因植株和3株野生型小麦展开的嫩叶上，每株接10头，培养10天后，统计叶片上蚜虫数量。结果如表1-3所示，与野生型对照相比，V-ATPase subunit E、COO2以及V-ATPase subunit E+COO2双基因RNAi转基因小麦植株获得了显著提高的抗蚜性。
V-ATPase subunit E，COO2以及V-ATPase subunit E+COO2双基因RNAi转基因小麦植株抗虫效果比较，V-ATPase subunit E+COO2双基因RNAi转基因小麦植株抗虫效果最佳。
表1 V-ATPase subunit E转基因小麦对蚜虫的抗性鉴定
Figure PCTCN2018114295-appb-000001
Figure PCTCN2018114295-appb-000002
表2 COO2转基因小麦对蚜虫的抗性鉴定
Figure PCTCN2018114295-appb-000003
表3 V-ATPase subunit E+COO2双基因转基因小麦对蚜虫的抗性鉴定
Figure PCTCN2018114295-appb-000004
Figure PCTCN2018114295-appb-000005
结果显示，五个转V-ATPase subunit E和COO2双基因RNAi载体T1代小麦植株上蚜虫数量显著低于野生型对照，并且与V-ATPase subunit E和COO2RNAi载体T1代小麦植株抗虫效果相比，效果更佳。
上述具体实施可由本领域技术人员在不背离本发明原理和宗旨的前提下以不同的方式对其进行局部调整，本发明的保护范围以权利要求书为准且不由上述具体实施所限，在其范围内的各个实现方案均受本发明之约束。
Figure PCTCN2018114295-appb-000006
Figure PCTCN2018114295-appb-000007
Figure PCTCN2018114295-appb-000008
Figure PCTCN2018114295-appb-000009
Figure PCTCN2018114295-appb-000010
Figure PCTCN2018114295-appb-000011

Claims (10)

  1. 一种核苷酸序列，其特征在于，所述核苷酸序列选自如下序列：
    1)SEQ ID NO:4-6中任一项所示的核苷酸序列；
    2)SEQ ID NO:4-6中任一项所示的核苷酸序列通过一个或几个核苷酸的取代、缺失、插入或添加衍生产生的核苷酸序列；
    3)包含与SEQ ID NO:4-6中任一项具有至少80％以上同源性的核苷酸序列。
  2. 一种双链RNA，其特征在于，由如权利要求1所述的核苷酸序列和其反向互补序列组成。
  3. 含有如权利要求1所述的核苷酸序列或如权利要求2所述的双链RNA分子的重组表达载体。
  4. 如权利要求3所述的重组表达载体，其特征在于，还包括能在植物中转录的启动子。
  5. 如权利要求1所述的核苷酸序列或如权利要求2所述的双链RNA在增强植物抗有害生物能力中的应用。
  6. 如权利要求5所述的应用，其特征在于，所述有害生物为蚜虫。
  7. 如权利要求6所述的应用，其特征在于，所述蚜虫为桃蚜(Myzus persicae)、豆长管蚜(Acyrthosiphon pisum)、麦双尾蚜(Diuraphis noxia)、高粱蚜(Melanaphis sacchari)、黄伪毛蚜(Sipha flava)和棉蚜(Aphis gossypii)中的一种或多种。
  8. 如权利要求5所述的应用，其特征在于，所述植物为双子叶植物、单子叶植物或裸子植物；可选地，所述植物为拟南芥、水稻、小麦、玉米、棉花、大豆、油菜、高粱、烟草、菊花、青菜、白菜、萝卜或番茄。
  9. 如权利要求5所述的应用，其特征在于，包括如下步骤：
    1)构建含有如权利要求1所述的核苷酸序列或如权利要求2所述的双链RNA分子的重组表达载体；
    2)将所述重组表达载体转入农杆菌，利用所述农杆菌侵染植物幼胚；
    3)通过抗生素筛选得到抗性植物幼苗。
  10. 一种双基因融合增强植物抗蚜性的方法，其特征在于，用V-ATPase subunit E基因、COO2基因以及V-ATPase subunit E基因和COO2基因部分序列融合片段分别构建得到具有抗蚜虫功能的基因，通过构建表达载体并将其分别转入农杆菌，以该农杆菌侵染小麦幼胚，分别在小麦中表达产生V-ATPase subunit E、COO2基因以及V-ATPase subunit E和COO2双基因的dsRNA，从而达到趋避蚜虫的目的；可选地，所述的表达载体，通过构建玉米ubiquitin-1启动子驱动V-ATPase subunit E，COO2基因以及V-ATPase subunit E基因和COO2融合基因沉默载体，即RNAi载体；可选地，所述的ubiquitin-1启动子具体为：组成型启动子；可选地，所述的侵染，即将小麦幼胚转移至农杆菌菌液中侵染后置于黑暗条件下的培养基上培养得到幼胚愈伤组织，进而经抗性再生植株筛选后得到小麦抗性苗；可选地，所述的抗蚜性包括：1)在防治蚜虫中的应用或在制备防治蚜虫产品中的应用；2)在促进蚜虫死亡中的应用或在制备促进蚜虫死亡产品中的应用；3)在抑制蚜虫生长中的应用或在制备抑制蚜虫生长产品中的应用。
PCT/CN2018/114295 2017-11-07 2018-11-07 一种核苷酸序列及其在增强植物抗有害生物能力中的应用 WO2019091390A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US16/760,447 US11208668B2 (en) 2017-11-07 2018-11-07 Nucleotide sequence and application thereof in enhancing plant pest resistance
AU2020100833A AU2020100833A4 (en) 2017-11-07 2020-05-25 Nucleotide sequence and application thereof in enhancing plant pest resistance

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711082638.4 2017-11-07
CN201711082638.4A CN107937424A (zh) 2017-11-07 2017-11-07 双基因融合增强植物抗蚜性的方法

Related Child Applications (1)

Application Number Title Priority Date Filing Date
AU2020100833A Division AU2020100833A4 (en) 2017-11-07 2020-05-25 Nucleotide sequence and application thereof in enhancing plant pest resistance

Publications (1)

Publication Number Publication Date
WO2019091390A1 true WO2019091390A1 (zh) 2019-05-16

Family

ID=61934402

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/114295 WO2019091390A1 (zh) 2017-11-07 2018-11-07 一种核苷酸序列及其在增强植物抗有害生物能力中的应用

Country Status (3)

Country Link
US (1) US11208668B2 (zh)
CN (2) CN107937424A (zh)
WO (1) WO2019091390A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107937424A (zh) 2017-11-07 2018-04-20 上海交通大学 双基因融合增强植物抗蚜性的方法
CN111394371B (zh) * 2020-03-31 2022-01-28 山西大学 飞蝗V-ATPase-V1结构域基因及其dsRNA在害虫防治中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206668A (zh) * 2011-04-28 2011-10-05 上海交通大学 增强植物抗蚜性的方法
CN103088024A (zh) * 2013-01-09 2013-05-08 中国农业科学院作物科学研究所 两种dsRNA及其组合在控制蚜虫危害中的应用
CN107937424A (zh) * 2017-11-07 2018-04-20 上海交通大学 双基因融合增强植物抗蚜性的方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003018822A1 (en) * 2001-08-22 2003-03-06 Monsanto Technology Llc An improved efficiency (agrobacterium)-mediated wheat transformation method
EP1971688B1 (en) * 2006-01-12 2012-03-14 Devgen NV Dsrna as insect control agent
US8299318B2 (en) * 2007-07-05 2012-10-30 Ceres, Inc. Nucleotide sequences and corresponding polypeptides conferring modulated plant characteristics
CN101851622B (zh) * 2010-01-15 2012-10-10 中国科学院微生物研究所 一种利用rna干扰技术培育抗虫性提高的转基因植物的方法及其专用dna片段
CN103571871B (zh) * 2011-04-28 2015-05-20 上海交通大学 增强植物抗蚜性的方法
CN105861513A (zh) * 2016-05-16 2016-08-17 中国农业科学院作物科学研究所 通过植物介导的RNAi培育抗蚜虫转基因小麦的方法及载体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206668A (zh) * 2011-04-28 2011-10-05 上海交通大学 增强植物抗蚜性的方法
CN103088024A (zh) * 2013-01-09 2013-05-08 中国农业科学院作物科学研究所 两种dsRNA及其组合在控制蚜虫危害中的应用
CN107937424A (zh) * 2017-11-07 2018-04-20 上海交通大学 双基因融合增强植物抗蚜性的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 20 May 2004 (2004-05-20), ANONYMOUS: "ID0AAA6AH06RM1 ApMS Acyrthosiphon pisum cDNA clone ID0AAA6AH06 5', mRNA sequence", XP055607879, retrieved from NCBI Database accession no. CN 763138.1 *
DATABASE NUCLEOTIDE 23 May 2016 (2016-05-23), ANONYMOUS: "Acyrthosiphon pisum V-type proton ATPase subunit E-like (LOC100165126), mRNA", XP055607875, retrieved from NCBI Database accession no. NM 001162178.2 *
GAO, CHAOBAO: "Study on the use of RNAi technology to resist cotton aphid", AGRICULTURE, CHINA MASTER S THESES FULL- TEXT DATABASE, 15 May 2012 (2012-05-15), pages D046 - 69, ISSN: 1674-0246 *
REBIJITH, K. B. ET AL.: "Diet-Delivered dsRNAs for Juvenile Hormone-Binding Protein and Vacuolar ATPase-H Implied Their Potential in the Management of the Melon Aphid (Hemiptera: Aphididae", ENVIRONMENTAL ENTOMOLOGY, vol. 45, no. 1, December 2015 (2015-12-01) - 1 February 2016 (2016-02-01), pages 268 - 275, XP055607884, ISSN: 0046-225X, DOI: 10.1093/ee/nvv178 *

Also Published As

Publication number Publication date
US20200283792A1 (en) 2020-09-10
US11208668B2 (en) 2021-12-28
CN107937424A (zh) 2018-04-20
CN109988761B (zh) 2023-09-19
CN109988761A (zh) 2019-07-09

Similar Documents

Publication Publication Date Title
CN101182523B (zh) 植物花药特异性启动子及其应用
WO2016138819A1 (zh) 杀虫蛋白的用途
CN105624177A (zh) 一种抗虫融合基因、编码蛋白、载体及其应用
CN101358190A (zh) 一种人工合成的对鳞翅目害虫表达高毒力蛋白的基因序列及其应用
CN111424022A (zh) 大丽轮枝菌VdEG抗病原菌靶基因片段及其干扰载体和应用
CN111315218B (zh) 杀虫蛋白的用途
CN113913457B (zh) 一种抑制或杀灭桃蛀螟的方法及其应用
WO2019091390A1 (zh) 一种核苷酸序列及其在增强植物抗有害生物能力中的应用
González et al. Efficient regeneration and Agrobacterium tumefaciens mediated transformation of recalcitrant sweet potato (Ipomoea batatas L.) cultivars
CN109735538B (zh) 一种提高森林草莓叶片再生效率的载体及其制备方法和应用
CN105755020B (zh) 三七丝裂原活化蛋白激酶激酶基因PnMAPKK1及其应用
Maligeppagol et al. Genetic transformation of chilli (Capsicum annuum L.) with Dreb1A transcription factor known to impart drought tolerance
CN103409460A (zh) 转化玉米的方法
WO2016101684A1 (zh) 杀虫蛋白的用途
WO2016184397A1 (zh) 杀虫蛋白的用途
CN110982817A (zh) 一种抗小麦黄花叶病毒的amiRNA及其应用
Aasim et al. Production of herbicide-resistant cowpea (Vigna unguiculata L.) transformed with the bar gene
CN103409445A (zh) 抗草甘膦基因mtp-smg2-epsps及其在培育抗草甘膦玉米中的应用
Ahmed et al. An efficient Agrobacterium-mediated genetic transformation method of lettuce (Lactuca sativa L.) with an aphidicidal gene, Pta (Pinellia ternata Agglutinin)
AU2020100833A4 (en) Nucleotide sequence and application thereof in enhancing plant pest resistance
CN109234307B (zh) 杀虫蛋白的用途
CN102250947B (zh) 一种植物不育系和恢复系的制备方法和应用
WO2013010368A1 (zh) 水稻通气组织形成关键基因OsLSD2的应用
CN101993484B (zh) 一种耐逆性相关蛋白及其编码基因与应用
CN107586324A (zh) TabZIP15蛋白及其编码基因与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18875360

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 08/10/2020)

122 Ep: pct application non-entry in european phase

Ref document number: 18875360

Country of ref document: EP

Kind code of ref document: A1