WO2015067194A1 - Method for controlling pests - Google Patents

Abstract

The present application relates to a method for controlling oriental armyworm pests, comprising contacting oriental armyworm pests with a Cry1A protein. The present application controls oriental armyworm pests by means of a Cry1A protein produced in plants which can kill the oriental armyworm pests; compared with the methods of agricultural control, chemical control and physical control used in the prior art, the present application is for protecting plants during the entire growth period, protecting the whole plant to prevent and control encroachments by oriental armyworm pests, and has no pollution, no residue, a stable effect, and is thorough, simple, convenient and economic.

Description

Method of controlling pests

The present application claims the priority of the Chinese Patent Application No. CN201310681139.2 filed on Dec. 12, 2013, and the Chinese Patent Application No. CN201310556674.5 filed on Nov. 11, 2013.

Technical field

The present application relates to a method of controlling pests, and more particularly to a method for controlling a plant of the oriental armyworm by using a CrylA protein expressed in a plant.

Background technique

Mythimna seperata belongs to the family Lepidoptera, and is an omnivorous, migratory and intermittent outbreak of pests mainly caused by wheat, corn, sorghum, rice and other food crops and pastures. The Xinjiang area has not yet occurred, and other places are harmful. Oriental sticky insects like to eat leaves, after the third age can bite the entire leaf into a nick, or eat the heart leaves, forming a heartless seedling. During the gluttony period of five to six years, all the above-ground parts of the seedlings can be eaten up, or the leaves of the whole plant can be eaten only to leave the veins, resulting in severe reduction of production or even rejection; the oriental armyworm can also damage the ears in addition to the favorite leaves.

Maize and rice are important food crops in China. In the past two years, the damage of oriental armyworms has been in an outbreak. In 2012, the second generation of oriental armyworms harmed corn area to 64.95 million mu. In 2013, it harmed 58 million mu of corn. The greater harm has seriously affected the living conditions of the local population. In order to control oriental armyworms, the main control methods commonly used are: agricultural control, chemical control and biological control.

Agricultural control is to comprehensively coordinate the multi-factors of the whole farmland ecosystem, regulate crops, pests, environmental factors, and create a farmland ecological environment that is conducive to crop growth and is not conducive to the occurrence of oriental armyworms. For example, before the adult spawning period, 8-10 roots of intact and unscented straws are selected into a small number, 30-50 per acre, and replaced every 5-7 days (if the grass is soaked with the agent, the reduction can be reduced. The number of times) can significantly reduce the density of insects in the field; it can also be used for feeding ducks during the occurrence of larvae. Because agricultural control must obey the requirements of crop layout and increase production, the application has certain limitations and cannot be used as an emergency measure. It seems to be powerless when the oriental armyworm breaks out.

Chemical control, that is, pesticide control, is the use of chemical pesticides to kill pests. It is an important part of the comprehensive management of oriental armyworms. It is characterized by rapid, convenient, simple and high economic benefits, especially in the case of oriental armyworms. In the case, it is an essential emergency measure. At present, the chemical control method is mainly a liquid spray. However, chemical control also has its limitations. If used improperly, it will lead to crops. The phytotoxicity, pests produce resistance, as well as killing natural enemies, polluting the environment, causing damage to farmland ecosystems and pesticide residues posing a threat to the safety of people and animals.

Physical control mainly relies on the response of pests to various physical factors in environmental conditions, and uses various physical factors such as light, electricity, color, temperature and humidity, and mechanical equipment to induce pests, radiation infertility and other methods to control pests. At present, the most widely used is the frequency-vibration insecticidal lamp trapping, which utilizes the phototaxis of pest adults, uses light at close range, uses waves at a long distance, attracts insects close to each other, and has excellent control effect on adults of oriental armyworms; Insect lamps need to clean the dirt on the high-voltage power grid every day, otherwise it will affect the insecticidal effect; and in the thunderstorm days, the lights cannot be turned on, and there is also the danger of electric shocks in the operation; in addition, the one-time investment in installing the lamps is large.

In order to solve the limitations of agricultural control, chemical control and physical control in practical applications, scientists have discovered that insect-resistant genes encoding insecticidal proteins can be transferred into plants, and some insect-resistant transgenic plants can be obtained to control plant pests. Cry1A insecticidal protein is one of many insecticidal proteins and is an insoluble parasporal crystal protein produced by Bacillus thuringiensis subsp. kurstaki (B.t.k.).

The Cry1A 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 with the Cry1A gene have been shown to be resistant to Lepidoptera pests such as corn borer, cotton bollworm, and fall armyworm. However, it is rare to control the damage of plants by oriental armyworms by producing transgenic plants expressing the Cry1A protein. Report.

Summary of the invention

The present application provides a method for controlling pests. For the first time, a method for controlling the damage of oriental armyworms to plants by producing transgenic plants expressing Cry1A protein is provided, and technical defects such as agricultural control, chemical control and physical control in the prior art are effectively overcome.

A first aspect of the present application relates to a method of controlling oriental armyworm pests, wherein the method comprises contacting an oriental armyworm pest with a Cry1A protein.

In a further embodiment, the Cry1A protein is present in a plant cell that produces the Cry1A protein, the oriental armyworm pest being contacted with the Cry1A protein protein by ingesting the plant cell.

In a further embodiment, the Cry1A is present in a transgenic plant producing the Cry1A protein, the oriental armyworm pest contacting the Cry1A protein by ingesting tissue of the transgenic plant, the oriental armyworm after contact Inhibition of pest growth and/or exposure leads to the East Mucoid pests die, thereby achieving control of plants that are harmful to the oriental armyworm.

In some embodiments, the Cry1A protein is one or more of a Cry1Ab protein, a Cry1Ac protein, a Cry1Ah protein, or a Cry1A.105 protein.

The transgenic plant can be in any growth period.

The tissue of the transgenic plant is selected from one or more of the following: leaves, stems, fruits, tassels, ears, anthers, and filaments.

The control of the plants against the oriental armyworm does not change due to changes in the location and/or planting time.

The plant is selected from the group consisting of corn, rice, wheat, sorghum, millet, barley or grasses, preferably the plant is corn or rice.

The step prior to the contacting step is the planting of a plant containing a polynucleotide encoding the Cry1A protein.

In some embodiments, the amino acid sequence of the Cry1A protein comprises: 1) the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, 2) and SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 has at least 70% homology and has an insecticidal activity against oriental armyworm, such as at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or 3) SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: An amino acid sequence obtained by substituting, deleting or adding one or more amino acid residues and having insect resistance to oriental armyworm, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50 amino acid residues.

In some embodiments, the nucleotide sequence encoding the Cry1A protein comprises: 1) the nucleotide sequence set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, 2) and SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 has a nucleotide sequence of at least about 75% homology and encodes an amino acid sequence having insecticidal activity against oriental armyworm, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, 3) under stringent conditions with SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 nucleotide sequence which hybridizes and encodes an amino acid sequence having insecticidal activity against oriental armyworm, 4) is different from SEQ ID NO: 4, SEQ ID NO due to codon degeneracy : 5 or a nucleotide sequence of SEQ ID NO: 6 encoding an amino acid sequence having insecticidal activity against oriental armyworm.

In some embodiments, the plant further comprises at least one second nucleotide different from the nucleotide encoding the Cry1A protein.

In a further embodiment, 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.

In a further embodiment, the second nucleotide encodes a Cry1F protein, a Vip3A protein, or a Cry2Ab.

In a still further embodiment, the second nucleotide comprises the nucleotide sequence set forth in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

In other embodiments, the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.

A second aspect of the present application relates to the use of a Cry1A protein for controlling oriental armyworm pests.

In some embodiments, the Cry1A protein is one or more of a Cry1Ab protein, a Cry1Ac protein, a Cry1Ah protein, or a Cry1A.105 protein.

In a further embodiment, the amino acid sequence of the Cry1A protein comprises: 1) the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, 2) and SEQ ID NO: SEQ ID NO: 2 or SEQ ID NO: 3 has at least 70% homology and has an insecticidal activity against oriental armyworm, such as at least 70%, 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or 3) SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 An amino acid sequence obtained by substituting, deleting and/or adding one or more amino acid residues and having insect resistance to oriental armyworms, such as 1, 2, 3, 4, 5, 6, 7, 8 9, 10, 15, 20, 30, 50 amino acid residues.

In a further embodiment, the nucleotide sequence encoding the Cry1A protein comprises: 1) the nucleotide sequence set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, 2) and SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 has a nucleotide sequence of at least about 75% homology and encodes an amino acid sequence having insecticidal activity against oriental armyworm, such as at least 75%, 80% , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, 3) under strict conditions and SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 nucleotide sequence which hybridizes and encodes an amino acid sequence having insecticidal activity against oriental armyworm, 4) is different from SEQ ID NO: 4, SEQ ID due to codon degeneracy NO: 5 or a nucleotide sequence of SEQ ID NO: 6 encoding an amino acid sequence having insecticidal activity against oriental armyworm.

In some embodiments, the Cry1A protein controls oriental armyworm pests by effecting expression of the Cry1A protein in plant cells and by inoculating the plant cells with the Cry1A protein by oriental armyworm pests.

In some embodiments, the Cry1A protein controls oriental armyworm pests by effecting expression of the Cry1A protein in the transgenic plant and contacting the tissue of the transgenic plant with the Cry1A protein by the oriental armyworm pest.

The transgenic plant can be in any growth period.

The tissue of the transgenic plant is selected from one or more of the following: leaves, stems, fruits, tassels, ears, anthers, and filaments.

The Cry1A protein controls the oriental armyworm pests to not change due to changes in planting location and/or planting time.

The plant is selected from the group consisting of corn, rice, wheat, sorghum, millet, barley or grasses, preferably the plant is corn or rice.

In some embodiments, the plant further comprises at least one second nucleotide different from the nucleotide encoding the Cry1A protein.

In a further embodiment, 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.

In a further embodiment, the second nucleotide encodes a Cry1F protein, a Vip3A protein, or a Cry2Ab protein.

In a still further embodiment, the second nucleotide comprises the nucleotide sequence set forth in S SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

In some embodiments, the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.

A third aspect of the present application relates to a method of preparing a plant cell, a transgenic plant or a part of a transgenic plant against an oriental armyworm pest, which comprises introducing a coding nucleotide sequence of a Cry1A protein into the plant cell, a transgenic plant or a transgene In a part of the plant, preferably, the coding nucleotide sequence of the Cry1A protein is introduced into the genome of the part of the plant cell, the transgenic plant or the transgenic plant.

In some embodiments, the portion of the transgenic plant is a propagation material or a non-propagating material.

The propagation material refers to the fruit, seed or callus of the plant.

The non-propagating material refers to a leaf, a stem, a tassel, an ear, an anther or a filament of a plant that does not have the ability to reproduce.

In some embodiments, the Cry1A protein is one or more of a Cry1Ab protein, a Cry1Ac protein, a Cry1Ah protein, or a Cry1A.105 protein.

In a further embodiment, the amino acid sequence of the Cry1A protein comprises: 1) the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, 2) and SEQ ID NO: SEQ ID NO: 2 or SEQ ID NO: 3 has at least 70% homology and has an insecticidal activity against oriental armyworm, such as at least 70%, 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or 3) SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 An amino acid sequence obtained by substituting, deleting and/or adding one or more amino acid residues and having insect resistance to oriental armyworms, such as 1, 2, 3, 4, 5, 6, 7, 8 9, 10, 15, 20, 30, 50 amino acid residues.

In a further embodiment, the nucleotide sequence encoding the Cry1A protein comprises: 1) the nucleotide sequence set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, 2) and SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 has a nucleotide sequence of at least about 75% homology and encodes an amino acid sequence having insecticidal activity against oriental armyworm, such as at least 75%, 80% , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, 3) under stringent conditions with SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: A nucleotide sequence of an amino acid sequence which hybridizes to 6 and encodes an insect-resistant activity against oriental armyworm.

The plant is selected from the group consisting of corn, rice, wheat, sorghum, millet, barley or grasses, preferably the plant is corn or rice.

In some embodiments, the method further comprises introducing at least one second nucleotide different from the nucleotide encoding the Cry1A protein into the plant cell, the transgenic plant, or a portion of the transgenic plant, preferably At least one second nucleotide different from the nucleotide encoding the Cry1A protein is introduced into the genome of the part of the plant cell, transgenic plant or transgenic plant.

In some embodiments, 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.

In a further embodiment, the second nucleotide encodes a Cry1F protein, a Vip3A protein, or a Cry2Ab protein.

In a still further embodiment, the second nucleotide comprises the nucleotide sequence set forth in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

In other embodiments, the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.

In some embodiments, the coding nucleotide is introduced into the plant cell, the transgenic plant by Agrobacterium-mediated transformation, microprojection bombardment, direct DNA uptake into protoplasts, electroporation or whisker silicon mediated DNA introduction. Or a portion of the transgenic plant, preferably Agrobacterium-mediated transformation.

A fourth aspect of the present application relates to the plant cell, transgenic plant or transgenic plant part of the oriental armyworm pest obtained by the method of the above third aspect.

A fifth aspect of the present application relates to the use of a Cry1A protein for the preparation of a plant cell, a transgenic plant or a part of a transgenic plant against an oriental armyworm pest. The definitions of "Cry1A protein", "anti-Oriental armyworm pests", "plants", "plant cells", "transgenic plants", "parts of transgenic plants" and their extensions referred to in this aspect are as defined above. .

A sixth aspect of the present application relates to a method of cultivating a plant for controlling oriental armyworm pests, comprising:

Planting at least one plant seed, the genome of the plant seed comprising a polynucleotide sequence encoding a Cry1A protein;

Planting the plant seed into a plant;

The plants are grown under conditions in which artificially inoculated oriental armyworm pests and/or oriental armyworm pests are naturally harmful, and plants with reduced plant damage and/or plants having no polynucleotide sequence encoding the Cry1A protein are harvested and/or Or plants with increased plant yield.

The definitions of "Cry1A protein", "plant" and their extensions referred to in this aspect are as above Limited in terms of the description. The meaning of "controlling oriental armyworm pests" is similar to the meaning of "anti-eastern armyworm pests", and is specifically limited as described above.

In the present application, expression of a Cry1A protein in a transgenic plant can 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. In addition, one plant (first parent) can express Cry1A protein by genetic engineering operation, and the second plant (second parent) can express Cry-like insecticidal protein and/or Vip-like insecticidal protein by genetic engineering operation. 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 (RNAi) refers to the phenomenon of highly-specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. Therefore, RNAi technology can be used in this application to specifically knock out or shut down the expression of a particular gene in a target insect pest.

DRAWINGS

1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing a Cry1Ab-01 nucleotide sequence of the method for controlling pests of the present application;

2 is a flow chart showing the construction of a recombinant expression vector DBN100124 containing the Cry1Ab-01 nucleotide sequence of the method for controlling pests of the present application;

3 is a diagram showing damage of a leaf of a transgenic maize plant inoculated with oriental armyworm according to the method for controlling pests of the present application;

Figure 4 is a diagram showing the damage of leaves of transgenic rice plants inoculated with oriental armyworms according to the method for controlling pests of the present application.

detailed description

Mythimna seperata and Spodoptera frugiperda belong to the family Lepidoptera, and are omnivorous pests, but they are obviously fascinated by grasses, most commonly corn, rice, sorghum and so on. Despite this, the oriental armyworm and the autumn armyworm (also known as the grasshopper moth) belong to the genus Zygophyllum and the genus Heliothis, which are biologically distinct and distinct species. At least the following major differences exist. :

1. The distribution area is different. Oriental armyworms are mainly distributed in Asia and Australia. At present, there are 27 countries and islands. In addition to Xinjiang, China has not been affected, and other places have harm. Autumn armyworms are mainly distributed overseas, including Canada, Mexico, the United States, Argentina, Bolivia, Brazil, Chile, Colombia, Ecuador, French Guiana, Guyana, Paraguay, Peru, Suriname, Uruguay, Venezuela and throughout Central America and the Caribbean. Sea area and about 36 degrees south latitude In most parts of the south, there are no reports of the presence of autumn armyworms in China.

2. Different habits. Oriental sticky insects like to eat leaves, after the third age, the whole leaf can be bitten into a nick, or eat the heart leaves, forming a heartless seedling; five to six years of gluttony, can eat all the above ground parts of the seedlings, or will The leaves of the whole plant are eaten leaving only the veins, causing severe reduction or even rejection; the oriental armyworm can also damage the ears. The autumn larvae feeding on the leaves can cause defoliation, and then transfer to the damage; sometimes a large number of larvae are cut by roots, cutting off the stems of seedlings and young plants; on larger crops, such as ear of corn, larvae can be drilled When feeding corn leaves, there are a lot of holes; when the young larvae feed, the veins are window-like; the old larvae can cut the 30-day-old seedlings along the base like the cutting roots; when the population is large, the larvae Marching, spreading in groups; often in weeds when the environment is favorable.

3. Different morphological characteristics.

1) Different egg morphology: The oriental eggworm has a hemispherical shape with a diameter of 0.5 mm. It is milky white at the time of initial production and has a reticular ridge on the surface. It is yellowish brown to dark brown before hatching. The single layer of egg granules is arranged in rows and blocks. In the sheath of the leaf sheath or in the dead leaf, the eggs are often rolled up at the tip of the rice and millet leaves. The autumn armyworm egg is semi-spherical, and the egg mass is concentrated on the surface of the leaf. Each egg contains 100-300 eggs, sometimes forming a Z layer. The surface of the egg block has a protective layer formed by the gray hair of the female belly.

2) The morphology of the larvae is different: the mature larvae of the oriental armyworm are 36-40mm in length, the body color is yellowish brown to dark green; the head is reddish brown, the head cover has a net text, the forehead is flat, and the head has a brown-black "eight" pattern; The midline of the back is white, the black lines on both sides, and the red line on the back line. The autumn armyworm larvae are all green, with black lines and spots when they first hatch; they remain green or pale yellow when growing, and have black back center lines and valve lines; if dense (large population density, food shortage), The larvae of the last instar are almost black during the migration period; the mature larvae are 35-40 mm in length, with yellow inverted Y-shaped spots on the head, and black scallops with native bristles (two bristles on each side of the midline); There are 4 black spots in a square arrangement in the last section; the larvae have 6 ages and even 5 occasions.

3) Different forms of mites: Oriental worms are reddish-brown, with a body length of 19-23 mm. There are horizontal horseshoe-shaped engravings on the back of the 5th, 6th and 7th sections of the abdomen, and 4 thorns on the hips. The autumn armyworm is brown, shiny and 18-20mm long.

4) Different forms of adult worms: Adult bodyworms have pale yellow or light gray-brown body length, body length 17-20mm, wingspan 35-45mm, filaments in tentacles, anterior wings grayish brown, yellow or orange, many changes; The line often only has a few black spots, the ring pattern and the kidney pattern are brownish yellow, and the boundary is not significant. There is a white point at the back end of the kidney pattern, and there is a black point on each side; the outer horizontal line is a black line; the Asian edge line From the apex angle to the M2 (the second midrib); the edge is a row of black spots; the hind wings are dark brown, gradually fade to the base. The autumn armyworm is stout, gray-brown, with wingspan 32-38mm; the female's fore wing is gray to gray-brown, but the male fore wing is darker, with dark spots and light-colored dark lines; the hind wings are white, and the hind wings are brown and Transparent; male anterior wing light-colored round, wing scorpion showing obvious gray caudal protrusion; micro-worm external genital holding the valve square, the end of the grip is not missing; female mating sac without mating piece.

4. Growth habits and occurrence patterns are different. The oriental armyworm is a typical migratory pest. From March to mid-August, the airflow moves from the south to the north. From late August to September, it moves southward with the northerly flow. The country goes from south to north every year. 8-2 generations occur; in the eastern half of China, 6-8 generations occur in the south of 27 degrees north latitude, which causes more damage to the late rice generation and winter damage in the autumn. The north latitude 27-33 degree occurs in 1 year. In the 6th generation, there were more generations of late rice in the autumn; 4-5 generations occurred in the 33-36 degree north latitude in 1 year, which occurred in the spring to damage the wheat generation; the latitude 36-39 degrees in the north 3-4 generation occurred in 3-4 generations. There are more generations in autumn, which are harmful to wheat, corn, millet, rice, etc.; 2-3 generations occur in the north of 39 degrees north latitude, which occurs in summer generations, which harms wheat, millet, corn, sorghum and pasture; The monthly isotherm 0°C (north of 33 degrees north latitude) cannot be wintered and moved from the south every year; the January isotherm 0-8°C (north latitude 33-27 degrees north) is mostly larvae or pupa in rice blast and paddy fields.越, straw piles, stalks, lotus buds, weeds, etc., wintering, the southern half of the larvae in the wheat field weeds overwinter, but the number is small; January, etc. The temperature of 8 °C (about 27 degrees north latitude) can be propagated all year round, mainly in the winter wheat field. The adults of autumn armyworms can move, and can spread a considerable distance on their own. Vegetable or fruit entrainment of larvae is an important international communication method.

Based on the above, it can be determined that the oriental armyworm and the autumn armyworm are two kinds of pests, and the relationship is far away, and it is impossible to mate to produce offspring.

The genome of a plant, plant tissue or plant cell as referred to in this application refers to any genetic material within a plant, plant tissue or plant cell, and includes the nucleus and plastid and mitochondrial genomes.

As used in the present application, "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.

The term "control" and/or "control" as used herein means that the oriental armyworm pest is in contact with the Cry1A protein, and the growth of the oriental armyworm pest is inhibited and/or causes death after contact. Further, the oriental armyworm pests are in contact with the Cry1A protein by ingesting plant tissues, and all or part of the oriental armyworm pests are inhibited from growing and/or causing death after 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. In addition, plants and/or plant seeds containing a polynucleotide sequence encoding a Cry1A protein that control oriental armyworm pests, under conditions in which artificially inoculated oriental armyworm pests and/or oriental armyworm pests are naturally harmful, and non-transgenic Wild-type plants have reduced plant damage compared to specific manifestations including, but not limited to, improved stem resistance, and/or increased kernel weight, and/or increased yield, and the like. The "control" and / or "control" effects of Cry1A protein on oriental armyworms can exist independently and are not attenuated and/or disappeared by other substances that can "control" and/or "control" the oriental armyworm pests. . Specifically, any tissue of a transgenic plant (containing a polynucleotide sequence encoding a Cry1A protein) is simultaneously and / Or, asynchronously, the presence and/or production of a Cry1A protein and/or another substance that can control the oriental armyworm pest, the presence of the other substance does not affect the "control" of the Cry1A protein to the oriental armyworm. And/or the "control" effect does not result in the "control" and/or "control" effect being completely achieved by the other substance, regardless of the Cry1A protein. In general, in Daejeon, the process of feeding on plant tissues by oriental armyworms is short-lived and difficult to observe with the naked eye. Therefore, under the conditions of artificial inoculation of oriental armyworm pests and/or oriental armyworm pests, such as genetic modification. Any tissue of a plant (containing a polynucleotide sequence encoding a Cry1A protein) is present in dead oriental armyworm pests, and/or oriental armyworm pests on which growth inhibition is inhibited, and/or with non-transgenic wild-type plants The method and/or use of the present application is achieved by the method and/or use of the present invention, that is, by contacting the oriental armyworm pest with the Cry1A protein to achieve a method and/or use for controlling oriental armyworm pests.

The polynucleotides and/or nucleotides described herein form a complete "gene" encoding a protein or polypeptide in a desired host cell. One of skill in the art will readily recognize that the polynucleotides and/or nucleotides of the present application can be placed under the control of regulatory sequences in a host of interest.

As is well known to those skilled in the art, 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 application includes 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 application 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 application hybridizes to the Cry1A gene of the present application under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Cry1A gene of the present application. A nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present application, 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. In the present application, 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. Similarly, 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. In order to make a nucleic acid The molecule can act as a primer or probe, only to ensure that it is sufficiently complementary in sequence to form a stable double-stranded structure at the particular solvent and salt concentration employed.

In the present application, 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. For example, 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. Further, the temperature conditions in the washing step can be raised from a low temperature strict room temperature of about 22 ° C to about 65 ° C under highly stringent conditions. Both the temperature conditions and the salt concentration can be changed, or one of them remains unchanged while the other variable changes. Preferably, the stringent conditions described herein may be specific hybridization with SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 at 65 ° C in a 6 x SSC, 0.5% SDS solution, and then The membrane was washed once with 2 x SSC, 0.1% SDS, and 1 x SSC, 0.1% SDS.

Thus, sequences having insect resistance and hybridizing under stringent conditions to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 of the present application are included in the present application. These sequences are at least about 40%-50% homologous to the sequences of the present application, about 60%, 65% or 70% homologous, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93. Sequence homology of %, 94%, 95%, 96%, 97%, 98%, 99% or greater.

The genes and proteins described in this application 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. By "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 the protein of the claims against the oriental armyworm pest.

A "fragment" or "truncated" sequence of a DNA molecule or protein sequence as referred to in this application 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 application can derive equivalent proteins and/or genes encoding these equivalent proteins from B.t. isolates and/or DNA libraries. There are a number of ways to obtain the insecticidal proteins of the present application. For example, antibodies to the pesticidal proteins disclosed and claimed herein can be used to identify and isolate other proteins from protein mixtures. In particular, antibodies may be caused by protein portions that are most constant in protein and most different from other B.t. proteins. These antibodies can then be used to specifically identify a characteristically active equivalent protein by immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) or western blotting. Antibodies raised in the present application 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.

Due to the abundance of the genetic code, a variety of different DNA sequences can encode the same amino acid sequence. It is within the skill of the art to produce alternative DNA sequences that encode the same or substantially the same protein. These different DNA sequences are included within the scope of the present application. 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.

Substitutions, deletions or additions of amino acid sequences in the present application are conventional in the art, and it is preferred that such amino acid changes are: small changes in properties, ie, conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, 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.

Examples of 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. L. Hill, "Protein", published at the 1979 New York Academic Press. The most common interchanges are 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 and Asp/Gly, and their opposite interchanges.

It will be apparent to those skilled in the art that such substitutions can occur outside of the regions that are important for molecular function and still produce active polypeptides. For polypeptides of the present application, amino acid residues necessary for their activity and thus selected for unsubstitution 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). 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).

In the present application, Cry1A protein includes, but is not limited to, Cry1Ab, Cry1A.105 or Cry1Ah protein, or an insecticidal fragment or functional region having at least 70% homology with the amino acid sequence of the above protein and having insect resistance to oriental armyworm. .

Thus, amino acid sequences having some homology to the amino acid sequences shown in Sequences 1, 2 and/or 3 are also included in the present application. These sequences are typically greater 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 sequence of the present application. Preferred polynucleotides and proteins of the present application may also be defined according to a more specific range of identity and/or similarity. For example, the sequence of the examples of the present application 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 as described herein include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory operably linked to the Cry1A protein, Vip3A protein, and other Cry-like proteins. sequence.

The promoter is a promoter expressible in a plant, and 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. Alternatively, 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. Alternatively, 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. Examples of 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 small RNA viral leader sequence, such as an EMCV leader Column (5' non-coding region of encephalomyocarditis virus); potato Y virus leader sequence, such as MDMV (maize dwarf mosaic virus) leader sequence; human immunoglobulin heavy chain binding protein (BiP); 苜蓿 mosaic virus shell Non-translated leader sequence of protein mRNA (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.

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. , Purple Jasmine Mosaic Virus (MMV) enhancer, Night fragrant yellow leaf curl virus (CmYLCV) enhancer, Multan cotton leaf curl virus (CLCuMV), Acanthus yellow mottle virus (CoYMV) and peanut chlorotic line flower Leaf virus (PCLSV) enhancer.

For monocot applications, the introns include, but are not limited to, maize hsp70 introns, maize ubiquitin introns, Adh introns 1, sucrose synthase introns, or rice Actl introns. For dicotyledonous applications, 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 α-tubulin gene. Polyadenylation signal sequence.

As used herein, "operably linked" refers to the joining of nucleic acid sequences that allow one sequence to provide the function required for the linked sequence. As used herein, "operably linked" can be such that a promoter is ligated to a 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. If 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. Alternatively, if 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 sequences that provide replication (ie, bacterial origins of replication, autonomously replicating sequences, centromeric sequences).

As used herein, "insecticidal" or "insect-resistant" means toxic to crop pests, thereby achieving "control" and/or "control" of crop pests. Preferably, said "insecticide" or "insect-resistant" means killing crop pests. More specifically, the target insect is an oriental armyworm pest.

The Cry1A protein in this application is toxic to oriental armyworm pests. The plants of the present application, particularly sorghum and maize, contain exogenous DNA in their genome, the exogenous DNA comprising a nucleotide sequence encoding a Cry1A protein, and the oriental armyworm in contact with the protein by ingesting plant tissue, contacting Post-Oriental mucoid pest growth is inhibited and eventually leads to death. Inhibition refers to death or sub-lethal death. 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. In addition, the plant substantially eliminates the need for chemical or biological insecticides that are insecticides against oriental armyworm pests targeted by the Cry1A 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.

Different tests can be applied to determine the insecticidal effect of ICP in plants. The target insects in this application are mainly oriental armyworms.

In the present application, the Cry1A protein may have the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3 in the Sequence Listing. In addition to the coding region comprising the Cry1A protein, other elements may be included, such as a protein encoding a selectable marker.

Furthermore, an expression cassette comprising a nucleotide sequence encoding a Cry1A protein of the present application 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.

In the present application, the foreign DNA is introduced into a plant, such as a gene encoding the Cry1A protein or an expression cassette or a recombinant vector, and the conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-launch bombardment, Direct DNA uptake into protoplast, electroporation or whisker silicon-mediated DNA introduction.

The present application provides a method of controlling pests, which has the following advantages:

1. Internal cause prevention. The prior art mainly controls the damage of oriental armyworm pests through external effects, ie, external factors, such as agricultural control, chemical control and physical control; and the present application controls the oriental stick by producing a Cry1A protein capable of killing oriental armyworms in plants. Insect pests, that is, through internal factors.

2, no pollution, no residue. Although the chemical control methods used in the prior art have played a role in controlling the damage of oriental armyworm pests, they also bring pollution, damage and residue to human, livestock and farmland ecosystems; use this application to control oriental armyworm pests. The method can eliminate the above-mentioned adverse consequences.

3. Prevention and control during the whole growth period. The methods used in the prior art for controlling oriental armyworm pests are all phased, and the present application protects plants from the whole growth period, and the transgenic plants (Cry1A protein) can be germinated, grown, flowered, and the results can be Avoid being attacked by oriental armyworms.

4. Whole plant control. The methods used in the prior art for controlling oriental armyworm pests are mostly local, such as foliar application; and the present application protects whole plants, such as leaves, stems, tassels of transgenic plants (Cry1A protein), Ears, anthers, and filaments are all resistant to oriental sticky insects.

5, the effect is stable. The frequency-vibration insecticidal lamp used in the prior art not only needs to clean the dirt of the high-voltage power grid every day, but also cannot be used in thunderstorm days; the present application is to express the Cry1A protein in plants, effectively overcoming the frequency-vibration killing. The effect of the insect lamp is affected by external factors, and the control effect of the transgenic plant (Cry1A protein) of the present application is stable at different locations, at different times, and in different genetic backgrounds.

6, simple, convenient and economical. The one-time input of the frequency-vibration insecticidal lamp used in the prior art is large, and the operation is improper and there is a danger of electric shock wounding; this application only needs to plant a transgenic plant capable of expressing the Cry1A protein, and does not need other measures. , which saves a lot of manpower, material resources and financial resources.

7, the effect is thorough. The method for controlling oriental armyworm pests used in the prior art has an incomplete effect and only serves to alleviate the effect; and the transgenic plant (Cry1A protein) of the present application can cause a large number of deaths of the newly hatched oriental larvae, and is small. The developmental progress of some surviving larvae was greatly inhibited. After 3 days, the larvae were still in the initial hatching state, all of which were obviously dysplastic, and had stopped development, while the transgenic plants were generally only slightly damaged.

The technical solutions of the present application are further described in detail below through the accompanying drawings and embodiments.

Example

The technical solution of the method for controlling pests of the present application is further illustrated by specific embodiments below.

First Example, Acquisition and Synthesis of Cry1A Gene

1. Obtain the Cry1A nucleotide sequence

The amino acid sequence of Cry1Ab-01 insecticidal protein (818 amino acids), as shown in SEQ ID NO: 1 in the Sequence Listing; Cry1Ab-01 encoding the amino acid sequence (818 amino acids) corresponding to the Cry1Ab-01 insecticidal protein Nucleotide sequence (2457 nucleotides) as shown in SEQ ID NO: 4 in the Sequence Listing. The amino acid sequence of Cry1Ab-02 insecticidal protein (615 amino acids), as shown in SEQ ID NO: 2 in the Sequence Listing; encoding the amino acid sequence corresponding to the Cry1Ab-02 insecticidal protein (615 The amino acid) Cry1Ab-02 nucleotide sequence (1848 nucleotides) is shown in SEQ ID NO: 5 of the Sequence Listing.

The amino acid sequence of Cry1A.105 insecticidal protein (1177 amino acids), as shown in SEQ ID NO: 3 in the Sequence Listing; Cry1A.105 encoding the amino acid sequence (1177 amino acids) corresponding to the Cry1A.105 insecticidal protein Nucleotide sequence (3534 nucleotides) as shown in SEQ ID NO: 6 in the Sequence Listing.

2. Obtain a Vip nucleotide sequence

A Vip3Aa nucleotide sequence (2370 nucleotides) encoding the amino acid sequence (789 amino acids) of the Vip3Aa insecticidal protein, as set forth in SEQ ID NO: 7 of the Sequence Listing.

3. Obtain a Cry-like nucleotide sequence

Cry1Fa nucleotide sequence (1818 nucleotides) encoding the amino acid sequence (605 amino acids) of the Cry1Fa insecticidal protein, as shown in SEQ ID NO: 8 in the Sequence Listing; amino acid sequence encoding the Cry2Ab insecticidal protein (634 The amino acid) Cry2Ab nucleotide sequence (1905 nucleotides) is shown in SEQ ID NO: 9 of the Sequence Listing.

4. Synthesize the above nucleotide sequence

The Cry1Ab-01 nucleotide sequence (as shown in SEQ ID NO: 4 in the Sequence Listing), the Cry1Ab-02 nucleotide sequence (as shown in SEQ ID NO: 5 in the Sequence Listing), and the Cry1A. 105 nucleotide sequence (as shown in SEQ ID NO: 6 in the Sequence Listing), the Vip3Aa nucleotide sequence (as shown in SEQ ID NO: 7 in the Sequence Listing), the Cry1Fa nucleotide sequence (such as The nucleotide sequence of SEQ ID NO: 8 in the list and the Cry2Ab nucleotide sequence (as shown in SEQ ID NO: 9 in the Sequence Listing) was synthesized by Nanjing Kingsray Biotechnology Co., Ltd. The 5' end of the synthesized Cry1Ab-01 nucleotide sequence (SEQ ID NO: 4) is also ligated with an NcoI cleavage site, and the 3' of the Cry1Ab-01 nucleotide sequence (SEQ ID NO: 4) The SpeI cleavage site is also ligated to the end; the 5' end of the synthesized Cry1Ab-02 nucleotide sequence (SEQ ID NO: 5) is further ligated with an NcoI cleavage site, and the Cry1Ab-02 nucleotide sequence The 3' end of (SEQ ID NO: 5) is also ligated with a SpeI cleavage site; the 5' end of the synthesized Cry1A.105 nucleotide sequence (SEQ ID NO: 6) is also ligated with an NcoI cleavage site The 3' end of the Cry1A.105 nucleotide sequence (SEQ ID NO: 6) is further ligated with a HindIII cleavage site; the 5' end of the synthesized Vip3Aa nucleotide sequence (SEQ ID NO: 7) Also ligated with a ScaI cleavage site, the 3' end of the Vip3Aa nucleotide sequence (SEQ ID NO: 7) is also ligated with a SpeI cleavage site; the synthesized Cry1Fa nucleotide sequence (SEQ ID NO: The 5' end of 8) is also ligated with an AscI cleavage site, and the 3' end of the Cry1Fa nucleotide sequence (SEQ ID NO: 8) is further ligated with a BamHI cleavage site; the synthesized Cry2Ab nucleotide The 5' end of the sequence (SEQ ID NO: 9) is also ligated with an NcoI cleavage site, the Cry2A The 3' end of the b nucleotide sequence (SEQ ID NO: 9) is also ligated with a SpeI cleavage site.

Second embodiment, construction of recombinant expression vector and recombinant expression vector for transformation of Agrobacterium

1. Construction of a recombinant cloning vector containing the Cry1A gene

The synthetic Cry1Ab-01 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. The construction process is shown in Figure 1 (wherein Amp represents the ampicillin resistance gene; f1 represents the origin of replication of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; and T7 is initiated by T7 RNA polymerase). Cry1Ab-01 is the Cry1Ab-01 nucleotide sequence (SEQ ID NO: 4); 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 μl E. coli T1 competent cells, 10 μl 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-β-D-galactoside) and X -gal (5-bromo-4-chloro-3-indolyl-β-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. 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 μl of ice (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose. , pH 8.0) suspension; add 150 μl of freshly prepared solution II (0.2 M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 4 times, mix, set on ice for 3-5 min; add 150 μl ice cold Solution III (4M potassium acetate, 2M acetic acid), mix well immediately, place on ice for 5-10 min; centrifuge at 5 ° C, 12000 rpm for 5 min, add 2 volumes of absolute ethanol to the supernatant, mix After homogenization, let it stand at room temperature for 5 min; centrifuge at 5 ° C, 12000 rpm for 5 min, discard the supernatant, and wash the precipitate with 70% ethanol (V/V) and dry it; add 30 μl of RNase (20 μg/ml). 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.

After the extracted plasmid was identified by KpnI and BglI digestion, the positive clone was verified by sequencing, and the result showed that the Cry1Ab-01 nucleotide sequence inserted into the recombinant cloning vector DBN01-T was represented by SEQ ID NO: 4 in the sequence listing. The nucleotide sequence, ie the Cry1Ab-01 nucleotide sequence, was correctly inserted.

According to the above method for constructing the recombinant cloning vector DBN01-T, the synthesized Cry1Ab-02 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein Cry1Ab-02 was Cry1Ab-02. Nucleotide sequence (SEQ ID NO: 5). The Cry1Ab-02 nucleotide sequence in the recombinant cloning vector DBN02-T was correctly inserted by restriction enzyme digestion and sequencing.

According to the above method for constructing the recombinant cloning vector DBN01-T, the synthesized Cry1A.105 The nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN03-T, wherein Cry1A.105 was the Cry1A.105 nucleotide sequence (SEQ ID NO: 6). The Cry1A.105 nucleotide sequence in the recombinant cloning vector DBN03-T was correctly inserted by restriction enzyme digestion and sequencing.

According to the above method for constructing the recombinant cloning vector DBN01-T, the synthesized Vip3Aa nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN04-T, wherein Vip3Aa was a Vip3Aa nucleotide sequence (SEQ ID NO: 7). The correct insertion of the Vip3Aa nucleotide sequence in the recombinant cloning vector DBN04-T was confirmed by restriction enzyme digestion and sequencing.

According to the above method for constructing the recombinant cloning vector DBN01-T, the synthesized Cry1Fa nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN05-T, wherein Cry1Fa is a Cry1Fa nucleotide sequence (SEQ ID NO: 8). The Cry1Fa nucleotide sequence in the recombinant cloning vector DBN05-T was correctly inserted by restriction enzyme digestion and sequencing.

According to the above method for constructing the recombinant cloning vector DBN01-T, the synthesized Cry2Ab nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN06-T, wherein the Cry2Ab was a Cry2Ab nucleotide sequence (SEQ ID NO: 9). The Cry2Ab nucleotide sequence in the recombinant cloning vector DBN06-T was correctly inserted by restriction enzyme digestion and sequencing.

2. Construction of a recombinant expression vector containing the Cry1A gene

Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases NcoI and SpeI, respectively, and the cut Cry1Ab-01 nucleotide sequence fragment was inserted. Between the NcoI and SpeI sites of the expression 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 DBN100124 is constructed. The construction process is shown in Figure 2 (Kan: Kanamycin gene; RB: right border; Ubi: maize Ubiquitin (ubiquitin) gene promoter (SEQ ID NO: 10); Cry1Ab-01: Cry1Ab-01 nucleotide sequence (SEQ ID NO: 4); Nos : terminator of the nopaline synthase gene (SEQ ID NO: 11); PMI: phosphomannose isomerase gene (SEQ ID NO: 12); LB: left border).

The recombinant expression vector DBN100124 was transformed into E. coli T1 competent cells by heat shock method, and the heat shock conditions were: 50 μl of E. coli T1 competent cells, 10 μl of plasmid DNA (recombinant expression vector DBN100124), and cultured at 42 ° C for 30 seconds; 37 ° C Water bath for 1 hour (shake at 100 rpm); then in LB solid plate containing 50 mg/L kanamycin (tryptone 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 of the recombinant expression vector DBN100124 between NcoI and SpeI was sequenced. The nucleotide sequence shown by SEQ ID NO: 4 in the table, that is, the Cry1Ab-01 nucleotide sequence.

According to the above method for constructing the recombinant expression vector DBN100124, the Cry1Ab-02 nucleotide sequence excised from the recombinant cloning vector DBN02-T by NcoI and SpeI was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100053. The nucleotide sequence in the recombinant expression vector DBN100053 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 5 in the sequence listing, that is, the Cry1Ab-02 nucleotide sequence, and the Cry1Ab-02 nucleotide sequence was digested and sequenced. The Ubi promoter and the Nos terminator can be ligated.

According to the above method for constructing the recombinant expression vector DBN100124, the Cry1Ab-01 nucleotide sequence and the Vip3Aa nucleotide sequence excised by the recombinant cloning vectors DBN01-T and DBN04-T, respectively, were digested with NcoI and SpeI, ScaI and SpeI, respectively. The expression vector DBNBC-01 was obtained to obtain a recombinant expression vector DBN100003. The nucleotide sequence in the recombinant expression vector DBN100003 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 4 and SEQ ID NO: 7 in the sequence listing, namely Cry1Ab-01 nucleotide sequence and Vip3Aa nucleoside. The acid sequence, the Cry1Ab-01 nucleotide sequence and the Vip3Aa nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.

According to the above method for constructing the recombinant expression vector DBN100124, the Cry1Ab-02 nucleotide sequence and the Cry1Fa nucleotide sequence excised by the recombinant cloning vectors DBN02-T and DBN05-T, respectively, were digested with NcoI and SpeI, AscI and BamHI, respectively. The expression vector DBNBC-01 was obtained to obtain a recombinant expression vector DBN100075. The nucleotide sequence in the recombinant expression vector DBN100075 contains the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 8 in the sequence listing, namely the Cry1Ab-02 nucleotide sequence and the Cry1Fa nucleoside. The acid sequence, the Cry1Ab-02 nucleotide sequence and the Cry1Fa nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.

According to the above method for constructing the recombinant expression vector DBN100124, the Cry1A.105 nucleotide sequence excised by NcoI and HindIII digestion recombinant cloning vector DBN03-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100029. The nucleotide sequence in the recombinant expression vector DBN100029 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing, that is, the Cry1A.105 nucleotide sequence, and the Cry1A.105 nucleotide sequence was digested and sequenced. The Ubi promoter and the Nos terminator can be ligated.

According to the above method for constructing the recombinant expression vector DBN100124, the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence excised by the recombinant cloning vectors DBN03-T and DBN06-T, respectively, were digested with NcoI and HindIII, NcoI and SpeI, respectively. 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: 6 and SEQ ID NO: 9 in the sequence listing, namely the Cry1A.105 nucleotide sequence and the Cry2Ab nucleoside. The acid sequence, the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.

3. Recombinant expression vector transforming Agrobacterium

The recombinant expression vectors DBN100124, DBN100053, DBN100003, DBN100075, DBN100029 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 μL. Agrobacterium LBA4404, 3 μL of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated in LB tube at a temperature of 28 ° C, 200 rpm After culturing for 2 hours, it was applied to LB plates containing 50 mg/L of rifampicin and 100 mg/L of kanamycin until a positive monoclonal was grown, and the monoclonal culture was picked and the plasmid was extracted. Recombinant expression vectors DBN100124 and DBN100075 were used with restriction endonucleases AhdI and StyI, and recombinant expression vectors DBN100053 and DBN100003 were treated with restriction endonucleases AhdI and XhoI, and restriction endonucleases StyI and XhoI were used to express recombinant expression vectors DBN100029 and DBN100076. After digestion and enzyme digestion, the results showed that the recombinant expression vectors DBN100124, DBN100053, DBN100003, DBN100075, DBN100029 and DBN100076 were completely correct.

Third embodiment, obtaining and verifying corn plants transferred into Cry1A gene

1. Obtain corn plants that have been transferred into the Cry1A gene.

The immature embryo of the aseptically cultured maize variety Heisei 31 (Z31) was co-cultured with the Agrobacterium described in the third embodiment in accordance with the conventional Agrobacterium infection method to construct the second embodiment. T-DNA in recombinant expression vectors DBN100124, DBN100053, DBN100003, DBN100075, DBN100029 and DBN100076 (including promoter sequence of maize Ubiquitin gene, Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide sequence, Cry1A.105 nucleoside) The acid sequence, the Vip3Aa nucleotide sequence, the Cry1Fa nucleotide sequence, the Cry2Ab nucleotide sequence, the PMI gene and the Nos terminator sequence were transferred into the maize genome to obtain a maize transformed into the Cry1Ab-01 nucleotide sequence. Plant, maize plant transformed into Cry1Ab-02 nucleotide sequence, maize plant transformed into Cry1Ab-01-Vip3Aa nucleotide sequence, maize plant transformed into Cry1Ab-02-Cry1Fa nucleotide sequence, transferred to Cry1A.105 The maize plant of the nucleotide sequence and the maize plant transformed into the nucleotide sequence of Cry1A.105-Cry2Ab; and the wild type maize plant was used as a control.

For Agrobacterium-mediated transformation of maize, briefly, immature immature embryos are isolated from maize, and the immature embryos are contacted with Agrobacterium suspension, wherein Agrobacterium can express Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide The sequence, the Cry1Ab-01-Vip3Aa nucleotide sequence, the Cry1Ab-02-Cry1Fa nucleotide sequence, the Cry1A.105 nucleotide sequence, and/or the Cry1A.105-Cry2Ab nucleotide sequence are delivered to at least one of the young embryos Cells (Step 1: Infection step), in which the immature embryos are preferably immersed in an Agrobacterium suspension (OD ₆₆₀ = 0.4-0.6, infecting medium (MS salt 4.3 g/L, MS vitamin, casein 300 mg) /L, sucrose 68.5g / L, glucose 36g / L, acetosyringone (AS) 40mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg / L, pH 5.3)) To initiate vaccination. The immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step). Preferably, 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. In the "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). Preferably, 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. Next, 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). Preferably, 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). Incubation with oxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8) resulted in selective growth of transformed cells. Then, 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.

2. TaqMan was used to verify the maize plants transferred to the Cry1A gene.

Maize plants transfected with Cry1Ab-01 nucleotide sequence, maize plants transfected with Cry1Ab-02 nucleotide sequence, maize plants transfected with Cry1Ab-01-Vip3Aa nucleotide sequence, and transferred into Cry1Ab-02-Cry1Fa The maize plants of the nucleotide sequence, the maize plants transformed with the Cry1A.105 nucleotide sequence, and the leaves of the maize plants transformed with the Cry1A.105-Cry2Ab nucleotide sequence were used as samples, and were extracted with Qiagen's DNeasy Plant Maxi Kit. The genomic DNA was used to detect the copy number of the Cry1A gene, the Cry1Fa gene, the Vip3Aa gene and the Cry2Ab gene by Taqman probe fluorescent quantitative PCR. At the same time, the wild type corn plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was set to repeat 3 times and averaged.

The specific methods for detecting the copy number of Cry1A gene, Cry1Fa gene, Vip3Aa gene and Cry2Ab gene are as follows:

Step 11. The maize plants transformed into the Cry1Ab-01 nucleotide sequence, the maize plants transformed into the Cry1Ab-02 nucleotide sequence, the maize plants transformed into the Cry1Ab-01-Vip3Aa nucleotide sequence, and the Cry1Ab- 02-Cry1Fa nucleotide sequence of maize plants, transferred into Cry1Ab-02-Cry1Fa nucleotide Sequence of maize plants, maize plants transfected with Cry1A.105 nucleotide sequence, maize plants transfected with Cry1A.105-Cry2Ab nucleotide sequence, and wild-type maize plants, each containing 100 mg of liquid nitrogen in a mortar Grind into 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 / μl;

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:

The following primers and probes were used to detect the Cry1Ab-01 nucleotide sequence:

Primer 1 (CF1): CGAACTACGACTCCCGCAC is shown in SEQ ID NO: 13 in the Sequence Listing;

Primer 2 (CR1): GTAGATTTCGCGGGTCAGTTG is shown in SEQ ID NO: 14 in the Sequence Listing;

Probe 1 (CP1): CTACCCGATCCGCACCGTGTCC is shown in SEQ ID NO: 15 in the Sequence Listing;

The following primers and probes were used to detect the Cry1Ab-02 nucleotide sequence:

Primer 3 (CF2): TGCGTATTCAATTCAACGACATG is shown in SEQ ID NO: 16 in the Sequence Listing;

Primer 4 (CR2): CTTGGTAGTTCTGGACTGCGAAC as shown in SEQ ID NO: 17 in the Sequence Listing;

Probe 2 (CP2): CAGCGCCTTGACCACAGCTATCCC as shown in SEQ ID NO: 18 in the Sequence Listing;

The following primers and probes were used to detect the Vip3Aa nucleotide sequence:

Primer 5 (VF1): ATTCTCGAAATCTCCCCTAGCG is shown in SEQ ID NO: 19 in the Sequence Listing;

Primer 6 (VR1): GCTGCCAGTGGATGTCCAG is shown in SEQ ID NO: 20 in the Sequence Listing;

Probe 3 (VP1): CTCCTGAGCCCCGAGCTGATTAACACC as shown in SEQ ID NO: 21 in the Sequence Listing;

The following primers and probes were used to detect the Cry1Fa nucleotide sequence:

Primer 7 (CF3): CAGTCAGGAACTACAGTTGTAAGAGGG as in the sequence listing SEQ ID NO: 22;

Primer 8 (CR3): ACGCGAATGGTCCTCCACTAG is shown in SEQ ID NO: 23 in the Sequence Listing;

Probe 4 (CP3): CGTGCAAGAATGTCTCCTCCCGTGAAC as shown in SEQ ID NO: 24 in the Sequence Listing;

The following primers and probes were used to detect the Cry1A.105 nucleotide sequence:

Primer 9 (CF4): GCGCATCCAGTTCAACGAC is shown as SEQ ID NO: 25 in the Sequence Listing;

Primer 10 (CR4): GTTCTGGACGGCGAAGAGTG as shown in SEQ ID NO: 26 in the Sequence Listing;

Probe 5 (CP4): TGAACAGCGCCCTGACCACCG is shown in SEQ ID NO: 27 in the Sequence Listing;

The following primers and probes were used to detect the Cry2Ab nucleotide sequence:

Primer 11 (CF5): CTGATACCCTTGCTCGCGTC is shown in SEQ ID NO: 28 in the Sequence Listing;

Primer 12 (CR5): CACTTGGCGGTTGAACTCCTC is shown in SEQ ID NO: 29 in the Sequence Listing;

Probe 6 (CP4): CGCTGAGCTGACGGGTCTGCAAG as shown in SEQ ID NO: 30 in the Sequence Listing;

The PCR reaction system is:

The 50× primer/probe mixture contained 45 μl of each primer at a concentration of 1 mM, 50 μl of a probe at a concentration of 100 μM, and 860 μl of 1×TE buffer, and stored at 4° C. in an amber tube.

The PCR reaction conditions are:

Data were analyzed using SDS2.3 software (Applied Biosystems).

The results showed that the Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide sequence, Cry1Ab-01-Vp3Aa nucleotide sequence, Cry1Ab-02-Cry1Fa nucleotide sequence, Cry1A.105 The nucleotide sequence and the Cry1A.105-Cry2Ab nucleotide sequence have been integrated into the genome of the tested maize plant, and the maize plant transformed into the Cry1Ab-01 nucleotide sequence was transferred to the Cry1Ab-02 nucleotide. Sequence of maize plants, maize plants transfected with Cry1Ab-01-Vip3Aa nucleotide sequence, maize plants transfected with Cry1Ab-02-Cry1Fa nucleotide sequence, maize plants transferred to Cry1A.105 nucleotide sequence, and transferred The maize plants of the Cry1A.105-Cry2Ab nucleotide sequence obtained transgenic maize plants containing a single copy of the Cry1A gene, the Cry1Fa gene, the Vip3Aa gene and/or the Cry2Ab gene.

Fourth embodiment, detection of insect resistance of transgenic corn plants

A maize plant transformed with the Cry1Ab-01 nucleotide sequence, a maize plant transformed with the Cry1Ab-02 nucleotide sequence, a maize plant transformed with the Cry1Ab-01-Vip3Aa nucleotide sequence, and transferred into a Cry1Ab-02-Cry1Fa nucleus Maize sequence glycoside plants, maize plants transfected with Cry1A.105 nucleotide sequence, maize plants transfected with Cry1A.105-Cry2Ab nucleotide sequence, wild-type maize plants and non-transgenic maize plants identified by Taqman Oriental armyworms tested for insect resistance.

Maize plants transfected with Cry1Ab-01 nucleotide sequence, maize plants transfected with Cry1Ab-02 nucleotide sequence, maize plants transfected with Cry1Ab-01-Vip3Aa nucleotide sequence, and transferred into Cry1Ab-02-Cry1Fa a nucleotide sequence of a maize plant, a maize plant transformed with a Cry1A.105 nucleotide sequence, a maize plant transformed with a Cry1A.105-Cry2Ab nucleotide sequence, a wild-type maize plant, and a non-transgenic maize plant identified by Taqman (V3-V4) fresh leaves (heart leaves), rinsed with sterile water and blotted the water on the leaves with gauze, then remove the veins of the corn leaves, and cut into strips of about 1cm × 4cm, take Three cut long strips of leaves were placed on the filter paper at the bottom of a round plastic petri dish, which was wetted with distilled water, and 10 captive oriental armyworms (initial hatching larvae) were placed in each dish. The test dish is capped and placed in a square box with wet gauze on the bottom, and placed at a temperature of 25-28 ° C, a relative humidity of 70%-80%, and a photoperiod (light/dark) of 16:8 for 3 days. To calculate the death and leaf damage of oriental larvae, and calculate the oriental armyworm in each sample. The average mortality rate. A total of 3 lines (S1, S2 and S3) transfected into the Cry1Ab-01 nucleotide sequence were transferred into the Cry1Ab-02 nucleotide sequence (S4, S5 and S6) and transferred to Cry1Ab- A total of 3 strains (S7, S8 and S9) of the nucleotide sequence of 01-Vip3Aa were transferred into Cry1Ab-02-Cry1Fa nucleotide sequence (S10, S11 and S12) and transferred to Cry1A. A total of 3 strains of 105 nucleotide sequences (S13, S14 and S15), transferred to the Cry1A.105-Cry2Ab nucleotide sequence for a total of 3 strains (S16, S17 and S18), identified by Taqman as non-transgenic There were 1 strain of (NGM1) and 1 strain of wild type (CK1); 5 strains were selected from each strain, and each plant was repeated 6 times. The results are shown in Table 1 and Figure 3.

Table 1. Results of insect resistance test of inoculated oriental worms on transgenic maize plants

The results in Table 1 indicate that maize plants transfected with the Cry1Ab-01 nucleotide sequence, maize plants transfected with the Cry1Ab-02 nucleotide sequence, maize plants transfected with the Cry1Ab-01-Vip3Aa nucleotide sequence, and transferred into Cry1Ab Maize plant with -02-Cry1Fa nucleotide sequence, transferred to Cry1A.105 nucleotide sequence The test insect mortality of maize plants and maize plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence was about 80% or more; while the test insect mortality of non-transgenic maize plants and wild-type maize plants identified by Taqman was generally Below 20%.

The results in Figure 3 indicate that maize plants transfected with the Cry1Ab-01 nucleotide sequence, maize plants transfected with the Cry1Ab-02 nucleotide sequence, and transferred to the Cry1Ab-01-Vip3Aa nucleotide were compared to wild-type maize plants. Sequence of maize plants, maize plants transfected with Cry1Ab-02-Cry1Fa nucleotide sequence, maize plants transfected with Cry1A.105 nucleotide sequence, and maize plants transfected with Cry1A.105-Cry2Ab nucleotide sequence The control effect of larvae is almost one hundred percent, and very few surviving larvae also basically stop development, and the maize plants that have been transferred into the Cry1Ab-01 nucleotide sequence, the maize plants that have been transferred into the Cry1Ab-02 nucleotide sequence, and transferred to Cry1Ab-01 a maize plant having a nucleotide sequence of Vip3Aa, a maize plant transformed with a Cry1Ab-02-Cry1Fa nucleotide sequence, a maize plant transformed with a Cry1A.105 nucleotide sequence, and a nucleotide sequence transferred into a Cry1A.105-Cry2Ab Maize plants are generally only slightly damaged, with only a small amount of pinhole damage on the leaves.

Thus, the maize plant transformed with the Cry1Ab-01 nucleotide sequence, the maize plant transformed with the Cry1Ab-02 nucleotide sequence, the maize plant transformed with the Cry1Ab-01-Vip3Aa nucleotide sequence, and the Cry1Ab-02- were transferred. The maize plant of the Cry1Fa nucleotide sequence, the maize plant transformed with the Cry1A.105 nucleotide sequence, and the maize plant transformed with the Cry1A.105-Cry2Ab nucleotide sequence all showed high activity against oriental armyworm, this activity. It is enough to have an adverse effect on the growth of oriental armyworms so that they can be controlled.

Fifth embodiment, obtaining and verifying rice plants transferred into Cry1A gene

1. Obtain rice plants that have been transferred into the Cry1A gene.

The callus of the aseptically cultivated indica rice variety Nipponbare is co-cultured with the Agrobacterium described in the third embodiment in accordance with the conventional Agrobacterium infection method to construct the recombinant expression vector constructed in the second embodiment. T-DNA in DBN100124, DBN100053, DBN100003, DBN100075, DBN100029 and DBN100076 (including the promoter sequence of maize Ubiquitin gene, Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide sequence, Cry1A.105 nucleotide sequence, The Vip3Aa nucleotide sequence, the Cry1Fa nucleotide sequence, the Cry2Ab nucleotide sequence, the PMI gene and the Nos terminator sequence were transferred into the rice genome, and the rice plant transformed into the Cry1Ab-01 nucleotide sequence was obtained. Rice plants into the Cry1Ab-02 nucleotide sequence, rice plants transfected with the Cry1Ab-01-Vip3Aa nucleotide sequence, rice plants transfected with the Cry1Ab-02-Cry1Fa nucleotide sequence, and transferred into Cry1A.105 nucleotides Sequence rice plants and rice plants transformed into the Cry1A.105-Cry2Ab nucleotide sequence; wild type rice plants were used as controls.

For Agrobacterium-mediated rice transformation, rice seeds were briefly inoculated in induction medium (N6 salt, N6 vitamin, casein 300 mg/L, sucrose 30 g/L, 2,4-dichlorophenoxyacetic acid (2, 4-D) 2 mg/L, plant gel 3 g/L, pH 5.8), callus was induced from rice mature embryos (step 1: callus induction step), after which callus was preferably used, with Agrobacterium Suspension contact with callus, Agrobacterium tumefaciens can express Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide sequence, Cry1Ab-01-Vip3Aa nucleotide sequence, Cry1Ab-02-Cry1Fa nucleotide sequence, Cry1A.105 nucleotide sequence and/or The Cry1A.105-Cry2Ab nucleotide sequence is delivered to at least one cell on the callus (step 2: infection step). In this step, the callus is preferably immersed in the Agrobacterium suspension (OD660=0.3, infecting medium (N6 salt, N6 vitamin, casein 300 mg/L, sucrose 30 g/L, glucose 10 g/L, acetosyringone) (AS) 40 mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 2 mg/L, pH 5.4)) to initiate infection. The callus was co-cultured with Agrobacterium for a period of time (3 days) (Step 3: co-cultivation step). Preferably, the callus is in a solid medium (N6 salt, N6 vitamin, casein 300 mg/L, sucrose 30 g/L, glucose 10 g/L, acetosyringone (AS) 40 mg/L, 2 after the infection step). 4-Dichlorophenoxyacetic acid (2,4-D) 2 mg/L, plant gel 3 g/L, pH 5.8). After this co-cultivation phase, there is a "recovery" step. In the "recovery" step, restore the medium (N6 salt, N6 vitamins, casein 300mg / L, sucrose 30g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 2mg / L, plant condensation At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in the gel 3 g/L, pH 5.8), and no selection agent for the plant transformant is added (step 4: recovery step). Preferably, the callus is cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells. Next, the inoculated callus is cultured on a medium containing a selective agent (mannose) and the grown transformed callus is selected (step 5: selection step). Preferably, the callus is in a selective solid medium (N6 salt, N6 vitamin, casein 300 mg/L, sucrose 10 g/L, mannose 10 g/L, 2,4-dichlorophenoxyacetic acid (2). , 4-D) 2 mg / L, plant gel 3 g / L, pH 5.8) culture, resulting in selective growth of transformed cells. Then, the callus regenerates the plant (step 6: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (N6 differentiation medium and MS rooting medium) Recycled plants.

The selected resistant callus was transferred to the N6 differentiation medium (N6 salt, N6 vitamin, casein 300 mg/L, sucrose 20 g/L, 6-benzylaminoadenine 2 mg/L, nafacetic acid 1 mg/L, Plant gel 3g/L, pH 5.8) was cultured and differentiated at 25 °C. The differentiated seedlings were transferred to the MS rooting medium (MS salt, MS vitamin, casein 300 mg/L, sucrose 15 g/L, plant gel 3 g/L, pH 5.8), and cultured at 25 ° C to about 10 cm. High, moved to the greenhouse to grow to firm. In a greenhouse, culture is carried out at 30 ° C per day.

2. TaqMan was used to verify the rice plants transferred into the Cry1A gene.

Rice plants transfected into Cry1Ab-01 nucleotide sequence, rice plants transfected with Cry1Ab-02 nucleotide sequence, rice plants transfected with Cry1Ab-01-Vip3Aa nucleotide sequence, and transferred into Cry1Ab-02-Cry1Fa Rice plants with nucleotide sequence, rice plants transfected with Cry1A.105 nucleotide sequence, and rice plants transfected with Cry1A.105-Cry2Ab nucleotide sequence were approximately 100 mg as samples and extracted with Qiagen's DNeasy Plant Maxi Kit. Its genomic DNA was detected by Taqman probe real-time PCR for detection of Cry1A gene, Cry1Fa gene, Vip3Aa gene and Cry2Ab. The copy number of the gene. At the same time, the wild type rice plants were used as a control, and the detection and analysis were carried out according to the method of verifying the corn plants transferred to the Cry1A gene by TaqMan in the above third embodiment. The experiment was set to repeat 3 times and averaged.

The results showed that Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide sequence, Cry1Ab-01-Vp3Aa nucleotide sequence, Cry1Ab-02-Cry1Fa nucleotide sequence, Cry1A.105 nucleotide sequence and Cry1A. The 105-Cry2Ab nucleotide sequence has been integrated into the genome of the tested rice plant, and the rice plant transformed into the Cry1Ab-01 nucleotide sequence, the rice plant transformed into the Cry1Ab-02 nucleotide sequence, and transferred into Rice plants with Cry1Ab-01-Vip3Aa nucleotide sequence, maize plants transfected with Cry1Ab-02-Cry1Fa nucleotide sequence, rice plants transfected with Cry1A.105 nucleotide sequence and transferred into Cry1A.105-Cry2Ab nucleoside Transgenic rice plants containing a single copy of the Cry1A gene, the Cry1Fa gene, the Vip3Aa gene and/or the Cry2Ab gene were obtained from the acid sequence rice plants.

Sixth embodiment, detection of insect resistance of transgenic rice plants

A rice plant transformed into a Cry1Ab-01 nucleotide sequence, a rice plant transformed into a Cry1Ab-02 nucleotide sequence, a rice plant transformed into a Cry1Ab-01-Vip3Aa nucleotide sequence, and transferred into a Cry1Ab-02-Cry1Fa nucleus a rice plant with a nucleotide sequence, a rice plant transformed with a Cry1A.105 nucleotide sequence, a rice plant transformed with a Cry1A.105-Cry2Ab nucleotide sequence, a wild type rice plant, and a non-transgenic rice plant identified by Taqman Oriental armyworms tested for insect resistance.

Rice plants transfected into Cry1Ab-01 nucleotide sequence, rice plants transfected with Cry1Ab-02 nucleotide sequence, rice plants transfected with Cry1Ab-01-Vip3Aa nucleotide sequence, and transferred into Cry1Ab-02-Cry1Fa a rice plant with a nucleotide sequence, a rice plant transformed with a Cry1A.105 nucleotide sequence, a rice plant transformed with a Cry1A.105-Cry2Ab nucleotide sequence, a wild type rice plant, and a non-transgenic rice plant identified by Taqman Fresh leaves (slurry period), rinsed with sterile water and blotted the water on the leaves with gauze, then remove the veins of the rice leaves, and cut into strips of about 1cm × 4cm, take 1 piece of cut length The strips of leaves are placed on the filter paper at the bottom of a circular plastic petri dish, which is wetted with distilled water, and 10 artificially reared oriental armyworms (initial hatching larvae) are placed in each dish, and the insect test dishes are covered. Put it into the square box with wet gauze at the bottom, and place it for 3 days at a temperature of 25-28 ° C, relative humidity 70%-80%, photoperiod (light/dark) 16:8, and count the oriental larvae Death and leaf damage, calculate the level of oriental armyworm in each sample Mortality. A total of 3 lines (S19, S20 and S21) transferred into the Cry1Ab-01 nucleotide sequence were transferred into the Cry1Ab-02 nucleotide sequence (S22, S23 and S24) and transferred to Cry1Ab- A total of 3 strains (S25, S26 and S27) of the nucleotide sequence of 01-Vip3Aa were transferred into Cry1Ab-02-Cry1Fa nucleotide sequence (S28, S29 and S30) and transferred to Cry1A. A total of 3 strains of 105 nucleotide sequences (S31, S32 and S33), transferred to the Cry1A.105-Cry2Ab nucleotide sequence of a total of 3 strains (S34, S35 and S36), identified by Taqman as non-transgenic (NGM2) has a total of 1 strain, One strain of wild type (CK2) was selected; 5 strains were selected from each strain, and each plant was repeated 6 times. The results are shown in Table 2 and Figure 4.

Table 2. Results of insect-resistant experiments on inoculation of oriental armyworms by transgenic rice plants

The results in Table 2 indicate that a rice plant transformed with the Cry1Ab-01 nucleotide sequence, a rice plant transformed with the Cry1Ab-02 nucleotide sequence, and a rice plant transformed with the Cry1Ab-01-Vip3Aa nucleotide sequence were transferred. The test insect mortality rate of rice plants into the Cry1Ab-02-Cry1Fa nucleotide sequence, rice plants transferred to the Cry1A.105 nucleotide sequence, and rice plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence was about 80%. Or above; and the test insect mortality of rice plants and wild-type rice plants identified as non-transgenic by Taqman is generally less than 10%.

The results in Figure 4 indicate that a rice plant transformed with the Cry1Ab-01 nucleotide sequence, a rice plant transformed with the Cry1Ab-02 nucleotide sequence, and a Cry1Ab-01-Vip3Aa nucleotide were transferred to the wild type rice plant. Sequence of rice plants, rice plants transformed into Cry1Ab-02-Cry1Fa nucleotide sequence, rice plants transformed into Cry1A.105 nucleotide sequence, and rice plants transformed into Cry1A.105-Cry2Ab nucleotide sequence The control effect of larvae is almost one hundred percent, and very few surviving larvae also basically stop development, and rice plants that have been transferred into the Cry1Ab-01 nucleotide sequence, rice plants that have been transferred into the Cry1Ab-02 nucleotide sequence, and transferred to Cry1Ab-01 a rice plant having a nucleotide sequence of Vip3Aa, a rice plant transformed with a Cry1Ab-02-Cry1Fa nucleotide sequence, a rice plant transformed with a Cry1A.105 nucleotide sequence, and a nucleotide sequence transferred into a Cry1A.105-Cry2Ab Rice plants are generally only slightly damaged, with only a small amount of pinhole damage on the leaves.

Thus, a rice plant transformed with the Cry1Ab-01 nucleotide sequence, a rice plant transformed with the Cry1Ab-02 nucleotide sequence, a rice plant transformed with the Cry1Ab-01-Vip3Aa nucleotide sequence, and transformed into Cry1Ab-02- were confirmed. Rice plants with Cry1Fa nucleotide sequence, rice plants transformed with Cry1A.105 nucleotide sequence, and rice plants transformed with Cry1A.105-Cry2Ab nucleotide sequence all showed high activity against oriental armyworm, this activity It is enough to have an adverse effect on the growth of oriental armyworms so that they can be controlled.

The above experimental results also showed that the maize plant transformed with the Cry1Ab-01 nucleotide sequence, the maize plant transformed with the Cry1Ab-02 nucleotide sequence, the maize plant transformed with the Cry1Ab-01-Vip3Aa nucleotide sequence, and the Cry1Ab- Maize plants with 02-Cry1Fa nucleotide sequence, maize plants transfected with Cry1A.105 nucleotide sequence, maize plants transfected with Cry1A.105-Cry2Ab nucleotide sequence, and rice transformed into Cry1Ab-01 nucleotide sequence Plant, rice plant transformed into Cry1Ab-02 nucleotide sequence, rice plant transformed into Cry1Ab-01-Vip3Aa nucleotide sequence, rice plant transformed into Cry1Ab-02-Cry1Fa nucleotide sequence, transferred to Cry1A.105 The rice plant of the nucleotide sequence and the rice plant transformed with the Cry1A.105-Cry2Ab nucleotide sequence are obviously resistant to oriental armyworm because the plant itself can produce the Cry1A protein, and therefore, it is well known to those skilled in the art according to the Cry1A protein. The same toxic effect on oriental armyworm can produce a similar transgenic plant that can express Cry1A protein and can be used to control the damage of oriental armyworm. The Cry1A protein in the present application includes, but is not limited to, the Cry1A 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 Cry1A protein, such as Vip3A protein, Cry1Fa protein or Cry2Ab protein and the like.

In summary, the method for controlling pests of the present invention controls the oriental armyworm pests by producing a Cry1A protein capable of killing oriental armyworms in plants; and the agricultural control methods and chemistry used in the prior art. Compared with the physical control method, the present application protects plants from the whole growth period and the whole plant to prevent the damage of oriental armyworm pests, and has no pollution and no residue, and the effect is stable, thorough, simple, convenient and economical.

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to be limiting, although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technology of the present application can be applied. Modifications or equivalents are made without departing from the spirit and scope of the technical solutions of the present application.

Claims (19)

A method of controlling oriental armyworm pests, wherein the method comprises contacting an oriental armyworm pest with a Cry1A protein. The method for controlling oriental armyworm pests according to claim 1, wherein the Cry1A protein is present in a plant cell producing the Cry1A protein, and the oriental armyworm pest ingests the plant cell and the Cry1A protein contact. The method for controlling oriental armyworm pests according to claim 2, wherein said Cry1A protein is present in a transgenic plant producing said Cry1A protein, said oriental mucoid pest by feeding said tissue of said transgenic plant The Cry1A protein is contacted, and the growth of the oriental armyworm pest is inhibited and/or contacted after the contact, resulting in the death of the oriental armyworm pest to achieve control of the plant against the oriental armyworm. The method for controlling oriental armyworm pests according to any one of claims 1 to 3, wherein the Cry1A protein is one or more of a Cry1Ab protein, a Cry1Ac protein, a Cry1Ah protein or a Cry1A.105 protein. A method of controlling oriental armyworm pests according to claim 3 or 4, wherein the transgenic plants can be in any growth period. The method for controlling oriental armyworm pests according to claim 3 or 4, wherein the tissue of the transgenic plant is selected from one or more of the following: leaves, stems, fruits, tassels, ears, anthers and filigrees. . The method of controlling oriental armyworm pests according to claim 3 or 4, wherein the control of the plants belonging to the oriental armyworm is not changed by the change of the planting place and/or the planting time. The method for controlling oriental armyworm pests according to any one of claims 2 to 7, wherein the plant is selected from the group consisting of corn, rice, wheat, sorghum, millet, barley or grass, preferably, the plant is Corn or rice. The method of controlling oriental armyworm pests according to any one of claims 1 to 8, wherein the step prior to the contacting step is planting a plant containing a polynucleotide encoding the Cry1A protein. The method for controlling oriental armyworm pests according to any one of claims 1 to 9, wherein the amino acid sequence of the Cry1A protein comprises: 1) SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The amino acid sequence shown, 2) an amino acid sequence having at least 70% homology to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and having insect resistance to Oriental armyworm, or 3) SEQ ID NO: 1, amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, which has been substituted, deleted and/or added with one or more amino acid residues and has an insect resistance activity against oriental armyworm Preface Column. The method for controlling oriental armyworm pests according to claim 10, wherein the nucleotide sequence of the Cry1A protein comprises: 1) SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: a nucleotide sequence, 2) a nucleotide having at least about 75% homology to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 and encoding an amino acid sequence having insecticidal activity against oriental armyworm Sequence, 3) a nucleotide sequence that hybridizes to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 under stringent conditions and encodes an amino acid sequence having insecticidal activity against oriental armyworm, 4) due to the password Subdegeneracy differs from the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 encoding an amino acid sequence having insecticidal activity against oriental armyworm. The method of controlling oriental armyworm pests according to any one of claims 2 to 11, wherein the plant further comprises at least one nucleotide different from the nucleotide encoding the Cry1A protein. The method for controlling oriental armyworm pests according to claim 12, wherein said second nucleotide encodes a Cry-like insecticidal protein, a Vip-like insecticidal protein, a protease inhibitor, a lectin, an alpha-amylase or Peroxidase. The method of controlling oriental armyworm pests according to claim 13, wherein the second nucleotide encodes a Cry1F protein, a Vip3A protein or a Cry2Ab protein. The method of controlling an oriental armyworm pest according to claim 14, wherein the second nucleotide comprises the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: . The method of controlling oriental armyworm pests according to claim 12, wherein the second nucleotide is a dsRNA which inhibits an important gene in a target insect pest. A method of preparing a plant cell, transgenic plant or part of a transgenic plant against an oriental armyworm pest, comprising introducing a coding nucleotide sequence of a Cry1A protein into a part of the plant cell, transgenic plant or transgenic plant, preferably The nucleotide sequence encoding the Cry1A protein is introduced into the genome of the part of the plant cell, transgenic plant or transgenic plant. Use of the Cry1A protein in the preparation of parts of plant cells, transgenic plants or transgenic plants against oriental armyworm pests. A method of cultivating a plant for controlling oriental armyworm pests, comprising: Planting at least one plant seed, the genome of the plant seed comprising a polynucleotide sequence encoding a Cry1A protein; Planting the plant seed into a plant; The plants are grown under conditions in which artificially inoculated oriental armyworm pests and/or oriental armyworm pests are naturally harmful, and plants with reduced plant damage and/or plants having no polynucleotide sequence encoding the Cry1A protein are harvested and/or Or plants with increased plant yield.

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