WO2016029765A1 - 杀虫蛋白的用途 - Google Patents

杀虫蛋白的用途 Download PDF

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WO2016029765A1
WO2016029765A1 PCT/CN2015/085047 CN2015085047W WO2016029765A1 WO 2016029765 A1 WO2016029765 A1 WO 2016029765A1 CN 2015085047 W CN2015085047 W CN 2015085047W WO 2016029765 A1 WO2016029765 A1 WO 2016029765A1
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plant
protein
cockroach
cry2ab
pest
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PCT/CN2015/085047
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English (en)
French (fr)
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李建勇
杨旭
张爱红
张欣馨
李梅
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北京大北农科技集团股份有限公司
北京大北农科技集团股份有限公司生物技术中心
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Publication of WO2016029765A1 publication Critical patent/WO2016029765A1/zh

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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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • 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
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof

Definitions

  • the present invention relates to the use of a pesticidal protein, and in particular to the use of a Cry2Ab protein to control a plant of the cockroach by expressing it in a plant.
  • Sesamia inferens belongs to the family Lepidoptera, and is an omnivorous pest. In addition to harming corn, it also harms grass crops such as rice, sugar cane, wheat and sorghum. It is widely distributed in central and southeastern parts of China, especially in Shaanxi. Most of the rice areas south of Henan. The larvae of the cockroaches are invaded into the stems of the crops, which can cause the death of the dead seedlings or the whole plant. The pupils are generally larger, and a large amount of worms are discharged from the stems, and the sorghum and wheat husks are heavier, and the summer corn is heavy. It occurs more heavily than spring corn.
  • Corn and sorghum are important food crops in China.
  • the annual food loss caused by the big cockroaches is huge, and even more affects the living conditions of the local population.
  • the main prevention methods commonly used are: agricultural control, chemical control and biological control.
  • Agricultural control is the comprehensive coordinated management of the multi-factors of the entire farmland ecosystem, regulating crops, pests, environmental factors, and creating a farmland ecological environment that is conducive to crop growth and is not conducive to the occurrence of large locusts.
  • large-scale wintering hosts reforming farming systems, planting large-scale varieties, planting traps and intercropping measures to reduce the damage of large cockroaches.
  • the application has certain limitations and cannot be used as an emergency measure. It seems to be powerless when the amnesty breaks out.
  • Chemical control that is, pesticide control
  • the chemical control methods mainly include granules, toxic soil, liquid spray, and wintering adults in the fumigation straw.
  • chemical control also has its limitations. If improper use, it will lead to phytotoxicity of crops, resistance to pests, killing natural enemies, polluting the environment, destroying farmland ecosystems and threatening the safety of humans and animals. Adverse consequences.
  • Biological control is the use of certain beneficial organisms or biological metabolites to control the population of pests to reduce or eliminate pests. It is characterized by safety for people and animals, less pollution to the environment, and These pests can achieve long-term control; however, the effects are often unstable, and the same investment is required regardless of the severity of the disease.
  • Cry2Ab insecticidal protein is one of many insecticidal proteins and is an insoluble parasporal crystal protein produced by Bacillus thuringiensis.
  • the Cry2Ab protein is ingested by insects into the midgut, and the protoxin is dissolved in the alkaline pH environment of the insect midgut.
  • the N- and C-termini of the protein are digested with alkaline protease to convert the protoxin into an active fragment; the active fragment binds to the receptor on the upper surface of the epithelial cell membrane of the insect and is inserted into the intestinal membrane, causing perforation of the cell membrane and destroying the inside and outside of the cell membrane. Changes in osmotic pressure and pH balance disrupt the insect's digestive process and ultimately lead to death.
  • Plants transgenic to the Cry2Ab gene have been shown to be resistant to lepidoptera pests such as corn borer and cotton bollworm. However, there have been no reports on the control of plant damage by large transgenic plants by producing transgenic plants expressing the Cry2Ab protein.
  • the object of the present invention is to provide a use of a pesticidal protein, for the first time, to provide a method for controlling the damage of the plant to the plant by producing a transgenic plant expressing the Cry2Ab protein, and effectively overcoming the prior art agricultural control, chemical control and biological control, etc. Technical flaws.
  • the present invention provides the following technical solutions, specifically:
  • the invention provides a method of controlling a cockroach pest comprising contacting a cockroach pest with a Cry2Ab protein;
  • the Cry2Ab protein is present in a plant cell producing the Cry2Ab protein, the large cockroach pest being contacted with the Cry2Ab protein by ingesting the plant cell;
  • the Cry2Ab protein is present in a transgenic plant producing the Cry2Ab protein, the large cockroach pest is contacted with the Cry2Ab protein by ingesting the tissue of the transgenic plant, and the growth of the cockroach pest is inhibited after contact And / or lead to death, in order to achieve control of plants that are harmful to the cockroach.
  • the transgenic plant is in any growth period;
  • the tissue of the transgenic plant is a leaf, a stem, a tassel, an ear, an anther or a filament; and/or
  • the control of the plants of the giant salamander does not change due to changes in the location and/or planting time.
  • the plant is corn, rice, sorghum, wheat, millet, cotton, reed, sugar cane, white peony, broad bean or canola.
  • the step before the contacting step is planting a plant containing a polynucleotide encoding the Cry2Ab protein.
  • the amino acid sequence of the Cry2Ab protein has the amino acid sequence shown in SEQ ID NO: 1.
  • the nucleotide sequence of the Cry2Ab protein has the nucleotide sequence shown in SEQ ID NO: 2.
  • the plant may further comprise at least one second nucleotide different from the nucleotide encoding the Cry2Ab protein;
  • the second nucleotide encodes a Cry-like insecticidal protein, a Vip-like insecticidal protein, a protease inhibitor, a lectin, an alpha-amylase or a peroxidase;
  • the second nucleotide encodes a Cry1A.105 protein
  • the second nucleotide has the nucleotide sequence set forth in SEQ ID NO:3.
  • the second nucleotide is a dsRNA which inhibits an important gene in a target insect pest.
  • the invention provides the use of a Cry2Ab protein for controlling a pest of the cockroach.
  • the invention provides the use of a plant cell, plant or plant part of a transgenic Cry2Ab gene to control a large pest.
  • the invention provides a method of producing a plant that controls a pest of the cockroach comprising introducing a polynucleotide sequence encoding a Cry2Ab protein into the genome of the plant.
  • the present invention provides a method of producing a plant seed for controlling a cockroach pest comprising first hybridizing a first plant obtained by the method as described above to a second plant to produce a polynucleotide sequence encoding a Cry2Ab protein. seed.
  • the invention provides a plant seed for controlling a cockroach pest, the seed being a seed comprising a polynucleotide sequence encoding a Cry2Ab protein.
  • the present invention provides a method of cultivating a plant that controls a pest of the cockroach, comprising:
  • the plants are grown under conditions in which the artificial inoculation of the giant salamander pests and/or the giant salamander pests are naturally harmful, and the plants are harvested with reduced plant damage and/or have a yield compared to other plants that do not have the polynucleotide sequence encoding the Cry2Ab protein. Increased plant yield of plants.
  • the present invention provides a plant for controlling cockroach pests produced by culturing according to the method described above.
  • the invention provides a plant cell, plant or plant part that controls a cockroach pest comprising a polynucleotide sequence encoding a Cry2Ab protein in the genome of the plant cell, plant or plant part.
  • plant cell plant or plant part as described above, wherein the plant is selected from the group consisting of corn, rice, sorghum, wheat, millet, cotton, reed, sugar cane, alfalfa, broad bean or canola, And wherein the plant part is a seed, a leaf, a stem, a tassel, an ear, an anther, a filament, a root or any part thereof.
  • Contact means that insects and/or pests touch, stay and/or ingest plants, plant organs, plant tissues or plant cells, and the plants, plant organs, plant tissues or plant cells can It is a pesticidal protein expressed in the body, and may also be a microorganism having a pesticidal protein on the surface of the plant, plant organ, plant tissue or plant cell and/or having a pesticidal protein.
  • control and/or “control” in the present invention means that the cockroach pest is contacted with the Cry2Ab protein, and growth of the cockroach pest is inhibited and/or causes death after contact. Further, the giant cockroach pest is in contact with the Cry2Ab protein by feeding the plant tissue, and all or part of the cockroach pest growth is inhibited and/or causes death after the contact. Inhibition refers to sublethal death, that is, it has not been killed but can cause certain effects in growth, behavior, behavior, physiology, biochemistry and organization, such as slow growth and/or cessation. At the same time, the plants should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product.
  • plants and/or plant seeds containing a polynucleotide sequence encoding a Cry2Ab protein that control the pests of the giant salamander, and non-transgenic wild-type plants under conditions in which the artificial inoculation of the pests of the giant salamander and/or the pests of the giant salamander are naturally occurring Specific manifestations include, but are not limited to, improved stem resistance, and/or increased kernel weight, and/or increased yield, etc., as compared to reduced plant damage.
  • the "control" and/or “control” effects of the Cry2Ab protein on the cockroach can exist independently and are not attenuated and/or disappeared by other substances that can "control" and/or "control” the cockroach pest.
  • any tissue of a transgenic plant (containing a polynucleotide sequence encoding a Cry2Ab protein) is present and/or asynchronously, present and/or produced, Cry2Ab Protein and/or another substance that can control the cockroach pest, the presence of the other substance neither affects the "control” and/or “control” effect of the Cry2Ab protein on the cockroach, nor does it result in the "”
  • the "control” and/or “control” effect is achieved entirely by the other substance, regardless of the Cry2Ab protein.
  • Daejeon the process of feeding plant tissues by large pests is short-lived and difficult to observe with the naked eye.
  • expression of a Cry2Ab protein in a transgenic plant may be accompanied by expression of one or more Cry-like insecticidal proteins and/or Vip-like insecticidal proteins. Co-expression of such more than one insecticidal toxin in the same transgenic plant can be achieved by genetic engineering to allow the plant to contain and express the desired gene.
  • one plant (the first parent) can express the Cry2Ab protein by genetic engineering operation
  • the second plant second parent
  • Progeny plants expressing all of the genes introduced into the first parent and the second parent are obtained by hybridization of the first parent and the second parent.
  • RNA interference refers to the phenomenon of highly-specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. Therefore, in the present invention, RNAi technology can be used to specifically knock out or shut down the expression of a specific gene in a target insect pest.
  • Sesamia inferens and Helicoverpa armigera Hubner belong to the family Lepidoptera, the two major species are distinctly distinct from the cotton bollworm, and at least the following major differences exist:
  • Daphnia is an omnivorous pest, but it is obviously annoyed by grasses, most commonly corn, rice, sorghum, sugar cane, etc.; and cotton bollworm is an important drill-bit pest of cotton buds, mainly feeding buds, flowers, bells, but also Eat young leaves.
  • Datun is widely distributed in the central and southeastern parts of China, especially in most of the southern and southern corn producing areas of Shaanxi and Henan. In addition to China, Datun has also distributed rice, corn and sugar cane in Southeast Asia, including Vietnam. , Laos, India, etc.; and cotton bollworms are widely distributed in China and around the world, China's cotton areas and vegetable growing areas have occurred, the Yellow River Basin cotton area, the Yangtze River valley cotton area is seriously affected; in recent years, Xinjiang cotton area also has occur.
  • Euphorbia is a borer pest, and the larva breaks into the stem of the crop, which can cause death of the dead seedling or the whole plant.
  • the pupil is generally large, and a large amount of worm droppings are discharged from the stem, and are often sandwiched between the sheath and the stem.
  • the leaves and sheaths of the leaves become yellow; the newly hatched larvae do not disperse, the inside of the cluster sheaths, the foraging leaf sheaths and the young stems; after the 3rd instar larvae, the scattered neighbors are scattered and can be transferred.
  • the damage is 5-6 strains. At this time, it is a serious damage period of Datun.
  • the shape of the egg is different: the egg of the big cockroach is round and round, grayish yellow after the initial white, and the surface has fine vertical and horizontal lines, which are concentrated or scattered, often arranged in 2-3 rows; and the eggs of the cotton bollworm It is hemispherical, about 0.5 mm, the top is slightly raised, the surface is covered with vertical and horizontal lines, the vertical lines are 12 from the top, and the middle 2 longitudinal lines are sandwiched between 1-2 short lines and 2-3 inches, so from the middle Look at 26-29 vertical lines, milky white.
  • the morphology of the larvae is different: the larvae of the late larvae are about 30mm in length, 4 heads are reddish brown to dark brown, and the back of the abdomen is pale purple, a total of 5-7 years old; while the cotton bollworm larvae are 6 years old, sometimes 5 years old ( Feeding pea seedlings, sunflower disk), old mature 6-year-old worms are about 40-50mm long, head yellow-brown with inconspicuous markings, larva body color changeable, divided into 4 types: a) body color reddish, topline , the back line is brown, the valve line is white, the hair is black; b) the body color is yellow and white, the back line, the sub-back line is light green, the valve line is white, the hairs are the same as the body color; c) the body color is light green, the back line, The sub-back line is not obvious, the valve line is white, the hairs are the same as the body color; d) the body color is dark green, the back line and the sub-
  • the shape of the cockroach is different: the cockroach has a length of 13-18mm, is thick and sturdy, reddish brown, has a grayish white powder on the abdomen, and has three hooked spines on the hip spine; and the cotton worm has a length of 17-20mm, a spindle shape, and a red Brown to dark brown, a pair of spurs at the end of the abdomen, the base of the thorn is open; the valve is larger, the perforated tube has a higher cylindrical protrusion, and the fifth and seventh quarter of the abdomen are semicircular, thick and thin; Into the soil 5-15cm phlegm, the outside was bandit.
  • the morphology of adult worms is different: the adult female moth is 15mm long, the wings are about 30mm, the head and chest are light yellowish brown, the abdomen is light yellow to grayish white; the antennae are silky, the front wings are nearly rectangular, light grayish brown, and the middle is black.
  • the four points are arranged in a square shape; the male moth is about 12 mm long, with a wingspan of 27 mm, and the antennae are dentate; and the adult cotton bollworm is a gray-brown medium-sized moth with a body length of 15-20 mm, a wingspan of 31-40 mm, a compound eye sphere, and a green color;
  • the female moth is auburn to taupe, the male moth is grayish gray, and the anterior and posterior wings of the cotton bollworm can be used as a model of the adult larvae.
  • the front wing has a dark gray band outside the outer horizontal line, and there are 7 small white spots and kidney lines on the belt.
  • the ring pattern is dark brown, the hind wings are gray, and there is a dark brown band along the outer edge. There are two connected white spots in the center of the broadband, and the front edge of the hind wing has a one-month brown spot.
  • the inner side of the 2 and 3rd leaf sheaths can account for more than 80% of the egg production.
  • Each female can lay 240 eggs, the egg duration is 12 days, the 2nd and 3rd generations are 5-6 days; the larval stage is about 30 days, the second generation is about 28 days, the third generation is about 32 days; the third generation is about 32 days; .
  • the female moth is weak in flying, and the spawning is concentrated. Near the insect source, the density of the insect population is large and harmful.
  • the algebras of cotton bollworms vary from year to year depending on the region. In Laizhou City, Shandong province, four generations occur each year.
  • the eggs are mostly produced on the back of the leaves, and are also produced on the front, top core, petiole, tender stems or other plants such as weeds; weeds have hatch habits after hatching, and newly hatched larvae have
  • the group has a restriction on eating habits.
  • Two or three heads, three or five heads are on the front or back of the leaves, and the heads are arranged in the leaf margins and feed inward from the leaf margins. As a result, the leaves are eaten, leaving only the main veins and petioles, or netted withered.
  • the age of the insects is not neat; the most suitable temperature for the occurrence of cotton bollworm is 25-28 ° C, the relative humidity is 70-90%; the second and third generations are the most serious, serious insects
  • the density is 98 heads/louvers, the rate of insects is 60-70%, the individual plots are 100%, the damaged leaves are more than 1/3, the leaf yield is 20%, the quality is reduced by at least 1 grade, and the growth of seedlings has great influence.
  • the big cockroach and the cotton bollworm belong to the family Lepidoptera, but there are many differences in external morphology and harm, and the relationship between them is far away, and they cannot be mated to produce offspring. Therefore, it can be determined that the big cockroach and the cotton bollworm are two different pests, indicating that the receptors bound to the Bt toxin on the upper surface of the intestinal epithelial cell membrane are also different, but a certain type of insecticidal crystal protein can only be specific The receptor protein binding, that is, the Bt toxin is specific to the target pest, it can be seen that the method of using the Cry2Ab protein to control the giant cockroach pest is not obvious.
  • the genome of a plant, plant tissue or plant cell as used in the present invention refers to a plant or a plant. Any genetic material within a tissue or plant cell, and includes the nucleus and plastid and mitochondrial genomes.
  • polynucleotides and/or nucleotides described herein form a complete "gene" encoding a protein or polypeptide in a desired host cell.
  • polynucleotides and/or nucleotides of the invention can be placed under the control of regulatory sequences in a host of interest.
  • DNA typically exists in a double stranded form. In this arrangement, one chain is complementary to the other and vice versa. Since DNA is replicated in plants, other complementary strands of DNA are produced. Thus, the invention encompasses the use of the polynucleotides exemplified in the Sequence Listing and their complementary strands.
  • a "coding strand” as commonly used in the art refers to a strand that binds to the antisense strand.
  • To express a protein in vivo one strand of DNA is typically transcribed into a complementary strand of mRNA that is used as a template to translate the protein. mRNA is actually transcribed from the "antisense" strand of DNA.
  • a “sense” or “encoding” strand has a series of codons (codons are three nucleotides, three reads at a time to produce a particular amino acid), which can be read as an open reading frame (ORF) to form a protein or peptide of interest.
  • the present invention also includes RNA and PNA (peptide nucleic acid) having comparable functions to the exemplified DNA.
  • the nucleic acid molecule or fragment thereof of the present invention hybridizes under stringent conditions to the Cry2Ab gene of the present invention. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Cry2Ab gene of the present invention.
  • a nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present invention, if two nucleic acid molecules can form an anti-parallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules are capable of specifically hybridizing each other. If two nucleic acid molecules exhibit complete complementarity, one of the nucleic acid molecules is said to be the "complement" of the other nucleic acid molecule.
  • nucleic acid molecules when each nucleotide of one nucleic acid molecule is complementary to a corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to exhibit "complete complementarity".
  • Two nucleic acid molecules are said to be “minimally complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under at least conventional "low stringency” conditions and bind to each other.
  • two nucleic acid molecules are said to be “complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under conventional "highly stringent” conditions and bind to each other.
  • Deviation from complete complementarity is permissible as long as such deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a nucleic acid molecule In order for a nucleic acid molecule to function as a primer or probe, it is only necessary to ensure that it is sufficiently complementary in sequence to allow for the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.
  • a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing to a complementary strand of another matched nucleic acid molecule under highly stringent conditions.
  • Suitable stringent conditions for promoting DNA hybridization for example, treatment with 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by washing with 2.0 x SSC at 50 ° C, these conditions are known to those skilled in the art. It is well known.
  • the salt concentration in the washing step can be selected from about 2.0 x SSC under low stringency conditions, 50 ° C to about 0.2 x SSC, 50 ° C under highly stringent conditions.
  • the temperature conditions in the washing step can be raised from a low temperature strict room temperature of about 22 ° C to a highly stringent condition of about 65 °C. Both the temperature conditions and the salt concentration can be changed, or one of them remains unchanged while the other variable changes.
  • the stringent conditions of the present invention may be specific hybridization with SEQ ID NO: 2 at 65 ° C in 6 x SSC, 0.5% SDS solution, followed by 2 x SSC, 0.1% SDS and 1 x SSC. 0.1% SDS was washed once each time.
  • sequence having insect resistance and hybridizing to SEQ ID NO: 2 of the present invention under stringent conditions is included in the present invention.
  • These sequences are at least about 40%-50% homologous to the sequences of the invention, about 60%, 65% or 70% homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93.
  • genes and proteins described in the present invention include not only specific exemplary sequences, but also portions and/or fragments that retain the insecticidal activity characteristics of the proteins of the specific examples (including internal and/or end ratios compared to full length proteins). Deletions), variants, mutants, substitutions (proteins with alternative amino acids), chimeras and fusion proteins.
  • variant or “variant” is meant a nucleotide sequence that encodes the same protein or an equivalent protein encoded with insecticidal activity.
  • the "equivalent protein” refers to a protein having the same or substantially the same biological activity as that of the protein of the claims.
  • a “fragment” or “truncated” sequence of a DNA molecule or protein sequence as used in the present invention refers to a portion of the original DNA or protein sequence (nucleotide or amino acid) involved or an artificially engineered form thereof (eg, a sequence suitable for plant expression)
  • the length of the aforementioned sequence may vary, but is of sufficient length to ensure that the (encoding) protein is an insect toxin.
  • Genes can be modified and gene variants can be easily constructed using standard techniques. For example, techniques for making point mutations are well known in the art. Further, for example, U.S. Patent No. 5,605,793 describes a method of using DNA reassembly to generate other molecular diversity after random fragmentation. Fragments of full-length genes can be made using commercial endonucleases, and exonucleases can be used according to standard procedures. For example, nucleotides can be systematically excised from the ends of these genes using enzymes such as Bal31 or site-directed mutagenesis. A gene encoding an active fragment can also be obtained using a variety of restriction enzymes. Active fragments of these toxins can be obtained directly using proteases.
  • the present invention may derive equivalent proteins and/or genes encoding these equivalent proteins from B.t. isolates and/or DNA libraries.
  • antibodies to the pesticidal proteins disclosed and claimed herein can be used to identify and isolate other proteins from protein mixtures.
  • antibodies may be caused by protein portions that are most constant in protein and most different from other B.t. proteins.
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies of the proteins disclosed herein or equivalent proteins or fragments of such proteins can be readily prepared using standard procedures in the art. Genes encoding these proteins can then be obtained from microorganisms.
  • the "substantially identical" sequence refers to a sequence which has an amino acid substitution, deletion, addition or insertion but does not substantially affect the insecticidal activity, and also includes a fragment which retains insecticidal activity.
  • Substitution, deletion or addition of an amino acid sequence in the present invention is a conventional technique in the art, and it is preferred that such an amino acid change is: a small change in properties, that is, a conservative amino acid substitution that does not significantly affect the folding and/or activity of the protein; a small deletion, Typically a deletion of about 1-30 amino acids; a small amino or carboxy terminal extension, such as a methionine residue at the amino terminus; and a small linker peptide, for example about 20-25 residues in length.
  • conservative substitutions are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine, hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small molecules Amino acids (such as glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions that generally do not alter a particular activity are well known in the art and have been described, for example, by N. Neurath and R.
  • substitutions can occur outside of the regions that are important for molecular function and still produce active polypeptides.
  • amino acids from the polypeptides of the invention that are essential for their activity and are therefore selected for unsubstitution they can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • the latter technique introduces a mutation at each positively charged residue in the molecule, and detects the insecticidal activity of the resulting mutant molecule, thereby determining an amino acid residue important for the activity of the molecule.
  • the substrate-enzyme interaction site can also be determined by analysis of its three-dimensional structure, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, eg, de Vos et al., 1992, Science 255). : 306-312; Smith et al, 1992, J. Mol. Biol 224: 899-904; Wlodaver et al, 1992, FEBS Letters 309: 59-64).
  • the Cry2Ab protein includes, but is not limited to, a Cry2Ab protein, or an insecticidal fragment or functional region having at least 70% homology with the amino acid sequence of the above protein and having insecticidal activity against Euphorbia.
  • amino acid sequence having a certain homology with the amino acid sequence shown in SEQ ID NO: 1 is also included in the present invention.
  • sequences are typically more than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%, similar to the sequences of the present invention.
  • the preferred multicore of the invention may also be defined in terms of a more specific range of identity and/or similarity. Glycosylates and proteins.
  • the sequence of the example of the present invention is 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% identity and/or similarity.
  • Regulatory sequences of the invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the Cry2Ab protein.
  • the promoter is a promoter expressible in a plant
  • the "promoter expressible in a plant” refers to a promoter which ensures expression of a coding sequence linked thereto in a plant cell.
  • a promoter expressible in a plant can be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the maize Ubi promoter, the promoter of the rice GOS2 gene, and the like.
  • a promoter expressible in a plant may be a tissue-specific promoter, ie the promoter directs the expression level of the coding sequence in some tissues of the plant, such as in green tissue, to be higher than other tissues of the plant (through conventional The RNA assay is performed), such as the PEP carboxylase promoter.
  • a promoter expressible in a plant can be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter that directs a wound-inducible expression pattern means that when the plant is subjected to mechanical or wounding by insect foraging, the expression of the coding sequence under the control of the promoter is significantly improved compared to normal growth conditions.
  • wound-inducible promoters include, but are not limited to, promoters of protease inhibitory genes (pin I and pin II) and maize protease inhibitory genes (MPI) of potato and tomato.
  • the transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a particular organelle or cell compartment, and for the receptor protein, the transit peptide can be heterologous, for example, using a coding chloroplast transporter
  • the peptide sequence targets the chloroplast, or targets the endoplasmic reticulum using the 'KDEL' retention sequence, or the CTPP-targeted vacuole using the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potato virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin protein heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.
  • EMCV leader sequence 5' non-coding region of encephalomyocarditis virus
  • a potato virus group leader sequence such as a MDMV (maize dwarf mosaic virus) leader sequence
  • MDMV human immunoglobulin protein heavy chain binding protein
  • AdMV alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • the enhancer includes, but is not limited to, a cauliflower mosaic virus (CaMV) enhancer, a figwort mosaic virus (FMV) enhancer, a carnation weathering ring virus (CERV) enhancer, and a cassava vein mosaic virus (CsVMV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • CERV carnation weathering ring virus
  • CsVMV cassava vein mosaic virus
  • MMV Purple Jasmine Mosaic Virus
  • MMV Yellow Jasmine Mosaic Virus
  • CmYLCV Night fragrant yellow leaf curl virus
  • CLCuMV Multan cotton leaf curl virus
  • CoYMV Acanthus yellow mottle virus
  • PCLSV peanut chlorotic line flower Leaf virus
  • the intron includes, but is not limited to, corn hsp70 inclusion Sub, corn ubiquitin intron, Adh intron 1, sucrose synthase intron or rice Act1 intron.
  • the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.
  • the terminator may be a suitable polyadenylation signal sequence that functions in plants, including but not limited to, a polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene. a polyadenylation signal sequence derived from the protease inhibitor II (pin II) gene, a polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and a gene derived from the ⁇ -tubulin gene. Polyadenylation signal sequence.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • operably linked refers to the joining of nucleic acid sequences that allow one sequence to provide the function required for the linked sequence.
  • the "operably linked” in the present invention may be such that the promoter is ligated to the sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter.
  • Effective ligation when a sequence of interest encodes a protein and is intended to obtain expression of the protein means that the promoter is ligated to the sequence in a manner that allows efficient translation of the resulting transcript.
  • the linker of the promoter to the coding sequence is a transcript fusion and it is desired to effect expression of the encoded protein, such ligation is made such that the first translation initiation codon in the resulting transcript is the start codon of the coding sequence.
  • the linkage of the promoter to the coding sequence is a translational fusion and it is desired to effect expression of the encoded protein, such linkage is made such that the first translation initiation codon and promoter contained in the 5' untranslated sequence Linked and linked such that the resulting translation product is in frame with the translational open reading frame encoding the desired protein.
  • Nucleic acid sequences that may be "operably linked” include, but are not limited to, sequences that provide for gene expression functions (ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, poly Adenylation site and/or transcription terminator), sequences that provide DNA transfer and/or integration functions (ie, T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide options Sexually functional sequences (ie, antibiotic resistance markers, biosynthetic genes), sequences that provide for the function of scoring markers, sequences that facilitate sequence manipulation in vitro or in vivo (ie, polylinker sequences, site-specific recombination sequences) and provision The sequence of the replication function (ie, the origin of replication of the bacteria, the autonomously replicating sequence, the centromeric sequence).
  • gene expression functions ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein
  • Insecticide as used in the present invention means toxic to crop pests. More specifically, the target insect is a large insect pest.
  • the Cry2Ab protein is toxic to the cockroach pest.
  • the plants of the present invention particularly maize and sorghum, contain exogenous DNA in their genome, the exogenous DNA comprising a nucleotide sequence encoding a Cry2Ab protein, which is contacted with the protein by feeding plant tissue, after contact The growth of the pests of the giant salamander is inhibited and/or causes death.
  • the plants should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product.
  • the plant substantially eliminates the need for chemical or biological insecticides that are insecticides against the giant cockroach pests targeted by the Cry2Ab protein.
  • the expression level of insecticidal crystal protein (ICP) in plant material can be detected by various methods described in the art, for example, by using specific primers to quantify the mRNA encoding the insecticidal protein produced in the tissue, or directly specific The amount of insecticidal protein produced is detected.
  • the target insects in the present invention are mainly large cockroaches.
  • the Cry2Ab protein may have the amino acid sequence shown by SEQ ID NO: 1 in the Sequence Listing.
  • other elements may be included, such as a coding region encoding a second insecticidal nucleotide, a protein encoding a selectable marker, or a protein conferring herbicide resistance.
  • an expression cassette comprising a nucleotide sequence encoding a Cry2Ab protein of the invention may also be expressed in a plant together with at least one protein encoding a herbicide resistance gene including, but not limited to, oxalic acid Phospho-resistant genes (such as bar gene, pat gene), benthamiana resistance genes (such as pmph gene), glyphosate resistance genes (such as EPSPS gene), bromoxynil resistance gene, sulfonylurea Resistance gene, resistance gene to herbicide tortoise, resistance gene to cyanamide or glutamine synthetase inhibitor (such as PPT), thereby obtaining high insecticidal activity and weeding Agent-resistant transgenic plants.
  • oxalic acid Phospho-resistant genes such as bar gene, pat gene
  • benthamiana resistance genes such as pmph gene
  • glyphosate resistance genes such as EPSPS gene
  • bromoxynil resistance gene sulfonylurea Resistance gene
  • a foreign DNA is introduced (introduced) into a plant, such as a gene encoding the Cry2Ab protein or an expression cassette or a recombinant vector
  • conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro Launch bombardment, direct DNA uptake into protoplasts, electroporation or whisker silicon-mediated DNA introduction.
  • the prior art mainly controls the harm of the giant cockroach pest through external action, ie, external cause, such as agricultural control, chemical control and biological control; and the present invention controls the cockroach pest by producing a Cry2Ab protein capable of killing the big cockroach in the plant. That is, through internal factors to prevent and cure.
  • the effect is stable.
  • the expression in the object is basically stable and consistent, and the control effect of the transgenic plant (Cry2Ab protein) of the present invention is stable at different locations, at different times, and in different genetic backgrounds.
  • the invention only needs to plant the transgenic plant capable of expressing the Cry2Ab protein without using other measures. , which saves a lot of manpower, material resources and financial resources.
  • the effect is thorough.
  • the method for controlling cockroach pests used in the prior art has an effect that is incomplete and only serves to alleviate the effect; whereas the transgenic plant of the present invention (Cry2Ab protein) can cause a large number of deaths of the newly hatched larvae and survive for a small portion.
  • the progress of larval development was greatly inhibited. After 3 days, the larvae were still in the initial hatching state, all of which were obviously dysplastic, and had stopped developing.
  • the transgenic plants were generally only slightly damaged.
  • Figure 1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing a Cry2Ab nucleotide sequence for use of the insecticidal protein of the present invention
  • Figure 2 is a flow chart showing the construction of a recombinant expression vector DBN100033 containing a Cry2Ab nucleotide sequence for use of the insecticidal protein of the present invention
  • Figure 3 is a diagram showing the leaf damage of inoculated sorghum of transgenic maize plants for use of the insecticidal protein of the present invention.
  • Cry2Ab insecticidal protein (634 amino acids), as shown in SEQ ID NO: 1 in the Sequence Listing; Cry2Ab nucleotide sequence (1905 nucleotides) encoding the amino acid sequence corresponding to the Cry2Ab insecticidal protein , as shown in SEQ ID NO: 2 in the Sequence Listing.
  • the Cry1A.105 nucleotide sequence (3534 nucleotides) encoding the amino acid sequence (1177 amino acids) of the Cry1A.105 insecticidal protein is shown in SEQ ID NO: 3 of the Sequence Listing.
  • the Cry2Ab nucleotide sequence (as shown in SEQ ID NO: 2 in the Sequence Listing) and the Cry1A.105 nucleotide sequence (as shown in SEQ ID NO: 3 in the Sequence Listing) are manufactured by Nanjing Kingsray Biotech Co., Ltd.
  • the synthetic Cry2Ab nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the procedure was carried out according to the Promega product pGEM-T vector specification to obtain a recombinant cloning vector DBN01-T.
  • Cry2Ab is the Cry2Ab nucleotide sequence (SEQ ID NO: 2); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cells by heat shock method (Transgen, Beijing, China, CAT: CD501) under heat shock conditions: 50 ⁇ l E. coli T1 competent cells, 10 ⁇ l plasmid DNA (recombinant) Cloning vector DBN01-T), water bath at 42 ° C for 30 seconds; shaking culture at 37 ° C for 1 hour (shake at 100 rpm), coated with IPTG (isopropylthio- ⁇ -D-galactoside) and X -gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) ampicillin (100 mg/L) in LB plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L, adjusted to pH 7.5 with NaOH) was grown overnight.
  • heat shock method Transgen, Beijing, China, CAT: CD501
  • White colonies were picked and cultured in LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, ampicillin 100 mg/L, pH adjusted to 7.5 with NaOH) at 37 °C. overnight.
  • the plasmid was extracted by alkaline method: the bacterial solution was centrifuged at 12000 rpm for 1 min, the supernatant was removed, and the precipitated cells were pre-cooled with 100 ⁇ l of ice (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose.
  • the TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was dissolved in the precipitate; the RNA was digested in a water bath at 37 ° C for 30 min; and stored at -20 ° C until use.
  • the Cry2Ab nucleotide sequence inserted into the recombinant cloning vector DBN01-T was the nucleus represented by SEQ ID NO: 2 in the sequence listing.
  • the nucleotide sequence, the Cry2Ab nucleotide sequence, was correctly inserted.
  • the synthesized Cry1A.105 The nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein Cry1A.105 was a Cry1A.105 nucleotide sequence (SEQ ID NO: 3).
  • the Cry1A.105 nucleotide sequence in the recombinant cloning vector DBN02-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the recombinant cloning vector DBN01-T and the expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases NcoI and SpeI, respectively, and the excised Cry2Ab nucleotide sequence fragment was inserted into the expression.
  • NcoI and SpeI sites of the vector DBNBC-01 the construction of the vector by conventional enzymatic cleavage method is well known to those skilled in the art, and the recombinant expression vector DBN100033 is constructed, and the construction process thereof is shown in Fig.
  • the recombinant expression vector DBN100033 was transformed into E. coli T1 competent cells by heat shock method.
  • the heat shock conditions were: 50 ⁇ l of E. coli T1 competent cells, 10 ⁇ l of plasmid DNA (recombinant expression vector DBN100033), 42 ° C water bath for 30 seconds; 37 ° C oscillation Incubate for 1 hour (shake shake at 100 rpm); then LB solid plate containing 50 mg/L kanamycin (trypeptin 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g) /L, adjust the pH to 7.5 with NaOH and incubate at 37 °C for 12 hours, pick white colonies, in LB liquid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, Kanamycin 50 mg/L was adjusted to pH 7.5 with NaOH and incubated overnight at 37 °C.
  • the plasmid was extracted by an alkali method.
  • the extracted plasmids were digested with restriction endonucleases NcoI and SpeI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the NcoI and SpeI sites of the recombinant expression vector DBN100033 was SEQ ID in the sequence listing. NO: The nucleotide sequence shown by 2, that is, the Cry2Ab nucleotide sequence.
  • the recombinant expression vector DBN100033 NcCl and SpeI, NcoI and HindIII were respectively digested into the recombinant cloning vectors DBN01-T and DBN02-T, and the Cry2Ab nucleotide sequence and the Cry1A.105 nucleotide sequence were inserted.
  • the expression vector DBNBC-01 was obtained to obtain a recombinant expression vector DBN100076.
  • the nucleotide sequence in the recombinant expression vector DBN100076 contains the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing, namely the Cry2Ab nucleotide sequence and the Cry1A.105 nucleoside.
  • the acid sequence, the Cry2Ab nucleotide sequence and the Cry1A.105 nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the recombinant expression vectors DBN100033 and DBN100076 which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium LBA4404, 3 ⁇ L of plasmid DNA ( Recombinant expression vector); The cells were placed in liquid nitrogen for 10 minutes, and warmed at 37 ° C for 10 minutes. The transformed Agrobacterium LBA4404 was inoculated into LB tubes and incubated at a temperature of 28 ° C and a rotation speed of 200 rpm for 2 hours, and applied to a 50 mg/L rifle.
  • the immature embryo of the aseptically cultured maize variety Heisei 31 was co-cultured with the Agrobacterium described in the third embodiment in accordance with the conventional Agrobacterium infection method to construct the second embodiment.
  • the T-DNAs in the recombinant expression vectors DBN100033 and DBN100076 (including the promoter sequence of the maize Ubiquitin gene, the Cry2Ab nucleotide sequence, the Cry1A.105 nucleotide sequence, the PMI gene and the Nos terminator sequence) were transferred into the maize genome.
  • Maize plants transformed with the Cry2Ab nucleotide sequence and maize plants transfected with the Cry2Ab-Cry1A.105 nucleotide sequence were obtained; wild type maize plants were used as controls.
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with an Agrobacterium suspension, wherein the Agrobacterium is capable of expressing the Cry2Ab nucleotide sequence and/or Cry1A.105 nucleotides
  • infecting medium MS salt 4.3
  • the immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
  • the immature embryo is in solid medium after the infection step (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, acetosyringone (AS) 100 mg/L) It was cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step.
  • the medium was restored (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg /
  • At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in L, agar 8 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step).
  • the immature embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the inoculated immature embryos are cultured on a medium containing a selective agent (mannose) and the grown transformed callus is selected (step 4: selection step).
  • the immature embryo is screened in solid medium with selective agent (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 5 g/L, mannose 12.5 g/L, 2,4-dichlorobenzene).
  • MS salt 4.3 g/L MS vitamin, casein 300 mg/L, sucrose 5 g/L, mannose 12.5 g/L, 2,4-dichlorobenzene
  • oxyacetic acid (2,4-D) 1 mg/L
  • agar 8 g/L, pH 5.8 resulted in selective growth of transformed cells.
  • the callus regenerates the plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) Recycled plants.
  • the selected resistant callus was transferred to the MS differentiation medium (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, 6-benzyl adenine 2 mg/L, mannose) 5g/L, agar 8g/L, pH 5.8), cultured and differentiated at 25 °C.
  • the differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, indole-3-acetic acid 1 mg/L, agar 8 g/L, pH 5 .8) Above, culture at 25 ° C to a height of about 10 cm, and move to a greenhouse to grow to firmness. In the greenhouse, the cells were cultured at 28 ° C for 16 hours and then at 20 ° C for 8 hours.
  • TaqMan was used to verify the maize plants transferred into the Cry2Ab gene.
  • the specific method for detecting the copy number of the Cry2Ab gene is as follows:
  • Step 11 Take 100 mg of each of the maize plants transformed with the Cry2Ab nucleotide sequence, the maize plants transformed with the Cry2Ab-Cry1A.105 nucleotide sequence, and the wild-type maize plants, respectively, and grind into liquid nitrogen in the mortar. Homogenate, take 3 replicates per sample;
  • Step 12 Extract the genomic DNA of the above sample using Qiagen's DNeasy Plant Mini Kit, and refer to the product manual for the specific method;
  • Step 13 Determine the genomic DNA concentration of the above sample using NanoDrop 2000 (Thermo Scientific).
  • Step 14 adjusting the genomic DNA concentration of the above sample to the same concentration value, the concentration value ranges from 80 to 100 ng / ⁇ l;
  • Step 15 The Taqman probe real-time PCR method is used to identify the copy number of the sample, and the sample with the known copy number is used as a standard, and the sample of the wild type corn plant is used as a control, and each sample has 3 replicates, and the average is taken. Value; the fluorescent PCR primers and probe sequences are:
  • Probe 1 CGCTGAGCTGACGGGTCTGCAAG as SEQ ID in the Sequence Listing NO: 9;
  • Probe 2 TGAACAGCGCCCTGACCACCG is shown in SEQ ID NO: 12 in the Sequence Listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mixture contained 45 ⁇ l of each primer at a concentration of 1 mM, 50 ⁇ l of a probe at a concentration of 100 ⁇ M, and 860 ⁇ l of 1 ⁇ TE buffer, and stored at 4° C. in an amber tube.
  • the PCR reaction conditions are:
  • Maize plants transformed with the Cry2Ab nucleotide sequence maize plants transfected with the Cry2Ab-Cry1A.105 nucleotide sequence, wild-type maize plants, and maize plants identified as non-transgenic by Taqman were tested for insect resistance.
  • the leaves are rinsed with sterile water and the water on the leaves is blotted dry with gauze.
  • the corn leaves are then removed from the veins and cut into strips of about 1 cm x 4 cm. Two long strips of cut leaves are placed in a circle. On the filter paper at the bottom of the plastic dish, the filter paper is moistened with distilled water.
  • total score 100 ⁇ mortality + [100 ⁇ Mortality +90 ⁇ (number of initial hatcheries/total worms)+60 ⁇ (number of initial hatching-negative control insects/total worms)+10 ⁇ (number of negative control insects/total worms)]+100 ⁇ (1 - blade damage rate).
  • Table 1 The results in Table 1 indicate that the total scores of the maize plants transformed with the Cry2Ab nucleotide sequence and the maize plants transfected with the Cry2Ab-Cry1A.105 nucleotide sequence were all around 260 points or more; The total score of the transgenic maize plants and the wild-type maize plants is generally about 30 minutes, which is significantly lower than the corn plants transferred into the Cry2Ab nucleotide sequence and the nucleotide sequence transferred into the Cry2Ab-Cry1A.105. Corn plants.
  • the results in Figure 3 indicate that the Cry2Ab nucleotide sequence was transferred to the wild-type maize plant.
  • Maize plants and maize plants transfected with Cry2Ab-Cry1A.105 nucleotide sequence can cause a large number of deaths of the newly hatched larvae, and greatly inhibit the development of a small number of surviving larvae. After 3 days, the larvae are still initially hatched.
  • the maize plants that have been transferred to the Cry2Ab nucleotide sequence and the maize plants that have been transferred into the Cry2Ab-Cry1A.105 nucleotide sequence are generally only slightly damaged, with only a small amount of pinhole-like lesions on the leaves, and their leaf damage. The rate is around 3%, with significantly reduced plant damage.
  • the Cry2Ab protein of the present invention includes, but is not limited to, the Cry2Ab protein of the amino acid sequence given in the specific embodiment, and the transgenic plant can also produce at least one second insecticidal protein different from the Cry2Ab protein, such as a Vip-like protein or a Cry-like protein. protein.
  • the use of the insecticidal protein of the present invention controls the giant salamander pest by producing a Cry2Ab protein capable of killing the giant salamander in the plant; the present invention is compared with the agricultural control method and the chemical control method used in the prior art.
  • the whole growth period and the protection of whole plants are carried out to prevent the infestation of the pests of the giant salamander, and there is no pollution, no residue, the effect is stable, thorough, simple, convenient and economical.

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Abstract

本发明涉及一种杀虫蛋白的用途，所述控制大螟害虫的方法包括：将大螟害虫与Cry2Ab蛋白接触。本发明通过转基因植物体内产生能够杀死大螟的Cry2Ab蛋白来控制大螟害虫。与现有技术使用的农业防治方法、化学防治方法和生物防治方法相比，本发明对植物进行全生育期、全植株的保护以防治大螟害虫的侵害，且无污染、无残留，效果稳定、彻底，简单、方便、经济。

Description

杀虫蛋白的用途
相关申请的交叉引用
本申请要求2014年8月27日提交的第201410428956.1号中国发明专利申请的优先权，所述申请以引用的方式整体并入本文。
技术领域
本发明涉及一种杀虫蛋白的用途，特别是涉及一种Cry2Ab蛋白质通过在植物中表达来控制大螟为害植物的用途。
背景技术
大螟(Sesamia inferens)属鳞翅目夜蛾科，为杂食性害虫，除为害玉米外，还为害水稻、甘蔗、小麦、高梁等禾本科作物，广泛分布于我国中部与东南部，特别是陕西、河南以南的大部稻区。大螟幼虫蛀入作物茎内为害，可造成枯心苗或整株死亡，其蛀孔一般较大，并有大量虫粪排出茎外，以低洼地及麦套玉米地发生重，且夏玉米发生重于春玉米。
玉米和高粱是中国重要的粮食作物，每年因大螟造成的粮食损失巨大，更甚者影响到当地人口的生存状况。为了防治大螟，人们通常采用的主要防治方法有：农业防治、化学防治和生物防治。
农业防治是把整个农田生态系统多因素的综合协调管理，调控作物、害虫、环境因素、创造一个有利于作物生长而不利于大螟发生的农田生态环境。如利用处理大螟越冬寄主、改革耕作制度、种植抗大螟品种、种植诱集田和间作等措施降低大螟的为害。因农业防治必须服从作物布局和增产的要求，应用有一定的局限性，不能作为应急措施，在大螟爆发时就显得无能为力。
化学防治即农药防治，是利用化学杀虫剂来杀灭害虫，是大螟综合治理的重要组成部分，它具有快速、方便、简单和高经济效益的特点，特别是大螟大发生的情况下，是必不可少的应急措施，它可以在大螟造成为害前将其消灭。目前化学防治方法主要有颗粒剂、撒毒土、药液喷雾、封垛熏蒸秸秆垛内越冬成虫等。但化学防治也有其局限性，如使用不当往往会导致农作物发生药害、害虫产生抗药性，以及杀伤天敌、污染环境，使农田生态系统遭到破坏和农药残留对人、畜的安全构成威胁等不良后果。
生物防治是利用某些有益生物或生物代谢产物来控制害虫种群数量，以达到降低或消灭害虫的目的。其特点是对人、畜安全，对环境污染少，对某 些害虫可达到长期控制的目的；但是效果常不稳定，并且不论大螟发生轻重均需同样投资进行。
为了解决农业防治、化学防治和生物防治在实际应用中的局限性，科学家们经过研究发现将编码杀虫蛋白的抗虫基因转入植物中，可获得一些抗虫转基因植物以防治植物虫害。Cry2Ab杀虫蛋白是众多杀虫蛋白中的一种，是由苏云金芽孢杆菌产生的不溶性伴孢结晶蛋白。
Cry2Ab蛋白被昆虫摄入进入中肠，毒蛋白原毒素被溶解在昆虫中肠的碱性pH环境下。蛋白N-和C-末端被碱性蛋白酶消化，将原毒素转变成活性片段；活性片段和昆虫中肠上皮细胞膜上表面上受体结合，插入肠膜，导致细胞膜出现穿孔病灶，破坏细胞膜内外的渗透压变化及pH平衡等，扰乱昆虫的消化过程，最终导致其死亡。
已证明转Cry2Ab基因的植株可以抵抗玉米螟、棉铃虫等鳞翅目(Lepidoptera)害虫的侵害，然而，至今尚无关于通过产生表达Cry2Ab蛋白的转基因植株来控制大螟对植物危害的报道。
发明内容
本发明的目的是提供一种杀虫蛋白的用途，首次提供了通过产生表达Cry2Ab蛋白的转基因植株来控制大螟对植物危害的方法，且有效克服现有技术农业防治、化学防治和生物防治等技术缺陷。
为实现上述目的，本发明提供了以下技术方案，具体地：
一方面，本发明提供一种控制大螟害虫的方法，包括将大螟害虫与Cry2Ab蛋白接触；
优选地，所述Cry2Ab蛋白存在于产生所述Cry2Ab蛋白的植物细胞中，所述大螟害虫通过摄食所述植物细胞与所述Cry2Ab蛋白接触；
进一步优选地，所述Cry2Ab蛋白存在于产生所述Cry2Ab蛋白的转基因植物中，所述大螟害虫通过摄食所述转基因植物的组织与所述Cry2Ab蛋白接触，接触后所述大螟害虫生长受到抑制和/或导致死亡，以实现对大螟危害植物的控制。
进一步地，根据如前所述的控制大螟害虫的方法，所述转基因植物处于任意生育期；和/或
所述转基因植物的组织为叶片、茎秆、雄穗、雌穗、花药或花丝；和/或
所述对大螟危害植物的控制不因种植地点和/或种植时间的改变而改变。
进一步地，根据如前所述的控制大螟害虫的方法，所述植物为玉米、水稻、高粱、麦、粟、棉花、芦苇、甘蔗、茭白、蚕豆或油菜。
进一步地，根据如前所述的控制大螟害虫的方法，所述接触步骤之前的步骤为种植含有编码所述Cry2Ab蛋白的多核苷酸的植物。
进一步地，根据如前所述的控制大螟害虫的方法，所述Cry2Ab蛋白的氨基酸序列具有SEQ ID NO：1所示的氨基酸序列；
优选地，所述Cry2Ab蛋白的核苷酸序列具有SEQ ID NO：2所示的核苷酸序列。
进一步地，根据如前所述的控制大螟害虫的方法，所述植物还可以包括至少一种不同于编码所述Cry2Ab蛋白的核苷酸的第二种核苷酸；
优选地，所述第二种核苷酸编码Cry类杀虫蛋白质、Vip类杀虫蛋白质、蛋白酶抑制剂、凝集素、α-淀粉酶或过氧化物酶；
进一步优选地，所述第二种核苷酸编码Cry1A.105蛋白
最优选地，所述第二种核苷酸具有SEQ ID NO：3所示的核苷酸序列。
更进一步地，根据如前所述的控制大螟害虫的方法，所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
进一步地，根据如前所述的控制大螟害虫的方法，其中所述大螟害虫在作物田地中。
另一方面，本发明提供一种Cry2Ab蛋白质控制大螟害虫的用途。
又一方面，本发明提供一种转Cry2Ab基因的植物细胞、植物或植物部分控制大螟害虫的用途。
再一方面，本发明提供一种产生控制大螟害虫的植物的方法，包括向所述植物的基因组中引入编码Cry2Ab蛋白的多核苷酸序列。
还一方面，本发明提供一种产生控制大螟害虫的植物种子的方法，包括将由如前所述方法获得的第一植株与第二植株杂交，从而产生含有编码Cry2Ab蛋白的多核苷酸序列的种子。
另一方面，本发明提供一种控制大螟害虫的植物种子，所述种子为含有编码Cry2Ab蛋白的多核苷酸序列的种子。
又一方面，本发明提供一种培养控制大螟害虫的植物的方法，包括：
种植至少一粒植物种子，所述植物种子的基因组中包括编码Cry2Ab蛋白的多核苷酸序列；
使所述植物种子长成植株；
使所述植株在人工接种大螟害虫和/或大螟害虫自然发生危害的条件下生长，收获与其他不具有编码Cry2Ab蛋白的多核苷酸序列的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株。
再一方面，本发明提供根据如前所述方法培养产生的控制大螟害虫的植物。
还一方面，本发明提供一种控制大螟害虫的植物细胞、植物或植物部分，所述植物细胞、植物或植物部分的基因组中包含编码Cry2Ab蛋白的多核苷酸序列。
进一步地，根据如前所述的植物细胞、植物或植物部分，其中所述植物选自由以下组成的组：玉米、水稻、高粱、麦、粟、棉花、芦苇、甘蔗、茭白、蚕豆或油菜，并且其中所述植物部分为种子、叶片、茎秆、雄穗、雌穗、花药、花丝、根或其任何部分。
本发明中所述的“接触”，是指昆虫和/或害虫触碰、停留和/或摄食植物、植物器官、植物组织或植物细胞，所述植物、植物器官、植物组织或植物细胞既可以是其体内表达杀虫蛋白，还可以是所述植物、植物器官、植物组织或植物细胞的表面具有杀虫蛋白和/或具有产生杀虫蛋白的微生物。
本发明术语“控制”和/或“防治”是指大螟害虫与Cry2Ab蛋白接触，接触后大螟害虫生长受到抑制和/或导致死亡。进一步地，大螟害虫通过摄食植物组织与Cry2Ab蛋白接触，接触后全部或部分大螟害虫生长受到抑制和/或导致死亡。抑制是指亚致死，即尚未致死但能引起生长发育、行为、生理、生化和组织等方面的某种效应，如生长发育缓慢和/或停止。同时，植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成。此外，含有编码Cry2Ab蛋白的多核苷酸序列的控制大螟害虫的植物和/或植物种子，在人工接种大螟害虫和/或大螟害虫自然发生危害的条件下，与非转基因的野生型植株相比具有减弱的植物损伤，具体表现包括但不限于改善的茎秆抗性、和/或提高的籽粒重量、和/或增产等。Cry2Ab蛋白对大螟的“控制”和/或“防治”作用是可以独立存在的，不因其它可“控制”和/或“防治”大螟害虫的物质的存在而减弱和/或消失。具体地，转基因植物(含有编码Cry2Ab蛋白的多核苷酸序列)的任何组织同时和/或不同步地，存在和/或产生，Cry2Ab 蛋白和/或可控制大螟害虫的另一种物质，则所述另一种物质的存在既不影响Cry2Ab蛋白对大螟的“控制”和/或“防治”作用，也不能导致所述“控制”和/或“防治”作用完全由所述另一种物质实现，而与Cry2Ab蛋白无关。通常情况下，在大田，大螟害虫摄食植物组织的过程短暂且很难用肉眼观察到，因此，在人工接种大螟害虫和/或大螟害虫自然发生危害的条件下，如转基因植物(含有编码Cry2Ab蛋白的多核苷酸序列)的任何组织存在死亡的大螟害虫、和/或在其上停留生长受到抑制的大螟害虫、和/或与非转基因的野生型植株相比具有减弱的植物损伤，即为实现了本发明的方法和/或用途，即通过大螟害虫与Cry2Ab蛋白接触以实现控制大螟害虫的方法和/或用途。
在本发明中，Cry2Ab蛋白在一种转基因植物中的表达可以伴随着一个或多个Cry类杀虫蛋白质和/或Vip类杀虫蛋白质的表达。这种超过一种的杀虫毒素在同一株转基因植物中共同表达可以通过遗传工程使植物包含并表达所需的基因来实现。另外，一种植物(第1亲本)可以通过遗传工程操作表达Cry2Ab蛋白质，第二种植物(第2亲本)可以通过遗传工程操作表达Cry类杀虫蛋白质和/或Vip类杀虫蛋白质。通过第1亲本和第2亲本杂交获得表达引入第1亲本和第2亲本的所有基因的后代植物。
RNA干扰(RNA interference，RNAi)是指在进化过程中高度保守的、由双链RNA(double-stranded RNA，dsRNA)诱发的、同源mRNA高效特异性降解的现象。因此在本发明中可以使用RNAi技术特异性剔除或关闭目标昆虫害虫中特定基因的表达。
大螟(Sesamia inferens)与棉铃虫(Helicoverpa armigera Hubner)虽然同属鳞翅目夜蛾科，但是大螟与棉铃虫在生物学上是清晰的、截然不同的两个物种，至少存在以下主要区别：
1、食性不同。大螟为杂食性害虫，但明显嗜好禾本科，最常为害玉米、水稻、高粱、甘蔗等；而棉铃虫是棉花蕾铃期重要钻蛀性害虫，主要蛀食蕾、花、铃，也取食嫩叶。
2、分布区域不同。大螟广泛分布于我国中部与东南部，特别是陕西、河南以南的大部稻区及西南玉米产区；除中国外，大螟在东南亚种植水稻、玉米及甘蔗的国家也有分布，包括越南、老挝、印度等；而棉铃虫广泛分布在中国及世界各地，中国棉区和蔬菜种植区均有发生，黄河流域棉区、长江流域棉区受害较重；近年来，新疆棉区也时有发生。
3、为害习性不同。大螟属钻蛀性害虫，幼虫蛀入作物茎内为害，可造成枯心苗或整株死亡，其蛀孔一般较大，并有大量虫粪排出茎外，多夹在叶鞘和茎秆之间，受害后的叶片、叶鞘部都变为黄色；刚孵化出的幼虫，不分散，群集叶鞘内侧，蛀食叶鞘和幼茎；幼虫3龄以后，分散迁害邻株，可转 害5-6株不等，此时是大螟的严重为害期，早春10℃以上的温度来得早，则大螟发生早；靠近村庄的低洼地及麦套玉米地发生重；春玉米发生偏轻，夏玉米发生较重。而棉铃虫初龄幼虫取食嫩叶，其后为害蕾、花、铃，多从基部蛀入蕾、铃，在内取食，并能转移为害，转移时间多在夜间和清晨；受害幼蕾苞叶张开、脱落，被蛀青铃易受污染而腐烂；老熟幼虫吐丝下垂，多数入土作土室化蛹，以蛹越冬。
4、形态特征不同。
1)卵的形态不同：大螟的卵扁圆形，初白色后变灰黄色，表面具细纵纹和横线，聚生或散生，常排成2-3行；而棉铃虫的卵呈半球形，约0.5毫米，顶部微隆起，表面布满纵横纹，纵纹从顶部看有12条，中部2纵纹间夹有1-2条短纹且多2-3岔，所以从中部看有26-29条纵纹，乳白色。
2)幼虫的形态不同：大螟末龄幼虫体长约30mm，粗4头红褐色至暗褐色，腹部背面淡紫红色，共5-7龄；而棉铃虫幼虫共有6龄，有时5龄(取食豌豆苗，向日葵花盘的)，老熟6龄虫长约40-50mm，头黄褐色有不明显的斑纹，幼虫体色多变，分4个类型：a)体色淡红，背线、亚背线褐色，气门线白色，毛突黑色；b)体色黄白，背线、亚背线淡绿，气门线白色，毛突与体色相同；c)体色淡绿，背线、亚背线不明显，气门线白色，毛突与体色相同；d)体色深绿，背线、亚背线不太明显，气门淡黄色；气门上方有一褐色纵带，是由尖锐微刺排列而成；幼虫腹部第1、2、5节各有2个毛突特别明显。
3)蛹的形态不同：大螟的蛹长13-18mm，粗壮，红褐色，腹部具灰白色粉状物，臀棘有3根钩棘；而棉铃虫的蛹长17-20mm，纺锤形，赤褐至黑褐色，腹末有一对臀刺，刺的基部分开；气门较大，围孔片呈筒状突起较高，腹部第5-7节的点刻半圆形，较粗而稀；入土5-15cm化蛹，外被土茧。
4)成虫的形态不同：大螟成虫雌蛾体长15mm，翅展约30mm，头部、胸部浅黄褐色，腹部浅黄色至灰白色；触角丝状，前翅近长方形，浅灰褐色，中间具小黑点4个排成四角形；雄蛾体长约12mm，翅展27mm，触角栉齿状；而棉铃虫成虫为灰褐色中型蛾，体长15-20mm，翅展31-40mm，复眼球形，绿色；雌蛾赤褐色至灰褐色，雄蛾青灰色，棉铃虫的前后翅，可作为夜蛾科成虫的模式，其前翅外横线外有深灰色宽带，带上有7个小白点，肾纹，环纹暗褐色，后翅灰白，沿外缘有黑褐色宽带，宽带中央有2个相连的白斑，后翅前缘有1个月牙形褐色斑。
5、生长习性和发生规律不同。大螟一年发生2-4代，随海拔的升高而减少，随温度的升高而增加。如云贵高原年生2-3代，江苏、浙江年生3-4代，江西、湖南、湖北、四川年生4代，福建、广西及云南开远年生4-5代， 广东南部、台湾年生6-8代。在温带以老熟幼虫在寄生残体(如茭白、水稻等作物茎秆或根茬)内或近地面的土壤中越冬，次年3月中旬(气温高于10℃)开始化蛹，15℃时羽化，4月上旬交尾产卵，3-5天达高峰期，4月下旬为孵化高峰期。成虫白天潜伏，常栖息在株间，傍晚开始活动，趋光性较弱，寿命5天左右。雌蛾交尾后2-3天开始产卵，3-5天达高峰期，喜在玉米苗上和地边产卵，多集中在玉米茎秆较细、叶鞘抱合不紧的植株靠近地面的第2节和第3节叶鞘的内侧，可占产卵量的80％以上。每雌可产卵240粒，卵历期一代为12天，2、3代为5-6天；幼虫期一代约30天，二代约28天，三代约32天；蛹期为10-15天。雌蛾飞翔力弱，产卵较集中，靠近虫源的地方，虫口密度大，为害重。而棉铃虫发生的代数因年份因地区而异，在山东省莱州市每年发生4代，九月下旬成长幼虫陆续下树入土，在苗木附近或杂草下5-10cm深的土中化蛹越冬；立春气温回升15℃以上时开始羽化，4月下旬至5月上旬为羽化盛期，成虫出现第一代在6月中下旬，第二代在7月中下旬，第三代在8月中下旬至9月上旬至10月上旬尚有棉铃虫出现，成虫有趋光性，羽化后即在夜间闪配产卵，卵散产，较分散，一头雌蛾一生可产卵500-1000粒，最高可达2700粒，卵多产在叶背面，也有产在正面、顶芯、叶柄、嫩茎上或农作的、杂草等其它植物上；幼虫孵化后有取食卵壳习性，初孵幼虫有群集限食习性，二三头、三五头在叶片正面或背面，头向叶缘排列、自叶缘向内取食，结果叶片被吃光，只剩主脉和叶柄，或成网状枯萎，造成干叶；1-2龄幼虫沿柄下行至银杏苗顶芽处自一侧蛀食或沿顶芽处下蛀入嫩枝，造成顶梢或顶部簇生叶死亡，危害十分严重；3龄前的幼虫食量较少，较集中，随着幼虫生长而逐渐分散，进入4龄食量大增，可食光叶片，只剩叶柄；幼虫7-8月份为害最盛；棉铃虫有转移危害的习性，一只幼虫可危害多株苗木；各龄幼虫均有食掉蜕下旧皮留头壳的习性，给鉴别虫龄造成一定困难，虫龄不整齐；棉铃虫发生的最适宜温度为25-28℃，相对湿度70-90％；第二代、第三代为害最为严重，严重地片虫口密度达98头/百叶，虫株率60-70％，个别地片达100％，受害叶片达1/3以上，影响叶产量20％，质量下降至少1个等级，苗木生长量影响很大。
综合上述，大螟与棉铃虫虽然同属鳞翅目夜蛾科，但是仅在外部形态和为害习性上就存在诸多方面的不同，且二者亲缘关系较远，无法交配产生后代。因此，可确定大螟与棉铃虫是两种不同的害虫，从而说明二者中肠上皮细胞膜上表面上与Bt毒素结合的受体也是不同的，但是某一类杀虫晶体蛋白只能与特定的受体蛋白结合，即Bt毒素对靶标害虫专一性强，可见将Cry2Ab蛋白用于控制大螟害虫的方法是非显而易见的。
本发明中所述的植物、植物组织或植物细胞的基因组，是指植物、植物 组织或植物细胞内的任何遗传物质，且包括细胞核和质体和线粒体基因组。
本发明中所述的多核苷酸和/或核苷酸形成完整“基因”，在所需宿主细胞中编码蛋白质或多肽。本领域技术人员很容易认识到，可以将本发明的多核苷酸和/或核苷酸置于目的宿主中的调控序列控制下。
本领域技术人员所熟知的，DNA典型的以双链形式存在。在这种排列中，一条链与另一条链互补，反之亦然。由于DNA在植物中复制产生了DNA的其它互补链。这样，本发明包括对序列表中示例的多核苷酸及其互补链的使用。本领域常使用的“编码链”指与反义链结合的链。为了在体内表达蛋白质，典型将DNA的一条链转录为一条mRNA的互补链，它作为模板翻译出蛋白质。mRNA实际上是从DNA的“反义”链转录的。“有义”或“编码”链有一系列密码子(密码子是三个核苷酸，一次读三个可以产生特定氨基酸)，其可作为开放阅读框(ORF)阅读来形成目的蛋白质或肽。本发明还包括与示例的DNA有相当功能的RNA和PNA(肽核酸)。
本发明中核酸分子或其片段在严格条件下与本发明Cry2Ab基因杂交。任何常规的核酸杂交或扩增方法都可以用于鉴定本发明Cry2Ab基因的存在。核酸分子或其片段在一定情况下能够与其他核酸分子进行特异性杂交。本发明中，如果两个核酸分子能形成反平行的双链核酸结构，就可以说这两个核酸分子彼此间能够进行特异性杂交。如果两个核酸分子显示出完全的互补性，则称其中一个核酸分子是另一个核酸分子的“互补物”。本发明中，当一个核酸分子的每一个核苷酸都与另一个核酸分子的对应核苷酸互补时，则称这两个核酸分子显示出“完全互补性”。如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在至少常规的“低度严格”条件下退火且彼此结合，则称这两个核酸分子为“最低程度互补”。类似地，如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在常规的“高度严格”条件下退火且彼此结合，则称这两个核酸分子具有“互补性”。从完全互补性中偏离是可以允许的，只要这种偏离不完全阻止两个分子形成双链结构。为了使一个核酸分子能够作为引物或探针，仅需保证其在序列上具有充分的互补性，以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。
本发明中，基本同源的序列是一段核酸分子，该核酸分子在高度严格条件下能够和相匹配的另一段核酸分子的互补链发生特异性杂交。促进DNA杂交的适合的严格条件，例如，大约在45℃条件下用6.0×氯化钠/柠檬酸钠(SSC)处理，然后在50℃条件下用2.0×SSC洗涤，这些条件对本领域技术人员是公知的。例如，在洗涤步骤中的盐浓度可以选自低度严格条件的约2.0×SSC、50℃到高度严格条件的约0.2×SSC、50℃。此外，洗涤步骤中的温度条件可以从低度严格条件的室温约22℃，升高到高度严格条件的约65 ℃。温度条件和盐浓度可以都发生改变，也可以其中一个保持不变而另一个变量发生改变。优选地，本发明所述严格条件可为在6×SSC、0.5％SDS溶液中，在65℃下与SEQ ID NO：2发生特异性杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜1次。
因此，具有抗虫活性并在严格条件下与本发明SEQ ID NO：2杂交的序列包括在本发明中。这些序列与本发明序列至少大约40％-50％同源，大约60％、65％或70％同源，甚至至少大约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更大的序列同源性。
本发明中所述的基因和蛋白质不但包括特定的示例序列，还包括保存了所述特定示例的蛋白质的杀虫活性特征的部分和/片段(包括与全长蛋白质相比在内和/或末端缺失)、变体、突变体、取代物(有替代氨基酸的蛋白质)、嵌合体和融合蛋白。所述“变体”或“变异”是指编码同一蛋白或编码有杀虫活性的等价蛋白的核苷酸序列。所述“等价蛋白”是指与权利要求的蛋白具有相同或基本相同的抗大螟害虫的生物活性的蛋白。
本发明中所述的DNA分子或蛋白序列的“片段”或“截短”是指涉及的原始DNA或蛋白序列(核苷酸或氨基酸)的一部分或其人工改造形式(例如适合植物表达的序列)，前述序列的长度可存在变化，但长度足以确保(编码)蛋白质为昆虫毒素。
使用标准技术可以修饰基因和容易的构建基因变异体。例如，本领域熟知制造点突变的技术。又例如美国专利号5605793描述了在随机断裂后使用DNA重装配产生其它分子多样性的方法。可以使用商业化核酸内切酶制造全长基因的片段，并且可以按照标准程序使用核酸外切酶。例如，可以使用酶诸如Bal31或定点诱变从这些基因的末端系统地切除核苷酸。还可以使用多种限制性内切酶获取编码活性片段的基因。可以使用蛋白酶直接获得这些毒素的活性片段。
本发明可以从B.t.分离物和/或DNA文库衍生出等价蛋白和/或编码这些等价蛋白的基因。有多种方法获取本发明的杀虫蛋白。例如，可以使用本发明公开和要求保护的杀虫蛋白的抗体从蛋白质混合物鉴定和分离其它蛋白。特别地，抗体可能是由蛋白最恒定和与其它B.t.蛋白最不同的蛋白部分引起的。然后可以通过免疫沉淀、酶联免疫吸附测定(ELISA)或western印迹方法使用这些抗体专一地鉴定有特征活性的等价蛋白。可使用本领域标准程序容易的制备本发明中公开的蛋白或等价蛋白或这类蛋白的片段的抗体。然后可以从微生物中获得编码这些蛋白的基因。
由于遗传密码子的丰余性，多种不同的DNA序列可以编码相同的氨基酸序列。产生这些编码相同或基本相同的蛋白的可替代DNA序列正在本领 域技术人员的技术水平内。这些不同的DNA序列包括在本发明的范围内。所述“基本上相同的”序列是指有氨基酸取代、缺失、添加或插入但实质上不影响杀虫活性的序列，亦包括保留杀虫活性的片段。
本发明中氨基酸序列的取代、缺失或添加是本领域的常规技术，优选这种氨基酸变化为：小的特性改变，即不显著影响蛋白的折叠和/或活性的保守氨基酸取代；小的缺失，通常约1-30个氨基酸的缺失；小的氨基或羧基端延伸，例如氨基端延伸一个甲硫氨酸残基；小的连接肽，例如约20-25个残基长。
保守取代的实例是在下列氨基酸组内发生的取代：碱性氨基酸(如精氨酸、赖氨酸和组氨酸)、酸性氨基酸(如谷氨酸和天冬氨酸)、极性氨基酸(如谷氨酰胺、天冬酰胺)、疏水性氨基酸(如亮氨酸、异亮氨酸和缬氨酸)、芳香氨基酸(如苯丙氨酸、色氨酸和酪氨酸)，以及小分子氨基酸(如甘氨酸、丙氨酸、丝氨酸、苏氨酸和甲硫氨酸)。通常不改变特定活性的那些氨基酸取代在本领域内是众所周知的，并且已由，例如，N.Neurath和R.L Hill在1979年纽约学术出版社(Academic Press)出版的《Protein》中进行了描述。最常见的互换有Ala/Ser，Val/Ile，Asp/Glu，Thu/Ser，Ala/Thr，Ser/Asn，Ala/Val，Ser/Gly，Tyr/Phe，Ala/Pro，Lys/Arg，Asp/Asn，Leu/Ile，Leu/Val，Ala/Glu和Asp/Gly，以及它们相反的互换。
对于本领域的技术人员而言显而易见地，这种取代可以在对分子功能起重要作用的区域之外发生，而且仍产生活性多肽。对于由本发明的多肽，其活性必需的并因此选择不被取代的氨基酸残基，可以根据本领域已知的方法，如定点诱变或丙氨酸扫描诱变进行鉴定(如参见，Cunningham和Wells，1989，Science 244：1081-1085)。后一技术是在分子中每一个带正电荷的残基处引入突变，检测所得突变分子的抗虫活性，从而确定对该分子活性而言重要的氨基酸残基。底物-酶相互作用位点也可以通过其三维结构的分析来测定，这种三维结构可由核磁共振分析、结晶学或光亲和标记等技术测定(参见，如de Vos等，1992，Science 255：306-312；Smith等，1992，J.Mol.Biol224：899-904；Wlodaver等，1992，FEBS Letters 309：59-64)。
在本发明中，Cry2Ab蛋白包括但不限于Cry2Ab蛋白，或者与上述蛋白的氨基酸序列具有至少70％同源性且对大螟具有杀虫活性的杀虫片段或功能区域。
因此，与序列1所示的氨基酸序列具有一定同源性的氨基酸序列也包括在本发明中。这些序列与本发明序列类似性/相同性典型的大于60％，优选的大于75％，更优选的大于80％，甚至更优选的大于90％，并且可以大于95％。也可以根据更特定的相同性和/或类似性范围定义本发明的优选的多核 苷酸和蛋白质。例如与本发明示例的序列有49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性和/或类似性。
本发明中所述调控序列包括但不限于启动子、转运肽、终止子，增强子，前导序列，内含子以及其它可操作地连接到所述Cry2Ab蛋白的调节序列。
所述启动子为植物中可表达的启动子，所述的“植物中可表达的启动子”是指确保与其连接的编码序列在植物细胞内进行表达的启动子。植物中可表达的启动子可为组成型启动子。指导植物内组成型表达的启动子的示例包括但不限于，来源于花椰菜花叶病毒的35S启动子、玉米Ubi启动子、水稻GOS2基因的启动子等。备选地，植物中可表达的启动子可为组织特异的启动子，即该启动子在植物的一些组织内如在绿色组织中指导编码序列的表达水平高于植物的其他组织(可通过常规RNA试验进行测定)，如PEP羧化酶启动子。备选地，植物中可表达的启动子可为创伤诱导启动子。创伤诱导启动子或指导创伤诱导的表达模式的启动子是指当植物经受机械或由昆虫啃食引起的创伤时，启动子调控下的编码序列的表达较正常生长条件下有显著提高。创伤诱导启动子的示例包括但不限于，马铃薯和西红柿的蛋白酶抑制基因(pin I和pin II)和玉米蛋白酶抑制基因(MPI)的启动子。
所述转运肽(又称分泌信号序列或导向序列)是指导转基因产物到特定的细胞器或细胞区室，对受体蛋白质来说，所述转运肽可以是异源的，例如，利用编码叶绿体转运肽序列靶向叶绿体，或者利用‘KDEL’保留序列靶向内质网，或者利用大麦植物凝集素基因的CTPP靶向液泡。
所述前导序列包含但不限于，小RNA病毒前导序列，如EMCV前导序列(脑心肌炎病毒5’非编码区)；马铃薯Y病毒组前导序列，如MDMV(玉米矮缩花叶病毒)前导序列；人类免疫球蛋白质重链结合蛋白质(BiP)；苜蓿花叶病毒的外壳蛋白质mRNA的不翻译前导序列(AMV RNA4)；烟草花叶病毒(TMV)前导序列。
所述增强子包含但不限于，花椰菜花叶病毒(CaMV)增强子、玄参花叶病毒(FMV)增强子、康乃馨风化环病毒(CERV)增强子、木薯脉花叶病毒(CsVMV)增强子、紫茉莉花叶病毒(MMV)增强子、夜香树黄化曲叶病毒(CmYLCV)增强子、木尔坦棉花曲叶病毒(CLCuMV)、鸭跖草黄斑驳病毒(CoYMV)和花生褪绿线条花叶病毒(PCLSV)增强子。
对于单子叶植物应用而言，所述内含子包含但不限于，玉米hsp70内含 子、玉米泛素内含子、Adh内含子1、蔗糖合酶内含子或水稻Act1内含子。对于双子叶植物应用而言，所述内含子包含但不限于，CAT-1内含子、pKANNIBAL内含子、PIV2内含子和“超级泛素”内含子。
所述终止子可以为在植物中起作用的适合多聚腺苷酸化信号序列，包括但不限于，来源于农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)基因的多聚腺苷酸化信号序列、来源于蛋白酶抑制剂II(pin II)基因的多聚腺苷酸化信号序列、来源于豌豆ssRUBISCO E9基因的多聚腺苷酸化信号序列和来源于α-微管蛋白(α-tubulin)基因的多聚腺苷酸化信号序列。
本发明中所述“有效连接”表示核酸序列的联结，所述联结使得一条序列可提供对相连序列来说需要的功能。在本发明中所述“有效连接”可以为将启动子与感兴趣的序列相连，使得该感兴趣的序列的转录受到该启动子控制和调控。当感兴趣的序列编码蛋白并且想要获得该蛋白的表达时“有效连接”表示：启动子与所述序列相连，相连的方式使得得到的转录物高效翻译。如果启动子与编码序列的连接是转录物融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得得到的转录物中第一翻译起始密码子是编码序列的起始密码子。备选地，如果启动子与编码序列的连接是翻译融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得5’非翻译序列中含有的第一翻译起始密码子与启动子相连结，并且连接方式使得得到的翻译产物与编码想要的蛋白的翻译开放读码框的关系是符合读码框的。可以“有效连接”的核酸序列包括但不限于：提供基因表达功能的序列(即基因表达元件，例如启动子、5’非翻译区域、内含子、蛋白编码区域、3’非翻译区域、聚腺苷化位点和/或转录终止子)、提供DNA转移和/或整合功能的序列(即T-DNA边界序列、位点特异性重组酶识别位点、整合酶识别位点)、提供选择性功能的序列(即抗生素抗性标记物、生物合成基因)、提供可计分标记物功能的序列、体外或体内协助序列操作的序列(即多接头序列、位点特异性重组序列)和提供复制功能的序列(即细菌的复制起点、自主复制序列、着丝粒序列)。
本发明中所述的“杀虫”是指对农作物害虫是有毒的。更具体地，目标昆虫是大螟害虫。
本发明中Cry2Ab蛋白对大螟害虫具有毒性。本发明中的植物，特别是玉米和高粱，在其基因组中含有外源DNA，所述外源DNA包含编码Cry2Ab蛋白的核苷酸序列，大螟害虫通过摄食植物组织与该蛋白接触，接触后大螟害虫生长受到抑制和/或导致死亡。同时，植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成。此外，该植物可基本消除对化学或生物杀虫剂的需要(所述化学或生物杀虫剂为针对Cry2Ab蛋白所靶向的大螟害虫的杀虫剂)。
植物材料中杀虫晶体蛋白(ICP)的表达水平可通过本领域内所描述的多种方法进行检测，例如通过应用特异引物对组织内产生的编码杀虫蛋白质的mRNA进行定量，或直接特异性检测产生的杀虫蛋白质的量。
可以应用不同的试验测定植物中ICP的杀虫效果。本发明中目标昆虫主要为大螟。
本发明中，所述Cry2Ab蛋白可以具有序列表中SEQ ID NO：1所示的氨基酸序列。除了包含Cry2Ab蛋白的编码区外，也可包含其他元件，例如编码第二种杀虫核苷酸、编码选择性标记的蛋白质或赋予除草剂抗性的蛋白质的编码区。
此外，包含编码本发明Cry2Ab蛋白的核苷酸序列的表达盒在植物中还可以与至少一种编码除草剂抗性基因的蛋白质一起表达，所述除草剂抗性基因包括但不限于，草胺膦抗性基因(如bar基因、pat基因)、苯敌草抗性基因(如pmph基因)、草甘膦抗性基因(如EPSPS基因)、溴苯腈(bromoxynil)抗性基因、磺酰脲抗性基因、对除草剂茅草枯的抗性基因、对氨腈的抗性基因或谷氨酰胺合成酶抑制剂(如PPT)的抗性基因，从而获得既具有高杀虫活性、又具有除草剂抗性的转基因植物。
本发明中，将外源DNA导入(引入)植物，如将编码所述Cry2Ab蛋白的基因或表达盒或重组载体导入植物细胞，常规的转化方法包括但不限于，农杆菌介导的转化、微量发射轰击、直接将DNA摄入原生质体、电穿孔或晶须硅介导的DNA导入。
本发明提供了一种杀虫蛋白的用途，具有以下优点：
1、内因防治。现有技术主要是通过外部作用即外因来控制大螟害虫的危害，如农业防治、化学防治和生物防治；而本发明是通过植物体内产生能够杀死大螟的Cry2Ab蛋白来控制大螟害虫的，即通过内因来防治。
2、无污染、无残留。现有技术使用的化学防治方法虽然对控制大螟害虫的危害起到了一定作用，但同时也对人、畜和农田生态系统带来了污染、破坏和残留；使用本发明控制大螟害虫的方法，可以消除上述不良后果。
3、全生育期防治。现有技术使用的控制大螟害虫的方法都是阶段性的，而本发明是对植物进行全生育期的保护，转基因植物(Cry2Ab蛋白)从发芽、生长，一直到开花、结果，都可以避免遭受大螟的侵害。
4、全植株防治。现有技术使用的控制大螟害虫的方法大多是局部性的，如叶面喷施；而本发明是对整个植株进行保护，如转基因植物(Cry2Ab蛋白)的叶片、茎秆、雄穗、雌穗、花药、花丝等都是可以抵抗大螟侵害的。
5、效果稳定。现有技术使用的喷施农药的方法需要直接喷施到作物表面，容易造成喷施不均匀或漏喷等情况；本发明是使所述Cry2Ab蛋白在植 物体内进行表达，表达量基本上稳定一致，且本发明转基因植物(Cry2Ab蛋白)的防治效果在不同地点、不同时间、不同遗传背景也都是稳定一致的。
6、简单、方便、经济。由于大螟特殊的隐蔽发生与危害特征，导致对其危害的监测和防治较为困难，大大地增加了种植成本；本发明只需种植能够表达Cry2Ab蛋白的转基因植物即可，而不需要采用其它措施，从而节省了大量人力、物力和财力。
7、效果彻底。现有技术使用的控制大螟害虫的方法，其效果是不彻底的，只起到减轻作用；而本发明转基因植物(Cry2Ab蛋白)可以造成初孵大螟幼虫的大量死亡，且对小部分存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，都是明显的发育不良，且已停止发育，转基因植物大体上只受到轻微损伤。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图的简要说明
图1为本发明杀虫蛋白的用途的含有Cry2Ab核苷酸序列的重组克隆载体DBN01-T构建流程图；
图2为本发明杀虫蛋白的用途的含有Cry2Ab核苷酸序列的重组表达载体DBN100033构建流程图；
图3为本发明杀虫蛋白的用途的转基因玉米植株接种大螟的叶片损伤图。
实施发明的最佳方式
下面通过具体实施例进一步说明本发明杀虫蛋白的用途的技术方案。
第一实施例、Cry2Ab基因的获得和合成
1、获得Cry2Ab核苷酸序列
Cry2Ab杀虫蛋白质的氨基酸序列(634个氨基酸)，如序列表中SEQ ID NO：1所示；编码相应于所述Cry2Ab杀虫蛋白质的氨基酸序列的Cry2Ab核苷酸序列(1905个核苷酸)，如序列表中SEQ ID NO：2所示。
2、获得Cry1A.105核苷酸序列
编码Cry1A.105杀虫蛋白质的氨基酸序列(1177个氨基酸)的Cry1A.105核苷酸序列(3534个核苷酸)，如序列表中SEQ ID NO：3所示。
3、合成上述核苷酸序列
所述Cry2Ab核苷酸序列(如序列表中SEQ ID NO：2所示)和所述Cry1A.105核苷酸序列(如序列表中SEQ ID NO：3所示)由南京金斯瑞生物科技有限公司合成；合成的所述Cry2Ab核苷酸序列(SEQ ID NO：2)的5’ 端还连接有NcoI酶切位点，所述Cry2Ab核苷酸序列(SEQ ID NO：2)的3’端还连接有SpeI酶切位点；合成的所述Cry1A.105核苷酸序列(SEQ ID NO：3)的5’端还连接有NcoI酶切位点，所述Cry1A.105核苷酸序列(SEQ ID NO：3)的3’端还连接有HindIII酶切位点。
第二实施例、重组表达载体的构建及重组表达载体转化农杆菌
1、构建含有Cry2Ab基因的重组克隆载体
将合成的Cry2Ab核苷酸序列连入克隆载体pGEM-T(Promega，Madison，USA，CAT：A3600)上，操作步骤按Promega公司产品pGEM-T载体说明书进行，得到重组克隆载体DBN01-T，其构建流程如图1所示(其中，Amp表示氨苄青霉素抗性基因；f1表示噬菌体f1的复制起点；LacZ为LacZ起始密码子；SP6为SP6 RNA聚合酶启动子；T7为T7 RNA聚合酶启动子；Cry2Ab为Cry2Ab核苷酸序列(SEQ ID NO：2)；MCS为多克隆位点)。
然后将重组克隆载体DBN01-T用热激方法转化大肠杆菌T1感受态细胞(Transgen，Beijing，China，CAT：CD501)，其热激条件为：50μl大肠杆菌T1感受态细胞、10μl质粒DNA(重组克隆载体DBN01-T)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)，在表面涂有IPTG(异丙基硫代-β-D-半乳糖苷)和X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)的氨苄青霉素(100毫克/升)的LB平板(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，用NaOH调pH至7.5)上生长过夜。挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，氨苄青霉素100mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒：将菌液在12000rpm转速下离心1min，去上清液，沉淀菌体用100μl冰预冷的溶液I(25mM Tris-HCl，10mM EDTA(乙二胺四乙酸)，50mM葡萄糖，pH8.0)悬浮；加入200μl新配制的溶液II(0.2M NaOH，1％SDS(十二烷基硫酸钠))，将管子颠倒4次，混合，置冰上3-5min；加入150μl冰冷的溶液III(3M醋酸钾，5M醋酸)，立即充分混匀，冰上放置5-10min；于温度4℃、转速12000rpm条件下离心5min，在上清液中加入2倍体积无水乙醇，混匀后室温放置5min；于温度4℃、转速12000rpm条件下离心5min，弃上清液，沉淀用浓度(V/V)为70％的乙醇洗涤后晾干；加入30μl含RNase(20μg/ml)的TE(10mM Tris-HCl，1mM EDTA，PH8.0)溶解沉淀；于温度37℃下水浴30min，消化RNA；于温度-20℃保存备用。
提取的质粒经EcoRI和XhoI酶切鉴定后，对阳性克隆进行测序验证，结果表明重组克隆载体DBN01-T中插入的所述Cry2Ab核苷酸序列为序列表中SEQ ID NO：2所示的核苷酸序列，即Cry2Ab核苷酸序列正确插入。
按照上述构建重组克隆载体DBN01-T的方法，将合成的所述Cry1A.105 核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN02-T，其中，Cry1A.105为Cry1A.105核苷酸序列(SEQ ID NO：3)。酶切和测序验证重组克隆载体DBN02-T中所述Cry1A.105核苷酸序列正确插入。
2、构建含有Cry2Ab基因的重组表达载体
用限制性内切酶NcoI和SpeI分别酶切重组克隆载体DBN01-T和表达载体DBNBC-01(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的Cry2Ab核苷酸序列片段插到表达载体DBNBC-01的NcoI和SpeI位点之间，利用常规的酶切方法构建载体是本领域技术人员所熟知的，构建成重组表达载体DBN100033，其构建流程如图2所示(Kan：卡那霉素基因；RB：右边界；Ubi：玉米Ubiquitin(泛素)基因启动子(SEQ ID NO：4)；Cry2Ab：Cry2Ab核苷酸序列(SEQ ID NO：2)；Nos：胭脂碱合成酶基因的终止子(SEQ ID NO：5)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO：6)；LB：左边界)。
将重组表达载体DBN100033用热激方法转化大肠杆菌T1感受态细胞，其热激条件为：50μl大肠杆菌T1感受态细胞、10μl质粒DNA(重组表达载体DBN100033)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)；然后在含50mg/L卡那霉素(Kanamycin)的LB固体平板(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，用NaOH调pH至7.5)上于温度37℃条件下培养12小时，挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，卡那霉素50mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒。将提取的质粒用限制性内切酶NcoI和SpeI酶切后鉴定，并将阳性克隆进行测序鉴定，结果表明重组表达载体DBN100033在NcoI和SpeI位点间的核苷酸序列为序列表中SEQ ID NO：2所示核苷酸序列，即Cry2Ab核苷酸序列。
按照上述构建重组表达载体DBN100033的方法，将NcoI和SpeI、NcoI和HindIII分别酶切重组克隆载体DBN01-T和DBN02-T切下的所述Cry2Ab核苷酸序列和Cry1A.105核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100076。酶切和测序验证重组表达载体DBN100076中的核苷酸序列含有为序列表中SEQ ID NO：2和SEQ ID NO：3所示核苷酸序列，即Cry2Ab核苷酸序列和Cry1A.105核苷酸序列，所述Cry2Ab核苷酸序列和所述Cry1A.105核苷酸序列可以连接所述Ubi启动子和Nos终止子。
3、重组表达载体转化农杆菌
对己经构建正确的重组表达载体DBN100033和DBN100076用液氮法转化到农杆菌LBA4404(Invitrgen，Chicago，USA，CAT：18313-015)中，其转化条件为：100μL农杆菌LBA4404、3μL质粒DNA(重组表达载体)； 置于液氮中10分钟，37℃温水浴10分钟；将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时，涂于含50mg/L的利福平(Rifampicin)和100mg/L的卡那霉素(Kanamycin)的LB平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，用限制性内切酶AhdI和XhoI对重组表达载体DBN100033和DBN100076酶切后进行酶切验证，结果表明重组表达载体DBN100033和DBN100076结构完全正确。
第三实施例、转入Cry2Ab基因的玉米植株的获得及验证
1、获得转入Cry2Ab基因的玉米植株
按照常规采用的农杆菌侵染法，将无菌培养的玉米品种综31(Z31)的幼胚与第二实施例中3所述的农杆菌共培养，以将第二实施例中2构建的重组表达载体DBN100033和DBN100076中的T-DNA(包括玉米Ubiquitin基因的启动子序列、Cry2Ab核苷酸序列、Cry1A.105核苷酸序列、PMI基因和Nos终止子序列)转入到玉米染色体组中，获得了转入Cry2Ab核苷酸序列的玉米植株和转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株；同时以野生型玉米植株作为对照。
对于农杆菌介导的玉米转化，简要地，从玉米中分离未成熟的幼胚，用农杆菌悬浮液接触幼胚，其中农杆菌能够将Cry2Ab核苷酸序列和/或Cry1A.105核苷酸序列传递至幼胚之一的至少一个细胞(步骤1：侵染步骤)，在此步骤中，幼胚优选地浸入农杆菌悬浮液(OD660＝0.4-0.6，侵染培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖68.5g/L、葡萄糖36g/L、乙酰丁香酮(AS)40mg/L、2，4-二氯苯氧乙酸(2，4-D)1mg/L，pH5.3))中以启动接种。幼胚与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，幼胚在侵染步骤后在固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)100mg/L、2，4-二氯苯氧乙酸(2，4-D)1mg/L、琼脂8g/L，pH5.8)上培养。在此共培养阶段后，可以有一个选择性的“恢复”步骤。在“恢复”步骤中，恢复培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、2，4-二氯苯氧乙酸(2，4-D)1mg/L、琼脂8g/L，pH5.8)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素)，不添加植物转化体的选择剂(步骤3：恢复步骤)。优选地，幼胚在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，接种的幼胚在含选择剂(甘露糖)的培养基上培养并选择生长着的转化愈伤组织(步骤4：选择步骤)。优选地，幼胚在有选择剂的筛选固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖5g/L、甘露糖12.5g/L、2，4-二氯苯氧乙酸(2，4-D)1mg/L、琼脂8g/L，pH5.8)上培养，导致转化的细胞选择性生长。然后，愈伤组织再生成植物(步骤5： 再生步骤)，优选地，在含选择剂的培养基上生长的愈伤组织在固体培养基(MS分化培养基和MS生根培养基)上培养以再生植物。
筛选得到的抗性愈伤组织转移到所述MS分化培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、6-苄基腺嘌呤2mg/L、甘露糖5g/L、琼脂8g/L，pH5.8)上，25℃下培养分化。分化出来的小苗转移到所述MS生根培养基(MS盐2.15g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、吲哚-3-乙酸1mg/L、琼脂8g/L，pH5.8)上，25℃下培养至约10cm高，移至温室培养至结实。在温室中，每天于28℃下培养16小时，再于20℃下培养8小时。
2、用TaqMan验证转入Cry2Ab基因的玉米植株
分别取转入Cry2Ab核苷酸序列的玉米植株和转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株的叶片约100mg作为样品，用Qiagen的DNeasy Plant Maxi Kit提取其基因组DNA，通过Taqman探针荧光定量PCR方法检测Cry2Ab基因和Cry1A.105基因的拷贝数。同时以野生型玉米植株作为对照，按照上述方法进行检测分析。实验设3次重复，取平均值。
检测Cry2Ab基因拷贝数的具体方法如下：
步骤11、分别取转入Cry2Ab核苷酸序列的玉米植株、转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株和野生型玉米植株的叶片各100mg，分别在研钵中用液氮研成匀浆，每个样品取3个重复；
步骤12、使用Qiagen的DNeasy Plant Mini Kit提取上述样品的基因组DNA，具体方法参考其产品说明书；
步骤13、用NanoDrop 2000(Thermo Scientific)测定上述样品的基因组DNA浓度；
步骤14、调整上述样品的基因组DNA浓度至同一浓度值，所述浓度值的范围为80-100ng/μl；
步骤15、采用Taqman探针荧光定量PCR方法鉴定样品的拷贝数，以经过鉴定已知拷贝数的样品作为标准品，以野生型玉米植株的样品作为对照，每个样品3个重复，取其平均值；荧光定量PCR引物和探针序列分别是：
以下引物和探针用来检测Cry2Ab核苷酸序列：
引物1(CF1)：CTGATACCCTTGCTCGCGTC如序列表中SEQ ID NO：7所示；
引物2(CR1)：CACTTGGCGGTTGAACTCCTC如序列表中SEQ ID NO：8所示；
探针1(CP1)：CGCTGAGCTGACGGGTCTGCAAG如序列表中SEQ ID  NO：9所示；
以下引物和探针用来检测Cry1A.105核苷酸序列：
引物3(CF2)：GCGCATCCAGTTCAACGAC如序列表中SEQ ID NO：10所示；
引物4(CR2)：GTTCTGGACGGCGAAGAGTG如序列表中SEQ ID NO：11所示；
探针2(CP2)：TGAACAGCGCCCTGACCACCG如序列表中SEQ ID NO：12所示；
PCR反应体系为：
Figure PCTCN2015085047-appb-000001
所述50×引物/探针混合物包含1mM浓度的每种引物各45μl，100μM浓度的探针50μl和860μl 1×TE缓冲液，并且在4℃，贮藏在琥珀试管中。
PCR反应条件为：
Figure PCTCN2015085047-appb-000002
利用SDS2.3软件(Applied Biosystems)分析数据。
实验结果表明，Cry2Ab核苷酸序列和Cry2Ab-Cry1A.105核苷酸序列均己整合到所检测的玉米植株的染色体组中，而且转入Cry2Ab核苷酸序列的玉米植株和转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株均获得了含有单拷贝Cry2Ab基因的转基因玉米植株。
第四实施例、转基因玉米植株的抗虫效果检测
将转入Cry2Ab核苷酸序列的玉米植株、转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株、野生型玉米植株和经Taqman鉴定为非转基因的玉米植株对大螟进行抗虫效果检测。
分别取转入Cry2Ab核苷酸序列的玉米植株、转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株、野生型玉米植株和经Taqman鉴定为非转基因的玉米植株(V3-V4期)的新鲜叶片，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后将玉米叶片去除叶脉，同时剪成约1cm×4cm的长条状，取2片剪后的长条状叶片放入圆形塑料培养皿底部的滤纸上，所述滤纸用蒸馏水润 湿，每个培养皿中放10头人工饲养的大螟(初孵幼虫)，虫试培养皿加盖后，在温度25-28℃、相对湿度70％-80％、光周期(光/暗)16：8的条件下放置3天后，根据大螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：总分＝100×死亡率+[100×死亡率+90×(初孵虫数/接虫总数)+60×(初孵-阴性对照虫数/接虫总数)+10×(阴性对照虫数/接虫总数)]+100×(1-叶片损伤率)。转入Cry2Ab核苷酸序列的共3个株系(S1、S2和S3)，转入Cry2Ab-Cry1A.105核苷酸序列的共3个株系(S4、S5和S6)，经Taqman鉴定为非转基因的(NGM)共1个株系，野生型的(CK)共1个株系；从每个株系选3株进行测试，每株重复6次。结果如表1和图3所示。
表1、转基因玉米植株接种大螟的抗虫实验结果
Figure PCTCN2015085047-appb-000003
表1的结果表明：转入Cry2Ab核苷酸序列的玉米植株和转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株的生测总分均在260分左右或以上；而经Taqman鉴定为非转基因的玉米植株和野生型玉米植株的生测总分一般在30分左右，显著低于所述转入Cry2Ab核苷酸序列的玉米植株和所述转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株。
图3的结果表明：与野生型玉米植株相比，转入Cry2Ab核苷酸序列的 玉米植株和转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株可以造成大螟初孵幼虫的大量死亡，且对小部分存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，且转入Cry2Ab核苷酸序列的玉米植株和转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株大体上只受到极轻微损伤，叶片上仅为极少量针孔状损伤，其叶片损伤率均在3％左右，具有显著减弱的植物损伤。
由此证明转入Cry2Ab核苷酸序列的玉米植株和转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株都显示出高抗大螟的活性，这种活性足以对大螟的生长产生不良效应从而使其得以控制。
上述实验结果还表明转入Cry2Ab核苷酸序列的玉米植株和转入Cry2Ab-Cry1A.105核苷酸序列的玉米植株对大螟的防治显然是因为植物本身可产生Cry2Ab蛋白，所以，本领域技术人员熟知的，根据Cry2Ab蛋白对大螟的相同毒杀作用，可产生类似的可表达Cry2Ab蛋白的转基因植株能够用于防治大螟的危害。本发明中Cry2Ab蛋白包括但不限于具体实施方式中所给出氨基酸序列的Cry2Ab蛋白，同时转基因植株还可以产生至少一种不同于Cry2Ab蛋白的第二种杀虫蛋白质，如Vip类蛋白、Cry类蛋白。
综上所述，本发明杀虫蛋白的用途通过植物体内产生能够杀死大螟的Cry2Ab蛋白来控制大螟害虫；与现有技术使用的农业防治方法和化学防治方法相比，本发明对植物进行全生育期、全植株的保护以防治大螟害虫的侵害，且无污染、无残留，效果稳定、彻底，简单、方便、经济。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。
Figure PCTCN2015085047-appb-000004
Figure PCTCN2015085047-appb-000005
Figure PCTCN2015085047-appb-000006
Figure PCTCN2015085047-appb-000007
Figure PCTCN2015085047-appb-000008
Figure PCTCN2015085047-appb-000009
Figure PCTCN2015085047-appb-000010
Figure PCTCN2015085047-appb-000011
Figure PCTCN2015085047-appb-000012
Figure PCTCN2015085047-appb-000013
Figure PCTCN2015085047-appb-000014

Claims (17)

  1. 一种控制大螟害虫的方法，其特征在于，包括将大螟害虫与Cry2Ab蛋白接触；
    优选地，所述Cry2Ab蛋白存在于产生所述Cry2Ab蛋白的植物细胞中，所述大螟害虫通过摄食所述植物细胞与所述Cry2Ab蛋白接触；
    进一步优选地，所述Cry2Ab蛋白存在于产生所述Cry2Ab蛋白的转基因植物中，所述大螟害虫通过摄食所述转基因植物的组织与所述Cry2Ab蛋白接触，接触后所述大螟害虫生长受到抑制和/或导致死亡，以实现对大螟危害植物的控制。
  2. 根据权利要求1所述的控制大螟害虫的方法，其特征在于，所述转基因植物处于任意生育期；和/或
    所述转基因植物的组织为叶片、茎秆、雄穗、雌穗、花药或花丝；和/或
    所述对大螟危害植物的控制不因种植地点和/或种植时间的改变而改变。
  3. 根据权利要求1或2所述的控制大螟害虫的方法，其特征在于，所述植物为玉米、水稻、高粱、麦、粟、棉花、芦苇、甘蔗、茭白、蚕豆或油菜。
  4. 根据权利要求1至3任一项所述的控制大螟害虫的方法，其特征在于，所述接触步骤之前的步骤为种植含有编码所述Cry2Ab蛋白的多核苷酸的植物。
  5. 根据权利要求1至4任一项所述的控制大螟害虫的方法，其特征在于，所述Cry2Ab蛋白的氨基酸序列具有SEQ ID NO:1所示的氨基酸序列；
    优选地，所述Cry2Ab蛋白的核苷酸序列具有SEQ ID NO:2所示的核苷酸序列。
  6. 根据权利要求1至5任一项所述的控制大螟害虫的方法，其特征在于，所述植物还可以包括至少一种不同于编码所述Cry2Ab蛋白的核苷酸的第二种核苷酸；
    优选地，所述第二种核苷酸编码Cry类杀虫蛋白质、Vip类杀虫蛋白质、蛋白酶抑制剂、凝集素、α-淀粉酶或过氧化物酶；
    进一步优选地，所述第二种核苷酸编码Cry1A.105蛋白
    最优选地，所述第二种核苷酸具有SEQ ID NO:3所示的核苷酸序列。
  7. 根据权利要求6所述的控制大螟害虫的方法，其特征在于，所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
  8. 根据权利要求1-7任一项所述的方法，其中所述大螟害虫在作物田 地中。
  9. 一种Cry2Ab蛋白质控制大螟害虫的用途。
  10. 一种转Cry2Ab基因的植物细胞、植物或植物部分控制大螟害虫的用途。
  11. 一种产生控制大螟害虫的植物的方法，其特征在于，包括向所述植物的基因组中引入编码Cry2Ab蛋白的多核苷酸序列。
  12. 一种产生控制大螟害虫的植物种子的方法，其特征在于，包括将由权利要求11所述方法获得的第一植株与第二植株杂交，从而产生含有编码Cry2Ab蛋白的多核苷酸序列的种子。
  13. 一种控制大螟害虫的植物种子，其特征在于，所述种子为含有编码Cry2Ab蛋白的多核苷酸序列的种子。
  14. 一种培养控制大螟害虫的植物的方法，其特征在于，包括：
    种植至少一粒植物种子，所述植物种子的基因组中包括编码Cry2Ab蛋白的多核苷酸序列；
    使所述植物种子长成植株；
    使所述植株在人工接种大螟害虫和/或大螟害虫自然发生危害的条件下生长，收获与其他不具有编码Cry2Ab蛋白的多核苷酸序列的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株。
  15. 根据权利要求14所述的方法培养产生的控制大螟害虫的植物。
  16. 一种控制大螟害虫的植物细胞、植物或植物部分，其特征在于，所述植物细胞、植物或植物部分的基因组中包含编码Cry2Ab蛋白的多核苷酸序列。
  17. 根据权利要求16所述的植物细胞、植物或植物部分，其中所述植物选自由以下组成的组：玉米、水稻、高粱、麦、粟、棉花、芦苇、甘蔗、茭白、蚕豆或油菜，并且其中所述植物部分为种子、叶片、茎秆、雄穗、雌穗、花药、花丝、根或其任何部分。
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