CN1266616A - Method for control of insects - Google Patents
Method for control of insects Download PDFInfo
- Publication number
- CN1266616A CN1266616A CN 00100699 CN00100699A CN1266616A CN 1266616 A CN1266616 A CN 1266616A CN 00100699 CN00100699 CN 00100699 CN 00100699 A CN00100699 A CN 00100699A CN 1266616 A CN1266616 A CN 1266616A
- Authority
- CN
- China
- Prior art keywords
- corn
- asiatic
- mon810
- corn borer
- borer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
A method for controlling the Asian corn borer features that the transgenic insect-resisting corn plants, especially the MON810, which can express the CrylAb protein in body, are planted. The Asian corn borers can die from eating the tissue of said plants. Its advantages are no limitation of planting time and places, high effect, simple method, low cost and no environmental pollution.
Description
The present invention relates to a kind of method to the crop pests control, particularly relate to by planting a kind of transgenic insect-resistant corn plant and prevent and treat the cause harm method of corn of Asiatic corn borer, specifically, the present invention relates to a kind of soil bacteria bacillus thuringiensis,Bt storehouse Stuckey subspecies (Bacillus thuringiensis subsp.kurstaki that natural world exists that derives from that expresses in the corn that is used in, B.t.k.) the CryIAb albumen in is prevented and treated the cause harm method of corn of Asiatic corn borer, more particularly, the transgenic insect-resistant corn strain MON810 that the present invention relates to can to express in a kind of its body CryIAb albumen among the B.t.k. is used to prevent and treat the cause harm method of corn of Asiatic corn borer; The present invention also relates to described transgenic insect-resistant corn, particularly transgenic insect-resistant corn strain MON810 is as the cause harm purposes of corn of control Asiatic corn borer.
Corn borer all has the generation of causing harm in all corn producing regions of China, is the first insect on the Maize Production.The underproduction 7～10% of being injured of general time spring corn, the outbreak year underproduction is more than 30%.Summer corn since the season of growth short, because of the cause harm loss that causes of corn borer bigger.Corn is Chinese the third-largest cereal crops, and year cultivated area is more than 2,000 ten thousand hectares.By 4,500 kilograms of calculating of the average per unit area yield of per hectare, annual because of the corn borer underproduction of causing harm reaches 60～9,000,000,000 kilograms, amount to about 20～3,000,000,000 yuan of RMB.
Asiatic corn borer experiences ovum, larva, pupa and 4 worm attitudes of adult throughout one's life.Larva is cause harm in life unique worm attitude of crop of corn borer.Asiatic corn borer belongs to typical facultative diapause insect, can take place in 1 year 1～7 generation from north to south, but only take place two on behalf of evil in the whole growing of corn, and promptly generation lobus cardiacus phase and fringe phase are from generation to generation.As in northern spring corn area, first generation ovum is contained the phase in the lobus cardiacus mid-term of corn growth, so also cry generation lobus cardiacus phase.Near the arteries and veins, the blade that launches leaf with the plant middle and upper part fully is many to the ovum fecund in vacuum side of blade.Larva had both showed hiding habit once hatching, climb into lobus cardiacus clump moth food lobus cardiacus or get the floral leaf that the mesophyll of food young leaflet tablet causes pin hole or window paper shape in the impeller clearance, if 3 ages, above larva moth was worn the not expansion lobus cardiacus of vertical volume, round appears when then blade is extracted out.In general, blade slightly is injured does not have obvious influence to corn growth.But if plantation sense worm kind, and the snout moth's larva generation is heavier, and when blade seriously was injured, the tassel after then being injured can not normally be extracted out, and plant height can significantly reduce.During larval feeding lobus cardiacus tissue development to 3 front and back in age, corn enters the phase of forming ears.The tender tassel of children's this moment has more attraction to snout moth's larva than lobus cardiacus, gets food so larva is all transferred to tassel.Take out male back larva and taken out of the lobus cardiacus clump.Larva continues to get the food tassel before tassel scatters.When tassel scatters gradually, larva promptly loses and meets its above-mentioned main taxis conditions needed, the necessary subenvironment of promptly surviving, except that the aging larva of minority can pupate on tassel, all larvas begin downward transfer, because the larva of this moment was 4～5 ages basically, its characteristic of " boring moth " is brought into play in beginning significantly.Eat into the tassel handle except that a part and grow to aging or downward once more the transfer, because the tassel handle is very thin, generally can only hold 1～2 mature larva, therefore more larva must get over the continuation of tassel handle and shift downwards, seeks position preferably and bores moth.Wherein, female fringe tight knot and up and down the joint be the position that subjects to attack most.The female fringe of corn this moment germinates, after being eaten into as near stipes it, can influence its normal development significantly even end and grow.If first fringe is injured early, then second fringe can continue to grow, but can not be long to the due size of first fringe.If second fringe is not in time reached maturity and bored moth and stasi by snout moth's larva, then this strain corn has no harvest more.In addition, because snout moth's larva is met wind and very easily causes down folding at plant middle and lower part moth stem, make loss bigger.First brood of larvae or on tassel, or in the tassel handle, or in the stem stalk, grow to aging, except that entering diapause, sub-fraction becomes the worm source in the coming year, and major part is pupated, is sprouted wings and produces first generation adult.
Second generation ovum (adult produces the first generation) the Sheng phase is contained the phase at corn capillament, also cries the fringe phase from generation to generation, and ovum still fecund also produces on stem stalk and bract once in a while in the middle and upper part vacuum side of blade.Newly hatched larvae focuses mostly on hiding in the filigree base portion on female fringe top greatly, gets the young tender seed that the anthophagy silk is got the female tip of the spike of food then, also has minority to hide and gets pollen or the axil tissue that dyspepsia is deposited at the axil place.Larvae development begins occur to bore the moth behavior after 3 ages, or directly eats into into cob from the fringe head, or continue and get the seed that food is being in the milk, or shifts once more moth downwards from female fringe top and go into female fringe, female fringe handle or stem stalk.This moment, corn female fringe its due size that reached full growth and entered milk ripe stage.Therefore fringe phase borer pest can not influence female fringe size, normally is in the milk but influence corn, thereby reduces thousand kernel weight.If female fringe handle is eaten hollow, can cause fringe folding and come off.The larval feeding seed often causes the infection of corncob, stem rot except that directly causing the production loss, has more increased the weight of production loss and quality and has descended.
The preventing and controlling of Chinese maize snout moth's larva start from the fifties.The main method of preventing and treating that it adopted has: cultural control, chemical control, physical control, sex pheromone control and biological control etc.
Cultural control is to be whole agricultural ecological multifactorial comprehensive coordination management, and regulation and control crop, insect, environmental factor are created a farmland ecological environment that helps plant growth and be unfavorable for the snout moth's larva generation.As utilize processing corn borer overwintering host, reform cropping system, the anti-snout moth's larva kind of plantation, plantation to lure measures such as collection field and intercropping to force down causing harm of corn borer.Must obey the requirement of crop allocation and volume increase because of cultural control, using has certain limitation, can not be as emergency measure, and it is powerless just to seem when borer pest breaks out.
Chemical control is a pesticide control, be to utilize chemical insecticide to come kill pests, it is the important component part of the corn borer comprehensive regulation, it has the characteristics of quick-acting, convenient, easy and high economic benefit, particularly under the situation of the big generation of insect, the emergency measure that is absolutely necessary, it can insect cause cause harm before with its elimination.Granule as present popularizing area maximum is prevented and treated method, and effect is the most stable and reliable, but the instrument of efficiently executing granule still reaches a standard far away, has a strong impact on the performance of its effect.Spread chemical control methods such as pesticide-clay mixture, medicine liquid spray, the interior winter generation adult of the stifling stalk buttress of dichlorvos envelope buttress in addition in addition.But chemical control also has its limitation, tend to cause crops generation poisoning, insect to develop immunity to drugs as improper use, and killed natural enemies, contaminated environment, make field ecosystem destroyed with residue of pesticide to the safety of people, animal adverse consequences such as constitute a threat to.
Physical control utilizes methods such as various physical factors such as light, electricity, look, temp. and humidity etc. and plant equipment are trapped and killed, steriliation by irradiation to come pest control mainly according to the reaction of insect to various physical factors in the environmental condition.At present most widely used is the high-pressure sodium lamp trapping, it utilizes the overwinter generation larva to concentrate on the characteristics of surviving the winter in the maize straw buttress in the village village, at the winter generation adult emergence period, in the village, village high-pressure sodium lamp is set in flakes on a large scale, the corn borer adult is trapped and killed, and control efficiency is obvious.High-pressure sodium lamp must be to guarantee can use in the village, village of power supply continuously night, and operation acquires a certain degree of difficulty.
The sex pheromone control is to utilize the nubilalis sex pheromone of synthetic directly to trap and kill corn borer male worm and the mating of interference snout moth's larva, reduces the female moth fertilization rate in field, alleviates borer pest.
Trapping method wherein is the nubilalis sex pheromone that utilizes synthetic in the mating habitat of corn borer such as the male moths of trapping corn borer such as good wheatland of growing way or vegetable plot.The shortcoming of this method is that the management of basin trapper is taken a lot of work, and must large tracts of land use just effective in flakes.
Isotropic method wherein is when overwinter generation corn borer adult eclosion 10%, hangs on the crop with 4500～6000 of sex pheromone vaporizers in its mating place per hectare, disturbs the mating of snout moth.The usability pheromones is prevented and treated generation corn borer, and method is easy, and is respond well, free from environmental pollution, do not injure natural enemy.Weak point is to lure the input of core to take a lot of work.
Biological control is to utilize some beneficial organism or biological metabolic product to come the Pest Control population quantity, to reach the purpose of forcing down or eliminating destructive insects.Be characterized in that environmental pollution is few to people, animal safety, can reach the purpose of long-term control to some insect.Modal biological control has oophagous trichogrammae and white muscardine fungi etc.But oophagous trichogrammae control is subjected to the weather factor affecting big, and effect is often unstable, no matter and snout moth's larva weight takes place all needs same investment to carry out.
Transgenic insect-resistant corn is exactly to change the means of external source anti insect gene by plant genetic engineering over to maize cell, regeneration plant then, thereby the pest-resistant characteristic that corn acquisition itself is not had.
Bacillus thuringiensis,Bt cries bacillus thuringiensis (Bacillus thuringiensis) again, is a kind of Gram-positive soil bacillus that can form gemma, can form parasporal crystal in its sporulation phase in thalline.These parasporal crystals are mainly by (27～140KD) protein are formed, and have highly single-minded pest-resistant effect, and are nontoxic to people and animals and non-target insect.The crystalline protein (Cry) that different strains produces shows special insecticidal activity to the variety classes insect, and up to the present, several ten thousand thuringiensis strain bacillus strains have been isolated in the whole world.
Cry albumen can be divided into I, II, III, IV class according to the difference of its pest-resistant spectrum, the anti-Lepidoptera of I class, anti-Lepidoptera of II class and diptera, the anti-coleoptera of III class, the anti-diptera of IV class, all kinds of in again according to some groups that how much are divided into of amino acid identity.For example, between the cryI gene, amino acid identity is 82%～90% the cryIA that is classified as; 55%～71% be classified as cryIB; CryIC is different each other with cryID, also is different from cryIA.In the cryIA gene, according to the zymogram of restriction enzyme and the size of molecular weight, be divided into again: cryIAa, 4.5kb; CryIAb, 5.3kb; CryIAc, 6.6kb subclass gene.
CryIAb albumen is that (it is insoluble crystalline protein to it is found that a kind of for Bacillusthuringiensis subsp.kurstaki, B.t.k.) companion's spore crystalline protein of Chan Shenging by bacillus thuringiensis,Bt storehouse Stuckey subspecies.Crystalline protein is formed with the form of proteinogen toxin.The pesticidal of CryIAb crystalline protein requires albumen to be absorbed.In insect gut, because high pH value, the proteinogen dissolving is combined closely by the effect of enzyme and the active nucleus of albumen, no longer by the proteasome degradation in the insect gut.Core protein is in the lepidopterous insects enteron aisle and specific receptors bind is arranged, and inserts goldbeater's skin, forms ion specificity passage.These biochemical reactions have been upset digestion process, cause insect death.The digestive tract of non-target insect, mammal, birds and fish organize do not contain can with the protein bound acceptor of CryIAb, therefore, CryIAb albumen can not destroyedly digest, thus also avirulence.
Along with the development of molecular genetic techniques, isolated the gene of multiple desinsection crystalline protein and measured its dna sequence dna.On crops such as soybean, corn, potato, some genetic engineering plant have been created with these genes.Proved the infringement of anti-multiple Lepidoptera (Lepidoptera) insect of this gene engineering plant.Yet, still have nothing to do so far in controlling the report that Asiatic corn borer is caused harm to corn by producing the transgenic corn plant of expressing CryIAb albumen.
Owing to use the different of cry gene kind, differences such as controlling element that construction of expression vector uses such as promotor, intron, terminator cause pest-resistant kind of its gained transformant and degree variant with company in the different experiments chamber.The pest-resistant corn strain of MON810 is a transgenic product of utilizing modern molecular biology technique to develop by U.S. Monsanto Company, and it is expressed in vivo is CryIAb among the B.t.k., and its trade name Chinese is for protecting rich corn, and English is YieldGard.Its product has carried out commercialization in the U.S., can buy at American market.
Though the pest-resistant corn strain of MON810 has carried out commercialization in the U.S., its controlling object is European corn borer (Ostrinia nubilalis), and controlling object of the present invention is Asiatic corn borer (Ostriniafurnacalis).
European corn borer (Ostrinia nubilalis) and Asiatic corn borer (Ostrinia furnacalis) are though all be subordinate in Insecta, Lepidoptera, Pyralidae, and all be the boring pest of seeding corn and other crops of causing harm, but both be clearly biologically, distinct two species, at least there are the following main distinction (plant protection journal, 1988, Vol.15 (3): 145-152; Agricultural encyclopedia-insect roller, 1990,456-459):
1, in the Chinese maize main producing region, Asiatic corn borer is the primary pest of corn of causing harm, and does not also find the cause harm evidence of corn of European corn borer
Chinese maize snout moth's larva integrated control research cooperative groups proves through for many years investigation and research:
Asiatic corn borer mainly is distributed in the wide geographic area in Chinese eastern half Heilungkiang to Guangdong, is the major pest of crops such as corn, millet, Chinese sorghum.The main corn producing region of China all in this scope, comprising: 17 provinces and cities (autonomous region) such as Heilungkiang, Jilin, Liaoning, Beijing, Hebei, Henan, Hubei, Guangdong, Sichuan, Yunnan, Jiangsu, Ningxia, Guizhou, the Inner Mongol, Shandong, Gansu.
Up to the present European corn borer only finds to be distributed in the Yining in Xinjiang, the Huhehaote in the Inner Mongol, the Yongning in Ningxia and Zhangjiakou, the Lu Tai in Hebei.Except that corn is caused harm in the Yining in Xinjiang, other regional main parasitic is in Siberian cocklebur and hemp etc., the evidence of the corn that also do not have to find to cause harm.
2, in the U.S., Asiatic corn borer is non-existent
Asiatic corn borer is distributed in temperate zone, Asia and areas such as the torrid zone, Australia and Micronesia, Oceania.External in removing, Asiatic corn borer is distributed widely in Japan, Southeast Asia and Australia, and there is no the report that relevant European corn borer exists in above-mentioned area.
European corn borer is distributed in Europe, North America, West Africa and little Ya Xiya, does not see the report that has Asiatic corn borer to exist in these areas.
3, the two is to the habit difference that causes harm of corn
It is reported that the European corn borer larva before four ages is hidden in leaf sheath when causing harm in the corncob phase and stem stalk intersection is caused harm; And many on the female fringe of corn, cause harm filigree and the tender seeds when causing harm of the Asiatic corn borer corncob phase before four ages.
4, the two is different on form
The evaluation of most of insect populations finally is to decide by the structure of dissecting the genitalia that compares its hero or female insects.That is to say, have many insect populations closely similar in appearance, but sexual organ are different, reality is diverse species; Vice versa.
Chinese maize snout moth's larva integrated control research cooperative groups has proved that through years of researches the Asiatic corn borer of taking place in China is and distinct two species of European corn borer on form, the two difference below existing on male genitalia (clasping spine abdomen) feature:
● the general tool 3-4 of Asia kind big thorn or 2 big thorns and 1 spinule, mean is more than european race.
● the head of district is in stingless district for Asia kind tool thorn, and european race is opposite.
● the two section length relation of both sides clasping spine abdomen is inconsistent.
5, exist reproduction to isolate between the two
Biological fundamental difference between not of the same race is the isolation that exists therebetween in the reproduction, can not mating produce the offspring with normal fertility between promptly not of the same race.Chinese maize snout moth's larva integrated control research cooperative groups used for 5 years, to surplus 1300 to picking up from the different regions corn borer of (comprising Austria), carry out multiple combination mating.Test is found, all are all isolated with there is reproduction from Austrian European corn borer from the corn borer on the corn of corn producing region, Eastern China, and only corn borer and the European corn borer from areas such as Yining corn and Ningxia and Zhangjiakou Siberian cockleburs do not have the reproduction isolation.Therefore, exist reproduction to isolate between corn borer of taking place in the Chinese maize main producing region and the European corn borer.
6, the two is to the reaction difference of sex pheromone
The sex pheromone chemical constitution of insect of the same race is not different.Insect sex pheromone is made up of multiple compound.And with the isolation of kind with the different property finished of mixing ratio of compound.Existing report proof, the chemical constitution of Eurasian two kinds of nubilalis sex pheromones is completely different.
Chinese maize snout moth's larva integrated control research cooperative groups used for 5 years, and that has carried out Eurasian two kinds of nubilalis sex pheromones in 34 areas of 20 provinces and cities lures the moth activity test.The result shows that the Asiatic corn borer of taking place in China does not react to the European corn borer sex pheromone.
7, sex pheromone chemical composition of the two and structure difference
The chemical composition of sex pheromone and the proportioning of each component are one of important indicators of distinguishing the different insects population.Analysis result to the nubilalis sex pheromone structure shows, the chemical composition of the sex pheromone of the Asiatic corn borer of taking place in China is suitable, anti-12-tetradecene alcohol acetic ester, proportioning is 47: 53, and the chemical composition of the sex pheromone of European corn borer is 97: 3 suitable, an anti-11-tetradecene alcohol acetic ester, and the two is different.
In sum, because there are the different of all many-sides in Asiatic corn borer with European corn borer, therefore, it is not to be conspicuous that transgenic insect-resistant corn of the present invention is used to prevent and treat the cause harm method of corn of Asiatic corn borer.
The present invention utilizes transgenic insect-resistant corn to prevent and treat causing harm of Asiatic corn borer.Itself can express CryIAb albumen this transgenic insect-resistant corn, thereby makes corn produce the ability of opposing Asiatic corn borer.
Therefore, the purpose of this invention is to provide a kind of transgenic insect-resistant corn that can express CryIAb albumen, particularly transgenic insect-resistant corn strain MON810, prevent and treat the cause harm method of corn of Asiatic corn borer.
Another object of the present invention is to provide a kind of described transgenic insect-resistant corn, particularly transgenic insect-resistant corn strain MON810 as the cause harm purposes of corn of control Asiatic corn borer.
The present invention relates to a kind of cause harm method of corn of Asiatic corn borer of preventing and treating, it is characterized in that, by planting a kind of transgenic insect-resistant corn plant that in its body, can express CryIAb albumen, thereby make the picked-up this plant tissue Asiatic corn borer get the food and growth be suppressed, and finally cause death, realize the cause harm control of corn of Asiatic corn borer.
According to the cause harm method of corn of control of the present invention Ostrinia furnacalis; CryIAbCryIAb：CryIAb,CryIAb 1 MDNNPNINEC IPYNCLSNPE VEVLGGERIE TGYTFIDISL SLTQFLLSEF 51 VPGAGFVLGL VDIIWGIFGP SQWDAFLVQI EQLINQRIEE FARNQAISRL101 EGLSNLYQIY AESFREWEAD PTNPALREEM RIQFNDMNSA LTTAIPLFAV151 QNYQVPLLSV YVQAANLHLS VLRDVSVFGQ RWGFDAATIN SRYNDLTRLI201 GNYTDHAVRW YNTGLERVWG PDSRDWIRYN QFRRELTLTV LDIVSLFPNY251 DSRTYPIRTV SQLTREIYTN PVLENFDGSF RGSAQGIEGS IRSPHLMDIL301 NSITIYTDAH RGEYYWSGHQ IMASPVGFSG PEFTFPLYGT MGNAAPQQRI351 VAQLGQGVYR TLSSTLYRRP FNIGINNQQL SVLDGTEFAY GTSSNLPSAV401 YRKSGTVDSL DEIPPQNNNV PPRQGFSHRL SHVSMFRSGF SNSSVSIIRA451 PMFSWIHRSA EFNNIIPSSQ ITQIPLTKST NLGSGTSWK GPGFTGGDIL501 RRTSPGQIST LRVNITAPLS QRYRVRIRYA STTNLQFHTS IDGRPINQGN551 FSATMSSGSN LQSGSFRTVG FTTPFNFSNG SSVFTLSAHV FNSGNEVYID601 RIEFVPAEVT FEAEYDLERA QKAVNELFTS SNQIGLKTDV TDYHIDQVSN651 LVECLSDEFC LDEKKELSEK VKHAKRLSDE RNLLQDPNFR GINRQLDRGW701 RGSTDITIQG GDDVFKENYV TLLGTFDECY PTYLYQKIDE SKLKAYTRYQ751 LRGYIEDSQD LEIYLIRYNA KHETVNVPGT GSLWPLSAPS PIGKCAHHSH801 HFSLDIDVGC TDLNEDLGVW VIFKIKTQDG HERLGNLEFL EGRAPLVGEA851 LARVKRAEKK WRDKREKLEW ETNIVYKEAK ESVDALFVNS QYDRLQADTN901 IAMIHAADKR VHSIREAYLP ELSVIPGVNA AIFEELEGRI FTAFSLYDAR951 NVIKNGDFNN GLSCWNVKGH VDVEEQNNHR SVLVVPEWEA EVSQEVRVCP1001 GRGYILRVTA YKEGYGEGCV TIHEIENNTD ELKFSNCVEE EVYPNNTVTC1051 NDYTATQEEY EGTYTSRNRG YDGAYESNSS VPADYASAYE EKAYTDGRRD1101 NPCESNRGYG DYTPLPAGYV TKELEYFPET DKVWIEIGET EGTFIVDSVE1151 LLLMEE
According to the cause harm method of corn of control of the present invention Asiatic corn borer, wherein said transgenic insect-resistant corn is transgenic insect-resistant corn strain MON810, it comprises any MON810 inbred line, crossbreed, synthetic variety or its colony, for example, inbred line LH185MON810 and Mo17MON810 etc., crossbreed LH198 * LH185MON810, B73 * Mo17MON810 etc.
According to the cause harm method of corn of control of the present invention Asiatic corn borer, wherein said Asiatic corn borer is caused harm whole plant that the control of corn is meant transgenic insect-resistant corn to the cause harm control of corn of Asiatic corn borer; Described whole plant comprises that to the control of Asiatic corn borer its blade, cane, tassel, flower pesticide and filigree etc. are to the cause harm control of corn of Asiatic corn borer;
According to the method for control of the present invention Asiatic corn borer, wherein said Asiatic corn borer is caused harm time of infertility that the control of corn is meant transgenic insect-resistant corn to Asiatic corn borer the cause harm control of corn of corn of causing harm; The described time of infertility, the control to Asiatic corn borer comprised the control to Asiatic corn borer such as its seedling stage, young leaves phase, the phase of forming ears, tasseling stage, filigree phase;
According to the method for control of the present invention Asiatic corn borer, wherein said the cause harm control of corn of Asiatic corn borer is meant that not the change because of the plantation place changes, the change because of implantation time does not change;
In addition, according to the cause harm method of corn of control of the present invention Asiatic corn borer, wherein said control to Asiatic corn borer harm corn is meant the production-increasing function that transgenic insect-resistant corn shown, the raising of kernel weight, the improvement of cane resistance etc. under land for growing field crops artificial infection Asiatic corn borer condition, with and the production-increasing function that under condition is caused harm in land for growing field crops Asiatic corn borer abiogenesis, is shown, the raising of kernel weight, the improvement of cane resistance etc.
The invention still further relates to described transgenic insect-resistant corn particularly transgenic insect-resistant corn strain MON810 as the cause harm purposes of corn of control Asiatic corn borer.
The present invention and existing the most tangible difference to the Asiatic corn borer control, i.e. the beneficial effect that the present invention produced is: the present invention has tangible production-increasing function.Corn is Chinese important crops, sowing area is more than 2,000 ten thousand hectares throughout the year, the average per unit area yield of per hectare about 5,000 kilogram, if all replant pest-resistant corn MON810, amount of increase in production 19.6% with this experiment is calculated (embodiment 6), can be China every year and increases income nearly 20,000,000,000 kilograms of grain, amounts to about 16,000,000,000 yuan of RMB; Even the amount of increase in production with 10% is calculated, also can be China every year and increase income nearly 10,000,000,000 kilograms of grain, amount to about 8,000,000,000 yuan of RMB.As seen the present invention has important social benefit and economic benefit.It is incomparable that this is that any Asiatic corn borer of adopting at present prevents and treats method.
The method of the chemical insecticide control Asiatic corn borer that present stage uses, though the control Asiatic corn borer has been played certain effect to causing harm of corn, but also brought harm simultaneously, used method of the present invention, can eliminate these harm human body, domestic animal and soil itself.
The existing Asiatic corn borer method of preventing and treating mainly is to be that external cause reaches the cause harm control of corn of Asiatic corn borer by external action.And the present invention prevents and treats Asiatic corn borer by producing the CryIAb albumen that can kill Asiatic corn borer in the plant corpus, promptly prevents and treats by internal cause;
It is not the time of infertility that existing Asiatic corn borer prevents and treats method, and the present invention is the protection of corn being carried out the time of infertility, and pest-resistant corn strain MON810 is from the seedling stage of corn growth; to the young leaves phase; to the phase of forming ears,, all exempt from the infringement of Asiatic corn borer to phase of weaving silk and even maturing stage.This is that the existing Asiatic corn borer method of preventing and treating is beyond one's reach;
The existing control that the method for preventing and treating of Asiatic corn borer is not existed so-called whole plant, and the present invention protects the whole plant of corn, the blade of pest-resistant corn strain MON810, stem stalk, flower pesticide, filigree etc. all are anti-Asiatic corn borer, and this also is that present Asiatic corn borer dispensary is insurmountable;
Existing Asiatic corn borer prevents and treats method, its effect is unsettled, and the present invention is stable to the cause harm control efficiency of corn of Asiatic corn borer, pest-resistant corn strain MON810 to the control efficiency of Asiatic corn borer in the different location, different time, different genetic background all be stable and consistent;
Existing Asiatic corn borer prevents and treats method, its effect is halfway, only play and alleviate effect, pest-resistant corn strain MON810 of the present invention is almost absolutely the control efficiency of just incubating the Asiatic corn borer larva, the larva bodily form of surviving extremely individually is minimum, all be tangible depauperation, and stasi, be difficult to again corn be caused cause harm;
Compare with the existing Asiatic corn borer method of preventing and treating, the present invention has simply, makes things convenient for, economical and environment do not produced advantage such as pollution.Adopt technology of the present invention, only the pest-resistant corn strain MON810 that needs plantation can express CryIAb albumen gets final product, and does not need to adopt other measure, thereby has saved a large amount of human and material resources and financial resources, also can not produce environment and pollute;
The difference of the present invention and the control of existing Asiatic corn borer is that also the present invention can make the performance of corn kernel proterties and stem stalk improve;
The difference of the present invention and the control of existing Asiatic corn borer also is, though the amino acid sequence of the active CryIAb albumen of MON810 corn strain performance is in full accord with the albumen that bacillus thuringiensis,Bt as microorganism insecticide produces, both are widely different.Because thuricade-1 need directly spray application to crop surface, therefore just caused activated crystalline protein (comprising CryIAb albumen) in environment, to be degraded, the duplication of production and the repeated application that cause insecticide, and, increased cost greatly for the practical application in agricultural production brings difficulty.And the present invention expresses this CryIAb albumen in plant corpus, has avoided biological insecticides in shortcomings such as natural world instabilities effectively, has saved the man power and material.
Below accompanying drawing is briefly explained:
Restriction site that Fig. 1 uses for the plasmid schematic diagram of PV-ZMBK07 and PV-ZMGT10: Southern analyzes and position (representing with base-pair) thereof are as shown in this figure.
Fig. 2 is the Southern analysis chart of corn strain MON810DNA, is to insert the segments goal analysis.Swimming lane 1 and swimming lane 2 are respectively the NdeI digestion products of following DNA, and PV-ZMBK07 is a hybridization probe.The DNA of swimming lane 1:MON818; The DNA of swimming lane 2:MON810.
The symbol of the dna fragmentation size that → expression draws from molecular weight marker;
≈ represents the symbol of the dna fragmentation size that draws from molecular weight marker and plasmid digestion product;
* the symbol of representing the background band.
Fig. 3 is the Southern analysis chart of corn strain MON810DNA, promptly to the analysis of CryIAb gene.Swimming lane 1, swimming lane 2 and swimming lane 3 are respectively the NcoI/EcoRI digestion products of following DNA, and the cryIAb gene is a hybridization probe.Swimming lane 1: about 50pg PV-ZMBK07; The DNA of swimming lane 2:MON818; The DNA of swimming lane 3:MON810.
The symbol of the size of → expression dna fragmentation that molecular weight marker draws from ethidium bromide (EB) stained gel;
The symbol of the dna fragmentation size that-expression draws from the plasmid digestion product;
≈ represents the symbol of the dna fragmentation size that draws from molecular weight marker and plasmid digestion product;
* represents to close on the hybridization zone of swimming lane.This symbol only occurs at swimming lane 1, because depart from swimming lane 1 during its DNA electrophoresis at an angle.
Fig. 4 is the Southern analysis chart of corn strain MON810DNA, promptly to the analysis of CP4 ESPSP gene and gox gene.Swimming lane 1, swimming lane 2, swimming lane 3 and swimming lane 4 are respectively the NcoI/BamHI digestion products of following DNA: swimming lane 1 and swimming lane 3 are plasmid PV-ZMBK07 and PV-ZMGT10 of about 50pg, swimming lane 2 and swimming lane 4 are DNA of MON810, the hybridization probe of swimming lane 1 and swimming lane 2 is CP4 ESPSP genes, and the hybridization probe of swimming lane 3 and swimming lane 4 is gox genes;
The symbol of → expression dna fragmentation the size that molecular weight marker draws from ethidium bromide (EB) stained gel;
The symbol of the dna fragmentation size that-expression draws from the plasmid digestion product.
Fig. 5 is the Southern analysis chart of corn strain MON810DNA.Swimming lane 1-6 is respectively the NcoI/EcoRI digestion product of following DNA: swimming lane 1 and swimming lane 4 are plasmid PV-ZMBK07 of about 50pg; Swimming lane 2 and swimming lane 5 are DNA of MON818, and swimming lane 3 and swimming lane 6 are DNA of MON810; The probe of swimming lane 1-3 is the nptII zone; The hybridization probe of swimming lane 4-6 is the ori-pUC zone.
The symbol of → expression dna fragmentation the size that molecular weight marker draws from ethidium bromide (EB) stained gel;
The symbol of the dna fragmentation size that-expression draws from the plasmid digestion product.
For further specifying the present invention, the basic condition of the transgenic insect-resistant corn strain MON810 of the described CryIAb of expression albumen is carried out following description:
1. purpose gene cryIAb
The cryIAb gene is the pest-resistant purpose gene that inserts in the pest-resistant corn strain of MON810, derives from the soil bacteria bacillus thuringiensis storehouse Stuckey subspecies (Bacillu sthuringiensis subsp.kurstaki, B.t.k.) that nature exists. The cryIAb gene that is used for the PV-ZMBK07 plasmid vector of conversion is structural gene, and length is 3468 nucleotides. Compare with the cryIAb among the former B.t.k., its gene order process changes, thereby the expression of CryIAb albumen in corn improved greatly.
2. the CryIAb albumen of purpose gene cryIAb coding
The B.t.k.HD-1[CryIAb of purpose gene cryIAb coding] albumen is made up of 1156 amino acid, and this albumen produces and a kind ofly contains approximately 600 and amino acid whosely have an antitryptic protein product with external in the plant body after trypsase is processed. The albumen that the structure of the CryIAb albumen of MON810 corn strain coding and the activity that shows thereof and Dipel produce is in full accord.
The amino acid sequence of CryIAb albumen is identical with the CryIAb albumen that nature exists.
3. the structure of plasmid vector:
The generation of the pest-resistant corn strain of MON810 uses two kinds of plasmid: PV-ZMBK07[to contain the cryIAb gene] and PV-ZMGT10[contain CP4EPSPS and gox gene]. The genetic constitution of two kinds of plasmid carriers is listed in table 1 and table 2, and the genetic map of two kinds of plasmid carriers is seen Fig. 1.
The cryIAb gene that the PV-ZMBK07 plasmid vector shows is under the control of CaMV35S promoter E35S of enhancing, and this promoter size is about 0.6Kb. Between E35S promoter and cryIAb gene is introne from the 0.8Kb of corn hsp70 (heat shock protein) gene, and its effect is to improve gene transcription level. Hsp70 introne back is that size is the cryIAb gene of 3.46Kb. The cryIAb gene is inserted into the nopaline synzyme 3 of 0.26Kb ' terminal non-translated sequence, NOS3 ', and this sequence provides mRNA poly-adenosine signal.
The marker gene of relevant PV-ZMGT10 plasmid vector sequence and the narration of function thereof are intellectual purely, because the pest-resistant corn strain of MON810 had not been found the related gene sequence of PV-ZMGT10 carrier. The PV-ZMGT10 plasmid contains gox and CP4EPSPS marker gene, and they have participated in chloroplaset transfer CTP1 and CTP2 peptide. Two code areas are all under the control that contains CaMV35S promoter, corn hsp70 introne and NOS3 ' terminator sequence.
The CP4EPSPS gene is separated from agrobacterium (Agrobacteriumsp.) CP4 bacterial strain, and showing behind the introduced plant has powerful resistance to the glyphosate inhibitory action. Its activity has been blocked in glyphosate and purpose enzyme EPSPS combination, and EPSPS is an enzyme in the aromatic amino acid biosynthesis pathway. Compare with most of EPSPS, CP4EPSPS has powerful tolerance to the inhibitory action of glyphosate, and the height catalytic action is arranged. Because the continuous action that the EPSPS of tolerance enzyme is arranged has been satisfied in the existence of aromatic, the CP4EPSPS albumen that plant cell is expressed has resistance to the glyphosate in the culture medium.
CP4EPSPS gene in PV-ZMGT10 contains in the chloroplaset of separating from mouseearcress EPSPS and to shift peptide CTP2, and it is CP4EPSPS albumen guiding chloroplaset, i.e. position and the ArAA synthetic site of EPSPS in plant. The size of carrying the CP4EPSPS gene of CTP2 is about 1.7Kb. CP4EPSPS gene cluster (by the promoter of 3 ' terminal sequence) is incorporated in the gox gene cluster.
Encoding, make the gox gene that metabolic alterations takes place glyphosate oxidoreductase (GOX) for glyphosate is to adopt clone technology to obtain from the LBAA bacterial strain of achromobacter (newly belonging to brown Flavobacterium). GOX albumen is integrated in the plasmid that contains chloroplaset transfer peptide sequence CTP1. CTP1 is from the mouseearcress ribulose-1,5-bisphosphate, a little subunit gene of 5-diphosphonic acid carboxylase (SSU1A) gene. GOX enzyme degradation of glyphosate makes it be converted into aminomethyl phosphonic acid and glyoxalic acid.
The α district of the lacZ gene of coding beta-galactosidase appears among PV-ZMBK07 and the PV-ZMGT10 in the presence of the promoter of bacterium control. A polylinker is contained in this zone, allows compatible gene to clone in plasmid vector. Be 0.65KbpUC plasmid (ori-pUC) replication origin after the lacZ-α district, this plasmid also can copy in Escherichia coli.
II type neomycin phosphotransferase (nptII) gene after the ori-pUC zone. This enzyme has drug resistance (for example kanamycins and neomycin) to aminoglycoside antibiotics, is used to when making up plasmid as resistance marker. The sequence of coding nptII gene is to derive from prokaryotes transposons Tn5 under the condition of the bacterium promoter existence of itself.
DNA composition list in the table 1:PV-ZMBK07 plasmid
| Gene element E35S hsp70 cryIAb NOS3 ' lacZ ori-pUC nptII | Size Kb 0.61 0.80 3.46 0.26 0.24 0.65 0.79 | Function cauliflower mosaic virus (CaMV) promoter tape copy enhancer zone. From the introne of corn hsp70 (heat shock protein) gene, effect is to improve gene transcription level. This gene code consists of the CryIAb protein product consistent with natural product. Nopaline synthase gene 3 ' end non-translational region stops transcribing, the domination polyadenylation. One fragment is from E.coli lacl coding promoter Plac sequence, and another section comes from the sequence of pUC119 coding beta-D-galactosidase or LacZ albumen. Be the replication origin of pUC plasmid, allow plasmid in Escherichia coli, to copy. II type neomycin phosphotransferase gene. This enzyme has drug resistance to aminoglycoside antibiotics, can be used as the bacterium mark of selecting to contain plasmid. |
DNA composition list in the table 2:PV-ZMGT10 plasmid
| Gene title E35S hsp70 CTP2 CP4EPSPS CTP1 gox NOS3 ' lacZ ori-pUC nptII | Size Kb 0.61 0.80 0.31 1.40 0.26 1.30 0.26 0.24 0.65 0.79 | Function cauliflower mosaic virus (CaMV) promoter tape copy enhancer zone. From the introne of corn hsp70 (heat shock protein) gene, effect is to improve gene transcription level. Chloroplaset shifts peptide, is to separate from mouseearcress EPSPS, and effect is that CP4EPSPS albumen is imported the synthetic site of aromatic amino acid in the chloroplaset. The gene of coding CP4EPSPS albumen is separated from agrobacterium CP4 bacterial strain, is used for transformant to the selection of glyphosate. Chloroplaset shifts peptide, is ribulose-1,5-bisphosphate from mouseearcress, and the little subunit gene on the 5-diphosphonic acid carboxylase gene is separated, and GOX albumen can be imported in the chloroplaset site that aromatic amino acid is synthetic. This gene is glyphosate coding, makes glyphosate oxidoreductase (GOX) that metabolism take place, and separates and gets from the LBAA bacterial strain of achromobacter (newly belonging to brown Flavobacterium). The district of nopaline synthase gene 3 ' terminal untranslated stops transcribing, the domination poly-adenosine. One fragment is from E.coli lacl coding promoter Plac sequence, and another fragment comes from the sequence of pUC119 coding beta-D-galactosidase or LacZ albumen. Be the replication origin of pUC plasmid, allow plasmid in Escherichia coli, to copy. II type neomycin phosphotransferase gene. This enzyme has drug resistance to aminoglycoside antibiotics, can be used as the bacterium mark of selecting to contain plasmid. |
4. acceptor material
The initial corn acceptor that imports the purpose gene is Hi-II, is to come from an offspring who is produced by A188 and two corn hybridizations between selfed lines of B73. A188 and B73 are respectively the self-mating systems of Univ Minnesota-Twin Cities USA and Iowa State University's seed selection, can buy on U.S. seed market. This receptor is organizing cultivation stage that stronger power of regeneration is arranged.
5. conversion method
After conversion carrier and acceptor system build up, just can operate by suitable conversion method, external source purpose gene cryIAb is imported acceptor Hi-II. The conversion method that this research is adopted is particle bombardment.
With calcium chloride and spermidine DNA is deposited on the particulate of tungsten and gold. The particulate that is wrapped with DNA is dropped on the plastics larger vector, make it pass through the gun barrel height with the explosive force of gunpowder and accelerate. Larger vector hits the plastics baffle plate, and the flight of larger vector is hindered, but is wrapped with the particulate continuation flight of DNA. Particulate penetrates the target plant cell, and the DNA precipitation is with the cell chromosomal integration.
6. transformant screening regeneration
To from no transformed cells, filter out transformant after the conversion.Cell is incubated at and contains auxin 2, grows on the tissue culture medium (TCM) of 4-D.Though containing to encode, the dna solution that is used to transform constitutes the gene (for example CP4EPSPS and gox gene) that glyphosate is had tolerance, be convenient in containing the medium of glyphosate, select genetically-altered cells, but these genes do not exist in the plant of the pest-resistant strain of MON810.Therefore, the cell that draws in the MON810 strain might be " person of slipping through the net " in the glyphosate selection course.In stage afterwards, do not having under the situation of glyphosate, callus plantlet is chemically examined, and identifies whether to have the CryIAb protein product.
7. the identification and analysis of transfer-gen plant
Be incorporated in the MON810 strain DNA by analysis so that determine the insert number (number of binding site in the maize chromosome group) of each gene and duplicate quantity.Adopt the Southern blotting during evaluation, analyze the genomic DNA that from contrast milpa and pest-resistant strain milpa leaf tissue, separates.
The generation of the pest-resistant corn strain of MON810 is by the particle gun method, and used dna solution contains two kinds of plasmid: PV-ZMBK07[and contains the cryIAb gene] and PV-ZMGT10[contain CP4EPSPS and gox gene].The MON810 corn strain is not incorporated the PV-ZMGT10 plasmid sequence into.This strain comprises the DNA that an about 5.5KbNdeI fragment is integrated, and wherein contains an E35S promotor, hsp70 intron and cryIAb gene.In the main chain of nptII gene and PV-ZMBK07 plasmid is not incorporated into.This strain does not contain CP4EPSPS, gox or nptII gene, does not contain the plasmid main chain of PV-ZMGT10 plasmid yet.
| Genetic element | Corn strain MON810 |
| The CryIAb gene | Have |
| The CP4ESPSP gene | No |
| The gox gene | No |
| ????nptII/ori-pUC | No |
The A.Southern results of hybridization
Plasmid PV-ZMBK07 has the cryIAb gene, and plasmid PV-ZMGT10 has CP4EPSPS gene and gox gene.The position of these two kinds of plasmid carriers of PV-ZMBK07 and PVZMGT10 and the restriction site used in Southern hybridization as shown in Figure 1.
The DNA of MON818 and MON810 plant is used for Southern hybridization after through the digestion of multiple restriction enzyme, enter the gene of corn gene group when the particle bombardment to identify.Specifically, be exactly to detect the number (number of integration site in the corn gene group) that inserts fragment, the copy number of each gene and integrality thereof.
The digestion result of B.NdeI
The purpose of NdeI digestion experiment is to determine the number of plasmid DNA insertion fragment in corn strain MON810.Plasmid PV-ZMBK07 and PV-ZMGT10 do not have the NdeI site.Therefore, this enzyme cuts outside the insertion fragment effectively, discharges a fragment that contains the corn gene group DNA that inserts DNA and close on.The contrast DNA of MON818 and the DNA of MON810 are through NdeI digestion, and PV-ZMBK07 is hybridized as probe with plasmid, and the result as shown in Figure 2.The DNA of MON818 (swimming lane 1) produces the band of about 21.0Kb of a very bright but diffusion; This is a background band, because it all exists in MON818 and MON810.MON810 produces the band (swimming lane 2) of a treaty 5.5Kb.This result proves that pest-resistant corn strain MON810 contains the dna fragmentation of an integration.The DNA that inserts adds the corn gene group DNA up to the NdeI restriction site that closes on, and 5.5Kb is arranged approximately.
C. insert the composition of fragment
1) integration of .cryIAb gene
The DNA of MON818 and MON810 digests with NcoI/EcoRI, to produce the cryIAb gene, uses the cryIAb gene as probe in Southern hybridization.Experimental result is shown in Fig. 3 (swimming lane 1-3).Positive hybridization contrast (swimming lane 1) produces the fragment of 1 3.46Kb, the size of the cryIAb gene that is equivalent to expect (with reference to the plasmid schematic diagram of figure 1).Because do not have the genomic DNA of contrast in the plasmid DNA, it is bigger than its actual molecular weight that this band seems.The DNA of MON818 (swimming lane 2) does not produce band, and this expected results with check clone is identical.MON810 (swimming lane 3) produces the band of a treaty 3.1Kb.
2) integration of .CP4ESPSP gene
Plasmid DNA (PV-ZMBK07 and PV-ZMGT10) and pest-resistant corn strain MON810DNA digest with NcoI/BamHI, to produce the CP4ESPSP gene, use the CP4ESPSP gene as probe in Southern hybridization.Experimental result is shown in Fig. 4 (swimming lane 1-2).The hybrid dna of two kinds of plasmids of about 50pg (swimming lane 1) produces the band of 1 3.1Kb, the size of the CP4ESPSP fragment that is equivalent to expect, and this is with identical from plasmid schematic diagram (PV-ZMGT10 Fig. 1) results estimated.The DNA of MON810 (swimming lane 2) does not have the fragment with the CP4ESPSP probe hybridization, proves that the pest-resistant strain MON810 of corn does not have the CP4ESPSP gene.
3) integration of .gox gene
Plasmid DNA (PV-ZMBK07 and PV-ZMGT10) and pest-resistant corn strain MON810DNA digest with NcoI/BamHI, to produce the gox gene, use the gox gene as probe in Southern hybridization.Experimental result is shown in Fig. 4 (swimming lane 3-4).The hybrid dna of two kinds of plasmids of about 50pg (swimming lane 3) produces the NcoI/NcoI fragment of 1 treaty 3.1Kb, the size of the gox fragment that is equivalent to expect, and this is with identical from plasmid schematic diagram (PV-ZMGT10 Fig. 1) results estimated.The DNA of MON810 (swimming lane 4) does not have the fragment with the gox probe hybridization, proves that pest-resistant corn strain MON810 does not have the gox gene.
4). the integration of carrier framework
The DNA of plasmid PV-ZMBK07, check clone MON818 and pest-resistant corn strain MON810 digests with NcoI/EcoRI, to produce the nptII/ori-pUC skeleton, uses the nptII gene as probe in Southern hybridization.Experimental result is shown in Fig. 5 (swimming lane 1-3).The DNA of the plasmid PV-ZMBK07 of about 50pg produces two bands (swimming lane 1) that are respectively 2.5Kb and 1.8Kb.2.5Kb and the band of 1.8Kb the size (with reference to figure 1) that is equivalent to expect from the fragment of carrier PV-ZMBK07.The DNA of MON818 (swimming lane 2) does not produce band, and this expected results with the check clone that is not transformed is identical.The DNA of MON810 (swimming lane 3) does not produce band, proves that the frame sequence of carrier is not integrated into pest-resistant corn strain MON810.
The Southern hybond membrane is cleaned the back and is used the ori-pUC zone as hybridization probe.The DNA of plasmid PV-ZMBK07 and PV-ZMGT10 (swimming lane 4) has the band of 1 1.8Kb.1.8Kb band the size (with reference to figure 1) that is equivalent to expect from the fragment of PV-ZMBK07 skeleton.The DNA of MON818 (swimming lane 5) does not produce band, and this expected results with the check clone that is not transformed is identical.The DNA of MON810 (swimming lane 6) does not produce band, proves that the frame sequence of carrier is not integrated into pest-resistant corn strain MON810.The band that lacks with oripUC probe and nptII probe hybridization proves that pest-resistant corn strain MON810 does not contain any frame sequence.
8. transgenic progeny heredity, phenotypic stability is identified
The pest-resistant corn strain allelomorph of MON810 separates and stability data passes through and compiles, and shows the data consistent with the insertion point of cryIAb genomic DNA single-activity.
Transgene and to the stability of corn borer resistance in the hybridization of seven generations with product subsequently is commercial all confirms in using.
9. backcross transformation
Adopt conventional hybridization and back cross breeding method, as donor, just can obtain the pest-resistant corn of MON810 of our required genetic background with the pest-resistant corn of any MON810.
For a better understanding of the present invention, we are set forth as follows some technology that adopted among the following embodiment:
1. the source of corn borer:
The all research that relates to the artificial infection Asiatic corn borer of present embodiment, used Asiatic corn borer are Hengshui Prefecture, the Hebei province population that survives the winter.Adopt the no agar meridic diet of invention of Plant Protection institute, Chinese Academy of Agricultral Sciences corn borer research department and gain the national patent and the artificial mass rearing method of Asiatic corn borer of Zhou Darong (1980) standard to raise.Testing used Asiatic corn borer pieces of an egg and newly hatched larvae is high-quality.
2. indoor biometrics method
The first step: according to the requirement of following prescription, the basal feed that preparation is used to raise Asiatic corn borer.After the mixing, place food mixer to stir each 1 minute (in case continuous stirring is overheated) 3 times.
Composition consumption (g)
1 soybean meal 15.0
2 corn flour 18.0
3 brewer's yeasts 9.0
4 glucose (or edible white sugar) 7.5
5 vitamins 0.5
6 erythromycin 0.15
7 sorbic acids 0.5
8 JSMD (agar substitute) 10.0
Second step: take by weighing and put into jar after 45g is cut into fragment for the tissue of identifying; Get the 90ml running water, pour jar into after adding 4 (about 0.2ml) 40% formaldehyde; Start is stirred 3 times, and each 1 minute, 30 seconds at interval.
The 3rd step: one, two liang of step gains is mixed in beaker, and it is standby to make two kinds of test feeds that contain MON810 and non-MON810 corn tissue.
The 4th step: will test pack into the dactylethrae of 2.5cm * 8.0cm of feed branch, every pipe 6g respectively connects and just incubates 10 of corn borer larvaes, with the stifled tight mouth of pipe of the rubber stopper that studs with the copper yarn, puts that cultivation 7-1 observed after 0 day under 28-30 ℃ of constant temperature, the 70-80%RH condition.Investigation survival larva number also claims its body weight.
3. improved indoor biometrics method
On former indoor biometrics method, done further improvement.The fresh MON810 that will adopt back from the field and blade, tassel and the filigree etc. of non-MON810 corn shred respectively, put into mortar again.Pour an amount of liquid nitrogen flash freezer into, grind pulping after, with its with a certain amount of through finished no agar meridic diet mixing respectively, make that to test feed standby.
Get a normal transparent spool box, cover at it and make a call to the circular hole that a diameter is 5mm, and glue a slice stainless steel yarn sheet, make testing cassete in the inboard.Every part of test feed branch is installed to 100 testing cassetes, the about 2g of every box is incubated the Asiatic corn borer larva at the beginning of connecing one, and per 10 boxes are one group, be placed on 28-30 ℃ of constant temperature, RH and be under the condition of 70-80% and raise, check after 8 days and to write down live borer population and worm of every group Asiatic corn borer heavy.
4. the food leaf-size class is other
9 grades of grade scales that adopt international corn borer cooperative groups to formulate.
1～3 grade: worm channel needle prick shape (claiming pin hole again).
1 grade: rare, dispersion,
2 grades: moderate quatity,
3 grades: a large amount of.
4～6 grades: worm channel match end size.
4 grades: rare, dispersion,
5 grades: moderate quatity,
6 grades: a large amount of.
7～9 grades: worm channel is greater than match end.
7 grades: rare, dispersion,
8 grades: moderate quatity,
9 grades: a large amount of.Wherein, 1～2.9 grade is high anti-type; 3～4.9 grades is anti-type; 5～6.9 grades be in anti-type; 7～9 grades are sense worm type (surpass 8 grades and be high sense type).
Prove that CryIAb albumen that pest-resistant corn strain MON810 expresses makes the anti-Asiatic corn borer of its blade in blade.
Following respectively the experiment in 1997 of embodiment 1 carried out in Plant Protection institute, Chinese Academy of Agricultral Sciences corn borer research department (Beijing).For examination material MON810 corn is crossbreed B73 * Mo17MON810, but not MON810 is the conventional hybridization kind B73 * Mo17 of its homotype.B73 is by the seed selection of U.S. clothing Aiwa state university, and Mo17 is by the seed selection of the state university of Missouri, USA, and Mo17MON810 is formed by backcross transformation by U.S. Holden ' s basis seeds company.B73 * Mo17MON810 and B73 * Mo17 can buy from U.S. Holden ' s basis seeds company (Holden ' s Foundation Seeds L.L.C., Williamsburg, Iowa, USA).
Experiment 1: give birth to the survey method and show, the CryIAb albumen that pest-resistant corn strain MON810 expresses in new leaf texture makes its anti-Asiatic corn borer of new leaf texture.
Take by weighing the fresh lobus cardiacus tissue of 45g corn, adopt the indoor biometrics method to identify.Observe and find, in the control tube of non-MON810, very easily see well-developed 3～4 instar larvaes, then do not see 3～4 instar larvaes in each pipe of MON810.In each pipe of MON810, visible is not have to grow promptly dead larva attached on the test feed of green and on the tube wall of dactylethrae.
Experiment 2: the field identifies and to show, the CryIAb albumen that pest-resistant corn strain MON810 expresses in the corn young leaves blade in mid-term makes the corn young leaves anti-Asiatic corn borer of blade in mid-term.
Sow on April 23rd, 1997 for MON810 and the non-MON810 corn identified, identify that the lobus cardiacus at corn carried out mid-term June 17 and 20 days, every strain connects 2 on blackhead ovum (every about 40-50 grain), in investigation result on June 28, totally 2 indexs: the one, international food leaf grade, the 2nd, every strain larva survives number.The results are shown in Table 3, clearly show splendid control efficiency: on the MON810 milpa, do not find the survival larva.
Table 3: the corn lobus cardiacus phase is to the resistance result of Asiatic corn borer
Experiment 3: the CryIAb albumen that pest-resistant corn strain MON810 expresses in the different blades of corn young leaves phase makes the anti-Asiatic corn borer of the different blades table 4 of corn young leaves phase: different blades of lobus cardiacus phase are to the resistance of Asiatic corn borer
Grow to lobus cardiacus during mid-term the MON810 of field growing and non-MON810 milpa, mark its 5th blade from bottom to top, flush with ground cuts off plant and fetches then.Further with whole blades by indicating phyllotaxy from bottom to top respectively, and neat stem stalk attachment region cuts piecewise, is divided into 5 groups, i.e. the 5th, 6 leaves, the 7th, 8 leaves, the 9th, 10 leaves, the tender leaf of the hard-pressed bale tassel bud that the 11st, 12 leaves and last 2-3 sheet are not put on display as yet.All each 5 groups of blades of cutting from 6 strain MON810 and the non-MON810 corn of 6 strains are shredded, mix by group respectively, handle and connect worm by the processing of indoor biometrics method and observe.The results are shown in table 4.
Table 4 result clearlys show the ability that all CryIAb that contain from the MON810 blade and the lobus cardiacus tissue of differing heights all have to be enough to kill almost all for the examination newly hatched larvae.In whole MON810 blade feeding trials, in 1000 newly hatched larvaes being inoculated, only find 1 obvious depauperation, the minimum worm alive of the bodily form, show to rest on 2 age grade sections, rather than all reached for 3～4 ages as non-MON810 processing contrast.
Experiment 4: two anti-Asiatic corn borer of leaf of topmost of the young tender tassel of the feasible parcel of the CryIAb albumen that pest-resistant corn strain MON810 expresses in two leaves of topmost of the young tender tassel of parcel
To fetch the laboratory at the MON810 and the non-MON810 milpa overground part of tasseling stage, two of topmosts that wrapping tassel will be cut.It is the same to give birth to the survey method, and the result shows that two leaves of topmost that wrap up young tender tassel all contain enough CryIAb toxalbumin, and the newly hatched larvae lethality reaches 100% (seeing Table 5).
Table 5: last two leaves of parcel tassel are to the resistance of Asiatic 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 |
| ????21 | ????9 | ????0 |
| ????22 | ????10 | ????0 |
| ????23 | ????9 | ????0 |
| ????24 | ????8 | ????0 |
| ????25 | ????4 | ????0 |
| ????26 | ????10 | ????0 |
| ????27 | ????10 | ????0 |
| ????28 | ????8 | ????0 |
| ????29 | ????10 | ????0 |
| ????30 | ????9 | ????0 |
| Add up to | ????262 | ????0 |
| On average | ????8.7333 | ????0 |
Experiment 5: the CryIAb albumen that pest-resistant corn strain MON810 expresses in two leaves of tasseling stage parcel tassel makes tasseling stage wrap up two anti-Asiatic corn borer of leaf of tassel
To fetch the laboratory at the MON810 and the non-MON810 milpa overground part of tasseling stage, two blades that wrapping tassel will be cut.Measure its resistance according to improved indoor biometrics method, the results are shown in table 6.Can see that from table 6 processing of MON810 maize leaf contains enough CryIAb albumen, the Asiatic corn borer larval mortality is respectively 99% and 100%, but not MON810 only is 8.73%; And MON810 and non-MON810 survival larva body weight are widely different.
Table 6: the insecticidal activity of revealing two blades of male phase parcel tassel
Handle larva number (head) survival borer population gross weight lethality worm weight/feed: tissue meets (%) average (mg) average 1: 1 non-MON810 11 8 0.1822 27.27 8.73 22.7750 21.0664 of borer population survival borer population (g)
10???????????8???????????0.2357????20?????????????29.4625
11???????????11??????????0.2061????0??????????????18.7364
12???????????12??????????0.2065????0??????????????17.2083
10???????????10??????????0.1769????0??????????????17.6900
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
10???????????8???????????0.1947????20?????????????24.33751∶1??MON810??????10???????????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?????????????????????1003∶2??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
Experiment 6: 5 following CryIAb albumen that launch to express in the blades of two leaves that pest-resistant corn strain MON810 tasseling stage wraps up tassel make tasseling stage wrap up 5 following anti-Asiatic corn borer of expansion blade of two leaves of tassel
To fetch the laboratory at the MON810 and the non-MON810 milpa overground part of tasseling stage, and launch blades and cut wrapping below two blades of tassel 5.Measure its resistance according to improved indoor biometrics method, the results are shown in table 7.Can see that from table 7 processing of MON810 maize leaf contains enough CryIAb albumen, the Asiatic corn borer larval mortality is respectively 98% and 100%, but not MON810 only is 3% and 6.91%; And MON810 and non-MON810 survival larva body weight are widely different.
Table 7: 5 following insecticidal activities of launching blade of two leaves of revealing male phase parcel tassel
Handle larva number (head) survival borer population gross weight lethality worm weight/feed: tissue meets (%) average (mg) average 3: 2 non-MON810 88 0.178 03 22.2500 19.8464 of borer population survival borer population (g)
10???????8???????0.137?????20?????????????17.1250
11???????11??????0.2049????0??????????????18?6273
10???????10??????0.184?????0??????????????18.4000
10???????10??????0.2143????0??????????????21.4300
10???????10??????0.1754????0??????????????17.5400
10???????9???????0.1703????10?????????????18.9222
10???????10??????0.2186????0??????????????21?8600
10???????10??????0.2264????0??????????????22.6400
10???????10??????0.1967????0??????????????19.6700??3∶2??MON810??????10???????1???????0.0000????90??????98?????????0.0??????????0.0
10???????0?????????????????100
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 1001: 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
12???????12??????0.299?????0???????????????24.9167
11???????10??????0.2259????9.1?????????????22.5900
10???????8???????0.1743????20??????????????21.7875
10???????10??????0.1987????0???????????????19.8700
10???????10??????0.2358????0???????????????23.58001∶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
Prove that the CryIAb albumen of expressing in the pest-resistant corn strain MON810 tassel makes the anti-Asiatic corn borer of its tassel.
Following respectively the experiment in 1997 of this embodiment carried out in Plant Protection institute, Chinese Academy of Agricultral Sciences corn borer research department (Beijing).For examination material MON810 corn is crossbreed B73 * Mo17MON810, but not MON810 is the conventional hybridization kind B73 * Mo17 of its homotype.B73 is by the seed selection of U.S. clothing Aiwa state university, and Mo17 is by the seed selection of the state university of Missouri, USA, and Mo17MON810 is formed by backcross transformation by U.S. Holden ' s basis seeds company.B73 * Mo17MON810 and B73 * Mo17 can buy from U.S. Holden ' s basis seeds company (Holden ' s Foundation Seeds L.L.C., Williamsburg, Iowa, USA).
Experiment 1:
The authenticate technology of tassel resistance is that 2 blackhead ovum are placed on the young tender tassel, puts a pollination bag then and tightens sack to reduce escape or to be pecked at by birds.On July 4th, 1997 was inoculated each 26 strain of corn of MON810 and non-MON810, and July 16, investigation survived the larva number, the results are shown in Table 8.The tassel significant difference of MON810 and non-MON810, MON810 maize male ears contain enough CryIAb albumen, do not find the survival larva, and larval mortality reaches 100%.
Table 8:MON810 tassel is to the resistance of Asiatic corn borer
| ????18 | ????12 | ????0 |
| ????19 | ????16 | ????0 |
| ????20 | ????6 | ????0 |
| ????21 | ????11 | ????0 |
| ????22 | ????13 | ????0 |
| ????23 | ????19 | ????0 |
| ????24 | ????11 | ????0 |
| ????25 | ????21 | ????0 |
| ????26 | ????7 | ????0 |
| Add up to | ????365 | ????0 |
| On average | ????14.04 | ????0 |
Experiment 2:
The indoor biometrics method is the same, just blends amount of water minimizing 10ml when organizing, because of fresh tassel water content is many far beyond lobus cardiacus.The result shows that the MON810 maize male ears contains enough CryIAb albumen, and the newly hatched larvae lethality reaches 100% (seeing Table 9).
Table 9: the tassel bud is to the resistance of Asiatic 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 |
| ??21 | ????9 | ????0 |
| ??22 | ????7 | ????0 |
| ??23 | ????10 | ????0 |
| ??24 | ????9 | ????0 |
| ??25 | ????8 | ????0 |
| ??26 | ????8 | ????0 |
| ??27 | ????7 | ????0 |
| ??28 | ????9 | ????0 |
| ??29 | ????9 | ????0 |
| ??30 | ????7 | ????0 |
| Add up to | ????251 | ????0 |
| On average | ????8.3667 | ????0 |
Experiment 3: the CryIAb albumen of expressing in the tassel before the loose powder makes the anti-Asiatic corn borer of tassel before the loose powder
Adopt the indoor measurement method of aforementioned improved, the result shows, contain the feed of tassel tissue before the MON810 corn loose powder and can kill Asiatic corn borer newly hatched larvae more than 93%, the larvae development of surviving stagnation (seeing Table 10), as seen it contains enough CryIAb albumen.
Table 10: the tassel before the loose powder is to the resistance of Asiatic corn borer
Handle a larva number survival borer population gross weight lethality worm weight/feed: tissue meets (%) average (mg) average 3: 2 non-MON810 10 10 0.1501 0 1.25 15.01 17.3226 of borer population survival borer population (g)
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????????????????19.623∶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 0.101: 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????????????????15.711∶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
Prove that CryIAb albumen that pest-resistant corn strain MON810 expresses makes the anti-Asiatic corn borer of its flower pesticide in flower pesticide.
Tested in 1997 and carry out in Plant Protection institute, Chinese Academy of Agricultral Sciences corn borer research department (Beijing).For examination material MON810 corn is crossbreed B73 * Mo17MON810, but not MON810 is for early generation is MC-37.B73 is by the seed selection of U.S. clothing Aiwa state university, and Mo17 is by the seed selection of the state university of Missouri, USA, and Mo17MON810 is formed by backcross transformation by U.S. Holden ' s basis seeds company.B73 * Mo17MON810 can buy from U.S. Holden ' s basis seeds company (Holden ' s Foundation Seeds L.L.C., Williamsburg, Iowa, USA).Non-MON810 corn, is provided by Plant Protection institute, Chinese Academy of Agricultral Sciences corn borer research department for being MC-37 for early.
The collection of flower pesticide: entangle the tassel of loose powder phase with pollination bag, second day shake tassel separates the flower pesticide that is scattered in the bag with sub-sieve and pollen, take back indoor.It is the same to give birth to the survey method.
The result shows that the flower pesticide of MON810 corn contains enough CryIAb albumen, and the feed that contains MON810 corn flower pesticide can kill 100% Asiatic corn borer newly hatched larvae (seeing Table 11).
Table 11: the insecticidal activity of flower pesticide
Handle larva number survival borer population gross weight lethality worm weight/head
Meet (%) average (mg) average MON810 50 100 100 of borer population survival borer population (g)
5??????????0??????????????100
5??????????0??????????????100
5??????????0??????????????100
5??????????0??????????????100
5??????????0??????????????100
5??????????0??????????????100
5??????????0??????????????100
5??????????0??????????????100
5??????????0??????????????100MC-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
Prove that CryIAb albumen that pest-resistant corn strain MON810 expresses makes the anti-Asiatic corn borer of its filigree in filigree.
Following respectively the experiment in 1997 among this embodiment carried out in Plant Protection institute, Chinese Academy of Agricultral Sciences corn borer research department (Beijing).For examination material MON810 corn is crossbreed B73 * Mo17MON810, but not MON810 is the conventional hybridization kind B73 * Mo17 of its homotype.B73 is by the seed selection of U.S. clothing Aiwa state university, and Mo17 is by the seed selection of the state university of Missouri, USA, and Mo17MON810 is formed by backcross transformation by U.S. Holden ' s basis seeds company.B73 * Mo17MON810 and B73 * Mo17 can buy from U.S. Holden ' s basis seeds company (Holden ' s Foundation Seeds L.L.C., Williamsburg, Iowa, USA).
Experiment 1:
Pick up from the MON810 and the fresh filigree of non-MON810 corn of field bunchy, dipping is once so that keep fresh in clear water earlier, and slightly putting into diameter behind the airing is 11.5cm, in the plastic tank of high 10.5cm, filter paper of jar heelpiece connects 2 blackhead ovum (about 30/piece) on every jar the filigree.Then, on every plastic tank, cover identical slack tank of button, but have 4 equidistant holes at the tank skin middle part, each Kong Jun studs with stainless steel yarn in case escape in larva hatching back, after connecing ovum, use adhesive tape bonding in aggregates for two jars up and down, place to cultivate after 6 days under 28-30 ℃ of constant temperature, the 70-80%RH condition and observe.
The result shows that raise after 6 days, the filigree of MON810 corn is still fresh intact, and the filigree that contrasts non-MON810 corn is all eaten up, only surplus dark-brown ight soil and chip, and a larva alive also be can not see in the process tank of MON810, and the difference of the two is extremely obvious.As seen, contain enough CryIAb albumen in the filigree of MON810 corn.
Experiment 2:
Choose newly hatched larvae, inoculating gun (Bazooka) the inoculation newly hatched larvae that adopts CIMMYT entomologist doctor Mihm invention is on the fresh filigree that corn has just spued.Investigation result sees Table 12.As seen, the filigree of MON810 corn and non-MON810 corn shows utmost point evident difference, therefore, contains enough CryIAb albumen in the filigree of MON810 corn.
The resistance of table 12:MON810 corn capillament
| Strain number | Larvae alive quantity (head/strain) | |
| 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 |
| ????21 | ????14 | ????0 |
| ????22 | ????13 | ????0 |
| ????23 | ????15 | ????0 |
| ????24 | ????29 | ????0 |
| ????25 | ????21 | ????0 |
| ????26 | ????21 | ????0 |
| ????27 | ????32 | ????0 |
| ????28 | ????14 | ????0 |
| ????29 | ????24 | ????0 |
| ????30 | ????27 | ????0 |
| Add up to | ????304 | ????4 |
| On average | ????10.13 | ????0.19 |
Experiment 3:
Tested in 1997 and carry out in Plant Protection institute, Chinese Academy of Agricultral Sciences corn borer research department, experimental technique adopts indoor improved living survey method.The results are shown in table 13.The MON810 corn capillament contains enough CryIAb albumen, can kill Asiatic corn borer newly hatched larvae more than 96% with its feed prepared, and the larvae development of surviving is stagnated.
Table 13: filigree is to the resistance of Asiatic corn borer
Handle a larva number survival borer population gross weight lethality worm weight/feed: tissue meets (%) average % (mg) average 3: 2 non-10 9 0.1379 10 13.75 15.32 12.16 of borer population survival borer population (g)
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???????????????????100???98.75??????????????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 1001: 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???????????13.041∶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
Embodiment 5
Proof under land for growing field crops artificial infection Asiatic corn borer condition, the production-increasing function that CryIAb albumen is showed in pest-resistant corn strain MON810.
Tested in 1998 and draw and educate 4 places, center and carry out being positioned at Shenyang Academy of Agricultural Sciences, Henan Academy of Agricultural Sciences, Jilin Academy of Agricultural Science and Jilin Province's new crop varieties that Chinese northern corn master plants the district.Amount to 14 corn hybrid seeds for the examination material, comprise 6 pairs of homotype crossbreed and two local main breeds (contrast).With LH be the crossbreed of assembly can buy by U.S. Holden ' s basis seeds company (Holden ' sFoundation Seeds L.L.C., Williamsburg, Iowa, USA), other homotype crossbreed can have been bought from U.S. Monsanto Company.Local contrast seed can have been bought in China local seed market.Each is made up of 1 pest-resistant crossbreed of MON810 and a non-MON810 conventional hybridization kind the homotype crossbreed, the difference of the two is that the pest-resistant crossbreed of MON810 can express CryIAb albumen, but not the MON810 crossbreed then can not, and other genetic constitution is consistent.What obviously, showed between the homotype crossbreed is not both owing to contain or do not contain the result of CryIAb albumen.
In whole corn growth season, connect worm and divide and carry out for three times.In the young leaves phase, two Asiatic corn borer blackhead ovum (about 50～60/piece) are placed in the top young leaves of plant for the first time; For the second time taking out forming ears the phase before male, upper blade is inoculated two Asiatic corn borer blackhead ovum; For the third time in the phase of weaving silk, two Asiatic corn borer blackhead ovum are seeded in the fresh filigree that corn just spued.Like this, every strain corn inoculum concentration adds up to and is about 300～360 Asiatic corn borer blackhead ovum.Yield result sees Table 14.
Table 14 shows that the MON810 crossbreed of the CryIAb albumen of can encoding is the non-MON810 crossbreed of its homotype relatively, is connecing under the worm condition, shows tangible effect of increasing production.Table 14 as can be seen, all single to the homotype crossbreed in all test sites, all show as the volume increase of MON810 crossbreed, what amount of increase in production was minimum is 6.7%, the highest is 89.5%.On all 4 test sites, the output per hectare of 6 non-MON810 crossbreed on average only is 7103 kilograms, and their 6 homotype MON810 crossbreed output per hectare on average then reaches 9102 kilograms, and per hectare produces more food 1999 kilograms, amount of increase in production 28.1%.4 test points, the amount of increase in production of the non-relatively MON810 crossbreed of MON810 crossbreed, Academy of Agricultural Sciences is the highest with Henan, reaches 69.1%, Shenyang Academy of Agricultural Sciences takes second place, and is 29.5%, is the Jilin Academy of Agricultural Science secondly, be 25.1%, minimum educates the center for drawing, and also has 15.5%.Difference all shows as volume increase although the amount of increase in production of the non-relatively MON810 of MON810 is with the difference of 6 pairs of test hybrid combinations, and amplitude is 19.8%～32.9%.Can draw, compare with the non-MON810 corn hybrid seed that does not contain coding CryIAb albumen, the MON810 corn hybrid seed yield level that contains coding CryIAb albumen is higher, the raising of output be since CryIAb albumen to due to the resistance of Asiatic corn borer.
Table 14:1998 Meng Shan is MON810 compliance test result test yield result table
| The crossbreed title | Pedigree | The academy of agricultural sciences, Shenyang | The homotype volume increase | The academy of agricultural sciences, Henan | The homotype volume increase | The academy of agricultural sciences, Jilin | The homotype volume increase | Draw and educate the center | The homotype volume increase | Each point is average | The homotype volume increase | |
| ????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 | |||||||
| The MON810 crossbreed is average | ????9063 | ????29.5 | ????5993 | ????69.1 | ????9398 | ????25.1 | ????11954 | ????15.5 | ????9102 | ????28.1 | ||
| Non-MON810 crossbreed is average | ????7001 | ????3544 | ????7514 | ????10354 | ????7103 | |||||||
Draw and educate the center: Jilin Province's new crop varieties draws educates the center
Homotype volume increase: the amount of increase in production of the relative homotype conventional hybridization of MON810 crossbreed kind
Embodiment 6
Proof under condition is caused harm in land for growing field crops Asiatic corn borer abiogenesis, the production-increasing function that CryIAb albumen is shown in pest-resistant corn strain MON810.
Test to draw at Shenyang Academy of Agricultural Sciences, Shandong Agricultural University, Dandong Academy of Agricultural Sciences, Jilin Academy of Agricultural Science and Jilin Province's new crop varieties and educate 5 places, center and carry out in 1998.These places suffer causing harm of Asiatic corn borer throughout the year, and cause certain production loss.Amount to 22 corn hybrid seeds for the examination material, wherein form by 10 pairs of homotype crossbreed for 20, promote mainly corn hybrid seed (contrast) for the locality for other 2.With LH be the crossbreed of assembly can buy by U.S. Holden ' s basis seeds company (Holden ' sFoundation Seeds L.L.C., Williamsburg, Iowa, USA), other homotype crossbreed can have been bought from U.S. Monsanto Company.Local contrast seed can have been bought in China local seed market.The homotype crossbreed is as described in the embodiment 5.Test is to carry out under the natural conditions of land for growing field crops, and all plant artificially do not inoculate Asiatic corn borer, and causing harm of all Asiatic corn borer all is to take place under the natural conditions of land for growing field crops.Yield result is listed in table 15.
As known from Table 15, each, all shows as MON810 crossbreed output and is higher than the non-MON810 crossbreed of its homotype in all test sites the homotype crossbreed.On all 5 test sites, the output per hectare of 10 non-MON810 crossbreed on average only is 8066 kilograms, and their 10 homotype MON810 crossbreed output per hectare on average then reaches 9645 kilograms, and per hectare produces more food 1579 kilograms, amount of increase in production 19.6%.For 5 test points, the amount of increase in production of the non-relatively MON810 crossbreed of MON810 crossbreed, the highest with Dandong Academy of Agricultural Sciences, reach 46.3%, minimum is the Jilin Academy of Agricultural Science, also have 9.8%, Shenyang Academy of Agricultural Sciences, Shandong Agricultural University and drawing educate centre bit placed in the middle between, be respectively 22.6%, 15.0% and 13.4%.Difference all shows as volume increase although the amount of increase in production of the non-relatively MON810 of MON810 is with the difference of 10 pairs of test hybrid combinations, and amplitude is 12.3%～26.4%.This test can draw, and compares with the non-MON810 corn hybrid seed that does not contain coding CryIAb albumen, and the MON810 corn hybrid seed output that contains coding CryIAb albumen is higher, the increase of output be since CryIAb albumen to due to the resistance of Asiatic corn borer.
Table 15:1998 Meng Shan is MON810 crossbreed comparative test yield result table
| The crossbreed title | Pedigree | The academy of agricultural sciences, Shenyang | The homotype volume increase | Shandong Agricultural University | The homotype volume increase | Academy of agricultural sciences, Dandong | The homotype volume increase | The academy of agricultural sciences, Jilin | The homotype volume increase | Draw and educate the center | The homotype volume increase | Each point is average | The homotype volume increase | |
| ????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 | ||||||||
| The MON810 crossbreed is average | ????9038 | ????22.6 | ????6656 | ????15.0 | ????9210 | ????46.3 | ????11070 | ????9.8 | ????12253 | ????13.4 | ????9645 | ????19.6 | ||
| Non-MON810 crossbreed is average | ????7372 | ????5788 | ????6293 | ????10079 | ????10800 | ????8066 | ||||||||
Draw and educate the center: Jilin Province's new crop varieties draws educates the center
Homotype volume increase: the amount of increase in production of the relative homotype conventional hybridization of MON810 crossbreed kind
Embodiment 7
Proof, under condition was caused harm in land for growing field crops Asiatic corn borer abiogenesis, CryIAb albumen made that in the expression of pest-resistant corn strain MON810 its kernel weight increases, cane resistance improvement (resistant to lodging, minimizing stem rot), and little to other The Characters influence.
Test method the results are shown in following table 16 with embodiment 6.
By seeing in the table 16, the CryIAb albumen that the MON810 crossbreed contains not only makes the thousand kernel weight of corn kernel improve, simultaneously because the improvement of cane quality makes its stem rot minimizing, the reduction of lodging property.With regard to thousand kernel weight, average 348 grams of MON810 crossbreed, but not average 321 grams of MON810 crossbreed, the MON810 crossbreed is improved 27 grams, reaches 8.4%.On these proterties that stem rot, root lodge and stem lodges, the MON810 crossbreed on average is respectively 9%, 5% and 0%, and corresponding non-MON810 crossbreed then is 19%, 9% and 1%.Obviously, this is because on the milpa that contains CryIAb albumen, the minimizing of Asiatic corn borer and reduction that the physics that causes and physiology destroy and the result that produces.
Also can see by table 16, with regard to plant height, the fringe position is high and breeding time these tree characteristics and the fertility proterties, 6 MON810 crossbreed average out to 269cm, 100cm and 110 days, the non-MON810 crossbreed of corresponding 6 homotypes are respectively 268cm, 99cm and 110 days, and the two is obviously difference not.Main leaf diseases and fringe portion proterties,, tassel row number thick, seed-producing rate as graywall, leaf blight, helminthosporium maydis, spike length, fringe, the two has the minute differences (87% pair 86%) except that seed-producing rate, and the two is identical for all the other The Characters.
Table 16:1998 Meng Shan is the main proterties result of MON810 crossbreed comparative test
| The crossbreed title | Pedigree | Plant height | The fringe position is high | Breeding time | Head smut | Smut | Stem rot | Rough dwarf disease | Graywall | Leaf blight | Helminthosporium maydis | Root example volt | The stem lodging | Empty bar rate | Spike length | Fringe is thick | Tassel row number | Seed-producing rate | Thousand kernel weight |
| ????cm | ????cm | My god | ????％ | ????％ | ??％ | ??％ | Level | Level | Level | ????％ | ????％ | ????％ | ????cm | ????cm | ????％ | ????9 | |||
| 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 |
| The MON810 crossbreed is average | ????269 | ????100 | ????110 | ????0 | ????0 | ????9 | ????11 | ????2 | ????0 | ????1 | ????5 | ????0 | ????2 | ????20 | ????5 | ????15 | ????87 | ????348 | |
| Non-MON810 crossbreed is average | ????268 | ????99 | ????110 | ????2 | ????0 | ????19 | ????11 | ????2 | ????0 | ????1 | ????9 | ????1 | ????5 | ????20 | ????5 | ????15 | ????86 | ????321 | |
Embodiment 8
Proof manually connects in the land for growing field crops under the condition of worm Asiatic corn borer, and the expression of CryIAb albumen in pest-resistant corn strain MON810 makes that its kernel weight increases, cane resistance improvement (resistant to lodging, minimizing stem rot), and little to other The Characters influence.
Test method is with experimental example 5, and observed result sees Table 17.
As can be seen from Table 17, connect under the worm condition in artificial heavy dose, can express that the MON810 crossbreed of CryIAb albumen and non-MON810 crossbreed are high at plant height, fringe position, not have big difference on the main proterties such as breeding time, leaf blight, helminthosporium maydis, rough dwarf disease, graywall, spike length, fringe are thick, tassel row number and seed-producing rate.Yet at grain on the heavy and stem stalk resistance, MON810 shows as not only that thousand kernel weight increases, and plant cane resistance strengthens (reduction of lodging property, stem rot minimizing).The mean value of the thousand kernel weight of MON810 crossbreed, lodging degree and three proterties of stem rot is respectively 330 grams, 12% and 7%, but not the analog value of MON810 crossbreed is respectively 313 grams, 20% and 12%.Obviously, this is because on the milpa that contains CryIAb albumen, the minimizing of Asiatic corn borer and reduction that the physics that causes and physiology destroy and the result that produces.
Table 17:1998 Meng Shan MON810 crossbreed compliance test result tests main proterties table as a result
| The crossbreed title | Pedigree | Plant height | The fringe position is high | Breeding time | Big spot | Stigma | Stem rot | Black powder | The silk dust-brand | Rough dwarf disease | Greyness | Lodging | Spike length | Fringe is thick | Tassel row number | Seed-producing rate | Thousand kernel weight |
| ????cm | ????cm | My god | Level | Level | ????％ | Level | Level | ????％ | Level | ????％ | ??cm | ??cm | ????％ | ????9 | |||
| ?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 | ????1?11 | ????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 | ????11?1 | ????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 | ????1?10 | ????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 |
| The MON810 crossbreed is average | ????260 | ????99 | ????110 | ????1 | ????2 | ????12 | ????0 | ????0 | ????21 | ????2 | ????7 | ????19 | ????5 | ????15 | ????86 | ????330 | |
| Non-MON810 crossbreed is average | ????257 | ????96 | ????109 | ????1 | ????2 | ????20 | ????1 | ????0 | ????19 | ????2 | ????12 | ????19 | ????5 | ????15 | ????86 | ????313 | |
Embodiment 9
Proof connects under the worm condition in the land for growing field crops, and the CryIAb that pest-resistant corn strain MON810 expresses in blade, cane and plant makes the anti-Asiatic corn borer of its blade, cane and plant.
Test method is with embodiment 5, the mensuration project comprise individual plant worm channel number, food leaf-size class not, individual plant length of tunnel and the strain rate that causes harm.Observed result sees Table 18.
As shown in Table 18,6 pairs of homotype crossbreed that all are participated in the experiment, on 4 index characters, though the change up and down with the difference of combination of its numerical values recited, the degree that causes harm of MON810 Asiatic corn borer that crossbreed suffers is all far below the non-MON810 crossbreed of its homotype.6 MON810 crossbreed of participating in the experiment individual plant worm channel number, food leaf-size class not, on individual plant length of tunnel and these 4 index characters of strain rate of causing harm, be respectively 0.04,0.52 grade, 0.03cm and 1.67%, corresponding 6 non-MON810 then are 5.48,3.92 grades, 11.09cm and 85%, and non-MON810 crossbreed is 137 times, 7 times, 370 times and 50 times of MON810 crossbreed.4 index characters of local two conventional check varieties are respectively 5.81,2.39 grades, 13.56cm and 85.4%, compare and can find with non-MON810 crossbreed, and the two gradient of infection to Asiatic corn borer is consistent.As seen, the suffered Asiatic corn borer of MON810 crossbreed of codified CryIAb albumen is caused harm and is starkly lower than two local contrasts and the homotype crossbreed that does not contain coding CryIAb albumen.Therefore, table 18 shows, is manually connecing under the worm processing, and the causing harm of blade, stem stalk and the suffered Asiatic corn borer of whole plant of containing the CryIAb protein maize hybrid will not contain the corn hybrid seed of CryIAb albumen well below those.Obviously, this comes from the control efficiency of CryIAb albumen to Asiatic corn borer.
Table 18:1998 Meng Shan MON810 crossbreed compliance test result tests main harm proterties table as a result
| The crossbreed title | The pedigree source | Individual plant worm channel number | The food leaf-size class is other | The individual plant length of tunnel | Harm strain rate |
| Individual | Level | ????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 | ????1.99 | ????15.42 | ????87.5 | |
| CK2 | ????5.54 | ????2.79 | ????11.69 | ????83.3 | |
| The MON810 crossbreed is average | ????0.04 | ????0.52 | ????0.03 | ????1.67 | |
| Non-MON810 crossbreed is average | ????5.48 | ????3.92 | ????11.09 | ????85.00 | |
The comprehensive analyse shows of embodiment 5 and example 6, the CryIAb albumen that pest-resistant corn strain MON810 expresses has showed tangible production-increasing function.Under the field condition that Asiatic corn borer is caused harm naturally, average 9645 kilograms of MON810 crossbreed output per hectare, and the non-MON810 crossbreed of their homotypes only is 8066 kilograms, MON810 crossbreed per hectare produces more food 1579 kilograms, amount of increase in production 19.6%.Under the field condition of artificial infection Asiatic corn borer, the output per hectare of non-MON810 crossbreed on average only is 7103 kilograms, their homotype MON810 crossbreed output per hectare on average then reaches 9102 kilograms, and per hectare produces more food 1999 kilograms, amount of increase in production 28.1%.Therefore, broadly, as long as the place that has Asiatic corn borer to cause harm, the pest-resistant corn of plantation MON810, its expressed CryIAb albumen all should make corn yield increasing.
Embodiment 1 is to the comprehensive analyse shows of example 9, and the MON810 corn strain produces the expression of CryIAb albumen of toxic action, and the difference because of the plantation place is different to Asiatic corn borer, and the variation because of implantation time does not change.The experiment of carrying out in Plant Protection institute, Chinese Academy of Agricultral Sciences corn borer research department (Beijing) in 1997 shows, the expression of the CryIAb albumen of MON810 corn strain is enough to prevent and treat Asiatic corn borer causing harm to it.Henan Academy of Agricultural Sciences in the summer corn district (Zhengzhou), Shandong Agricultural University (Tai'an) and Shenyang Academy of Agricultural Sciences (Shenyang), Dandong City Academy of Agricultural Sciences (Dandong), Jilin Academy of Agricultural Science (Gongzhuling) and Jilin Province's new crop varieties in the spring corn district in 1998 draw educates center (Gongzhuling) and also all shows, no matter be that the land for growing field crops Asiatic corn borer is caused harm under the condition naturally, or the land for growing field crops manually connects under the condition of worm Asiatic corn borer, and the CryIAb albumen that the MON810 corn strain is expressed also all is enough to prevent and treat Asiatic corn borer causing harm to corn.Therefore, broadly, at any time and the place, as long as the cause harm place of corn of Asiatic corn borer is arranged, the pest-resistant corn of plantation MON810, the CryIAb albumen that it produced all should be enough to prevent and treat Asiatic corn borer causing harm to corn.
Transgenic insect-resistant corn of the present invention obviously is because corn itself can produce CryIAb albumen to the control of Asiatic corn borer, and the MON810 corn only is a carrier of CryIAb protein expression, therefore, those of ordinary skill in the art, according to the identical toxic action of CryIAb albumen to Asiatic corn borer, can produce the transgenic insect-resistant corn that similarly can express CryIAb albumen, be used to prevent and treat Asiatic corn borer causing harm corn.
The CryIAb albumen that is applicable to this invention is the CryIAb polypeptide of the given amino acid sequence of this paper.
By the introduction of previous embodiment, we are not difficult to find out unique distinction of the present invention.
The present invention has provided a kind of cause harm new method of corn of Asiatic corn borer of preventing and treating, and it is to have utilized the modern biology technology to make the expression in the comfortable plant corpus of the toxigenous CryIAb egg of Asiatic corn borer is realized.Broadly, this described method of preventing and treating is not only to corn, and any crop that caused harm by Asiatic corn borer is not always the case.These crops of being caused harm by Asiatic corn borer include, but is not limited to cotton, millet, Chinese sorghum etc.Because as long as CryIAb albumen is at these crop expression in vivo, just can prevent and treat Asiatic corn borer causing harm to it.
Claims (17)
1, a kind of cause harm method of corn of Asiatic corn borer of preventing and treating, it is characterized in that, it is by planting a kind of transgenic insect-resistant corn plant that can express CryIAb albumen in vivo, thereby make the picked-up this plant tissue Asiatic corn borer get the food and growth be suppressed, and finally cause death, realize the cause harm control of corn of Asiatic corn borer.
2, method according to claim 1 is characterized in that, described CryIAb albumen has the amino acid sequence of described CryIAb albumen.
3, method according to claim 1 is characterized in that, described transgenic insect-resistant corn is transgenic insect-resistant corn strain MON810.
4, method according to claim 3 is characterized in that, described transgenic insect-resistant corn strain MON810 comprises any MON810 inbred line, crossbreed, synthetic variety or colony.
5, according to each described method of claim 1 to 4, it is characterized in that, described Asiatic corn borer is caused harm whole plant that the control of corn is meant transgenic insect-resistant corn to the cause harm control of corn of Asiatic corn borer.
6, method according to claim 5 is characterized in that, the whole plant of described transgenic insect-resistant corn comprises that to the cause harm control of corn of Asiatic corn borer its blade, cane, tassel, flower pesticide and filigree are to the cause harm control of corn of Asiatic corn borer.
7, according to each described method of claim 1 to 4, it is characterized in that, described Asiatic corn borer is caused harm time of infertility that the control of corn is meant transgenic insect-resistant corn to the cause harm control of corn of Asiatic corn borer.
8, method according to claim 7, it is characterized in that described transgenic insect-resistant corn time of infertility comprises that to the cause harm control of corn of Asiatic corn borer its seedling stage, young leaves phase, the phase of forming ears, tasseling stage, filigree phase are to the cause harm control of corn of Asiatic corn borer.
According to each described method of claim 1 to 4, it is characterized in that 9, described control to Asiatic corn borer does not change because of the change in plantation place.
10, according to each described method of claim 1 to 4, it is characterized in that, described the cause harm control of corn of Asiatic corn borer is not changed because of the change of implantation time.
11, according to each described method of claim 1 to 4, it is characterized in that, described the cause harm control of corn of Asiatic corn borer is meant the production-increasing function that transgenic insect-resistant corn is shown under land for growing field crops artificial infection Asiatic corn borer condition.
12, according to each described method of claim 1 to 4, it is characterized in that, described the cause harm control of corn of Asiatic corn borer is meant the raising of the kernel weight that transgenic insect-resistant corn is shown under land for growing field crops artificial infection Asiatic corn borer condition.
13, according to each described method of claim 1 to 4, it is characterized in that, described the cause harm control of corn of Asiatic corn borer is meant the improvement of the cane resistance that transgenic insect-resistant corn is shown under land for growing field crops artificial infection Asiatic corn borer condition.
14, according to each described method of claim 1 to 4, it is characterized in that, described the cause harm control of corn of Asiatic corn borer is meant the production-increasing function that transgenic insect-resistant corn is shown under condition is caused harm in land for growing field crops Asiatic corn borer abiogenesis.
15, according to each described method of claim 1 to 4, it is characterized in that, described the cause harm control of corn of Asiatic corn borer is meant the raising of the kernel weight that transgenic insect-resistant corn is shown under condition is caused harm in land for growing field crops Asiatic corn borer abiogenesis.
16, according to each described method of claim 1 to 4, it is characterized in that, described the cause harm control of corn of Asiatic corn borer is meant the improvement of the cane resistance that the anti-corn of transgenosis is shown under condition is caused harm in land for growing field crops Asiatic corn borer abiogenesis.
17, each described transgenic insect-resistant corn of claim 1 to 4 is as the cause harm purposes of corn of control Asiatic corn borer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001006991A CN1158012C (en) | 2000-01-28 | 2000-01-28 | Method for control of insects |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001006991A CN1158012C (en) | 2000-01-28 | 2000-01-28 | Method for control of insects |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1266616A true CN1266616A (en) | 2000-09-20 |
| CN1158012C CN1158012C (en) | 2004-07-21 |
Family
ID=4575620
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB001006991A Expired - Lifetime CN1158012C (en) | 2000-01-28 | 2000-01-28 | Method for control of insects |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1158012C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101919410A (en) * | 2010-01-20 | 2010-12-22 | 西北农林科技大学 | Enhanced CpGV virus preparation and preparation method thereof |
| CN109874752A (en) * | 2019-04-03 | 2019-06-14 | 中国农业科学院植物保护研究所 | A kind of diapause regulation of the endoparasitoid Microcentrus cingulum and storage method |
-
2000
- 2000-01-28 CN CNB001006991A patent/CN1158012C/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101919410A (en) * | 2010-01-20 | 2010-12-22 | 西北农林科技大学 | Enhanced CpGV virus preparation and preparation method thereof |
| CN101919410B (en) * | 2010-01-20 | 2013-07-17 | 西北农林科技大学 | Enhanced CpGV virus preparation and preparation method thereof |
| CN109874752A (en) * | 2019-04-03 | 2019-06-14 | 中国农业科学院植物保护研究所 | A kind of diapause regulation of the endoparasitoid Microcentrus cingulum and storage method |
| CN109874752B (en) * | 2019-04-03 | 2021-04-09 | 中国农业科学院植物保护研究所 | Diapause regulation and storage method for waistband long body hornets |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1158012C (en) | 2004-07-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sétamou et al. | Evaluation of lectin-expressing transgenic sugarcane against stalkborers (Lepidoptera: Pyralidae): effects on life history parameters | |
| CN106916844B (en) | Insect-resistant and glyphosate-resistant expression vector, plasmid and application thereof | |
| AU2014350741B2 (en) | Method for controlling pest | |
| CN103421816B (en) | Killing gene and purposes thereof | |
| CN104946631A (en) | Corn transformation event 'double resistance 12-5' and specificity identification method thereof | |
| CN106591352B (en) | Insecticidal protein combinations and methods of managing insect resistance | |
| CN111606984B (en) | Plant insect-resistant protein and coding gene and application thereof | |
| CN1321067A (en) | Method of controlling cutworm pests | |
| CN103718895A (en) | Method for controlling injurious insects | |
| CN101044855A (en) | Agricultural chemical composition containing nimbin and abamectin and its application | |
| CN1375009A (en) | Insect-resistant rice plants | |
| CN103719137A (en) | Pest control method | |
| CN105219781A (en) | The method of cultivation of pest-resistant transgenic rice T1c-19 | |
| CN102584961B (en) | Anti-insect protein Cry1A.401 and expression vector and application thereof | |
| CN110195049B (en) | Brown planthopper eye color gene NlGCHI and encoding protein and application thereof | |
| CN109776659B (en) | Application of cry2Ah-vp gene in anti-myxobolus | |
| US10626411B2 (en) | Gene for encoding Bacillus thuringiensis crystal proteins, and use thereof | |
| CN108795940A (en) | A method of effectively preventing lepidoptera pest with RNAi | |
| CN102851297B (en) | Myzuspersicae hunchback gene cDNA and application thereof | |
| CN1158012C (en) | Method for control of insects | |
| CN1064684A (en) | The polypeptide that has desinsection to render a service | |
| CN106749566B (en) | Insecticidal protein combinations and methods of managing insect resistance | |
| CN106701790A (en) | Insecticidal protein-encoding nucleotide sequence and application thereof | |
| Homrich et al. | Agronomic performance, chromosomal stability and resistance to velvetbean caterpillar of transgenic soybean expressing cry1Ac gene | |
| CN1357553A (en) | Heliangine and its encoding gene and application in insect-resisting plant gene engineering |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term | ||
| CX01 | Expiry of patent term |
Granted publication date: 20040721 |