AU2014350741A1

AU2014350741A1 - Method for controlling pest

Info

Publication number: AU2014350741A1
Application number: AU2014350741A
Authority: AU
Inventors: Guowei JIAO; Yuejing Kang; Cong TIAN; Dengyuan Wang; Yunzhu Zhang
Original assignee: Beijing Dabeinong Technology Group Co Ltd; Beijing Dabeinong Biotechnology Co Ltd
Current assignee: Beijing Dabeinong Biotechnology Co Ltd
Priority date: 2013-11-15
Filing date: 2014-11-13
Publication date: 2016-06-02
Anticipated expiration: 2034-11-13
Also published as: CN103719136A; AU2014350741B2; AR098426A1; WO2015070778A1

Abstract

A method for controlling spodoptera litura pests, the spodoptera litura pests contacting the Vip3A protein; and the spodoptera litura pests are controlled via the Vip3A protein generated in a plant and capable of killing the spodoptera litura.

Description

Method for Controlling Pests

The present application claims the priority of Chinese patent application No. 201310573441.6 filed on November 15, 2013 with an invention title “Method for Controlling Pests”, the full text of which is incorporated here by reference.

Field of the Invention

The present application relates to a method for controlling pests, particularly to a method for controlling harm to plant caused by Spodoptera litura by using Vip3A protein expressed in plant.

Background of the Invention

Spodoptera litura belongs to noctuidae family, lepidoptera order. It is an omnivorous and gluttonous pest and is harm to many kinds of hosts. In addition to maize and soybean, it may also be harm to melons, eggplants, beans, shallots, leeks, spinach as well as cruciferae plant, grain and economic crops etc. in total of nearly 100 families and more than 300 species of plant. Spodoptera litura is distributed all over the world and occurs in various parts of China, mainly in Yangtze River basin and Yellow River basin. Pest Spodoptera litura is mainly harm to whole plant when they are larvae and live on leaf backs in group and gnaw at leaves when they are in low instar. After the third instar, they will scatter and be harm to leaves and tender stems. Mature larvae may eat into fruit.

Maize and soybean are important food crops in China. Every year, huge grain loss is caused by Spodoptera litura. In more serious cases, it may affect the living condition of local people. In order to control Spodoptera litura, people typically adopt the following main control methods: agricultural control, chemical control and physical control.

Agricultural control is comprehensive coordination and management of multiple factors of the whole farmland ecosystem to regulate and control crops, pests and environmental factors so as to create a farmland ecological environment that is favorable to crop growth but unfavorable to occurrence of Spodoptera litura. For example, weed removal, soil turnover and sunning after harvesting or irrigation can destroy or worsen the pupation sites of Spodoptera litura so as to help reduce pest sources; or for example, egg masses and clustered newly hatched larvae are removed by the way during management to reduce pest sources. As agricultural control mostly adopts preventive measures, its application is limited and cannot be used as an emergency measure. In case of breakout of Spodoptera litura, it is powerless.

Chemical control, i.e.: control by pesticide, uses chemical insecticides to kill pests. It is an important part of comprehensive control of Spodoptera litura. It is characterized by high speed, convenience, easiness and high economic benefit, particularly during outbreak of Spodoptera litura, it is an indispensable emergency measure. It may wipe out Spodoptera litura before it causes harm. At present, the method of chemical control mainly refers to spray of drugs. However, chemical control also has limitation. If it is not used properly, it may cause phytotoxicity to crops, drug resistance of pests, as well as may natural enemies being reduced and wounded, environmental pollution, which will casue bad consequences such as the destruction of farmland ecosystem and threat to the safety of human and livestock due to pesticide residue .

Physical control is to control pests by using physical factors, such as: light, electricity, color, humidity, temperature and etc as well as mechanical equipment to trap and kill the pests and sterilize the pests by irradiation based on the response of pests to physical factors in the environmental conditions. The methods widely applied at present mainly include: attracting moths with lamp, syrupacetiacid bait trap, and trapping and killing moths by dipping and sprinkling 500 dipterex using willow branch. Although the above methods show a control effect to some extent, they have certain difficulty in operation.

In order to solve the limitation of agricultural control, chemical control and physical control in practical application, scientists discovered in research that when insecticidal genes coding insecticidal proteins are transformed into a plant, some insecticidal transgenic plants may be obtained to control plant pests. Vip3 A insecticidal protein is one of the numerous insecticidal proteins. It is a specific protein generated by Bacillus cereus.

Vip3A protein has the effect of poisoning and killing sensitive insects by activating programmed death of apoptosis types of cells. Vip3A protein is hydrolysed into four main protein product in the intestinal of the insect, wherein, only one protein hydrolysate (33KD) is the core structure representing the toxicity of Vip3A protein. Vip3A protein binds the epithelial cells of midgut of sensitive insects, thereby to start the programmed death, which can result in dissolution of epithelial cells of midgut so as to cause death of the insects. Vip3 A protein does not produce any disease to the non-sensitive insects, so does not result in apoptosis of epithelial cells of midgut and dissolution.

It has been proved that plant genetically modified by Vip3 A may resist the encroachment of Agrotis ypsilon Rottemberg, Spodoptera frugiperda, sesamia inferens, heliothis zea and other Lepidoptera pests. However, by now there is no report on controlling harm of Spodoptera litura to plant through generating transgenic plant expressing Vip3 A protein.

Summary of the Invention

The object of the present application is to provide a method for controlling pests, particularly provide a method for controlling harm of Spodoptera litura to plant through a generating transgenic plant expressing Vip3A protein for the first time, which effectively overcame the technical defects of agricultural control, chemical control, physical control and etc in prior art.

The first aspect of the present application relates to a method for controlling pest Spodoptera litura, wherein pest Spodoptera litura contacts with Vip3 A protein.

In some embodiments, the Vip3 A protein is Vip3 Aa protein.

In further embodiments, the Vip3Aa protein exists in a plant cell generating the Vip3Aa protein, and the pest Spodoptera litura contacts with the Vip3Aa protein through intaking the plant cell.

In further embodiments, the Vip3Aa protein exists in a transgenic plant generating the Vip3 Aa protein, the pest Spodoptera litura contacts with the Vip3 Aa protein through intaking the tissue of the transgenic plant, and after the contact, the growth of the Pest Spodoptera litura is inhibited and/or the pest Spodoptera litura dies, so as to achieve control over the harm of Spodoptera litura to plant.

The transgenic plant may be in any growth period.

The tissue of the transgenic plant is selected from leaves, stalks, tassels, female spikes, anthers, filaments and fruits.

The control over the harm of Spodoptera litura to plant is not changed with the change of planting location and/or planting time.

The plant is selected from maize, soybean, cotton, sweet potato, taro, lotus, sesbania, tobacco, beet, cabbage and eggplant. Preferably, the plant is selected from maize and soybean. A step before the contact step is planting the plant containing polynucleotide coding the Vip3 Aa protein.

In some embodiments, the amino acid sequence of the Vip3Aa protein includes: 1) an amino acid sequence as shown by SEQ ID NO: 1 or SEQ ID NO: 2, 2) an amino acid sequence having at least 70% of homology with SEQ ID NO: 1 or SEQ ID NO: 2 and having insecticidal activity against pest Spodoptera litura, for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or 3) an amino acid sequence obtained from the amino acid sequence shown by SEQ ID NO: 1 or SEQ ID NO: 2 through substitution, deletion and/or addition of one or multiple amino acid residues and having insecticidal activity against pest Spodoptera litura, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50 amino acid residues.

In some embodiments, the coding nucleotide sequence of the Vip3Aa protein includes: 1) a nucleotide sequence shown by SEQ ID NO: 3 or SEQ ID NO: 4, 2) a nucleotide sequence having at least about 75% of homology with SEQ ID NO: 3 or SEQ ID NO: 4 and coding the amino acid sequence having insecticidal activity against pest Spodoptera litura, for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, 3) a nucleotide sequence hybridizing with SEQ ID NO: 3 or SEQ ID NO: 4 under strict conditions and coding the amino acid sequence having insecticidal activity against pest Spodoptera litura, 4) a nucleotide sequence different from SEQ ID NO: 3 or SEQ ID NO: 4 due to codon degeneracy and coding the amino acid sequence having insecticidal activity against pest Spodoptera litura.

In some embodiments, the plant also contains at least a second nucleotide different from the nucleotide coding the Vip3 Aa protein.

In further embodiments, the second nucleotide codes Cry type insecticidal protein, Vip type insecticidal protein, protease inhibitor, lectin, α-amylase or peroxidase.

In further embodiments, the second nucleotide codes Cry 1 Ab protein, CrylFa protein or Cry IBa protein.

In still further embodiments, the second nucleotide has the nucleotide sequence shown by SEQ ID NO: 5 or SEQ ID NO: 6.

In some other embodiments, the second nucleotide is a dsRNA inhibiting an important gene in target insects and pests.

The second aspect of the present application relates to the use of Vip3A protein for controlling pest Spodoptera litura.

In some embodiments, the Vip3 A protein is Vip3Aa protein.

In further embodiments, the amino acid sequence of the Vip3Aa protein includes: 1) an amino acid sequence as shown by SEQ ID NO: 1 or SEQ ID NO: 2, 2) an amino acid sequence having at least 70% of homology with SEQ ID NO: 1 or SEQ ID NO: 2 and having insecticidal activity against pest Spodoptera litura, for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or 3) an amino acid sequence obtained from the amino acid sequence shown by SEQ ID NO: 1 or SEQ ID NO: 2 through substitution, deletion and/or addition of one or multiple amino acid residues and having insecticidal activity against pest Spodoptera litura, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50 amino acid residues.

In further embodiments, the coding nucleotide sequence of the Vip3Aa protein includes: 1) a nucleotide sequence shown by SEQ ID NO: 3 or SEQ ID NO: 4, 2) a nucleotide sequence having at least about 75% of homology with SEQ ID NO: 3 or SEQ ID NO: 4 and coding the amino acid sequence having insecticidal activity against pest Spodoptera litura, for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, 3) a nucleotide sequence hybridizing with SEQ ID NO: 3 or SEQ ID NO: 4 under strict conditions and coding the amino acid sequence with insecticidal activity against pest Spodoptera litura, 4) a nucleotide sequence different from SEQ ID NO: 3 or SEQ ID NO: 4 due to codon degeneracy and coding the amino acid sequence having insecticidal activity against pest Spodoptera litura.

In some embodiments, Vip3A protein realizes control of pest Spodoptera litura by the following method: allowing Vip3A protein to be expressed in a plant cell and allowing the pest Acronycta rumicis to intake the plant cells, so as to contact with the Vip3A protein.

In some embodiments, Vip3A protein realizes control of pest Spodoptera litura by the following method: allowing Vip3 A protein to be expressed in a transgenic plant and allowing the pest Spodoptera litura to intake the tissues of the transgenic plant, so as to contact with the Vip3 A protein.

The transgenic plant may be in any growth period.

The tissue of the transgenic plant is selected from leaves, stalks, fruits, tassels, female spikes, anthers and filaments.

The control of the Vip3 A protein over pest Spodoptera litura is not changed with the change of planting location and/or planting time.

The plant is selected from maize, soybean, cotton, sweet potato, taro, lotus, sesbania, tobacco, beet, cabbage and eggplant. Preferably, the plant is selected from maize and soybean.

In some embodiments, the second nucleotide is dsRNA inhibiting important genes in target insects and pests.

The third aspect of the present application relates to a method for preparing plant cell, transgenic plant or part of transgenic plant controlling pest Spodoptera litura. The method includes introducing the coding nucleotide sequence of Vip3A protein into the plant cell, transgenic plant or part of the transgenic plant, preferably, introducing the coding nucleotide sequence of Vip3 A protein into the genome of plant cell, transgenic plant or part of transgenic plant.

In some embodiments, the part of transgenic plant is propagating materials or nonpropagating materials.

The propagating materials refer to plant fruits, seeds or calli.

The non-propagating materials refer to plant leaves, stalks, tassels, female spikes, anthers or filaments without fertility.

In some embodiments, the Vip3 A protein is Vip3Aa protein.

In further embodiments, the amino acid sequence of the Vip3Aa protein includes: 1) an amino acid sequence shown by SEQ ID NO: 1 or SEQ ID NO: 2, 2) an amino acid sequence having at least 70% of homology with SEQ ID NO: 1 or SEQ ID NO: 2 and having insecticidal activity against pest Spodoptera litura, for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or 3) an amino acid sequence obtained from the amino acid sequence shown by SEQ ID NO: 1 or SEQ ID NO: 2 through substitution, deletion and/or addition of one or multiple amino acid residues and having insecticidal activity against pest Spodoptera litura, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50 amino acid residues.

In further embodiments, the coding nucleotide sequence of the Vip3Aa protein includes: 1) a nucleotide sequence shown by SEQ ID NO: 3 or SEQ ID NO: 4, 2) a nucleotide sequence having at least about 75% of homology with SEQ ID NO: 3 or SEQ ID NO: 4 and coding the amino acid sequence having insecticidal activity against pest Spodoptera litura, for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, 3) a nucleotide sequence hybridizing with SEQ ID NO: 3 or SEQ ID NO: 4 under strict conditions and coding the amino acid sequence having insecticidal activity against pest Spodoptera litura, 4) a nucleotide sequence different from SEQ ID NO: 3 or SEQ ID NO: 4 due to codon degeneracy and coding the amino acid sequence having insecticidal activity against pest Spodoptera litura.

In some embodiments, the method also includes introducing at least a second nucleotide different from the nucleotide coding the Vip3 A protein into the plant cells, transgenic plant or parts of transgenic plant, preferably, introducing at least a second nucleotide different from the nucleotide coding the Vip3 A protein into the genomes of the plant cells, transgenic plant or parts of transgenic plant.

In some embodiments, the second nucleotide codes Cry type insecticidal protein, Vip type insecticidal protein, protease inhibitor, lectin, α-amylase or peroxidase.

In further embodiments, the second nucleotide codes Cry 1 Ab protein, CrylFa protein, or Cry IBa protein.

In some other embodiments, the second nucleotide is dsRNA inhibiting important genes in target insects and pests.

In some embodiments, the coding nucleotide is introduced into the plant cell, transgenic plant or part of transgenic plant through agrobacterium tumefaciens-mediated transformation, trace emission bombardment, direct ingestion of DNA into protoplast, electroporation or silica whisker mediated DNA introduction, preferably, agrobacterium tumefaciens-mediated transformation.

The fourth aspect of the present application relates to the plant cell, transgenic plant or part of transgenic plant controlling pest Spodoptera litura obtained by the method in the foregoing third aspect.

The fifth aspect of the present application relates to the use of Vip3A protein in preparing the plant cell, transgenic plant or part of transgenic plant controlling pest Spodoptera litura. “Vip3A protein”, “control/controlling pest Spodoptera litura”, “plant”, “plant cells”, “transgenic plant” and “part of transgenic plant” related in this aspect as well as their extended content are defined as the foregoing aspects.

The sixth aspect of the present application relates to a method for cultivating plant which control pest Spodoptera litura, including: planting at least a kind of plant seed of which genome includes the polynucleotide sequence coding Vip3 A protein; making the plant seeds grow into a plant; making the plant grow under the condition that pest Spodoptera litura does harm through artificial inoculation and/or natural occurrence, and harvesting plant with weakened plant damage and/or increased plant yield compared to other plant without polynucleotide sequence coding Vip3 A protein. “Vip3A protein” and “plant” related in this aspect as well as their extended content are defined as the foregoing aspects. The meaning of “control pest Spodoptera litura” is similar to the meaning of “resist pest Spodoptera litura". The concrete definitions are described above.

In the present application, the expression of Vip3A protein in one transgenic plant also may be accompanied with the expression of one or multiple Vip type and/or Cry type insecticidal proteins. This co-expression of more than one insecticidal protein in a same transgenic plant may be realized by genetic engineering, which makes the plant contain and express the needed gene. In addition, one plant (the first parent) may express Vip3A protein through genetic engineering, and the second plant (the second parent) may express Vip type and/or Cry type insecticidal proteins through genetic engineering. The offspring plant expressing all the genes of the first parent and the second parent introduced are obtained through hybridization between the first parent and the second parent. RNA interference (RNAi) refers to the phenomenon of highly effective and specific degradation of highly conserved homological mRNA induced by double-stranded RNA (dsRNA) in the evolution process. Therefore, in the present application, RNAi technology may be used to specifically remove or close the expression of specific gene in target insects and pests.

In some aspects, the present application also relates to the following content:

Item 1: A method for controlling pest Spodoptera litura, characterized in that it allowing pest Spodoptera litura to contact with Vip3 A protein.

Item 2: The method for controlling pest Spodoptera litura according to Item 1, characterized in that the Vip3 A protein is Vip3 Aa protein.

Item 3: The method for controlling pest Spodoptera litura according to Item 2, characterized in that the Vip3Aa protein exists in a plant cell generating the Vip3Aa protein and the pest Spodoptera litura contacts with the Vip3Aa protein through intaking the plant cell.

Item 4: The method for controlling pest Spodoptera litura according to Item 3, characterized in that the Vip3Aa protein exists in a transgenic plant generating Vip3Aa protein, the pest Spodoptera litura contacts with the Vip3Aa protein through intaking the tissue of the transgenic plant, the growth of the pest Spodoptera litura is inhibited after the contact, which eventually results in death, so as to achieve control over the harm of Spodoptera litura to plant.

Item 5: The method for controlling pest Spodoptera litura according to Item 4, characterized in that the transgenic plant may be in any growth period.

Item 6: The method for controlling pest Spodoptera litura according to Item 4, characterized in that the tissue of the transgenic plant may be leaves, stalks, tassels, female spikes, anthers, filaments or fruits.

Item 7: The method for controlling pest Spodoptera litura according to Item 4, characterized in that the control over the harm of Spodoptera litura to plant is not changed with the change of planting location.

Item 8: The method for controlling pest Spodoptera litura according to Item 4, characterized in that the control over the harm of Spodoptera litura to plant is not changed according to the change of planting time.

Item 9: The method for controlling pest Spodoptera litura according to any one of Items 3 to 8, characterized in that the plant is selected from maize, soybean, cotton, sweet potato, taro, lotus, sesbania, tobacco, beet, cabbage and eggplant.

Item 10: The method for controlling pest Spodoptera litura according to any one of Items 3 to 9, characterized in that a step before the contact step is planting a plant containing polynucleotide which codes the Vip3 Aa protein.

Item 11: The method for controlling pest Spodoptera litura according to any one of Items 2 to 10, characterized in that the amino acid sequence of the Vip3Aa protein has the amino acid sequence shown by SEQ ID NO: 1 or SEQ ID NO: 2.

Item 12: The method for controlling pest Spodoptera litura according to Item 11, characterized in that the nucleotide sequence of the Vip3Aa protein has the nucleotide sequence shown by SEQ ID NO: 3 or SEQ ID NO: 4.

Item 13: The method for controlling pest Spodoptera litura according to any one of Items 3 to 12, characterized in that the plant may also generate at least a second nucleotide different from the Vip3 Aa protein.

Item 14: The method for controlling pest Spodoptera litura according to Item 13, characterized in that the second nucleotide may code Cry type insecticidal protein, Vip type insecticidal protein, protease inhibitor, lectin, α-amylase or peroxidase.

Item 15: The method for controlling pest Spodoptera litura according to Item 14, characterized in that the second nucleotide may code Cry 1 Ab protein, CrylFa protein, or Cry IBa protein.

Item 16: The method for controlling pest Spodoptera litura according to Item 15, characterized in that the second nucleotide contains the nucleotide sequence shown by SEQ ID NO: 5 or SEQ ID NO: 6.

Item 17: The method for controlling pest Spodoptera litura according to Item 13, characterized in that the second nucleotide is a dsRNA inhibiting an important genes in target insects and pests.

Item 18: Use of Vip3 A protein for controlling pest Spodoptera litura.

Brief Description of Drawings FIG. 1 is a constructional flow diagram of recombinant cloning vector DBN01-T containing Vip3Aa-01 nucleotide sequence according to the pest control method of the present application; FIG. 2 is a constructional flow diagram of recombinant expressing vector DBN100066 containing Vip3Aa-01 nucleotide sequence according to the pest control method of the present application; FIG. 3 is a constructional flow diagram of recombinant expressing vector DBN100002 containing Vip3Aa-01 nucleotide sequence according to the pest control method of the present application; FIG. 4 is a diagram showing the insecticidal effect of transgenic maize inoculating Spodoptera litura according to the pest control method of the present application; FIG. 5 is a diagram showing the insecticidal effect of transgenic soybean inoculating Spodoptera litura according to the pest control method of the present application.

Detailed Description of the Embodiments

Spodoptera litura and Spodoptera frugiperda both belong to noctuidae family, lepidoptera order. They are both omnivorous pests and harm to maize, soybean, cotton and sweet potato. Nevertheless, Spodoptera litura and Spodoptera frugiperda are definitely different species biologically and have the following main differences: 1. Different feeding habits: Spodoptera litura is an omnivory and overintaking pest and does harm intermittently and rampantly. Spodoptera litura does harm to hosts widly, intaking nearly 300 kinds of plant, such as, sweet potato, cotton, taro, lotus, sesbania, soybeans, tobacco, sugar beet as well as cruciferous and solanaceae vegetables etc. Spodoptera frugiperda is a polyphagous pest, but obviously favorite to graminaceous, commonly does harm to weeds, maize, paddy, sorghum, sugar cane, also does harm to cotton, cruciferae, cucurbitaceae, peanuts, alfalfa, onions, phaseolus, sweet potatoes, tomatoes and other solanaceae plant (aubergine, tobacco, capsicum), a variety of ornamental plant (compositae, carnations, geranium). 2. Different distribution regions: Spodoptera litura is distributed all over the world, and occurs in all parts of China, mainly in Jiangxi, Jiangsu, Hunan, Hubei, Zhejiang and Anhui of the Yangtze River basin as well as the provinces such as Henan, Hebei and Shandong of the Yellow River basin. Spodoptera frugiperda is mainly distributed outside China, including Canada, Mexico, the United States, Argentinia, Bolivia, Brazil, Chile, Colombia, Ecuador, French Guiana, Guyana, Paraguay, Peru, Surinam, Uruguay and Venezuela of America Continent as well as the whole Central America and Caribbean area. While there is no report that Spodoptera frugiperda exists in China. 3. Different damage habits: Pest Spodoptera liturns do harm to whole plant in larvae stage, live on leaf backs in group and gnaw at lower epidermis and mesophyll, only leaving upper epidermis in form of transparent spots when they are in early instar. After third instar, they will scatter and do harm to leaves and tender stems. After fourth instar, they become gluttonous and gnaw at leaves, only leaving main veins: mature larvae may eat into fruits; their feeding habits are omnivorous and do harm to all organs. They are gluttonous when they are in old-aging, so they are very dangerous pests. While Spodoptera frugiperda is fed with leaves which can result in fallen leaves, after which, they will transfer the harm. Sometimes, a large number of larvae do harm by cutting root way, cutting the stems of seedling and young plant. On some larger crops, such as corncob, the larvae can do harm by drilling in. When they are fed with maize leaves, they will leave a large number of holes. After being eaten by low age larvae, the veins will represent a window screening shape. Like rootworms, the elder larvae can cut off the seedlings with 30 days from the roots. When the population quantity is large, the larvae are like marching, expanding by group. When the environment is advantageous, they generally stay in weeds. 4. Different morphological characteristics 1) Different egg morphology: The eggs of Spodoptera litura are in a flat semi-sphere shape. When they are just laid, the eggs are yellowish white and later become dark gray, stick together in a shape of block, covered with brown fluffs. In comparison, the eggs of Spodoptera frugiperda are in a shape of hemisphere, the egg masses are laid together on the surface of leaves, each egg mass contains 100-300 eggs, sometimes it presents as a Z layer, the surfaces of the egg masses have banding protective layers formed by the gray hairs of Female insect abdomen. 2) Different larva morphology: The larvae of Spodoptera litura are 33-50mm long. Their heads are black brown. The color of chest is variable, from earthy yellow to blackish green. Small white dots are scattered all over the body surface. There is a pair of half-moon black spots in a shape similar to a triangle in Winter Solstice. The larvae normally have six instars. In comparison, the whole bodies of the larvae of Spodoptera frugiperda are green when they are newly horned, and have black lines and spots. When growing, the whole bodies thereof still keep green and become pale yellow, and have black dorsal lines and spiracular lines. When in dense (the pupulationg denstity is large and food are short), the last stage larvae are almost black when they are in migratory phase. The bodies of elder larvae are 35-40 mm long, their heads have spots presenting a yellow and inverted "Y". Primary setaes inserted in the black dorsal seta in group (both sides of dorsal line on each joint have two setaes). The terminal joint of abdomen have four black spots arrayed in a square. The larvae have six stadiums, or rarely have five stadiums. 3) Different pupal morphology: The pupae of Spodoptera litura are 15-20mm long. They are cylindrical and red brown. The tail has a pair of short spines. In comparison, the pupae of Spodoptera frugiperda are brown and shiny, and have 18-20 mm long. 4) Different adult morphology: The adults of Spodoptera litura are about 14-20mm long, the wing span is 35-46mm, the body is dark brown, the back surface of the chest has white clustered hair, the fore wings are grayish brown and have many patterns, the internal and external transverse lines are white and in wavy shape, and in the middle, there are obvious white diagonal and wide bands. For this reason, it is called Spodoptera litura. In comparison, the adults of Spodoptera frugiperda are hairchested and taupe brown, the wing span thereof is 32-38 mm. The fore wings of females are from gray to taupe brown, but the fore wings of males are blacker, and have black spots and light-colored dark lines. The underwings are white, and the nervures of underwings are brown and transparent. Genitalia of micro insect holds valva square, the end claspers of the claspers is missing; the female copulatory bursa has no mating piece 5. Different growth habits and occurrence regulations: Spodoptera litura has four generations (North China)-nine generations (Guangdong). Generally, mature larvae or pupae overwinter in weeds on field ridges. In Guangzhou, there is no overwintering in a real sense. In the region north of the Yangtze River basin, the pests are likely to be frozen to death in winter, so overwintering is not a final conclusion. It is speculated that the local pests may be migrated from South China; it outbreaks mostly in July-August in the Yangtze River basin, and mostly in August-September in Yellow River basin. The adults are nocturnal, have strong flying power, phototaxis and chemotaxis and are particularly sensitive to sugar, vinegar, alcohol and other fermentations. Every female moth can lay 3-5 masses of eggs. Each mass has about 100-200 eggs. The eggs are mostly laid at the forks of leaf veins on leaf back. More eggs are laid in luxuriant and green crops. They are laid in piles. The egg masses are often covered with scaly hair, so they can be easily found. The temperature suitable for egg hatching is about 24 °C. The larval stage is 14-20 days at air temperature of 25 °C. The newly hatched larvae have the habit of living and doing harm in group. After third instar, they scatter. Mature larvae have nocturnality and syncope. In the day, they mostly hide in soil seams. At dusk, they craw out and look for food. Once they are frightened, they will curl up and feign death. When foodstuff is inadequate or improper, the larvae may migrate by group to the fields nearby to do harm to the fields. Therefore, they also are commonly known as “army worms”. The soil humidity suitable for pupation is about 20% of water content in soil. The pupal stage is 11-18 days. Spodoptera litura is a thermophic pest and resistant to high temperature and intermittently doing rampant harm. The temperature suitable for growth in every insect stage is 28-30°C, but the pests can also live normally under high temperature (33-40°C). They have weak resistance to cold. They basically cannot survive long-time at low temperature of around 0 °C in winter. Generally speaking, hot years and seasons are favorable to their growth and reproduction. Low temperature likely causes mass death of pupae. This pest is omnivorous, but the foodstuff situations, such as different hosts, even a same host in different growth stages or different organs, and the sufficiency or deficiency of foodstuff have obvious influence on its growth and reproduction. The fields with interplanting, multiple crop indexes or excessive close planting are favorable to its occurrence. Its natural enemies include braconid and polyhedrosis virus parasitic in larvae. In comparison, Spodoptera frugiperda has the ability of migratory flight, and can spreads itself for quite a distance. Vegetable or fruit with larva is an important way of international communication. Spodoptera frugiperda has one time of migratory flight in America regularly in one year 1, spreading to the whole United States. At prophase of oviposition (development of sexual maturity), they spread widely. In the United States, the adult can borrow a low airflow to diffuse from Mississippi to Canada in less than 30 hours. Late summer or early fall, larvae normally migrate in group. As a result, local diffusion successfully can help reduce larval mortalit.

To summarize, Spodoptera litura and Spodoptera frugiperda are different pests, have distant ties of consanguinity and cannot mate each other and generate later generations.

The genomes of the plant, plant tissue or plant cell described in the present application refer to any genetic material in the plant, plant tissue or plant cell and include cell nuclei, plasmids and genomes of mitochondria.

The “contact” in the present application refers to that insects and/or pests touch, stay and/or intake plant, plant organs, plant tissues or plant cells. The plant, plant organs, plant tissues or plant cells refer to they can expresse insecticidal proteins in vivo or the surface of the plant, plant organ, plant tissue or plant cell has insecticidal proteins or microorganisms generating insecticidal proteins.

Terms “control” and/or “prevent” in the present application refers to that pest Spodoptera litura comes into contact with Vip3A protein, and after the contact, the growth of pest Spodoptera litura is inhibited and/or the pest Spodoptera litura dies. Further, the pest Spodoptera litura contacts with Vip3A protein through intaking plant tissues. After the contact, the growth of all or some of the pest Spodoptera litura is inhibited and/or all or some of them die. Inhibition refers to sub-lethality, i.e.: it does not refer to lethal, but it may arouse certain effects in such aspects such as growth and development, behavior, physiology, biochemistry and tissues, for example: the growth and development is slow and/or stops. Meanwhile, the plant should be normal in morphology and can be cultured under conventional methods in order to use them for consumption and/or generation of products.

Besides, compared with non-transgenic wild plant, the plant and/or plant seeds controlling pest Spodoptera litura which contain polynucleotide sequence coding Vip3A protein have weakened plant damage under the condition that the pest Spodoptera litura does harm through artificial inoculation and/or natural occurrence. The concrete manifestation includes but without limitation: improved stalk resistance, and/or increased grain weight and/or yield. The “control” and/or “prevent” function of Vip3A protein over the pset Spodoptera litura may exist independently and will not abate and/or disappear due to the existence of other substances which can “control” and/or “prevent” pest Spodoptera litura. Specifically, if the tissues of transgenic plant (containing polynucleotide sequence coding Vip3A protein) simultaneously and/or asynchronously contain and/or generate Vip3 A protein and/or another substance which can control pest Spodoptera litura, then the existence of another substance will neither affect the “control” and/or “prevent” function of Vip3 A protein over Spodoptera litura, nor can result in that the “control” and/or “prevent” function is realized completely by another substance, while irrelevance with Vip3A protein. Under normal conditions, on farmland, the ingestion process of plant tissues by pest Spodoptera litura is short and can hardly be observed by naked eyes, therefore, under the condition that pest Spodoptera litura does harm through artificial inoculation and/or natural occurrence, if any tissue of transgenic plant (containing polynucleotide sequence coding Vip3A protein) has dead pest Spodoptera litura, and/or pest Spodoptera litura whose growth is inhibited stays on them, and/or plant damage is weakened compared with non-transgenic wild plant, it means the method and use of the present application are realized, i.e.: the method and/or use for controlling the pest Spodoptera litura is realised through the contact between pest Spodoptera litura and Vip3A protein.

The polynucleotide and/or nucleotide in the present application form complete “gene” and code proteins or polypeptides in the needed host cells. Those skilled in the art can easily know the polynucleotide and/or nucleotide in the present application can be located under the control of the regulating sequence in the target host.

As well known to those skilled in the art, DNA typically exists in form of double strands. In this arrangement, one strand is complementary with the other strand, vice versa. As DNA replicates and generates other complementary strands of DNA in plant, the present application includes the use of polynucleotides listed in the sequence listing as well as their complementary strands. The “coding strand” commonly used in the art refers to the strand bound with antisense strand. In order to express proteins in vivo, typically one strand of DNA is transcribed into a complementary strand of mRNA, which as a template translates proteins. In fact, mRNA is transcribed from “antisense” strand of DNA. “Sense” or “coding” strand contains a series of codons (a codon consists of three nucleotides. When three nucleotides are read in a time, a specific amino acid may be generated). It may be used as an open reading frame (ORF) to form target proteins or peptides. The present application also includes RNA and PNA (peptide nucleic acid) with functions equivalent to the exemplified DNA.

The nucleic acid molecule or its fragments in the present application is hybridized with Vip3Aa gene of the present application under strict conditions. Any conventional method for hybridization or amplification of nucleic acid may be used to identify the existence of Vip3 Aa gene of the present application. Under specific condition, nucleic acid molecule or its fragments can be specifically hybridized with other nucleic acid molecules. In the present application, if two nucleic acid molecules can form an antiparallel double-strand nucleic acid structure, it can be said that these two nucleic acid molecules can be specifically hybridized with each other. If two nucleic acid molecules show complete complementation, then one nucleic acid molecule is “complementation” of the other nucleic acid molecule. In the present application, when every nucleotide of a nucleic acid molecule is complementary with corresponding nucleotide of another nucleic acid molecule, then it can be said that these two nucleic acid molecules show “complete complementation”. If two nucleic acid molecules can be hybridized with each other in enough stability, thereby allowing them to anneal and bond with each other at least under conventional “lowly strict” condition, then it can be said that these two nucleic acid molecules are “complementary at the minimum level”. Similarly, if two nucleic acid molecules can be hybridized with each other in enough stability, thereby allowing them to anneal and bond with each other at least under conventional “highly strict” condition, then it can be said that these two nucleic acid molecules have “complementarity”. It is allowable to deviate from complete complementarity as long as the deviation does not completely prevent the two molecules from forming a double-strand structure. In order to allow one nucleic acid molecule can be used as primer or probe, only sufficient complementarity on the sequence needs to be guaranteed so as to form a stable double-strand structure in the adopted specific solvent and at specific salt concentration.

In the present application, the basically homologous sequence is a fragment of nucleic acid molecule. Under highly strict condition, this nucleic acid molecule can be specifically hybridized with the complementary strand of another matched fragment of nucleic acid molecule. The appropriate strict conditions promoting DNA hybridization, such as: trintaking with 6. 0 x NaCl/Sodium citrate (SSC) at about 45 °C, and then washing with 2. 0 χ SSC at 50°C, are known to those skilled in the art. For example, in the washing step, salt concentration can be selected from about 2.Ox SSC 50 °C under lowly strict condition to about 0.2xSSC 50°C under highly strict condition. Besides, the temperature condition in the washing step may be raised from room temperature ~22°C under lowly strict condition to ~65 °C under highly strict condition. Both temperature conditions and salt concentration may be changed. Alternatively, one may remain unchanged and the other variable is changed. Preferably, the strict conditions mentioned in the present application may be: specificly hybridizing with SEQ ID NO: 3 or SEQ ID NO: 4 at 65 °C in 6xSSC 0.5% SDS solution and washing the membrane once with each 2xSSC 0.1%SDS and lx SSC 0.1% SDS .

Therefore, the sequences having insecticidal activity and hybridized with SEQ ID NO: 3 and/or SEQ ID NO: 4 of the present application under strict conditions are included in the present application. These sequences have at least about 40%-50% homology, about 60%, 65% or 70% homology, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology with the sequence of the present application.

The genes and proteins in the present application not only include specific exemplary sequences but also include parts and/or fragments (they are included in full-length protein and/or terminal is deleted), variants, mutants, substitutions (proteins containing substituted amino acid), chimeras and fusion proteins, which maintain the insecticidal activity characteristics of the proteins in specific examples. The “variants” or “variation” refers to the nucleotide sequence coding a same protein or coding equivalent protein with insecticidal activity. The “equivalent protein” refers to the protein with bioactivity against pest Spodoptera litura being same or basically same as that of the proteins claimed.

The “fragments” or “truncatures” of DNA molecules or protein sequences in the present application refer to one part or artificially modified form (for example, the sequences suitable for expression in a plant) of original DNA or protein sequence (nucleotide or amino acid) involved. The length of the foregoing sequences may exsit variable, but it shall be enough to ensure the (coded) proteins are insect toxins.

By using standard technologies, genes may be modified and gene variants can be easily constructed. For example, those skilled in the art know very well the technology for making point mutation. For another example, American patent NO. 5605793 describes a method by using DNA reassembly to generate other molecular diversity after random fracture. Commercial endonuclease may be used to generate fragments of full-length gene, and exonuclease may be used according to a standard procedure. For example, enzymes, such as: Bal3l or site directed mutagenesis, may be used to systematically knock out nucleotides from the end of these genes. Alternatively, multiple restriction endonucleases may be used to acquire the genes with coding active fragments. These active fragments can be directly obtained by using protease.

The present application may derive equivalent proteins and/or genes coding these equivalent proteins from B. t. isolates and/or DNA library. The insecticidal proteins of the present application may be obtained by multiple methods. For example, the antibodies of the insecticidal proteins disclosed and claimed by the present application may be used to identify and separate other proteins from protein mixture. Specially, the antibodies may be produced by proteins most constant and most different from other B.t. proteins. Then by immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) or western blot method, these antibodies are used to exclusively identify equivalent proteins with characteristic activity. The standard procedures in the art may be used to easily prepare the proteins, or equivalent proteins, or the antibodies of the fragements of such proteins disclosed in the present application. Then from microorganisms, the genes coding these proteins may be obtained.

Due to the redundancy of genetic codons, different DNA sequences may code a same amino acid sequence. Generating substitutable DNA sequences coding same or basically same proteins is within the technical level of those skilled in the art. These different DNA sequences are included in the scope of the present application. The “basically same” sequences refer to the sequences with substitution, deletion, addition or insertion of amino acids, but not affecting the insecticidal activity in essence, also including fragments retaining insecticidal activity.

The substitution, deletion or addition of amino acids in the present application is conventional technique in the art. Preferably, such change of amino acid is: small change of characteristics, conservative amino acid substitution not significantly affecting protein folding and/or activity; small deletion, typically deletion of about 1-30 amino acids; small extension of amino or carboxyl terminal, for example: one methionine residue is extended from amino terminal; small linker peptide, for example, about 20-25 residues long.

An example of conservative substitution is substitution occurring in 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 and asparaginate), hydrophobic amino acids (such as: leucine, isoleucine and valine), aromatic amino acids (such as: phenylalanine, tryptophan and tyrosine), and micromolecular amino acids (such as: glycine, alanine, serine, threonine and methionine). Those amino acid substitutions normally not changing specific activity are known to all in the art and have been described for example in ‘Protein” published by N. Neurath and R. L. Hill, Academic Press, 1979. The most popular 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, as well as their reverse interchanges.

It is apparent to those skilled in the art that this substitution may occur outside the regions where can play an important role on molecular functions, and still generate active polypeptides. For the polypeptides of the present application, the amino acid residue needed for its activity and thereby not select to be substituted may be identified by methods known in the art, such as: site directed mutagenesis or alanine scanning mutagenesis (for example, refer to Cunningham and Wells, 1989, Science 244: 1081-1085). The latter technology is to introduce mutation at every residue with positive charges in the molecule and detect the insecticidal activity of the mutated molecule, thereby determining amino acid residue important to the activity of this molecule. The substrate-enzyme interaction site may also be determined through analysis of three-dimensional structure. This three-dimensional structure may be analysed by NMR analysis, crystallography, photoaffinity labeling and other techniques (refer to, for example, 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, Vip3A protein includes without limitation: Vip3Aal, Vip3Afl, Vip3Aall, Vip3Aal9, Vip3Ahl, Vip3Adl, Vip3Ael or Vip3Aa20 protein, or insecticidal fragments or functional regions having at least 70% homology with the amino acid sequence of the foregoing proteins and having insecticidal activity against Spodoptera litura.

Therefore, the amino acid sequences with certain homology with the amino acid sequences shown by sequence 1 and/or 2 are also included in the present application. Typically, the similarity/identity of these sequences with the sequences in the present application is greater than 60%, preferably greater than 75%, more preferably greater than 80%, still more preferably greater than 90%, and may be greater than 95%. Alternatively, the preferred polynucleotides and proteins in the present application may be defined according to a more specific range of identity and/or similarity. For example, there 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 with the sequences shown in the present application.

The regulating sequences in the present application include without limitation: promoter, transit peptides, terminator, enhancer, leader sequence, intron and other regulating sequences which can be operably linked to the Vip3 A protein and Cry type protein.

The promoter is a promoter expressible in plant. The “promoter expressible in plant” refers to the promoter which ensures the coding sequence linked to it is expressed in plant cells. The promoters expressible in plant may be constitutive promoters. The examples of the promoters guiding constitutive expression in plant include without limitation: 35S promoter originating from cauliflower mosaic virus, maize ubi promoter, paddy GOS2 gene promoter and so on. Alternatively, the promoter expressible in plant may be a promoter with tissue specificity. In other words, under the guidance of this promoter, for example PEP carboxylase promoter, the expression level of coding sequences in some tissues of plant such as green tissues is higher than the expression level in other tissues (it may be determined through conventional RNA test). Alternatively, the promoter expressible in plant may be a wound-induced promoter. Wound-induced promoter or promoter guiding the expression mode of wound inducement refers to that when a plant is subjecting to a wound caused by machine or insect gnawing, the expression of the coding sequence under regulation of the promoter is increased significantly compared to normal growth conditions. The examples of wound-induced promoters include without limitation: promoters of potato and tomato protease inhibitor genes (pin I and pin II) and the promoter of maize protease inhibitor genes (MPI).

The transit peptides (also known as secretive signal sequence or guiding sequence) guide transgenic products to specific organelles or cell compartments. To receptor proteins, the transit peptides may be heterogenous. For example, the transit peptide sequence coding chloroplast is used to target chloroplast, or ‘KDEL’ retention sequence is used to target endoplasmic reticulum, or CTPP of lectin gene of barley is used to target vacuole.

The leader sequences include without limitation, leader sequence of small RNA virus, such as: leader sequence of EMCV (5’ noncoding region of encephalomyocarditis virus); leader sequence of potyvirus group, such as: leader sequence of MDMV (maize dwarf mosaic virus); heavy-chain binding protein of human immunoglobulin (BiP); leader sequence of untranslated mRNA of coat protein of alfamovirus (AMV RNA4); leader sequence of tobacco mosaic virus (TMV).

The enhancers include without limitation: cauliflower mosaic virus (CaMV) enhancer, figwort mosaic virus (FMV) enhancer, carnation etched ring virus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer, mirabilis mosaic virus (MMV) enhancer, cestrum yellow leaf curl virus (CmYLCV) enhancer, Multan cotton leaf curl multan virus (CLCuMV), commelina yellow mottle virus (CoYMV) and peanut chlorotic streak virus (PCLSV) enhancer.

For the application of monocotyledon plant, the introns include without limitation: maize hsp70 intron, maize ubiquitin intron, Adh intron 1, sucrose synthase intron or paddy Actl intron. For the application of dicotyledonous plant, the introns include without limitation: CAT-1 intron, pKANNIBAL intron, PIV2 intron and “super ubiquitin” intron.

The terminators may be appropriate polyadenylation signal sequences playing a role in plant and include without limitation: polyadenylation signal sequence originating from nopaline synthase (NOS) gene of Agrobactedum tumefaciens, polyadenylation signal sequence originating from trypsin inhibitor II (pin II) gene, polyadenylation signal sequence originating from pea ssRUBISCO E9 gene and polyadenylation signal sequence originating from a-tubulin gene. “Effective linkage” in the present application denotes the binding of nucleic acid sequences. The binding enables a sequence to provide the functions needed to bound sequences. The “effective linkage” in the present application may be linkage between promoter and interesting sequence so that the transcription of this interesting sequence is controlled and regulated by this promoter. When the interesting sequence codes protein and the expression of the protein is wanted, “effective linkage” denotes linkage between the promoter and the sequence. The linkage mode makes the obtained transcripts be translated efficiently. If the linkage between promoter and coding sequence is fusion of transcripts and the expression of coded protein wants to be realized, establish this kind of linkage, which makes the first translation initiation codon in the obtained transcripts be the initiation codon of the coding sequence. Alternatively, If the linkage between promoter and coding sequence is translation fusion and the expression of the coded protein wants to be realized, establish this kind of linkage, which makes the first translation initiation codon in 5’ untranslated sequence link with the promoter, and the linkage mode makes the relationship between the obtained translation product and the translation ORF coding the protein wanted conform to the reading frame. The nucleic acid sequences which can be “effectively linked” include without limitation: the sequence providing the function of gene expression (i.e.: gene expression elements, such as: promoter, 5’ untranslated region, intron, protein coding region, 3’ untranslated region, polyadenylation site and/or transcription terminator), the sequence providing the function of DNAtransfer and/or integration (i.e.: T-DNAborder sequence, site-specific recombinase recognition site and integrase recognition site), the sequence providing selective function (i.e.: antibiotic resistance marker and biosynthesis gene), the sequence providing the function of scorable marker, the sequence assisting sequence operation in vivo or in vitro (i.e.: multi-joint sequence, site-specific recombination sequence) and the sequence providing the function of replication (i.e.: origin of bacterial replication, autonomously replicating sequence and centromeric sequence).

The “insect killing” or “insect resistant” in the present application means toxic to crop pests, thereby to achieve “controlling” and/or “preventing” of crop pests. Preferably, the “insect killing” or “insect resistant” refers to killing crop pests. More specifically, the target insect is pest Spodoptera litura.

Vip3A protein in the present application is toxic to pest Spodoptera litura. The genomes of the plant in the present application, in particular soybean and maize, contain exogenous DNA, which contains nucleotide sequence coding Vip3A protein. Pest Spodoptera litura contacts with this protein through intaking plant tissues. After the contact, the growth of pest Spodoptera litura is inhibited and death is eventually caused. Inhibition refers to lethality or sub-lethality. Meanwhile, the plant should be normal in morphology and can be cultured by conventional methods in order to be used for consumption and/or generation of products. Further, the plant may basically eliminate the need for chemical or biological insecticides (the chemical or biological insecticides are the insecticides against pest Spodoptera litura targeted by Vip3 A protein).

In plant material, the expression level of insecticidal protein may be detected by various methods described in the art, for example, quantifying mRNA of coded insecticidal protein generated in tissues through applying specific primers, or directly and specially detecting the amount of generated insecticidal protein.

The insecticidal effect of insecticidal protein in plant may be determined by applying different tests. The target insects in the present application are mainly Spodoptera litura.

In the present application, the Vip3A protein may have the amino acid sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 2 in the sequence listing. In addition to including the coding region of Vip3Aprotein, other elements may also be included, such as: the elements coding the selective labeled protein.

Further, the expression cassette containing the nucleotide sequence coding Vip3 A protein of the present application may also be expressed together with at least one gene coding herbicide resistance protein. The herbicide resistance gene includes without limitation: phosphinothricin resistance gene (such as: bar gene and pat gene), phenmedipham resistance gene (such as: pmph gene), glyphosate resistance gene (such as: EPSPS gene), bromoxynil resistance gene, sulfonylurea resistance gene, herbicide dalapon resistance gene, cyanamide resistance gene or glutamine synthetase inhibitor (such as: PPT) resistance gene, thereby obtaining transgenic plant with both high insecticidal activity and herbicide resistance.

In the present application, exogenous DNA is introduced into plant. For example, the gene or expression cassette or recombinant vector coding the Vip3A protein is introduced into plant cells. The conventional transformation methods include without limitation: agrobacterium tumefaciens-mediated transformation, trace emission bombardment, direct ingestion DNA into protoplast, electroporation or silica whisker mediated DNA introduction.

The present application provides a method for controlling pests, which has the following advantages: 1. Control through internal cause: The prior art controls the harm of pest Spodoptera litura mainly through external action, i.e.: external cause, for example, agricultural control, chemical control and physical control. While the present application controls pest Spodoptera litura through generating Vip3A protein which can kill Spodoptera litura inside plant, i.e.: controls pest Spodoptera litura through internal cause. 2. No pollution and no residue: The chemical control method used in prior art plays certain role in controlling the harm of pest Spodoptera litura, but in the same time, it also causes pollution, destruction and residue to human, livestock and farmland ecosystem. Using the method for controlling pest Spodoptera litura provided in the present application may eliminate the foregoing bad consequences. 3. Control throughout all growth period: All the methods for controlling pest Spodoptera litura used in prior art are staged, while the present application protects the plant throughout all growth period so that transgenic plant (Vip3 A protein) can be free from the encroachment of Spodoptera litura from sprouting, growth and till blooming and fruit. 4. Control over whole plant: The method for controlling pest Spodoptera litura used in the prior art mostly are localised, for example foliage spray, while the present application protects whole plant, for example, the leaves, stalks, tassels, female spikes, anthers, filaments, fruits and so on of transgenic plant (Vip3A protein) all may resist the harm of Spodoptera litura. 5. Effect stability: Both agricultural control method and physical control method used in the prior art need to utilize environmental conditions to control pests and have many variable factors. The present application makes the Vip3A protein be expressed inside the plant and effectively avoids the defect of unstable environmental conditions. Further, the control effect of transgenic plant (Vip3A protein) provided by the present application is stable and consistent in different places, different time and different genetic backgrounds. 6. Simple, convenient and economical: The physical control method in the prior art havs certain difficulties on agricultural production and operation, while the present application only needs to plant the transgenic plant which can express Vip3 A protein and does not need to take other measures, thereby saving a large amount of human, material and financial resources. 7. Thorough effect: The method for controlling pest Spodoptera litura used in the prior art does not have a thorough effect and only plays a role of mitigation, while transgenic plant (Vip3 A protein) provided by the present application may cause mass death of newly hatched larvae of Spodoptera litura and significantly inhibit the growth of the small portion survived larvae. 3 days later, larvae are basically in a newly hatching state or a state between new hatching and negative control, all suffer maldevelopment and have stopped development. The transgenic plant largely suffer mild damage.

The technical solution of the method for controlling the pest provided by the present application is further described below by referring to specific examples.

Examples

Example 1: Obtain and synthesize Vip3 A gene 1. Obtain Vip3 A nucleotide sequence

The amino acid sequence of Vip3Aa-01 insecticidal protein (789 amino acids), is shown by SEQ ID NO: 1 in the sequence listing; Vip3Aa-01 nucleotide sequence encoding corresponding amino acid sequence (789 amino acids) of the Vip3Aa-01 insecticidal protein (2370 nucleotides), is shown by SEQ ID NO: 3 in the sequence listing. The amino acid sequence of Vip3Aa-02 insecticidal protein (789 amino acids), is shown by SEQ ID NO: 2 in the sequence listing; Vip3Aa-02 nucleotide sequence encoding correspondin amino acid sequence (789 amino acids) of the Vip3Aa-02 insecticidal protein (2370 nucleotides), is shown by SEQ ID NO: 4 in the sequence listing. 2. Obtain Cry lAand CrylF nucleotide sequences

Cry lAb nucleotide sequence encoding the amino acid sequence (818 amino acids) of Cry lAb insecticidal protein (2457 nucleotides), is shown by SEQ ID NO: 5 in the sequence listing; CrylFa nucleotide sequence encoding the amino acid sequence (605 amino acids) of CrylFa insecticidal protein (1818 nucleotides), is shown by SEQ ID NO: 6 in the sequence listing. 3. Synthesize the foregoing nucleotide sequences

The Vip3Aa-01 nucleotide sequence (as shown by SEQ ID NO: 3 in the sequence listing), the Vip3Aa-02 nucleotide sequence (as shown by SEQ ID NO: 4 in the sequence listing), the Cry lAb nucleotide sequence (as shown by SEQ ID NO: 5 in the sequence listing) and the CrylFa nucleotide sequence (as shown by SEQ ID NO: 6 in the sequence listing) were synthesized by GenScript (Nanjing) Co., Ltd.; 5’-end of the synthesized Vip3Aa-01 nucleotide sequence (SEQ ID NO: 3) is also linked with Seal restriction site, and 3’-end of the Vip3Aa-01 nucleotide sequence (SEQ ID NO: 3) is also linked with Spel restriction site; 5’-end of the synthesized Vip3Aa-02 nucleotide sequence (SEQ ID NO: 4) is also linked with Seal restriction site, and 3’-end of the Vip3Aa-02 nucleotide sequence (SEQ ID NO: 4) is also linked with Spel restriction site; 5’-end of the synthesized Cry lAb nucleotide sequence (SEQ ID NO: 5) is also linked with Ncol restriction site, and 3’-end of the Cry lAb nucleotide sequence (SEQ ID NO: 5) is also linked with Spel restriction site; 5’-end of the synthesized CrylFa nucleotide sequence (SEQ ID NO: 6) is also linked with AscI restriction site, and 3’-end of the CrylFa nucleotide sequence (SEQ ID NO: 6) is also linked with BamHI restriction site.

Example 2: Constructing recombinant expression vector and transforming agrobacteria with the recombinant expression vector 1. Construct recombinant cloning vector containing Vip3 A gene

The synthesized Vip3Aa-01 nucleotide sequence is linked into cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600). The operating steps are conducted according to the pGEM-T vector manual provided by Promega, so as to obtaine a recombinant cloning vector DBN01-T. Its construction flow diagram is shown in FIG. 1 (wherein, Amp stands for ampicillin resistance gene; fl stands for replication origin of phage fl; LacZ stands for the initiation codon of LacZ; SP6 stands for SP6 RNA polymerase promoter; T7 stands for T7 RNA polymerase promoter; Vip3Aa-01 stands for Vip3Aa-01 nucleotide sequence (SEQ ID NO: 3); MCS stands for multiple cloning site).

Then the recombinant cloning vector DBN01-T is used to transform Escherichia coli T1 competent cells (Transgen, Beijing, China, CAT: CD501) by heat shock method. The heat shock conditions are: keeping 50pl of Escherichia coli T1 competent cells and 10μ1 of plasmid DNA (recombinant cloning vector DBN01-T) in a 42°C water bath for 30s; shaking and cultivating at 37°C for 1 hour (shaking on a shaking table at lOOrpm), and growing overnight on ampicillin (lOOmg/L) LB plate (tryptone lOg/L, yeast extract 5g/L, NaCl lOg/L and agar 15g/L; adjusting pH to 7.5 by NaOH) coating with IPTG (isopropyl-P-D-thiogalactoside) and X-gal (5-bromine-4-chlorine-3-indol-P-D- galactoside) on the surface. Picking up white colonies, and cultivating them overnight in LB liquid culture (tryptone lOg/L, yeast extract 5g/L, NaCl lOg/L and ampicillin lOOmg/L; adjusting pH to 7.5 by NaOH) at 37°C. Extracting its plasmid by alkaline method: centrifuging bacteria solution at 12000rpm for lmin, removing the supemate, suspending the precipitate thalli with ΙΟΟμΙ solution I precooled by ice (25mM Tris-HCl, lOmM EDTA (Ethylene Diamine Tetraacetic Acid), 50mM glucose, pH8. 0); adding 150μ1 newly prepared solution II (0.2M NaOH, 1% SDS (lauryl sodium sulfate)), turning the tube up and down for 4 times, mixing them and placing the mixture on ice for 3-5min: adding 150μ1 ice-cooled solution III (4M potassium acetate, 2M acetic acid), immediately thoroughly mixing them well, placing the obtained solution on ice for 5-10min; centrifuging it at 4°C and 12000rpm for 5min, adding 2X volumes of absolute elthyl alcohol into the supemate, mixing them well, and then keeping the obtained solution at room temperature for 5min; centrifuging it at 4°C and 12000rpm for 5min, discarding the supernate, washing the precipitate with 70% (V/V) ethanol and then drying it in the air; adding 30μ1 of TE (lOmM Tris-HCl, ImM EDTA, pH8. 0) containing RNase (20pg/ml) to dissolve the precipitate; keeping it in a 37°C water bath for 30min, and digesting RNA; keeping it at -20 °C for future use.

After that the extracted plasmid is digested and identified by EcoRV and SphI, positive clone is validated through sequencing. The result indicates that the Vip3Aa-01 nucleotide sequence inserted in the recombinant cloning vector DBN01-T is the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, i.e.: Vip3Aa-01 nucleotide sequence is correctly inserted.

According to the foregoing method for constructing recombinant cloning vector DBN01-T, the synthesized Vip3Aa-02 nucleotide sequence is linked onto cloning vector pGEM-T to obtain recombinant cloning vector DBN02-T, wherein Vip3Aa-02 is Vip3Aa-02 nucleotide sequence (SEQ ID NO: 4). Enzyme digestion and sequencing validate that the Vip3Aa-02 nucleotide sequence in the recombinant cloning vector DBN02-T is correctly inserted.

According to the foregoing method for constructing recombinant cloning vector DBN01-T, the synthesized Cry 1 Ab nucleotide sequence is linked onto cloning vector pGEM-T to obtain recombinant cloning vector DBN03-T, wherein Cry lAb is Cry lAb nucleotide sequence (SEQ ID NO: 5). Enzyme digestion and sequencing validate that the Cry lAb nucleotide sequence in the recombinant cloning vector DBN03-T is correctly inserted.

According to the foregoing method for constructing recombinant cloning vector DBN01-T, the synthesized CrylFa nucleotide sequence is linked onto cloning vector pGEM-T to obtain recombinant cloning vector DBN04-T, wherein CrylFa is CrylFa nucleotide sequence (SEQ ID NO: 6). Enzyme digestion and sequencing validate that the CrylFa nucleotide sequence in the recombinant cloning vector DBN04-T is correctly inserted. 2. Construct recombinant expression vector containing Vip3 A gene

Restriction endonuclease Seal and Spel are used to digest recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (can be provided by CAMBIA organization)) respectively. The cut fragments of Vip3Aa-01 nucleotide sequence are inserted into the sites between Seal and Spel of expression vector DBNBC-01. Applying a conventional digestion method to construct vector is well known to those skilled in the art. Construct recombinant expression vector DBN100066. The construction flow is shown in FIG. 2 (Kan: kanamycin gene; RB: right border; Ubi: maize ubiquitin gene promoter (SEQ ID NO: 7); Vip3Aa-01; Vip3Aa-01 nucleotide sequence (SEQ ID NO: 3); Nos: terminator of nopaline synthase gene (SEQ ID NO: 8); PMI: phosphomannose isomerase gene (SEQ ID NO: 9); LB: left border).

The recombinant expression vector DBN100066 is used to transform Escherichia coli T1 competent cells by heat shock method. The heat shock conditions are: keeping 50μ1 of Escherichia coli T1 competent cells and 10μ1 of plasmid DNA (recombinant expression vector DBN100066) in a 42 °C water bath for 30s; shaking and cultivating at 37°C for 1 hour (shaking on a shaking table at lOOrpm); then culturing on an LB solid-plate (tryptone lOg/L, yeast extract 5g/L, NaCl lOg/L, agar 15g/L; adjusting pH to 7.5 by NaOH) containing 50mg/L kanamycin at 37°C for 12h, picking up white colonies, and culturing them overnight in an LB liquid medium (tryptone lOg/L, yeast extract 5g/L, NaCl lOg/L and kanamycin 50mg/L; adjusting pH to 7.5 by NaOH) at 37°C; Extracting its plasmid by alkaline method. Digest the extracted plasmid by restriction endonuclease Seal and Spel and then identify it, sequence and identify the positive clones. The result indicates that the nucleotide sequence of recombinant expression vector DBN100066 between sites Seal and Spel is the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, i.e.: Vip3Aa-01 nucleotide sequence.

According to the foregoing method for constructing recombinant expression vector DBN100066, the Vip3Aa-01 nucleotide sequence and Cry lAb nucleotide sequence cut off from recombinant cloning vectors DBN01-T and DBN03-T digested by Seal and Spel, Ncol and Spel are inserted into expression vector DBNBC-01 to obtain recombinant expression vector DBN100003. Digestion and sequencing validate that the nucleotide sequences in recombinant expression vector DBN100003 contain the nucleotide sequences shown by SEQ ID NO: 3 and SEQ ID NO: 5 in the sequence listing, i.e.: Vip3Aa-01 nucleotide sequence and Cry 1 Ab nucleotide sequence, which may link the Ubi promoter and Nos terminator.

According to the foregoing method for constructing recombinant expression vector DBN100066, the Vip3Aa-02 nucleotide sequence and CrylFa nucleotide sequence cut off from recombinant cloning vectors DBN02-T and DBN04-T digested by Seal and Spel, AscI and BamHI are inserted into expression vector DBNBC-01 to obtain recombinant expression vector DBN100276. Digestion and sequencing validate that the nucleotide sequences in recombinant expression vector DBN100276 contain nucleotide sequences shown by SEQ ID NO: 4 and SEQ ID NO: 6 in the sequence listing, i.e.: Vip3Aa-02 nucleotide sequence and CrylFa nucleotide sequence, which may link the Ubi promoter and Nos terminator.

According to the foregoing method for constructing recombinant expression vector DBN100066, restriction endonuclease Seal and Spel are used to digest recombinant cloning vector DBN01-T and expression vector DBNBC-02 (vector backbone: pCAMBIA2301 (can be provided by CAMBIA organization)) respectively. The cut fragments of Vip3Aa-01 nucleotide sequence are inserted into the sites between Seal and Spel of expression vector DBNBC-02. Utilizing a conventional digestion method to construct vector is well known to those skilled in the art. Construct recombinant expression vector DBN100002. The construction flow is shown in FIG. 3 (Kan: kanamycin gene; RB: right border; Ubi: maize Ubiquitin gene promoter (SEQ ID NO: 7): Vip3Aa-01: Vip3Aa-01 nucleotide sequence (SEQ ID NO: 3); Nos: terminator of nopaline synthase gene (SEQ ID NO: 8); PAT: glufosinate acetyltransferase gene (SEQ ID NO: 22); LB: left border).

According to the foregoing method for constructing recombinant expression vector DBN100002, the Vip3Aa-01 nucleotide sequence and Cry lAb nucleotide sequence cut off from recombinant cloning vector DBN01-T and DBN03-T digested by Seal and Spel, Ncol and BamHI are inserted into expression vector DBNBC-02 to obtain recombinant expression vector DBN100321. Digestion and sequencing validate that the nucleotide sequences in recombinant expression vector DBN100321 contain nucleotide sequences shown by SEQ ID NO: 3 and SEQ ID NO: 5 in the sequence listing, i.e.: Vip3Aa-01 nucleotide sequence and Cry 1 Ab nucleotide sequence, which may link the Ubi promoter and Nos terminator.

According to the foregoing method for constructing recombinant expression vector DBN100002, the Vip3Aa-02 nucleotide sequence and CrylFa nucleotide sequence cut off from recombinant cloning vector DBN02-T and DBN04-T digested by Seal and Spel, AscI and BamHI are inserted into expression vector DBNBC-02 to obtain recombinant expression vector DBN100013. Digestion and sequencing validate that the nucleotide sequences in recombinant expression vector DBN100013 contain the nucleotide sequences shown by SEQ ID NO: 4 and SEQ ID NO: 6 in the sequence listing, i.e.: Vip3Aa-02 nucleotide sequence and CrylFa nucleotide sequence, which may link the Ubi promoter and Nos terminator. 3. Transform agrobacteria with recombinant expression vector

The correctly constructed recombinant expression vector DBN100066, DBN100003, DBN100276, DBN100002, DBN100321 and DBN100013 are transformed into agrobacteria LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method. The transformation conditions are: placing 100pL of agrobacteria LBA4404 and 3pL of plasmid DNA (recombinant expression vector) in liquid nitrogen for 10 min, and a 37°C warm bath for 10 min; inoculating the transformed agrobacteria LBA4404 to a LB test tube, culturing it at 28 °C and 200rpm for 2 h, coating it onto an LB plate containing 50mg/L Rifampicin and lOOmg/L Kanamycin until positive monoclone grow out, picking up the monoclone to culture it and extract plasmid thereof, after using restriction endonuclease Styl and Aatll to digest recombinant expression vector DBN100066, DBN100003, DBN100276, DBN100002, DBN100321 and DBN100013, then validate the digestion. The results indicate that the structures of recombinant expression vector DBN100066n DBN100003n DBN100276n DBN100002n DBN100321 andDBN100013 are completely correct.

Example 3: Obtain and validate the maize plant transformed with Vip3 A gene 1. Obtain the maize plant transformed with Vip3 A gene

According to conventional agrobacteria infection method, the aseptically cultured young embryos of maize breed Zong31 (Z31) and the agrobacteria in Item 3 of Example 2 are cocultured so as to transfer the T-DNA (including the promoter sequence of maize Ubiquitin gene, Vip3Aa-01 nucleotide sequence, Vip3Aa-02 nucleotide sequence, Cry lAb nucleotide sequence, CrylFa nucleotide sequence, PMI gene and Nos terminator sequence) in recombinant expression vector DBN100066, DBN100003 and DBN100276 constructed in Item 2 of Example 2 into maize chromosome complement and obtain maize plant transformed with Vip3Aa-01 nucleotide sequence, maize plant transformed with Vip3Aa-01-CrylAb nucleotide sequence, maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence. Meanwhile, wild maize plants are used as controls.

As for agrobacterium mediated maize transformation, simply speaking, separat immature young embryos from maize and allow contact between the young embryos and agrobacteria suspension, wherein agrobacteria can transfer Vip3Aa-01 nucleotide sequence, Vip3Aa-01-CrylAb nucleotide sequence and/or Vip3Aa-02-CrylFa nucleotide sequence to at least one cell of one of the embryos (step 1: infection step). In this step, young embryos preferably infect into agrobacteria suspension (OD660=0.4-0.6, infection medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2,4-di chi or ophenoxy acetic acid (2, 4-D) lmg/L, pH5.3)) in order to initiate inoculation. The young embryos and agrobacteria are co-cultured for a period of time (3 days) (step 2: coculture step). Preferably, after the infection step, the young embryos are cultured on a solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose lOg/L, acetosyringone (AS) lOOmg/L, 2, 4- dichlorophenoxyacetic acid (2, 4-D) lmg/L, agar 8g/L, pH5.8); after this co-culture stage, there may be a selective “recovery” step. In the “recovery” step, the recovery medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 2, 4- dichlorophenoxyacetic acid (2, 4-D) lmg/L, agar 8g/L, pH5.8) contains at least one antibiotic (cephalosporin) known to inhibit the growth of agrobacteria, and no selective agent of plant transformant is added (step 3: recovery step). Preferably, young embryos are cultured on a solid medium containing antibiotic, but no selective agent, to eliminate agrobacteria and provide a recovery period for the infected cells. Then, the inoculated young embryos are cultured on a culture medium containing selective agent (mannose) and select the growing transformed calli (step 4: selection step). Preferably, the young embryos are cultured on a screening solid medium containing selective agent (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 5g/L, mannose 12.5 g/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) lmg/L, agar 8g/L, pH5.8), resulting in selective growth of the transformed cells. Then, calli are regenerated into plant (step 5: regeneration step). Preferably, the calli growing on a medium containing selective agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) to regenerate plant.

The resistant calli obtained from screening are transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyladenine 2mg/L, mannose 5g/L, agar 8gm, pH5.8), and cultured it to differentiate at 25°C. The seedlings obtained from differentiation are transferred to the MS rooting culture (MS salt 2.15g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, indol-3-acetic acid lmg/L, agar 8g/L, pH5.8), and cultured at 25 °C till a height of about 10cm, then transferred to a greenhouse and cultured till fructification. In the greenhouse, they are cultured at 28 °C for 16 h and then at 20 °C for 8 h each day. 2. Use TaqMan to validate maize plant transformed with Vip3 A gene

About lOOmg of the leaves are taken from the maize plant transformed with Vip3Aa-01 nucleotide sequence, the maize plant transformed with Vip3Aa-01-CrylAb nucleotide sequence and the maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence respectively as samples. Their genome DNAs are extracted by Qiagen DNeasy Plant Maxi Kit. The copy numbers of Vip3A gene, Cryl A gene and CrylF gene are detected by Taqman probe fluorescent quantitation PCR method. Meanwhile, wild maize plants are used as controls. Detection and analysis are conducted according to the above method. The experiment is repeated three times and their average value is adopted.

The copy numbers of Vip3Agene, Cryl A gene and CrylF gene are detected by the following method:

Step 11: Weigh 100 mg of the leaves of the maize plant transformed with Vip3Aa-01 nucleotide sequence, the maize plant transformed with Vip3Aa-01-Cry lAb nucleotide sequence, the maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence and wild maize plant respectively, grind them with liquid nitrogen into homogenate in a mortar respectively, and do the above step three times for each sample.

Step 12: Use Qiagen DNeasy Plant Mini Kit to extract the genome DNAs of the foregoing samples. For concrete method, please refer to product manual thereof.

Step 13: Use NanoDrop 2000 (Thermo Scientific) to determine genome DNAs concentration of the foregoing samples.

Step 14: Adjust the genome DNAs concentration of the foregoing samples to a same concentration value. The range of the concentration values is 80-100ng/pl;

Step 15: Identify the copy number of each sample by Taqman probe fluorescent quantitation PCR method, use the identified samples with known copy number as standard substances, and the samples of wild maize plant as controls. Do the above step three times for each sample and adopt the average value. The sequences of primers and probe for fluorescent quantitation PCR are:

The following primers and probes are used to detect Vip3Aa-01 nucleotide sequence:

Primer 1 (VF1): ATTCTCGAAATCTCCCCTAGCG as shown by SEQ ID NO: 10 in the sequence listing;

Primer 2 (VR1): GCTGCCAGTGGATGTCCAG as shown by SEQ ID NO: 11 in the sequence listing;

Probe 1 (VP1): CTCCTGAGCCCCGAGCTGATTAACACC as shown by SEQ ID NO: 12 in the sequence listing;

The following primers and probes are used to detect Vip3 Aa-02 nucleotide sequence:

Primer 3 (VF2): ATTCTCGAAATCTCCCCTAGCG as shown by SEQ ID NO: 13 in the sequence listing;

Primer 4 (VR2): GCTGCCAGTGGATGTCCAG as shown by SEQ ID NO: 14 in the sequence listing;

Probe 2 (VP2): CTCCTGAGCCCCGAGCTGATTAACACC as shown by SEQ ID NO: 15 in the sequence listing;

The following primers and probes are used to detect Cryl Ab nucleotide sequence:

Primer 5 (CF1): CGAACTACGACTCCCGCAC as shown by SEQ ID NO: 16 in the sequence listing;

Primer 6 (CR1): GTAGATTTCGCGGGTCAGTTG as shown by SEQ ID NO: 17 in the sequence listing;

Probe 3 (CPI): CTACCCGATCCGCACCGTGTCC as shown by SEQ ID NO: 18 in the sequence listing;

The following primers and probes are used to detect CrylFa nucleotide sequence:

Primer 7 (CF2): CAGTCAGGAACTACAGTTGTAAGAGGG as shown by SEQ ID NO: 19 in the sequence listing;

Primer 8 (CR2): ACGCGAATGGTCCTCCACTAG as shown by SEQ ID NO: 20 in the sequence listing;

Probe 4 (CP2): CGTCGAAGAATGTCTCCTCCCGTGAAC as shown by SEQ ID NO: 21 in the sequence listing; PCR reaction system:

Jump Start™ Taq Ready Mix™ (Sigma) 1 Opl 50xprimer/probe mixture lpl

Genome DNA 3μ1

Water (ddH20) 6μ1

The 50xprimer/probe mixture contains 45μ1 of each primer at concentration of ImM, 50μ1 of ΙΟΟμΜ probe and 860μ1 of 1 xTE buffer solution and is stored in an amber test tube at 4°C. PCR reaction conditions:

Step Temperature Time 21 95 °C 5 min 22 95 °C 30 sec 23 60 °C 1 min 24 Go back to step 22, repeat it 40 times

Analyze data using SDS2.3 software (Applied Biosystems).

The experimental results indicate that Vip3Aa-01 nucleotide sequence, Vip3Aa-01-CrylAb nucleotide sequence and Vip3Aa-02-CrylFa nucleotide sequence all have been integrated to the chromosome complements of the detected maize plant, and the maize plant transformed with Vip3Aa-01 nucleotide sequence, the maize plant transformed with Vip3Aa-01-CrylAb nucleotide sequence and the maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence all have obtained transgenic maize plant containing a single copy of Vip3 A gene, CrylAgene and/or CrylF gene.

Example 4: Detect the insecticidal effect of transgenic maize plant

Detect the insecticidal effect aginst Spodoptera litura of the maize plant transformed with Vip3Aa-01 nucleotide sequence, the maize plant transformed with Vip3Aa-01-CrylAb nucleotide sequence, the maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence, wild maize plant and non-transgenic maize plant as identified by Taqman.

Fresh leaves (interior leaves) are taken from the maize plant transformed with Vip3Aa-01 nucleotide sequence, the maize plant transformed with Vip3Aa-01-CrylAb nucleotide sequence, the maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence, wild maize plant and non-transgenic maize plant as identified by Taqman (V3-V4 stages) respectively and washed with sterile water. The water on the leaves is sucked dry by gauze. Then the veins of the maize leaves are removed and the leaves are cut into long strips of about lcmx4cm. Two leaf strips are put on the filter paper at the bottom of a round plastic culture disk. The filter paper is moistened with distilled water. 10 artificially fed Pest Spodoptera lituras (newly hatched larvae) are put into each culture dish. The culture dishes with pests are covered and then put into a square box with wet gauze at its bottom and rest for 3 days under the conditions of 26-28°C, RH 70%-80% and photoperiod (light/dark) 16: 8. Based on three indexes: development progress and mortality of Spodoptera litura larvae and leaf damage rate, the total score of resistance is obtained: Total score= 100/mortality+[ 100/mortality+90/(number of newly hatched larvae/ total number of inoculating larvae)+60x(number of newly hatched larvae - number of pests in negative controls/total number of inoculating larvae)+10x(number of pests in negative controls/total number of inoculating larvae)]+100x(l-leaf damage rate). There are 3 strains (SI, S2 and S3) transformed with Vip3Aa-01 nucleotide sequence, 3 strains (S4, S5 and S6) transformed with Vip3Aa-01-CrylAb nucleotide sequence, 3 strains (S7, S8 and S9) transformed with Vip3Aa-02-CrylFa nucleotide sequence, a strain identified by Taqman to be non-transgenic (NGM1) and a wild strain (CK1); three plant are selected from each strain to do test and each plant is tested six times. The results are shown in Table 1 and FIG. 4.

Table 1 Results of insecticidal experiments of transgenic maize plant inoculated with Spodoptera litura

The results in Table 1 indicate: the total scores of the maize plant transformed with Vip3Aa-01 nucleotide sequence, the maize plant transformed with Vip3Aa-01-CrylAb nucleotide sequence and the maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence in bioassay all reach full mark 300 points; in comparison, the total scores of non-transgenic maize plant identified by Taqman and wild maize plant in bioassay are around 15 points in general.

The results in FIG. 4 indicate: compared with wild maize plant, the controlling effects on newly hatched larvae of the maize plant transformed with Vip3Aa-01 nucleotide sequence, the maize plant transformed with Vip3Aa-01-CrylAb nucleotide sequence and the maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence are nearly 100%, and the leaves of maize plant transformed with Vip3Aa-01 nucleotide sequence, the maize plant transformed with Vip3Aa-01-CrylAb nucleotide sequence and the maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence almost have no damage.

Thus it is proved that the maize plant transformed with Vip3Aa-01 nucleotide sequence, the maize plant transformed with Vip3Aa-01-CrylAb nucleotide sequence and the maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence all show high activity against Spodoptera litura and this activity is enough to generate harmful effect on the growth of Spodoptera litura, so as to control it.

Example 5: Obtain and validate the soybean plant transformed with Vip3 A gene 1. Obtain the soybean plant transformed with Vip3 A gene

According to conventional agrobacteria infection method, the aseptically cultured cotyledonary node tissues of soybean breed ZHONGHUANG 13 and the agrobacteria in Item 3 of Example 2 are co-cultured so as to transfer the T-DNA (including the promoter sequence of maize Ubiquifin gene, Vip3Aa-01 nucleotide sequence, Vip3Aa-02 nucleotide sequence, Cry lAb nucleotide sequence, CrylFa nucleotide sequence, PAT gene and Nos terminator sequence) in recombinant expression vector DBN100002, DBN100321 and DBN100013 constructed in Item 2 of Example 2 into soybean chromosome complement and obtain soybean plant transformed with Vip3Aa-01 nucleotide sequence, soybean plant transformed with Vip3Aa-01-CrylAb nucleotide sequence and soybean plant transformed with Vip3Aa-02-CrylFa nucleotide sequence. Meanwhile, wild soybean plants are used as controls.

As for agrobacterium mediated soybean transformation, simply speaking, germinate mature soybean seeds on a soybean germination medium (B5 salt 3.1g/L, B5 vitamin, sucrose 20g/L, agar 8g/L, pH5.6), inoculate the seeds on the germination medium and culture them under the following conditions: 25±1°C temperature; photoperiod (light/dark) 16/8h. After 4-6 days’ germination, selecte swollen sterilized soybean seedlings at bright green cotyledonary nodes and cut off the hypocotyledonary axis from the location 3-4mm below the cotyledonary node, the cotyledon is cut open longitudinally and apical bud, lateral bud and seed root are removed. The back of a scalpel is used to wound the cotyledonary node. Allow Agrobacteria suspension to contact with the wounded cotyledonary node tissues. The agrobacteria has the ability to transfer the sequences to the wounded cotyledonary node tissues (step 1: infection step). In this step, cotyledonary node tissues are preferably infected into agrobacteria suspension (OD660=0.5-0.8, infection medium (MS salt 2.15g/L, B5 vitamin, sucrose 20g/L, glucose lOg/L, acetosyringone (AS) 40mg/L, 2-morpholineethanesulfonic acid (MES) 4g/L, zeatin (ZT) 2mg/L, pH5.3) to initiate inoculation. Cotyledonary node tissues and agrobacteria are co-cultured for a period of time (3 days) (step 2: co-culture step). Preferably, after this infection step, cotyledonary node tissues are cultured on a solid medium (MS salt 4.3g/L, B5 vitamin, sucrose 20g/L, glucose lOg/L, 2-morpholineethanesulfonic acid (MES) 4g/L, zeatin 2mg/L, agar 8g/L, pH5.6). After this co-culture stage, there may be a selective “recovery” step. In the “recovery” step, the recovery medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholineethanesulfonic acid (MES) lg/L, sucrose 30g/L, zeatin (ZT) 2mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid lOOmg/L, aspartic acid lOOmg/L, pH5.6) contains at least one antibiotic (cephalosporin) known to inhibit the growth of agrobacteria, and no selective agent of plant transformant is added (step 3: recovery step). Preferably, the tissue blocks regenerated by the cotyledonary node are cultured on a solid medium containing antibiotic, but no selective agent, to eliminate agrobacteria and provide a recovery period for the infected cells. Then, the tissue blocks regenerated by the cotyledonary node are cultured on a culture medium containing selective agent (phosphinothricin) and select the growing transformed calli (step 4: selection step). Preferably, the tissue blocks regenerated by the cotyledonary node are cultured on a screening solid medium containing selective agent (B5 salt 3.lg/L, B5 vitamin, 2-morpholineethanesulfonic acid (MES) lg/L, sucrose 30g/L, 6-benzyladenine (6-BAP) lmg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid lOOmg/L, aspartic acid lOOmg/L, phosphinothricin 6mg/L, pH5.6), resulting in selective growth of the transformed cells. Then, the transformed cells regenerate into plant (step 5: regeneration step). Preferably, the tissue blocks regenerated by the cotyledonary node grown on a culture medium containing selective agent are cultured on a solid medium (B5 differentiation medium and B5 rooting culture ) to regenerate plant.

The resistant tissue blocks obtained from screening are transferred to the B5 differentiation medium (B5 salt 3. lg/L, B5 vitamin, 2-morpholineethanesulfonic acid (MES) lg/L, sucrose 30g/L, zeatin (ZT) lmg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin lmg/L, auxin lmg/L, phosphinothricin 6mg/L, pH5.6) and cultured them to differentiate at 25 °C. The seedlings obtained from differentiation are transferred to the B5 rooting culture (B5 salt 3. lg/L, B5 vitamin, 2-morpholineethanesulfonic acid (MES) lg/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indol-3-butyric acid (IBA) lmg/L), and cultured on the rooting medium at 25 °C till a height of about 10cm, then transferred to a greenhouse and cultured till fructification. In the greenhouse, they are cultured at 26 °C for 16 h and then at 20 °C for 8 h each day. 2. Use TaqMan to validate soybean plant transformed with Vip3 A gene

About lOOmg of the leaves are taken from the soybean plant transformed with Vip3Aa-01 nucleotide sequence, the soybean plant transformed with Vip3Aa-01-CrylAb nucleotide sequence and the soybean plant transformed with Vip3Aa-02-CrylFa nucleotide sequence respectively as samples. Their genome DNAs are extracted by Qiagen DNeasy Plant Maxi Kit. The copy numbers of Vip3A gene, Cryl A gene and CrylF gene are detected by Taqman probe fluorescent quantitation PCR method. Meanwhile, wild soybean plant are used as controls. Detection and analysis are conducted according to the foregoing method for using TaqMan to validate the maize plant transformed with Vip3A gene in Item 2 of Example 3.

The experiment is repeated three times and their average value is adopted.

The experimental results indicate Vip3Aa-01 nucleotide sequence, Vip3Aa-01-CrylA nucleotide sequence and Vip3Aa-02-CrylFa nucleotide sequence all have been integrated into the chromosome complements of the detected soybean plant, and the soybean plant transformed with Vip3Aa-01 nucleotide sequence, the soybean plant transformed with Vip3Aa-01-Cryl Ab nucleotide sequence and the soybean plant transformed with Vip3Aa-02-CrylFa nucleotide sequence all have obtained transgenic soybean plant containing a single copy of Vip3Agene, Cry 1A gene and/or CrylF gene.

Example 6: Detect the insecticidal effect of transgenic soybean plant

Detect insecticidal effect aginst Spodoptera litura of the soybean plant transformed with Vip3Aa-01 nucleotide sequence, the soybean plant transformed with Vip3Aa-01-CrylAb nucleotide sequence, the soybean plant transformed with Vip3Aa-02-CrylFa nucleotide sequence, wild soybean plant and non-transgenic soybean plant as identified by Taqman.

Fresh leaves are taken from the soybean plant transformed with Vip3Aa-01 nucleotide sequence, the soybean plant transformed with Vip3Aa-01-CrylAb nucleotide sequence, the soybean plant transformed with Vip3Aa-02-CrylFa nucleotide sequence, wild soybean plant and non-transgenic soybean plant as identified by Taqman (three leaves stage) respectively and washed with sterile water. The water on the leaves is sucked dry by gauze. Meanwhile the leaves are cut into squares of about 2cmx2cm. A cut square leaf is put on the filter paper at the bottom of a round plastic culture disk. The filter paper is moistened with distilled water. 10 artificially fed Pest Spodoptera lituras (newly hatched larvae) are put into each culture dish. The culture dishes with pests are covered and then put into a square box with wet gauze at its bottom and rest for 3 days under the conditions of 26-28 °C, RH 70%-80% and photoperiod (light/dark) 16: 8. Based on three indexes: development progress and mortality of Spodoptera litura larvae and leaf damage rate, the total score of resistance is obtained: Total score= 100/mortal ity+[100/mortality+90/ (number of newly hatched larvae/total number of inoculating larvae)+60/(number of newly hatched larvae - number of pests in negative controls/total number of inoculating larvae)+l Ox (number of pests in negative controls/total number of inoculating larvae)]+100x(l-leaf damage rate). There are 3 strains (S10, Sll and S12) transformed with Vip3Aa-01 nucleotide sequence, 3 strains (S13, S14 and S15) transformed with Vip3Aa-01-CrylAb nucleotide sequence, 3 strains (S16, S17 and SI8) transformed with Vip3Aa-02-CrylFa nucleotide sequence, a strain identified by Taqman to be non-transgenic (NGM2) and a wild strain (CK2); three plant are selected from each strain to do test and each plant is tested six times. The results are shown in Table 2 and FIG. 5.

Table 2 Results of insecticidal experiments of transgenic soybean plant inoculated with Spodoptera litura

The results in Table 2 indicate: the total scores of the soybean plant transformed with Vip3Aa-01 nucleotide sequence, the soybean plant transformed withVip3Aa-01-CrylAb nucleotide sequence and the soybean plant transformed with Vip3Aa-02-CrylFa nucleotide sequence in bioassay are all around full mark 300 points; in comparison, the total scores of non-transgenic soybean plant as identified by Taqman and wild soybean plant in bioassay are around 50 points in general.

The results in FIG. 5 indicate: compared with wild soybean plant, the controlling effect of the soybean plant transformed with Vip3Aa-01 nucleotide sequence, the soybean plant transformed with Vip3Aa-01-Cryl Ab nucleotide sequence and the soybean plant transformed with Vip3Aa-02-CrylFa nucleotide sequence to the newly hatched larvae is almost 100%. The larvae surviving in an extremely small portion basically stop development, and the soybean plant transformed with Vip3Aa-01 nucleotide sequence, the soybean plant transformed with Vip3Aa-01-Cryl Ab nucleotide sequence and the soybean plant transformed with Vip3Aa-02-CrylFa nucleotide sequence are only subjected to slight damage by and large, there is only very little pinhole-like mild damage on leaves, the leaf damage rates thereof are all at or below 3%.

Thus it is proved that the soybean plant transformed with Vip3Aa-01 nucleotide sequence, the soybean plant transformed with Vip3Aa-01-CrylAb nucleotide sequence and the soybean plant transformed with Vip3Aa-02-CrylFa nucleotide sequence all show high activity against Spodoptera litura and this activity is enough to generate harmful effect on the growth of Spodoptera litura, so as to control it.

The above experimental results also indicate that the maize plant transformed with Vip3Aa-01 nucleotide sequence, the maize plant transformed with Vip3Aa-01-CrylAb nucleotide sequence, the maize plant transformed with Vip3Aa-02-CrylFa nucleotide sequence, the soybean plant transformed with Vip3Aa-01 nucleotide sequence, the soybean plant transformed with Vip3Aa-01-CrylAb nucleotide sequence and the soybean plant transformed with Vip3Aa-02-CrylFa nucleotide sequence can control Spodoptera litura apparently because the plant themselves can generate Vip3 A protein. Therefore, it is well known to those skilled in the art that based on the same toxic action of Vip3 A protein to Spodoptera litura, transgenic plant that may generate similar expressive Vip3A protein can be used to control the harm of Spodoptera litura. The Vip3 A protein in the present application includes without limitation the Vip3A protein given amino acid sequence in embodiments. Meanwhile, the transgenic plant may also generate at least a second-type insecticidal protein different from Vip3 A protein, such as: Cryl A protein, CrylF protein and CrylB protein.

To summarize, the pest controlling method of the present application controls pest Spodoptera litura through generating Cry IF protein which can kill Spodoptera litura inside plant. Compared with agricultural control method, chemical control method and physical control method used in the prior art, the present application protects the whole plant throughout all growth period to control the encroach of pest Spodoptera litura, and the pest controlling method of the present application has no pollution and residue, the effect is stable and thorough, and is simple, convenient and economical.

Lastly it should be noted that the foregoing examples are intended to describe the technical solution of the present application without limitation, although the present application has been elaborated by referring to preferred exampls, those skilled in the art should understand that modifications or equivalent replacements can be made to the technical solution of the present application as long as such modifications or replacements do not depart from the spirit and scope of the technical solution of the present application.

Claims

Claims What is claimed is:

1. A method for controlling pest Spodoptera litura, wherein the pest Spodoptera litura contacts with a Vip3 A protein.
2. The method for controlling pest Spodoptera litura according to claim 1, wherein the Vip3 A protein is Vip3Aa protein.
3. The method for controlling pest Spodoptera litura according to claim 2, wherein the Vip3Aa protein exists in a plant cell generating the Vip3Aa protein, and the pest Spodoptera litura contacts with the VipSAa protein through intaking the plant cell.
4. The method for controlling pest Spodoptera litura according to claim 3, wherein the Vip3Aa protein exists in a transgenic plant generating Vip3Aa protein, the pest Spodoptera litura contacts with the VipSAa protein through intaking the tissues of the transgenic plant, and the growth of the pest Spodoptera litura is inhibited and/or the pest Spodoptera litura dies after the contact, so as to achieve control over the harm of Spodoptera litura to plant.
5. The method for controlling pest Spodoptera litura according to claim 4, wherein the transgenic plant may be in any growth period.
6. The method for controlling pest Spodoptera litura according to claim 4, wherein the tissue of the transgenic plant is selected from leaves, stalks, tassels, female spikes, anthers, filaments and fruits.
7. The method for controlling pest Spodoptera litura according to claim 4, wherein the control over the harm of Spodoptera litura to plant is not changed with the change of planting location and/or planting time.
8. The method for controlling pest Spodoptera litura according to any one of claims 3 to 7, wherein the plant is selected from maize, soybean, cotton, sweet potato, taro, lotus, sesbama, tobacco, beet, cabbage and eggplant, preferably, the plant is selected from maize and soybean.
9. The method for controlling pest Spodoptera litura according to any one of claims 2 to 8, wherein a step before the contact step is planting the plant containing polynucleotide coding the Vip3Aa protein.
10. The method for controlling pest Spodoptera litura according to any one of claims 2 to 9, wherein the amino acid sequence of the Vip3Aa protein includes: 1) an amino acid sequence shown by SEQ ID NO: 1 or SEQ ID NO: 2, 2) an amino acid sequence having at least 70% of homology with SEQ ID NO: 1 or SEQ ID NO: 2 and having insecticidal activity against pest Spodoptera litura, or 3) an amino acid sequence obtained from foe amino acid sequence shown by SEQ ID NO: 1 or SEQ ID NO: 2 through substitution, deletion and/or addition of one or more amino acid residues and having insecticidal activity against pest Spodoptera litura.
11. The method for controlling pest Spodoptera titura according to claim 10, wherein the nucleotide sequence coding the Vip3Aa protein includes: 1) a nucleotide sequence shown by SEQ ID NO: 3 or SEQ ID NO: 4, 2) a nucleotide sequence having at least about 75% of homology with SEQ ID NO: 3 or SEQ ID NO: 4 and coding an amino add sequence having insecticidal activity against pest Spodoptera litura, 3} a nucleotide sequence hybridizing with SEQ ID NO: 3 or SEQ ID NO: 4 under strict conditions and coding the amino acid sequence having insecticidal activity against pest Spodoptera litura, 4) a nucleotide sequence different from SEQ ID NO: 3 or SEQ ID NO: 4 due to codon degeneracy and coding the amino acid sequence having insecticidal activity against pest Spodoptera litura.
12. The method for controlling pest Spodoptera litura according to any one of claims 3 to 11, wherein the plant also contains at least a second nucleotide different from the nucleotide coding the Vip3Aa protein.
13. The method for controlling pest Spodoptera litura according to claim 12, wherein the second nucleotide codes Cry type insecticidal protein, Vip type insecticidal protein, protease inhibitor, lectin, α-amylase or peroxidase.
14. The method for controlling pest Spodoptera litura according to claim 13, wherein the second nucleotide codes Cry lAb protein. Cry IPa protein or Cry IBa protein.
15. The method for controlling pest Spodoptera litura according to claim 14, wherein the second nucleotide has a nucleotide sequence shown by SEQ ID NO: 5 or SEQ ID NO: 6.
16. The method for controlling pest Spodoptera litura according to claim 12, wherein the second nucleotide is a dsRNA inhibiting an important gene in target insect and pest.
17. A method for preparing plant cell, transgenic plant or part of transgenic plant controlling pest Spodoptera litura, including introducing a nucleotide sequence coding Vip3A protein into the plant cell, transgenic plant or part of transgenic plant, preferably, introducing a nucleotide sequence coding Vrp3A protein into the genome of plant cell, transgenic plant or part of transgenic plant.
18. Use of Vip3A protein in preparing plant cell, transgenic plant or part of transgenic plant controlling pest Spodoptera litura.
19. A method for cultivating a plant which controls pest Spodoptera litura, including: planting at least one plant seed whose genome contains a polynucleotide sequence coding Vip3A protein; making the plant seed grow' into a plant; making tire plant grow under the condition that pest Spodoptera litura does harm through artificial inoculation and/or natural occurrence, and harvesting plant with weakened plant damage and/or increased plant yield compared to other plant without polynucleotide sequence coding Vip3 A protein.