CN103045497A - Soft-rot Erwinia with plant immune activation effect, immune activation protein extracted and application thereof - Google Patents

Abstract

The invention relates to soft-rot Erwinia with a plant immune activation effect, an immune activation protein extracted and an application thereof. The soft-rot Erwinia is preserved Chinese Common Microorganism Culture Preservation Management Center on 15th, August, 2012, with the preserving number of CGMCCNO.6433. The soft-rot Erwinia obtained in the invention can induce plants to generate resistance response so as to have the immune activation to plants. Through verification, material in the soft-rot Erwinia with the immune activation effect is a protein generated, wherein the protein is named EccPR protein for plant immune activation, so that resistance gene expression in plants is remarkably improved, thereby effectively inducing plants to have resistance effect to pathogenic bacteria.

Description

A kind of soft rot Erwinia, immuno-stimulating albumen and application thereof from wherein extracting with plant immunization activation

Technical field

The present invention relates to a kind of soft rot Erwinia, immuno-stimulating albumen and application thereof from wherein extracting with plant immunization activation.

Background technology

China is the premier large agricultural country in the world today, but owing to many-sided reason, frequency and area that great disease occurs obviously increase, and the production loss that causes is all about 10%.Hebei province staple crops, cash crop such as wheat, corn, cotton etc. and booth vegetable every year, due to illness the loss of insect pest also was huge, can reach billions of units according to estimates.In order to tackle serious corps diseases, the agricultural producer uses chemical pesticide in a large number.Using also of chemical pesticide made huge contribution to the control of corps diseases really.But using in a large number of chemical pesticide also showing serious negative impact.Therefore, in the world research, development and production safely, have no side effect, development priority that environment amenable biotechnological formulation becomes new industry.Along with the cries such as biological control, green agriculture grow to even greater heights, the research of plant disease-resistant immunological technique becomes the important research field of in recent years rising.

The disease-resistant activator of plant immunization is some biologies and abiotic factor, physiological status that can the inducing plant uniqueness, plant can be better or the physiological status of faster or not only good but also fast allotment defense response, make the state of plant defense disease and pest be in critical, reach and be triggered at any moment, when disease when infecting plant, plant can excite the resistance level of plant integral body at short notice by means of inducible factor, transfer the immunological competence of whole plant, the expression of activated plant self disease-resistant related gene, infecting of opposing pathogen, self does not work to pathogenic bacteria, pathogenic bacteria is developed immunity to drugs, and consumption is low, efficiently, the plant disease-resistant spectrum of inducing is wide, disease-resistant longer duration, this Sustainable Control system for Plant diseases has great importance.

Soft rot Erwinia is a kind of widely pathogenic bacterium, mainly occurs on Chinese cabbage, Caulis et Folium Brassicae capitatae, cauliflower, the potatoes and other crops mainly cause harm leaf, stem and stem tuber.The leaf Lao Ye near the ground that catches an illness falls ill first, and sick section is irregular dun scab, rots during high humidity.Stem catches an illness and starts from wound more, spreads to stem again, and myeloid tissue is rotted in the rear stem, the tool stench, and sick stem top branches and leaves are wilted sagging, the leaf flavescence.Stem tuber is caught an illness and many is caused by the cortex wound, just is water soaking mode, and the disintegration of rear potato block organization gives out a foul smell.

Summary of the invention

Purpose of the present invention provides a kind of soft rot Erwinia, immuno-stimulating albumen and application thereof from wherein extracting with plant immunization activation.

The technical scheme that the present invention takes:

A kind of Erwinia carotovora subsp.carotovora with plant immunization activation Erwinia carotovoraSubsp. CarotovoraStrain Ecc 601 is preserved in Chinese common micro-organisms culture presevation administrative center on August 15th, 2012, and deposit number is CGMCC No. 6433.

Described soft rot Erwinia Erwinia carotovoraSubsp. CarotovoraThe immuno-stimulating albumen that extracts among the strain Ecc 601, its extracting method is as follows:

(1) the mini-Tn5-Km transposon is inserted soft rot Erwinia Erwinia carotovoraSubsp. CarotovoraAmong the strain Ecc 601, filter out that can not to make tobacco leaf anaphylaxis occur be the mutant bacteria that occurs necrotic plaque on the blade;

(2) utilize Km label on the transposon mini-Tn5-Km, the dna fragmentation by two ends, transposon insertion point in the RACE-PCR amplification said mutation bacterium after the order-checking splicing, obtains dna sequence dna;

(3) dna sequence dna that step (2) is obtained is connected on the pET30a (+), and it is transferred among the intestinal bacteria E.coli BL21, obtains engineering bacteria E.coli BL21(pET30a-EccPR);

(4) with engineering bacteria E.coli BL21(pET30a-EccPR) inducing culture obtains immuno-stimulating albumen with plant immunization activation.

The application of described immuno-stimulating albumen.

Beneficial effect of the present invention: the soft rot Erwinia that the present invention obtains can produce the resistance reaction by inducing plant itself, and plant is played Immunestimulatory effect.Empirical tests, the material that plays Immunestimulatory effect in this soft rot Erwinia is a kind of albumen of its generation, and with its called after EccPR albumen, this albumen is used for plant immunization and activates, the interior tolerant gene expression of plant materials is obviously improved, thereby inducing plant play the resistance effect to pathogenic bacteria effectively.

Embodiment

The present invention's screening obtains a kind of soft rot Erwinia with plant immunization activation Erwinia carotovoraSubsp. CarotovoraStrain Ecc 601, this bacterial strain can not make Arabidopis thaliana catch an illness, and this bacterium is inoculated in tobacco leaf during as the pathogen infection Arabidopis thaliana, and tobacco leaf can anaphylaxis, necrotic plaque occurs.

The present invention is from soft rot Erwinia Erwinia carotovoraSubsp. CarotovoraThe process of extracting EccPR albumen among the strain Ecc 601 is as follows:

(1) the mini-Tn5-Km transposon is inserted soft rot Erwinia Erwinia carotovoraSubsp. CarotovoraObtain the sudden change strain library among the strain Ecc 601, filter out and to make tobacco leaf anaphylactoid mutant bacteria occur Erwinia carotovora subsp. Carotovora strain Ecc 601108, namely the insertion of transposon has destroyed soft rot Erwinia in this bacterial strain Erwinia carotovoraSubsp. CarotovoraCan make plant pathogenic bacteria be produced the structure of the functional unit of resistance among the strain Ecc 601;

(2) utilize Km label on the transposon mini-Tn5-Km, by the dna fragmentation at RACE-PCR amplification two ends, transposon insertion point, after the order-checking splicing, obtain dna sequence dna;

(3) dna sequence dna that step (2) is obtained is connected on the pET30a (+), and it is transferred among the intestinal bacteria E.coli BL21, obtains engineering bacteria E.coli BL21(pET30a-EccPR);

(4) with engineering bacteria E.coli BL21(pET30a-EccPR) inducing culture obtains immuno-stimulating albumen EccPR with plant immunization activation, and its detailed process is:

With engineering bacteria E.coli BL21(pET30a-EccPR) single bacterium colony provokes and carry out incubated overnight in the LB substratum under 37 ℃, with it as mother's kind, second day is planted mother and is inoculated in the liquid LB substratum with 1% inoculum size, under 37 ℃, cultivated 5-7 hour, the IPTG that adds 0.4mM in its nutrient solution carries out inducing culture, after inducing 6-8 hour, carry out this nutrient solution centrifugal, the thalline that obtains is then with its aqueous suspension and boiled 10-15 minute, after centrifugal with the supernatant lyophilize, acquisition contains the crude protein of 70-80% fermentation immuno-stimulating albumen EccPR, if obtain pure immuno-stimulating albumen EccPR, then it is crossed chromatography column and can obtain sterling immuno-stimulating albumen EccPR.

The EccPR albumen that the present invention obtains produces in the disease resistance at inducing plant and uses:

Be that the EccPR immuno-stimulating protein solution of 1mg/ml sprays application to respectively on Arabidopis thaliana, cucumber and the tobacco with concentration, sprayed once in per 7 days that spray Tot Prot is the 20-25mg/ strain at every turn, altogether sprays 3 times.After spraying for the last time 20 days, at Arabidopis thaliana inoculation pseudomonas syringae DC3000, inoculate wilt cucumber respectively, at tobacco inoculation soft rot Erwinia Erwinia carotovora subsp. Carotovora strain Ecc.Do contrast with the plant individual that does not spray EccPR immuno-stimulating protein solution simultaneously.Cultivate after 72 hours, find on the plant that does not spray activator EccPR, all to catch an illness, and the rate of catching an illness is 20% on the plant that sprays, and these plants that catch an illness have all produced anaphylaxis.Pathogenic bacteria number in the plant leaf of processing with activator EccPR has reduced by 45% than untreated, and corresponding tolerant gene expression all is significantly improved: PR1 improves 7.3 times, PR5 improves 1.8 times, PDF1.2 improves 11.7 times, Thi2.1 improves 21.1 times, and VSP2.1 improves 43.4 times.These explanations EccPR immuno-stimulating albumen is a kind of effective resistance inductor, and effectively inducing plant plays the resistance effect to pathogenic bacteria, and this using method can produce resistance by best inducing plant.

EccPR albumen also has the effect that promotes seed germination and Promoting plant growth:

Immuno-stimulating albumen EccPR sterling is dissolved in the water, prepares respectively the solution of different concns, strength of solution is respectively 0.01mg/ml, 0.1mg/ml, 1mg/ml, 10mg/ml, 100mg/ml.The immuno-stimulating albumen EccPR of these different concns was soaked respectively Arabidopis thaliana, cucumber and tobacco seed after 2 hours, the normal seed of cultivating, the concentration that found that immuno-stimulating albumen EccPR is during more than or equal to 0.1 mg/ml, can promote this three kinds of Seed germination speed, improve the germination rate of these three kinds of seeds.And find that 1mg/ml improves the Seed germination rate and reaches 21%.

Arabidopis thaliana, cucumber and tobacco seedling are cultivated in containing the MS liquid nutrient medium of EccPR immuno-stimulating albumen that final concentration is 1mg/ml respectively, cultivate after 20 days, the root of Arabidopis thaliana, cucumber and tobacco is long to increase respectively 12%, 5%, 10%, and fresh weight increases respectively 16%, 8%, 12%.These explanations EccPR albumen can promote the growth of plant.

Claims (3)

1. soft rot Erwinia with plant immunization activation Erwinia carotovoraSubsp. CarotovoraStrain Ecc 601 is characterized in that it is preserved in Chinese common micro-organisms culture presevation administrative center on August 15th, 2012, and deposit number is CGMCC No. 6433.

2. by soft rot Erwinia claimed in claim 1 Erwinia carotovoraSubsp. CarotovoraThe immuno-stimulating albumen that extracts among the strain Ecc 601 is characterized in that its extracting method is as follows:

(1) the mini-Tn5-Km transposon is inserted soft rot Erwinia Erwinia carotovoraSubsp. CarotovoraAmong the strain Ecc 601, filter out that can not to make tobacco leaf anaphylaxis occur be the mutant bacteria that occurs necrotic plaque on the blade;

(2) utilize Km label on the transposon mini-Tn5-Km, the dna fragmentation by two ends, transposon insertion point in the RACE-PCR amplification said mutation bacterium after the order-checking splicing, obtains dna sequence dna;

(3) dna sequence dna that step (2) is obtained is connected on the pET30a (+), and it is transferred among the intestinal bacteria E.coli BL21, obtains engineering bacteria E.coli BL21(pET30a-EccPR);

(4) with engineering bacteria E.coli BL21(pET30a-EccPR) inducing culture obtains immuno-stimulating albumen with plant immunization activation.

3. the application of immuno-stimulating albumen claimed in claim 2.