WO2012163084A1 - 基于RNAi技术防治害虫的新方法 - Google Patents
基于RNAi技术防治害虫的新方法 Download PDFInfo
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- WO2012163084A1 WO2012163084A1 PCT/CN2012/000731 CN2012000731W WO2012163084A1 WO 2012163084 A1 WO2012163084 A1 WO 2012163084A1 CN 2012000731 W CN2012000731 W CN 2012000731W WO 2012163084 A1 WO2012163084 A1 WO 2012163084A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
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- 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
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- 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
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- 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.
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- 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 present invention is in the field of biotechnology and agrochemicals; more specifically, the present invention relates to a novel method for controlling pests based on RNAi technology. Background technique
- the perennial damage area of Plutella xylostella accounts for about 10% of the total area of vegetable cultivation (2010 vegetable planting area is 33.33 million hm 2 , Plutella xylostella is 2.53 million hm 2 ; 2009 vegetable planting area is 30 million hm 2 , Plutella xylostella is 2.7 million Hm 2 ), the loss is about 20 ⁇ 30%. If the prevention is not timely or the measures are unreasonable, it may cause the harvest. At present, the control of Asian corn borer, cotton bollworm and diamondback moth is still dominated by chemical control, but it has caused great damage to the ecological environment and food security production. Many hazards of chemical control have made people have to seek better pest control methods. Although biological control can play a preventive role, it is not effective because of its slow effect, and it is not recognized by the public because of its environmental impact.
- RNAi phenomenon has developed rapidly since its discovery in 1991. Studies have shown that through the specific gene RNAi, target genes of targeted interference species can be reached, and some physiological phenomena appear, which can achieve the purpose of studying gene function. At the same time, this phenomenon is extremely high. The specificity, that is, the interference effect of homologous genes on different species is not obvious, so it is an ideal pest control system.
- RNAi technology has been reported for pest control.
- Baum et al. (2007) demonstrated that v-ATPase has a lethal effect on Western corn rootworm WCR Diabrotica virgifera virgifera LeConte and can be used for field control.
- RNAi technology is feasible as a new pest control method, but previous studies have also shown that RNAi-based pest control has Many problems need to be solved, such as: 1) how to quickly and high-throughput target genes; 2) how to apply them easily to field production; 3) resistance problems; 4) safety issues, etc. Therefore, applying RNAi technology In the field of pest control, it is also necessary to solve the above problems. First, it is necessary to obtain enough and effective target genes, so that a large number of target genes can be controlled by different combinations and different periods using different genes, which can effectively prevent pest resistance. The emergence of sex.
- the present invention also provides insecticidal preparations and methods for obtaining insect-species-specific and inter-species broad-spectrum types using different fragments of the same gene. Summary of the invention
- an isolated polypeptide selected from the group consisting of:
- polypeptide is a polypeptide of the amino acid sequence of SEQ ID NO: 2.
- an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:
- the polynucleotide is selected from the group consisting of - (1) a polynucleotide of the sequence of SEQ ID NO: 1;
- the encoded protein has the same function as the polynucleotide encoded by the polynucleotide of (1) or (2).
- a vector comprising the polynucleotide is provided.
- a genetically engineered host cell preferably a non-reproductive or reproductive cell, comprising the vector, or a polynucleoside thereof integrated in the genome thereof acid.
- polynucleotide fragment having a sequence of 10% or more (preferably 15% or more; more preferably 20%) of the polynucleotide sequence described above More preferably 25./.; more preferably 30%) sequence identity.
- polynucleotide fragment is selected from the group consisting of:
- a vector comprising one, two or more of said polynucleotide fragments is provided.
- a genetically engineered host cell comprising the vector, or one or two or more of the polynucleotide fragments integrated in the genome of the genome.
- the use of the polynucleotide or polynucleotide fragment is provided for use as a suppressor or silencing target for the preparation of an interfering molecule that specifically interferes with the expression of a pest gene or inhibits the growth of a pest
- the pest gene is selected from the group consisting of a storage protein, a white gene; or as a target for the preparation of an insecticidal (such as lepidopteran) preparation.
- the storage chalk gene is selected from the group consisting of Asian corn borer storage white gene or cotton bollworm storage white gene or Plutella xylostella storage white gene;
- the lepidopteran insect is selected from the group consisting of Asian corn borer or cotton bollworm or diamondback moth;
- the use of the polypeptide is provided for use as a target for screening or preparing insecticidal formulations.
- the preparation is selected from, but not limited to, a nucleic acid inhibitor, a protein inhibitor, an antibody, a ligand, a hydrazine hydrolase, a protein binding molecule.
- nucleic acid inhibitor is selected from the group consisting of
- dsRNA antisense nucleic acid, small interfering RNA, microRNA; or
- nucleic acid inhibitor selected from the group consisting of
- polynucleotide or polynucleotide fragment, or a transcript thereof for inhibiting or silencing a target dsRNA, an antisense nucleic acid, a small interfering RNA, a microRNA; wherein any of the dsRNAs, antisense When nucleic acids, small interfering RNAs, and microRNAs are ingested by pests, they inhibit the growth of the harmful organisms; or
- RNA wherein any of the dsRNA, antisense nucleic acid, small interfering RNA, and microRNA are ingested by a pest, and the pest gene is selected from the storage of the white gene; or
- the nucleic acid inhibitor is a dsRNA produced by expression of the polynucleotide, wherein When the dsRNA is ingested by a pest, it inhibits the growth of the pest.
- the nucleic acid inhibitor is dsRNA having the following structure:
- Seq' E 3 ⁇ 4 is an RNA sequence corresponding to the polynucleotide of any one of claims 2 or 3 or a sequence fragment thereof, or a nucleotide sequence selected from the sequence defined in claim 2 or 3.
- Seq' s i5 is a sequence substantially complementary to Seq
- X ' is absent; or located Seq' ⁇ and Seq 'spacer sequence between s, the spacer sequence is Seq' and Seq ' ⁇ not complementary;
- nucleic acid inhibitor is a construct comprising the following structure:
- Seq E ft is a polynucleotide of any one of claims 2 or 3 or a sequence fragment thereof, or a core comprising at least a portion of a nucleotide sequence selected from the sequence defined in claim 2 or 3 Glycosidic acid sequence;
- Seq S (3 ⁇ 4 is a sequence that is substantially complementary to Seq «;
- X is a spacer sequence located between Seq j ⁇ and Seq ⁇ , and the spacer sequence is not complementary to Seq ia and Seq.
- the construct may form the following structure -
- Seq Seq s and X are as described above.
- the construct is an expression vector (plasmid).
- the polynucleotide or polynucleotide fragment can serve as a target for inhibiting mRNA transcription of the polynucleotide or inhibiting expression of a polypeptide encoded by the polynucleotide.
- the polynucleotide or polynucleotide fragment can be used as a target for the preparation of insecticidal formulations.
- a host cell e.g., a bacterium, a fungus, a yeast, a plant cell
- the nucleic acid inhibitor such as the construct
- nucleic acid inhibitor or the host cell described for the preparation of an insect control preparation.
- an insect control preparation (such as a pesticide preparation or a pesticide composition) comprising: a safe and effective amount of a substance selected from the group consisting of: the nucleic acid inhibitor (e.g., 10-50 (g) /mL) or the host cell; And; a pharmaceutically acceptable carrier.
- it further comprises at least one insecticide selected from the group consisting of chemical insecticides, potato glycoside, Bacillus thuringiensis, insecticidal white, pathogenic bacilli, white bacillus, light bacillus Insecticidal white, Bacillus licheniformis, and B. sphaericus insecticidal protein.
- insecticides selected from the group consisting of chemical insecticides, potato glycoside, Bacillus thuringiensis, insecticidal white, pathogenic bacilli, white bacillus, light bacillus Insecticidal white, Bacillus licheniformis, and B. sphaericus insecticidal protein.
- a method of controlling a pest that interferes with expression of a pest gene is selected, the pest gene being selected from the group consisting of a storage white gene.
- the method comprises: feeding or/or spraying a pest of the nucleic acid inhibitor of any one of claims 7-10 or the host cell of claim 5 or 11.
- a method of killing an insect comprising: reducing expression of the polypeptide in the insect; or reducing the polynucleotide or polynucleotide in the insect Transcription of the corresponding mRNA of the fragment.
- the method of providing comprises: applying the nucleic acid inhibitor or the host cell to (eg, spraying) a subject to be controlled (eg, a plant, particularly an insect-infested plant; or an insect) ).
- a subject to be controlled eg, a plant, particularly an insect-infested plant; or an insect
- nucleic acid inhibitor or the host cell in another aspect of the invention, there is provided the feeding of the nucleic acid inhibitor or the host cell to an insect or a spray insect.
- the method comprises: expressing in the plant an interfering molecule that specifically interferes with expression of the pest gene; the insect gene is selected from the group consisting of a storage protein gene.
- a method of preparing an insect resistant plant comprising: introducing the nucleic acid inhibitor into a plant.
- a plant or a seed thereof which plant is obtained by transformation of said polynucleotide.
- the polynucleotide is expressed as a dsRNA in a plant cell.
- the pest is selected from the group consisting of: insects, mites, fungi, yeasts, molds, bacteria, nematodes, weeds and parasites, and saprophytic plants.
- the pest is an insect, including but not limited to: a Lepidoptera, a Coleoptera, a Hemiptera, and a Diptera.
- the insect pest is selected from the group consisting of lepidopteran insects, preferably corn borer or cotton bollworm or diamondback moth.
- a method of preparing an insect resistant plant comprising: introducing the nucleic acid inhibitor into a plant.
- the method of preparing an insect resistant plant is:
- step (1) (2) contacting a plant cell or tissue or organ with Agrobacterium in step (1) to transfer the nucleic acid inhibitor into a plant cell or tissue or organ.
- the method for preparing an insect-resistant plant further comprises: (3) selecting a plant cell or tissue or organ into which the nucleic acid inhibitor has been introduced;
- step (3) Regenerating the plant cell or tissue or organ in step (3).
- the insects have a significant decrease in survival or death after eating the insect-resistant plants.
- Other aspects of the invention will be apparent to those skilled in the art from this disclosure.
- FIG. 1 Different domains of lethal efficiency at different times after dsRNA treatment of Asian corn borer.
- EYFP is an enhanced yellow fluorescent white gene as a foreign gene control.
- CK is a blank control and is a normal artificially reared Asian corn borer.
- Ostrinia furnacalis Asian corn snail.
- Figure 1 The lethality of the cotton bollworm's own SP gene dsRNA against cotton bollworm.
- FIG. 13 Schematic representation of the pART27-dsRNA vector. detailed description
- the inventors have found extensively a target gene useful for controlling lepidopteran insects, and based on the nucleic acid sequence of the target gene, a nucleic acid inhibitor or a host expressing a nucleic acid inhibitor can effectively kill lepidopteran insects. . the term
- isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
- the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
- Those skilled in the art will be able to purify the protein using standard ruthenium purification techniques.
- a substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.
- the "plant” is not particularly limited as long as the “plant” is easily attacked by insects such as lepidopterans, such as various crops, flower plants, or forestry plants.
- the plant may be, for example, without limitation: a dicot, a monocot, or a gymnosperm.
- the plants include (but are not limited to): cotton, wheat, barley, rye, rice, corn, sorghum, beets, apples, pears, plums, peaches, apricots, cherries, strawberries, raspberries, blackberries , beans, lentils, peas, soybeans, rapeseed, mustard, opium, olean, sunflower, coconut, castor oil plant, cocoa beans, peanuts, gourd, cucumber, watermelon, flax, hemp, jute, citrus, lemon, Grapefruit, spinach, chicory, asparagus, cabbage, Chinese cabbage, pakchoi, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugar cane, tea, pepper, grape Trees, ramie, bananas, natural rubber trees and ornamental plants.
- insect refers to an insect that includes a storage protein gene in any genome.
- the insect may be a plant-feeding insect that feeds on a plant, for example, it may be a scutellaria, isoptera, coleoptera, diptera, hymenoptera, lepidoptera, orthoptera.
- Insects or agricultural pests of the order Hemiptera, Helicoptera for example, the species of the genus Heliconia, the genus Heliconia, the genus Heliconia, the genus Diptera, the cotton leaf moth, the larvae, the yellow Moth, Moth, Moth, Maternaria, Moth, Moth, Moth, Moth, Moth, Moth, Moth, Moth, Coleoptera, apple-shaped moth, Coleoptera, Corn genus, Spodoptera frugiperda, Diamond, genus, genus, genus, genus, genus, genus, genus, genus, broad-winged Small moth, Moth, Moth, American white moth, Moth, Moth, Moth, Moth, Moth, Lepidoptera, Brassica, Tobacco, Moth, Toad Genus, European corn borer, ultra-small moth, small eyed moth, red bollworm, cotton bollworm
- the "insect” is an insect that is harmful to plants
- the patent application of the Chinese Patent Application No. 200680042821.5 and US 2009/0306189 Al, WO 2007/080127 A2, AU 2006/335978 AL US 2009/0285784 Al The application text is a reference for the invention, and the description of insects on pages 41-47 of CN101365795 can be incorporated into the description of the present invention.
- lepidoptera refers to a gene (fragment) including any one of SEQ ID NO: 3-5, SEQ ID NO: 17, and SEQ ID NO: 20-21 in any genome. a sequence or a homologous gene thereof or a hybrid thereof a gene of lepidopteran insects, and these genes are genes in the insects necessary for the growth or survival of the insect.
- the lepidoptera includes moths, examples include: Tineidae and Oecophoridae such as Tineola bisselliella (Common Clothes Moth), and Cocoonidae (Pyralidae) such as Pyralis farinalis (Meal Moth), Asian corn borer, and Noctuidae such as cotton bollworm, and the genus Moth, such as the diamondback moth.
- RNA interference refers to a phenotype in which some RNAs efficiently and specifically block the expression of specific genes in the body, promote mRNA degradation, and induce cells to exhibit specific gene deletions. Called RNA intervention or intervention. RNA interference is a highly specific gene silencing mechanism at the mRNA level.
- interfering RNA or “dsRNA” refers to an RNA molecule that is capable of degrading specific mRNAs with mRNAs of homologous complementary sequences, a process known as the RNA interference pathway.
- substantially complementary means that the sequences of the nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (e.g., a stem-loop structure).
- a secondary structure e.g., a stem-loop structure.
- at least 70% of the nucleotides of the two "substantially complementary" nucleotide sequences are complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%.
- mismatched nucleotides there may be up to 7 mismatched nucleotides between two sufficiently complementary molecules; preferably, up to 6 mismatched nucleotides; more preferably, up to 5 mismatched nucleosides Acid; further preferred, having up to 4 mismatched nucleotides, such as having 0, 1, 2, 3, 4 mismatched nucleotides.
- a "complementary" sequence generally refers to a sequence that converts a sequence in the 5'-3' direction to its 3'-5' direction (eg, 5'ATCG 3'->GCTA), and then takes its complementary sequence (such as GCTA-5'CGAT 3,).
- a “stem loop” structure also referred to as a “hairpin” structure, refers to a nucleic acid molecule that forms a secondary structure comprising a double-stranded region (stem), said double-stranded region Formed by two regions of the nucleic acid molecule (on the same molecule), the two regions are flanked by double-stranded portions; they also include at least one "loop" structure, including non-complementary nucleic acid molecules, ie, single-stranded regions. Even if the two regions of the nucleic acid molecule are not fully complementary, the double-stranded portion of the nucleic acid can remain in a double-stranded state.
- insertions, deletions, substitutions, etc. may result in non-complementation of a small region or the formation of a stem-loop structure or other form of secondary structure by itself, however, the two regions may still be substantially complementary and are foreseeable Interaction occurs in the manner, forming a double-stranded region of the stem-loop structure.
- Stem loop structures are well known to those skilled in the art, and typically after obtaining a nucleic acid having a nucleotide sequence of a primary structure, one skilled in the art will be able to determine whether the nucleic acid is capable of forming a stem-loop structure.
- nucleic acid inhibitor refers to a class of substances having the activity of controlling lepidopteran insects obtained by preparing a target gene or a fragment thereof or a truncated form useful for controlling lepidopteran insects according to the present invention.
- the "nucleic acid inhibitor” is, for example, some interfering molecule, including dsRNA (also known as double-stranded RNA, double-stranded ribonucleic acid or double-stranded ribonucleotide sequence), antisense nucleic acid, small interfering RNA, microRNA, etc. , or can A construct that expresses or forms the dsRNA, antisense nucleic acid, small interfering RNA, or microRNA.
- operably linked refers to a spatial arrangement of the functionality of two or more nucleic acid regions or nucleic acid sequences.
- the promoter region is placed at a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is introduced by the promoter region such that the promoter region is “operably linked” to the nucleic acid sequence.
- an RNA sequence corresponding to a polynucleotide (or DNA) sequence refers to an RNA sequence which is "AU” if the DNA sequence is "AT”.
- containing includes “including”, “consisting essentially of”, “consisting essentially of”, and “consisting of”; “mainly composed of", “consisting essentially of” and “consisting of” belonging to “contains” , the underlying concept of "with” or “including”. Storage of chalk and its coding gene
- a target gene of the present invention which is a storage white gene.
- Storage ⁇ white is one of the hexamer ⁇ white families.
- the sp-related genes in insects generally have cylindrical structures, consisting of 7-8 polypeptide chains of about 50KD; there is a copper binding site. According to the amino acid structure, it can be divided into storage proteins rich in proline (> 4% methionine) and storage whites rich in aromatic amino acids (>15% aromatic amino), and spions rich in aromatic amino acids.
- proline-rich sp does not contain glycosylation sites, and sequence identity of different species is between 40% and 90% (40°/. sequence identity is considered to be higher homology).
- insects such as the corn borer OfSP gene, H. arm SP, Plutella xylostella PxSP1, PxSP2
- insect storage protein genes from insects are key genes involved in insect development or growth, and the expression of these genes is down-regulated or Inhibition can cause problems with insect growth (eg, abnormal phlegm or eggs that cannot be hatched) or death.
- genes can be prepared to inhibit the growth and development of corn borer or cotton bollworm or diamondback moth.
- the above genes may also be derived from other organisms whose functions have been established in the literature, their nucleotide sequences are highly homologous to the corn borer gene or their gene functions are similar to those of the corn borer gene, and these heights are foreseen.
- a homologous gene or a functionally similar gene can be used as a target to prepare specific interfering molecules and inhibit the growth of other organisms.
- highly homologous or “highly homologous” as used herein, when referring to a nucleic acid sequence, means that under stringent conditions SEQ ID NO: 1, SEQ ID NO: 16 > SEQ ID NO: 18-19 A nucleotide sequence that hybridizes to any sequence or its complement.
- such highly homologous sequences are exemplified by a control nucleotide sequence set forth in any of SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 18-19, or a complement thereof
- identity refers to the relationship at the nucleotide level between sequences.
- a "percent identity” is determined by comparing the optimally aligned sequences (eg, two or more) in a comparison window, wherein the sequence portion of the comparison window is aligned with the reference sequence of the optimal sequence Ratios can include insertions or deletions. The reference sequence does not contain an insertion or deletion.
- the reference window is selected from at least 10 contiguous nucleotides to about 50, about 100 or to about 150 nucleotides, preferably about 50 to 150 nucleotides.
- the "percent identity” is then calculated by determining the number of nucleotides that are consistent between the sequences in the window and dividing the number by the number of nucleotides in the window and multiplying by 100.
- the present inventors further studied target gene fragments which can effectively down-regulate these target genes, and finally found six target gene fragments whose nucleotide sequences are selected from SEQ ID NOs: 3-5, SEQ ID NO. : 17. The sequence shown in any one of SEQ ID NOS: 20-21.
- Fragments or truncated forms of the target gene are also included in the present invention, as long as the fragments or truncated forms are used to prepare the nucleic acid inhibitor, the nucleic acid inhibitor also has a killing scale. The activity of the winged insect.
- the inventors have unexpectedly discovered that these target gene fragments or their truncated bodies play an important role in insects, and that their inhibition, interference or silencing will result in a significant decrease in the survival rate of insects or direct death. Therefore, various nucleic acid inhibitors can be designed based on these target gene fragments for use in pest control.
- the invention also provides a polynucleotide set (set) comprising the sequences set forth in SEQ ID NO: 3-5, SEQ ID NO: 17, SEQ ID NO: 20-21.
- a nucleic acid inhibitor e.g., construct or dsRNA
- 1-6 polynucleotides can be obtained from the polynucleotide set.
- a nucleic acid inhibitor or a host expressing a nucleic acid inhibitor can be separately prepared based on a sequence of a plurality of polynucleotides (preferably, for example, 6), which can achieve a broader spectrum and more when applied simultaneously (or mixedly). Effectively kills lepidopteran insects.
- Nucleic acid inhibitor e.g., construct or dsRNA
- the nucleic acid inhibitor is a construct of some interfering molecule, eg, dsRNA, antisense nucleic acid, small interfering RNA or microRNA, or may express or form the dsRNA, antisense nucleic acid, small interfering RNA or micro RNA construct. More preferably, it is a dsRNA or a construct that can express the dsRNA.
- a construct for expressing a dsRNA, an antisense nucleic acid, a small interfering RNA or a microRNA can be designed. Accordingly, the present invention provides an artificially constructed construct. Designing the constructs according to the genes provided by the present invention and their sequences is well known to those skilled in the art, and the construction can usually be made
- the product contains an intron sequence (not complementary to the flanking sequences), and the complementary gene sequences are ligated at both ends, and after entering the cell, a "stem loop" structure can be produced, and the "stem” portion can form dsRNA, antisense nucleic acid.
- small interfering RNA or microRNA this dsRNA, antisense nucleic acid, small interfering RNA or microRNA can be particularly effective in inhibiting the expression of the gene of interest.
- the construct contains at least one structure as shown below -
- nucleotide sequence of Seq ⁇ ⁇ 5 is selected from the sequence or sequence fragment shown in any one of claims 2 or 3, and the Seq ⁇ is a nucleotide sequence substantially complementary to Seq ⁇ ;
- X is a spacer sequence located between Seq ⁇ and Seq ⁇ , and the spacer sequence is not complementary to Seq ⁇ and Seq s ;
- the construct is located on an expression vector.
- the present invention also encompasses a vector comprising the construct.
- the expression vector typically also contains a promoter operably linked to the construct, an origin of replication and/or a marker gene, and the like. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
- the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance .
- Vectors comprising the appropriate gene sequences described above, as well as appropriate promoters or control sequences, can be used to transform a suitable host.
- the host may be any host suitable for carrying the expression vector and capable of expressing a nucleic acid inhibitor.
- the host is Escherichia coli, fungus, yeast, plant cells, animal cells, and the like.
- Transformation of the host with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art, depending on the type of plant.
- competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
- Another method is to use MgCl 2 , if desired, the conversion can also be carried out by electroporation. Transformation of fungal and yeast cells, plant cells, animal cells is also well known to those skilled in the art.
- a host carrying the construct or expression vector and capable of expressing a nucleic acid inhibitor can be directly applied to a subject (such as a plant) to be controlled to achieve the purpose of controlling lepidopteran insects.
- the "stem"-like portion of the stem-loop structure described above is formed by the interaction of Seq ⁇ ⁇ and Seq ⁇ and can be processed to form a nucleic acid inhibitor.
- the formed nucleic acid inhibitor has the following structure: Seq'iH to one - -,,,
- Seq' is selected from the RNA sequence or sequence fragment corresponding to the sequence of any of claims 2 or 3; Seq' s is a sequence substantially complementary to Seq'.
- X' is a spacer sequence located between Seq ⁇ and Seq, ⁇ , which is not complementary to Seq' ⁇ and Seq'sfi .
- the ⁇ ' sequence can be excised in vitro or without excision. After the dsRNA enters the insect body, it is processed and excised by an enzyme (such as nuclease Dicer) in the insect.
- the nucleic acid inhibitor can be directly applied to a subject (e.g., a plant) to be controlled to achieve the purpose of controlling lepidopteran insects.
- the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
- the polypeptides of the invention may be naturally purified products, either chemically synthesized or produced recombinantly from prokaryotic or eukaryotic hosts (e.g., plants, bacteria, yeast, insect cells).
- the polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.
- the invention also encompasses fragments, derivatives and analogs of the storage protein 2.
- fragment refers to a polypeptide that substantially retains the same biological function or activity of the storage buffer of the present invention.
- the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (Hi) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example a polypeptide formed by fusion of a polyethylene glycol) or (iv) an additional amino acid sequence fused to the polypeptide sequence (eg, a pre-sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or fusion) protein).
- conservative or non-conservative amino acid residues preferably conservative amino acid residues
- substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptid
- the term "storage protein 2" refers to a polypeptide of the sequence of SEQ ID NO: 2 or a variant thereof. These variations include (but are not limited to): several (usually 1 - 50, preferably 1 - 30, more preferably 1 - 20, optimally 1-10, still more preferably 1 -8 or 1-5) amino acid deletions, insertions and/or substitutions, and addition or deletion of one or several at the C-terminus and/or N-terminus (usually within 20, preferably within 10, More preferably, it is 5 or less amino acids.
- the function of the white matter is generally not altered.
- the addition or subtraction of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the white matter.
- the term also encompasses active fragments and active derivatives of storage protein 2.
- Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, mutagenized mutants, DNA encoded by DNA that hybridizes to storage protein 2 DNA under high or low stringency conditions. White and so on.
- the invention also provides other polypeptides, such as fusion albumin comprising storage chalk 2 or a fragment thereof.
- the present invention also encompasses soluble fragments of the storage protein 2.
- the fragment has at least about 20 contiguous amino acids of the storage protein 2 sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, optimally at least about 100 consecutive amino acids.
- the invention also provides an analog of the storage protein 2 or polypeptide.
- the difference between these analogues and natural storage of chalk 2 can be The difference in amino acid sequence may also be a difference in the modification form that does not affect the sequence, or both.
- These polypeptides include natural or induced genetic variants. Mutant variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
- Modifications include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to enhance their resistance to chalky hydrolysis or to optimize solubility properties.
- "storage 22 2 conservative variant polypeptide” means having up to 20, preferably up to 10, more preferably up to 5, optimally compared to the amino acid sequence of SEQ ID NO: 2. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.
- the survival rate of the insect can be significantly reduced, and it can be used for screening or preparing the target of the insecticidal preparation. Therefore, based on the target cockroach, the design can be designed.
- insecticidal agents such as protein inhibitors, can act to kill insects after being applied to a subject.
- the target albumin or a fragment thereof or a variant thereof is also included in the present invention, and as long as the fragment or variant is used to prepare the chalk inhibitor, the chalk inhibitor has a kill scale The activity of the winged insect.
- the present invention also provides polynucleotides encoding the storage sputum 2 of the present invention or a conservative variant polypeptide thereof.
- the polynucleotide of the present invention may be in the form of DNA or RNA.
- DNA forms include C DNA, genome
- DNA or synthetic DNA can be single-stranded or double-stranded.
- the DNA can be a coding strand or a non-coding strand.
- the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
- degenerate variant in the present invention refers to a nucleic acid sequence which encodes a purine having the sequence of SEQ ID NO.-2 but differs from the coding region of SEQ ID NO: 1.
- Polynucleotides encoding the mature polypeptide of SEQ ID NO: 2 include: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and Non-coding sequence.
- the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
- Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
- an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
- Shorter fragments derived from the target genes of the present invention are also included in the present invention as a basis for designing nucleic acid inhibitors. It is well understood by those skilled in the art that some shorter sequences can also be used as nucleic acid inhibitors, for example, some siRNA sequences are often only 18 bp.
- the invention further relates to hybridization to the sequences described above and having at least 15%; preferably at least 18%, more preferably at least 25°/ between the two sequences. More preferably at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical Sex polynucleotide.
- the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent (stringent) conditions.
- stringent (strict) conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 °C; or (2) Addition of a denaturing agent, such as 50% 0 ') formamide, 0.1% calf serum / 0.1%. Ficoll, 42 °C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 80%, preferably at least 90%, more preferably 95% or more.
- the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 2.
- nucleic acid fragments that hybridize to the sequences described above.
- a "nucleic acid fragment” is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
- Nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to identify and/or isolate polynucleotides encoding storage ⁇ 2.
- the full-length nucleotide sequence encoding the sputum 2 of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
- primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
- the library is used as a template to amplify the relevant sequences. When the sequence is long, it often needs to be done Two or more PCR amplifications are performed, and then the amplified fragments are spliced together in the correct order.
- synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
- a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
- DN A sequence encoding the ruthenium (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis.
- the DN A sequence can then be introduced into various existing DN A molecules (e.g., vectors) and cells known in the art.
- mutations can also be introduced into the chalk sequence of the present invention by chemical synthesis.
- the above-described polynucleotide encoding the sputum white 2 of the present invention can be used as a target gene for preparing an insecticidal preparation.
- the present inventors have found that the target gene or fragment thereof of the present invention plays an important role in insects, and that inhibition, interference or silencing thereof causes a significant decrease in the survival rate of insects or direct death. Therefore, based on the target gene, a plurality of insecticidal preparations can be designed, for example, a nucleic acid inhibitor can be designed, which can be used to kill insects after being applied to a subject.
- the target gene or fragment thereof or truncated form or gene variant or fragment variant is also included in the invention, as long as the fragment or truncated form or variant is used to prepare the nucleic acid inhibitor,
- the nucleic acid inhibitor also has the activity of killing lepidopteran insects.
- the invention also relates to a vector comprising a polynucleotide of the invention, or a fragment or variant thereof, and a host cell genetically engineered using the vector of the invention or the storage sputum 2 coding sequence, and recombinantly produced to produce the invention A method of polypeptide.
- polynucleotide sequence of the present invention can be utilized to express or produce recombinant storage protein 2 by conventional recombinant DNA techniques. Generally there are the following steps:
- the storage protein 2 polynucleotide sequence can be inserted into a recombinant expression vector.
- recombinant expression vector refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art.
- any plasmid and vector can be used as long as it can replicate and stabilize in the host.
- An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
- Expression vector for translation control signals include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like.
- the DNA sequence can be operably linked to an appropriate promoter in an expression vector to refer to mRNA synthesis.
- the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
- the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent ⁇ White (GFP), or kanamycin or ampicillin resistance for E. coli.
- selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent ⁇ White (GFP), or kanamycin or ampicillin resistance for E. coli.
- Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
- the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
- a prokaryotic cell such as a bacterial cell
- a lower eukaryotic cell such as a yeast cell
- a higher eukaryotic cell such as a plant cell.
- Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.
- an enhancer sequence is inserted into the vector.
- An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, used as a promoter to enhance transcription of a gene.
- Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
- the host is a prokaryote such as E. coli
- competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
- Another method is to use MgCl 2 . Conversion can also be carried out by electroporation if desired.
- the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
- the transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, rice immature embryo transformation method and the like.
- Agrobacterium transformation or gene gun transformation such as leaf disc method, rice immature embryo transformation method and the like.
- plants can be regenerated using conventional methods.
- Insecticide Insecticide
- Any substance which is prepared based on the target protein or its fragment or truncated form provided by the present invention and which has activity against lepidopteran can be used as an insecticidal preparation for controlling lepidopteran insects.
- Such preparations are, for example, protein inhibitors, proteolytic enzymes, protein binding molecules, antibodies, ligands. Any substance which inhibits the activity or expression of the target protein is included in the present invention.
- the nucleic acid inhibitor is preferably a construct of some interfering molecule, for example, dsRNA, antisense nucleic acid, small interfering RNA or microRNA, or can express or form the dsRNA, antisense nucleic acid, small interfering RNA or micro RNA construct. More preferably, it is a dsRNA or a construct that can express the dsRNA.
- constructs for expressing dsRNA, antisense nucleic acid, small interfering RNA or microRNA can be designed. Accordingly, the present invention provides an artificially constructed construct. Designing the constructs according to the genes provided by the present invention and their sequences is well known to those skilled in the art, and the constructs can generally be made to comprise an intron sequence (not complementary to the flanking sequences;) The complementary gene sequence, after entering the cell, produces a "stem loop" structure, and the "stem” portion can form dsRNA, antisense nucleic acid, small interfering RNA or microRNA, such dsRNA, antisense nucleic acid, small interfering RNA Or microRNA can be particularly effective in inhibiting the expression of a gene of interest.
- the nucleic acid inhibitor has the following structure:
- Seq' is an RNA sequence corresponding to the polynucleotide sequence encoding the storage protein 2 or a fragment thereof; Seq, i5 is a sequence substantially complementary to Seq, ⁇ .
- the nucleic acid inhibitor can be directly applied to a subject (such as a plant) to be controlled to achieve the purpose of controlling lepidopteran insects.
- the Seq' is an RNA corresponding to the DNA selected from any one of SEQ ID NO: 3-5 SEQ ID NO: 17 SEQ ID NO: 20-21. More preferably, it is an RNA corresponding to the DNA selected from any one of SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 17, or SEQ ID NO: 20-21, the former (SEQ ID NO: 3) It can be used to develop a specific preparation for controlling Asian corn borer; the latter (SEQ ID NO: 5) is more suitable for the development of a broad-spectrum preparation for controlling lepidopteran pests.
- the construct contains at least one of the structures shown below:
- Seq ⁇ is a polynucleotide encoding the storage protein 2 or a fragment thereof, and Seq « is substantially complementary nucleotide sequence with Seq;
- X is a spacer sequence located between Seq ⁇ and Seq ⁇ , and the spacer sequence is not complementary to Seq ⁇ and Seq s ;
- the structure can be further cleaved and processed to form an interfering molecule (RNA molecule) in a double-stranded form, thereby exerting a gene silencing effect.
- RNA molecule interfering molecule
- the construct may be prepared in a form that forms more than one stem-loop structure, for example, may comprise two or more stem-loop structures.
- the construct is located on an expression vector.
- the present invention also encompasses a vector comprising the construct.
- the expression vector typically also contains a promoter operably linked to the construct, an origin of replication and/or a marker gene, and the like. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
- the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance .
- a host carrying the construct or expression vector and capable of expressing a nucleic acid inhibitor can be directly administered to a need Control objects (such as plants) to achieve the purpose of controlling lepidopteran insects.
- the nucleic acid inhibitor of the present invention can be obtained by a method of in vitro chemical synthesis in addition to cell expression using a construct. It will be understood that any method which can form the nucleic acid inhibitors of the present invention can be used in the present invention. Genetically modified plant
- the present invention also relates to a method of controlling a pest by a plant or a method of improving the insect resistance of a plant, which comprises introducing the nucleic acid inhibitor into a plant.
- the transgene of a plant is a technique well known to those skilled in the art, and for example, methods such as Agrobacterium transformation or gene gun transformation can be used, such as leaf disc method, immature embryo transformation method and the like.
- methods such as Agrobacterium transformation or gene gun transformation can be used, such as leaf disc method, immature embryo transformation method and the like.
- plants can be regenerated by conventional methods to obtain plants having insect resistance.
- the basic principle of the method for controlling pests by plants or improving the insect resistance of plants is: using plants as a medium to make insects interfere with interfering RNA which can interfere with insect storage of white gene expression, Thereby inhibiting the growth of insects or killing insects.
- interfering RNA refers to a substance having insect-control activity obtained by preparing or processing (such as being processed in vivo) based on the insect storage white gene or a fragment thereof (trunform form) provided as a target provided by the present invention.
- the "interfering RNA” includes, for example, dsR A, antisense nucleoside acid, small interfering RNA, micro RNA, and the like.
- a transgenic method is used to express a double-stranded RNA (dsR A) of an insect gene (full length or part) in a plant to form a high-abundance interfering RNA in a plant, when the insect feeds
- This transgenic plant also ingests a large amount of interfering RNA, which can inhibit the expression of the insect gene after entering the insect, and interfere with the normal growth and development of the insect and even cause its death.
- plants can be effectively improved in resistance to insects.
- the application of RNA interference technology to transgenic plants and the development of new transgenic resistant plants are of great significance for the development of agriculture.
- a method for improving the insect resistance of a plant comprises:
- step (1) (2) contacting a plant cell or tissue or organ with Agrobacterium in step (1) to transfer said nucleic acid inhibitor to a plant cell or tissue or organ;
- step (3) Regenerating the plant cell or tissue or organ in step (3).
- the method can be carried out using any suitable conventional means, including reagents, temperatures, pressure conditions, and the like.
- a medium such as Ying-Bo
- nucleic acid inhibitors interfering molecules
- Mao et al Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol (NATURE BIOTECHNOLOGY VOLUME 25
- composition and method for controlling lepidopteran insects The invention also provides a formulation (e.g., a pesticidal composition) comprising a safe and effective amount of a host (cell) or a nucleic acid inhibitor (eg, a nucleic acid inhibitor) that carries the construct or expression vector and is capable of expressing a nucleic acid inhibitor : 10- 50 (Vg / ml; preferably 20-20 (Vg / ml; more preferably 30- 100 ⁇ 8 / ⁇ 1); pesticide and a pharmaceutically acceptable carrier.
- a formulation e.g., a pesticidal composition
- a formulation e.g., a pesticidal composition
- a formulation e.g., a pesticidal composition
- a formulation e.g., a pesticidal composition
- a formulation e.g., a pesticidal composition
- a formulation e.g., a pesticidal composition
- a formulation e.g.
- the term "containing” means that the various ingredients can be used together in the mixture or composition of the present invention. Therefore, the terms “consisting mainly of” and “consisting of” are included in the term “contains”.
- the "pesticide-acceptable" component is suitable for agricultural use and does not cause excessive adverse side effects (such as toxicity, irritation, and allergic reaction) to humans or animals (other than lepidopteran insects). That is, a substance with a reasonable benefit/risk ratio.
- the "pesticide-acceptable carrier” is for transferring a host carrying the construct or expression vector of the present invention and capable of expressing a nucleic acid inhibitor or a nucleic acid inhibitor of the present invention to a lepidopteran insect Acceptable solvents, suspending agents or excipients.
- the pharmaceutically acceptable carrier can be a liquid or a solid, preferably a carrier which retains the host or nucleic acid inhibitor activity to a greater extent.
- the dosage form of the preparation may be various, including but not limited to: aqueous solution, suspending agent, wettable powder, emulsifiable concentrate, emulsion, sprayable solution, aqueous dispersion, powder, granule Agent, or microcapsules. It is to be understood that as long as the host of the present invention carrying the construct or expression vector and capable of expressing the nucleic acid inhibitor or the nucleic acid inhibitor of the present invention can be delivered to the lepidopteran in keeping all or part of the activity, It is all desirable. Those which are easy to deliver are preferred, for example, the pesticidal composition is a liquid spray, or a spray.
- the adjuvant is an auxiliary component which functions as an auxiliary regulating function, for example, it may be a surfactant, an adhesion aid or other type of auxiliary agent.
- the active ingredient in the concentrated pesticide composition ie, the host carrying the construct or the expression vector and capable of expressing the nucleic acid inhibitor or the nucleic acid inhibitor of the present invention
- the pesticide composition is diluted and used in the actual content of active ingredient is low, such as those containing a nucleic acid inhibitor 20-10 ( ⁇ g / m l, is administered in an amount of 50-1000 ⁇ 1 / 300-500 head, or under
- the nucleic acid inhibitor content of the host (cell) expression carrying the construct or the expression vector and capable of expressing the nucleic acid inhibitor, a certain amount of the host (cell) is administered with reference to the nucleic acid inhibitor application amount.
- Common components such as chemical agents, synergists, trace elements, stabilizers, binders, wetting agents, dispersants, emulsifiers, penetrants, oximes, solvents, fillers, etc.
- the pesticide composition of the present invention It may also contain other active insecticides or microbicides.
- suitable solid diluents include, but are not limited to, diatomaceous earth, corn hulls, tricalcium phosphate, cork powder, clays such as kaolin, bentonite or attapulgite, and water-soluble polymers.
- the solid compositions may also contain one or more compatible wetting agents, dispersing agents, emulsifying agents or pigments which may also act as diluents in the solid state.
- Such a solid composition may be in the form of a powder, granule or wettable powder.
- the powder is usually obtained by grinding, and granules, tablets or bricks are obtained by granulation or tableting.
- the liquid composition may be in the form of a solution, a suspension and an emulsion, or it may be coated in a natural or synthetic polymer. And may contain a wetting agent, a dispersing agent or an emulsifier.
- emulsions, suspensions or solutions can be prepared with aqueous, organic or water-organic diluents to prepare water soluble polymers (and mixtures of the above diluents).
- the diluent may contain, for example, the above-mentioned ionic or nonionic wetting agent, dispersing agent or emulsifier or a mixture thereof.
- Desirable formulation adjuvants for use in the compositions of the present invention are also known, for example: Watkins “Insecticide Powder Thinner and Carrier Handbook” 2nd Edition, Darland Books, Caldwell N. I; HvOlphen, "Clay Colloid Chemistry” 2nd ed., J. Wiley & Sons, NY, Marsden, “Solvent Guide” 2nd Edition, Interscience, N Y. 1950; McCutcheon's, “Detergents and Emulsifiers", MC Publ. Corp., Ridgewood NJ; Sisley and Wood, “Encyclopedia of Surfactants", Chem Publ. Co. Inc., NY I 964; Schonfelt, " GrenzflachenGermanin
- the wettable powder can be uniformly dispersed in water.
- the wettable powder may further contain a non-environmentally hazardous substance such as a wetting agent, a dispersing agent, and a diluent.
- the powder may be prepared by grinding the active material together with a solid material such as finely pulverized talc, kaolin, bentonite or the like, or a solid material such as diatomaceous earth.
- the granules may be prepared by spraying the active substance particles onto the inert substance particles, or applying the active substance solution to a carrier (for example, sand, kaolin or inert substance) through a binder (for example, polyvinyl alcohol, sodium polyacrylate, or mineral oil). Particle) surface.
- a suitable active substance can be prepared into granules as in the preparation of fertilizer granules.
- the transport of insect control dsRNA to the surface of the plant by spray application provides another means of protecting plants.
- bacteria engineered to produce and accumulate dsRNA can be fermented, and the fermented product is formulated as a spray product compatible with common agricultural applications.
- the formulation may include: suitable gums and wetting agents for efficient leaf coverage, and UV protectants for protecting dsRNA from UV damage.
- suitable gums and wetting agents for efficient leaf coverage and UV protectants for protecting dsRNA from UV damage.
- UV protectants for protecting dsRNA from UV damage.
- formulations for soil applications can include a granular formulation that acts as a bait for larvae of soil harmful insects (e.g., corn rootworm).
- the inventors have unexpectedly discovered that the nucleic acid inhibitor can be invaded from the insect body into the insect by infiltration. Therefore, insect control can be achieved by applying various nucleic acid inhibitors or compositions containing various nucleic acid inhibitors to insect body surfaces.
- the modes of application include, but are not limited to, spraying, coating, and dropping onto the surface of an insect.
- the nucleic acid inhibitor is selected from the group consisting of: dsRNA, antisense nucleic acid, small interfering RNA, microRNA; or a construct capable of expressing or forming the dsRNA, antisense nucleic acid, small interfering RNA, microRNA.
- the present invention also provides a method for controlling lepidopteran insects, the method comprising: inhibiting said host (cell) or said nucleic acid carrying said construct or expression vector capable of expressing a nucleic acid inhibitor
- the substance is applied to a subject to be controlled (such as a plant, in particular a plant infested by a lepidopteran insect; or the insect itself).
- the pesticidal composition can be formulated as a spray so that it can be directly sprayed for pest control.
- the dsRNA can be directly sprayed for pest control, and the purified dsRNA is added to the penetrant or the sputum to spray the field for pest control.
- the invention aims at the prevention and control of lepidopteran pests, and develops a target gene and an effective fragment (domain thereof) which can be used for field control of insects based on RNAi technology, and proves that the simple inhibition of the nucleic acid inhibitor can be suppressed by spraying.
- the effect of gene expression ultimately leads to the death of lepidopteran pests, thereby achieving the purpose of prevention and treatment, which is convenient, rapid, accurate and pollution-free.
- the present invention first revealed and confirmed a novel target gene which can be used as a target for controlling insects.
- the present invention discloses for the first time that different target domains of genes can be used to synthesize specific and broad-spectrum target genes for controlling lepidopteran pests, and it is also proved that dsRNAs obtained based on these gene domains can be directly applied. Pest control.
- the pest control method is convenient, fast, accurate, and can well solve the pest resistance and environmental pollution problems currently encountered in pest control.
- the present invention is based on RNAi technology for the purpose of insecticidal, which is a novel high-efficiency, fixed-point control, pollution-free technology; the obtained dsRNA can be directly applied to the field for pest control.
- RNA samples with concentration ⁇ 300 ⁇ 8 / ⁇ 1, total ⁇ 6 g, and OD260/280 of 1.8 ⁇ 2.2.
- the polyA-bearing mRNA was isolated using magnetic beads with oligo-dT, and then the first strand of cDNA was synthesized using a random 6-mer and Invitrogen's Superscript II reverse transcriptase kit.
- the target gene-specific primer (OfSPF: CATGGGGACACAGTTGAGCTTC (SEQ ID NO: 14); OfSPR: TGCTGTAAATCTAGCCTCAGTCA (SEQ ID NO: 15)) was used for amplification, and the obtained gene fragment was purified and ligated to (made by Takara) PMD-18
- the transformation into ToplO strain, blue-white spot screening, positive strain sequencing, and the sequencing result of the full length sequence of Asian corn borer storage white 2 are as follows (SEQ ID NO: 1;
- the inventors selected three domains of the storage protein 2 gene (SP-N, SP-M, SP-C, the sequence is shown in Table 2), and amplified.
- the primers required for the three domains are shown in Table 3.
- EYFP is an enhanced yellow fluorescent white gene as a foreign gene control.
- the DNA sequences of the three domains SP-N, SP-M, and SP-C of the purine white 2 gene were amplified by the primers described in Table 3, respectively, as templates for the synthesis of dsRNA.
- the target gene dsRNA was synthesized using the MEGAscript® T7 Kit (available from AMBION, product number AM 1334) to obtain dsRNA for bioassay.
- the brief principles and steps are as follows: First, the total RNA of the Asian corn borer larvae tissue is used as a template to obtain cDNA by reverse transcription, and then the domains of different OfSP genes are amplified by the dsRNA synthesis primers listed in Table 3, and the products are purified. Then, using the purified product as a template, in vitro transcription using the Kit kit, obtaining dsRNA, removing the DNA in the product, and purifying the desired dsRNA.
- the specific steps are as follows:
- the in vitro transcription reaction is completed.
- the enzymatic reaction system contains a small amount of template DNA, which needs to be digested by DNasel. Dnasel is added to the above reaction solution, mixed uniformly, and incubated for 37 min for 30 min.
- reaction was terminated by adding lOO L and 15 L ammonium acetate;
- dsSP-N The final dsRNAs were named as: dsSP-N, dsSP-M dsSP-C, and the results of electrophoresis identification are shown in Figure 4. It can be seen that the desired dsRNA was successfully obtained.
- Example 3 Silencing effect of dsRNA on the genes of Asian corn borer
- the present inventors directly sprayed dsSP-C (dissolved in ddH 2 0) on the first instar larva of Asian corn borer, and observed changes in gene expression.
- the gene expression on days 0, 1, 3, and 5 after storage of the sputum white 2 gene (O yP) is shown in Fig. 5.
- the inventors directly obtained the dsRNA (dsSP-N, dsSP-M, dsSP-C dissolved in dd 0) directly sprayed on the Asian corn borer.
- the lethal concentration of LC 5 on the 7th day of dsSP-N is the standard concentration, the concentration is 41.55 g/mL
- the concentration is 41.55 g/mL
- the inventors directly sprayed the obtained dsRNA (dsSP-N dsSP-M, dsSP-C dissolved in dd3 ⁇ 40) onto the newly hatched larva of Asian corn borer.
- the lethal medium concentration LC 5 o of dsSP-N on day 7 was used as the standard concentration, the concentration was 41.55 ⁇ g/mL), and the lethality of the larvae on day 1, 3, 5, and 7 after spraying was observed.
- dsSP-N, dsSP-M and dsSP-C synthesized from the three domains of the target gene of the present invention were sprayed on Asian corn borer, respectively. After that, their lethal rates on day 7 were 74.82%, 73.13%, and 76.69%, respectively, while the blank control (CK) and the exogenous gene control (EYFP) had no significant lethal effect.
- Example 7 The lethal rate of cotton bollworm by the dsRNA of SP gene of Helicoverpa armigera
- the method described in the present invention is applied to the study of cotton bollworm.
- the nucleotide sequence of the gene PxSP1 homologous to the corn borer sp gene in Helicoverpa armigera is shown in SEQ ID NO.
- the present inventors prepared a dsRNA of a gene homologous to the corn borer sp gene (H.arm-SP) in the cotton bollworm, and the gene fragment homologous to the corn borer sp gene is as follows (SEQ ID NO: 17):
- the synthesis of the target gene dsRNA was carried out by referring to the method provided in Example 2 using MEGAscript® T7 Kit (available from AMBION, product number AM1334).
- the primer sequences are as follows:
- the present inventors directly obtained a dsRNA (dsH.arm-SP, dissolved in dd3 ⁇ 40) of a gene homologous to the corn borer sp gene (d.H.arm-SP) in the cotton-instar worm, directly sprayed on the newly hatched larva of the cotton bollworm (
- the lethal concentration LC 5 of Asian corn borer (Of) dsSP-N on day 7 was used as the standard concentration, the concentration was 41.55 wg/mL), and the first, third, fifth, and seventh days (d) larvae were observed after spraying. The fatal situation.
- Example 8 The lethal rate of dsRNA of SP gene of Plutella xylostella to P. xylostella Using the method of the present invention, it is applied to the research of Plutella xylostella.
- the nucleotide sequence of the gene PxSP l homologous to the corn borer sp gene in Plutella xylostella is shown in SEQ ID NO: 18; the nucleotide sequence of the gene PxSP2 homologous to the corn borer sp gene in Plutella xylostella is shown in FIG.
- TGTACCTCAACCGCGACG A CAAACTCAG( GTA C' TTCACTGAAGACATCGACCTGAACA CC
- Example 2 The method of Example 2 with reference to embodiments provided by MEGAscript® T7 Kit (available from AMBION company, producing f Num AMI 334) was synthesized dsRNA of target genes.
- the primer sequences are as follows:
- Px SP1 DSF TAATACGACTCACTATAGGGAGAACGTCGACCGCCAGATGAAGGA (SEQ ID NO: 32) Px SP1 DSR TMTACGACTCACTATAGGGAGATTGTGCAGGGTCATCCAGGGCA (SEQ ID NO: 33) Px SP2 DSF TAATACGACTCACTATAGGGAGAACGTCGACCGCCAGATGAAGGA (SEQ ID NO: 34) Px SP2 DSR TAATACGACTCACTATAGGGAGAACACCGTTGTGCAGCGCCAT (SEQ ID NO. 35)
- the present inventors directly obtained the dsRNAs of P. xylostella SP 1 and SP2 (dsPxSP1 and dsPxSP2 dissolved in ddH 2 0, respectively) directly onto the newly hatched larvae of Plutella xylostella (the lethal concentration in the 7th day of Asian corn borer dsSP-N)
- the LC50 was the standard concentration at a concentration of 41.55 g/mL), and the lethality of the larvae on days 1, 3, and 5 after the spraying was observed.
- dsRNAs synthesized using the double-stranded RNA (PxSP l and PxSP2) of the two target genes of Plutella xylostella of the present invention were sprayed on the newly hatched larvae of Plutella xylostella, respectively, on the fifth day.
- the lethal rates were 58.00% and 54.67%, respectively, and the mortality rate of Plutella xylostella was tested by dsSP of corn borer.
- the 5-day mortality rate was 14.67%. There was no significant difference from the control.
- Example 9 application of dsRNA
- the Asian corn borer gene has the most homologous relationship with the cotton bollworm, but the Asian corn borer SP-C is utilized.
- the dsRNA can achieve the same insecticidal effect in cotton bollworm, and it is expected that it will have a similar effect on other lepidopteran pests.
- Example 10 SP-N, SP-M, SP-C truncated form and its effect
- the present inventors designed a truncated form of dsRNA fragment based on SP-N (SEQ ID NO: 3), SP-M (SEQ ID NO: 4) and SP-C (SEQ ID NO: 5). as follows:
- SP-N-M1 dsRNA The synthesis and purification method is as in Example 2, except that the target gene fragment is position 1-420 of SEQ ID NO: 3.
- SP-N-M2 dsRNA The synthesis and purification method was as in Example 2, except that the target gene fragment was at positions 10-420 of SEQ ID NO: 3.
- the SP-M-M1 dsRNA synthesis and purification method was as in Example 2 except that the target gene fragment was position 1-615 of SEQ ID NO: 4.
- the SP-M-M2 dsRNA synthesis and purification method was as in Example 2 except that the target gene fragment was at positions 10-615 of SEQ ID NO: 4.
- SP-C-M1 dsRNA synthesis and purification method as in Example 2, except that the target gene fragment is SEQ ID NO:
- the SP-C M2 dsRNA; synthesis and purification method was as in Example 2, except that the target gene fragment was at position 10-603 in SEQ ID NO: 5.
- Example 11 Construction and Transformation of Transgenic Vectors
- the desired dsRNA vector as shown by pART27-dsRNA in ge 13, includes a 35S promoter, a reverse gene fragment (ie, Anti-sense), an intron of the PDK (pyruvate phosphokinase) gene (ie, Intron, approximately 742 bp), a forward gene fragment (ie Sense C1) and an OCS terminator, as well as the NOS promoter, ⁇ ⁇ (neomycin phosphotransferase gene) and NOS terminator.
- a reverse gene fragment ie, Anti-sense
- an intron of the PDK (pyruvate phosphokinase) gene ie, Intron, approximately 742 bp
- a forward gene fragment ie Sense C1
- OCS terminator as well as the NOS promoter, ⁇ ⁇ (neomycin phosphotransferase gene) and NOS terminator.
- the expression vector was digested with Not I by a binary vector pKANNIBAL vector (purchased from the Chinese plasmid vector strain cell strain gene collection) containing the Sense-Intron-Antisense sequence, and then ligated into the pART27 vector which was also digested with Not I. From Chinese plasmid The vector strain cell line gene collection center was constructed and obtained.
- the gene-specific primers dsGFP-2F-Kpn I and dsGFP_2R_Xho I; ds0fSP_C2F_Kpn I and ds0fSP_C2R-Xho I containing Kpn I and Xho I cleavage sites were treated with high-fidelity enzyme KOD, respectively, with GFP and Asian corn borer.
- cDN A was used as a template for PCR amplification, and the corresponding dsGFP-2 and dsOf SP-C2 fragments were cloned.
- the cloned fragment was double digested with Kpn I and Xho I and inserted into the corresponding cloning site (Kpn I /Xho I ) on the pKANNIBAL vector which already contained the forward dsGFP-1 and dsOfSP-C 1 fragments. between.
- the constructed pKANNIBAL/dsGFP and pKANNIBAL/dsOfSP-C vectors were digested with Not I, respectively, and pART27 was digested with Not l, and the digested dsGFP and dsOfSP_C fragments were inserted into pART27 vector, respectively.
- Recombinant expression vectors carrying the corresponding fragments of interest, respectively, are referred to as 35 S : dsGFP and 35S : : dsOfSP-C expression vectors, respectively.
- Agrobacterium tumefaciens The transformation of Agrobacterium tumefaciens is carried out by freeze-thaw method.
- a single colony GV3101, LBA4404 or EHA105 (all purchased from Invitrogen), 3ml LB medium (containing 50 ⁇ ⁇ / ⁇ 1 Kan kanamycin and 25 g / ml rifamycin Rif), 28. C, 220 rpm, culture to OD600 0.5 (about 6 hours). Place on ice for 30 minutes, centrifuge at 5000 g for 5 minutes at 4 °C. Resuspend in 1 mL of 0.15 M NaCl. 4. C, 5000 g was centrifuged for 5 minutes.
- transgene of Arabidopsis thaliana, tobacco, and maize is taken as an example, and the transformation of other plants can also be referred to herein.
- the flower bud part of the plant is soaked in the bacterial liquid for 5 seconds, placed in a plastic pot, moisturized, protected from light for 16 to 24 hours, and then grown in the greenhouse to flowering and seeding.
- the TO generation seeds were treated with spring water for 4 days at 4 °C, treated with 20% bleach for 15 minutes, and washed with sterile water for 3 ⁇ times.
- Agrobacterium LBA4404 containing the gene of interest was cultured overnight (cultured overnight at 28 ° C to OD 600 2.0).
- the sterile tobacco leaves are cut to a size of about 1.0 cm 2 and soaked in Agrobacterium culture medium for 5 to 10 minutes.
- the excess Agrobacterium culture medium was aspirated with sterile filter paper, and the soaked tobacco leaves were spread on 1/2 MS steroid medium and cultured in the dark for two days. Then, the leaves were moved to MS (containing 1 mg/L 6-BA)
- the newly grown small shoots were transferred to MS medium without 6-BA, and fresh MS was replaced every 10 to 15 days until it was rooted and moved to the soil for planting.
- Rif's YEP liquid medium was incubated at 28 °C to OD600 0.6, and centrifuged at 8000 rpm for 5 minutes to collect Agrobacterium.
- the bacteria were suspended in the infested medium and acetosyringone (AS) 100 ⁇ /L and 0.05% surfactant Silwet L-77 were resuspended in the suspension before Agrobacterium transformation.
- AS acetosyringone
- Silwet L-77 surfactant Silwet L-77 were resuspended in the suspension before Agrobacterium transformation.
- Pick the neat and full corn seeds soak them in 70% ethanol for 2 ⁇ 5 minutes, then soak them in 0.1% HgC12 for 10 ⁇ 5 minutes, then rinse them with sterile water for 3 ⁇ 5 times.
- the T3 homozygous transgenic plants obtained in Example 12 were selected and screened by antibiotics.
- RNA sample of the transgenic plants was extracted by a conventional method, and lOx electrophoresis sample solution was added to the RNA sample, mixed, placed at 65 ° C for 10 minutes, and cooled on ice. Electrophoresis was performed using 15% TBE-urea PAGE gel, ⁇ ⁇ running buffer. Pre-electrophoresis for 5 to 10 minutes before loading and rinse the lane with running buffer to remove the urea precipitated from the well. The amount of each lane was 10-20 total RNA, and the electric field strength was 20 V/cm, which ended when the bromophenol blue dye migrated to the bottom of the gel. The gel was equilibrated in l xTBE for 10 minutes.
- Hofer Semi-Dry Transfer Units Amersham, Cat. 80-621 1-86. Transfer conditions: 40 mA (about 7-8 V), 2 to 4 hours, Hybond-N+ (Amersham, Cat. RPN303B) nylon membrane.
- Electrophoresis gel storage solution 15% polyacrylamide (30% Acyl/bis, 19:1, Huajing, Cat. W443), 8M urea, ⁇ ⁇ ;
- Probe labeling 25 ng of purified PCR product was used as a template labeling probe. The probe was labeled using Prime-a-Gene Labeling System (Promega, Cat. U1 100). 37 ° C water bath for 1 hour. The labeled probe was placed in boiling water for 5 minutes and immediately placed on ice for later use.
- Pre-hybridization and hybridization were used (Clontech's ExpressHyb system): The nylon membrane was placed in a hybridization tube and wetted with 6xSSC to ensure no air bubbles between the membrane and the tube wall. Pour 6x SSC, add 5 mL of hybridization solution, and pre-hybridize at 37 °C for 60 minutes. After the pre-hybridization is completed, replace 5 mL of fresh hybridization solution, add the probe, mix, and hybridize overnight.
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Abstract
本发明涉及基于RNAi技术防治害虫的新方法。揭示了一个对于防治鳞翅目昆虫有用的靶标基因及其编码的蛋白,基于该靶标基因的核酸序列制备核酸抑制物或表达核酸抑制物的宿主,可有效地杀灭鳞翅目昆虫。本发明还提供了利用同一基因的不同片段,获得昆虫种类特异型和种间广谱型的杀虫制剂和方法。
Description
基于 RNAi技术防治害虫的新方法
技术领域
本发明属于生物技术和农药学领域; 更具体地, 本发明涉及基于 RNAi技术防治害虫 的新方法。 背景技术
亚洲玉米螺 {Ostrinia furnacalis Guen6e)禾口棉铃虫 (HeZ cove/pa armigera Hubner)是 重要世界性农业害虫, 亚洲玉米螟主要危害玉米、高粱、 向日葵等重要的粮食经济作物, 棉铃虫主要危害棉花和蔬菜等 20多科 200余种重要农作物。 小菜蛾常年危害面积占蔬 菜种植总面积的 10%左右 (2010蔬菜种植面积 3333万 hm2, 小菜蛾发生面积达 253万 hm2 ; 2009菜种植面积 3000万 hm2 ,小菜蛾发生面积达 270万 hm2 ),造成的损失在 20~30% 左右, 如果防治不及时或者措施不合理可能造成绝收。 目前对亚洲玉米螟、 棉铃虫和小 菜蛾的防治, 仍然是以化学防治为主, 但是其对生态环境及粮食安全生产造成了巨大的 损害。 化学防治诸多危害已经使得人们不得不寻求更好的害虫治理办法, 生物防治虽然 可以起到防治作用, 但是由于其见效期慢, 效果不明显, 受环境影响较大而得不到大众 认可。
RNAi现象自从 1991年发现以来迅速发展, 研究表明, 通过特异基因的 RNAi , 可 以达到定向干涉物种的靶基因, 出现某些生理现象, 达到研究基因功能的目的, 同时, 这种现象有着极高的特异性, 即同源基因对不同物种起的干涉效果并不明显, 因此是一 种理想的害虫防治体系。
目前, 利用 RNAi技术进行害虫防治已有报道, Baum等 (2007)证明 v-ATPase对玉 米根蛮叶甲 (Western corn rootworm WCR Diabrotica virgifera virgifera LeConte)有致死效 果, 可以用于田间防治; 同年, mao等通过将 P450的 dsRNA 入棉花中, 可以 致棉 铃虫 (cotton bollworm Helicoverpa ami gera)死亡; Tian(2009)证实通过词喂的方法可以 ^
ejc g«" (Hiibner))的死亡, 一系列的现有技术都表明, RNAi技术 作为一种新型的害虫防治方法是可行的, 但是, 之前的研究也表明, 基于 RNAi技术的 害虫防治还有很多问题需要解决, 如; 1)如何快速高通量的发掘靶标基因; 2)如何简便 的应用于田间生产; 3)抗性问题; 4)安全性问题等等。 因此, 将 RNAi技术应用于害虫 防治领域, 还需要解决上述问题。 首先需要获得足够多的、 有效的靶标基因, 这样, 大 量的靶标基因通过不同的组合方式, 不同的时期使用不同的基因进行防治, 可有效地避 免害虫抗性的出现。
因此, 利用 RNAi技术的害虫防治集中在靶标基因的获得以及如何方便、 有效地应 用于田间这两个急需解决的问题, 本发明旨在为解决这些问题提供途径。 此外, 本发明 还提供了利用同一基因的不同片段, 可以获得昆虫种类特异型和种间广谱型的杀虫制剂 和方法。
发明内容
本发明的目的在于提供一种基于 R Ai技术防治害虫的新方法。
在本发明的第一方面, 提供一种分离的多肽, 该多肽选自下组:
(a) SEQ ID NO: 2氨基酸序列的多肽;
(b) 将 SEQ ID NO: 2氨基酸序列经过一个或多个氨基酸残基的取代、 缺失或添加而 形成的, 且具有 (a)多肽功能的由 (a)衍生的多肽。
在另一优选例中, 该多肽是 SEQ ID NO: 2氨基酸序列的多肽。
在本发明的另一方面, 提供一种分离的多核苷酸, 它包含一核苷酸序列, 该核苷酸序 列选自下组:
(a) 编码所述多肽的多核苷酸;
(b) 与多核苷酸 (a)互补的多核苷酸。
在另一优选例中, 该多核苷酸选自下组的一种- (1) SEQ ID NO: 1所示序列的多核苷酸;
(2) SEQ ID NO: 1中第 41-2308位的序列的多核苷酸;
(3) 与 (1)或 (2)限定的序列在严格条件下杂交的且编码的蚩白与 (1)或 (2)的多核苷酸 编码的蚩白功能相同的多核苷酸;
(4) 与(1)或 (2)限定的序列在具有 80%以上 (较佳地 90%以上; 更佳地 95%; 更佳地 98%; 更佳地 99%) 的序列相同性且编码的蛋白与(1)或 (2)的多核苷酸编码的蛋白功能相 同的多核苷酸。 在本发明的另一方面, 提供一种载体, 它含有所述的多核苷酸。
在本发明的另一方面, 提供一种遗传工程化的宿主细胞 (较佳地, 为非生殖性或繁殖 性的细胞), 含有所述的载体, 或其基因组中整合有所述的多核苷酸。
在本发明的另一方面,提供一种多核苷酸片段,所述多核苷酸片段的序列与前面所述 的多核苷酸序列具有 10%以上 (较佳地 15%以上;更佳地 20%;更佳地 25。/。;更佳地 30%) 的序列相同性。
在另一优选例中, 该多核苷酸片段选自下组的一种:
(i) SEQ ID NO: 1、 SEQ ID NO: 3-5、 SEQ ID NO: 17, SEQ ID NO: 20-21任一所示 序列的多核苷酸;
(ii) 与(i)限定的序列在严格条件下杂交的任一多核苷酸或互补序列, 其中包含所 述的任一条核苷酸的 dsRNA被有害生物摄取后, 会抑制所述有害生物的生长;
(iii) 与 (i ) 限定的序列具有至少 70% (优选至少 75%、 80%、 85。/。、 90%, 更优选至 少 95%、 96%, 97% 98%、 99%) 以上的序列一致性的任一多核苷酸或互补序列, 其中 包含所述的任一条核苷酸的 dsRNA被有害生物摄取后, 会抑制所述有害生物的生长;
(iv) 包含 (i)限定的任一所示序列之至少 17-21个连续核苷酸的序列或互补序列, 其 中包含所述的任一条核苷酸的 dsRNA被有害生物摄取后, 会抑制所述有害生物的生长; (V) 与 (i)或 (ii)或 (Hi)或(iv)限定的序列在严格条件下杂交的且编码的蛋白结构域与 (i)或 (ii)或 (iii)的多核苷酸片段编码的蚩白结构域功能相同的多核苷酸片段;
(vi) 与 (i)或 (ii)或 (iii)或 (iv) 限定的序列在具有 80%以上 (较佳地 90%以上; 更佳地
95%; 更佳地 98%; 更佳地 99%) 的序列相同性且编码的蚩白结构域与 (i)或 (ii)或 (iii)的多 核苷酸片段编码的蚩白结构域功能相同的多核苷酸片段。
在本发明的另一方面, 提供一种载体, 它含有所述的多核苷酸片段中的一种、 二种或 多种。
在本发明的另一方面, 提供一种遗传工程化的宿主细胞, 它含有所述的载体, 或其基 因组中整合有所述的多核苷酸片段中的一种、 二种或多种。
在本发明的另一方面, 提供所述的多核苷酸或多核苷酸片段的用途, 用于作为制备 特异性干扰有害生物基因表达或抑制有害生物生长的干扰分子的抑制或沉默靶标, 所述 的有害生物基因选自贮藏蛋白蚩白基因; 或用于作为制备杀昆虫 (如鳞翅目昆虫)的制剂 的靶标。
在另一优选例中,所述的贮藏蚩白基因选自亚洲玉米螟贮藏蚩白基因或棉铃虫贮藏蚩 白基因或小菜蛾贮藏蚩白基因;
所述的鳞翅目昆虫选自亚洲玉米螟或棉铃虫或小菜蛾;
在本发明的 一方面, 提供所述的多肽的用途, 用于作为筛选或制备杀昆虫的制剂 的靶标。
在另一优选例中, 所述的制剂选自 (但不限于): 核酸抑制物, 蛋白抑制剂, 抗体, 配 体, 蚩白水解酶, 蛋白结合分子。
在另一优选例中, 所述的核酸抑制物选自:
dsRNA、 反义核酸、 小干扰 RNA、 微小 RNA; 或
能表达或形成所述 dsRNA、 反义核酸、 小干扰 RNA、 微小 RNA的构建物。
在本发明的另一方面, 提供一种核酸抑制物, 选自:
(1) 以所述的多核苷酸或多核苷酸片段, 或它们的转录本为抑制或沉默靶标的 dsRNA、 反义核酸、 小干扰 RNA、 微小 RNA; 其中所述的任一 dsRNA、 反义核酸、 小 干扰 RNA、 微小 RNA被有害生物摄取后, 会抑制所述有害生物的生长; 或
(2)以有害生物基因作为抑制或沉默靶标的 dsRNA、 反义核酸、 小干扰 RNA、 微小
RNA,其中所述的任一 dsRNA、反义核酸、小干扰 RNA、微小 RNA被有害生物摄取后, 会抑制所述有害生物的生长, 所述的有害生物基因选自贮藏蚩白基因; 或
(3) 能表达或形成 (1)、 (2 ) 所述 dsRNA、 反义核酸、 小干扰 RNA、 微小 RNA的构 建物。
在另一优选例中, 所述的核酸抑制物是所述的多核苷酸表达产生的 dsRNA, 其中所
述 dsRNA被有害生物摄取后, 会抑制所述有害生物的生长。
在另一优选例中, 所述的核酸抑制物是 dsRNA, 其具有如下结构:
Seq '正向」
¾ ',' X,
反向" — 或 Scq X - Scq
其中,
Seq'E ¾是与权利要求 2或 3任一所述的多核苷酸所对应的 RNA序列或它们的序列片 段, 或包含与选自权利要求 2或 3中所限定序列的核苷酸序列之至少一部分互补的核苷 酸序列所对应的 RNA序列;
Seq's i5为与 Seq, 基本上互补的序列;
X'为无;或为位于 Seq' ^和 Seq's 之间的间隔序列,所述间隔序列与 Seq' 和 Seq' ^不互补;
II 表示在 Seq' 和 Seq'^之间形成的氢键。
在另一优选例中, 所述的核酸抑制物是构建物, 所述构建物含有以下结构:
Seq -X-Seq
其中, Seq E ft是权利要求 2或 3任一所述的多核苷酸或它们的序列片段, 或包含与 选自权利要求 2或 3中所限定序列的核苷酸序列之至少一部分互补的核苷酸序列;
Seq S(¾为与 Seq «基本上互补的序列;
X为位于 Seq j ^和 Seq ^之间的间隔序列, 并且所述间隔序列与 Seq ia和 Seq 向不 互补。
在另一优选例中, 所述的构建物可形成以下结构-
Seq正向
' ' X
Seq反向
其中,
Seq Seq s向和 X的定义如上述,
II 表示在 Seq 和 Seq 之间形成的氢键。
在另一优选例中, 所述的构建物是表达载体 (质粒)。
在另一优选例中, 所述的多核苷酸或多核苷酸片段可作为抑制该多核苷酸的 mRNA 转录或抑制该多核苷酸编码的多肽的表达的靶标。
在另一优选例中, 所述的多核苷酸或多核苷酸片段可作为制备杀昆虫的制剂的靶标。 在本发明的另一方面, 提供一种宿主细胞 (如细菌、 真菌、 酵母、 植物细胞), 其包含 所述的核酸抑制物 (如所述的构建物)。
在本发明的另一方面, 提供所述的核酸抑制物或所述的宿主细胞的用途, 用于制备 防治昆虫的制剂。
在本发明的另一方面,提供一种防治昆虫的制剂 (如农药制剂或农药组合物),其包含: 安全有效量选自以下的物质: 所述的核酸抑制物 (如 10-50( g/mL)或所述的宿主细胞; 以
及; 农药学上可接受的载体。
在另一优选例中,其还包含至少一种选自以下的杀虫剂:化学杀虫剂、马铃薯糖蚩白、 苏云金芽孢杆菌杀虫蚩白、 致病杆菌杀虫蚩白、 光杆状菌杀虫蚩白、 侧孢芽孢杆菌杀虫 蚩白和球形芽孢杆菌杀虫蛋白。
在本发明的另一方面, 提供一种防治有害生物的方法, 干扰有害生物基因表达, 所述 的有害生物基因选自贮藏蚩白基因。
在另一优选例中, 所述方法包括: 将权利要求 7-10任一所述的核酸抑制物或权利要 求 5或 11所述的宿主细胞喂食和 /或喷洒有害生物。
在本发明的另一方面, 提供一种杀灭昆虫的方法, 所述方法包括: 降低所述昆虫中所 述的多肽的表达;或降低所述昆虫中所述的多核苷酸或多核苷酸片段相应的 mRNA的转 录。
在另一优选例中, 提供所述方法包括: 将所述的核酸抑制物或所述的宿主细胞施用 于 (如喷洒)需要防治的对象 (如植物, 特别是被昆虫侵害的植物; 或昆虫)。
在本发明的另一方面, 提供将所述的核酸抑制物或所述的宿主细胞喂食昆虫或喷洒 昆虫。
在另一优选例中, 所述方法包括: 在植物体内表达特异性干扰有害生物基因表达的 干扰分子; 所述的昆虫基因选自贮藏蛋白基因。
在本发明的另一方面, 提供一种制备抗昆虫的植物的方法, 所述方法包括: 将所述 的核酸抑制物 入到植物中。
在本发明的另一方面, 提供一种植物或其种子, 所述植物由所述的多核苷酸转化获 得。
在另一优选例中, 所述多核苷酸在植物细胞中表达为 dsRNA。
在另一优选例中, 所述有害生物是选自: 昆虫、 螨、 真菌、 酵母、 霉菌、 细菌、 线 虫、 杂草和寄生虫以及腐生植物。
在另一优选例中, 所述的有害生物是昆虫, 包括但不限于: 鳞翅目昆虫、 鞘翅目昆 虫、 半翅目昆虫、 以及双翅目昆虫。
在另一优选例中, 所述的昆虫有害生物选自鳞翅目昆虫, 优选为玉米螟或棉铃虫或 小菜蛾。
在本发明的另一方面, 提供一种制备抗昆虫的植物的方法, 所述方法包括: 将所述 的核酸抑制物 入到植物中。
在另一优选例中, 所述制备抗昆虫的植物的方法是:
(1) 提供携带表达载体的农杆菌, 所述的表达载体含有所述的核酸抑制物;
(2) 将植物细胞或组织或器官与步骤 (1)中的农杆菌接触, 从而使所述的核酸抑制物 转入植物细胞或组织或器官。
在另一优选例中, 所述制备抗昆虫的植物的方法还包括:
(3) 选择出转入了所述的核酸抑制物的植物细胞或组织或器官; 和
(4) 将步骤 (3)中的植物细胞或组织或器官再生成植物。
在另一优选例中, 昆虫食用所述的抗昆虫的植物后, 存活率显著下降或死亡。 本发明的其它方面由于本文的公开内容, 对本领域的技术人员而言是显而易见的。 附图说明
图 1、 不同结构域 dsRNA处理亚洲玉米螟后不同时间的致死效率。 其中, EYFP为 增强型黄色荧光蚩白基因, 作为外源基因对照。 CK为空白对照, 为正常人工饲养的亚 洲玉米螟。
图 2、 不同结构域 dsRNA处理棉铃虫后不同时间的致死效率。
图 3、亚洲玉米螟 ΟβΡ基因与其它鳞翅昆虫基因的同源比对结果。其中, Trichoplusia ni: 粉纹夜蛾; Hyalophora cecropia-. 亥1 J克罗普斯査蛾; Choristonetira f miferana-. 云杉 卷叶饿; Helicoverpa zea-.谷实夜蛾; Helicoverpa armigera-.棉铃虫; Sesa ia nonagrioides-. 粉 螟; Omphisa fuscidentalis-. 竹蠹螺; Hyphantria cunea-. 美国白蛾; Plodia interpunctella: 印度谷螟; Plutella xylostell -. 小菜蛾; Spodoptera litura.- 斜紋夜蛾;
Ostrinia furnacalis: 亚洲玉米螺。
图 4、 dsSP-N、 dsSP-M、 dsSP-C的电泳鉴定结果。 其中 M为分子量标记。
图 5、 dsSP-C施用于亚洲玉米螟 1龄幼虫后第 0、 1、 3、 5天 (d)的基因表达情况。 其中, CK为空白对照。
图 6、 不同结构域 dsRNA处理亚洲玉米螟后不同时间的致死效率。 其中, EYFP为 增强型黄色荧光蛋白基因, 作为外源基因对照。 CK为空白对照, 为正常人工饲养的亚 洲玉米螟。
图 7、 经 dsSP喷洒处理后的玉米螟幼虫不能正常化蛹。
图 8、 经 dsSP喷洒处理后的玉米螟成虫不能正常产卵。
M 9、 经 dsSP喷洒处理后的棉铃虫幼虫不能正常化蛹。
图 10、 经 dsSP喷洒处理后的棉铃虫成虫不能正常产卵。
图 1 1、 棉铃虫自己 SP基因的 dsRNA对棉铃虫的致死率。
图 12、 玉米螟及小菜蛾自己的 SP基因的 dsRNA对小菜蛾的致死率。
图 13、 pART27-dsRNA载体的示意图。 具体实施方式
本发明人经过广泛的研究, 找到了一个对于防治鳞翅目昆虫有用的靶标基因, 基于 该靶标基因的核酸序列制备核酸抑制物或表达核酸抑制物的宿主, 可有效地杀灭鳞翅目 昆虫。
术语
如本文所用, "分离的" 是指物质从其原始环境中分离出来 (;如果是天然的物质, 原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯 化的, 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开, 则为分离 纯化的。 本领域的技术人员能用标准的蚩白质纯化技术纯化蛋白。 基本上纯的多肽在非 还原聚丙烯酰胺凝胶上能产生单一的主带。
如本文所用, 所述的 "植物 "没有特别的限制, 只要所述"植物"是易于被昆虫 (如 鳞翅目昆虫)侵害的,如各种农作物、花卉植物、或林业植物等。所述的植物比如可以是(不 限于): 双子叶植物、 单子叶植物、 或裸子植物。 更具体地, 所述的植物包括 (但不限于): 棉花、 小麦、 大麦、 黑麦、 水稻、 玉米、 高梁、 甜菜、 苹果、 梨、 李、 桃、 杏、 樱桃、 草莓、 木莓、 黑莓、 豆、 扁豆、 豌豆、 大豆、 油菜、 芥、 罂莱、 齐墩果、 向日葵、 椰子、 蓖麻油植 物、 可可豆、 花生、 葫芦、 黄瓜、 西瓜、 亚麻、 ***、 黄麻、 柑桔、 柠檬、 葡萄柚、 菠菜、 苘苣、 芦笋、 洋白菜、 大白菜、 小白菜、 胡萝卜、 洋葱、 土豆、 西红柿、 青椒、 鳄梨、 桂皮、 樟脑、 烟叶、 坚果、 咖啡、 茄子、 甘蔗、 茶叶、 胡椒、 葡萄树、 蚝麻草、 香蕉、 天然橡胶树 和观赏植物等。
如本文所用, 所述的 "昆虫"是指任何基因组中包括有贮藏蛋白基因的昆虫。 较佳地, 所述昆虫可以是一种能以植物为食的植食性昆虫, 比如其可以是弹尾目、 等翅目、 鞘翅目、 双翅目、 膜翅目、 鳞翅目、 直翅目、 半翅目、 缕翅目的昆虫或农业害虫, 具体例如是长翅卷 蛾属种、 褐带卷蛾属、 透翅蛾属、 地老虎属、 棉叶波纹夜蛾、 黎豆夜蛾、 黄卷蛾属、 带卷蛾 属、 夜蛾属、 玉米楷夜蛾、 粉斑蟆、 桃蛀果蛾、 蟆属、 色卷蛾属、 葡萄果蠹蛾、 卷叶蟆属、 云卷蛾属、 鞘蛾属、 苹果异型小卷蛾、 卷叶蛾属、 玉米蟆属、 展叶松夜蛾、 金刚钻属、 粉蟆 属、 花小卷蛾属、 黄毒蛾属、 切根虫属、 食心虫属、 广翅小卷蛾、 夜蛾属、 菜蟆、 美国白蛾、 番茄蠹蛾、 旋纹潜叶蛾、 细蛾属、 毒蛾属、 潜叶蛾属、 天幕毛虫属、 甘蓝夜蛾、 烟草天蛾、 尺蠖属、 欧洲玉米蟆、 超小卷蛾属、 小眼夜蛾、 红铃虫、 棉铃虫、 马铃薯块茎蛾菜粉蝶、 粉 蝶属、 小菜蛾、 巢蛾属、 白野蟆属、 大蟆属、 卷叶蛾属、 粘虫属、 透翅蛾属、 带蛾属、 卷蛾 属、 粉纹夜蛾、 树巢蛾属、 叩甲属、 象甲属、 甜菜隐食甲、 甜菜茎跳甲、 谷象属、 实象属、 皮蠹属、 叶甲属、 瓢虫属、 马铃薯甲虫、 稻象甲属、 金龟属、 谷盗属、 耳喙象属、 丽金龟属、 跳甲属、 蠹属、 金龟子、 米象属、 麦蛾属、 粉甲属、 拟谷盗属、 斑皮蠹属、 非蠊属、 蠊属、 蝼蛄属、 马得拉非蠊、 飞蝗属、 大蠊属、 蚱蜢属、 白蚁属、 蓟马属、 条蓟马属、 单蓟马属、 棕黄蓟马、 棉蓟马、 非洲桔硬蓟马。 更佳地, 所述的 "昆虫"是对植物有害的昆虫, 中国专 利申请号为 200680042821.5 的专利申请文本及 US 2009/0306189 Al, WO 2007/080127 A2, AU 2006/335978 AL US 2009/0285784 Al的申请文本作为发明的参考文献, CN101365795 中 41-47页对于昆虫的描述可以纳入本发明的说明书内容。
如本文所用,所述的 "鳞翅目昆虫" 是指任何基因组中包括有 SEQ ID NO: 3-5、 SEQ ID NO: 17、 SEQ ID NO: 20-21任一所示的基因(片段)序列或其同源基因或与其杂交的
基因的鳞翅目昆虫, 且这些基因在所述的昆虫中作为该昆虫生长或生存所必需的基因。 所述的鳞翅目包括蛾 (moths), 实例包括: 谷蛾科 (Tineidae)和织蛾科 (Oecophoridae)比如衣蛾 (Tineola bisselliella) (普通衣蛾 (Common Clothes Moth)), 和螟蛾科 (Pyralidae)比如紫斑谷螟 (Pyralis farinalis) (膳食飞蛾 (Meal Moth)), 亚洲玉米螟, 和夜蛾科比如棉铃虫, 和菜蛾科比 如小菜蛾等。
如本文所用, 术语" RNA干扰 (RNA interference, RNAi) "是指一些 RNA可以高效、 特异地阻断体内特定基因的表达, 促使 mRNA降解, 诱使细胞表现出特定基因缺失的表 型, 其也称为 RNA干预或者干涉。 RNA干扰是高度特异的在 mRNA水平上的基因沉默机 制。
如本文所用, 术语 "干扰 RNA" 或 " dsRNA"是指一种 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)。
如本文所用, "基本上互补"是指核苷酸的序列是足够互补的, 可以以一种可预见 的方式发生相互作用, 如形成二级结构 (如茎环结构)。 通常, 两条 "基本上互补" 的核 苷酸序列互相之间至少有 70%的核苷酸是互补的; 优选的, 至少有 80%的核苷酸是互补 的; 更优选的, 至少有 90%的核苷酸是互补的; 进一步优选的, 至少有 95%的核苷酸是 互补的; 如 98%、 99%或 100%。 一般地, 两条足够互补的分子之间可以具有最多 7个 不匹配的核苷酸; 优选的, 具有最多 6个不匹配的核苷酸; 更优选的, 具有最多 5个不 匹配的核苷酸; 进一步优选的, 具有最多 4个不匹配的核苷酸, 如具有 0、 1、 2、 3、 4 个不匹配的核苷酸。
如本文所用, "互补" 的序列通常是指将 5'-3'方向的序列转换为其 3 '- 5'方向的序 列 (如 5'ATCG 3'→GCTA), 然后再取其互补序列(如 GCTA—5'CGAT 3,)。
如本文所用, "茎环 "结构也被称作 "发夹"结构, 是指一种核酸分子, 其可形成 一种包括双链区域 (茎部)的二级结构, 所述的双链区域由该核酸分子的两个区域 (位于同 一分子上)形成, 两个区域分列双链部分的两侧; 其还包括至少一个 "环"结构, 包括非 互补的核酸分子, 即单链区域。 即使该核酸分子的两个区域不是完全互补的, 核酸的双 链部分也可保持双链状态。 例如, ***、 缺失、 取代等可 致一个小区域的不互补或该 小区域自身形成茎环结构或其它形式的二级结构, 然而, 该两个区域仍可基本上互补, 并在可预见的方式中发生相互作用, 形成茎环结构的双链区域。 茎环结构是本领域技术 人员所熟知的, 通常在获得了一条具有一级结构的核苷酸序列的核酸后, 本领域技术人 员能够确定该核酸是否能形成茎环结构。
如本文所用, 所述的 "核酸抑制物"是指基于本发明的对于防治鱗翅目昆虫有用的 靶标基因或其片段或截短形式制备获得的、 具有防治鳞翅目昆虫活性的一类物质的总 称。 所述的 "核酸抑制物"例如是一些干扰分子, 包括 dsRNA (又称为, 双链 RNA、 双 链核糖核酸或双链核糖核苷酸序列)、 反义核酸、 小干扰 RNA、 微小 RNA等, 或是可以
表达或形成所述 dsRNA、 反义核酸、 小干扰 RNA或微小 RNA的构建物。
如本文所用, 所述的 "可操作 (性)地连接"或 "操作性连接"是指两个或多个核酸区 域或核酸序列的功能性的空间排列。 例如: 启动子区被置于相对于目的基因核酸序列的 特定位置, 使得核酸序列的转录受到该启动子区域的引 , 从而, 启动子区域被 "可操 作地连接" 到该核酸序列上。
如本文所用, "与多核苷酸 (或 DNA)序列对应的 RNA序列 " 是指一种 RNA序列, 若 DNA序列是 " AT"则 RNA序列是 " AU" 。
如本文所用, 所述的 "含有", "具有 "或"包括"包括了 "包含"、 "主要由 ... ... 构成" 、 "基本上由 ......构成" 、 和 "由 ... ...构成" ; "主要由 ... ...构成" 、 "基本上 由 ... ...构成" 和 "由 ......构成"属于 "含有" 、 "具有"或 "包括" 的下位概念。 贮藏蚩白及其编码基因
为了寻找对于防治有害生物有用的靶标基因, 本发明人经过了广泛而深入的研究, 最终找到了本发明的靶标基因, 所述的靶标基因为贮藏蚩白基因。
贮藏蚩白(SP)是属于六聚体蚩白家族的其中一种, 昆虫中的 sp 相关基因一般具有 cylindrical structures, 由 7-8个约 50KD的 polypeptide chains 组成的结构; 有 1个铜结 合位点, 根据氨基酸结构又可分为富含蚩氨酸 (> 4% methionine) 的贮藏蛋白和富含芳 香基的氨基酸(>15% aromatic amino ) 的贮藏蚩白, 与富含芳香基氨基酸的 sp相比, 富 含蚩氨酸的 sp不含有糖基化位点, 不同物种序列一致性在 40%-90%之间 (40°/。序列一 致性被认为是同源较高) 。
本发明人深入研究后发现, 来自昆虫的贮藏蛋白基因 (例如玉米螟 OfSP基因、 棉铃 虫 H.arm SP, 小菜蛾 PxSPl、 PxSP2) 是昆虫发育或生长相关的关键基因, 这些基因的 表达下调或抑制可以 致昆虫生长发生问题(例如化蛹异常或产的卵不能孵化)或死亡。
以上述基因作为抑制或沉默靶标, 可制备特异性干扰分子, 抑制玉米螟或棉铃虫或 小菜蛾生长发育。上述基因还可以是来自其它生物,所述基因的功能己被建立于文献中, 其核苷酸序列与玉米螟基因具有高度同源性或其基因功能与玉米螟基因功能相似, 可预 见这些高度同源基因或功能相似基因可作为靶标, 制备特异性干扰分子, 抑制其他生物 的生长。
本文中使用的术语"高度同源"或者"高度同源性",在提到核酸序列时,表示在严谨条 件下与 SEQ ID NO: 1、 SEQ ID NO: 16> SEQ ID NO: 18-19任何序列或其互补序列能杂 交的核苷酸序列。 在严谨条件下与 SEQ ID NO: 1、 SEQ ID NO: 16、 SEQ ID NO: 18-19 或其互补序列能杂交的序列, 是允许两条序列间发生反平行比对的序列, 然后两条序列 能在严谨条件下以相反链上的对应碱基形成氢键, 以形成二体分子, 其在严谨条件下是 足够稳定的,使用本领域公知方法能探测到。优选地,此类高度同源的序列与 SEQ ID NO: 1、 SEQ ID NO: 16、 SEQ ID NO: 18-19任何序列所示的对照核苷酸序列或其互补序列具
有大约 40%至大约 50%的序列一致性, 或者优选地, 大约 65 %至大约 70 %的序列一致 性, 或者更优选地, 大约 80% 至大约 85 %的序列一致性, 或者最优选地, 大约 90 %至 大约 95 %的序列一致性, 至大约 99%的序列一致性。
用于确定序列一致性的方法是本领域常规的, 其包括使用 Blast软件和 EMBOSS软 #(The European Molecular Biology Open Software Suite(2000), Rice, P.Longden, Land Bleasby, A. Trends in Genetics 16, (6)pp276-277)。本文使用的术语 "一致性"是指序列之 间在核苷酸水平上的关系。 通过在比较窗中对最优比对的序列 (例如, 两条或更多)进行 比较来确定"一致性百分比", 其中所述比较窗中的序列部分与最优序列比对的参考序列 相比可包含***或缺失。 所述参考序列不包含***或缺失。 所述参考窗选自至少 10个 连续核苷酸至约 50、 约 100或者至约 150个核苷酸, 优选约 50〜150个核苷酸。 然后, 通过测定所述窗中的序列之间一致的核苷酸数目并将该数目除以所述窗中的核苷酸数 并乘以 100来计算 "一致性百分比"。
针对上述靶基因, 本发明人进一步研究了可有效下调这些靶基因的靶标基因片段, 最终找到了 6个靶标基因片段,它们的核苷酸序列选自 SEQ ID NO: 3-5、 SEQ ID NO: 17、 SEQ ID NO: 20-21任一所示的序列。
所述的靶标基因 (片段)的片段或截短形式也包括在本发明中,只要这些片段或截短形 式在被用于制备所述的核酸抑制物后, 该核酸抑制物也具有杀灭鳞翅目昆虫的活性。
本发明人意外地发现, 这些靶标基因片段或它们的截短体在昆虫体内发挥着重要的 作用, 它们被抑制、 干扰或沉默将 致昆虫存活率的显著下降或直接死亡。 因此, 可基 于这些靶标基因片段来设计各种核酸抑制物, 从而用于害虫的防治。
本发明还提供了一个多核苷酸集 (集合), 所述多核苷酸集包括 SEQ ID NO: 3-5、 SEQ ID NO: 17、 SEQ ID NO: 20-21所示的序列。当需要制备防治鱗翅目昆虫的核酸抑制物 (如 构建物或 dsRNA)或其制剂时, 可从所述的多核苷酸集中获取 1-6条多核苷酸。 可基于 多个多核苷酸的序列 (较佳地, 例如是 6)来分别制备核酸抑制物或表达核酸抑制物的宿 主, 当同时 (或混合)应用时, 可达到更为广谱、 更为有效的杀灭鳞翅目昆虫的效果。 核酸抑制物
任何基于本发明提供的靶标基因 (片段)或其片段或截短形式制备获得的、具有防治鳞 翅目昆虫活性的物质都可作为核酸抑制物, 用于防治鳞翅目昆虫。 所述等核酸抑制物较 佳的是一些干扰分子, 例如, dsRNA、 反义核酸、 小干扰 RNA或微小 RNA的构建物, 或可以表达或形成所述 dsRNA、 反义核酸、 小干扰 RNA或微小 RNA的构建物。 更佳 地是 dsRNA或可以表达所述 dsRNA的构建物。
根据本发明所提供的基因及其序列, 可设计出用于表达 dsRNA、 反义核酸、 小干扰 RNA或微小 RNA的构建物。 因此, 本发明提供了一种人工构建的构建物。 根据本发明 提供的基因及其序列来设计所述的构建物是本领域技术人员可了解的, 通常可使该构建
物包含一个内含子序列 (与两侧序列不互补), 两端连接上互补的基因序列, 入细胞后, 能产生 "茎环" 结构, 并且 "茎"状部分能够形成 dsRNA、 反义核酸、 小干扰 RNA或 微小 RNA, 这种 dsRNA、 反义核酸、 小干扰 RNA或微小 RNA能特别有效的抑制目的 基因的表达。
作为本发明的一种优选方式, 所述的构建物含有至少一个如下所示的结构-
Seq —X_Seq反向
其中, Seq Ε ί5的核苷酸序列选自权利要求 2或 3任一所示的序列或序列片段, Seq Ε 向与 Seq ^为基本上互补的核苷酸序列;
X为位于 Seq ^和 Seq ^之间的间隔序列, 并且所述间隔序列与 Seq ^和 Seq s向不 互补;
式 I所示的结构在可形成如下所示的二级结构:
Seq 向
" _ X
Seq反向 ■ 。
该结构可进一步被剪切、 加工形成双链形式的干扰分子, 从而发挥基因沉默的作用。 所述的构建物可以制备成可形成多于 1个茎环结构的形式,例如,可以包含 2个或 2 个以上的茎环结构。
通常, 所述的构建物位于表达载体上。 因此, 本发明还包括一种载体, 它含有所述 的构建物。 所述的表达载体通常还含有与所述的构建物操作性相连的启动子、 复制起点 和 /或标记基因等。本领域的技术人员熟知的方法能用于构建本发明所需的表达载体。这 些方法包括体外重组 DNA技术、 DNA合成技术、 体内重组技术等。 所述的表达载体优 选地包含一个或多个选择性标记基因, 以提供用于选择转化的宿主细胞的表型性状, 如 卡那霉素、 庆大霉素、 潮霉素、 氨苄青霉素抗性。
包含上述的适当基因序列以及适当启动子或者控制序列的载体, 可以用于转化适当 的宿主。 在本发明的方法中, 所述的宿主可以是任何适合于携带所述表达载体并能够表 达出核酸抑制物的宿主。 例如, 所述的宿主为大肠杆菌, 真菌, 酵母, 植物细胞, 动物 细胞等。
用重组 DNA转化宿主可用本领域技术人员熟知的常规技术进行,具体视植物种类的 不同而定。 当宿主为原核生物如大肠杆菌时, 能吸收 DNA的感受态细胞可在指数生长 期后收获, 用 CaCl2法处理, 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2 如果需要, 转化也可用电穿孔的方法进行。 真菌和酵母细胞、 植物细胞、 动物细胞的转 化也是本领域技术人员熟知的。
携带所述构建物或表达载体并能够表达出核酸抑制物的宿主能够直接施用于需要防 治的对象 (如植物), 以达到防治鳞翅目昆虫的目的。 以上所述的茎环结构的 "茎"状部分即由 Seq ί ^和 Seq ^相互作用形成, 可被加工 形成核酸抑制物。 所形成的核酸抑制物具有如下结构:
Seq'iH向一- -、、
Seq,反 -- 其中, Seq'^选自权利要求 2或 3任一所示序列对应的 RNA序列或序列片段; Seq' s向为与 Seq'^基本上互补的序列。 X'为位于 Seq^和 Seq,^之间的间隔序列, 所述间 隔序列与 Seq'^ ^和 Seq'sfi不互补。Χ'序列可在体外进行切除,也可不进行切除,待 dsRNA 进入昆虫体内后由昆虫体内的酶 (如核酸酶 Dicer)加工切除。
所述的核酸抑制物可直接施用于需要防治的对象 (如植物),以达到防治鳞翅目昆虫的 目的。
本发明的多肽 (蚩白)可以是重组多肽、 天然多肽、 合成多肽, 优选的是重组多肽。 本 发明的多肽可以是天然纯化的产物, 或是化学合成的产物, 或使用重组技术从原核或真 核宿主 (例如, 植物、 细菌、 酵母、 昆虫细胞)中产生。 根据重组生产方案所用的宿主, 本发明的多肽可以是糖基化的, 或可以是非糖基化的。 本发明的多肽还可包括或不包括 起始的甲硫氨酸残基。
本发明还包括贮藏蛋白 2的片段、 衍生物和类似物。 如本文所用, 术语 "片段" 、 "衍生物"和 "类似物"是指基本上保持本发明的贮藏蚩白 2相同的生物学功能或活性 的多肽。 本发明的多肽片段、 衍生物或类似物可以是 (i)有一个或多个保守或非保守性氨 基酸残基 (优选保守性氨基酸残基)被取代的多肽, 而这样的取代的氨基酸残基可以是也 可以不是由遗传密码编码的,或 (ii)在一个或多个氨基酸残基中具有取代基团的多肽, 或 (Hi)成熟多肽与另一个化合物 (比如延长多肽半衰期的化合物,例如聚乙二醇)融合所形成 的多肽, 或 (iv)附加的氨基酸序列融合到此多肽序列而形成的多肽 (如前 序列或分泌序 列或用来纯化此多肽的序列或蛋白原序列, 或融合蛋白)。根据本文的定义这些片段、衍 生物和类似物属于本领域熟练技术人员公知的范围。
在本发明中, 术语 "贮藏蛋白 2 "指 SEQ ID NO: 2序列的多肽或其变异形式。 这些 变异形式包括 (但并不限于): 若干个 (通常为 1 -50个, 较佳地 1-30个, 更佳地 1-20个, 最佳地 1-10个, 还更佳如 1-8个或 1 -5个)氨基酸的缺失、 ***和 /或取代, 以及在 C末 端和 /或 N末端添加或缺失一个或数个 (通常为 20个以内, 较佳地为 10个以内, 更佳地 为 5个以内)氨基酸。 例如, 在本领域中, 用性能相近或相似的氨基酸进行取代时, 通常 不会改变蚩白质的功能。 又比如, 在 C末端和 /或 N末端添加或减少一个或数个氨基酸 通常也不会改变蚩白质的功能。 该术语还包括贮藏蛋白 2的活性片段和活性衍生物。
多肽的变异形式包括: 同源序列、 保守性变异体、 等位变异体、 天然突变体、 诱 突变体、 在高或低的严紧度条件下能与贮藏蛋白 2 DNA杂交的 DNA所编码的蚩白等。 本发明还提供了其他多肽, 如包含贮藏蚩白 2或其片段的融合蚩白。 除了几乎全长的多 肽外, 本发明还包括了贮藏蛋白 2的可溶性片段。 通常, 该片段具有贮藏蛋白 2序列的 至少约 20个连续氨基酸, 通常至少约 30个连续氨基酸, 较佳地至少约 50个连续氨基 酸, 更佳地至少约 80个连续氨基酸, 最佳地至少约 100个连续氨基酸。
本发明还提供贮藏蛋白 2或多肽的类似物。 这些类似物与天然贮藏蚩白 2的差别可
以是氨基酸序列上的差异, 也可以是不影响序列的修饰形式上的差异, 或者兼而有之。 这些多肽包括天然或诱 的遗传变异体。 诱 变异体可以通过各种技术得到, 如通过辐 射或暴露于诱变剂而产生随机诱变, 还可通过定点诱变法或其他已知分子生物学的技 术。类似物还包括具有不同于天然 L-氨基酸的残基 (如 D-氨基酸)的类似物, 以及具有非 天然存在的或合成的氨基酸 (如 β、 氨基酸)的类似物。 应理解, 本发明的多肽并不限 于上述例举的代表性的多肽。
修饰 (通常不改变一级结构)形式包括:体内或体外的多肽的化学衍生形式如乙酰化或 羧基化。修饰还包括糖基化。修饰形式还包括具有磷酸化氨基酸残基 (如磷酸酪氨酸, 磷 酸丝氨酸,磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蚩白水解性能或优化了溶 解性能的多肽。
在本发明中, "贮藏蚩白 2保守性变异多肽"指与 SEQ ID NO: 2的氨基酸序列相比, 有至多 20个, 较佳地至多 10个, 更佳地至多 5个, 最佳地至多 3个氨基酸被性质相似 或相近的氨基酸所替换而形成多肽。 这些保守性变异多肽最好根据表 1进行氨基酸替换 而产生。
本发明所述的贮藏蚩白 2在昆虫体内的表达被降低后, 昆虫的存活率可显著降低, 可以用于筛选或制备杀昆虫的制剂的靶标, 因此, 基于该靶标蚩白, 可设计出多种的 杀虫制剂, 例如蛋白抑制剂, 其可在被施用于防治对象后, 起到杀灭昆虫的作用。
所述的靶标蚩白或其片段或其变异体也包括在本发明中, 只要这些片段或变异体在 被用于制备所述的蚩白抑制物后, 该蚩白抑制物也具有杀灭鳞翅目昆虫的活性。
表 1
本发明的多核苷酸可以是 DNA形式或 RNA形式。 DNA形式包括 CDNA、 基因组
DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链或非编 码链。 编码成熟多肽的编码区序列可以与 SEQ ID NO: 1所示的编码区序列相同或者是 简并的变异体。 如本文所用, "简并的变异体"在本发明中是指编码具有 SEQ ID NO.- 2 序列的蚩白, 但与 SEQ ID NO: 1所示的编码区序列有差别的核酸序列。
编码 SEQ ID NO: 2的成熟多肽的多核苷酸包括: 只编码成熟多肽的编码序列; 成熟 多肽的编码序列和各种附加编码序列; 成熟多肽的编码序列 (和任选的附加编码序列)以 及非编码序列。
术语 "编码多肽的多核苷酸 "可以是包括编码此多肽的多核苷酸, 也可以是还包括 附加编码和 /或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体,其编码与本发明有相同的氨基酸序列的多肽 或多肽的片段、 类似物和衍生物。 此多核苷酸的变异体可以是天然发生的等位变异体或 非天然发生的变异体。 这些核苷酸变异体包括取代变异体、 缺失变异体和***变异体。 如本领域所知的, 等位变异体是一个多核苷酸的替换形式, 它可能是一个或多个核苷酸 的取代、 缺失或***, 但不会从实质上改变其编码的多肽的功能。
来源于本发明的靶标基因的较短的片段也被包含在本发明中, 作为设计核酸抑制物 的基础。本领域人员均了解,一些较短的序列也可用于作为核酸抑制物,例如一些 siRNA 序列往往仅有 1 8bp。本发明还涉及与上述的序列杂交且两个序列之间具有至少 15% ; 较 佳地至少 18%,更佳地至少 25°/。,更佳地至少 50%, 更佳地至少 70%, 更佳地至少 80% , 更佳地至少 90%, 更佳地至少 95%, 更佳地至少 98%, 更佳地至少 99%相同性的多核苷 酸。 本发明特别涉及在严格 (严紧)条件下与本发明所述多核苷酸可杂交的多核苷酸。 在 本发明中, "严格 (严紧)条件"是指: (1 )在较低离子强度和较高温度下的杂交和洗脱, 如 0.2 X SSC , 0. 1%SDS, 60 °C ; 或 (2)杂交时加有变性剂, 如 50%0 ')甲酰胺, 0. 1%小 牛血清 /0. 1 /。Ficoll, 42 °C等; 或 (3)仅在两条序列之间的相同性至少在 80%以上, 较好至 少 90%以上, 更好是 95%以上时才发生杂交。 并且, 可杂交的多核苷酸编码的多肽与 SEQ ID NO: 2所示的成熟多肽有相同的生物学功能和活性。
本发明还涉及与上述的序列杂交的核酸片段。 如本文所用, "核酸片段" 的长度至 少含 15个核苷酸, 较好是至少 30个核苷酸, 更好是至少 50个核苷酸, 最好是至少 100 个核苷酸以上。核酸片段可用于核酸的扩增技术 (如 PCR)以确定和 /或分离编码贮藏蚩白 2的多核苷酸。
本发明的编码贮藏蚩白 2的核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组 法或人工合成的方法获得。 对于 PCR扩增法, 可根据本发明所公开的有关核苷酸序列, 尤其是开放阅读框序列来设计引物, 并用巿售的 cDNA库或按本领域技术人员己知的常 规方法所制备的 cDNA库作为模板, 扩增而得有关序列。 当序列较长时, 常常需要进行
两次或多次 PCR扩增, 然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列, 就可以用重组法来大批量地获得有关序列。 这通常是将其 克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外, 还可用人工合成的方法来合成有关序列, 尤其是片段长度较短时。 通常, 通 过先合成多个小片段, 然后再进行连接可获得序列很长的片段。
目前, 已经可以完全通过化学合成来得到编码本发明蚩白 (或其片段, 或其衍生物) 的 DN A序列。然后可将该 DN A序列引入本领域中己知的各种现有的 DN A分子 (或如载 体)和细胞中。 此外, 还可通过化学合成将突变引入本发明蚩白序列中。
本发明上述的编码贮藏蚩白 2的多核苷酸可用于作为制备杀虫制剂的靶标基因。 本 发明人发现, 本发明的靶标基因或其片段在昆虫体内发挥着重要的作用, 它们被抑制、 干扰或沉默将 致昆虫存活率的显著下降或直接死亡。 因此, 基于该靶标基因, 可设 计出多种的杀虫制剂, 例如设计核酸抑制物, 其可在被施用于防治对象后, 起到杀灭昆 虫的作用。
所述的靶标基因或其片段或截短形式或基因变异体或片段变异体也包括在本发明 中, 只要这些片段或截短形式或变异体在被用于制备所述的核酸抑制物后, 该核酸抑制 物也具有杀灭鳞翅目昆虫的活性。
本发明也涉及包含本发明的多核苷酸或其片段或变异体的载体, 以及用本发明的载 体或贮藏蚩白 2编码序列经基因工程产生的宿主细胞, 以及经重组技术产生本发明所述 多肽的方法。
通过常规的重组 DNA技术,可利用本发明的多聚核苷酸序列来表达或生产重组的贮 藏蚩白 2。 一般来说有以下步骤:
(1) .用本发明的编码贮藏蛋白 2的多核苷酸 (或变异体), 或用含有该多核苷酸的重组 表达载体转化或转 合适的宿主细胞;
(2) .在合适的培养基中培养宿主细胞;
(3).从培养基或细胞中分离、 纯化蚩白质。
本发明中, 贮藏蛋白 2多核苷酸序列可***到重组表达载体中。 术语 "重组表达载 体"指本领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒 或其他载体。 总之, 只要能在宿主体内复制和稳定, 任何质粒和载体都可以用。 表达载 体的一个重要特征是通常含有复制起点、 启动子、 标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含贮藏蚩白 2编码 DNA序列和合适的转录
/翻译控制信号的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成技术、 体内重 组技术等。 所述的 DNA序列可有效连接到表达载体中的适当启动子上, 以指 mRNA 合成。 表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外, 表达载体优选地包含一个或多个选择性标记基因, 以提供用于选择转化的宿 主细胞的表型性状, 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗性以及绿色荧光蚩
白 (GFP), 或用于大肠杆菌的卡那霉素或氨苄青霉素抗性。
包含上述的适当 DNA序列以及适当启动子或者控制序列的载体,可以用于转化适当 的宿主细胞, 以使其能够表达蚩白质。
宿主细胞可以是原核细胞, 如细菌细胞; 或是低等真核细胞, 如酵母细胞; 或是高 等真核细胞, 如植物细胞。 代表性例子有: 大肠杆菌, 链霉菌属、 农杆菌; 真菌细胞如 酵母; 植物细胞等。
本发明的多核苷酸在高等真核细胞中表达时, 如果在载体中***增强子序列时将会 使转录得到增强。 增强子是 DNA的顺式作用因子, 通常大约有 10到 300个碱基对, 作 用于启动子以增强基因的转录。
本领域一般技术人员都淸楚如何选择适当的载体、 启动子、 增强子和宿主细胞。 用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核 生物如大肠杆菌时, 能吸收 DNA的感受态细胞可在指数生长期后收获, 用 CaCl2法处 理, 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如果需要, 转化也可用 电穿孔的方法进行。 当宿主是真核生物, 可选用如下的 DNA转染方法: 磷酸钙共沉淀 法, 常规机械方法如显微注射、 电穿孔、 脂质体包装等。 转化植物也可使用农杆菌转化 或基因枪转化等方法, 例如叶盘法、 水稻幼胚转化法等。 对于转化的植物细胞、 组织或 器官可以用常规方法再生成植株。 杀虫制剂
任何基于本发明提供的靶标蚩白或其片段或截短形式制备获得的、 具有防治鳞翅目 昆虫活性的物质都可作为杀昆虫的制剂, 用于防治鳞翅目昆虫。 所述的制剂例如蛋白抑 制剂, 蛋白水解酶, 蛋白结合分子, 抗体, 配体。 任何可以抑制该靶标蚩白的活性或表 达的物质, 都被包含在本发明中。
任何基于本发明提供的靶标基因或其片段或截短形式制备获得的、 具有防治鳞翅目 昆虫活性的物质都可作为核酸抑制物, 用于防治鳞翅目昆虫。 所述的核酸抑制物较佳的 是一些干扰分子, 例如, dsRNA、 反义核酸、 小干扰 RNA或微小 RNA的构建物, 或可 以表达或形成所述 dsRNA、 反义核酸、 小干扰 RNA或微小 RNA的构建物。 更佳地是 dsRNA或可以表达所述 dsRNA的构建物。
根据本发明所提倂的基因及其序列, 可设计出用于表达 dsRNA、 反义核酸、 小干扰 RNA或微小 RNA的构建物。 因此, 本发明提供了一种人工构建的构建物。 根据本发明 提供的基因及其序列来设计所述的构建物是本领域技术人员可了解的,通常可使该构建 物包含一个内含子序列 (与两侧序列不互补;), 两端连接上互补的基因序列, 入细胞后, 能产生 "茎环"结构, 并且 "茎" 状部分能够形成 dsRNA、 反义核酸、 小干扰 RNA或 微小 RNA, 这种 dsRNA、 反义核酸、 小干扰 RNA或微小 RNA能特别有效的抑制目的 基因的表达。
根据本发明的一种优选方式, 所述的核酸抑制物具有如下结构:
Seq
Seq
其中, Seq' 是与编码所述的贮藏蛋白 2 的多核苷酸序列或其片段所对应的 RNA 序列; Seq, i5为与 Seq,^基本上互补的序列。
所述的核酸抑制物可直接施用于需要防治的对象 (如植物), 以达到防治鳞翅目昆虫 的目的。
作为本发明的优选实施方式,所述的 Seq' 是选自 SEQ ID NO: 3-5 SEQ ID NO: 17 SEQ ID NO: 20-21任一所示的 DNA所对应的 RNA。更优选地,是选自 SEQ ID NO: 3或 SEQ ID NO: 5或 SEQ ID NO: 17, 或 SEQ ID NO: 20-21任一所示的 DNA所对应的 RNA, 前者 (SEQ ID NO: 3)可用于开发防治亚洲玉米螟的特异型制剂;而后者 (SEQ ID NO: 5)更适用 于开发防治鳞翅目害虫的广谱型制剂。
作为本发明的另一种可选择的方式, 所述的构建物含有至少一个如下所示的结构:
Seq -X"Seq
其中, Seq ^是编码所述的贮藏蛋白 2的多核苷酸或其片段, Seq «与 Seq 为基本 上互补的核苷酸序列;
X为位于 Seq ^和 Seq ^之间的间隔序列, 并且所述间隔序列与 Seq ^和 Seq s向不 互补;
式 I所示的结构在可形成如下所示的二级结构:
Seq正 —
X
Seq反向一 。
该结构可进一步被剪切、 加工形成双链形式的干扰分子 (RNA分子), 从而发挥基因 沉默的作用。
所述的构建物可以制备成可形成多于 1个茎环结构的形式, 例如, 可以包含 2个或 2 个以上的茎环结构。
通常, 所述的构建物位于表达载体上。 因此, 本发明还包括一种载体, 它含有所述 的构建物。 所述的表达载体通常还含有与所述的构建物操作性相连的启动子、 复制起点 和 /或标记基因等。本领域的技术人员熟知的方法能用于构建本发明所需的表达载体。这 些方法包括体外重组 DNA技术、 DNA合成技术、 体内重组技术等。 所述的表达载体优 选地包含一个或多个选择性标记基因, 以提供用于选择转化的宿主细胞的表型性状, 如 卡那霉素、 庆大霉素、 潮霉素、 氨苄青霉素抗性。
包含上述的适当基因序列以及适当启动子或者控制序列的载体,可以用于转化适当 的宿主。 在本发明的方法中, 所述的宿主可以是任何适合于携带所述表达载体并能够表 达出核酸抑制物的宿主。 例如, 所述的宿主为大肠杆菌, 真菌, 酵母, 植物细胞, 动物 细胞等。
携带所述构建物或表达载体并能够表达出核酸抑制物的宿主能够直接施用于需要
防治的对象 (如植物), 以达到防治鳞翅目昆虫的目的。
本发明所述的核酸抑制物除了利用构建物进行细胞表达外,还可以利用体外化学合 成的方法获得。 应理解, 任何可形成本发明所述的核酸抑制物的方法均可被应用于本发 明中。 转基因植物
本发明还涉及一种以植物为媒介防治有害生物的方法或改良植物的抗虫性的方法, 该方法包括将所述的核酸抑制物 入到植物中。 植物的转基因是本领域技术人员熟知的 技术, 例如可使用农杆菌转化或基因枪转化等方法, 例如叶盘法、 幼胚转化法等。 对于 转化的植物细胞、组织或器官可以用常规方法再生成植株,从而获得具有抗虫性的植物。
在本发明中,所述的以植物为媒介防治有害生物的方法或改良植物的抗虫性的基本原 理是: 以植物作为媒介, 使昆虫服食可干扰昆虫贮藏蚩白基因表达的干扰 RNA, 从而抑制昆 虫的生长或杀灭昆虫。 所述的 "干扰 RNA "是指基于本发明提供的作为靶标的昆虫贮藏蚩白 基因或其片段 (截短形式)制备或加工 (如被体内加工)获得的、 具有防治昆虫活性的一类物质 的总称。 所述的 "干扰 RNA "例如包括 dsR A、 反义核 (苷)酸、 小干扰 RNA、 微小 RNA等。 更具体地, 所述的原理为: 通过转基因的方法让植物体内表达昆虫基因 (全长或部分)的双链 RNA(dsR A), 在植物体内形成高丰度的干扰 RNA, 当昆虫取食这种转基因植物的同时也摄 入了大量的干扰 RNA, 所述的干扰 RNA在进入昆虫体内后能够抑制所述昆虫基因在表达, 干扰昆虫正常的生长发育甚至引起其死亡。利用所述原理, 可以有效地改良植物对于昆虫的 抵抗能力。将 RNA干扰技术应用到转基因植物上, 开发新型的转基因抗性植物, 对农业的发 展具有重大的意义。
优选的, 一种改良植物的抗虫性的方法包括:
(1) 提供携带表达载体的农杆菌, 所述的表达载体含有所述的核酸抑制物;
(2) 将植物细胞或组织或器官与步骤 ( 1)中的农杆菌接触, 从而使所述的核酸抑制物 转入植物细胞或组织或器官;
(3) 选择出转入了所述的核酸抑制物的植物细胞或组织或器官; 和
(4) 将步骤 (3)中的植物细胞或组织或器官再生成植物。
可采用任何适当的常规手段, 包括试剂、 温度、 压力条件等来实施所述的方法。 目前, 利用植物作为媒介, 通过使昆虫服食可干扰昆虫基因表达的核酸抑制物 (干扰 分子), 从而抑制昆虫的生长或杀灭昆虫己经是本领域人员熟知的技术了, 例如 Ying-Bo Mao等, Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol (NATURE BIOTECHNOLOGY VOLUME 25
NUMBER 1 1 NOVEMBER 2007) 中证明了该种方法的有效性。 组合物和防治鳞翅目昆虫的方法
本发明还提供了一种制剂 (如农药组合物),其含有安全有效量的携带所述构建物或表 达载体并能够表达出核酸抑制物的宿主 (细胞)或所述的核酸抑制物 (如: 10- 50(Vg/ml; 较佳地 20-20(Vg/ml; 更佳地 30- 100μ8/ιη1); 以及农药学上可接受的载体。
本发明中, 术语 "含有"表示各种成分可一起应用于本发明的混合物或组合物中。 因此, 术语 "主要由 ...组成"和 "由…组成"包含在术语 "含有" 中。
本发明中, "农药学上可接受的" 的成分是适用于农业用途而对人或动物(除鳞翅 目昆虫以外)、 植物没有过度不良副反应(如毒性、 刺激和***反应)的, 即有合理的效 益 /风险比的物质。
本发明中, "农药学上可接受的载体"是用于将本发明的携带所述构建物或表达载 体并能够表达出核酸抑制物的宿主或本发明的核酸抑制物传送给鳞翅目昆虫的可接受 的溶剂、 悬浮剂或赋形剂。 农药学上可接受的载体可以是液体或固体, 较佳的是能够较 高程度保持宿主或核酸抑制物活性的载体。
所述制剂(或农药组合物)的剂型可以是多种多样的, 包括但不限于: 水溶液, 悬浮 剂, 可湿粉剂, 可乳化浓缩物, 乳液, 可喷洒溶液, 水性分散系, 粉剂, 颗粒剂, 或微 胶囊。 应理解, 只要能够将本发明的携带所述构建物或表达载体并能够表达出核酸抑制 物的宿主或本发明的核酸抑制物在保持全部或部分活性的前提下递送到鳞翅目昆虫的 剂型都是可取的。 优选那些易于递送的剂型, 例如, 所述农药组合物是液体喷洒剂、 或 喷雾剂。
在本发明中, 所述的辅剂是一种辅助成分, 在组合物中起辅助调节功能, 比如其可 以是一种表面活性剂、 附着助剂或其它类型助剂。
浓缩型的农药组合物中活性成分 (即:携带所述构建物或表达载体并能够表达出核酸 抑制物的宿主或本发明的核酸抑制物)的含量较高, 如含有核酸抑制物 200-500μβ/ηι1, 而 稀释型农药和实际使用的组合物中活性成分含量较低, 如含有核酸抑制物 20-10(^g/ml, 施用量为 50-1000μ1/300-500头,或根据携带所述构建物或表达载体并能够表达出核酸抑 制物的宿主 (细胞)表达的核酸抑制物含量, 参照核酸抑制物施用量, 施用一定量的宿主 (细胞)。 此外, 还可以包含其他合适的化学剂、 增效剂、 微量元素、 稳定剂、 粘合剂、 润湿剂、 分散剂、 乳化剂、 渗透剂、 鞣化剂、 溶剂、 充填剂等常用组分。 本发明农药组 合物中还可以含有其它活性杀虫剂或杀微生物剂。
在制备农药组合物时, 合适的固体稀释剂包括但不限于: 硅藻土, 玉米壳, 磷酸三 钙, 软木粉, 高岭土、 膨润土或硅镁土等粘土, 和水溶性聚合物。
此外, 固体组合物还可以含有一种或多种相容性润湿剂, 分散剂, 乳化剂或色素, 这些成分在固态时也可起稀释剂的作用。
这样的固体组合物可以是粉剂, 颗粒剂或可湿粉剂的形式。 通常通过研磨获得粉剂, 通过造粒或压片获得颗粒剂、 片剂或砖型剂。
液体组合物的形式可以是溶液, 悬浮液和乳液, 也可以将其包在天然或合成聚合物
中, 并可以包含润湿剂、 分散剂或乳化剂。 这样的乳液、 悬浮液或溶液可用水性、 有机 或水 -有机稀释剂来制备水溶性聚合物 (以及上述稀释剂的混合物)。 此外, 所述稀释剂中 可含有例如以上所述离子型或非离子型的润湿剂、 分散剂或乳化剂或它们的混合物。
各种制剂的原理都是己知的, 并在例如以下文献中有所描述: Winnacker-Kuchler, " Chemische Technologie' '化学技术, Vol.7, C.Hauser Verlag Munich, 第 4版, 1986; van Valkenburg, "农药剂型" , Marcel Dekker N. Y., 第 2版, 1972-73 ; K. Martens, "喷雾干燥手册" , 第 3版, G. Goodwin Ltd. London。
用于本发明组合物的所需的配制辅剂, (例如惰性物质, 表面活性剂, 溶剂和其他添 加剂;),也是已知的,其描述例如: Watkins"杀虫粉剂稀释剂和载体手册 "第 2版, Darland Books, Caldwell N. I; H.v.Olphen, "粘土胶体化学 引 "第 2版, J. Wiley & Sons, N.Y. , Marsden, "溶剂指南"第 2版, Interscience, N Y. 1950; McCutcheon's, "除 垢剂和乳化剂年刊", MC Publ. Corp. , Ridgewood N.J. ; Sisley和 Wood, "表面活性剂 百科全书" , Chem Publ. Co. Inc. , N.Y. I 964; Schonfelt, " Grenzflachenaktive
Athylenoxidaddukte " 表面活性环氧乙院加成产物, Wiss.Verlagsgesell. , Stuttgart 1 76; Winnacker-Kuchler, " Chemische Technologie"化学技术, Vol.7 , C.Hauser Verlag Munich, 第 4版, 1986。
可湿粉剂可均匀分散于水。 除活性物质之外, 可湿粉剂还可包含润湿剂, 分散剂, 稀释剂等无环境公害的物质。 粉剂的制备可以是: 将活性物质与精细粉碎后的滑石、 高 岭土、 膨润土之类天然粘土或硅藻土等固体物质一同研磨。 颗粒剂的制备可以是用活性 物质喷涂吸附于惰性物质颗粒, 或将活性物质溶液通过粘合剂 (例如聚乙烯醇、聚丙烯酸 钠, 或矿物油)施加于载体 (例如砂、 高岭土或惰性物质颗粒)表面。 如果欲与化肥混合施 用, 则可将合适的活性物质象制备化肥颗粒那样制备成颗粒。
就控制植物中昆虫侵袭的目的而言, 通过喷雾应用将昆虫控制用的 dsRNA运送到植 物表面提供了另一种保护植物的手段。在这种情况下, 被工程改造以产生和积累 dsRNA 的细菌可被发酵, 发酵产品被配方为可与常见农业应用兼容的喷雾产品。 所述配方可以 包括: 高效叶片覆盖所需的合适的胶粘物和润湿剂, 以及用于保护 dsRNA免受 UV损 害的 UV保护剂。 此类添加剂是生物杀昆虫剂工业中常用的, 也是本领域技术人员公知 的。同样地,用于土壤应用的配方可以包括下述颗粒配方,其用作为土壤有害昆虫 (例如, 玉米根虫)幼虫的饵。
其中所述细菌或酵母细胞被灭活或被杀死, 例如通过热处理或机械处理。
本发明人意外地发现, 所述的核酸抑制物可通过渗透的方式由昆虫体表迸入到昆虫 体内。 因此可通过将各种核酸抑制物或含有各种核酸抑制物的组合物施用于昆虫体表来 达到防治昆虫的目的。 所述的施用方式包括但不限于喷洒、 涂覆、 滴加于昆虫的体表。 所述的核酸抑制物选自: dsRNA、 反义核酸、 小干扰 RNA、 微小 RNA; 或能表达或形 成所述 dsRNA、 反义核酸、 小干扰 RNA、 微小 RNA的构建物。
本发明还提供了一种防治鳞翅目昆虫的方法, 所述方法包括: 将所述的携带所述构 建物或表达载体并能够表达出核酸抑制物的宿主 (细胞)或所述的核酸抑制物施用于需要 防治的对象 (如植物, 特别是被鳞翅目昆虫侵害的植物; 或昆虫本身)。
也可直接用所述的核酸抑制物或含有该核酸抑制剂的农药组合物或表达所述核酸抑 制物的宿主细胞喂食昆虫或喷洒昆虫。 所述的农药组合物可被制成喷洒剂, 从而可直接 喷洒用于害虫防治。
具体地, 可合成 dsRNA直接喷洒, 用于害虫防治, 合成 dsRNA纯化物加入渗透剂 或鞣化剂喷洒田间用于害虫防治。
本发明针对鳞翅目害虫防治难题, 基于 RNAi技术开发了可用于田间防治昆虫的靶 标基因及其有效片段 (结构域), 并证明了通过喷雾这种简便的方法可以使核酸抑制物起 到抑制基因表达的效果, 最终 致鳞翅目害虫的死亡, 从而达到防治的目的, 所述方法 方便、 快捷、 准确且无公害。 本发明的主要优点在于:
(1) 本发明首次揭示以及确认了全新的, 可用于作为防治昆虫的靶点的靶标基因。
(2) 本发明首次揭示了利用基因的不同结构域可以分别合成特异型和广谱型的用于 防治鳞翅目害虫的靶标基因, 同时证明了基于这些基因结构域获得的 dsRNA可直接应 用于害虫防治。 所述害虫防治方法方便、 快捷、 准确, 并能很好地解决目前害虫防治中 所面临的害虫抗性和环境污染问题。
(3) 本发明基于 RNAi技术来实现杀虫的目的, 这是一种新型高效、 定点控制、 无公 害的技术; 获得的 dsRNA可直接应用于田间, 进行害虫防治。
(4) 筛选出物种特异的靶标基因片段, 将对非靶标的影响程度降到最低。
(5) 筛选出同类物种广谱型的靶标基因片段, 将防治成本降到最低。
(6) 开发成本低, 可快速用于生产。
(7) 使用方便、 环境兼容性好。 下面结合具体实施例, 进一步阐述本发明。 应理解, 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法, 通常按照常规 条件如 J.萨姆布鲁克等编著, 分子克隆实验指南, 科学出版社, 2002中所述的条件, 或 按照制造厂商所建议的条件。 除非另外说明, 否则百分比和份数按重量计算。
除非另行定义, 文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义 相同。 此外, 任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。 文中所 述的较佳实施方法与材料仅作示范之用。
实施例 1、 获得靶标基因信息
以亚洲玉米螟的分析为例, 方法步骤如下:
1. 亚洲玉米螟总 RNA的提取
采用常规 Trizol法提取,常规方法纯化, DNA酶处理,获得浓度≥300η8/μ1、总量≥6 g、 OD260/280为 1.8〜2.2的 Total RNA样品。
2. mRNA的分离及 cDNA的合成
用带有 oligo-dT的磁珠分离出带有 polyA的 mRNA,然后用随机 6聚物和 Invitrogen 的 Superscript II reverse transcriptase试剂盒合成 cDNA第一链。
3. 基因的扩增及测序
利用靶标基因特异的引物 (OfSPF: CATGGGGACACAGTTGAGCTTC(SEQ ID NO: 14); OfSPR: TGCTGTAAATCTAGCCTCAGTCA(SEQ ID NO: 15))进行扩增, 将获得的 基因片段纯化, 连接到 (Takara公司生产) PMD- 18载体中, 转化入 ToplO菌株, 蓝白斑 筛选, 阳性菌株测序, 亚洲玉米螟贮藏蚩白 2全长序列的测序结果如下 (SEQ ID NO: 1; 其
gcaaatgttcatcttgaaattacttaaccacgttctggagcccgtcatgtacaaggaaatcgaagatattggcaa
ctactactactacttccacgtcgactatccgttctggatgagagacgaatgcttcgacaagatcaggatcaggcg tttcgaacttactctgtacg atacgaaatgatcatcaaggaagctattgtcaagggattCRtt^aagtcaatggcttaagactggaactgaccaa
caaactggtcaatggcaagaacactattgtcaggaactccttcgacatgcacaacctggtccgcgaccgcatcat gacccgcRacctctRRaaRaaggttgacaccatcact acttcagagatctccttgtcaaggacctcagaaactt
ccacactggtttccccaccaggttgcttctRcccagaagtaaggttggaggtatgaagatgatgctgtacgtcat cgtgactcctctgaagttggtcgacaacgttgacctcaacattctggacaccaaccgcaaagacttcatgatgga cttcactcctaacatgaagtttgtcgatgttat^atcttccacaaKaagcaggtttgcgacatgaagactcgctg gaacaaatacgtgttgaagaactacgacatgtatgacaggaccatgatcaccgatgacacctccttcgtcgacat cgacatccacacgaagtcaatggtagaccatgacgtcaacRtgttcgacaattactaaatsKtaataatattcct aatttaataaacaaaaaaaaaaaaaa 编码的蚩白序列 (SEQ ID NO: 2; 755aa):
WNKYVL丽 DMYDRTMI TDDTSFVD I D I HTKSMVDHDVNVFDNY 实施例 2、 dsRNA 的合成
利用本发明所述的方法, 运用于玉米螟的研究当中, 本发明人选取贮藏蛋白 2基因 的三个结构域 (SP-N, SP-M, SP-C , 序列见表 2), 扩增该三个结构域所需引物见表 3。
表 2、 贮藏蚩白 2基因的三个结构域序列
caagaaccgcatgaacttcgtcgaactcgacagcttcctctacaaactggtcaatggcaagaaca ctattgtcaggaactccttcgacatgcacaacctggtccgcgaccgcatcatgacccgcgacctc
aaagacttcatgatggacttcaggtcgactgttcttttggacaagaggcctcttggtttcccatt cgaccgtcgcattgatgtagtgaagttcttcactcctaacatgaagtttgtcgatgttatgatct tccacaagaagcaggtttgcgacatgaagactcgctggaacaaatacgtgttgaagaactacgac at g t t gac ggac catgatc ccgatg(SEQ ID NO; 5) 表 3、 合成 dsRNA所用引物
SEQ ID
名称 引物序列
NO:
正向 6 TAATACGACTCACTATAGGGAGACTATTGGTTTTGGCCATTGCGG dsOfSP_N
反向 7 TAATACGACTCACTATAGGGAGATGACGGCTACAGTGAGAGCATA 正向 8 TAATACGACTCACTATAGGGAGATGACAAGCTTCGCTACTTCACC dsOfSP_M
反向 9 TAATACGACTCACTATAGGGAGAGTAGAAGACTGGGTCACGGAGA 正向 10 TAATACGACTCACTATAGGGAGAGACTGCGTCATCAGGATCTTCC dsOfSP_C
反向 11 TAATACGACTCACTATAGGGAGACATCGGTGATCATGGTCCTGTC 正向 12 TAATACGACTCACTATAGGGAGGACGACGGCAACTACAAG
EYFP
反向 13 TAATACGACTCACTATAGGGGAACTCCAGCAGGACCATGT 其中, EYFP为增强型黄色荧光蚩白基因, 作为外源基因对照。 以表 3所述的引物分别扩增贮藏蚩白 2基因的三个结构域 SP-N, SP-M, SP-C的 DNA 序列, 作为合成 dsRNA的模板。
利用 MEGAscript® T7 Kit (购自 AMBION 公司, 产品货号 AM 1334) 进行靶标基因 dsRNA的合成, 获得可用于生物测定的 dsRNA。 其简要原理及步骤是: 首先用亚洲玉 米螟幼虫组织的总 RNA为模板, 反转录获得 cDNA, 然后以表 3所列的 dsRNA合成引 物扩增不同 OfSP 基因的结构域, 对其产物进行纯化, 再以纯化产物为模板, 利用 Kit 试剂盒进行体外转录, 获得 dsRNA后, 再去除产物中的 DNA, 并进行纯化处理, 就可 以获得所需的 dsRNA。 具体操作步骤如下:
以前述获得的三个结构域的 DNA序列为模板, 按照 AM1334 Kit中的说明书进行 操作- 1) 体外转录获得 dsRNA
ATP 2 μL·
GTP 2
CTP 2 μL·
UTP 2 μL·
T7 Enzyme Mix 2
模板 DNA 0.1 μL
10X 反应缓冲液
Neclease-free Water 加至 20 μΐ
混合均匀, 短暂离心, 37°C孵育 4h。
2) 消除 DNA
体外转录反应结束, 酶化反应体系中含有少量模板 DNA, 需要在 DNasel作用下消 化掉, 在上述反应液中加入 Ι μί 的 Dnasel, 混合均匀, 37孵育 30 min。
3) dsRNA的纯化
上述反应体系中:
加入 lOO L和 15 L醋酸铵终止反应;
加入等体积酚 /氯仿 /异戊醇混和液 (25:24: 1):
4°C 15 OOO r/min, 离心 15 min, 取上层水相转移至新的 1.5 mL离心管中; 加入 140 (等体积) 氯仿 /异戊醇混和液 (24: ]);
加入 12μί (1/10体积) 3Μ 醋酸钠溶液 (pH5.5);
加入 2倍体积无水乙醇, -20Γ过夜沉淀 RNA;
12 000 r/min离心 15-30 min;
弃上清, 加入 70%乙醇 250μί, 室温轻轻吹打洗涤:
12 000 r/min离心 10 min, 弃上清, 室温干燥沉淀; 用适量 TE溶解沉淀。
最终获得的 dsRNA分别命名为: dsSP-N、 dsSP-M dsSP-C , 电泳鉴定结果见图 4, 可见成功获得了所需的 dsRNA。 实施例 3、 dsRNA对亚洲玉米螟的基因的沉默作用
本发明人将获得的 dsSP-C (溶于 ddH20)直接喷雾于亚洲玉米螟 1龄幼虫, 观察其基 因表达情况的变化。 喷雾后贮藏蚩白 2基因 ( O yP) 第 0、 1、 3、 5天的基因表达情况 见图 5。
从图 5的结果可以看出, 用双链 RNA处理后第 5天, 该基因的表达被显著抑制。 同 样地, 蚩白电泳鉴定发现, 贮藏蚩白 2的表达也被显著抑制。 实施例 4、 dsRNA对亚洲玉米螟的致死效率
利用本发明所述的方法, 运用于亚洲玉米螟的研究当中, 本发明人将获得的 dsRNA(dsSP-N、 dsSP-M、 dsSP-C分别溶于 dd 0)直接喷雾于亚洲玉米螟初孵幼虫上(以 dsSP- N第 7天的致死中浓度 LC5。为标准浓度,浓度为 41.55 g/mL),观察在喷药后第 1、 3、 5、 7、 9天 (d)幼虫的致死情况。
结果如图 1, 以本发明的靶标基因的三个结构域 (SP-N, SP-M, SP-C)为模板合成的 dsSP-N、 dsSP-M, dsSP-C分别地喷雾于亚洲玉米螟后, 它们在第 7天的致死率分别为 51.36%、 32.14% 51.43%, 而空白对照 (CK)和外源基因对照 (EYFP)没有显著致死效果。
分析结果显示, 三种 dsRNA的致死效率差异均显著, 其中针对 SP-N, SP-C两个结 构域的 dsRNA效果更优。 由此表明, dsRNA可以通过昆虫体表进入昆虫体内, 抑制靶 标基因, 起到杀虫作用, 所以基于本发明靶标基因的其它形式的核酸抑制物也可以通过 喷洒昆虫起到杀虫作用。 实施例 5、 亚洲玉米螟的 dsRNA对棉铃虫的致死效率
利用本发明所述的方法, 应用于棉铃虫的研究当中, 本发明人将获得的 dsRNA(dsSP- N、 dsSP-M, dsSP-C 分别溶于 dd¾0)直接喷雾于棉铃虫的初孵幼虫上(以 dsSP- N第 7天的致死中浓度 LC5Q为标准浓度,浓度为 41.55 g/mL),观察在喷药后第 1、 3、 5、 7、 9天 (d)幼虫的致死情况。
结果如图 2, 以本发明的靶标基因的三个结构域 (SP-N, SP-M, SP-C)为模板合成的 dsSP-N , dsSP-M、 dsSP-C分别地喷雾于棉铃虫的初孵幼虫上后, 它们在第 7天的致死 率分别为 13.33%、 19.85%、 45.24%。
分析结果显示, 三种 dsRNA均有一定的致死作用, 其中针对 SP-C结构域的 dsRNA 效果最优。 可见, SP-C可作为棉铃虫防治的较好的靶标结构域。
可能由于喷洒不均匀, 尽管 dsSP-C处理的死亡率仅有 45.24%。 但进一步的观察发 现, 即使在幼虫期没有立即死亡, 有一部分会出现化蛹异常而死亡, 结果见图 9。
再进一步观察, 还有个别能比较正常化蛹的虫子, 尽管也能羽化为成虫, 但是其所 产的卵却不能孵化出下一代的幼虫。 如图 10所示, 3-5天后, 未处理的对照已经转色, 将要孵化, 但处理过成虫所产的卵还未转色, 最终也没能孵化出幼虫。 实施例 6、 dsRNA对亚洲玉米螟的致死效率
利用本发明所述的方法, 运用于亚洲玉米螟的研究当中, 本发明人将获得的 dsRNA(dsSP-N dsSP-M、 dsSP-C分别溶于 dd¾0)直接喷雾于亚洲玉米螟初孵幼虫上(以 dsSP-N第 7天的致死中浓度 LC5o为标准浓度, 浓度为 41.55 μ g/mL), 观察在喷药后第 1、 3、 5、 7天 (d)幼虫的致死情况。
结果如图 6, 以本发明的靶标基因的三个结构域 (SP-N, SP-M, SP-C)为模板合成的 dsSP-N、 dsSP-M、 dsSP-C 分别喷雾于亚洲玉米螟后, 它们在第 7 天的致死率分别为 74.82%、 73.13%、 76.69%, 而空白对照 (CK)和外源基因对照 (EYFP)没有显著致死效果。
分析结果显示,三种 dsRNA 与对照相比的致死效率差异均达到显著水平,其中针对 SP-N, SP-C两个结构域的 dsRNA 效果更优。 由此表明, dsRNA 可以通过昆虫体表进 入昆虫体内, 抑制靶标基因, 起到杀虫作用, 所以基于本发明靶标基因的其它形式的核
酸抑制物也可以通过喷洒昆虫起到杀虫作用。
由于喷洒更均匀, 死亡率比前次实验 (见实施例 4, 最高仅为 51%) 有所提高, 死 亡率已经超过 70%, 进一步的观察发现, 即使在幼虫期没有立即死亡, 有一部分会出现 化蛹异常而最终死亡, 结果见图 7。
再进一步观察, 还有个别能比较正常化蛹的虫子, 尽管也能羽化为成虫, 但是其所 产的卵却不能孵化出下一代的幼虫。 如图 8所示, 处理过的成虫所产的卵发育异常, 不 能孵化。 实施例 7、 棉铃虫自身 SP基因的 dsRNA对棉铃虫的致死率
利用本发明所述的方法, 应用于棉铃虫的研究当中。 棉铃虫中与玉米螟 sp基因同源 的基因 PxSPl的核苷酸序列见 SEQ ID NO. 16。
(1) 棉铃虫中与玉米螟 sp基因同源的基因 (H.arm-SP)的 dsRNA的制备
本发明人制备了棉铃虫中与玉米螟 sp基因同源的基因 (H.arm-SP)的 dsRNA, 所针对 的玉米螟 sp基因同源的基因片段如下 (SEQ ID NO: 17):
AATTATGA ,CCAAAACCCCGGCCTT(CAGAGGAT AACACCCA .T' GGCCAAAAAACCAATTG( GTTG : TTCAACCTACAAATGGGACAACAATGTGGTGA T' CAGGTGGAATGCTACG, ACTTAAGAAGCGAAACCATGCCT CC TTGCTTAC TTCACTCA ■TTGGAACCTTTCAGC*
参照实施例 2中提供的方法, 利用 MEGAscript® T7 Kit (购自 AMBION 公司, 产品 货号 AM1334) 进行靶标基因 dsRNA的合成。 引物序列如下:
H.arni SP DSF TMTACGACTCACTATAGGGAGACGGTGAAATTATGACAACCGCTC(SEQ ID NO: 30) H.arm SP DSR TMTACGACTCACTATAGGGAGAGACGTAGTTGTTGTGGTACTGG(SEQ ID NO. 31 ) (2) 效果验证
本发明人将获得的棉龄虫中与玉米螟 sp 基因同源的基因(H.arm-SP)的 dsRNA ( dsH.arm-SP, 溶于 dd¾0)直接喷雾于棉铃虫的初孵幼虫上(以亚洲玉米螟 (Of) dsSP-N 第 7天的致死中浓度 LC5。为标准浓度, 浓度为 41.55 w g/mL),观察在喷药后第 1、 3、 5、 7天 (d)幼虫的致死情况。
结果如图 1 1 , 以本发明的靶标基因的为模板合成的 dsH.arm-SP喷雾于棉铃虫的初孵 幼虫上后, 在第 7 天的致死率为 73.33%。 实施例 8、 小菜蛾自身的 SP基因的 dsRNA对小菜娥的致死率
利用本发明所述的方法, 应用于小菜蛾的研究当中。 小菜蛾中与玉米螟 sp基因同源 的基因 PxSP l的核苷酸序列见 SEQ ID NO: 18; 小菜蛾中与玉米螟 sp基因同源的基因 PxSP2的核苷酸序列见 19。
(1) 小菜蛾中与玉米螟 sp基因同源的基因 (PxSPl、 PxSP2)的 dsRNA的制备 本发明人制备了小菜蛾中与玉米螟 sp基因同源的基因 (PxSPl、 PxSP2)的 dsRNA, 所 针对的小菜蛾 sp基因同源的基因片段如下:
dsPxspl(SEQ ID NO: 20):
ACGTCGACCGC C AGATGAAGGAGGTTGTCAAAGCTTTCCATCTCCTTT A TTTCGCAAA' ACTTCACCACr T TCCTCAAGACTGCCTGCTGGATGCGTCTCTACCTGAAC GAGGGCAT<
CAAAATAAGGCGTGGCGAAATCTGTTTATA CA T GATGCAACAACTGCTTG CCAGGCAl CCTTGAACGTCTTTCCAACGGTTTGGG CGA T TACTACCCTGTCCTGGAA CA AACCC.
CAAGAAGGGATTCGTGCCCTGGATGACCCTGCACAA DsPxsp2(SEQ ID NO: 21):
TGTACCTCAACCGCGACG A, CAAACTCAG( GTA C' TTCACTGAAGACATCGACCTGAACA CC TACTACTACTACTTCCACG JT' TGACTATCCGTTCT G< GATGAGGGATGACATCTTCAGGAAC GATTTCACTAAGACCAGG C' GTGGAGAAATCTGCI GC
CCGACACTACTTAGAGCG C T T PC' AA GCCl GATCACGACT
ACAAACCCATCAAGAAGG G AT T CGT GCCC TGG ATGGCGC: TGCACAACGGTGT
参照实施例 2中提供的方法, 利用 MEGAscript® T7 Kit (购自 AMBION 公司, 产 f 货号 AMI 334) 进行靶标基因 dsRNA的合成。 引物序列如下:
Px SP1 DSF TAATACGACTCACTATAGGGAGAACGTCGACCGCCAGATGAAGGA(SEQ ID NO: 32) Px SP1 DSR TMTACGACTCACTATAGGGAGATTGTGCAGGGTCATCCAGGGCA(SEQ ID NO: 33) Px SP2 DSF TAATACGACTCACTATAGGGAGAACGTCGACCGCCAGATGAAGGA(SEQ ID NO: 34) Px SP2 DSR TAATACGACTCACTATAGGGAGAACACCGTTGTGCAGCGCCAT(SEQ ID NO. 35)
(2) 效果验证
本发明人将获得的小菜蛾 SP 1和 SP2的 dsRNA(dsPxSPl和 dsPxSP2分别溶于 ddH20) 直接喷雾于小菜蛾的初孵幼虫上 (以亚洲玉米螟 dsSP-N 第 7 天的致死中浓度 LC50 为 标准浓度, 浓度为 41.55 g/mL), 观察在喷药后第 1、 3、 5天 (d)幼虫的致死情况。
结果如图 12, 以本发明的小菜蛾的 2个靶标基因的双链 RNA(PxSP l和 PxSP2)为模 板合成的 dsRNA分别地喷雾于小菜蛾的初孵幼虫上后, 它们在第 5天的致死率分别为 58.00%和 54.67%,并以玉米螟的 dsSP测试了对小菜蛾的致死率, 5天死亡率为 14.67%,
与对照无显著差异。 实施例 9、 dsRNA的应用
通过将玉米螟贮藏蚩白基因 ΟβΡ与其它昆虫的基因进行比对,从图 3的结果可以看 出, 亚洲玉米螟 基因与棉铃虫的同源关系相对最远, 但利用亚洲玉米螟 SP-C 的 dsRNA可以在棉铃虫中得到同样的杀虫效果, 可以预期, 它对鳞翅目其它害虫也会产生 相似的作用效果。 实施例 10、 SP-N、 SP-M、 SP-C截短形式及其效果
本发明人基于 SP-N (SEQ ID NO: 3)、 SP-M (SEQ ID NO: 4)和 SP-C(SEQ ID NO: 5) 设计了截短形式的 dsRNA片段。 如下:
SP-N- Ml dsRNA:合成和纯化方法如实施例 2,不同点在于靶标基因片段为 SEQ ID NO: 3中第 1-420位。
SP-N-M2 dsRNA:合成和纯化方法如实施例 2,不同点在于靶标基因片段为 SEQ ID NO: 3中第 10-420位。
SP-M-M1 dsRNA:合成和纯化方法如实施例 2,不同点在于靶标基因片段为 SEQ ID NO: 4中第 1-615位。
SP-M-M2 dsRNA:合成和纯化方法如实施例 2,不同点在于靶标基因片段为 SEQ ID NO: 4中第 10-615位。
SP-C-M1 dsRNA:合成和纯化方法如实施例 2,不同点在于靶标基因片段为 SEQ ID
NO: 5中第 1-603位。
SP-C M2 dsRNA;合成和纯化方法如实施例 2,不同点在于靶标基因片段为 SEQ ID NO: 5中第 10-603位。
如实施例 4的方法, 将以上获得的 6种 dsRNA分别喷洒于亚洲玉米螟和棉铃虫初孵 幼虫上 (浓度为 41.55 g/mL)。 在施用后第 7天, 这些 dsRNA对于幼虫的致死率均达到 10%以上。 实施例 11、 转基因载体的构建和转化
1、 35S::dsGFP和 35S::dsOfSP— C表达载体的构建
所需构建的 dsRNA载体如阁 13中 pART27-dsRNA所示, 包括一个 35S启动子, 一 个反向的基因片段 (即 Anti-sense) , 一个 PDK (丙酮酸磷酸激酶) 基因的内含子 (即 Intron, 约 742bp) , 一个正向的基因片段 (即 Sense C1 ) 和 OCS终止子, 以及 NOS启 动子, ΝΡΤ Π (新霉素磷酸转移酶基因) 和 NOS 终止子。 该表达载体是通过包含 Sense-Intron-Antisense序列的双元载体 pKANNIBAL载体 (购自中国质粒载体菌株细胞株 基因保藏中心)通过 Not I酶切后连入同样经过 Not I酶切的 pART27载体 (购自中国质粒
载体菌株细胞株基因保藏中心)而构建获得。
用含有 Hindlll和 Xba I 酶切位点的基因特异引物 dsGFP_lF—HindIII和 dsGFP lR Xba I; dsOfSP— C 1F— Hindlll和 dsOfSP— C 1 R— Xba I用高保真酶 KOD,分别以 GFP (来源于质粒 Pcambial 302, 参加 http:〃 www.cambia.org/daisy/bios/585. html , 该基因 不存在于亚洲玉米螟中) , 以及亚洲玉米螟的 cDNA为模板, 进行 PCR扩增, 克隆相 应的 dsGFP—l和 dsOfSP_C l片段。 再将克隆到的片段用 Hindin和 Xba I进行双酶切, 分别***到 pKANNIBAL 载体上的克隆位点 (HindIII/Xba I ) 之间, 此即为正向片段 ( Sense ) 。
用相同的方法将含有 Kpn I和 Xho I酶切位点的基因特异引物 dsGFP— 2F一 Kpn I和 dsGFP_2R_Xho I; ds0fSP_C2F_Kpn I和 ds0fSP_C2R— Xho I用高保真酶 KOD, 分别以 GFP以及亚洲玉米螟的 cDN A为模板,进行 PCR扩增,克隆相应的 dsGFP一 2和 dsOf SP— C2 片段。 再将克隆到的片段用 Kpn I和 Xho I进行双酶切, 进而***到相应的已经含有正 向 dsGFP—l和 dsOfSP— C 1片段的 pKANNIBAL载体上的克隆位点(Kpn I /Xho I )之间。
将构建好的 pKANNIBAL/dsGFP和 pKANNIBAL/dsOfSP— C载体分别用 Not I进行酶 切, 同时将 pART27用 Not l进行酶切, 分别将酶切下来的 dsGFP和 dsOfSP_C片段插 入到 pART27载体上,从而获得分别携带相应目的片段的重组表达载体,分别称为 35 S : : dsGFP和 35S : : dsOfSP— C表达载体。
前述制备过程中采用的引物如表 4。
表 4
2、 根癌农杆菌的转化
根癌农杆菌的转化采用冻融法。一个单菌落 GV3101、 LBA4404或 EHA105 (均购自 Invitrogen公司), 3ml LB培养基(含 50 μβ/ηι1 卡那霉素 Kan和 25 g/ml利福霉素 Rif), 28。C, 220 rpm , 培养到 OD600=0.5 (约 6小时) 。 在冰上放置 30分钟, 4°C, 5000 g 离心 5分钟。 重悬于 l OmL 0.15M NaCl。 4。C, 5000 g离心 5分钟。 重悬于 1 mL 20 mM
CaCl2, 50 μυ管分装, 液氮速冻, - 80°C保存感受态细胞。 混合含目的基因的双元载体 和 50 μΙ7管感受态细胞, 在冰上放置 30分钟, 液氮速冻 1分钟。 在 37°C水浴中 5分钟 使菌液融化, 力卩 l mL LB培养基, 28°C, 220 rpm, 培养 2~4小时。 取 50~100μ 涂 LB 培养基平板 (含 50 μδ/ηι1卡那霉素 Kan和 25 μ^ηιΐ利福霉素 Rif) 实施例 12、 植物的转化以及转基因后代的筛选
在本实施例中, 以拟南芥、 烟草以及玉米的转基因为例, 其它植物的转化也可以此 为参照。
1. 拟南芥的转基因
拟南芥植物的转化采用花芽浸泡法(floral dip)。一个含双元载体的单菌落 GV3101,
3 mL LB 培养基(50 g/ml卡那霉素 Kan、 50 μβ/ιη1庆大霉素 Gen和 25 μ^ηιΐ利福霉素 Rif) , 28°C, 220 rpm, 12小时。 2 mL菌液, 50 mL培养基 (50 g/ml 卡那霉素 Kan、 50 μ^ηιΐ 庆大霉素 Gen和 25 \aglm\利福霉素 Rif), 28。C, 220 rpm, 12小时。 4200 rpm, 15分钟。菌体重悬于 500 mL含 0.02 % Silwet L-77的 5% 蔗糖溶液中。植株花芽部分在 菌液中浸泡 5秒钟, 平放于塑料盆内, 保湿, 避光, 16~24小时, 然后温室生长至开花 结籽。 TO代种子在 4°C春花 2 4天, 用 20%漂白水处理 15分钟, 无菌水清洗 3~ 遍。 悬于 0.5 %的琼脂糖(55°C) ,铺在 0.6 %琼脂的 LB培养基(含 50 g/ml 卡那霉素 Kan), 22°C, 连续光照, 约 1周后, 绿色抗性苗移栽到营养土 (泥炭: 蛭石: 珍珠岩 =1: 1: 1) 中生长。
2. 烟草的转基因
将含有目的基因的农杆菌 LBA4404过夜培养 (28°C培养过夜, 至 OD600 2.0) 。 将无菌烟草叶片切割成约 1.0 cm2大小, 浸泡在农杆菌培养液中 5~10分钟。 用无菌滤纸 吸去多余的农杆菌培养液, 将浸泡过的烟草叶片铺在 1/2 MS 阖体培养基上, 暗培养两 天。 然后, 将叶片移至 MS (含 1 mg/L 6-BA) |≤|体筛选培养基(Kan', Cefr) , 隔 10〜15 天更换新鲜 MS培养基, 直至叶片伤口处长出小芽, 将新长出的小芽移至不含 6- BA的 MS培养基, 隔 10 15天更换新鲜 MS, 直至其长根后, 移至土壤中种植。
MS培养基: 4.4 g/L Murashige and Skoog basal medium (Sigma, Cat. M5519) , 蔗 糖 15 g/L, 0.8% 琼脂粉, MES 0.5 g/L, pH 5.7。
3. 玉米的转基因
将含有目的基因的农杆菌 EHA105在含 50 g/ml卡那霉素 Kan和 50 g/ml利福霉素
Rif的 YEP液体培养基中 28°C震荡培养至 OD600 0.6, 8000 rpm离心 5分钟收集农杆 菌。菌体重悬于侵染培养基中,在农杆菌转化前向重悬液中乙酰丁香酮(AS) 100 μηιοΙ/L 和 0.05 %的表面活性剂 Silwet L- 77。 挑选整齐饱满的玉米种子, 用 70 %的乙醇浸泡 2~5 分钟, 然后用 0.1 % HgC12浸泡 10~ 5分钟, 再用无菌水冲洗 3~5次。 将灭菌种子放在 无菌的三角瓶中, 加适量无菌水在培养箱中 25°C暗培养待萌发, 种子发芽后转入萌发
培养基上继续生长, 待苗长至 4 cm时剥离胚芽鞘和幼叶, 暴露出茎尖生长点, 用手术 刀轻微挫伤用于转化。将茎尖浸泡在重悬液中并置于真空干燥器中, 在 50 kPa压力侵染 7 min o 侵染结束后用无菌滤纸吸净种子上多余 ide菌液, 然后放在萌发培养基上, 25°C 黑暗条件下继续培养, 直至重新长出新叶片。 将长出新叶片的转化苗用清水洗掉菌体后 移栽到花盆中, 下部为土壤, 上部为 6^8 cm的蛭石。 2 3周后转化苗长至三叶期, 用 300 ppm的除草剂 (农达) 喷洒在玉米叶片上进行转化植株的筛选, 经过筛选后将存活 的植株移栽到温室中。 实施例 13、 转基因植物的分子生物学鉴定
在本实施例中,选取实施例 12获得的 T3代纯合株系转基因植物,采用抗生素筛选、
Northern杂交对转基因植株进行验证。
用于 Northern检测的转膜:
转膜: 采用常规方法提取转基因植物的总 RNA样品, 在 RNA样品中加入 lOx电泳 加样液,混匀, 65°C放置 10分钟, 冰上冷却。 电泳使用 15%TBE-urea PAGE胶, Ι χΤΒΕ 电泳缓冲液。 上样前预电泳 5~10分钟并用电泳缓冲液冲洗泳道, 除去上样孔中析出的 尿素。 每泳道加样量为 10~20 总 RNA, 电场强度 20 V/cm, 至溴酚蓝染料迁移至凝 胶的底部时结束。凝胶在 l xTBE中平衡 10分钟。采用半干电转移***(Hofer Semi-Dry Transfer Units, Amersham, Cat. 80-621 1-86 )转移 RNA。转移条件: 40 mA (约 7-8 V), 2~4小时, Hybond-N+ ( Amersham, Cat. RPN303B)尼龙膜。 尼龙膜经 6^SSC溶液短暂 漂洗, 紫外交眹 (120 mJ ) , 夹在两层滤纸之间, 80°C烘烤 1小时, 备用。
ΙΟχΤΒΕ: 0.9 M Tris, 0.9 M硼酸, 20 mM EDTA ( pH 8.0 ) ( Sigma, Cat. T4415 ) ; ΙΟχ电泳加样液: Ambion, Cat. 8546G;
电泳胶储存液: 15%聚丙烯酰胺(30% Acyl/bis, 19: 1 , 华舜, Cat. W443 ) , 8M尿 素, Ι χΤΒΕ;
15% TBE-urea PAGE胶( 10 mL): 10 mL电泳胶储存液, 80 10% 过硫酸铵, 5
TEMED, 混匀;
探针标记:取 25 ng纯化 PCR产物作为模板标记探针。探针的标记使用 Prime-a-Gene Labeling System ( Promega, Cat. U1 100) 。 37°C水浴 1小时。 标记探针在沸水中放置 5 分钟, 立即置于冰上, 备用。
预杂交和杂交采用(Clontech的 ExpressHyb体系):将尼龙膜放入杂交管中,以 6xSSC 润湿, 保证膜和管壁间没有气泡。 倒掉 6x SSC, 加入 5 mL杂交液, 37°C预杂交 60分 钟。 预杂交结束后, 更换 5 mL新鲜杂交液, 加入探针, 混匀, 杂交过夜。
洗膜与压片: 杂交结束后, 倒出杂交液。 在室温下, 用 2xSSC, 0.05% SDS , 洗膜 两次, 每次 5分钟, 然后用 0.2xSSC, 0.1%SDS , 在洗涤两次, 每次 20分钟。 用保鲜膜 包裹, 胶带固定, 压增感屏和 X-ray胶片, -70°C, 2天。 胶片用 D-72液显影。
Northern检测表达含 GFP和 OfSP— C基因序列 dsRNA的转基因拟南芥、烟草和玉米。 实施例 14、 检测表达 OfSPj 基因序列 dsRNA的转基因植物对玉米螟的影响 取生长一致的三日龄玉米螟, 分别喂以 dsGFP (对照) , dsOfSP— C转基因拟南芥、 烟草或玉米, 每天观察玉米螟幼虫的表型。 结果证明, 取食 dsOfSP—C转基因玉米的幼 虫死亡率显著高于取食 dsGFP的转基因玉米。 在本发明提及的所有文献都在本申请中引用作为参考, 就如同每一篇文献被 单独引用作为参考那样。 此外应理解, 在阅读了本发明的上述讲授内容之后, 本 领域技术人员可以对本发明作各种改动或修改, 这些等价形式同样落于本申请所 附权利要求书所限定的范围。
Claims
1. 一种分离的多肽, 其特征在于, 该多肽选自下组:
(a) SEQ ID NO: 2所示氨基酸序列的多肽;
(b) 将 SEQ ID NO: 2任一所示氨基酸序列经过一个或多个 (如 1 -20个; 较佳地 1-15 个; 更佳地 1-10个; 更佳地 1-5个; 如 3个)氨基酸残基的取代、 缺失或添加而形成的, 且具有 (a)多肽功能的由 (a)衍生的多肽。
2.—种分离的多核苷酸,其特征在于,它包含一核苷酸序列,该核苷酸序列选自下组; (a) 编码如权利要求 1所述多肽的多核苷酸;
(b) 与多核苷酸 (a)互补的多核苷酸。
3.—种分离的多核苷酸, 其特征在于, 它包含一核苷酸序列, 该多核苷酸选自下组的 一种- (a) SEQ ID NO SEQ ID NO: 3-5 > SEQ ID NO: 17、 SEQ ID NO: 20-21任一所示序 列的多核苷酸;
(b) 与 (a)限定的序列在严格条件下杂交的任一多核苷酸或互补序列, 其中包含所述 的任一条核苷酸的 dsRNA被有害生物摄取后, 会抑制所述有害生物的生长;
(c) 与 (a)限定的序列具有至少 70%以上的序列一致性的任一多核苷酸或互补序列, 其中包含所述的任一条核苷酸的 dsRNA被有害生物摄取后, 会抑制所述有害生物的生 长;
(d) 包含 (a)限定的任一所示序列之至少】7-21个连续核苷酸的序列或互补序列,其中 包含所述的任一条核苷酸的 dsRNA被有害生物摄取后, 会抑制所述有害生物的生长。 4. 一种载体, 其特征在于, 它含有权利要求 2或 3所述的多核苷酸中的一种、 二种 或多种。
5. 一种遗传工程化的宿主细胞, 其特征在于, 它含有权利要求 4所述的载体, 或其 基因组中整合有权利要求 2或 3所述的多核苷酸中的一种、 二种或多种。
6. 权利要求 2或 3任一所述的多核苷酸的用途, 用于作为制备特异性干扰有害生物 基因表达或抑制有害生物生长的干扰分子的抑制或沉默靶标, 所述的有害生物基因选自 贮藏蚩白基因。
7. 一种核酸抑制物, 选自:
(a) 以权利要求 2或 3任一所述的多核苷酸作为抑制或沉默靶标的 dsRNA、 反义核
酸、 小干扰 RNA、 微小 RNA, 其中所述核酸抑制物被害生物摄取后, 会抑制所述有害 生物的生长; 或
(b)以有害生物基因作为抑制或沉默靶标的 dsRNA、 反义核酸、 小干扰 RNA、 微小 RNA, 其中所述核酸抑制物被有害生物摄取后, 会抑制所述有害生物的生长, 所述的有 害生物基因选自贮藏萤白基因; 或
(c) 能表达或形成 (a)、 (b)所述 dsRNA、 反义核酸、 小干扰 RNA、 微小 RNA的构建 物。
8. 如权利要求 7所述的核酸抑制物, 其特征在于, 所述的核酸抑制物是权利要求 2 或 3所述的多核苷酸表达产生的 dsRNA, 其中所述的 dsRNA被有害生物摄取后, 会抑 制所述有害生物的生长。
9. 如权利要求 8所述的核酸抑制物,其特征在于,所述的 dsRNA,其具有如下结构:
Seq 'in -、、
ι,ι χ,
Seq,s向一'、一一, 或 Seq,正向 -X,-Seq,反向
其中,
Seq'Efi是与权利要求 2或 3任一所述的多核苷酸所对应的 RNA序列,或它们的序列 片段, 或包含与选自权利要求 2或 3中所限定序列的核苷酸序列之至少一部分互补的核 苷酸序列所对应的 RNA序列;
Seq,£Pl为与 Seq'^ ^基本上互补的序列;
X'为无;或为位于 Seq,Eia和 Seq' 之间的间隔序列,所述间隔序列与 Seq'^和 Seq' s向不互补;
10. 如权利要求 7所述的核酸抑制物, 其特征在于, 所述的核酸抑制物是构建物, 所 述构建物含有以下结构:
Seq 正向— X— Seq 反向',
其中,
Seq 是权利要求 2或 3任一所述的多核苷酸或它们的序列片段, 或包含与选自权 利要求 2或 3中所限定序列的核苷酸序列之至少一部分互补的核苷酸序列;
Seq 为与 Seq Ε 基本上互补的序列;
X为位于 Seq 和 Seq ^之间的间隔序列, 并且所述间隔序列与 Seq 和 Seq s向不 互补。 种宿主细胞, 其包含权利要求 7-10任一所述的核酸抑制物。
12. 权利要求 7-10任一所述的核酸抑制物或权利要求 5或 1 1所述的宿主细胞的用 途, 用于制备防治有害生物的制剂。
13. 一种防治有害生物的制剂, 其包含:
安全有效量选自以下的物质: 权利要求 7-10任一所述的核酸抑制物或权利要求 5或
1 1所述的宿主细胞; 以及;
农药学上可接受的载体。
14. 权利要求 12或 13中任一项的制剂, 其还包含至少一种选自以下的杀虫剂: 化学 杀虫剂、 马铃薯糖蚩白、 苏云金芽孢杆菌杀虫蛋白、 致病杆菌杀虫蚩白、 光杆状菌杀虫 蛋白、 侧孢芽孢杆菌杀 虫蚩白和球形芽孢杆菌杀虫蚩白。
15. 一种防治有害生物的方法, 其特征在于, 干扰有害生物基因表达, 所述的有害生 物基因选自贮藏蚩白基因。
16. 如权利要求 15所述的方法, 其特征在于, 所述方法包括: 将权利要求 7-10任一 所述的核酸抑制物或权利要求 5或 1 1所述的宿主细胞喂食 '和/或喷洒有害生物。
17. 如权利要求 15所述的方法, 其特征在于, 所述方法包括: 在植物体内表达特异 性干扰有害生物基因表达的干扰分子; 所述的有害生物基因选自贮藏蛋白基因。
18. 如权利要求 17所述的方法, 其特征在于, 所述方法包括: 将权利要求 7- 10任一 所述的核酸抑制物 入到植物中。 19. 一种植物或其种子, 其特征在于, 所述植物由权利要求 2或 3所述的多核苷酸转 化获得。
20. 如权利要求 19所述的植物, 其特征在于, 所述多核苷酸在植物细胞中表达为 dsRNA。
21. 如权利要求 3所述的多核苷酸, 或权利要求 7或 8所述的核酸抑制物, 或权利要 求 6或 12所述的用途, 或权利要求 13所述的制剂, 或权利要求 15-17所述的方法, 其 特征在于, 所述有害生物是选自: 昆虫、 螨、 真菌、 酵母、 霉菌、 细菌、 线虫、 杂草 和寄生虫以及腐生植物。
22. 如权利要求 3所述的多核苷酸, 或权利要求 7或 8所述的核酸抑制物, 或权利要
求 6或 12所述的用途, 或权利要求 13所述的制剂, 或权利要求 15- Π所述的方法, 其 特征在于, 所述的有害生物是昆虫, 包括但不限于: 鱗翅目昆虫、 鞘翅目昆虫、 半翅目 昆虫、 以及双翅目昆虫。
23. 如权利要求 3所述的多核苷酸, 或权利要求 7或 8所述的核酸抑制物,或权利要 求 6或 12所述的用途, 或权利要求 13所述的制剂, 或权利要求 15-17所述的方法, 其 特征在于, 所述的昆虫选自鳞翅目昆虫, 优选为玉米螟或棉铃虫或小菜蛾。
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Non-Patent Citations (6)
Title |
---|
DATABASE GENBANK 12 January 2010 (2010-01-12), FENG, C. ET AL.: "Ostrinia fumacalis storage protein mRNA, partial cds", accession no. U229935 * |
DATABASE GENBANK 15 April 2005 (2005-04-15), ZHENG, Y., accession no. AB55603 * |
DATABASE GENBANK 29 May 2011 (2011-05-29), ZHANG H. ET AL., accession no. EG19552 * |
DATABASE GENBANK 29 May 2011 (2011-05-29), ZHANG, H. ET AL.: "Ostrinia fumacalis storage protein mRNA, complete cds", accession no. E798635 * |
DATABASE GENBANK 4 August 2007 (2007-08-04), ASHFAQ, M. ET AL.: "Plutella xylostella PxSPl mRNA for methionine-rich storage proteinl, complete cds", accession no. B266595 * |
ZHOU, X. ET AL.: "RNA interference in the termite Reticulitermes flavipes through ingestion of double-stranded RNA.", INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY., vol. 38, August 2008 (2008-08-01), pages 805 - 815 * |
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