CN1158012C - Method for control of insects - Google Patents

Abstract

The present invention relates to a method for preventing and curing maize damage caused by Asian corn borers. Through transgenic insect resisting maize plants plated in the asiatic corn borers and capable of expressing CryIAb protein, particularly an insect resisting maize strain MON810, the feeding and the growth of the asiatic corn borers ingesting plant tissues are inhibited, and the asiatic corn borers are finally dead. The prevention and the cure of the asiatic corn borers by the method are carried out on all plants and all growth periods and are not changed because of planting time, planting places, etc. The yield of insect resisting maize is obviously improved, and at the same time, the representation of the grain characters and the haulm characters of the insect resisting maize are ameliorated. The method has the advantages of good effects, simplicity, convenience, economy, etc. and pollution is not generated on environment.

Description

A method of controlling pests

The present invention relates to a method for controlling crop pests, in particular to a method for preventing and treating corn borer damage to corn by planting a transgenic insect-resistant corn plant. Specifically, the present invention relates to a source for expression in corn The CryIAb protein in the soil bacterium Bacillus thuringiensis subsp.kurstaki subspecies (Bacillus thuringiensis subsp.kurstaki, B.t.k.) that exists in nature prevents and treats the method for corn borer damage corn, more specifically, the present invention relates to a kind of its body can The transgenic insect-resistant corn line MON810 expressing the CryIAb protein in B.t.k. is used for preventing and treating the corn borer damage to the method; the use of.

The corn borer is the largest pest in corn production and occurs in all corn producing areas in China. In general years, the damage of spring corn will reduce the yield by 7-10%, and in the year of severe occurrence, the yield will be reduced by more than 30%. Summer maize, due to its short growing season, suffered even more losses due to corn borer damage. Corn is the third largest grain crop in China, with an annual planting area of over 20 million hectares. Based on an average yield of 4,500 kilograms per hectare, the annual production loss due to corn borer damage is 6-9 billion kilograms, equivalent to about 2-3 billion yuan.

The Asian corn borer goes through four stages in its life: egg, larva, pupa and adult. The larvae are the only insect state in the corn borer's life that damages crops. The Asian corn borer is a typical facultative diapause insect, which can produce 1 to 7 generations a year from north to south, but only two generations in the whole growth period of maize, that is, the generation of the heart leaf stage and the generation of the ear stage. For example, in the northern spring corn area, the first generation of eggs is in the middle of the heart leaf stage of corn growth and development, so it is also called the heart leaf stage generation. Eggs are mostly produced near the midrib on the back of the leaves, most of which are fully expanded leaves in the middle and upper parts of the plant. Once hatched, the larvae show latent habits, climb into the heart leaf clump and eat the heart leaf, or eat the mesophyll of the young leaves in the gaps between the leaves, resulting in pinholes or window paper-like flowers and leaves. If the heart leaf is not unfolded, a row of holes will appear when the leaf is pulled out. Generally speaking, slight damage to the leaves has no obvious effect on the growth of corn. However, if the insect-susceptible varieties are planted, and borer occurrence is heavy, and the leaves are seriously damaged, the damaged tassels cannot be drawn out normally, and the plant height will be significantly reduced. When the larvae feed on the heart leaf tissue and develop to around the 3rd instar, the maize enters the budding stage. At this time, the tender tassels are more attractive to borers than the heart leaves, so all the larvae are transferred to the tassels for food. After tasseling, the larvae are carried out of the heart leaf clump. The larvae continue to feed on the tassel until the tassel disperses. When the tassels gradually disperse, the larvae lose the conditions required for the above-mentioned main tropism, that is, the small environment necessary for survival. Except for a few mature larvae that can pupate on the tassels, all larvae begin to move toward the tassels. Since the larvae are basically 4-5 instars at this time, they begin to obviously exert their "boring" characteristics. Except for a part of the tassel peduncle that bores into the tassel pedicle and develops to maturity or moves downward again, because the tassel pedicle is very thin, it generally can only accommodate 1 to 2 mature larvae, so more larvae must climb over the tassel pedicle and continue to move downward. Move down to find a more suitable place to drill. Among them, the ear insertion node and its upper and lower nodes are the most vulnerable parts to be attacked. At this time, the ear of corn has begun to develop. If the stem node near it is mothed, it will obviously affect its normal development or even stop its development. If the first ear is damaged earlier, the second ear can continue to develop, but it is impossible to grow to the proper size of the first ear. If the second ear does not mature in time and is bored by borers and stops growing, the corn plant will die out. In addition, because stem borers eat the stems in the middle and lower parts of the plants, it is easy to cause inversions when encountering wind, which makes the losses even greater. The first-generation larvae develop to maturity either on the tassel, or in the tassel stalk, or in the stalk, and most of them pupate and emerge to produce the first generation Generation of adults.

The second-generation eggs (produced by the first-generation adults) are in the peak period of corn filaments, also known as the ear stage generation. Eggs are still mostly produced on the back of the middle and upper leaves, and occasionally on the stalks and bracts. Most of the newly hatched larvae hide in the base of the filaments at the top of the ear, feed on the filaments and then the young grains on the top of the ear, and a few larvae hide in the leaf axils to feed on the accumulated pollen or leaf axil tissue. After the larvae develop to the 3rd instar, they begin to bore and bore into the cob directly from the ear head, or continue to eat the grains that are being filled, or move down from the top of the ear to bore into the ear, ear stalk or stalk. At this time, the ear of corn has fully developed to its proper size and has entered the stage of milk maturity. Therefore, borer damage at the ear stage will not affect the ear size, but it will affect the normal grain filling of corn, thereby reducing the thousand-grain weight. If the ear stalk is hollowed out, it will cause the ear to break and fall off. In addition to directly causing yield loss, larvae eating grains often cause infection of corn ear and stalk rot, which aggravates yield loss and quality decline.

The control of corn borer in China began in the 1950s. The main control methods used are: agricultural control, chemical control, physical control, sex pheromone control and biological control.

Agricultural control is the comprehensive and coordinated management of multiple factors in the entire farmland ecosystem, regulating crops, pests, and environmental factors, and creating a farmland ecological environment that is conducive to crop growth but not conducive to the occurrence of stem borers. For example, measures such as treating the overwintering host of corn borer, reforming the farming system, planting borer-resistant varieties, planting trap fields and intercropping to reduce the damage of corn borer. Because agricultural control must obey the requirements of crop layout and yield increase, its application has certain limitations and cannot be used as an emergency measure, and it appears powerless when borer damage breaks out.

Chemical control, that is, pesticide control, is to use chemical insecticides to kill pests, and is an important part of the comprehensive management of corn borer. It has the characteristics of quick effect, convenience, simplicity and high economic benefits, especially in the case of large-scale occurrence of pests , is an essential emergency measure, it can eliminate pests before they cause damage. For example, the granule control method, which is currently being promoted on the largest area, has the most stable and reliable effect. However, the tools for high-efficiency granule application are still far from passing the test, which seriously affects its function. In addition, there are chemical control methods such as spreading poisonous soil, spraying liquid medicine, and fumigation of dichlorvos sealing piles to overwinter in straw piles. However, chemical control also has its limitations. Improper use will often lead to chemical damage of crops, resistance of pests, and killing of natural enemies, polluting the environment, destroying the farmland ecosystem, and posing a threat to the safety of humans and animals due to pesticide residues, etc. Adverse consequences.

Physical control is mainly based on the response of pests to various physical factors in environmental conditions, using various physical factors such as light, electricity, color, temperature and humidity, etc., as well as mechanical equipment to trap and kill pests, radiation sterility and other methods to control pests. At present, the most widely used is high-pressure mercury lamp trapping. It takes advantage of the fact that overwintering larvae are concentrated in the corn straw piles in the village to overwinter. Adults of corn borer are trapped and killed, and the control effect is obvious. High-pressure mercury lamps must be used in villages that ensure continuous power supply at night, and the operation is difficult.

Sex pheromone control is to use artificially synthesized corn borer sex pheromone to directly trap and kill corn borer males and interfere with mating of borers, reduce the fertilization rate of female moths in the field, and reduce borer damage.

The trapping method is to use artificially synthesized corn borer sex pheromone to trap and kill the corn borer male moth in the mating habitat of the corn borer, such as a well-growing wheat field or a vegetable field. The disadvantage of this method is that the management of water basin traps is labor-intensive, and it must be used in large areas to be effective.

Wherein the fascination method is to hang 4500-6000 sex pheromone dispensers per hectare on the crops at the mating site when the overwintering generation corn borer adults emerge 10% to interfere with the mating of the borer moth. Using sex pheromone to prevent and control the first generation corn borer is simple and effective, without polluting the environment and harming natural enemies. The disadvantage is that it takes a lot of work to put in the lure.

Biological control is the use of certain beneficial organisms or biological metabolites to control the population of pests to achieve the purpose of depressing or eliminating pests. It is characterized by being safe to humans and animals, less polluting to the environment, and can achieve long-term control of certain pests. The most common biological control are Trichogramma and Beauveria bassiana. However, the control of Trichogramma is greatly affected by climate factors, and the effect is often unstable, and the same investment is required regardless of the severity of borer occurrence.

Transgenic insect-resistant corn is to transfer exogenous insect-resistant genes into corn cells through plant genetic engineering, and then regenerate plants, so that corn can obtain insect-resistant characteristics that it does not have.

Bacillus thuringiensis, also known as Bacillus thuringiensis, is a Gram-positive soil bacillus that can form spores, and can form parasporal crystals in the bacterium during the sporulation period. These parasporal crystals are mainly composed of (27-140KD) proteins, have highly specific anti-insect effects, and are non-toxic to humans, animals and non-target insects. The crystal protein (Cry) produced by different strains shows specific insecticidal activity to different kinds of insects. So far, tens of thousands of strains of Bacillus thuringiensis have been isolated all over the world.

Cry proteins can be divided into classes I, II, III, and IV according to their anti-insect spectrum, class I against Lepidoptera, class II against Lepidoptera and Diptera, class III against Coleoptera, and class IV against Diptera According to the order, each category is divided into several subcategories according to the degree of amino acid homology. For example, among the cryI genes, amino acid homology of 82%-90% is classified as cryIA; 55%-71% is classified as cryIB; cryIC and cryID are different from each other and also different from cryIA. In the cryIA gene, according to the zymogram and molecular weight of the restriction endonuclease, it is further divided into: cryIAa, 4.5kb; cryIAb, 5.3kb; cryIAc, 6.6kb subclass genes.

CryIAb protein is a parasporal crystal protein produced by Bacillus thuringiensis subsp. kurstaki (B.t.k.) discovered by people, and it is an insoluble crystal protein. Crystal proteins are composed in the form of proprotein toxins. Insecticidal properties of CryIAb crystal proteins require protein uptake. In the insect gut, due to the high pH value, the proprotein dissolves, binds tightly to the active core of the protein through the action of the enzyme, and is no longer degraded by the protease in the insect gut. The core protein binds to specific receptors in the intestine of Lepidoptera insects, inserts into the intestinal membrane, and forms ion-specific channels. These biochemical reactions disrupt the digestive process, leading to the death of the insect. The digestive tract tissues of non-target insects, mammals, birds, and fish do not contain receptors that can bind to the CryIAb protein. Therefore, the CryIAb protein cannot be destroyed and digested, so it is not toxic.

With the development of molecular genetic technology, genes of various insecticidal crystalline proteins have been isolated and their DNA sequences determined. These genes have been used to create genetically engineered plants in crops such as soybeans, corn, and potatoes. It has been proved that this genetically engineered plant is resistant to various Lepidoptera pests. However, there is no report about controlling the damage of corn borer to corn by producing transgenic corn plants expressing CryIAb protein.

Due to the different types of cry genes used by different laboratories and companies, the regulatory elements used to construct expression vectors such as promoters, introns, and terminators are different, resulting in differences in the types and degrees of insect resistance of the transformants obtained. MON810 insect-resistant corn strain is a transgenic product developed by Monsanto Corporation of the United States using modern molecular biology techniques. It expresses CryIAb in B.t.k. in vivo. Its trade name is Baofeng Corn in Chinese and YieldGard in English. Its products have been commercialized in the United States and are available in the American market.

Although the MON810 insect-resistant corn line has been commercialized in the United States, its control object is the European corn borer (Ostrinia nubilalis), while the control object of the present invention is the Asian corn borer (Ostrinia furnacalis).

Although both European corn borer (Ostrinia nubilalis) and Asian corn borer (Ostrinia furnacalis) belong to Insecta, Lepidoptera, Mothidae, and are both moth-eating pests that damage crops such as corn, but both are biologically There are at least the following main differences between two clear and distinct species (Journal of Plant Protection, 1988, Vo1.15(3): 145-152; Agricultural Encyclopedia—Insect Volume, 1990, 456-459):

1. In the main corn producing areas of China, the Asian corn borer is the main pest that damages corn, and no evidence of European corn borer harming corn has been found

After years of investigation and research, the China Corn Borer Integrated Prevention and Control Research Collaborative Group has proved that:

The Asian corn borer is mainly distributed in the vast area from Heilongjiang to Guangdong in the eastern half of China. It is a major pest of corn, millet, sorghum and other crops. China's main corn producing areas are all within this range, including: Heilongjiang, Jilin, Liaoning, Beijing, Hebei, Henan, Hubei, Guangdong, Sichuan, Yunnan, Jiangsu, Ningxia, Guizhou, Inner Mongolia, Shandong, Gansu and other 17 provinces and cities (autonomous regions) ).

European corn borer has only been found in Yining of Xinjiang, Hohhot of Inner Mongolia, Yongning of Ningxia, Zhangjiakou and Lutai of Hebei so far. Except for the damage to corn in Yining, Xinjiang, other areas mainly parasitize cocklebur and hemp, and no evidence of damage to corn has been found.

2. The Asian corn borer does not exist in the United States

The Asian corn borer is distributed in temperate and tropical Asia, Australia and Oceania Micronesia and other regions. Except for China, the Asian corn borer is widely distributed in Japan, Southeast Asia and Australia, but there is no report about the existence of the European corn borer in the above areas.

European corn borer is distributed in Europe, North America, West Africa and Asia Minor, and there is no report of the existence of Asian corn borer in these areas.

3. The damage habits of the two to corn are different

It is reported that the larvae of the European corn borer before the fourth instar hide at the junction of the leaf sheath and the stalk when the corn ear is damaged; while the Asian corn borer before the fourth instar mostly damage the filaments and tender leaves on the ear of corn. grain.

4. The two are different in form

The identification of most insect populations is ultimately determined by anatomical comparisons of the structures of the external genitalia of their male and female insects. That is, there are many insect populations that are very similar in appearance but have different genitalia and are actually completely different species; and vice versa.

After years of research, the China Corn Borer Integrated Control Research Collaborative Group has proved that the Asian corn borer that occurs in China is morphologically different from the European corn borer. The following differences exist:

·Asian species generally have 3-4 large spines or 2 large spines and 1 small spine, the average number is more than that of European species.

·The spiny area is longer than the non-thorny area in Asian species, and the opposite is true in European species.

·The relationship between the lengths of the two areas on both sides of the cradle abdomen is inconsistent.

5. There is reproductive isolation between the two

The fundamental difference between different species of organisms is that there is reproductive isolation between them, that is, different species cannot mate to produce offspring with normal reproductive ability. The Chinese Corn Borer Integrated Control Research Collaborative Group spent 5 years mating more than 1,300 pairs of corn borers collected from different regions (including Austria) in various combinations. The experiment found that all the corn borers from the corn-producing areas in eastern China were reproductively isolated from the European corn borer from Austria, and only the corn borers from Yining, Xinjiang, and Ningxia and Zhangjiakou cocklebur were asexual to the European corn borer. isolation. Therefore, there is reproductive isolation between O. corn borer and O. corn borer that occur in the main corn producing areas of China.

6. The two respond differently to sex pheromones

The chemical structures of sex pheromones of different insect species are different. Insect sex pheromones consist of a variety of compounds. Moreover, the isolation is accomplished with different types of compounds and different mixing ratios. It has been reported that the chemical structures of the sex pheromones of the two Eurasian corn borers are quite different.

The China Corn Borer Comprehensive Control Research Collaborative Group spent 5 years in 34 regions in 20 provinces and cities to conduct moth-attracting activity tests of two Eurasian corn borer sex pheromones. The results showed that the Asian corn borer that occurred in China did not respond to the sex pheromone of European corn borer.

7. The chemical composition and structure of sex pheromone are different

The chemical composition of sex pheromones and the ratio of each component are one of the important indicators to distinguish different insect populations. The analysis results of the sex pheromone structure of the corn borer showed that the chemical composition of the sex pheromone of the Asian corn borer in China was cis and trans 12-tetradecenyl acetate with a ratio of 47:53, while the European corn borer The chemical composition of the sex pheromone of the borer is 97:3 cis and trans 11-tetradecenyl acetate, which are different.

To sum up, since the Asian corn borer is different from the European corn borer in many respects, it is not obvious that the method of using the transgenic insect-resistant corn of the present invention to prevent and control corn caused by the Asian corn borer is not obvious.

The invention uses the transgenic insect-resistant corn to prevent and control the damage caused by the Asian corn borer. The transgenic insect-resistant corn itself can express CryIAb protein, so that the corn has the ability to resist the Asian corn borer.

Therefore, the object of the present invention is to provide a transgenic insect-resistant corn that can express CryIAb protein, especially the transgenic insect-resistant corn line MON810, to prevent and control the method of corn borer damage to corn.

Another object of the present invention is to provide the above-mentioned transgenic insect-resistant corn, especially the use of the transgenic insect-resistant corn line MON810 for preventing and controlling corn borer damage to corn.

The invention relates to a method for preventing and controlling corn borer damage to corn, which is characterized in that by planting a transgenic insect-resistant corn plant capable of expressing CryIAb protein in the body, the feeding and Growth inhibition, and eventually death, is achieved for the control of corn borer infestation by the Asian corn borer.

According to the method for preventing and treating corn borer damage to corn according to the present invention, wherein the CryIAb protein has the following amino acid sequence of the CryIAb protein:

The amino acid sequence of the CryIAb protein is as follows, which is identical to the naturally occurring CryIAb protein

1 MDNNPNINEC IPYNCLSNPE VEVLGGERIE TGYTPIDISL SLTQFLLSEF

51 VPGAGFVLGL VDIIWGIFGP SQWDAFLVQI EQLINQRIEE FARNQAISRL

101 EGLSNLYQIY AESFREWEAD PTNPALREEM RIQFNDMNSA LTTAIPLFAV

151 QNYQVPLLSV YVQAANLHLS VLRDVSVFGQ RWGFDAATIN SRYNDLTRLI

201 GNYTDHAVRW YNTGLERVWG PDSRDWIRYN QFRRELTLTV LDIVSLFPNY

251 DSRTYPIRTV SQLTREIYTN PVLENFDGSF RGSAQGIEGS IRSPHLMDIL

301 NSITIYTDAH RGEYYWSGHQ IMASPVGFSG PEFTFPLYGT MGNAAPQQRI

351 VAQLGQGVYR TLSSTLYRRP FNIGINNQQL SVLDGTEFAY GTSSNLPSAV

401 YRKSGTVDSL DEIPPQNNNV PPRQGFSHRL SHVSMFRSGF SNSSVSIIRA

451 PMFSWIHRSA EFNNIIPSSQ ITQIPLTKST NLGSGTSVVK GPGFTGGDIL

501 RRTSPGQIST LRVNITAPLS QRYRVRIRYA STTNLQFHTS IDGRPINQGN

551 FSATMSSGSN LQSGSFRTVG FTTPFNFSNG SSVFTLSAHV FNSGNEVYID

601 RIEFVPAEVT FEAEYDLERA QKAVNELFTS SNQIGLKTDV TDYHIDQVSN

651 LVECLSDEFC LDEKKELSEK VKHAKRLSDE RNLLQDPNFR GINRQLDRGW

701 RGSTDITIQG GDDVFKENYV TLLGTFDECY PTYLYQKIDE SKLKAYTRYQ

751 LRGYIEDSQD LEIYLIRYNA KHETVNVPGT GSLWPLSAPS PIGKCAHHSH

801 HFSLDIDVGC TDLNEDLGVW VIFKIKTQDG HERLGNLEFL EGRAPLVGEA

851 LARVKRAEKK WRDKREKLEW ETNIVYKEAK ESVDALFVNS QYDRLQADTN

901 IAMIHAADKR VHSIREAYLP ELSVIPGVNA AIFEELEGRI FTAFSLYDAR

951 NVIKNGDFNN GLSCWNVKGH VDVEEQNNHR SVLVVPEWEA EVSQEVRVCP

1001 GRGYILRVTA YKEGYGEGCV TIHEIENNTD ELKFSNCVEE EVYPNNTVTC

1051 NDYTATQEEY EGTYTSRNRG YDGAYESNSS VPADYASAYE EKAYTDGRRD

1101 NPCESNRGYG DYTPLPAGYV TKELEYFPET DKVWIEIGET EGTFIVDSVE

1151 LLLMEE

According to the method for preventing and controlling corn borer damage to corn according to the present invention, wherein the transgenic insect-resistant corn is the transgenic insect-resistant corn line MON810, which includes any MON810 inbred line, hybrid, integrated species or its population, for example, Inbred lines LH185MON810 and Mo17MON810, etc., hybrids LH198×LH185MON810, B73×Mo17MON810, etc.

According to the method for controlling corn borer damage to corn according to the present invention, wherein said prevention and control of corn borer damage corn refers to the prevention and treatment of corn damage damage to corn borer by the whole plant of transgenic insect-resistant corn; the whole plant The prevention and control of the Asian corn borer includes its leaves, stems, tassels, anthers and filaments, etc. to prevent and control the corn caused by the Asian corn borer;

According to the method for preventing and treating corn borer in accordance with the present invention, wherein said prevention and treatment of corn caused by corn borer refers to the prevention and treatment of corn damaged by corn borer during the whole growth period of transgenic insect-resistant corn; The control of the Asian corn borer during the growth period includes the control of the Asian corn borer at the seedling stage, new leaf stage, budding stage, tasseling stage, and filament stage;

According to the method for preventing and treating the Asian corn borer of the present invention, wherein the prevention and treatment of corn caused by the Asian corn borer refers to not changing due to the change of the planting location, and not changing due to the change of the planting time;

In addition, according to the method for preventing and controlling corn borer damage to corn according to the present invention, wherein the prevention and control of corn borer damage to corn refers to the yield-increasing effect of transgenic insect-resistant corn under the condition of artificial inoculation of corn borer in the field , increase in grain weight, improvement in stalk resistance, etc., as well as its effect on increasing yield, increase in grain weight, improvement in stalk resistance, etc.

The present invention also relates to the use of the transgenic insect-resistant corn, especially the transgenic insect-resistant corn line MON810, as a method for preventing corn from being damaged by the Asian corn borer.

The most obvious difference between the present invention and the existing one for the prevention and treatment of the Asian corn borer is that the beneficial effect produced by the present invention is: the present invention has obvious effect of increasing production. Corn is an important food crop in China. The annual planting area is more than 20 million hectares, and the average yield per hectare is about 5,000 kilograms. If all of them are replanted with insect-resistant corn MON810, the increase in yield in this experiment is 19.6% (Example 6). It can increase China's grain income by nearly 20 billion kilograms, which is equivalent to about 16 billion yuan; even with a 10% increase in production, it can increase China's grain income by nearly 10 billion kilograms, equivalent to about 8 billion yuan. It can be seen that the present invention has important social and economic benefits. This is unmatched by any control method of Asian corn borer currently used.

The method that the chemical insecticide used at the present stage prevents and treats the Asian corn borer, though has played a certain effect to the damage of the corn to control the Asian corn borer, but also brought the harm to human body, livestock and soil itself simultaneously, uses the method of the present invention method to eliminate these hazards.

The existing methods for preventing and controlling the Asian corn borer mainly achieve the prevention and control of the corn caused by the Asian corn borer through external effects, that is, external factors. And the present invention prevents and controls the Asian corn borer by producing the CryIAb protein capable of killing the Asian corn borer in the plant, that is, preventing and treating the Asian corn borer through internal causes;

None of the existing methods for preventing and controlling the Asian corn borer covers the whole growth period, but the present invention protects the whole growth period of corn. The insect-resistant corn strain MON810 grows from the seedling stage of corn growth to the new leaf stage to the budding stage. From the silking stage to the mature stage, it is free from the Asian corn borer. This is beyond the reach of the existing Asian corn borer control methods;

There is no so-called prevention and control of the whole plant in the existing control methods for the Asian corn borer, but the present invention protects the whole corn plant, and the leaves, stems, anthers, filaments, etc. This is also something that cannot be solved by the current control of Asian corn borer;

The effect of the existing methods for preventing and controlling the Asian corn borer is unstable, but the control effect of the present invention on the corn damaged by the Asian corn borer is stable. , Different genetic backgrounds are stable and consistent;

The existing methods for preventing and controlling the Asian corn borer are incomplete and only play a role in mitigating. The insect-resistant corn strain MON810 of the present invention has a control effect of almost 100% on the newly hatched Asian corn borer larvae, and very few surviving larvae are extremely small. Small, all of which are obviously stunted and have stopped growing, so it is difficult to cause damage to corn;

Compared with the existing method for preventing and controlling the Asian corn borer, the present invention has the advantages of simplicity, convenience, economy, no pollution to the environment, and the like. With the technology of the present invention, it is only necessary to plant the insect-resistant corn strain MON810 capable of expressing the CryIAb protein, and no other measures are required, thereby saving a lot of manpower, material and financial resources, and will not pollute the environment;

The difference between the present invention and the existing control of the Asian corn borer lies in that the present invention can improve the properties of corn grains and stalks;

The difference between the present invention and the existing control of the Asian corn borer is that although the amino acid sequence of the active CryIAb protein expressed by the MON810 corn strain is completely consistent with the protein produced by Bacillus thuringiensis as a microbial insecticide, the two are very different. big. Since Bacillus thuringiensis preparations need to be sprayed directly onto the crop surface, active crystallized proteins (including CryIAb proteins) are degraded in the environment, resulting in repeated production and repeated application of pesticides, and lay a foundation for agricultural production. Practical application brings difficulties and greatly increases the cost. However, in the present invention, the CryIAb protein is expressed in plants, which effectively avoids the shortcomings of biopesticides being unstable in nature, and saves manpower and material resources.

The accompanying drawings are briefly described below:

Figure 1 is a schematic diagram of the plasmids of PV-ZMBK07 and PV-ZMGT10: the restriction sites and their positions (expressed in base pairs) used in Southern analysis are shown in this figure.

Figure 2 is a Southern analysis diagram of the DNA of the maize line MON 810, which is the analysis of the number of inserts. Lanes 1 and 2 are the NdeI digestion products of the following DNAs, respectively, and PV-ZMBK07 is the hybridization probe. Lane 1: DNA of MON 818; Lane 2: DNA of MON 810.

→ A symbol indicating the size of a DNA fragment derived from a molecular weight marker;

≈ is a symbol denoting the size of DNA fragments derived from molecular weight markers and plasmid digests;

^* Denotes the symbol of the background band.

Figure 3 is a Southern analysis diagram of the DNA of the maize line MON 810, that is, the analysis of the CryIAb gene. Swimming lane 1, swimming lane 2 and swimming lane 3 are the NcoI/EcoRI digestion products of the following DNAs respectively, and the cryIAb gene is the hybridization probe. Lane 1: About 50 pg PV-ZMBK07; Lane 2: DNA of MON818; Lane 3: DNA of MON 810.

→ Symbol indicating the size of DNA fragments derived from molecular weight markers in ethidium bromide (EB)-stained gels;

- symbol indicating the size of DNA fragments derived from plasmid digests;

≈ is a symbol denoting the size of DNA fragments derived from molecular weight markers and plasmid digests;

^** indicates the hybridization area adjacent to the lane. This symbol appears only in lane 1 because its DNA deviates from lane 1 at an angle during electrophoresis.

Figure 4 is a Southern analysis diagram of the DNA of the maize line MON 810, that is, the analysis of the CP4 ESPSP gene and the gox gene. Swimming lane 1, swimming lane 2, swimming lane 3 and swimming lane 4 are the NcoI/BamHI digestion product of following DNA respectively: swimming lane 1 and swimming lane 3 are the plasmid PV-ZMBK07 and PV-ZMGT10 of about 50pg, swimming lane 2 and swimming lane 4 are the DNA of MON 810 , the hybridization probe of swimming lane 1 and swimming lane 2 is CP4 ESPSP gene, the hybridization probe of swimming lane 3 and swimming lane 4 is gox gene;

→ Symbols representing DNA fragment sizes derived from molecular weight markers in ethidium bromide (EB)-stained gels;

- Symbols indicating the size of DNA fragments derived from plasmid digests.

Figure 5 is a Southern analysis chart of the DNA of the maize line MON 810. Swimming lanes 1-6 are the NcoI/EcoRI digestion products of the following DNAs respectively: Swimming lanes 1 and 4 are about 50pg of the plasmid PV-ZMBK07; Swimming lanes 2 and 5 are the DNA of MON818, and Swimming lanes 3 and 6 are the DNA of MON810; Swimming lanes The probe for 1-3 is the nptII region; the hybridization probe for lanes 4-6 is the ori-pUC region.

→ Symbols representing DNA fragment sizes derived from molecular weight markers in ethidium bromide (EB)-stained gels;

- Symbols indicating the size of DNA fragments derived from plasmid digests.

To further illustrate the present invention, the basic situation of the transgenic insect-resistant corn line MON810 that can express the CryIAb protein is described as follows:

1. Target gene cryIAb

The cryIAb gene is an insect resistance gene inserted into the MON810 insect-resistant maize line, which is derived from the soil bacteria Bacillus thuringiensis subsp. kurstaki (B.t.k.) that exists in nature. The cryIAb gene in the PV-ZMBK07 plasmid vector used for transformation is a structural gene with a length of 3468 nucleotides. Compared with the cryIAb in the original B.t.k., its gene sequence has been changed, so that the expression level of the CryIAb protein in maize is greatly improved.

2. The CryIAb protein encoded by the target gene cryIAb

The B.t.k.HD-1[CryIAb] protein encoded by the target gene cryIAb consists of 1156 amino acids. After the protein is treated with trypsin, it produces a protein product containing about 600 amino acids with antitrypsin in the plant and in vitro. The structure and activity of the CryIAb protein encoded by the MON810 maize strain is completely consistent with the protein produced by Bacillus thuringiensis.

The amino acid sequence of the CryIAb protein is the same as that of the CryIAb protein existing in nature.

3. Construction of plasmid vector:

The MON810 insect-resistant maize line was generated using two plasmids: PV-ZMBK07 [containing the cryIAb gene] and PV-ZMGT10 [containing the CP4EPSPS and gox genes]. The genetic components of the two plasmid vectors are listed in Table 1 and Table 2, and the genetic maps of the two plasmid vectors are shown in Figure 1.

The cryIAb gene displayed by the PV-ZMBK07 plasmid vector is under the control of the enhanced CaMV35S promoter E35S, which is about 0.6Kb in size. Located between the E35S promoter and the cryIAb gene is a 0.8Kb intron from the maize hsp70 (heat shock protein) gene, which functions to increase the gene transcription level. The Hsp70 intron is followed by the cryIAb gene with a size of 3.46Kb. The cryIAb gene was inserted into the 0.26Kb untranslated sequence at the 3' end of nopaline synthase, NOS3', which provides the mRNA polyadenylation signal.

The description of the marker gene and its function related to the sequence of the PV-ZMGT10 plasmid vector is purely informative, because the relevant gene sequence of the PV-ZMGT10 vector has not been found out in the MON810 insect-resistant maize line. The PV-ZMGT10 plasmid contains the gox and CP4EPSPS marker genes, which are involved in the transfer of CTP1 and CTP2 peptides by chloroplasts. Both coding regions are under the control of sequences containing the CaMV35S promoter, maize hsp70 intron and NOS 3' terminator.

The CP4EPSPS gene was isolated from the Agrobacterium sp. CP4 strain, and when introduced into plants showed strong resistance to glyphosate inhibition. Glyphosate binds and blocks the activity of the target enzyme EPSPS, an enzyme in the biosynthetic pathway of aromatic amino acids. Compared with most EPSPS, CP4EPSPS has a strong tolerance to the inhibitory effect of glyphosate and is highly catalytic. The CP4EPSPS protein expressed by plant cells is resistant to glyphosate in the medium because the presence of aromatic compounds satisfies the continuous action of the tolerant EPSPS enzyme.

The CP4EPSPS gene in PV-ZMGT10 contains the chloroplast transfer peptide CTP2 isolated from Arabidopsis EPSPS, which directs the CP4EPSPS protein to the chloroplast, which is the position of EPSPS in plants and the site of aromatic amino acid synthesis. The size of the CP4EPSPS gene carrying CTP2 is about 1.7Kb. The CP4EPSPS gene family (promoter via the 3' end sequence) was integrated into the gox gene family.

The gox gene, which encodes glyphosate and causes glyphosate oxidoreductase (GOX) to undergo metabolic changes, is obtained from the LBAA strain of the genus Achromobacter (new genus Phryobacterium) by cloning technology. The GOX protein was integrated into a plasmid containing the chloroplast transfer peptide sequence CTP1. CTP1 is a small subunit gene from the Arabidopsis ribulose-1,5-bisphosphate carboxylase (SSU1A) gene. GOX enzymes degrade glyphosate, converting it to aminomethyl phosphate and glyoxylate.

The α region of the lacZ gene encoding β-galactosidase is present in PV-ZMBK07 and PV-ZMGT10 in the presence of a bacterially controlled promoter. This region contains a polylinker that allows compatible genes to be cloned in plasmid vectors. The lacZ-alpha region is followed by the origin of replication of the 0.65KbpUC plasmid (ori-pUC), which can also replicate in E. coli.

The ori-pUC region is followed by the neomycin phosphotransferase type II (nptII) gene. This enzyme is resistant to aminoglycoside antibiotics (such as kanamycin and neomycin) and is used as a resistance marker when constructing plasmids. The sequence encoding the nptII gene was derived from the prokaryotic transposon Tn5 in the presence of its native bacterial promoter.

Table 1: List of DNA components in the PV-ZMBK07 plasmid Gene component E35Shsp70cryIAbNOS3'lacZori-pUCnptII SizeKb0.610.803.460.260.240.650.79 Functional cauliflower mosaic virus (CaMV) promoter with replication enhancer region. An intron from the maize hsp70 (heat shock protein) gene, which acts to increase gene transcription levels. This gene encodes a CryIAb protein product that is consistent with the natural product. The 3' untranslated region of the nopaline synthase gene terminates transcription and governs polyadenylation. One fragment is from E.coli lacl encoding promoter Plac sequence, and the other is from pUC119 encoding β-D-galactosidase or LacZ protein sequence. Is the origin of replication of the pUC plasmid, allowing the plasmid to replicate in E. coli. Type II neomycin phosphotransferase gene. This enzyme is resistant to aminoglycoside antibiotics and can be used as a marker for selecting plasmid-containing bacteria.

Table 2: List of DNA components in the PV-ZMGT10 plasmid Gene Name E35Shsp70CTP2CP4EPSPSCTP1goxNOS3'lacZori-pUCnptII SizeKb0.610.800.311.400.261.300.260.240.650.79 Functional cauliflower mosaic virus (CaMV) promoter with replication enhancer region. An intron from the maize hsp70 (heat shock protein) gene, which acts to increase gene transcription levels. Chloroplast transfer peptide is isolated from Arabidopsis thaliana EPSPS, and its function is to introduce CP4EPSPS protein into the site of aromatic amino acid synthesis in chloroplast. The gene encoding CP4EPSPS protein is isolated from Agrobacterium CP4 strain and used for the selection of transformed cells to glyphosate. The chloroplast transfer peptide is isolated from the small subunit gene of the ribulose-1,5-bisphosphate carboxylase gene in Arabidopsis thaliana, and can introduce GOX protein into the chloroplast, the site of aromatic amino acid synthesis. This gene is encoded by glyphosate, which can metabolize glyphosate oxidoreductase (GOX), and it is isolated from the LBAA strain of Achromobacter (new genus Phryobacterium). The untranslated region at the 3' end of the nopaline synthase gene terminates transcription and governs polyadenylation. One fragment is from E. coli lacl encoding promoter Plac sequence, and the other fragment is from pUC119 encoding β-D-galactosidase or LacZ protein sequence. Is the origin of replication of the pUC plasmid, allowing the plasmid to replicate in E. coli. Type II neomycin phosphotransferase gene. This enzyme is resistant to aminoglycoside antibiotics and can be used as a marker for selecting plasmid-containing bacteria.

4. Receptor material

The initial maize recipient for introducing the target gene was Hi-II, which came from a progeny produced by crossing two maize inbred lines, A188 and B73. A188 and B73 are inbred lines selected by the University of Minnesota and Iowa State University respectively, and are available in the US seed market. The receptor has a strong regenerative capacity in tissue culture stage.

5. Conversion method

After the transformation vector and the receptor system are established, the exogenous target gene cryIAb can be introduced into the receptor Hi-II through appropriate transformation methods. The transformation method used in this study is gene gun method.

DNA was precipitated onto tungsten and gold particles using calcium chloride and spermidine. The DNA-coated particles are dropped on a large plastic carrier, and the explosive force of gunpowder is used to accelerate it through the barrel to a high degree. The large carrier hits the plastic baffle, and the flight of the large carrier is blocked, but the particles wrapped in DNA continue to fly. Microparticles penetrate target plant cells, DNA precipitates, and integrates with cell chromosomes.

6. Regeneration of Transformants Screening

After transformation, transformants should be screened from untransformed cells. Cells were grown in tissue culture medium containing auxin 2,4-D. Although the DNA solution used for transformation contains genes that encode tolerance to glyphosate (such as CP4EPSPS and gox genes), which facilitates the selection of genetically altered cells in glyphosate-containing media, these genes were not detected in MON810-resistant cells. Plants of the worm strain were not present. Therefore, it is possible that cells derived from the MON810 line were "missers" in the glyphosate selection process. At a later stage, callus regenerated plants in the absence of glyphosate were assayed for the presence of the CryIAb protein product.

7. Identification and analysis of transgenic plants

DNA integrated in the MON810 line was analyzed to determine the number of insertions (number of junctions within the maize genome) and the number of copies for each gene. Identification was performed using Southern blot analysis of genomic DNA isolated from leaf tissues of control maize plants and maize plants of insect-resistant lines.

The MON810 insect-resistant maize line was produced by the gene gun method, and the DNA solution used contained two plasmids: PV-ZMBK07 [containing the cryIAb gene] and PV-ZMGT10 [containing the CP4EPSPS and gox genes]. The MON810 maize line did not incorporate the PV-ZMGT10 plasmid sequence. The strain contains an approximately 5.5Kb NdeI fragment integrated DNA, which contains an E35S promoter, hsp70 intron and cryIAb gene. The nptII gene and the backbone of the PV-ZMBK07 plasmid were not integrated. This line does not contain the CP4EPSPS, gox or nptII genes, nor does it contain the plasmid backbone of the PV-ZMGT10 plasmid. genetic component Corn Line MON810 CryIAb gene have CP4ESPSP gene No gox gene No nptII/ori-pUC No

A. Southern hybridization result

Plasmid PV-ZMBK07 has cryIAb gene, and plasmid PV-ZMGT10 has CP4EPSPS gene and gox gene. The positions of the two plasmid vectors, PV-ZMBK07 and PVZMGT10, and the restriction sites used in Southern hybridization are shown in Figure 1 .

DNA from MON818 and MON810 plants, after digestion with various restriction enzymes, was used in Southern blots to identify genes that entered the maize genome upon particle bombardment. Specifically, it is to detect the number of inserts (the number of integration sites in the maize genome), the copy number of each gene and its integrity.

B. Digestion results of NdeI

The purpose of the Ndel digestion experiment was to determine the number of plasmid DNA inserts in maize line MON810. Neither plasmid PV-ZMBK07 nor PV-ZMGT10 has an NdeI site. Thus, the enzyme efficiently cuts outside the insert, releasing a fragment containing the insert DNA and adjacent maize genomic DNA. The control DNA of MON818 and the DNA of MON810 were digested with NdeI, and the plasmid PV-ZMBK07 was used as a probe for hybridization. The results are shown in FIG. 2 . DNA from MON818 (lane 1) yields a bright but diffuse band at approximately 21.0 Kb; this is a background band as it is present in both MON818 and MON810. MON810 produced a band of approximately 5.5Kb (lane 2). This result demonstrates that the insect-resistant maize line MON810 contains an integrated DNA segment. The inserted DNA plus the adjacent maize genomic DNA up to the NdeI restriction site is about 5.5 Kb.

C. Components of the Insert

1). Integration of cryIAb gene

The DNA of MON818 and MON810 was digested with NcoI/EcoRI to generate the cryIAb gene, which was used as a probe in Southern hybridization. The experimental results are shown in Figure 3 (lanes 1-3). The positive hybridization control (lane 1) yielded a 3.46Kb fragment, corresponding to the expected size of the cryIAb gene (see Figure 1 for a schematic diagram of the plasmid). Since there is no control genomic DNA in the plasmid DNA, this band appears larger than it actually is. DNA of MON818 (lane 2) produced no band, as expected for the control line. MON810 (lane 3) yielded a band of approximately 3.1 Kb.

2). Integration of CP4ESPSP gene

Plasmid DNA (PV-ZMBK07 and PV-ZMGT10) and insect resistant maize line MON810 DNA were digested with NcoI/BamHI to generate the CP4ESPSP gene, which was used as a probe in Southern hybridization. The experimental results are shown in Figure 4 (lanes 1-2). Approximately 50 pg of mixed DNA of the two plasmids (lane 1) yielded a 3.1 Kb band corresponding to the size of the expected CP4ESPSP fragment, as estimated from the plasmid schematic (PV-ZMGT10 in Figure 1). The DNA of MON810 (lane 2) has no fragments hybridizing with the CP4ESPSP probe, which proves that the corn insect-resistant line MON810 does not have the CP4ESPSP gene.

3). Integration of gox gene

Plasmid DNA (PV-ZMBK07 and PV-ZMGT10) and insect-resistant maize line MON810 DNA were digested with NcoI/BamHI to generate gox genes, which were used as probes in Southern hybridization. The experimental results are shown in Figure 4 (lanes 3-4). About 50 pg of mixed DNA of the two plasmids (lane 3) yielded a NcoI/NcoI fragment of about 3.1 Kb, which corresponds to the size of the expected gox fragment, which is consistent with the result estimated from the plasmid schematic (PV-ZMGT10 in Figure 1) same. The DNA of MON810 (lane 4) has no fragments that hybridize with the gox probe, proving that the insect-resistant maize line MON810 has no gox gene.

4). Integration of carrier backbone

The DNA of plasmid PV-ZMBK07, control line MON818, and insect-resistant maize line MON810 were digested with NcoI/EcoRI to generate the nptII/ori-pUC backbone, and the nptII gene was used as a probe in Southern hybridization. The experimental results are shown in Figure 5 (lanes 1-3). About 50 pg of DNA from plasmid PV-ZMBK07 yielded two bands of 2.5 Kb and 1.8 Kb respectively (lane 1). The 2.5Kb and 1.8Kb bands correspond to the expected size of the fragment from vector PV-ZMBK07 (cf. Figure 1). DNA of MON818 (lane 2) produced no band, as expected for the untransformed control line. The DNA of MON810 (lane 3) did not produce a band, demonstrating that the backbone sequence of the vector was not integrated into the insect-resistant maize line MON810.

The ori-pUC region was used as a hybridization probe after the Southern blot membrane was washed. The DNA of plasmids PV-ZMBK07 and PV-ZMGT10 (lane 4) had a band of 1.8 Kb. The 1.8 Kb band corresponds to the size expected for a fragment from the PV-ZMBK07 backbone (cf. Figure 1). DNA of MON818 (lane 5) produced no band, as expected for the untransformed control line. The DNA of MON810 (lane 6) did not produce a band, proving that the backbone sequence of the vector was not integrated into the insect resistant maize line MON810. The absence of bands hybridizing to the oripUC probe and the nptII probe demonstrates that the insect resistant maize line MON810 does not contain any backbone sequences.

8. Inheritance of transgenic offspring, identification of phenotypic stability

Allelic segregation and stability data of the insect-resistant maize line MON810 were collected and sorted out, indicating that they were consistent with the insertion point data of cryIAb genomic DNA single activity.

The stability of the transferred gene and its resistance to corn borer has been confirmed in seven generations of hybridization and subsequent commercial application of the product.

9. Backcross transfer

Using conventional hybridization and backcross breeding methods, any MON810 insect-resistant corn can be used as a donor to obtain the MON810 insect-resistant corn with the genetic background we need.

In order to better understand the present invention, we set forth as follows some technologies adopted in the following examples:

1. The source of corn borer:

In this example, all the researches involving the artificial inoculation of the Asian corn borer were the overwintering populations of the Asian corn borer in Hengshui, Hebei Province. The corn borer was raised using the agar-free semi-artificial diet invented by the Corn Borer Research Office of the Plant Protection Institute of the Chinese Academy of Agricultural Sciences and obtained the national patent, and the artificial large-scale feeding method of the Asian corn borer according to Zhou Darong's (1980) standard. The egg masses and newly hatched larvae of Ostrinia sativa used in the experiment were of high quality.

2. Indoor bioassay method

The first step: according to the requirements of the following formula, prepare the basic feed for raising the Asian corn borer. Once combined, place in a food blender and blend 3 times, 1 minute each (to prevent overheating with continuous stirring).

Ingredients Dosage (g)

1 soybean flour 15.0

2 corn flour 18.0

3 Brewer's yeast 9.0

4 glucose (or edible sugar) 7.5

5 vitamins 0.5

6 erythromycin 0.15

7 Sorbic acid 0.5

8 JSMD (agar substitute) 10.0

Step 2: Weigh 45g of the tissue for identification and cut it into pieces and put it into the mixing cup; take 90ml of tap water, add 4 drops (about 0.2ml) of 40% formaldehyde and pour it into the mixing cup; turn it on and stir 3 times, each time for 1 minute , with an interval of 30 seconds.

Step 3: Mix the results of Step 1 and Step 2 evenly in a beaker to prepare two test feeds containing MON810 and non-MON810 corn tissues for later use.

Step 4: Divide the test feed into 2.5cm×8.0cm finger tubes, 6g in each tube, each receive 10 newly hatched corn borer larvae, block the mouth of the tube with a rubber stopper inlaid with copper yarn, and place it at 28- Observation was carried out after culturing for 7-10 days at a constant temperature of 30°C and 70-80% RH. Investigate the number of surviving larvae and weigh them.

3. Improved indoor bioassay method

Further improvements were made on the original laboratory bioassay method. The leaves, tassels and filaments of fresh MON810 and non-MON810 corn collected from the field were chopped separately and put into a mortar. Pour in an appropriate amount of liquid nitrogen for quick freezing, grind it into a slurry, and then mix it with a certain amount of processed agar-free semi-artificial feed to make a test feed for later use.

Take an ordinary transparent film box, punch a round hole with a diameter of 5mm on the cover, and stick a piece of stainless steel gauze inside to make a test box. Divide each test feed into 100 test boxes, about 2g per box, and connect one newly hatched Ostrinia officinalis larvae, each 10 boxes as a group, and put them under the conditions of constant temperature of 28-30°C and RH of 70-80%. After 8 days, check and record the number of live worms and the weight of the Asian corn borer in each group.

4. Leaf-eating level

The 9-level grading standard developed by the International Corn Borer Collaborative Group was adopted.

Grades 1 to 3: wormhole needle-like (also known as pinhole).

Level 1: rare, scattered,

Level 2: Medium Quantity,

Level 3: Large amount.

Level 4-6: The size of a wormhole match head.

Level 4: rare, scattered,

Level 5: Medium Quantity,

Level 6: Lots of it.

Level 7-9: The wormhole is larger than the match head.

Level 7: rare, scattered,

Grade 8: Medium Quantity,

Level 9: Lots of it.

Among them, grades 1 to 2.9 are highly resistant; grades 3 to 4.9 are resistant; grades 5 to 6.9 are moderately resistant; grades 7 to 9 are susceptible (more than grade 8 is high).

Example 1

It was demonstrated that the CryIAb protein expressed in the leaves of the pest-resistant maize line MON810 made its leaves resistant to Ostrinia sativa.

The following experiments in Example 1 were carried out in 1997 at the Corn Borer Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (Beijing). The test material MON810 maize is the hybrid B73×Mo17 MON810, not the conventional hybrid B73×Mo17 whose isotype is MON810. B73 was bred by Iowa State University in the United States, Mo17 was bred by Missouri State University in the United States, and Mo17MON810 was bred by backcrossing by Holden's Basic Seed Company in the United States. B73xMo17MON810 and B73xMo17 are commercially available from Holden's Foundation Seeds L.LC., Williamsburg, Iowa, USA.

Experiment 1: Bioassays showed that the CryIAb protein expressed in the new leaf tissue of insect-resistant maize line MON810 made its new leaf tissue resistant to Ostrinia sativa.

Weigh 45g of fresh corn heart leaf tissue, and use the indoor bioassay method for identification. It was observed that well-developed 3rd to 4th instar larvae were easily seen in the non-MON810 control tubes, but no 3rd to 4th instar larvae were seen in the MON810 tubes. In the MON810 tubes, it was visible that non-developing or dead larvae were attached to the green test feed and to the tube walls of the finger tubes.

Experiment 2: Field tests showed that the insect-resistant corn line MON810 expressed CryIAb protein in the mid-new leaf leaves of corn to resist the Asian corn borer.

The MON810 and non-MON810 corns for identification were sown on April 23, 1997, and the identification was carried out on June 17 and 20 in the mid-heart leaf stage of the corn. Each plant received 2 pieces of blackhead eggs (about 40-50 grains per piece), The results of the survey on June 28 included two indicators: one is the international leaf-eating grade, and the other is the number of surviving larvae per plant. The results, shown in Table 3, clearly demonstrate an excellent control effect: no viable larvae were found on the MON810 corn plants.

Table 3: Results of resistance to Asian corn borer at the heart leaf stage of corn

Experiment 3: CryIAb protein expressed in different leaves of corn new leaf stage by insect-resistant corn line MON810 makes different leaves of corn new leaf stage resistant to Asian corn borer

Table 4: Resistance of different leaves at the heart-leaf stage to O. corn borer

When the MON810 and non-MON810 maize plants grown in the field developed to the mid-heart leaf stage, the fifth leaf from bottom to top was marked, and then the plants were cut off at the same level as the ground and retrieved. Further mark the phyllotaxy of all the leaves from bottom to top, and cut off the stems one by one, and divide them into 5 groups, that is, the 5th and 6th leaves, the 7th and 8th leaves, the 9th and 10th leaves, and the 9th and 10th leaves. 11, 12 leaves and the last 2-3 young leaves that are not yet exhibited tightly encased in tassel buds. All 5 groups of leaves cut from 6 plants of MON810 and 6 plants of non-MON810 corn were cut into pieces, mixed in groups, processed and observed by indoor bioassay method. The results are listed in Table 4.

The results in Table 4 clearly show that the CryIAb contained in the leaf and heart leaf tissues of all MON810 from different heights has the ability to kill almost all newly hatched larvae. In all the MON810 leaf feeding experiments, among the 1,000 newly hatched larvae inoculated, only one living worm was found to be obviously stunted and extremely small in size, showing that it stayed at the 2nd instar stage, not like the non-MON810 treated control. Up to 3 to 4 years old.

Experiment 4: The CryIAb protein expressed in the upper two leaves of the insect-resistant maize line MON810 makes the upper two leaves of the young tassel resistant to the Asian corn borer

The shoots of MON810 and non-MON810 maize plants at the tasseling stage were taken back to the laboratory, and the top two pieces enclosing the tassels were cut off. The bioassay method was the same as before, and the results showed that the two uppermost leaves that wrapped the young tassels contained enough CryIAb toxin protein, and the mortality rate of newly hatched larvae reached 100% (see Table 5).

Table 5: Resistance of the last two leaves of the tassel to O. corn borer

12 9 0 13 8 0 14 10 0 15 8 0 16 8 0 17 9 0 18 10 0 19 9 0 20 9 0 twenty one 9 0 twenty two 10 0 twenty three 9 0 twenty four 8 0 25 4 0 26 10 0 27 10 0 28 8 0 29 10 0 30 9 0 Total 262 0 average 8.7333 0

Experiment 5: Insect-resistant corn line MON810 expressed CryIAb protein in the two leaves wrapped tassels at the tasseling stage, making the two leaves wrapped tassels resistant to Asian corn borer

The shoots of MON810 and non-MON810 maize plants at the tasseling stage were taken back to the laboratory, and the two leaves enclosing the tassels were cut off. The resistance was determined according to the improved indoor bioassay method, and the results are listed in Table 6. It can be seen from Table 6 that the treatment of MON810 corn leaves contained enough CryIAb protein, and the larval mortality of Ostrinia officinalis was 99% and 100%, respectively, while that of non-MON810 was only 8.73%; and the difference in body weight of surviving larvae between MON810 and non-MON810 very big.

Table 6: Insecticidal activity of the two leaves wrapped tassels at the male stage

Treatment                                                                                                            

Feed: Tissue Number of Infested Insects Number of Surviving Insects (g) (%) Average (mg) Average

1:1 Non-MON810 11 8 0.1822 27.27 8.73 22.7750 21.0664

10 8 0.2357 20 29.4625

                                                                                                                             

12 12 0 2065 0 17.2083

                                                                                                         

10 10 0 1945 0 19.4500

10 9 0.1692 10 18.8000

10 9 0 1957 10 21.7444

10 10 0.2046 0 20.4600

                                                                                                  , 

1:1 MON810 10 0 0 100 99 0.0

10 0 100

10 0 100

10 0 100

10 0 100

10 1 0.0000 90 0.0

10 0 100

10 0 100

10 0 100

10 0 100

3:2 MON810 10 0 0 100 100

10 0 100

10 0 100

10 0 100

10 0 100

10 0 100

10 0 100

10 0 100

10 0 100

10 0 100

Experiment 6: The CryIAb protein expressed in the 5 unfolded leaves below the two leaves wrapping the tassel of the insect-resistant corn line MON810 at the tasseling stage made the five unfolded leaves below the two leaves wrapping the tassel resistant to the Asian corn borer

The shoots of MON810 and non-MON810 maize plants at the tasseling stage were taken back to the laboratory, and the five expanded leaves below the two leaves covering the tassels were cut off. The resistance was determined according to the improved indoor bioassay method, and the results are listed in Table 7. It can be seen from Table 7 that the treatment of MON810 corn leaves contained enough CryIAb protein, and the larval mortality of Ostrinia cerevisiae was 98% and 100%, respectively, while that of non-MON810 was only 3% and 6.91%; and MON810 and non-MON810 survived Larval body weights vary widely.

Table 7: Insecticidal activity of 5 unfolded leaves below the two leaves that wrap tassels at the male tassel stage

The total weight mortality of larvae (head) the number of survivors (heads) of the number of live insects/heads/head

Feed: Tissue Number of Infested Insects Number of Surviving Insects (g) (%) Average (mg) Average

3:2 Non-MON810 8 8 8 0.178 0 3 22.2500 19.8464

10 8 0.137 20 17.1250

11 11 0.2049 0 18.6273

10 10 0.184 0 18.4000

                                                                                , 

10 10 0.1754 0 17.5400

10 9 0.1703 10 18.9222

                                                                             ,

                                                                               , 

                                                                                                        , 

3:2 MON810 10 1 0.0000 90 98 0.0 0.0

10 0 0 100

10 0 0 100

10 0 0 100

10 0 0 100

10 0 0 100

10 1 0.0000 90 0.0

10 0 100

10 0 100

10 0 100

1:1 Non-MON810 10 8 0.18 20 6.91 22.5000 22.3414

10 10 0.2482 0 24.8200

10 10 0.2242 0 22.4200

10 8 0.1484 20 18.5500

10 10 0.2238 0 22.3800

                                                                                                                                                      , 

                                                                                                                                                      

10 8 0.1743 20 21.7875

10 10 0.1987 0 19.8700

10 10 0.2358 0 23.5800

1:1 MON810 10 0 100 100

10 0 100

10 0 100

10 0 100

10 0 100

10 0 100

10 0 100

10 0 100

10 0 100

10 0 100

Example 2

It was demonstrated that the CryIAb protein expressed in the tassels of insect-resistant maize line MON810 rendered the tassels resistant to O. corn borer.

The following experiments in this example were carried out in the Corn Borer Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (Beijing) in 1997. The test material MON810 maize is the hybrid B73×Mo17 MON810, not the conventional hybrid B73×Mo17 whose isotype is MON810. B73 was bred by Iowa State University in the United States, Mo17 was bred by Missouri State University in the United States, and Mo17MON810 was bred by backcrossing by Holden's Basic Seed Company in the United States. B73xMo17MON810 and B73xMo17 are commercially available from Holden's Foundation Seeds L.L.C., Williamsburg, Iowa, USA.

Experiment 1:

The identification technique of tassel resistance is to put 2 pieces of blackhead eggs on young tassels, then put a pollination bag and tie the mouth of the bag tightly to reduce escape or pecking by birds. On July 4, 1997, 26 corn plants of MON810 and non-MON810 were inoculated, and the number of surviving larvae was investigated on July 16. The results are shown in Table 8. The tassels of MON810 and non-MON810 were significantly different. The tassels of MON810 corn contained enough CryIAb protein, no surviving larvae were found, and the mortality of larvae reached 100%.

Table 8: Resistance of MON810 tassels to corn borer

18 12 0 19 16 0 20 6 0 twenty one 11 0 twenty two 13 0 twenty three 19 0 twenty four 11 0 25 twenty one 0 26 7 0 Total 365 0 average 14.04 0

Experiment 2:

The indoor bioassay method is the same as before, except that the amount of water added when crushing the tissue is reduced by 10ml, because the water content of fresh tassels is much higher than that of heart leaves. The results showed that MON810 maize tassels contained sufficient CryIAb protein, and the mortality rate of newly hatched larvae reached 100% (see Table 9).

Table 9: Resistance of tassel buds to corn borer

11 7 0 12 10 0 13 9 0 14 8 0 15 9 0 16 9 0 17 10 0 18 10 0 19 7 0 20 8 0 twenty one 9 0 twenty two 7 0 twenty three 10 0 twenty four 9 0 25 8 0 26 8 0 27 7 0 28 9 0 29 9 0 30 7 0 total 251 0 average 8.3667 0

Experiment 3: CryIAb protein expressed in pre-pollination tassels makes pre-pollination tassels resistant to O. corn borer

Using the aforementioned improved indoor measurement method, the results show that the feed containing the tassel tissue before the loose powder of MON810 corn can kill more than 93% of the newly hatched larvae of O. Sufficient CryIAb protein.

Table 10: Resistance of tassels before pollination to O. corn borer

                                                                                       

Feed: Tissue Number of Infested Insects Number of Surviving Insects (g) (%) Average (mg) Average

3∶2 Non-MON810 10 10 0.1501 0 1.25 15.01 17.3226

11 11 0.2123 0 19.30

10 10 0.1513 0 15.13

10 9 0.207 10 23.00

11 11 0.1572 0 14.29

10 10 0.1352 0 13.52

10 10 0.1871 0 18.71

10 10 0.1962 0 19.62

3:2 MON810 11 1 0.0002 90.91 93.98 0.20 0.1500

10 1 0.0001 90 0.10

11 1 0.0000 90.91 0.0

10 0 100

10 1 0.0002 90 0.20

10 0 100

10 0 100

10 1 0.0001 90 0.10

1:1 Non-MON810 10 9 0.09 10 2.50 10.00 14.2721

10 9 0.1905 10 21.17

10 10 0.1252 0 12.52

10 10 0.1402 0 14.02

10 10 0.1134 0 11.34

10 10 0.1598 0 15.98

10 10 0.1344 0 13.44

10 10 0.1571 0 15.71

1:1 MON810 11 2 0.0002 81.82 95.23 0.10 0.2333

10 0 100

10 1 0.0003 90 0.30

10 0 100

10 0 100

10 1 0.0003 90 0.30

10 0 100

10 0 100

Example 3

It was demonstrated that the CryIAb protein expressed in the anthers of insect-resistant maize line MON810 made its anthers resistant to Ostrinia sativa.

The experiment was carried out in 1997 in the Corn Borer Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (Beijing). The test material MON810 maize is the hybrid B73×Mo17MON810, and the non-MON810 is the early generation line MC-37. B73 was bred by Iowa State University in the United States, Mo17 was bred by Missouri State University in the United States, and Mo17MON810 was bred by Holden's Basic Seed Company in the United States through backcrossing. B73 x Mo17MON810 is commercially available from Holden's Foundation Seeds L.L.C., Williamsburg, Iowa, USA. The non-MON810 corn is the early generation line MC-37, provided by the Corn Borer Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

Collection of anthers: Cover the tassels in the pollination stage with pollination bags, shake the tassels the next day, separate the anthers scattered in the bags from the pollen with a sampling sieve, and bring them back indoors. The bioassay method was the same as before.

The results showed that the anthers of MON810 corn contained enough CryIAb protein, and the feed containing the anthers of MON810 corn could kill 100% of the newly hatched larvae of Ostrinia sativa (see Table 11).

       Table 11: Insecticidal activity of anthers

Treatment Number of larvae Total weight of surviving insects Mortality rate Insect weight/head

Infestation Number Surviving Insects (g) (%) Average (mg) Average

MON810 5 0 0 100 100

5 0 0 100

5 0 0 100

5 0 0 100

5 0 0 100

5 0 0 100

5 0 0 100

5 0 0 100

5 0 0 100

5 0 0 100

MC-37(CK) 5 3 0.0101 40 16 3.37 4.7682

5 5 0.0341 0 6.82

5 4 0.0204 20 5.10

5 5 0.0167 0 3.34

5 4 0.0171 20 4.28

5 5 0.0077 0 1.54

5 3 0.0228 40 7 60

5 4 0.0222 20 5.55

5 5 0.0172 0 3.44

5 4 0 0266 20 6.65

Example 4

It was demonstrated that the CryIAb protein expressed in the filaments of insect-resistant maize line MON810 made its filaments resistant to O. corn borer.

The following experiments in this example were carried out in 1997 at the Corn Borer Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (Beijing). The test material MON810 maize is the hybrid B73×Mo17 MON810, not the conventional hybrid B73×Mo17 whose isotype is MON810. B73 was bred by Iowa State University in the United States, Mo17 was bred by Missouri State University in the United States, and Mo17MON810 was bred by backcrossing by Holden's Basic Seed Company in the United States. B73xMo17MON810 and B73xMo17 are commercially available from Holden's Foundation Seeds L.L.C., Williamsburg, Iowa, USA.

Experiment 1:

Bunches of MON810 and non-MON810 fresh corn filaments collected from the field are soaked in clean water to keep them fresh, and then put into a plastic tank with a diameter of 11.5cm and a height of 10.5cm after a little drying, with a sheet at the bottom of the tank Filter paper, connect 2 pieces of blackhead eggs (about 30 pieces/piece) on the silk of each jar. Then, put an identical empty tank on each plastic tank, but there are 4 equidistant holes in the middle of the tank wall, each hole is inlaid with stainless steel yarn to prevent the larvae from escaping after hatching. Afterwards, glue them into a whole with adhesive tape, place them under constant temperature of 28-30°C and 70-80% RH, cultivate them for 6 days and then observe them.

The results showed that after 6 days of feeding, the filaments of the MON810 corn were still fresh and intact, while the filaments of the control non-MON810 corn had all been eaten up, leaving only dark brown feces and debris, and a live larva was also found in the treatment tank of MON810 No, the difference between the two is extremely obvious. It can be seen that the silk of MON810 corn contains enough CryIAb protein.

Experiment 2:

Select the newly hatched larvae, and use the inoculation gun (Bazooka) invented by CIMMYT entomologist Dr. Mihm to inoculate the newly hatched larvae on the fresh filaments just spit out from the corn. The survey results are shown in Table 12. It can be seen that the filaments of MON810 corn and non-MON810 corns show very obvious differences, therefore, the filaments of MON810 corn contain enough CryIAb protein.

Table 12: Resistance of MON810 corn filaments Plant number Number of surviving larvae (head/plant) Non-MON810 MON810 1 4 0 2 4 0 3 3 0 4 3 0 5 6 0 6 5 0 7 4 0 8 7 0 9 4 0 10 3 0 11 5 0 12 3 0 13 5 0 14 2 0 15 3 4 16 3 0 17 8 0 18 5 0 19 11 0 20 6 0 twenty one 14 0 twenty two 13 0 twenty three 15 0 twenty four 29 0 25 twenty one 0 26 twenty one 0 27 32 0 28 14 0 29 twenty four 0 30 27 0 total 304 4 average 10.13 0.19

Experiment 3:

The experiment was carried out in the Corn Borer Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences in 1997, and the experimental method used an improved indoor bioassay method. The results are listed in Table 13. The MON810 corn filaments contain enough CryIAb protein, and the feed formulated with it can kill more than 96% of the newly hatched larvae of Ostrinia officinalis, and the surviving larvae stagnate.

                                                                                      

Treatment Number of larvae Total weight of surviving insects Mortality rate Insect weight/head

Feed: Tissue Number of inoculated insects Number of surviving insects (g) (%) Average% (mg) Average

3:2 Non 10 9 0.1379 10 13.75 15.32 12.16

MON810

10 8 0.0884 20 11.05

10 9 0.1131 10 12.57

10 8 0.0861 20 10.76

10 6 0.0432 40 7.20

10 10 0.1543 0 15.43

10 10 0.089 0 8.90

10 9 0.1446 10 16.07

3:2 MON810 10 0 0 100 98.75 0.0

10 0 0 100

10 0 0 100

10 0 100

10 0 100

10 1 0.0000 90 0.0

10 0 100

10 0 100

1:1 Non 10 7 0.116 30 10.81 16.57 14.08

MON810

10 10 0.16 0 16.00

10 10 0.1734 0 17.34

10 9 0.1209 10 13.43

10 9 0.0897 10 9.97

10 10 0.1578 0 15.78

10 8 0.1003 20 12.54

11 10 0.1209 9.0909 12.09

11 9 0.1174 18.1818 13.04

1:1 MON810 11 2 0.0006 81.8182 96.18 0.30 0.27

10 0 100

10 1 0.0003 90 0.30

10 0 100

10 0 100

10 0 100

10 1 0.0002 90 0.20

10 0 100

10 0 100

10 0 100

Example 5

It was proved that CryIAb protein can increase the yield of insect-resistant maize line MON810 under the condition of artificially inoculating with Ostrinia sativa in the field.

The experiment was carried out in 1998 at 4 sites located in the main corn planting area of northern China, Shenyang Academy of Agricultural Sciences, Henan Academy of Agricultural Sciences, Jilin Academy of Agricultural Sciences and Jilin Provincial Crop New Variety Introduction Center. A total of 14 maize hybrids were tested, including 6 pairs of homohybrids and two local main varieties (control). Hybrids assembled with the LH line can be purchased from Holden's Foundation Seeds L.L.C., Williamsburg, Iowa, USA, and other homohybrids can be purchased from Monsanto, USA. Local control seeds are available in local seed markets in China. Each pair of isohybrids is composed of a MON810 insect-resistant hybrid and a non-MON810 conventional hybrid. Composition is consistent. Apparently, the differences shown between isohybrids were due to the presence or absence of the CryIAb protein.

Inoculation was performed in three stages throughout the corn growing season. For the first time, at the new leaf stage, two pieces of blackhead eggs (about 50-60 grains/piece) of the Asian corn borer were put into the upper new leaves of the plant; for the second time, at the budding stage before tasseling, the upper leaves were inoculated with two Asian corn borer black head eggs; the third time at the silking stage, two pieces of Asian corn borer black head eggs were inoculated in the freshly spun silk of corn. In this way, the inoculum amount of each corn plant is about 300 to 360 black-headed eggs of the corn borer in total. The yield results are shown in Table 14.

Table 14 shows that the MON810 hybrid that can encode the CryIAb protein exhibits a significant yield-increasing effect under inoculation conditions, compared with its isotype non-MON810 hybrid. It can be seen from Table 14 that all single pairs of isotype hybrids show increased yield of MON810 hybrids in all test locations, with the lowest yield increasing rate being 6.7% and the highest yield increasing rate being 89.5%. In all 4 test sites, the average yield per hectare of the 6 non-MON810 hybrids was only 7103 kg, while the average yield per hectare of their 6 isotype MON810 hybrids reached 9102 kg, and the grain yield increased by 1999 kg per hectare. 28.1%. In the 4 test points, the increase rate of MON810 hybrids relative to non-MON810 hybrids was highest in Henan Academy of Agricultural Sciences, 69.1%, followed by Shenyang Academy of Agricultural Sciences, 29.5%, followed by Jilin Academy of Agricultural Sciences, 25.1%, and the lowest There are also 15.5% for the introduction center. Although the range of yield increase of MON810 relative to non-MON810 was different with the 6 pairs of tested cross combinations, they all showed yield increases, ranging from 19.8% to 32.9%. It can be concluded that compared with non-MON810 maize hybrids not containing the CryIAb-encoding protein, the yield level of the MON810 maize hybrids encoding the CryIAb protein is higher, and the increased yield is due to the resistance of the CryIAb protein to O. corn borer.

Table 14: Output results of Monsanto MON810 effect verification test in 1998 hybrid name pedigree Shenyang Academy of Agricultural Sciences isotropic Henan Academy of Agricultural Sciences isotropic Jilin Academy of Agricultural Sciences isotropic Induction center isotropic average of each point isotropic kg/ha ±% kg/ha ±% kg/ha ±% kg/ha ±% kg/ha ±% FBH8-1 LH198×LH185MON810 10792 28.2 6375 48.8 9823 59.7 11150 11.5 9535 32.2 FNH8-1 LH198×LH185 8417 4283 6149 10000 7212 FBH8-2 LH200×LH185MON810 8959 16.2 4333 38.7 9356 6.7 12750 28.1 8850 19.8 FNH8-2 LH200×LH185 7709 3125 8767 9950 7388 FBH8-3 LH198×LH172MON810 9125 43.1 6050 82.4 9743 20.8 12325 10.0 9311 28.6 FNH8-3 LH198×LH172 6375 3317 8068 11200 7240 FBH8-4 LH200MON810×LH172 7292 50.8 5842 89.5 9188 27.0 11000 10.8 8330 32.9 FNH8-4 LH200×LH172 4834 3083 7232 9925 6269 FNA8-1 7051/Mo17 6959 4025 6846 11825 7414 FBA8-1 7051/Mo17MON810 9500 36.5 6892 71.2 8892 29.9 12825 8.5 9527 28.5 FNA8-2 7054/Mo17 7709 3433 8023 9225 7098 FBA8-2 7054/Mo17MON810 8709 13.0 6467 88.3 9384 17.0 11675 26.6 9059 27.6 CK1 8834 3067 9642 8500 7511 CK2 9125 4875 7962 9000 7740 MON810 hybrid average 9063 29.5 5993 69.1 9398 25.1 11954 15.5 9102 28.1 Average non-MON810 hybrids 7001 3544 7514 10354 7103

Introduction and Breeding Center; Jilin Provincial Crop New Variety Introduction and Breeding Center

Yield increase of the same type: the yield increase of the MON810 hybrid relative to the conventional hybrid of the same type

Example 6

It was proved that the CryIAb protein can increase the yield of the insect-resistant corn line MON810 under the condition of Ostrinia sativa naturally occurring in the field.

The experiment was carried out in Shenyang Academy of Agricultural Sciences, Shandong Agricultural University, Dandong Academy of Agricultural Sciences, Jilin Academy of Agricultural Sciences and Jilin Province Crop New Variety Introduction and Breeding Center in 1998. These sites are perennially infested by the Asian corn borer and cause some yield loss. A total of 22 maize hybrids were tested, 20 of which were composed of 10 pairs of isotype hybrids, and the other 2 were the main local maize hybrids (control). Hybrids assembled with the LH line can be purchased from Holden's Foundaion Seeds L.L.C., Williamsburg, Iowa, USA, and other homohybrids can be purchased from Monsanto, USA. Local control seeds are available in local seed markets in China. Homohybrids are as described in Example 5. The test was carried out under the natural conditions of the field. All the plants were not artificially inoculated with the Asian corn borer, and all the damage of the Asian corn borer occurred under the natural conditions of the field. Yield results are listed in Table 15.

It can be seen from Table 15 that each pair of homotype hybrids shows that the yield of MON810 hybrids is higher than that of its homotype non-MON810 hybrids in all test locations. In all five test sites, the average yield per hectare of the 10 non-MON810 hybrids was only 8,066 kg, while their average yield per hectare of the 10 isotype MON810 hybrids reached 9,645 kg, increasing grain yield by 1,579 kg per hectare. 19.6%. For the 5 test points, compared with non-MON810 hybrids, Dandong Academy of Agricultural Sciences had the highest yield increase rate of 46.3%, Jilin Academy of Agricultural Sciences had the lowest rate of 9.8%, Shenyang Academy of Agricultural Sciences, Shandong Agricultural University and the breeding center are in the middle, accounting for 22.6%, 15.0% and 13.4% respectively. Although the range of yield increase of MON810 relative to non-MON810 was different with the 10 pairs of tested hybrid combinations, they all showed yield increases, ranging from 12.3% to 26.4%. In this experiment, it can be concluded that compared with non-MON810 corn hybrids that do not contain the CryIAb protein, the yield of the MON810 corn hybrid that encodes the CryIAb protein is higher, and the increase in yield is due to the resistance of the CryIAb protein to the Asian corn borer. .

Table 15: 1998 Monsanto MON810 Hybrid Comparative Test Yield Results hybrid name pedigree Shenyang Academy of Agricultural Sciences isotropic Shandong Agricultural University isotropic Dandong Academy of Agricultural Sciences isotropic Jilin Academy of Agricultural Sciences isotropic Induction center isotropic average of each point isotropic kg/ha ±% kg/ha ±% kg/ha ±% kg/ha ±% kg/ha ±% kg/ha ±% VBH8-1 LH198×LH185MON810 11083 36.4 8350 21.9 10437 35.6 10876 6.7 12700 25.7 10689 24.4 VNH8-1 LH198×LH185 8126 6850 7698 10194 10100 8594 VBH8-2 LH200×LH185MON810 9708 20.1 7300 20.5 10389 18.4 11541 3.7 12275 7.9 10243 12.8 VNH8-2 LH200×LH185 8084 6058 8778 11125 11375 9084 VBH8-3 LH198×LH172MON810 7751 13 4 5650 0.9 8198 33.3 9828 0.0 11475 17.1 8580 12.3 VNH8-3 LH198×LH172 6834 5600 6151 9825 9800 7642 VBH8-4 LH200MON810×LH172 6875 5.8 7325 18.9 7476 62.7 11196 42.2 11850 15.6 8944 26.4 VNH8-4 LH200×LH172 6500 6158 4595 7873 10250 7075 VBA8-1 7054×Mo17MON810 9251 36.2 6825 15.8 8564 23.7 10259 5.9 11850 6.8 9350 15.7 VNA8-1 7054×Mo17 6792 5892 6921 9692 11100 8079 VBA8-2 7064×Mo17MON810 9125 27.3 6892 14.2 10984 77.7 11114 4.4 12275 19.8 10078 25.1 VNA8-2 7064×Mo17 7167 6033 6183 10647 10250 8056 VBA8-3 7051×Mo17MON810 9626 14.9 5742 31.0 8944 67.7 11852 5.1 13275 18.3 9888 21.8 VNA8-3 7051×Mo17 8376 4383 5333 11276 11225 8119 VBA8-4 7098×Mo17MON810 8000 9.7 6133 8.9 8444 107.4 10608 26.0 12400 7.1 9117 23.2 VNA8-4 7098×Mo17 7293 5633 4071 8416 11575 7398 VBA8-5 7195×Mo17MON810 8000 46.5 5967 6.2 8556 84.6 12314 12.8 11800 6.8 9327 23.8 VNA8-5 7195×Mo17 5459 5617 4635 10915 11050 7535 VBA8-6 7145×Mo17MON810 10959 20.6 6375 12.7 10103 18.0 11112 2.6 12625 12.0 10235 12.7 VNA8-6 7145×Mo17 9084 5658 8564 10826 11275 9081 CK1 9584 6792 5754 11396 9325 8570 CK2 8084 8058 7135 10492 8975 8549 MON810 hybrid average 9038 22.6 6656 15.0 9210 46.3 11070 9.8 12253 13.4 9645 19.6 Average non-MON810 hybrids 7372 5788 6293 10079 10800 8066

Introduction and Breeding Center: Jilin Provincial Crop New Variety Introduction and Breeding Center

Yield increase of the same type: the yield increase of the MON810 hybrid relative to the conventional hybrid of the same type

Example 7

It was proved that under the condition of natural occurrence of Ostrinia sativa in the field, the expression of CryIAb protein in the insect-resistant corn line MON810 resulted in increased grain weight, improved stalk resistance (lodging resistance, reduced stalk rot), and other traits. Has little effect.

The test method is the same as in Example 6, and the results are shown in Table 16 below.

It can be seen from Table 16 that the CryIAb protein contained in the MON810 hybrid not only increases the thousand-grain weight of corn kernels, but also reduces stem rot and lodging due to the improvement of stem quality. In terms of thousand-kernel weight, MON810 hybrids averaged 348 grams, while non-MON810 hybrids averaged 321 grams, and MON810 hybrids increased by 27 grams, or 8.4%. Stem rot, root lodging and stem lodging averaged 9%, 5% and 0% for MON810 hybrids, respectively, compared to 19%, 9% and 1% for the corresponding non-MON810 hybrids. Apparently, this is a consequence of the reduced physical and physiological damage caused by the reduction of O. corn borer in corn plants containing the CryIAb protein.

It can also be seen from Table 16 that in terms of plant height, ear height and growth period these plant traits and growth traits, the average of 6 MON810 hybrids is 269cm, 100cm and 110 days, and the corresponding 6 isotype non-MON810 hybrids The two species are 268cm, 99cm and 110 days respectively, and there is no significant difference between the two. The main leaf diseases and ear traits, such as gray spot, large leaf spot, small spot, ear length, ear thickness, ear row number, seed yield, except that there is a slight difference in the seed yield (87% vs. 86%), the rest of the traits are completely the same.

Table 16: 1998 Monsanto MON810 Hybrid Comparison Test Main Character Results hybrid name pedigree plant height spike height Growth Period Head smut Smut stem rot rough dwarf disease gray spot Great Spot small spot disease root lodging stem lodging Empty shot rate ear length ear thickness Ear row number Seed yield Thousand Kernel Weight cm cm sky % % % % class class class % % % cm cm % g VBH8-1 LH198×LH185MON810 286 110 111 0.0 0.0 3.6 16.9 2.0 0.0 0.8 5.2 0.0 1.6 21.0 5.0 14.2 87.2 385.7 VNH8-1 LH198×LH185 276 94 111 0.0 0.0 6.4 9.0 2.0 0.0 0.8 10.2 3.8 11.5 19.5 4.8 14.1 86.8 337.5 VBH8-2 LH200×LH185MON810 277 109 110 0.0 0.0 17.1 8.0 1.5 0.0 0.5 12.4 0.0 1.7 20.8 4.9 15.0 86.5 375.0 VNH8-2 LH200×LH185 267 100 111 13.3 0.0 10.3 6.5 1.5 0.0 1.3 12.6 1.7 4.9 20.1 5.0 15.6 87.8 350.0 VBH8-3 LH198×LH172MON810 233 84 108 0.0 0.0 30.2 8.9 3.0 0.0 1.8 0.9 0.0 0.0 17.8 5.0 16.6 89.2 286.9 VNH8-3 LH198×LH172 232 83 107 0.0 0.0 24.7 6.9 3 0 0.0 1.5 1.4 0.0 3.4 18.1 5.0 16.6 86.8 273.0 VBH8-4 LH200MON810×LH172 238 86 108 0.0 0.0 16.2 5.0 2.0 0.2 1.8 4.9 0.0 0.0 18.8 4.9 16.4 88.1 282.5 VNH8-4 LH200×LH172 229 87 108 0.0 0.0 13.4 8.9 2.0 0.2 1.8 4.0 0.0 1.8 18.3 4.8 17.4 85.0 260.2 VBA8-1 7054×Mo17MON810 259 98 110 0.0 0.0 2.9 5.9 2.0 0.0 1.8 3.2 0.0 0.0 22.0 4.6 14.1 88.2 353.9 VNA8-1 7054×Mo17 270 104 110 0.0 0.0 28.9 20.9 2.0 0.0 1.3 3.8 0.0 7.0 21.2 4.6 14.1 86.2 321.9 VBA8-2 7064×Mo17MON810 275 99 111 0.0 0.0 3.2 10.9 2.0 0.0 1.3 5.7 0.0 0.0 20.0 4.7 13.9 86.7 322.7 VNA8-2 7064×Mo17 271 102 110 3.1 0.0 23.7 15.8 2.0 0.2 1.3 15.5 1.7 0.0 20.2 4.7 15.2 86.3 311.1 VBA8-3 7051×Mo17MON810 281 96 110 0.0 0.0 5.9 14.0 1.0 0.2 1.3 5.7 0.0 5.3 21 4 4.7 14.1 85.3 367.2 VNA8-3 7051×Mo17 285 97 109 0.0 0.0 30.0 10.0 2.0 0.2 1.5 11.4 0.0 0.0 21.0 4.7 14.2 84.7 341.2 VBA8-4 7098×Mo17MON810 282 110 111 0.0 0.0 3.1 9.5 2.0 0.2 2.0 6 8 1.8 1.8 20.5 4.6 13.9 86.7 378.2 VNA8-4 7098×Mo17 281 113 110 0.0 0.0 27.1 6.5 2.0 0.0 1.5 6.9 0.0 1.8 20.4 4.7 13.8 84.2 336.8 VBA8-5 7195×Mo17MON810 276 104 112 0.0 0.0 4.7 9.6 1.0 0.2 1.8 5.3 0.0 7.0 21.8 4.7 14.6 86.8 339.3 VNA8-5 7195×Mo17 276 106 112 0.0 0.0 16.9 7.5 1.0 0.7 1.0 7.4 0.0 14.0 21.3 4.7 14.9 84.1 328.0 VBA8-6 7145×Mo17MON810 283 100 111 0.0 0.0 3.3 17.0 1.0 0.2 1.0 3.5 0.0 0.0 20.8 4.8 14.0 87.1 385.0 VNA8-6 7145×Mo17 290 104 111 0.0 0.0 12.1 16.5 1.0 0.2 1.8 12.9 0.0 3.4 20.4 4.8 14.1 86.4 345.7 MON810 hybrid average 269 100 110 0 0 9 11 2 0 1 5 0 2 20 5 15 87 348 Average non-MON80 hybrids 268 99 110 2 0 19 11 2 0 1 9 1 5 20 5 15 86 321

Example 8

It was proved that under the condition of artificial inoculation with O. corn borer in the field, the expression of CryIAb protein in the insect-resistant corn line MON810 made the grain weight increase and stem resistance improvement (lodging resistance, reduction of stem rot), while other traits have little effect.

The test method is the same as in Experimental Example 5, and the observation results are shown in Table 17.

It can be seen from Table 17 that under the condition of artificial high-dose inoculation, the MON810 hybrids that can express CryIAb protein and the non-MON810 hybrids have different effects on plant height, ear height, growth period, large spot disease, small spot disease, and coarse shrinkage. There was no big difference in main traits such as disease, gray spot, ear length, ear thickness, ear row number and seed yield. However, in terms of grain weight and stalk resistance, MON810 showed not only increased thousand-grain weight, but also enhanced plant stalk resistance (decreased lodging and reduced stalk rot). The mean values of 1000-kernel weight, lodging degree and stalk rot of MON810 hybrids were 330 g, 12% and 7%, respectively, while the corresponding values of non-MON810 hybrids were 313 g, 20% and 12%, respectively. Apparently, this is a consequence of the reduced physical and physiological damage caused by the reduction of O. corn borer in corn plants containing the CryIAb protein.

Table 17: 1998 Monsanto MON810 Hybrid Effectiveness Verification Test Main Character Result Table hybrid name pedigree plant height spike height Growth Period big spot small spot stem rot black powder silk smut rough dwarf disease gray spots lodging ear length ear thickness Ear row number Seed yield Thousand Kernel Weight cm cm sky class class % class class % class % cm cm % g FBH8-1 LH198×LH185MON810 281 111 110 1.0 1.2 4.0 0.0 0 10.0 2 5.7 18.9 4.70 14.1 85.2 380 FNH8-1 LH198×LH185 273 100 109 0.7 1.5 13.9 0.0 0 22 0 1 12.9 18.4 4.78 13.9 86.5 353 FBH8-2 LH200×LH185MON810 270 107 111 1.0 1.7 21.5 0.0 0 23.0 2 25.7 19.6 4.93 15.3 85.8 361 FNH8-2 LH200×LH185 258 96 111 0.7 1.7 9.9 6.0 0 23.0 2 17.7 19.2 4.88 16.2 86.6 333 FBH8-3 LH198×LH172MON810 232 84 108 1.0 2.5 17.4 0.0 0 26.0 4 1.7 18.4 4.72 16.6 87.4 279 FNH8-3 LH198×LH172 241 87 108 1.3 3 25.3 0.0 0 25.0 3 3.7 17.2 4.83 16.4 86.7 268 FBH8-4 LH200MON810×LH172 247 94 110 1.7 2.2 12.0 0.0 0 36.0 3 0.0 18.3 4.88 17.7 86 277 FNH8-4 LH200×LH172 245 89 109 2.0 2.5 34.7 1.8 0 16.0 2 0.6 17.6 4.83 16.6 85.1 266 FNA8-1 7051/Mo17 267 102 109 1.0 2.7 15.1 0.0 0 13.0 2 26.2 19.4 4.63 14.5 83.7 351 FBA8-1 7051/Mo17MON810 275 101 110 1.0 2.3 8.2 0.0 0 11.0 1 8.0 19.8 4.77 13.9 85.1 368 FNA8-2 7054/Mo17 256 99 110 1.0 2.7 21.0 0.0 0 17.0 3 10.1 19.5 4.57 14.0 87.9 307 FBA8-2 7054/Mo17MON810 256 96 111 1.7 2.3 10.8 0.0 1 6 17.0 2 1.3 21.5 4.45 14.0 85 316 MON810 hybrid average 260 99 110 1 2 12 0 0 twenty one 2 7 19 5 15 86 330 Average non-MON810 hybrids 257 96 109 1 2 20 1 0 19 2 12 19 5 15 86 313

Example 9

It was proved that CryIAb expressed in the leaves, stems and plants of insect-resistant maize line MON810 made its leaves, stems and plants resistant to Ostrinia sativa under field inoculation conditions.

The test method is the same as in Example 5, and the measurement items include the number of wormholes per plant, the level of leaf-eating, the length of the tunnel per plant and the rate of damaged plants. The observation results are shown in Table 18.

It can be seen from Table 18 that for the 6 pairs of isotype hybrids tested, although the values of the 4 index traits fluctuate with different combinations, the damage degree of the MON810 hybrid is far lower than that of the Asian corn borer. Non-MON810 hybrids of its homotype. The six MON810 hybrids tested were 0.04, 0.52, 0.03cm and 1.67% in terms of the four index traits of wormhole number per plant, leaf-eating level, tunnel length per plant and damage rate, respectively, while the corresponding The six non-MON810 are 5.48, 3.92, 11.09cm and 85%, and the non-MON810 hybrids are 137 times, 7 times, 370 times and 50 times that of the MON810 hybrids. The four index traits of the two local conventional control varieties were 5.81, 2.39 grade, 13.56cm and 85.4%. Compared with the non-MON810 hybrids, it can be found that the infection degree of the two varieties to Ostrinia sativa is consistent. It can be seen that the MON810 hybrid that can encode the CryIAb protein suffers significantly less damage from O. corn borer than the two local controls and the homohybrid that do not encode the CryIAb protein. Therefore, Table 18 shows that under the artificial inoculation treatment, the leaves, stalks and whole plants of the corn hybrids containing the CryIAb protein were far less damaged by the Asian corn borer than those without the CryIAb protein. Obviously, this comes from the control effect of the CryIAb protein on the Asian corn borer.

Table 18: 1998 Monsanto MON810 Hybrid Effect Verification Test Results of Main Hazardous Traits hybrid name Pedigree source Number of wormholes per plant Leafy grade Single plant tunnel length Hazard rate indivual class cm % FBH8-1 LH198×LH185MON810 0.00 0.41 0.00 0.0 FNH8-1 LH198×LH185 4.78 2.67 11.89 87.5 FBH8-2 LH200×LH185MON810 0.08 0.37 0.03 4.2 FNH8-2 LH200×LH185 4.43 4.04 11.22 85.8 FBH8-3 LH198×LH172MON810 0.06 0.52 0.07 3.3 FNH8-3 LH198×LH172 4.89 2.85 7.65 75.8 FBH8-4 LH200MON810×LH172 0.06 0.75 0.02 0.8 FNH8-4 LH200×LH172 5.34 5.19 9.54 86.7 FNA8-1 7051/Mo17 6.91 3.79 13.00 88.3 FBA8-1 7051/Mo17MON810 0.02 0.48 0.01 1.7 FNA8-2 7054/Mo17 6.54 5.00 13.23 85.8 FBA8-2 7054/Mo17MON810 0.02 0.58 0.02 0.0 CK1 6.08 1.992.79 15.42 87.5 CK2 5.54 11.69 83.3 MON810 hybrid average 0.04 0.52 0.03 1.67 Average non-MON810 hybrids 5.48 3.92 11.09 85.00

The comprehensive analysis of Examples 5 and 6 showed that the CryIAb protein expressed by the insect-resistant maize line MON810 exhibited a significant yield-increasing effect. Under the field conditions where the Asian corn borer is naturally infested, the average yield per hectare of the MON810 hybrids is 9645 kg, while their non-MON810 hybrids of the same type are only 8066 kg. Under field conditions artificially inoculated with the Asian corn borer, the average yield per hectare of the non-MON810 hybrids was only 7103 kg, while their isotype MON810 hybrids had an average yield of 9102 kg per hectare, with an increase of 1999 kg of grain per hectare, an increase of 28.1 %. Therefore, broadly speaking, as long as the Asian corn borer is infested, the CryIAb protein expressed in MON810 insect-resistant corn should increase the yield of corn.

The comprehensive analysis of Examples 1 to 9 shows that under the detection of various identification methods (indoor bioassay method, improved indoor bioassay method, field identification), the leaves, stalks, tassels, and anthers of the MON810 insect-resistant corn line The expression level of CryIAb protein in tissues such as silk and filaments is enough to kill almost all the newly hatched larvae of Ostrinia officinalis. Therefore, this is a whole plant's ability to prevent and control Ostrinia officinalis. This ability to kill pests is not only reflected in the mid-leaf stage (also known as the new leaf stage generation) and the silk peak stage (also called the ear stage generation) when the Asian corn borer is most likely to damage corn, but also in the seedling stage and budding stage. , mature stage, etc. Therefore, this is also a kind of control ability against Asian corn borer in the whole growth period. It can be seen that the CryIAb protein produced by MON810 insect-resistant corn is a kind of protection for the whole plant and the whole growth period of corn.

The comprehensive analysis of Examples 1 to 9 shows that the expression of the CryIAb protein that has a poisonous effect on the corn borer in the MON810 corn line does not vary due to different planting locations and does not change due to changes in planting time. In 1997, experiments conducted at the Corn Borer Laboratory of the Plant Protection Institute of the Chinese Academy of Agricultural Sciences (Beijing) showed that the expression of CryIAb protein in the MON810 corn line was sufficient to prevent and control the damage caused by the Asian corn borer. In 1998, Henan Academy of Agricultural Sciences (Zhengzhou), Shandong Agricultural University (Tai'an) in the summer corn area, Shenyang Academy of Agricultural Sciences (Shenyang), Dandong Academy of Agricultural Sciences (Dandong), and Jilin Academy of Agricultural Sciences (Gongzhuling) in the spring corn area in 1998 and the Jilin Province Crop New Variety Introduction and Breeding Center (Gongzhuling) have also shown that the CryIAb protein expressed by the MON810 corn line is also sufficient no matter under the condition of natural damage by the Asian corn borer in the field or under the condition of the artificial inoculation of the Asian corn borer in the field. Control of Asian corn borer damage to corn. Therefore, broadly speaking, at any time and place, as long as the Asian corn borer damages corn, the CryIAb protein produced by planting MON810 insect-resistant corn should be sufficient to prevent the Asian corn borer from harming corn.

The comprehensive analysis of Examples 1 to 9 shows that the expression of CryIAb protein in the insect-resistant maize line MON810 does not change due to changes in the genetic background of maize. The genetic background of the test material used by the Corn Borer Research Office of the Plant Protection Institute of the Chinese Academy of Agricultural Sciences is B73×Mo17MON810. Under the condition of artificial inoculation of the corn borer in the field, six hybrids of MON810 with different genetic backgrounds were tested (see Table 14), and under the natural damage condition of Ostrinia sativa in field, it is the different MON810 hybrids (seeing table 15) of ten genetic backgrounds that are tested, all these different MON810 hybrids of genetic backgrounds all show consistent control Good effect of Asian corn borer. This also broadly suggests that expression of the CryIAb protein is genetically dependent on whether or not it is the MON810 insect-resistant maize line. Therefore, any MON810 insect-resistant corn line, whether it is an inbred line, a hybrid, a population, a composite, etc., should be resistant to O. cornborer.

The prevention and control of the transgenic insect-resistant corn of the present invention to the Asian corn borer is obviously because corn itself can produce CryIAb protein, and MON810 corn is only a carrier for the expression of CryIAb protein. Therefore, those skilled in the art, according to the CryIAb protein to The same poisonous effect of the Asian corn borer can produce a similar transgenic insect-resistant corn that can express the CryIAb protein, which is used to prevent and control the damage of the Asian corn borer to corn.

CryIAb proteins suitable for use in this invention are CryIAb polypeptides of the amino acid sequence given herein.

Through the introduction of the foregoing embodiments, we can easily see the uniqueness of the present invention.

The present invention provides a new method for preventing and controlling corn borer damage to corn, which is realized by expressing the CryIAb protein that is toxic to the Asian corn borer in plants by using modern biological technology. Broadly speaking, this described method of control is true not only of corn, but of any crop affected by the Asian corn borer. These crops damaged by the Asian corn borer include (but are not limited to) cotton, millet, sorghum and the like. Because as long as the CryIAb protein is expressed in these crops, it can prevent the Asian corn borer from harming it.

Claims (17)

1. A method for preventing and treating corn borer damage to corn, characterized in that, by planting a transgenic insect-resistant corn plant that can express CryIAb protein in vivo, the Asian corn borer that ingests the plant tissue can feed and grow be suppressed, and eventually lead to death, to achieve the prevention and control of corn borer damage to corn. 2. The method according to claim 1, wherein said CryIAb protein has the amino acid sequence of said CryIAb protein. 3. The method according to claim 1, wherein the transgenic insect-resistant corn is a transgenic insect-resistant corn line MON810. 4. The method according to claim 3, characterized in that said transgenic insect-resistant corn line MON810 includes any MON810 inbred line, hybrid, composite or population. 5. The method according to any one of claims 1 to 4, characterized in that the prevention and control of corn harmed by the Asian corn borer refers to the control of the whole plant of the transgenic insect-resistant corn against the corn harmed by the Asian corn borer. 6. The method according to claim 5, characterized in that the prevention and control of the entire plant of the transgenic insect-resistant corn against corn borer damage includes its leaves, stems, tassels, anthers and filaments against the Asian corn borer. Hazardous corn control. 7. The method according to any one of claims 1 to 4, characterized in that the prevention and control of corn harmed by the Asian corn borer refers to the prevention and control of the corn harmed by the Asian corn borer during the whole growth period of the transgenic insect-resistant corn. 8. The method according to claim 7, characterized in that the whole growth period of the transgenic insect-resistant corn prevents and controls corn borer damage, including its seedling stage, new leaf stage, budding stage, tasseling stage, filament The prevention and control of Asian corn borer damage to corn in the future. 9. The method according to any one of claims 1 to 4, characterized in that the control of the Asian corn borer does not change due to the change of the planting location. 10. The method according to any one of claims 1 to 4, characterized in that the control of the damage to corn by the Asian corn borer does not change due to the change of planting time. 11. The method according to any one of claims 1 to 4, characterized in that, the prevention and control of corn harmed by the Asian corn borer refers to the performance of the transgenic insect-resistant corn under the condition of artificially inoculating the Asian corn borer in the field. Increase production. 12. The method according to any one of claims 1 to 4, characterized in that the prevention and control of the corn borer harmed by the corn borer refers to the performance of the transgenic insect-resistant corn under the condition of artificially inoculating the corn borer in the field. Increased kernel weight. 13. The method according to any one of claims 1 to 4, characterized in that, the prevention and control of corn harmed by the Asian corn borer refers to the performance of the transgenic insect-resistant corn under the condition of artificially inoculating the Asian corn borer in the field. Improvements to stem resistance. 14. The method according to any one of claims 1 to 4, characterized in that the prevention and control of corn harmed by the Asian corn borer refers to the behavior of the transgenic insect-resistant corn under the natural occurrence of the corn borer in the field. production-increasing effect. 15. The method according to any one of claims 1 to 4, characterized in that the prevention and control of corn borer damage to corn refers to the behavior of transgenic insect-resistant corn under the natural occurrence of corn borer damage in the field. increase in kernel weight. 16. The method according to any one of claims 1 to 4, characterized in that the prevention and control of corn borer damage to corn refers to the performance of transgenic resistant corn under the natural occurrence of corn borer damage in the field. Improvements to stem resistance. 17. The use of the transgenic insect-resistant corn according to any one of claims 1 to 4 to prevent corn from being harmed by the Asian corn borer.

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