CN100491529C - HrpN gene for encoding plant multifunction activity and broad spectrum resistance cell signal factor and its expression product and use - Google Patents

Abstract

The present invention discloses a kind of hrpN gene of cell signal factor encoding multifunctional activity and broad spectrum resistance and its expression product and application. The gene of the present invention is originated from Erwinia carotovora subsp.carotovora strain, has the nucleotide sequence of SEQ ID No.1. The gene has wide application foreground in agricultural breeding and transgenic plant related to regulating plant's growth and development and preventing and controlling diseases and pests. The gene coded protein has the amino acid sequence of SEQ ID No.2, and possesses several functional activities of inducing plant to generate broad spectrum disease resistance, pest expelling resistance and stress tolerance, promoting plant growth and development and raising yield, so that the relevant products may be used as biological control agent for plant growth and development regulation and disease and pest prevention and control.

Description

The hrpN gene of a kind of multifunctional activity of coded plant and broad spectrum resistante cell singal gene and expression product and application

Technical field

The invention belongs to the biological gene engineering field, relate to the hrpN gene and the expression product thereof of a kind of multifunctional activity of coded plant and broad spectrum resistante cell singal gene, and this gene and its expression product are in the application of aspects such as improvement plant quality.

Background technology

The microbial proteinous agricultural chemicals is that multiple kinds of crops is had the very class protein medicine of strong biological activity.Can be divided into traditional microbial proteinous agricultural chemicals and novel microorganism albumen agricultural chemicals again by mechanism of action difference.Tradition microbial proteinous agricultural chemicals mainly is the insecticidal crystal protein (ICP) from bacillus thuringiensis (Bt), novel microorganism albumen agricultural chemicals mainly is an albumen exciton class material, the difference of it and traditional microbial proteinous agricultural chemicals maximum is its directly kill pests and pathogen, but excite the disease-resistant, insect-prevention expression of gene of plant self, and promote growth and development of plants.At present the representative novel microorganism albumen agricultural chemicals of research report mainly contains allergen protein (Harpin), latent ground albumen (Cryptogein) and activator (Activator) etc., and is wherein of greatest concern, what application prospect was arranged most is Harpin proteinoid exciton.

Harpin albumen mainly is can excite the anaphylactoid protein of plant (30-40kda) by the bacteriogenic class of Erwinia (Erwinia), but the expression of series of genes in the inducing plant body, the growing system of activated plant self and defence system, thereby make plant can resist infecting of multiple diseases and coercing of adverse circumstance, and obtain to promote to grow and improve the effect of volume increase.1992, the Wei Zhongmin of U.S. Cornell University etc. at first clone this class allergen protein gene from pears fire epidemic disease Erwinias (E.amylovora), and the anaphylaxis that proposes the protein induced plant of Harpin first may be relevant with its disease resistance effect, promoted the rise of novel microorganism albumen pesticide research thus.Through about 10 years effort; U.S. Eden biotechnology company as background successfully develops and develops the novel microorganism albumen agricultural chemicals Messenger with broad-spectrum disease resistance insect protected function; this product to the good effect in field crop and cash crop disease-resistant yield-increasing aspect and to the remarkable contribution of green non-pollution agricultural, once obtained the presidential Green Chemistry challenge prize that the U.S. environment protection council issues with it twice.

Modern molecule plant pathology discloses, plant pathogenetic bacteria is existing, and it is pathogenic, the ability that also has inducing plant to produce disease resistance and promote to grow, and determine that the gene of this ability is hrp (the hypersensitive response and pathogenicity) gene cluster of plant pathogenetic bacteria.The hrp gene is determining plant pathogenetic bacteria to excite anaphylaxis (hypersensitive response on non-host plants, ability HR) and the basic parasitics on host plant or pathogenic (parasitism or pathogenicity onhost plants) on non-host plant.HR is a kind of partial, cell dead form of programming fast of plant, be the initiatively result of resistance of plant, its resistance effect not only shows the restriction of pathogenetic bacteria being infected expansion, and the further interior systemic acquired resistance reaction that produces similar immunologic mechanism of inducing plant body.

Continue (Wei ZM, et al.Science, 1992,257 (5066): 85-88) successfully clone and express hrpN such as Wei Zhongmin _EaAfter the gene, people are cloned into the gene of coding Harpin proteinoid again in succession from E.carotovora subsp carotovora, E.chrysanthemi, P.s.syringae, P.s.pv.glycinea, P.s.tomato, R.solanacearum during the nearly last ten years, and successful expression these albumen, as Harpin _Ecc, 36kda (Mukherjee A., Mol.Plant-Microbe Interact.1997,10:462-471.); Harpin _Ech, 36kda (Bauer D.W., Mol.Plant Microbe Interact, 1995,8:484-491); Harpin _Pss, 34.7kda; Harpin _Psg, 35.3kda; Harpin _Pst, 36.5kda (Preston G., Mol Plant-Microbe Interact., 1995,8:717); Harpin _Xoo, 15.3kda, Harpin _Xooc, 15.6kda (Wen Weigang, plant pathology, 2001,31 (4): 295-300) etc.

Up to now, all these Harpin proteinoids almost all have following characterization of molecules: (1) wetting ability; (2) to thermally-stabilised, 100 ℃ handle 10 minutes can inactivation; (3) to Proteinase K and ultraviolet-sensitive; (4) be rich in glycine and lack halfcystine; (5) water-soluble acid albumen; (6) no quaternary structure.

Nearest studies show that the Harpin proteinoid may play an important role in the signal path of plant.The researchist handles Arabidopis thaliana with Harpin, find that Cl, Ca, K, H, Cu, Zn ionic channel and various oxydase are activated in the Arabidopis thaliana, these oxydase comprise ACC synthetic enzyme, Vitamin C oxidase, Cu/Zn superoxide-dismutase, Cu molecular chaperones precursor or the like.It has been generally acknowledged that now, extensively there is the proteic acceptor of Harpin in the plant cell wall, be that conjugated protein (Harpin-binding proteins, HrBP), Harpin can be by combining the many signal paths in the activated plant with these receptor proteins HrBP for Harpin.These signal paths roughly can be divided into following three classes: the activation of the signal path that (1) is relevant with plant disease-resistant and expelling parasite comprises the approach of anaphylaxis and necrocytosis, ion-exchange passage, salicylate pathway, jasmonic approach, phenylalanine ammonia lyase mediation and the approach of some other resistant gene mediation; (2), comprise that general growth regulator, plumular axis extend transcription factor, growth transcription factor, ethylene reaction element conjugated protein system and sucrose synthase and other embryos form enzyme etc. with the activation that promotes the signal path that plant-growth is relevant; (3) activation of the signal path relevant with plant stress-resistance comprises heat shock protein(HSP), COR gene and salt tolerant zinc albumen etc.May also there be the signal path of a more special plant that does not rely on the NPR1 mediation in addition to bacterial resistance.

Though Harpin albumen derives from pathogenic bacteria, as the nonspecific elicitor protein factor of a class, but can cause non-host plant generation anaphylaxis, and induce the increase of its disease resistance, insect-repellent and resistance, and promote to grow, improve output etc.More what is interesting is, changeing hrpN _EaOn the pears of gene, Harpin _EaThe host bacterial parasite E.amylovora that produces fire blight of pear is also shown very strong resistance.

Summary of the invention

The hrpN gene and the expression product thereof that the purpose of this invention is to provide a kind of multifunctional activity of coded plant and broad spectrum resistante cell singal gene, and the application of hrpN gene and its expression product.

The hrpN gene of a kind of multifunctional activity of coded plant provided by the present invention and broad spectrum resistante cell singal gene, name is called hrpN _CSSY002Its gene coding region has 1071 Nucleotide altogether, its nucleotide sequence such as SEQ ID No.1, this gene is from carrot soft rot erwinia (Erwinia carotovora subsp.carotovora) CSSY002 bacterial strain, Radix Dauci Sativae, Chinese cabbage, celery etc. are the host plants of this bacillus, and this gene is present on the chromosomal DNA of CSSY002 bacterial strain with hrpN gene cluster form.This gene can be used for promoting growth and development of plants; Strengthen the broad spectrum resistance of plant, comprise resistance various bacteria, fungi and virus; Strengthen the insect-repellent of plant; Strengthen the resistance (plant is to the tolerance of arid, severe cold, poor environment such as saline and alkaline) of plant; Prolong the storage time of crop product; Plastotype effect to potted flower, nursery stock.This gene can also be as genetic resources in addition, grows the Agricultural biotechnologies breeding relevant with prevention and elimination of disease and pests, transgenic plant and has wide application prospects with aspects such as genetic recombination body that other desirable genes recombination to construct has a commercial value in coordinate plant growth.The genetic recombination body that contains this gene also can be applicable to coordinate plant growth and grows aspects such as the Agricultural biotechnologies breeding relevant with prevention and elimination of disease and pests, transgenic plant.

The gene hrpN of a kind of multifunctional activity of coded plant and broad spectrum resistante cell singal gene _CSSY002Coded albumen Harpin _CSSY002SEQ ID No.2 in its aminoacid sequence such as the sequence table, have 356 amino-acid residues, 36.64kda, iso-electric point pI:5.9 is rich in glycine (Gly) and shortage halfcystine (Cys), is water-soluble acid albumen, no quaternary structure, and have the basic molecule attribute and the biologic activity of Harpin proteinoid.

Harpin _CSSY002Protein promote growth and development of plants, strengthen the broad spectrum resistance (comprising resistance) of plant, the insect-repellent that strengthens plant, the resistance (plant is to the tolerance of arid, severe cold, poor environment such as saline and alkaline) that strengthens plant, prolongation crop product to various bacteria, fungi and virus storage time, aspect the plastotype effect of potted flower and nursery stock, have wide application prospects.Therefore, this protein can be mixed with biological products, is applied to above aspect.

The present invention is about the gene hrpN of a kind of multifunctional activity of coded plant and broad spectrum resistante cell singal gene _CSSY002And expression product Harpin _CSSY002Protein, will specifically explain by following content:

1, hrpN _CSSY002The source hrpN of gene _CSSY002From carrot soft rot erwinia (Erwinia carotovora subsp.carotovora) CSSY002 bacterial strain, Radix Dauci Sativae, Chinese cabbage, celery are its host plants to gene from gene.This gene is present on the chromosomal DNA of CSSY002 bacterial strain with hrpN gene cluster form.

2, hrpN _CSSY002The clone of gene and order-checking (1) bacterial strain at first screens on the LB flat board, and carries out the active and HR detection of extracellular protease.(2) with the mono-clonal bacterium colony of choosing, under 28 ℃ in the LB liquid nutrient medium shaking culture 12 hours, centrifugal collection thalline, with the genomic dna separating kit separate, the purification of bacterial chromosomal DNA.(3) bacterial chromosomal dna partly digests through Sau3A, makes up the genomic library of carrot soft rot erwinia (Erwinia carotovora subsp.carotovora) CSSY002 bacterial strain with plasmid vector pLARF5.HrpN with carrot soft rot erwinia (Erwinia carotovora subsp.carotovora) ECC71 bacterial strain _EccGene (Wei ZM, et al.Science, 1992,257 (5066): 85-88) make molecular probe, screen from the CSSY002 genomic library by clone hybridization and differentiate hrpN ⁺Plasmid obtains to comprise the 1.9kb DNAs fragment of the whole coding region of hrpN gene, and further this fragment subclone is advanced in the pBluescript carrier.(5) carry out nucleotide sequencing with universal primer T7, wherein have the reading frame (reading frame) of a 1071bp to be hrpN _CSSY002The coding region of gene, sequence such as SEQ ID No.1.

3, hrpN _CSSY002The pcr amplification of gene and purifying are according to hrpN _CSSY002The nucleotide sequence of gene, design one couple of PCR amplimer, i.e. P1:5 '-TGT GGATCCATGCTTAATTCTCTTGGTGGCGGAG-3 ' and P2:5 '-TGT AAGCTTTAGCTGGAGAGCTTCTTCAACCC-3 '.Wherein G/GATCC is the restriction enzyme site of the restriction enzyme BamHI of introducing, and A/AGCTT is the restriction enzyme site of the restriction enzyme HindIII of introducing.Chromosomal DNA with the CSSY002 bacterial strain of purifying is made template, carries out the PCR reaction by following amplification parameter: 95 ℃ of pre-sex change 5min earlier, 1 circulation of 94 ℃/2min then; 94 ℃/15sec+55 ℃/30sec+72 ℃/24 circulations of 15sec; 94 ℃/1min+55 ℃/2min+72 ℃/a 3min1 circulation.Amplified production carries out purifying with PCR product purification test kit.

4, hrpN _CSSY002The structure hrpN of engineering strain _CSSY002The pcr amplification product of gene is after restriction enzyme BamH I and HindIII digestion, and the clone enters the BamH I-HindIII site of high-expression vector pET28a (+).With pET28a (+)-hrpN _CSSY002Plasmid transformation receptor bacterium E.coli JM109 (DE3) contains pET28a (+)-hrpN by selecting to press to filter out _CSSY002The positive expression engineering strain of plasmid.

5, hrpN _CSSY002Expression of gene product and detection hrpN thereof _CSSY002The expression of gene product is Harpin _CSSY002Protein.To contain hrpN _CSSY002Gene overexpression plasmid pET28a (+)-hrpN _CSSY002Colibacillus engineering strain E.coli JM109 (DE3) 37 ℃ of incubation growth in being added with the LB liquid nutrient medium of 50ml/L Km, treat OD ₆₀₀After reaching 0.7 (being that bacterium enters logarithmic phase), add inductor IPTG again, continue to cultivate after 6 hours centrifugal collection bacterial cell to final concentration 1mM.Bacterial cell on the bacteria samples swimming lane of electrophoresis offset plate, can present a remarkable wide and dense 35.55kda band through ultrasonic disruption, centrifugal, 12%SDS-PAGE gel electrophoresis, and it is exactly hrpN _CSSY002Expression of gene product Harpin _CSSY002Protein accounts for about 50% of bacterioprotein total amount.With Harpin _CSSY002The further separation and purification of protein, and to Harpin _CSSY002Protein carries out the Westernblot analysis and HR detects.

According to hrpN _CSSY002The nucleotide sequence of gene is inferred, Harpin _CSSY002Proteinic aminoacid sequence such as SEQ ID.No2.This protein has following attribute: (1) is made up of 356 amino-acid residues; (2) molecular weight 36.64kda; (3) iso-electric point pI:5.9; (4) be rich in glycine Gly, lack halfcystine Cys; (5) special stable to temperature, tolerance still had activity in 10-15 minute in 100 ℃ of boiling water; (6) there is not quaternary structure.

6, Harpin _CSSY002The biological activity assay method of the Harpin proteinoid that proteinic biologic activity and application thereof are generally acknowledged at present mainly contains: (1) anaphylaxis (hypersensitive response, HR), usually do tested plant with tobacco, this also is quick, the easiest method of Harpin proteinoid biological activity assay; (2) serological reaction, promptly make serum antibody with the pure product of Harpin albumen of standard, with this antiserum(antisera) unknown sample is carried out Western blot then and analyze,, can accurately detect the Harpin proteinoid in the unknown sample by antibody-antigen recognition reaction.

Harpin _CSSY002Protein has following main biological function: (1) promotes growth and development of plants, improves output; (2) broad spectrum resistance of enhancing plant comprises the resistance to various bacteria, fungi and virus; (3) make the insect-repellent of plant acquisition to some harmful insect; (4) resistance of enhancing plant; (5) storage time of prolongation plant prod; (6) potted flower, nursery stock there is good plastotype effect.

Harpin _CSSY002Protein is strengthening plant disease-resistant, expelling parasite, degeneration-resistant and promote growth and improve good result aspect the output.Harpin _CSSY002Protein can not cause the resistance of pathogen, also change that can inducing plant genetic construction (genomic dna), and this protein belongs to natural organic protein, nature can be very fast by photolysis or be degraded by microorganisms, do not have residual public hazards, to people and animals bird fish nontoxicity, in use need not special safeguard procedures, be the following ideal environment protection biological product that substitutes high poison, high residue chemical pesticide and chemical regulator.

At present various crops, vegetables, fruit, flowers and various economic plants are comprised that the disease-resistant expelling parasite of tens of kind of plant such as tobacco, tealeaves and short long effect of increasing production test and the product ratio widely with regard to it.Existing test-results shows Harpin _CSSY002Albumen is obvious to disease-resistant expelling parasite and the short long effect of increasing production of cucumber, tomato, potato, capsicum, tobacco, corn, strawberry etc., simultaneously to improve certain plants such as tobacco, quality of tea leaves also has good effect, and contrast effect is also very remarkable in similar Harpin product.Harpin _CSSY002Albumen can directly apply to growth and development of plant and regulate and the prevention and control of plant diseases, pest control, comprises the maintenance on food crop, economic plants, vegetables, fruit, flowers and gardens and grassland etc.; Using method has seed soaking, dresses seed, dips in root, seed pelleting, plant injection, foliage-spray, flowers and fruits spraying etc.; Using dosage is different because of using method and kind, and general 5 μ g/ml-80 μ g/ml are effective concentration; Use the back and promptly have an effect in 2-12 hour, sustainable about 20 days of drug effect, but season of growth medication 2-3 time.

Employed engineering bacteria E.coli JM109 (DE3) bacterial strain and hrpN among the present invention _CSSY002The widespread use in molecular biology research and the medicine industry at home and abroad of efficient expression vector pET28a (+) plasmid of gene, environmentally safe reliable.

The shortenings that uses among the present invention: LB (bacteria culture medium, yeast powder 5g/L+ peptone 10g/L+NaCl10g/L, pH7.0); Km (kantlex); IPTG (isopropylthio-beta galactose glycosides); PMSF (phenylmethylsulfonyl fluoride); Tris (Tutofusin tris).

Embodiment

Embodiment one hrpN _CSSY002The clone of gene and order-checking

(1) hrpN _CSSY002Gene is from carrot soft rot erwinia (Erwinia carotovora subsp.carotovora) CSSY002 bacterial strain, and this gene is present on the chromosomal DNA of CSSY002 bacterial strain with hrpN gene cluster form.Carrot soft rot erwinia (Erwinia carotovora subsp.carotovora) CSSY002 bacterial strain is at first screened on the LB flat board, and carry out the active and HR detection of extracellular protease.(2) with the mono-clonal bacterium colony of choosing, under 28 ℃ in the LB liquid nutrient medium shaking culture 12 hours, centrifugal collection thalline, with the genomic dna separating kit separate, the purification of bacterial chromosomal DNA.(3) bacterial chromosomal dna partly digests through Sau3A, makes up the genomic library of carrot soft rot erwinia (Erwinia carotovora subsp.carotovora) CSSY002 bacterial strain with plasmid vector pLARF5.(4) hrpN of usefulness carrot soft rot erwinia (Erwinia carotovora subsp.carotovora) ECC71 bacterial strain _EccGene (Wei ZM, et al.Science, 1992,257 (5066): 85-88) make molecular probe, screen from the CSSY002 genomic library by clone hybridization and differentiate hrpN ⁺Plasmid obtains to comprise the 1.9kb DNAs fragment of the whole coding region of hrpN gene, and further this fragment subclone is advanced in the pBluescript carrier.(5) carry out nucleotide sequencing with universal primer T7, wherein have the reading frame (readingframe) of a 1071bp to be hrpN _CSSY002The coding region of gene, sequence such as SEQ ID No.1.

Embodiment two hrpN _CSSY002The related application of gene

(1) with gene recombination technology with hrpN _CSSY002Gene clone is advanced in the Ti-plasmids carrier of anti-kantlex, forms hrpN _CSSY002-Ti-plasmids; (2) transform agrobatcerium cell with the hrpNEccsl-Ti plasmid, screening contains hrpN _CSSY002The positive expression Agrobacterium of-Ti-plasmids; (3) the Radix Dauci Sativae protoplastis that will be in regeneration wall period with contain hrpN _CSSY002The Agrobacterium of-Ti-plasmids was cultivated 36-48 hour together altogether, and centrifuge washing goes containing the carrot cell clone that screening transforms on the selection substratum of kantlex behind the bacterium; (4) carrot cell that transforms is cloned in cultivates into regeneration plant on the division culture medium, promptly obtain Radix Dauci Sativae hrpN _CSSY002The gene transgenic plant, this transfer-gen plant with do not change hrpN _CSSY002The Radix Dauci Sativae regeneration plant of gene is compared, and has obvious growth advantage and anti-soft rot characteristic.

Embodiment three hrpN _CSSY002The application of gene-correlation genetic recombination body

(1) with gene recombination technology with hrpN _CSSY002Gene and hrpNEa gene fusion form hrpN _CSSY002-hrp NEa fusion gene; (2) with hrpN _CSSY002-hrpNEa fusion gene cloning advances among the efficient expression plasmid carrier pETa (+) of anti-kantlex, forms hrpN _CSSY002-hrpNEa-pETa (+) plasmid; (3) use hrpN _CSSY002-hrpNEa-pETa (+) plasmid transforms engineering bacteria JM109 (DE3), selects to depress screening and contains hrpN adding kantlex _CSSY002The positive expression bacterial strain of-hrpNEa-pETa (+) plasmid; (4) the fusion rotein Harpin that expresses with this fusion gene of fermentative Production _CSSY002-HarpinEa, and by SDS PAGE detection Expression of Fusion Protein situation, thereby the stronger and wider Harpin class fusion rotein exciton of sphere of action of biologic activity obtained.

Embodiment four Harpin _CSSY002Proteinic preparation and purifying

1 strain preparation and large scale fermentation

(1) preparation original seed: will have hrp _NCSSY002Engineering bacteria E.coli JM109 (DE3) bacterial strain of gene efficient expression plasmid pET28a (+) is rule 37 ℃ of overnight incubation on the inclined-plane of glycerine-LB+Km20 μ g/ml.(2) preparation is female plants: well-grown mono-clonal colony inoculation on the original seed inclined-plane is shaken in the bottle in the 250ml that is added with 100ml glycerine-LB+Km20 μ g/ml, in 37 ℃ of 110rpm shaking culture 12h.(3) preparation is produced kind: mother is planted bacterium liquid be inoculated in by 1% volume ratio in the 5L fermentor tank that is added with 3L glycerine-LB+Km25 μ g/ml, cultivate 12h under 37 ℃ of 110rpm and the 0.5L/min air flow condition.(4) large scale fermentation: after the fermentor tank more than the 50L being added the Km of the glycerine-LB of 70% volume and final concentration 40 μ g/ml, in-situ sterilization 30min (120 ℃, 1.1MPa); Insert the seeding bacterium by 1% volume ratio then, 37 ℃ of 200rpm and 1L/min air flow condition bottom fermentation are cultivated; Treat that bacterium enters logarithmic phase (OD ₆₀₀=0.7) after, regulates rotating speed and air flow, to guarantee yeasting competent oxygen supply (DO〉30%) is arranged, and carry out logarithm feed supplement by stages by specific program; Before the bacterium logarithmic phase finishes 1-2 hour, by millipore filtration adding inductor IPTG to final concentration 1mM; Continue to induce fermentation 4-6 hour, can play jar.This method can realize the high-density or the high-efficient culture of engineering bacteria.

2 Harpin _CSSY002Proteinic separation and purifying

Harpin _CSSY002Protein mainly exists with inclusion body (inclusion body) form in Bacillus coli cells.Industrial scale operation Harpin _CSSY002During albumen, adopt the broken preparation of 8M urea.In the laboratory in order to obtain further Harpin _CSSY002The pure product of protein matter, can operate by follow procedure: centrifugal (5,000rpm, 10min) 4-6 hour Bacillus coli cells is induced in collection through IPTG, after the abundant suspension of 20mMTris damping fluid (pH9.0), add the N,O-Diacetylmuramidase (100mg/ml) of 10 μ l/25ml, ice bath 10min, add 0.5ml/25mlPMSF (20mg/ml), the broken 10min of ultrasonic wave (20KHz) adds the PMSF of 0.5ml/25ml again, ultrasonic disruption 10min again behind the mixing, 10, the centrifugal 10min of 000rpm gets supernatant liquor in 100 ℃ of water-bath 10min, cooling back 12, the centrifugal 10min of 000rpm collects supernatant liquor.The Harpin that promptly contains higher degree in this supernatant liquor _CSSY002Protein, concentration reach 3600 μ g/ml.

Embodiment five Harpin _CSSY002Proteinic using method

Harpin _CSSY002Protein method in the use is various, can be because of handling the different differences to some extent of material, into treatment sites, treatment time and processing intent.Specifically can use by the method for following introduction:

(1) seed soaking: at first water is with Harpin _CSSY002Protein is made into the concentration of 30-60 μ g/ml, then seed is dipped in wherein 8-12 hour.Suitable crop or the vegetable seed of doing vernalization of this method.After seed soaking finishes, with seed from Harpin _CSSY002Take out in the protein seed-soaking liquid, can vernalization or sowing.Emerge after a week, the height of seedling comparison is according to increasing more than 10%, and increment also improves more than 10%, and seedling stage anti-samping off ability also obviously strengthen.

(2) seed dressing: seed is moistening a little, the Harpin of adding grain weight 0.1% _CSSY002Protein formulation is fully mixed the back sowing thoroughly.The neat seedling of back seedling of emerging is strong, strengthens the ability of anti-samping off in seedling stage.

(3) dip in root: be suitable for transplanting the crop that grows surely, as melon and fruit, vegetables, cotton, corn etc.At first with Harpin _CSSY002Protein formulation is made into the aqueous solution of 30 μ g/ml, and the root that will be transplanted crop seedling then was immersed in this solution 1-2 hour.After the transplanting, plant recovers growth soon, and transplanting seedling time shortens, and the plant growing way is powerful.

(4) foliar spray: the whole growth of crop all can spray season.With Harpin _CSSY002Protein formulation is made into the aqueous solution of 15-20 μ g/ml, in seedling stage of crop, tillering phase, initial bloom stage, fruit phase or the filling stage use of spraying just.General sprayable 2-3 time of crop growing season.After the spraying, can improve crop alimentary bulk-growth amount 15-30%, improve output 10-20%, and whole growth can save the chemical pesticide more than 80% season.Some crop such as cucumber can be kept out 60% gray mold, the mosaic virus that tobacco can be kept out 60-80%.Also have certain degeneration-resistant (drought resisting, cold-resistant, salt tolerant alkali etc.) and anthelminthic effect in addition.

Embodiment six Harpin _CSSY002Proteinic applicating example

Tens of kinds of crops, vegetables, fruit, flowers have been made Harpin at present _CSSY002Proteinic application effect test, though the performance degree is different on the Different Crop kind, Harpin _CSSY002The effect of aspects such as proteinic short long volume increase, disease-resistant expelling parasite, raising resistance and potted flower, nursery stock plastotype is significant.Lifting several examples below is illustrated:

(1) corn: choose 4 leaf phase maize seedling fields of two 5 fens ground sizes, be used separately as contrast and treatment samples piece.Treating piece is the Harpin with 15-20 μ g/ml in 4 leaf phases and 6 leaf phases respectively _CSSY002Protein soln has been made foliar spray, does not use any agricultural chemicals in addition, and the management of others is the same with the contrast piece.Find that in process of the test in a week after the medication, the average plant height comparison for the treatment of piece exceeds 10-15cm according to piece, the corn northern and southern leaf blight sickness rate is also compared according to the low 20-30% of piece; Treating piece is bloomed to compare according to piece and is shifted to an earlier date 5-10 days simultaneously, and the filling stage, two luxuriant rates were apparently higher than contrast.The results back is output relatively, and the treating piece comparison is shone the piece volume increase more than 12%.

(2) cucumber: choose 3 leaf phase cucumber seedling fields of two 5 fens ground sizes, be used separately as contrast, Harpin _CSSY002The sample piece that protein is handled.The concentration of albumen treatment solution is 20 μ g/ml.Treating piece carried out foliar spray in 3 leaf phases, 5 leaf phases and 7 leaf phases respectively, and other management is with contrast.Test-results shows: Harpin _CSSY002Protein is handled, and the comparison of blooming was according to 8 days in advance; The mosaic disease comparison is according to reducing 65%; The comparison of grey mold handle is according to reducing 30%; Yield increased increases more than 23%.

(3) strawberry: choose 3 leaf phase of the wing-rooms on either side of a one-story house strawberry seedling canopy of about 2 minutes ground sizes, compare respectively and Harpin _CSSY002Protein is handled.Handle the Harpin of seedling canopy _CSSY002The protein working concentration is 20 μ g/ml.3 leaf phases began foliar spray, and later every interval sprayed once in 15 days, amounted to 5 times.The result shows that the comparison of processing canopy is done sth. in advance about 10 according to canopy and bloomed; Individual plant flower, the contrast of fruit rate average specific increase 2-3; The gray mold incidence obviously is less than contrast; The fresh fruit colour generation is good, the fruit type is excellent, the malformed fruit rate is low, and yield increased is increased production more than 25%.In addition, weigh, find that the dry-matter accumulation of processing canopy is also compared according to increasing 1-2% by the equivalent fresh fruit is dried.

(4) Cyclamen persicum: Cyclamen persicum is the potted flower kind that liked by masses, but the mortality ratio that caused by bacterial soft rot is very high, dormancy during high temperature, and also due to illness the abnormal leaf, the flower that cause of viral disease had a strong impact on its ornamental value and commercial value.We have chosen 400 basin 3-4 monthly age Cyclamen persicums and have done to be divided into two groups at random for the examination material, compare respectively and Harpin _CSSY002Protein is handled.Harpin _CSSY002Proteinic working concentration is 20 μ g/ml.Treatment process is a foliar spray, sprays once in every interval 10-15 days.The result shows, after one week of medication, the leaf look of treatment group deepens, glossy, and that plant type also obviously becomes is tall and straight, compact, live and take off, and can break the high temperature dormancy; After the medication one month, viral disease and spire that deformity is curled begins to be extended into gradually normal morphology due to illness in the past, the spire of the control group more and more deformity that then becomes, the petal of leaving also is deformity and twists.In addition, Harpin _CSSY002Protein also has certain effect of driveing to the trialeurodes vaporariorum on the Cyclamen persicum blade.

(5) tobacco: tobacco is the aphid susceptible plants.Harpin with 25 μ g/ml concentration _CSSY002Protein formulation was soaked seed 24-48 hour to tobacco seed under the 250C condition, and compare seed soaking with the 5mM potassium phosphate buffer of pH6.5 and handle, be sowed at respectively then in three culture plates and sprout, grouping is transplanted in same big Tanaka after waiting to emerge, subsequently the number of observed and recorded infection every day aphid.Found that the mean number of control treatment group individual plant infection in the 1st, 2,3,6,7,8,9,10 days aphid is respectively: 0,3,3,6,7,9,13,17; Harpin _CSSY002The mean number of albumen treatment group individual plant infection in the 1st, 2,3,6,7,8,9,10 days aphid is respectively: 0,0,0,1,0,0,2,2.

(6) African Flower of Garden Balsam: African Flower of Garden Balsam is a kind of common showy flowers of herbaceous plants of viewing and admiring.Get the African Flower of Garden Balsam of 10 basin growth potential basically identicals, divide to deal with and contrast 2 groups, every group 5 basin, treatment group and control group are used the Harpin of 25 μ g/ml concentration respectively _CSSY002Protein formulation and water carry out foliar spray, spray altogether 2 times, and each 7 days at interval, other management condition unanimity.After treating 15 days, 2 groups of materials are stopped water and fertilizer management (promptly stopping to water and applying fertilizer), found that after 15 days, the control group plant begins to occur withered and yellow withered, the mosaic virus outburst, and mosaic virus does not take place in treatment group plant growing way health.

(7) strawberry fruit is fresh-keeping: get 100 of the fresh strawberries of firm collection, become to handle and contrast 2 groups by 50 1 components, treatment group is with 30 μ g/ml Harpin _CSSY002Protein solution soaked 10 minutes, and control group is contained in respectively in 2 ventilative corbeils then with pure water logging 10 minutes, above coated with gauze, placed for 1 week under the room temperature, the result shows that the control group strawberry is softening more than 90%, color is dun, wherein 10% seriously goes rotten, and treatment group strawberry 90% above color is vivid, and the fruit grain is tender and crisp, have only several grains to darken, the deliquescing of fruit grain.

Sequence table

＜110〉Pairun Biological Science and Technology Co., Ltd., Chengdu

＜120〉the hrpN gene of a kind of multifunctional activity of coded plant and broad spectrum resistante cell singal gene and expression product and application

<160>2

<170>PatentIn?Version?2.1

<210>1

<211>1071

<212>DNA

＜213〉carrot soft rot Erwinia (Erwinia carotovora subsp.carotovora)

<221>1-1071

<400>1

<210>2

<211>356

<212>PRT

＜213〉carrot soft rot Erwinia (Erwinia carotovora subsp.carotovora)

<400>2

Claims (9)

1. the hrpN gene of multifunctional activity of coded plant and broad spectrum resistante cell singal gene, it is characterized in that: the nucleotide sequence of this gene is the SEQ ID No.1 in the sequence table.

2. the described hrpN gene of claim 1 is grown the application aspect the Agricultural biotechnologies breeding relevant with prevention and elimination of disease and pests or the application of the transgenic plant aspect relevant with prevention and elimination of disease and pests with the coordinate plant growth growth in coordinate plant growth.

3. the described hrpN gene of claim 1 is in the application of following either side: (1) promotes growth and development of plants; (2) broad spectrum resistance of plant; (3) insect-repellent of plant; (4) resistance of plant; (5) storage time of prolongation crop product; (6) to the plastotype effect of potted flower, nursery stock.

4. the genetic recombination body that contains the described hrpN gene of claim 1.

5. the described genetic recombination body of claim 4 is grown the application aspect the Agricultural biotechnologies breeding relevant with prevention and elimination of disease and pests or the application of the transgenic plant aspect relevant with prevention and elimination of disease and pests with the coordinate plant growth growth in coordinate plant growth.

6. the Harpin protein of the hrpN genes encoding of multifunctional activity of coded plant and broad spectrum resistante cell singal gene, it is characterized in that: this proteinic aminoacid sequence is the aminoacid sequence of SEQ ID No.2 in the sequence table, has following attribute: (1) has 356 amino-acid residues; (2) molecular weight 36.64kda; (3) iso-electric point pI:5.9; (4) be rich in glycine (Gly), lack halfcystine (Cys); (5) to temperature-stable, tolerance still had activity in 10-15 minute in 100 ℃ of boiling water; (6) there is not quaternary structure.

7. contain the proteinic biological products of the described Harpin of claim 6.

8. the described Harpin protein of claim 6 is in the application of following either side: (1) promotes growth and development of plants; (2) broad spectrum resistance of plant; (3) insect-repellent of plant; (4) resistance of plant; (5) storage time of prolongation crop product; (6) to the plastotype effect of potted flower, nursery stock.

9. the proteinic biological products of the described Harpin of claim 7 are in the application of following either side: (1) promotes growth and development of plants; (2) broad spectrum resistance of plant; (3) insect-repellent of plant; (4) resistance of plant; (5) storage time of prolongation crop product; (6) to the plastotype effect of potted flower, nursery stock.