CN1357553A - Heliangine and its encoding gene and application in insect-resisting plant gene engineering - Google Patents

Abstract

The present invention relates to Jerusalen artichoke agglutinin protein, its encoding gene and plant expression carrier carrying out the said gene; and the present invention also relates to the method of utilizing jerusalen artichoke agglutinin gene to produce dinsert-resisting plant cell and plant.

Description

Helianthus tuberosus agglutinin, its encoding gene and the application in insect-resisting plant gene engineering thereof

Invention field

The present invention relates to helianthus tuberosus agglutinin albumen, its encoding gene carries the plant expression vector of said gene; Also relate to by said plant expression vector plant transformed cell with by the transgenic plant and the offspring thereof that homoptera pest is had resistance of these cell regenerationes, comprise plant seed and plant tissue; The invention particularly relates to the vegetables of paddy rice, cotton, wheat or the Cruciferae of homoptera pest-resisting.

Technical background

Phytohemagglutinin is found in 1888 the earliest, Stillmark has found a kind of cell agglutination factor in the castor seeds extract, has the erythrocytic effect of aggegation, multiple afterwards lectin constantly is found, all found phytohemagglutinin in unifacial leaf and the dicotyledons, be present in the storage organ and organ of multiplication of various plants.Phytohemagglutinin once was considered to a kind of typical seed albumen for a long time, this is owing to many lectins are to find in seed, in the vegetative organ of many plants, as blade, stem, the stem skin, bulb, stem tuber, bulb, root stock, root, fruit, the beggar room, all found phytohemagglutinin (Handbook of plant lectins:Van Damme et al. in phloem juice and the nectar, Published by John Willey and Sons Ltd, Chichester, England1997), the phytohemagglutinin of non-seed source is widely distributed equally.

When lectin was found in 1888, be named as hemagglutinin (haemagglutinin) according to its characteristic, subsequently very over a long time in continue to use this title always.Up to finding optionally certain type hemocyte in the aggegation ABO blood group system of some phytohemagglutinins, Lectin one speech just is suggested, be used to illustrate the phytohemagglutinin agglutination reaction selectional feature (Boyd andReguera, Jounal of Immunology 1949,62:333-339).Lectin one speech derives from Greek lgere, and the meaning is to select, but this title is very not strict, because it has comprised some protein that to have general agglutination activity list be not lectin simultaneously.Say the clear more precisely erythrocytic ability of lectin aggegation of hemagglutinin (haemagglutinin) speech from the strict sense, but because hemagglutinin possesses the ability of other cell of aggegation simultaneously, so lectin (agglutinin) speech is more suitable.Although the accurate meaning of haemagglutinin, lectin, agglutinin is also inequality, they are used to describe same class protein usually, and in three nouns, it is more relatively that lectin one speech uses.The initial definition of lectin is meant the carbohydrate-binding protein in non-immunity source, can the aggegation cell or the precipitation glycopolymers, Peumans and Van Damme (Peumans andVan Damme, Plant Physiol.1995,109:347-352) in nineteen ninety-five with phytohemagglutinin be defined as have at least one can with the vegetable-protein of special, the reversible bonded on-catalytic of monose or oligosaccharide structural domain, according to this definition, the vegetable-protein that a big class has different aggegation cells or precipitation glycoconjugate ability all is included in the category of lectin.

Homology and the relation on evolving thereof according to the lectin aminoacid sequence, phytohemagglutinin is divided into 7 families: the legume lectin element, the chitin binding lectin, the unifacial leaf mannose binding lectin, 2 type ribosome inactivating proteins, jackfruit element (jacalin) family, Curcurbitaceae phloem lectin and Amaranthaceae lectin (Peumans and Van Damme, Biotechnology and GeneticEngineering Reviews 1998,15:199-228).

Now the physiological action to phytohemagglutinin it be unclear that, and has determined their function in conjunction with activity and specificity thereof but relatively more consistent view is the sugar of lectin.Specificity studies show that the external source polysaccharide may be the most probable acceptors of many phytohemagglutinins, and the external source polysaccharide comprises chitin, the glycoconjugate on fungi and plant virus surface, insect and phytophagous animal intestinal cell surface.Based on the interaction of lectin with sugar, physiological action to lectin has many hypothesis, for example, intravital lectin of plant and sugared interaction are considered to relevant with the processes such as transportation, the accumulation of reserve substance, intercellular mutual work and fissional regulation and control of sugar.The mutual work of lectin and external source polysaccharide is considered to that plant/microorganism does mutually, the foundation of symbiotic relationship and determinative that external source biophylaxis mechanism is set up.People recognized that phytohemagglutinin not only worked in plant materials afterwards, as nitrogenous source, special recognition factor etc., and worked in the defensive raction of plant opposing adventive such as plant virus, insect, phytophagous animal.Many phytohemagglutinins are present in the storage organ, they both can be used as nitrogenous source, also can be subjected to harm as Buchner's bodies performance function plant, for example, accumulated a large amount of lectins in the bark of black locust tree and Williams Elder Twig, can cause the muroid poisoning, thereby be not subjected to the harm of animals such as rodents, and do not contain the kind of lectin in the bark, as the very easily harm of rodents (Peumans and Van Damme such as willow, willow, codlins, Biotechnologyand Genetic Engineering Reviews 1998,15:199-228)

The artificial breeding test shows, many phytohemagglutinins have toxicity to the chewing type insect, growth (the Murdock et al. that suppresses the bean weevil larva as wheat germ agglutinin (WGA), potato tuber lectin, peanut agglutinin, thorn apple phasin, Urtica (stinging nettle) lectin, Phytochemistry 1990,29:85-89; Huesing et al., Phytochemistry1991,30:3565-3568).Corn borer larvae is WGA, very low can the poisoning of purple Hupeh Bauhinia Root phasin dosage, the Zea mays root snout moth's larva can be killed by the Phytolacca lectin, suppressed to grow (Peumans and Van Damme by other several lectins, Biotechnology and GeneticEngineering Reviews 1998,15:199-228).Nearest work discovery piercing sucking insect such as coconut palm aleyrodid, leafhopper, aphid etc. are to some monocotyledons mannose binding lectin sensitivities, for example the insect of Snowdrop lectin of purifying (GNA) and commentaries on classics gna tobacco is fed and all shows, GNA has resistance to piercing sucking insect, but also can resist pathogenic nematodes (Hilder etal., Transgenic Research 1995,4:18-25).Phytohemagglutinin to the toxicity of Coleoptera, Diptera pest also have report (Gatehouse et al., Moluecular Breeding 1999,5:153-165).The above results explanation phytohemagglutinin has defense function in plant.

Phytohemagglutinin is still not fully aware of to the mechanism of insect and animal generation murder by poisoning, it is generally acknowledged phytohemagglutinin may by with glycoprotein, as the glycoconjugate of the chitin on insect peritrophic membrane surface, gastrointestinal epithelial cells or glycosylated digestive ferment etc. in conjunction with and influence the absorption of insect, animal to nutrition, the breeding of bacterium and bring out focus in the promoting digestion road, suppress growing, breeding of insect, finally reach desinsection or animal is produced the purpose of murder by poisoning.

In recent years, phytohemagglutinin is in the anti insect gene application in engineering, and particularly the application at Homoptera pest resistances such as aphids has been subjected to more and more many attention.That reports has anti-aphid active gene except that the phytohemagglutinin gene, also have ipt gene (isopentenyl transferase genes) and Mi gene (tomato nematicide gene), wherein using maximum is the phytohemagglutinin gene, comprises Snowdrop lectin (GNA) gene, companion's ConA (ConA) gene, three-coloured amaranth lectin (AHA) base.The plant of the commentaries on classics agglutinin gene of report has tobacco, paddy rice, potato, wheat, tomato etc., they show the vegetable garden noctuid, black peach aphid, the paddy rice brown paddy plant hopper, inhibition of insects such as grain aphid and resistance, for example, (Shi et al. such as Shi, Journal of Experimental Botany 1994,45:623-631) GNA is placed under the control of phloem specific promoter RSs1, transformation of tobacco, immunolocalization analysis revealed to GNA, GNA only expresses at phloem, GUS coloration result unanimity with RSs1-uidA, immunodetection to the black peach aphid honeydew proves that also the GNA that expresses in the transgene tobacco is transported in the phloem juice, think that phloem specific promoter RSs1 can be used to control insecticidal proteins,, and then prevent and treat the insect that sucks phloem juice as the specifically expressing of GNA at plant phloem.(Stoger et al. such as Stoger, Molucular Breeding 1999,5:65-73) GNA is placed under the control of constitutive promoter and phloem specific promoter, change wheat over to, the transgenic wheat that obtains has significantly reduced the fecundity of grain aphid, nymph quantity on average descends 50%, and the survival rate of aphid is 60% of contrast.(Gatehouse et al., Entomol.Exp.Appl., 1996 such as Gatehouse, 29:295-307) GNA is changed over to potato, the result shows that transgenic Rhizoma Solani tuber osi has significantly reduced the reproductivity of black peach aphid, and each female aphid lays eggs 4.1-4.2 every day, and contrast is 5.4.Rao etc. (Rao et al., Plant Journal 1998 15:469-477) changes GNA over to paddy rice, the insect test-results shows: the survival rate of paddy rice brown paddy plant hopper descends, and begins 25 of every strain inoculations, after 13 days, there are 7.6 on the transfer-gen plant, have 11.6 on the adjoining tree; Growth is suppressed, and after 13 days, the average every strain of transfer-gen plant has 0.8 hair to educate to the stage of maturity, and contrast is 6.4; Fecundity descends about 30%.Gatehouse etc. (Gatehouse et al., Moluecular Breeding1999,5:153-165) change ConA over to tomato after, the plant that ConA expresses has suppressed the growth of tomato exigua larvae, the degree of larva weight loss is greater than 45%; Make the fecundity of black peach aphid descend 45%.(Zhou Yonggang etc. such as Zhou Yonggang, the biotechnology journal, 2001,17:34-39) cDNA and the genomic fragment (AHAc and AHAg) of three-coloured amaranth agglutinin gene from Amaranthus prince's-feather (Amaranthus hypochondriacus), have been cloned, the difference transformation of tobacco, spot immune detects and shows, the proteic expression of AHA is arranged in the rotaring gene plant blade, worm test result to aphid shows: AHAc and AHAg are respectively 48.8% and 57.2% to the average inhibiting rate of aphid mouth density, the plant that has is up to more than 90%, they think that AHAg has better aphid resistance than AHAc, express the proteic transgene tobacco of AHA mainly by the breeding that suppresses aphid being influenced the formation of its colony, little to the influence of aphid mortality ratio.

Helianthus tuberosus agglutinin belongs to the plain family of jackfruit, constitutes a tetramer by 4 monomers, has the seminose binding specificity.To studies show that of its three-dimensional arrangement, three β lamellas of 12 chain formations are arranged in the β prismatic structure, the prismatical axle of the direction of β lamella and β parallel (Barre et al.Biochimie 2001 83:645-651).Helianthus tuberosus agglutinin forms eight aggressiveness when crystallization, eight aggressiveness are arranged in the form of a ring, and sugared binding site comprises 5 amino-acid residue (Gly ¹⁸, Gly ¹³⁵, Asp ¹³⁶, Val ¹³⁷, Asp ¹³⁹), they with seminose by 8 hydrogen bonds link to each other (Bourne et al., Structure1999,7:1473-1482).

In the disclosed content of the application, a lot of terms have been used.In application, " gene " is meant all or part of nucleic acid fragment of a kind of specific protein of coding, and comprises the regulating and controlling sequence of this front, coding region (5 ' non-coding region) and back (3 ' non-coding region)." foreign gene " is meant the gene that does not have in the host living beings under the normal circumstances, import by transgenosis; Also making a comment or criticism often is present in the host living beings, but is imported the gene of another locus outside its natural gene seat in its genome again, and this variation can cause a kind of appearance of one or several additional copy of encoding sequence of native gene.

" promotor " is meant that one section can combine and initial accurately and effectively dna sequence dna of transcribing with the trans factor that RNA polymerase and some other influences are transcribed." constitutive promoter " is to express in each etap of organism and different sites by the driving purposes gene, and expression level does not have a class promotor of notable difference." tissue-specific promoter " is meant the class promotor that the driving purposes gene is expressed in organism particular organization." organ specific promoters " is meant the class promotor that the driving purposes gene is expressed in the organism certain organs." inducible promoter " is meant that (as light, temperature, chemical substance etc.) induce a class promotor of driving purposes genetic expression down under certain ambient conditions.

" combined promoter " is between the different promoters or promotor and the common formation of regulating and controlling sequence.Controlling element comprises the dna sequence dna that can strengthen exogenous gene expression or regulatory gene various sources of expressive site in plant.The sequence that is intended to strengthen exogenous gene expression is paddy rice Actin1 introne 1, corn Ubiquitin introne 1, corn sucrose synthase gene Sh1 introne 1, maize alcohol dehydrogenase 1-S gene intron 1, corn sucrose synthase Sh1 exon and rice EPSP synthase gene first intron.

" enhanser " is to strengthen the active one section cis regulating and controlling sequence of promoter transcription." intron " is one section non transcribed dna sequence dna, and indivedual introns of some gene can strengthen the transcriptional activity of promotor.

" plant expression vector " is meant the carrier of energy driving purposes gene at plant interior expression." conversion of integral level " is meant with the organism to be the method for transformation of transformation receptor, and it comprises gene transformation, sexual cell infusion method, blastular or the ovary injection etc. of pollen tube passage method mediation.

" cDNA library " is to become the cDNA strand from the mRNA reverse transcription, becomes double-stranded cDNA molecule through the role transformation of archaeal dna polymerase, is inserted in the appropriate carriers then and cloning and the set that forms.

" degeneracy " is the phenomenon of the corresponding a kind of coded amino acid of a plurality of codons that causes of the slewability owing to the 3rd of genetic codon.

In this description, " plant " means any multi-cell organism that can carry out photosynthetic differentiation, and " vegetable cell " means and derive from plant and can form for example callus or differentiated tissues any cell of embryo or plant part or seed for example of indifferent tissue.

The present invention is active to tryptic inhibition by measuring jerusalem artichoke stem tuber cDNA library abduction delivering product, carried out library screening, 4 homologous cDNA sequences have been obtained, the aminoacid sequence of inferring and the mannose binding lectin of Jacalin family have homology, wherein with people (Van Damme et al. such as Van Damme, Eur.J.Biochem.1999 259:135-142) Bao Dao Heltuba has high homology, homology of nucleotide sequence is between 85.2%～97.8%, and the homology of amino acid levels is between 91.3%～97.9%.The cDNA sequence that obtains has been carried out abduction delivering in intestinal bacteria, the inhibition determination of activity result of bacterial expression product shows, this gene expression product has trypsin inhibition activity, thrombotest shows that also its sugar with lectin is in conjunction with activity, therefore it is considered herein that a kind of bifunctional protein of this genes encoding, sugar with lectin is in conjunction with activity and protease inhibiting activity, with its called after hta.

The objective of the invention is at following two facts: homoptera pests such as (1) aphid, planthopper, leafhopper are worldwide serious to plant hazard.The mouthpart of Homoptera insect is a pierce-suck type, digestive ferment level in its enteron aisle is very low, they obtain nutrition by sucking plant phloem juice, directly utilize free amino acid in the phloem as nitrogenous source, can't utilize the anti-system of strategy sucking pest (the Stoger et al. of Bt toxalbumin or proteinase inhibitor, Molecular Breeding 1999,5:65-73), thereby the plant that changes Bt, SCK gene does not have effect to sucking pest such as aphid.Homoptera pests such as aphid also cause indirect hazard by carrying various plants virus to plant except that directly endangering the plant by drawing phloem juice.Phytohemagglutinin has resistance to homoptera pest, on pest-resistant, has complementarity with Bt, SCK, complementarity comprises two aspects, the one, same Bt, the SCK complementation on pest-resistant spectrum, the 2nd, by lectin is absorbed, can strengthen Bt, SCK toxic action, reduce the probability that insect produces resistance lepidopterous insects.(2) being extensive use of of chemical insecticide except that causing environmental pollution, also can be killed beneficial insect when killing off the insect pests, and destroys the eubiosis.Utilize transgenic technology that killing gene is imported plant, make plant obtain insect resistance capacity, not only can reduce the usage quantity of agricultural chemicals, reduce agricultural chemicals and use the environmental pollution that causes, and can alleviate the artificial destruction of farmland ecosystem equilibrated, help the reconstruction and the recovery of the eubiosis, be beneficial to the Sustainable development of agriculture production.

Summary of the invention

An object of the present invention is to provide a kind of helianthus tuberosus agglutinin albumen.

Helianthus tuberosus agglutinin albumen of the present invention has SEQ ID NO:2, SEQ ID NO:4, aminoacid sequence shown in SEQ IDNO:6 or the SEQ ID NO:8.

Another object of the present invention provides the proteic gene of a kind of coding helianthus tuberosus agglutinin of the present invention.

The proteic gene of helianthus tuberosus agglutinin of the present invention has SEQ ID NO:1, SEQ ID NO:3, the nucleotide sequence shown in SEQ ID NO:5 or the SEQ ID NO:7.

The present invention also provides a kind of and has utilized the generation of helianthus tuberosus agglutinin gene to have the vegetable cell of insect-resistance and the method for plant, this method may further comprise the steps: (a) upstream extremity of helianthus tuberosus agglutinin gene is connected in after the nucleic acid fragment of promotor on sense orientation, its downstream end is connected in before the nucleic acid fragment of a kind of suitable regulating and controlling sequence that is used to control Transcription Termination of coding, and then makes up plant expression vector; (b) with the plant expression vector transformed plant cells that builds; (c) vegetable cell after will transforming under the suitable culture condition is regenerated, and obtains expressing the plant of goal gene.

In aforesaid method of the present invention, described promotor can comprise constitutive promoter, organ specific promoters, tissue-specific promoter, inducible promoter or the combined promoter of being made up of promotor and controlling element.Used method for transformation can comprise the method for transformation of method for transformation, electric shocking method or the integral level of agriculture bacillus mediated method for transformation, particle bombardment, protoplastis mediation.Described plant can be monocotyledons or dicotyledons, for example paddy rice, wheat, barley or tobacco, cotton, soybean, sweet potato, potato, Chinese cabbage, green vegetables, celery, green pepper.

The present invention also provides the plant expression vector that contains gene of the present invention.

Brief Description Of Drawings Fig. 1: the restriction enzyme mapping of plasmid pET16.Contain the T7 promotor, Amp resistance part and lacI portion

Divide.Fig. 2: the restriction enzyme mapping of plasmid pETHTA.Contain HTA gene cDNA complete sequence, the plasmid bone

Frame is from pET16.Fig. 3: the restriction enzyme mapping of plasmid pSPROK.P-35S is the CaMV 35S promoter, belongs to composing type

Promotor.Fig. 4: the restriction enzyme mapping of plasmid pCNPT-II.This plasmid can be used to transform dicotyledons, contains

Left and right sides border sequence, the plant selectable marker gene is a neomycin phosphotransferase gene.Bacterium

Selective marker is a kalamycin resistance.The plasmid skeleton is from pCAMBIA2300 Fig. 5: the restriction enzyme mapping of plasmid pCNHTA.This plasmid can be used to transform dicotyledons.P-35S

Be the CaMV35S promotor, be used to regulate and control hta and transcribe that the control of Tnos terminator is transcribed end

End.NptII is that neomycin phosphotransferase gene is as the selection markers gene.The plasmid skeleton comes

From pCNPT-II.Fig. 6: the PAGE electrophoretogram of pETHTA intestinal bacteria abduction delivering product.1: inductive not

The intestinal bacteria product that contains the pET16 empty carrier.2: inductive contains the big of pET16 empty carrier

The enterobacteria expression product.3,5,7,8: inductive contains the intestinal bacteria of pETHTA carrier

Reach product.4,6: the inductive intestinal bacteria that contain the pETHTA carrier do not reach product.Fig. 7: pETHTA intestinal bacteria abduction delivering product is to the column diagram of trypsin inhibition activity.Right

According to: the Δ that contains the e. coli expression product of pET16 empty carrier ₄₀₅Value.hta-a、hta-b、

Hta-c, hta-d: the Δ that contains the e. coli expression product of pETHTA expression vector respectively ₄₀₅

Value.Fig. 8: T ₀The PCR that generation is changeed the hta tobacco detects.1. positive plasmid pCNHTA, 2. non-transformed plant,

3～11. transfer-gen plant Fig. 9: T ₀In generation, changeed the Southern hybridization of hta genetic tobacco.1: adjoining tree, 2～12: change base

Because of plant.Figure 10: T ₀The Northern hybridization that generation is changeed the hta tobacco detects.1～4: the negative control plant, its

Surplus is transfer-gen plant.Figure 11: T ₀In generation, changeed the column diagram of a black peach aphid worm number on the hta tobacco.hta-a、hta-b、hta-c、hta-d

Be respectively the plant that changes the hta gene, CK is for changeing the adjoining tree of pCNPT-II.Figure 12: T ₀In generation, changeed the hta tobacco breeds average inhibiting rate to black peach aphid column diagram.hta-a、hta-b、

Hta-c, hta-d are respectively the plant that changes the hta gene, and CK plants for the contrast of changeing pCNPT-II

Strain.Figure 13: T ₀In generation, changeed the inhibition graphic representation of hta tobacco to the black peach aphid breeding.hta-a、hta-b、hta-c、

Hta-d is respectively the plant that changes the hta gene, and CK is for changeing the adjoining tree of pCNPT-II.Figure 14: T ₁In generation, changeed the column diagram of a black peach aphid worm number on the hta tobacco.Hta-b, hta-c are for changeing the hta base

The plant of cause, CK is for changeing the adjoining tree of pCNPT-II.Figure 15: T ₁In generation, changeed the hta tobacco breeds average inhibiting rate to black peach aphid column diagram.Hta-b, hta-c are

Change the plant of hta gene, CK-1, CK-2, CK-3 are three groups changes pCNPT-II's

Adjoining tree.Figure 16: T ₁In generation, changeed the influence of hta tobacco to the black peach aphid nymphal development.Hta-c, hta-b are the transgenosis cigarette

Grass, CK-1, CK-2, CK-3 are three groups of adjoining trees that change pCNPT-II.

Further illustrate the present invention below in conjunction with embodiment, and do not constitute restriction claim scope of the present invention.In an embodiment, used term and abbreviation are general term of those skilled in the art and abbreviation.Embodiment 1: the structure in jerusalem artichoke stem tuber cDNA library

Being implemented among the expression type phage vector λ TripIEx2 of jerusalem artichoke stem tuber cDNA library adopted the Smart of Clontech company ^TMCDNA library construction test kit carries out according to the method on the providing handbook.1.1RNA extraction

The method (Chomezynski and Sacchi, AnalyticalBoichemistry.1987,162:156～159) that the extraction of total RNA is pressed in the document is carried out, and improves a little.2.0g stem tuber (growing for 4 weeks) is pulverized in liquid nitrogen, transfer in the centrifuge tube of 40ml, add 5ml sex change liquid, mixing, add 0.5ml 2M NaAc (pH4.5), the 5ml water-saturated phenol, the 1ml chloroform, ice bath is 15 minutes behind the mixing, 10, centrifugal 10 minutes of 000g, supernatant adds the equal-volume Virahol, and-20 ℃ precipitate 1 hour, 10, behind centrifugal 10 minutes of the 000g, supernatant discarded, the LiCl washing precipitation of adding 1ml 4M, 10, abandon supernatant after 000g is centrifugal, precipitation is dissolved in the water of 1ml DEPC processing, adds the water-saturated phenol of 1 times of volume: chloroform: twice of primary isoamyl alcohol (25: 24: 1) extracting, supernatant adds 1/10 volume 3M NaAc (pH4.5), the dehydrated alcohol of 2 times of volumes ,-20 ℃ precipitate 1 hour, 10, centrifugal 10 minutes of 000g, precipitation with 70% washing with alcohol once is dissolved in after the drying in the water of DEPC processing of proper volume, and-70 ℃ of preservations are standby.1.2 cDNA first chain is synthetic

1 μ lRNA (the total RNA of about 1 μ g)

1 μ lSMART III primer

1 μ l CDS III/3 ' PCR primer

Sterilized water to the 5 μ l that adds RNase-free, behind the mixing, 72 ℃ of sex change 2 minutes, cooled on ice added after 2 minutes:

2μl?5×First?strand?buffer

1μl?DTT(20mM)

1μl?dNTP(10mM)

1 μ l MMLV reversed transcriptive enzyme (200U)

Mixing carried out reverse transcription reaction 1 hour under 42 ℃.1.3 cDNA second chain is synthetic

The product of getting among the 2 μ l 1.2 carries out the synthetic of cDNA second chain:

2μl?First-strand?cDNA

80μl?H2O

10μl?10×cDNA?PCR?buffer

2μl?dNTP(50mM)

2μl?5′PCR?primer

2μl3′PCR?primer

2μl?50×Advantage?cDNA?Polymerose?mix

In GeneAmp9600 (PE company), carry out PCR reaction with following condition: 95 ℃ of pre-sex change 60 seconds, 95 ℃ of sex change 30 seconds, 61 ℃ of renaturation 30 seconds, 72 ℃ were extended 6 minutes, and carried out 19 circulations.1.4 purifying, the enzyme of cDNA are cut, fractional separation and with being connected of carrier

The purifying of cDNA: get the product among the 50 μ l 1.3, add 2 μ l Proteinase Ks (20 μ g/ μ l), mixing, 45 ℃ of insulations are after 20 minutes, add 50 μ l water extended volume after, through isopyknic phenol: chloroform (50: 49: 1) extracting 2 times, add 10 μ l 3M NaAC (pH4.5), 1.3 μ l glycogen (20 μ g/ μ l), the dehydrated alcohol of 2 times of volumes ,-20 ℃ of precipitations are spent the night, 10, centrifugal 10 minutes of 000g, precipitation with 70% washing with alcohol once is dissolved in after the drying in the 79 μ l water.

Sfi I enzyme is cut:

79 μ l cDNA (product of previous step)

10μl?10×Sfi?I?buffer

10 μ l Sfi I enzymes

1μl?100×BSA

Mixing, 50 ℃ are incubated 2 hours.Add 2 μ l, 1% dimethylbenzene green grass or young crops.

The fractional separation of cDNA: the method for recommending in the by specification, separate through CHROMA SPIN-400 post, dropwise collect elutriant, get the distribution of 5 μ l electrophoresis detection cDNA, add 1/10 volume 3M NaAC (pH4.5), the dehydrated alcohol of 2 times of volumes after will comprising the the 1st, the 2nd, the 3rd merging of cDNA, 1.3-20 ℃ of precipitations of μ l glycogen (20 μ g/ μ l) are spent the night, 10, centrifugal 10 minutes of 000g is dissolved in the 7 μ l ultrapure waters after the precipitation drying.

CDNA is connected with carrier:

cDNA 0.5μl

Vector 1.0μl

ATP(10mM) 0.5μl

T ₄Dna ligase 0.5 μ l

Ultrapure water 2.0 μ l

Behind the mixing, spend the night in 16 ℃ of connections.1.5 connect product packing, library titre mensuration and expand numerous

Add 5 μ l in the 50 μ l packaging proteins (the λ DNA packaging system of Promega company) and connect product, mixing gently, 22 ℃ of insulations added 445 μ l phage buffer, 25 μ l chloroforms after 3 hours.Carry out the mensuration of library titre and expand numerous (molecular cloning: laboratory manual, Sambrook et al, New York:Cold Spring Harbor Laboratory Press, 1989) by the method in the molecular cloning.The one-level library that obtains comprises 1.5～2 * 10 ⁶Individual phage, recombination fraction is greater than 95%.Behind the amplified library packing be stored in-70 ℃ standby.Embodiment 2: jerusalem artichoke stem tuber cDNA library screening and the segmental acquisition of hta cDNA

In order to obtain to have the gene of trypsin inhibition activity, the present invention is active to tryptic inhibition by measuring library abduction delivering product, the step-sizing in the enterprising style of writing of 96 orifice plates storehouse.Get 10 ⁶Individually (contain 10mM MgSO from the phage in secondary library and an amount of host bacterium (XL1-Blue), LB substratum ₄0.2% maltose, 0.1mM IPTG) mix the back evenly distribute in each hole of 96 orifice plates, 37 ℃ in shaking table incubated overnight expand numerous after, the measuring method (vitality test of toolenzyme: Jiang Chuankui according to trypsin inhibitor activity, Shanghai: Science and Technology of Shanghai press, 1980, p105-107), measure the trypsin inhibition activity in each hole, concrete grammar is as follows: get 50 μ l split products from the every hole of 96 orifice plates, the corresponding adding in another 96 new orifice plates, add 50 μ lTrypsin solution (0.05mg/ml) simultaneously, (Tris-HCl pH8.0 contains 10mMCa to 100 μ l reaction buffers ²⁺), mixing, 30 ℃ of reactions were got 50 μ l reaction mixtures respectively and are transferred in another 96 orifice plate after 2 hours, added 50 μ l reaction buffers and 100 μ 10mM BApNA (contain 10mM Ca ²⁺, Sigma company), on microplate reader, measure under the 405nm changing value of photoabsorption in 5 minutes immediately, and compare, determine positive hole.Measure the titre in positive hole, get 10 ⁵Individual phage is used for the next round screening, each takes turns 10 times of all more last round of dilutions of used phage, up to the dilution of every hole when a phage is only arranged, get positive hole twice flat board in shop continuously, the one plaque of picking, infect intestinal bacteria BM25.8,, have the segmental phage of external source and partly be cyclized into and have ammonia benzyl resistance (Amp by the reorganization in LoxP site ^r) plasmid optionally.10 ⁶After individual phage is taken turns screening through 5, obtain 5 positive holes, therefrom chosen 3 difference, twice single plaque of dull and stereotyped picking in shop continuously, infect intestinal bacteria BM25.8, picking 24 resistance bacterium colonies extract plasmids, Sfi I restriction analysis inserts fragment and shows, wherein 23 clones' insertion clip size basically identical.After the order-checking cDNA sequence is shown that in NCBI (National Center for Biotechnology Information) result relatively the coded product and the phytohemagglutinin of this gene have homology, with its called after hta.The expression of embodiment 3:hta gene in intestinal bacteria and the structure of mensuration 3.1 coli expression carriers of trypsin inhibition activity

Coli expression carrier pET16 is provided by the heredity Huang Hualiang researcher of institute of the Chinese Academy of Sciences.Plasmid pET16 after adding the big fragment of Klenow and mending flat end, cuts with Xho I enzyme after Nco I enzyme is cut again, reclaims carrier part.Plasmid pTripHTA reclaims hta fragment part through Sma I/Xho I double digestion, and hta fragment and pET16 carrier are through T ₄16 ℃ of connections of dna ligase are spent the night, and connect product electric shocking method transformed into escherichia coli competent cell, and screening obtains recon pETHTA.Plasmid extracts, enzyme is cut, the recovery of DNA, purifying are all carried out (molecular cloning: laboratory manual, Sambrook et al, New York:Cold Spring Harbor Laboratory Press, 1989) by the method in the molecular cloning.3.2 colibacillary abduction delivering and SDS-PAGE electrophoresis

The pETHTA electric shock is changed in E.coli BL21 (DE3) bacterial strain, 3 single bacterium colonies of picking, incubated overnight in the LB liquid nutrient medium is transferred in the fresh LB liquid nutrient medium in 1: 50 ratio, is cultured to OD ₆₀₀=0.6～0.8, add IPTG to final concentration 0.1mM, abduction delivering is after 3 hours, and the thalline behind the centrifugal collection 3ml abduction delivering is resuspended in the 400 μ l deionized waters, and ultrasonic wave is broken bacterium, and 4 ℃, 10, the centrifugal 10min of 000g, supernatant is the electrophoresis sample.The SDS-PAGE protein electrophoresis carries out (molecular cloning: laboratory manual, Sambrook et al, New York:Cold Spring Harbor Laboratory Press, 1989) by the method in the molecular cloning.The result of protein electrophoresis shows to have specifically expressing HTA foreign protein in the intestinal bacteria product after inducing.3.3 the mensuration of trypsin inhibition activity

Get the supernatant 50 μ l in 3.2, press following mixed:

Standard pipe sample hose control tube damping fluid 100 100 100 water 350 300 300 bovine trypsins 50 50 50 samples 0 50 50

Annotate: unit: μ l

Damping fluid: 50mM Tris-HCl (pH8.0) contains 40mM Ca ²⁺

Bovine trypsin: 1mg/ml

Mixing, 25 ℃ are incubated 60 minutes, and (1mM contains 20mM Ca to get 100 μ l reaction mixtures adding 1ml substrate B ApNA ²⁺, Sigma company), read the photoabsorption at 405nm place immediately, read once every 30 seconds, read altogether 3 minutes, calculate Δ ₄₀₅° result shows: pETHTA intestinal bacteria product has tangible trypsin inhibition activity.Embodiment 4: the structure of dicotyledons expression vector pCNHTA

Select the expression of CaMV35S constitutive promoter and no terminator control hta in plant for use, make up plant expression vector.Process is as follows: at first Sma I/Sac I double digestion reclaims hta from carrier pTripHAT, inserts the Sma I/Sac I site of plasmid pSPROK, obtains recon pSRHTA.PSRHTA reclaims the 35Spromoter-hta-Tnos part through Pvu II/Bgl II double digestion, inserts the Sma I/BamH I site of plant expression vector pCNPT-II, obtains plant expression vector pCNHTA.Plasmid extracts, enzyme is cut, the recovery of DNA, purifying are all carried out (molecular cloning: laboratory manual, Sambrook et al, New York:Cold Spring Harbor LaboratoryPress, 1989) by the method in the molecular cloning.

Change plasmid pCNHTA electric shock over to root Agrobacterium LBA4404, transform the electric exciter that uses BIO-RAD company, competent preparation and the electricity method that swashs is carried out with reference to product description, at YEB (Rif25, Km50) dull and stereotyped last 28 ℃ of dark cultivations promptly have the bacterium colony of conversion to grow after 2 days, further extract plasmid and identified from transformed bacteria.Embodiment 5: tobacco transforms the acquisition of seedling

Utilize improvement Ye Panfa, by the agrobacterium-mediated transformation transformation of tobacco.Specific operation process is as follows, and the Agrobacterium LBA4404 that contains plant expression vector among the embodiment 4 is rule in containing on the corresponding antibiotic YEB solid medium, and 28 ℃ of lucifuges are cultured to the single bacterium colony (about 36h) that grows the about 1mm size of diameter.Get single bacterium colony and transfer once again on same solid medium, be cultured to growth animated period (about 36h).Get a little root Agrobacterium and transfer in the 20mlYEB liquid nutrient medium (contain corresponding microbiotic, use the 100ml triangular flask), in 28 ℃, 220rpm shaking culture spend the night (about 18h).Transferring with 2%～4% inoculum size next day does not contain in the antibiotic YEB liquid nutrient medium in 20ml, and to add Syringylethanone to final concentration be 100 μ M.Continue shaking culture 3～4h, arrive logarithmic growth during mid-term, be diluted to naked eyes with three times of liquid-based basal culture mediums and see the muddy (OD that gets final product slightly with upper volume ₆₀₀=0.1), prepares against the usefulness of conversion.Get complete unfolded tobacco aseptic seedling blade, (diameter 0.9cm) makes the leaf dish with punch tool, soaks 10min in bacterium liquid.Take out the leaf dish, after blotting with aseptic filter paper, change in the common culture medium that is coated with one deck aseptic filter paper in 25 ℃ dark cultivate 2～3 days after, the leaf dish is changed in the subculture medium.Illumination cultivation in subculture medium (light/dark cycle is 16/8h) changed the leaf dish over to screening culture medium after 3 days.Continue to cultivate after 7～10 days, change the leaf dish over to regeneration culture medium.Promptly can be observed that blade edge grows callus or the regeneration bud of growing thickly in 15～20 days, can grow to 1～2cm about one month, change in the new regeneration culture medium, treat that it is long during to 3～4cm, with scalper regeneration bud is downcut, and change in the root media.Regrowth is taken root on the root media that contains kantlex (50mg/L), treat after one month that root system development fully after, seedling is transferred to the greenhouse growth.Embodiment 6: tobacco transforms the extraction of the Molecular Identification 6.1 tobacco DNA of seedling

Get T ₀Fresh blade 0.3mg places mortar for tobacco, adds the liquid nitrogen grinding powder, adds 0.6ml again and is preheating to 60 ℃ CTAB damping fluid (30g/L CTAB, 1.4mol/L NaCl, 0.2% mercaptoethanol, 20mmol/L EDTA, 100mmol/L Tris-HCl, pH8.0).60 ℃ are incubated 30 minutes, and jog for several times therebetween.Add isopyknic chloroform then: primary isoamyl alcohol (24: 1) extracting once, supernatant liquor is transferred in the new centrifuge tube and to be added 2/3 times of volume Virahol, the precipitation of formation is DNA, (volume fraction is 76% ethanol, 10mM NH to add a little washing lotion ₄AC) washing precipitation once, dry back is with 500 μ l TE damping fluid (10M Tris-HCl (pH8.0), 1mM EDTA) dissolving DNAs.Add RNase A (final concentration 10mg/L) subsequently, 37 ℃ are incubated 30 minutes, use isopyknic phenol, phenol successively: chloroform: primary isoamyl alcohol (25: 24: 1), chloroform: each extracting of primary isoamyl alcohol (24: 1) once, water adds 2.5 times of volume dehydrated alcohol deposit D NA.Be dissolved in after the DNA drying in the 100 μ l sterilized waters.6.2T ₀PCR for transfer-gen plant detects

The DNA that gets among the 1 μ l6.1 is that template is carried out the PCR reaction.Comprise in the reaction system of 50 μ l: 5 μ l, 10 * PCR reaction buffer, each 1 μ l (10 μ M) of primer P1 (atggctgccagtgacattg), P2 (aggaacaactacgccacc), 1 μ l dna profiling, 4 μ l dNTP (2.5mM), 1U Taq enzyme is added sterilized water to cumulative volume 50 μ l.The PCR reaction conditions is as follows: 94 ℃ of pre-sex change 5 minutes, and 1 minute, 52 ℃ renaturation of 94 ℃ of sex change were extended 1.5 minutes for 1 minute, 72 ℃, carried out 30 circulations, and last 72 ℃ were extended 10 minutes.Get 10 μ l PCR products and carry out the agarose electrophoresis detected result.The result that PCR detects shows that the transgene tobacco genomic dna can amplify the single band onesize with target gene fragment.6.3T ₀Southern for transfer-gen plant detects

After an amount of EcoR I (TaKaRa) enzyme of DNA adding among about 20 μ g 6.1 was cut, the agarose gel electrophoresis through 1% separated, and 0.25N HCl depurination 10 minutes is transferred to Hybond-N with 0.4N NaOH ⁺On (Amersham pharmacia) film, the film after the transfer is washed in 2 * SSC, and 80 ℃ of vacuum are fixed 2 hours.65 ℃ of prehybridizations are 2 hours in the 0.5M sodium phosphate buffer that contains 7%SDS (W/V), add [α- ³²P] the hta cDNA probe of dCTP random priming mark hybridizes (Promega random primer labelling test kit).65 ℃ of hybridization are spent the night, and wash film for 65 ℃ among 0.1 * SSC (containing 0.1%SDS), press the X-ray sheet, radioactive automatic developing.Results of hybridization shows that hta has been incorporated in the tobacco gene group.6.4T ₀Northern for transfer-gen plant detects

Method in the extraction of the total RNA of transgene tobacco blade same 1.1.Total RNA of about 30 μ g transgene tobaccos is after containing 0.1% denaturing formaldehyde glue (1.2%) electrophoresis, transfer to (Amersham pharmacia) on the Hybond-XL film with 20 * SSC, film after the transfer was washed in water 10 minutes earlier, in 10 * SSC, washed again 10 minutes, after 80 ℃ of vacuum are fixed 2 hours, promptly can be used for hybridization, the method in the process same 6.3.The result of Northern hybridization shows that hta expresses in transgene tobacco.Embodiment 7: the aphid resistance of transgene tobacco is identified

Transgene tobacco is identified the resistance of black peach aphid and is entrusted agricultural insect system of Plant Protection institute, Chinese Academy of Agricultral Sciences to carry out.7.1 anti-aphid authentication method:

Every strain T ₀Insert 1-2 age with writing brush if 10 of aphids are connected on the tender leaf at plant top for transgene tobacco, keep certain distance in different cigarette strains, to shift between basin and the basin to avoid aphid.The cigarette strain places illumination cultivation indoor, photoperiod 16/8h, and 25 ℃ of temperature, the aphid quantity in investigation cigarette on the 3rd strain is added up 4 times altogether.

T ₀After the sterilization of 30% clorox, place MS substratum (plate) to send out seedling for the seed of transgene tobacco, wait to grow 2-3 sheet true leaf after, transfer in the MS substratum that contains kantlex (50mg/L) and screen, the seedling that normally takes root is regarded as T ₁For positive transfer-gen plant.After treating that the cigarette seedling grows 4～5 true leaves, connect one 2～3 black peach aphid about age on every tobacco seedling, added up the nymph number of once breeding and the development condition of nymph, after the statistics nymph is all taken out, add up 3 cycles altogether every 3 days.7.2 the statistical study of anti-aphid data

The statistical study of anti-aphid data is carried out (biostatistics: Li Chunxi etc., Science Press, second edition, Beijing, 2000) with reference to the statistical method of routine.The result of statistics shows, T ₀Commentaries on classics hta tobacco has obvious suppression effect (table 1,2,3) to the breeding of black peach aphid, and average inhibiting rate is between 46.9%～78%.。The significant difference of aphid offspring number (table 4,5,6) on statistical results show transfer-gen plant and the adjoining tree, T ₁For transfer-gen plant the average inhibiting rate that black peach aphid breeds is respectively 50.1% (hta-b) and 64.6% (hta-c), transfer-gen plant removes has the obvious suppression effect to the black peach aphid breeding, also inhibited to the growth as if aphid.Table 1 T ₀For black peach aphid progeny population number analysis of variance table source of variation df SS s on the transgene tobacco ²F F _0.01Between processing 4 16287.27 4071.82 14.07 ^*4.77 16 4630.54 289.41 total variation 20 20917.81 table 2 T in handling ₀For black peach aphid progeny population number difference opposite sex significance comparison sheet (duncan's new multiple range method) on the transgene tobacco

The gene mean number that the otherness significance transforms

á=0.05 á=0.01CK 93.75 a Ahta-c 49.75 b Bhta-b 22.67 c BChta-d 21.63 c Chta-a 20.5 c C table 3 T ₀LSR value (duncan's new multiple range method) M 234 5SSR for black peach aphid progeny population number on the transgene tobacco _0.053.00 3.14 3.24 3.30SSR _0.014.13 4.31 4.42 4.51LSR _0.0525.53 26.72 27.57 28.08LSR _0.0135.15 36.68 37.61 38.38 table 4 T ₁Between handling for black peach aphid progeny population number analysis of variance table source of variation df SS s2 F F0.01 on the transgene tobacco 4 6253.14 1563.29 26.87 ^*3.72 50 2908.61 58.17 total variation 54 9161.75 table 5 T in handling ₁For black peach aphid progeny population number difference opposite sex significance comparison sheet (duncan's new multiple range method) on the transgene tobacco

The gene mean number that the otherness significance transforms

á=0.05 á=0.01CK 34.73 a Ahta-b 16.59 b Bhta-c 12.47 b B table 6 T ₁LSR value (duncan's new multiple range method) M 234 5SSR0.05 2.84 2.99 3.08 3.15SSR0.01 3.79 3.96 4.06 4.20LSR0.05 6.53 6.88 7.08 7.25LSR0.01 8.72 9.11 9.34 9.66 for black peach aphid progeny population number

Sequence table＜110〉Institute of Genetics, Academia Sinica＜120〉helianthus tuberosus agglutinin gene and the application in insect-resisting plant gene engineering＜130 thereof〉I2001278＜160〉8＜170〉PatentIn version 3.1＜210〉1＜211〉775＜212〉DNA＜213〉Helianthus tuberosus＜220〉＜221〉CDS＜222, (48) .., (491)＜223〉＜400〉1cacaacataa gttacttaag aagctttgaa tttgcataga ccagaaa atg gct gcc 56

Met?Ala?Ala

1act?gac?att?gcg?gta?cag?gcc?gga?cca?tgg?gga?ggt?aat?ggt?gga?aaa 104Thr?Asp?Ile?Ala?Val?Gln?Ala?Gly?Pro?Trp?Gly?Gly?Asn?Gly?Gly?Lys

5 10 15cgc?tgg?ttg?caa?aca?gct?cgt?ggt?ggt?aag?att?act?tcg?atc?att?atc 152Arg?Trp?Leu?Gln?Thr?Ala?Arg?Gly?Gly?Lys?Ile?Thr?Ser?Ile?Ile?Ile20 25 30 35aaa?ggc?gga?act?tgt?att?ttc?tcc?atc?cag?ttc?gta?tat?aag?gac?aaa 200Lys?Gly?Gly?Thr?Cys?Ile?Phe?Ser?Ile?Gln?Phe?Val?Tyr?Lys?Asp?Lys

40 45 50gat?aat?att?gaa?tac?cat?tct?gga?caa?ttt?ggt?gtt?cag?ggt?gac?aaa 248Asp?Asn?Ile?Glu?Tyr?His?Ser?Gly?Gln?Phe?Gly?Val?Gln?Gly?Asp?Lys

55 60 65gct?gaa?aca?att?acc?ttt?gcg?gac?gat?gag?gac?atc?act?gcg?atc?agc 296Ala?Glu?Thr?Ile?Thr?Phe?Ala?Asp?Asp?Glu?Asp?Ile?Thr?Ala?Ile?Ser

70 75 80gga?act?ttc?gga?gca?tat?tat?cac?ttg?aca?gtt?gtt?aca?tcg?ctc?act 344Gly?Thr?Phe?Gly?Ala?Tyr?Tyr?His?Leu?Thr?Val?Val?Thr?Ser?Leu?Thr

85 90 95ttt?cag?acc?aac?aaa?aag?gtt?tat?ggg?cca?ttc?ggc?acg?gtg?gct?agt 392Phe?Gln?Thr?Asn?Lys?Lys?Val?Tyr?Gly?Pro?Phe?Gly?Thr?Val?Ala?Ser100 105 110 115tcg?agt?ttc?tca?ctg?cct?cta?act?aag?ggt?aag?ttt?gcc?gga?ttc?ttt 440Ser?Ser?Phe?Ser?Leu?Pro?Leu?Thr?Lys?Gly?Lys?Phe?Ala?Gly?Phe?Phe

120 125 130ggg?aac?agt?gga?gac?gtc?ctt?gac?tcg?att?ggt?ggc?gta?gtt?gtt?cct 488Gly?Asn?Ser?Gly?Asp?Val?Leu?Asp?Ser?Ile?Gly?Gly?Val?Val?Val?Pro

135 140 145taa?tcacaacgcc?tgatatgatg?aaaataagaa?gttaaggaaa?gacttcacgt 541ttgtaaaaga?gttgtgacat?tcagctcgta?tttgtgtgta?tgcatctttc?aatttcgtct 601ctcgtatttg?tgtgtatgca?tctttcaatt?tcgtctctcg?tttttctcgg?gaaataagct 661tttatgatat?attccaataa?gcccgagctt?gtcgtgcact?agacttctgt?tctccgcaaa 721aggttttaaa?tgatcatttt?ttcccaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaa 775<210>2<211>147<212>PRT<213>Helianthus?tuberosus<400>2Met?Ala?Ala?Thr?Asp?Ile?Ala?Val?Gln?Ala?Gly?Pro?Trp?Gly?Gly?Asn1 5 10 15Gly?Gly?Lys?Arg?Trp?Leu?Gln?Thr?Ala?Arg?Gly?Gly?Lys?Ile?Thr?Ser

20 25 30Ile?Ile?Ile?Lys?Gly?Gly?Thr?Cys?Ile?Phe?Ser?Ile?Gln?Phe?Val?Tyr

35 40 45Lys?Asp?Lys?Asp?Asn?Ile?Glu?Tyr?His?Ser?Gly?Gln?Phe?Gly?Val?Gln

50 55 60Gly?Asp?Lys?Ala?Glu?Thr?Ile?Thr?Phe?Ala?Asp?Asp?Glu?Asp?Ile?Thr65 70 75 80Ala?Ile?Ser?Gly?Thr?Phe?Gly?Ala?Tyr?Tyr?His?Leu?Thr?Val?Val?Thr

85 90 95Ser?Leu?Thr?Phe?Gln?Thr?Asn?Lys?Lys?Val?Tyr?Gly?Pro?Phe?Gly?Thr

100 105 110Val?Ala?Ser?Ser?Ser?Phe?Ser?Leu?Pro?Leu?Thr?Lys?Gly?Lys?Phe?Ala

115 120 125Gly?Phe?Phe?Gly?Asn?Ser?Gly?Asp?Val?Leu?Asp?Ser?Ile?Gly?Gly?Val

130 135 140Val?Val?Pro145<210>3<211>784<212>DNA<213>Helianthus?tuberosus<220><221>CDS<222>(48)..(491)<223><400>3cacaacataa?gttacttaag?aagctttgaa?tatgcataga?ccagaaa?atg?gct?gcc 56

Met?Ala?Ala

1agt?gac?att?ggg?gta?cag?gcc?gga?cca?tgg?gga?ggt?aat?ggt?gga?aaa 104Ser?Asp?Ile?Gly?Val?Gln?Ala?Gly?Pro?Trp?Gly?Gly?Asn?Gly?Gly?Lys

5 10 15cgc?tgg?ttg?caa?aca?gct?cat?ggc?ggt?aag?att?act?tcg?atc?att?atc 152Arg?Trp?Leu?Gln?Thr?Ala?His?Gly?Gly?Lys?Ile?Thr?Ser?Ile?Ile?Ile20 25 30 35aaa?ggc?gga?act?tgt?att?ttc?tcc?atc?cag?ttc?gta?tat?agg?gac?aaa 200Lys?Gly?Gly?Thr?Cys?Ile?Phe?Ser?Ile?Gln?Phe?Val?Tyr?Arg?Asp?Lys

40 45 50gat?aat?att?gaa?cac?cat?tct?gga?caa?ttt?ggt?gtt?cag?ggt?gac?aaa 248Asp?Asn?Ile?Glu?His?His?Ser?Gly?Gln?Phe?Gly?Val?Gln?Gly?Asp?Lys

55 60 65gct?gaa?aca?att?acc?ttt?gcg?gac?gat?gag?gac?atc?act?ggg?atc?agc 296Ala?Glu?Thr?Ile?Thr?Phe?Ala?Asp?Asp?Glu?Asp?Ile?Thr?Gly?Ile?Ser

70 75 80gga?act?ttc?gga?gca?tat?tat?cag?atg?aca?gtt?gtt?aca?tcg?ctc?act 344Gly?Thr?Phe?Gly?Ala?Tyr?Tyr?Gln?Met?Thr?Val?Val?Thr?Ser?Leu?Thr

85 90 95ttt?aag?acc?aac?aaa?aag?gtt?tat?ggg?cca?ttc?ggt?acg?gtg?gct?ggt 392Phe?Lys?Thr?Asn?Lys?Lys?Val?Tyr?Gly?Pro?Phe?Gly?Thr?Val?Ala?Gly100 105 110 115tcg?agt?ttc?tca?ctg?cct?cta?act?aag?ggt?aag?ttt?gcc?gga?ttc?ttt 440Ser?Ser?Phe?Ser?Leu?Pro?Leu?Thr?Lys?Gly?Lys?Phe?Ala?Gly?Phe?Phe

120 125 130gga?aac?agt?gga?gac?gtc?ctt?gac?tcg?att?ggt?ggc?gta?gtt?gtt?cct 488Gly?Asn?Ser?Gly?Asp?Val?Leu?Asp?Ser?Ile?Gly?Gly?Val?Val?Val?Pro

135 140 145taa?tcacaacgcc?tgatatgatg?aaaataagaa?gttaaggaaa?gacttcacgt 541ttgtaaaaga?gttgtgacat?tcagctcgta?tttgtgtgta?tgcatctttc?aatttcgtct 601ctcgtatttg?tgtgtatgca?tctttcaact?tcgtctctcg?tttttctcgg?gaaataagct 661tttatgatat?attacaataa?gcacgagctt?gtcgtgcact?agacttctgt?tctccgcaaa 721aggttctaaa?tgatcatttt?tatctaaaag?ttaacgaaaa?aaaaaaaaaa?aaaaaaaaaa 781aaa 784<210>4<211>147<212>PRT<213>Helianthus?tuberosus<400>4Met?Ala?Ala?Ser?Asp?Ile?Gly?Val?Gln?Ala?Gly?Pro?Trp?Gly?Gly?Asn1 5 10 15Gly?Gly?Lys?Arg?Trp?Leu?Gln?Thr?Ala?His?Gly?Gly?Lys?Ile?Thr?Ser

20 25 30Ile?Ile?Ile?Lys?Gly?Gly?Thr?Cys?Ile?Phe?Ser?Ile?Gln?Phe?Val?Tyr

35 40 45Arg?Asp?Lys?Asp?Asn?Ile?Glu?His?His?Ser?Gly?Gln?Phe?Gly?Val?Gln

50 55 60Gly?Asp?Lys?Ala?Glu?Thr?Ile?Thr?Phe?Ala?Asp?Asp?Glu?Asp?Ile?Thr65 70 75 80Gly?Ile?Ser?Gly?Thr?Phe?Gly?Ala?Tyr?Tyr?Gln?Met?Thr?Val?Val?Thr

85 90 95Ser?Leu?Thr?Phe?Lys?Thr?Asn?Lys?Lys?Val?Tyr?Gly?Pro?Phe?Gly?Thr

100 105 110Val?Ala?Gly?Ser?Ser?Phe?Ser?Leu?Pro?Leu?Thr?Lys?Gly?Lys?Phe?Ala

115 120 125Gly?Phe?Phe?Gly?Asn?Ser?Gly?Asp?Val?Leu?Asp?Ser?Ile?Gly?Gly?Val

130 135 140Val?Val?Pro145<210>5<211>752<212>DNA<213>Helianthus?tuberosus<220><221>CDS<222>(47)..(490)<223><400>5cacaacataa?gttacttaag?aagctttgaa?tttgcataac?cagaaa?atg?gct?gcc 55

Met?Ala?Ala

1agt?gac?att?ggg?gta?cag?gcc?gga?cca?tgg?gga?ggt?aat?ggt?gga?aaa 103Ser?Asp?Ile?Gly?Val?Gln?Ala?Gly?Pro?Trp?Gly?Gly?Asn?Gly?Gly?Lys

5 10 15cgc?tgg?ttg?caa?aca?gct?cat?ggc?ggt?aag?att?act?gcg?atc?att?atc 151Arg?Trp?Leu?Gln?Thr?Ala?His?Gly?Gly?Lys?Ile?Thr?Ala?Ile?Ile?Ile20 25 30 35aaa?ggc?gga?act?tgt?att?ttc?tcc?atc?cag?ttc?gta?tat?aag?gac?aaa 199Lys?Gly?Gly?Thr?Cys?Ile?Phe?Ser?Ile?Gln?Phe?Val?Tyr?Lys?Asp?Lys

40 45 50gat?aat?att?gaa?tac?ctt?tct?gga?caa?ttt?ggt?gtt?cag?ggt?gac?aaa 247Asp?Asn?Ile?Glu?Tyr?Leu?Ser?Gly?Gln?Phe?Gly?Val?Gln?Gly?Asp?Lys

55 60 65gct?gaa?aca?att?acc?ttt?gcg?gac?gat?gag?gac?atc?act?ggg?atc?agc 295Ala?Glu?Thr?Ile?Thr?Phe?Ala?Asp?Asp?Glu?Asp?Ile?Thr?Gly?Ile?Ser

70 75 80gga?act?ttc?gga?gca?tat?tat?cag?atg?aca?gtt?gtt?aca?tcg?ctc?act 343Gly?Thr?Phe?Gly?Ala?Tyr?Tyr?Gln?Met?Thr?Val?Val?Thr?Ser?Leu?Thr

85 90 95ttt?cag?acc?aac?aaa?aag?gtt?tat?ggg?cca?ttc?ggc?aca?gtg?gct?ggt 391Phe?Gln?Thr?Asn?Lys?Lys?Val?Tyr?Gly?Pro?Phe?Gly?Thr?Val?Ala?Gly100 105 110 115tcg?cgt?ttc?tca?ctg?cct?cta?act?aag?ggt?aag?ttt?gcc?gga?ttc?ttt 439Ser?Arg?Phe?Ser?Leu?Pro?Leu?Thr?Lys?Gly?Lys?Phe?Ala?Gly?Phe?Phe

120 125 130ggg?aac?agt?gga?gac?gtc?ctt?gac?tcg?att?ggt?ggc?gta?gtt?gtt?cct 487Gly?Asn?Ser?Gly?Asp?Val?Leu?Asp?Ser?Ile?Gly?Gly?Val?Val?Val?Pro

135 140 145taa?tcacaaccgc?ctgatatgat?gaaaataaga?agttaaggaa?agacttcacg 540tttgtaaaag?agttgtggca?ttcagctcgt?atgtgtgtgt?atgcatcttt?caatttcgtc 600tctcgttttt?ctcgggaaat?aagcttttat?gatatattcc?aataagcccg?agcttgtcgt 660gcactagact?tttgttctcc?gcaaaaggtt?ttaaatgatc?attaccatat?tattaatttc 720ccaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aa 752<210>6<211>147<212>PRT<213>Helianthus?tuberosus<400>6Met?Ala?Ala?Ser?Asp?Ile?Gly?Val?Gln?Ala?Gly?Pro?Trp?Gly?Gly?Asn1 5 10 15Gly?Gly?Lys?Arg?Trp?Leu?Gln?Thr?Ala?His?Gly?Gly?Lys?Ile?Thr?Ala

20 25 30Ile?Ile?Ile?Lys?Gly?Gly?Thr?Cys?Ile?Phe?Ser?Ile?Gln?Phe?Val?Tyr

35 40 45Lys?Asp?Lys?Asp?Asn?Ile?Glu?Tyr?Leu?Ser?Gly?Gln?Phe?Gly?Val?Gln

50 55 60Gly?Asp?Lys?Ala?Glu?Thr?Ile?Thr?Phe?Ala?Asp?Asp?Glu?Asp?Ile?Thr65 70 75 80Gly?Ile?Ser?Gly?Thr?Phe?Gly?Ala?Tyr?Tyr?Gln?Met?Thr?Val?Val?Thr

85 90 95Ser?Leu?Thr?Phe?Gln?Thr?Asn?Lys?Lys?Val?Tyr?Gly?Pro?Phe?Gly?Thr

100 105 110Val?Ala?Gly?Ser?Arg?Phe?Ser?Leu?Pro?Leu?Thr?Lys?Gly?Lys?Phe?Ala

115 120 125Gly?Phe?Phe?Gly?Asn?Ser?Gly?Asp?Val?Leu?Asp?Ser?Ile?Gly?Gly?Val

130 135 140Val?Val?Pro145<210>7<211>718<212>DNA<213>Helianthus?tuberosus<220><221>CDS<222>(48)..(491)<223><400>7cacaacataa?gttacttaag?aagctttgaa?tttgcacaga?ccagaaa?atg?gct?gcc 56

Met?Ala?Ala

1agt?gac?gtt?gcg?gta?cag?gcc?gga?cca?tgg?gga?ggt?aat?ggt?gga?aaa 104Ser?Asp?Val?Ala?Val?Gln?Ala?Gly?Pro?Trp?Gly?Gly?Asn?Gly?Gly?Lys

5 10 15cgc?tgg?ttg?caa?aca?gct?cat?ggt?ggt?aag?att?act?tcg?atc?att?atc 152Arg?Trp?Leu?Gln?Thr?Ala?His?Gly?Gly?Lys?Ile?Thr?Ser?Ile?Ile?Ile20 25 30 35aaa?ggc?gga?act?tgt?att?ttc?tcc?atc?cag?ttc?gca?tat?aag?gac?aaa 200Lys?Gly?Gly?Thr?Cys?Ile?Phe?Ser?Ile?Gln?Phe?Ala?Tyr?Lys?Asp?Lys

40 45 50gat?aat?att?gaa?tac?ctt?tct?gga?caa?ttt?ggt?gtt?cag?ggt?gac?aaa 248Asp?Asn?Ile?Glu?Tyr?Leu?Ser?Gly?Gln?Phe?Gly?Val?Gln?Gly?Asp?Lys

55 60 65gct?gaa?aca?att?acc?ttt?gcg?gac?aat?gag?gac?atc?act?gcg?atc?agc 296Ala?Glu?Thr?Ile?Thr?Phe?Ala?Asp?Asn?Glu?Asp?Ile?Thr?Ala?Ile?Ser

70 75 80gga?act?ttc?gga?gca?tat?tat?caa?atg?aca?gtt?gtt?aca?tcg?ctc?act 344Gly?Thr?Phe?Gly?Ala?Tyr?Tyr?Gln?Met?Thr?Val?Val?Thr?Ser?Leu?Thr

85 90 95ttt?cag?acc?aac?aaa?aag?gtt?tat?ggg?cca?ttc?ggc?acg?gtg?gct?agt 392Phe?Gln?Thr?Asn?Lys?Lys?Val?Tyr?Gly?Pro?Phe?Gly?Thr?Val?Ala?Ser100 105 110 115tcg?agt?ttc?tca?ctg?cct?cca?act?aag?ggt?aag?ttt?gcc?gga?ttc?ttt 440Ser?Ser?Phe?Ser?Leu?Pro?Pro?Thr?Lys?Gly?Lys?Phe?Ala?Gly?Phe?Phe

120 125 130ggg?aac?agt?gga?gac?gtg?ctt?gac?tcg?att?ggt?ggc?gta?gtt?gtt?cct 488Gly?Asn?Ser?Gly?Asp?Val?Leu?Asp?Ser?Ile?Gly?Gly?Val?Val?Val?Pro

135 140 145taa?tcacaacgcc?tgatatgatg?aaaataagaa?gttaaggaaa?gacttcacgt 541tcgtaaaaga?gttgtgacat?tcagctcgta?tttgtgtgta?tgcatctttc?aatttcgtct 601ctcgtatttg?tgtgtatggg?aaataagctt?ttatgatata?ttacaataag?cacgagcttg 661tcgtgcacta?gacttctgtt?ctccggtaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaa 718<210>8<211>147<212>PRT<213>Helianthus?tuberosus<400>8Met?Ala?Ala?Ser?Asp?Val?Ala?Val?Gln?Ala?Gly?Pro?Trp?Gly?Gly?Asn1 5 10 15Gly?Gly?Lys?Arg?Trp?Leu?Gln?Thr?Ala?His?Gly?Gly?Lys?Ile?Thr?Ser

20 25 30Ile?Ile?Ile?Lys?Gly?Gly?Thr?Cys?Ile?Phe?Ser?Ile?Gln?Phe?Ala?Tyr

35 40 45Lys?Asp?Lys?Asp?Asn?Ile?Glu?Tyr?Leu?Ser?Gly?Gln?Phe?Gly?Val?Gln

50 55 60Gly?Asp?Lys?Ala?Glu?Thr?Ile?Thr?Phe?Ala?Asp?Asn?Glu?Asp?Ile?Thr65 70 75 80Ala?Ile?Ser?Gly?Thr?Phe?Gly?Ala?Tyr?Tyr?Gln?Met?Thr?Val?Val?Thr

85 90 95Ser?Leu?Thr?Phe?Gln?Thr?Asn?Lys?Lys?Val?Tyr?Gly?Pro?Phe?Gly?Thr

100 105 110Val?Ala?Ser?Ser?Ser?Phe?Ser?Leu?Pro?Pro?Thr?Lys?Gly?Lys?Phe?Ala

115 120 125Gly?Phe?Phe?Gly?Asn?Ser?Gly?Asp?Val?Leu?Asp?Ser?Ile?Gly?Gly?Val

130 135 140Val?Val?Pro145

Claims (10)

1. helianthus tuberosus agglutinin albumen, it has SEQ ID NO:2, SEQ ID NO:4, aminoacid sequence shown in SEQID NO:6 or the SEQ ID NO:8.

2. coding is by the described proteic gene of claim 1.

3. according to the described gene of claim 2, it has following feature:

(a) has SEQ ID NO:1, SEQ ID NO:3, the nucleotide sequence shown in SEQ ID NO:5 or the SEQ ID NO:7;

(b) nucleotide sequence that causes because of the degeneracy of codon is different with the sequence described in the claim 3 (a), but still the proteic nucleic acid molecule of the described aminoacid sequence of coding claim 1;

(c) be cDNA molecule or genomic dna molecule.

4. one kind is utilized the generation of helianthus tuberosus agglutinin gene to have the vegetable cell of insect-resistance and the method for plant, and this method may further comprise the steps:

(a) upstream extremity of helianthus tuberosus agglutinin gene is connected on sense orientation after the nucleic acid fragment of promotor, its downstream end is connected in before a kind of nucleic acid fragment of the suitable regulating and controlling sequence that is used to control Transcription Termination, and then makes up plant expression vector;

(b) with the plant expression vector transformed plant cells that builds;

(c) vegetable cell after will transforming under the suitable culture condition is regenerated, and obtains expressing the plant of goal gene.

5. in accordance with the method for claim 4, described insect-resistance is meant the ability of homoptera pest-resisting.

6. in accordance with the method for claim 4, it is characterized in that described promotor comprises constitutive promoter, organ specific promoters, tissue-specific promoter, inducible promoter or the combined promoter of being made up of promotor and controlling element.

7. in accordance with the method for claim 4, it is characterized in that used method for transformation comprises the method for transformation of method for transformation, electric shocking method, pollen tube passage method or the integral level of agriculture bacillus mediated method for transformation, particle bombardment, protoplastis mediation.

8. in accordance with the method for claim 4, described plant is characterised in that this plant is monocotyledons or dicotyledons.

9. in accordance with the method for claim 8, described plant is paddy rice, wheat, barley, perhaps tobacco, cotton, soybean, sweet potato, potato, Chinese cabbage, green vegetables, celery, green pepper.

10. the plant expression vector that contains the described gene of claim 2.

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